University de Sherbrooke BASED CHEM OTHERAPY AND RADIATION: STUDIES ON CYTOTOXICITY,

University de Sherbrooke
CONCOMITANT TREATMENT OF COLORECTAL CANCER W ITH PLATINUMBASED CHEMOTHERAPY AND RADIATION: STUDIES ON CYTOTOXICITY,
PHARMACOKINETICS AND CONCOMITANT IN VITRO AND IN VIVO EFFECTS
By
Thititip TIPPAYAMONTRI
Departement de Medecine Nucleaire et Radiobiologie
Thesis submitted to the Faculte de medecine et des sciences de la santy
for the Degree of
Doctor of Philosophy (Ph.D.) in Radiation Sciences and Biomedical Imaging
Sherbrooke, Quebec, Canada
August 2013
Doctoral Committee:
Thesis Examiner
Prof. Thierry M. Muanza, McGill University
Thesis Examiner
Prof. Johannes van Lier, University de Sherbrooke
Thesis Examiner
Prof. David Mathieu, University de Sherbrooke
Thesis Supervisor
Prof. Rami Kotb, University de Sherbrooke
Thesis Supervisor
Prof. Leon Sanche, University de Sherbrooke
Thesis Supervisor
Prof. Benoit Paquette, University de Sherbrooke
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ABSTRACT
CONCOMITANT TREATMENT OF COLORECTAL CANCER WITH PLATINUMBASED CHEMOTHERAPY AND RADIATION: STUDIES ON CYTOTOXICITY,
PHARMACOKINETICS AND CONCOMITANT IN VITRO AND IN VIVO EFFECTS
By
Thititip Tippayamontri
Department of Nuclear Medicine and Radiobiology
Thesis submitted to the Facultd de medecine et des sciences de la sant6 for the graduation of
philisophiae doctor (Ph.D.) in sciences des radiations et imagerie biom&iicale, Faculty de
mddecine et des sciences de la sante, University de Sherbrooke, Sherbrooke, Quebec, Canada
J1H 5N4
Advances in curing rectal cancer came from successful chemoradiotherapy. Platinumbased drugs such as oxaliplatin have also been studied and integrated in treatment strategies
against rectal cancer. Although platinum-based drugs can act as radiosensitizers, their
radiosensitizing activity is limited by their narrow therapeutic index which avoids the dose
escalation. In addition, it is important also to optimize the schedule of drug administration
with radiation treatment to gain advantage of drug-radiation interactions and maximize tumor
response. We evaluated the new liposomal formulation of cisplatin and oxaliplatin
(Lipoplatin™ and Lipoxal™, respectively) that should increase the anticancer effectiveness
while minimizing the side effects.
We investigated different chemoradiation schedules to assess the best antitumor
efficacy with regard to our hypothesis of the “true” concomitant chemoradiotherapy which
consist in the addition of radiation at the time of maximum accumulation o f platinum in the
DNA of cancer cells. We performed in vitro studies using human colorectal carcinoma
HCT116, and in vivo using nude mice HCT116 xenograft. Pharmacokinetic studies on
platinum accumulation were measured by inductively coupled plasma mass spectrometry.
Regarding the results o f DNA-platinum concentrations, die synergy with radiation was
assessed for in vitro and in vivo studies. Cytotoxicity was determined by a colony formation
assay, while the resulting tumor growth delay in animal model was correlated to induction of
apoptosis and histophatology analyses. The synergism of combined treatments was evaluated
using the combination index method.
In this study, a radiosensitizing enhancement was observed with combining radiation
treatment with cisplatin, oxaliplatin and their liposomal formulations in both in vitro and in
vivo studies. Variations of platinum accumulation with incubation time in normal and tumor
tissues and in different cell compartments, as well as platinum-DNA were measured. A higher
level of synergism was observed when radiotherapy was performed in vitro at 8 h of exposure
and in vivo at 4 and 48 h after drug administration, which corresponded to the times of
maximal platinum binding to tumor DNA. These results were correlated to a highest induction
o f apoptosis and a low mitotic activity. In conclusion, the optimal treatment schedule of
chemoradiotherapy is dependent on the time interval between drug administration and
radiation, which was closely associated to the kinetics of platinum accumulation to DNA and
the intracellular concentration of the platinum drugs. Regarding our hypothesis, administered
radiotherapy to the time intervals of maximum synergism could improve efficacy of
chemoradiation treatment. This should be confirmed in clinical trials.
Keywords: Cisplatin, oxaliplatin, Lipoplatin™, Lipoxal™, radiotherapy, pharmacokinetic,
colorectal cancer, concomittance.
i
RESUME
Traitem ent concomitant du cancer rectal avec la chimiotherapie basee sur des derives de
platin et la radiotherapie: Etudes sur la cytotoxicity, la pharmacocinetique et l'effet
concomitant in vitro et in vivo
Par
Thititip Tippayamontri
Departement de medecine nucleaire et radiobiologie
Thyse pr6 sentye a la Faculty de mddecine et des sciences de la sante pour l’obtention du grade
de philisophiae doctor (Ph.D.) en sciences des radiations et imagerie biom&licale, Faculty de
mydecine et des sciences de la santy, University de Sherbrooke, Sherbrooke, Quybec, Canada
J1H 5N4
Les progrys des traitements du cancer colorectal proviennent principalement des
succys en chimio-radiothyrapie. Toutefois, 1’augmentation de l'activity radiosensibilisante du
cisplatine et de l'oxaliplatine est principalement limitye par leur toxicity ce qui limite la dose
administrye. Par consequent, l’optimisation de la syquence et de la cydule d’administration
entre la chimiothyrapie et la radiothyrapie devient essentielle pour maximiser l’interaction du
rayonnement ionisant et de la chimiothyrapie et ainsi optimiser la ryponse tumorale. Notre
objectif est de dyvelopper une cydule optimale de la chimio-radiothyrapie pour obtenir la
meilleure efficacity anti-tumorale tout en minimisant les effets secondaires aux tissus sains.
Pour atteindre ce dernier objectif, nous avons ygalement yvalud la nouvelle formulation
liposomale de cisplatine et d’oxaliplatine (Lipoplatin™ et Lipoxal™, respectivement) qui ont
pour but d'accroitre l'efficacity anticancyreuse tout en ryduisant les effets secondaires.
Nous avons ytudiy la syquence optimale de radio-chimiothyrapie en lien avec a notre
hypothyse de "vraie" concomitance qui favorise l'ajout de rayonnement au moment de
1'accumulation maximale de platine dans l'ADN des cellules cancyreuses. Notts avons effectuy
des ytudes in vitro utilisant le carcinome colorectal humain HCT116, et in vivo sur des souris
nue HCT116 xenogreffe. Les ytudes pharmacocinytiques sur 1'accumulation de platine ont yty
mesuryes par spectromytrie de masse couplee au plasma induit. La cytotoxicity a yty
dyterminye par un essai de formation de colonie, tandis que le retard de croissance tumorale
obtenue en modyie animal est corryie a l'apoptose et analyses histopathologiques. La synergie
des traitements combinys a yty evaluye en utilisant l'indice de combinaison.
Dans cette ytude, nous avons observy une amyiioration de la radiothyrapie combinye
avec le cisplatine, l'oxaliplatine et leurs formulations de liposomes a la fois in vitro et in vivo.
Des variations entre 1'accumulation du platine dans les cellules cancyreuses, de tissus
normaux et tumoraux, ainsi que des adduits du platine k ADN en fonction de la cydule
d'administration du mydicament ont yty observyes. Un fort effet concomitant in vitro a yty
observy lorsque la radiothyrapie a yty dyiivrye k 8 h et in vivo a 4 et 48 h apr£s l'administration
du mydicament, ce qui correspondait au temps de liaison maximale du platine k l'ADN
tumoral. L'augmentation de la radiosensibilisation a yty corryiye a une yiyvation de l’apoptose
et une ryduction de l’activity mitotique. La cydule de traitement optimal de la chimioradiothyrapie dypend de l'intervalle de temps entre l'administration de la radiation et de la
drogue, ce qui a yty ytroitement associy k la cinytique d’accumulation de platine k l’ADN et de
Jeurs concentrations intracellulaires. En conclusion, les meilleurs rysultats in vitro et in vivo
pourraient etre ultyrieurement confirmys en essai clinique pour valider ces concepts.
Mots-ciys:
Cisplatine,
oxaliplatine,
Lipoplatin™,
pharmacocinytique, cancer colorectal, concomittance
Lipoxal™,
radiothyrapie,
ACKNOWLEDGEMENTS
I would like to thank to the Department of Nuclear Medicine and Radiobiology,
Faculty of Medicine and Health Sciences, University de Sherbrooke, to give me the
opportunity to perform my Ph.D.
I would like to express my thankful to Prof. Leon Sanche, Prof. Benoit Paquette and
Dr. Rami Kotb, for their supervision, encouragement, and kindness throughout the course of
this work. I am thankful to Prof. Sherif Abou Elela, co-supervisor of my comiti
d'encadrement.
I would like to thank Dr. Theirry Muanza, Dr. David Mathieu, Prof. Johan E. Van
Lier, for having accepted to be on my thesis committee.
I wish to thank the professors of the Deparment of Nuclear Medicine and
Radiobiology for their great courses and suggestions. I am thankful to Dr. Ana-Maria CrousTsanaclis for her kindly advices and interesting discussions.
I am grateful to Gabriel Charest, Helene Therriault and Rosalie Lemay for their
advices and help to begin my experimental work in the laboratory. I also appreciated the help,
friendship and cheerful support of all my colleagues in our laboratory and in the department.
Most importantly, my warmest thanks go to my always-supporting family for their
generosity and understanding, their encouragement, and their great love.
This research was supported by the Canadian Institute of Health Research (grant #
81356). Prof. L6 on Sanche, Prof. Benoit Paquette and Dr. Rami Kotb are members of the
Centre de recherche Clinique-Etienne Lebel supported by the Fonds de la Recherche en Santd
du Quebec. Sanofi-Aventis Canada has offered a partial unrestricted grant to support this
project. Regulon has provided liposomal formulation drugs (Lipoplatin™ and Lipoxal™) for
this project.
TABLE OF CONTENTS
ABSTRACT..:............................................................
i
RESU M E.......................................
ii
ACKNOW LEDGEMENTS...............................................................................................
iii
LIST OF ILLUSTRATIONS (TABLES AND FIGURES).................................
vi
LIST OF ABBREVIATIONS/SYMBOLS................................................................................ x
CHAPTER I - INTRODUCTION...................................
1
Colorectal cancer..................................................
1
Platinum (Pt) compounds -Cisplatin and Oxaliplatin...................................................................6
History
................................................................................................................................6
Chemical structure............................................................
;.................... 7
Chemistry........................................................
Mechanism of action..................................................
8
:................................... 9
Interaction with the cell............................................................................................................9
Interaction with the D N A .......................................................................................................11
Liposomal formulation ofplatinum compounds.......................................................................... 15
Lipoplatin™ and Lipoxal™...................................................................................................16
Mechanism of action
.......................................................................................................17
Preclinical and clinical studies of Lipoplatin™ and Lipoxal™.......................................... 18
Chemoradiation treatment...................................
19
Objectives o f this study..........................................................................................................
28
CHAPTER II - ARTICLE NO. 1................................................
29
CHAPTER III - ARTICLE NO. 2 ........................................................................................... 51
CHAPTER IV - ARTICLE NO. 3 ........................................................................................... 80
CHAPTER V - ARTICLE NO.4
.............................................................................115
CHAPTER VI - DISCUSSION.............................................................................................. 147
The scope o f this thesis..............................................................................................
:.. 147
Cytotoxicity potential o f cisplatin and oxaliplatin.................................................................... 147
Pharmacokinetics o f platinum drugs......................................................
148
Platinum-based radiosensitizer and cell cycle progression to chemoradiotherapy
in human colorectal cancer HCT116 cell...................................................................................151
TXyf
Tk/f
Radiosensitizing activity o f Lipoplatin and Lipoxal ...........................................................156
Histopathological analysis...................................................................................... ..................156
Emerging strategies fo r improvement in chemoradiotherapy: Clinical aspects..................... 166
CHAPTER VII - CONCLUSIONS....................................
BLIBIOGRAPHY
171
.......................................................................................................173
v
LIST OF ILLUSTRATIONS (TABLES AND FIGURES)
CHAPTER I - INTRODUCTION
Table 1. Staging for colorectal cancer, percentage of occurrence and percentage of survival ..2
Table 2. Example of preclinical and clinical studies on oxaliplatin-based
chemoradiotherapy of colorectal cancer
- ......................................................................5-6
Figure 1. The chemical structures of cisplatin and oxaliplatin.................................................
7
Figure 2. The hydrolysis reaction scheme of cisplatin...........................................
8
Figure 3. The hydrolysis reaction scheme of oxaliplatin...............................................................9
Figure 4. Mechanism o f platinum uptake and efflux................................................................... 10
Figure 5. Formation of cisplatin- and oxaliplatin-DNA adducts................................................ 12
Figure 6 . Interaction of cisplatin and oxaliplatin with the DNA................................................. 13
Figure 7. Mechanism of cell death after platinum chemotherapy.............................................. 15
Figure 8 . Depiction of a liposomal formulation......................................
17
Figure 9. Liposome-cell interactions............................................................................................ 18
Figure 10. The theoretical framework for the prediction of the outcome of combined
treatments with cytotoxic chemotherapy and radiaotherapy...................................................... 21
Table 3. The mechanisms of chemotherapy and radiation interaction................................. .
22
Figure 11. Comparison of the yield of SSB and DSB as a function of different cisplatin
to plasmid DNA ratios................................................................................................................... 24
Table 4. Example of previous studies of combined treatment of cisplatin, oxaliplatin,
Lipoplatin™ and Lipoxal™ plus radiation............................................................................. 26-27
CHAPTER II - ARTICLE No.l
Figure 1. Chemical structures o f platinum-based drugs studied and schematic of
a liposome...................................
34
Table 1. IC50 values o f the platinum compounds for the HCT116 cells.....................................37
Figure 2, Time course of platinum derivatives accumulation in HCT116 cells........................ 38
Figure 3. Cell proliferation in presence of the platinum derivatives
!................................39
Figure 4. Amount of platinum bound to DNA after exposing the HCT116 cells to
platinum drugs for 4 and 24 h
............................................................................................. 40
Figure 5. Time course o f platinum binding to DNA after exposing the HCT116 cells .......... 41
Figure 6 . Distribution of platinum compounds in cytoplasm or bound to DNA...................... 42
CHAPTER III - ARTICLE No.2
Figure 1 . Dose response curve for radiotherapy alone on HCT116 cells................................. 61
Figure 2. Response curves for chemoradiotherapy on HCT116 cells at
8
h incubation time.. 62
Figure 3. Response curves for chemoradiotherapy on HCT116 cells at 48 h incubation
time..................................................................................................................................................63
Figure 4. The IC50 vialues of cisplatin, oxaliplatin and their liposomal formulation
(Lipoplatin™ and Lipoxal™) in combination with radiation..................................................... 64
Figure 5. The combination index (Cl) of chemoradiotherapy on HCT116 cells...................... 6 6
Figure 6 . Flow cytometric measurement of cell-cycle distribution of the HCT116 c e ll......... 69
TV/I
Figure 7. Induction of apoptosis by cisplatin, oxaliplatin, Lipoplatin
and Lipoxal
* rw
combination with radiation............................................................................................................ 70
CHAPTER IV - ARTICLE No.3
Table 1. Change in body weight of male nu/nu nude mice treated with i.v. oxaliplatin
or Lipoxal™ in combination with GK irradiation....................................................................... 91
Figure 1. The experiment setup for chemoradiotherapy in nu/nu nude mice model.................92
Figure 2. Pharmacokinetic profiles of platinum concentration in blood, tumor tissues
and normal tissues after a single i.v. injection of oxaliplatin or Lipoxal
TK/C
.......................... 95-96
Figure 3. Time dependence of platinum concentration in cytoplasm, nucleus and
platinum-DNA adducts obtained after a single i.v. injection of oxaliplatin or Lipoxal™
99
Figure 4. The tumor response after nu/nu nude mice were treated initially with
chemotherapy and followed by radiotherapy............................................................................. 10 0
Table 2. In vivo efficacy of platinum-based chemotherapy alone and in combination
with radiation treatment.............................
Figure 5. The combination index of chemoradiotherapy in nude mice model
101
...........103
Figure 6 . TUNEL assay after chemoradiotherapy of oxaliplatin and Lipoxal™.................... 104
CHAPTER V - ARTICLE No.4
Table 1. Change in body weight of male nu/nu nude mice treated with i.v. cisplatin or
TK/f
Lipoplatin
in combination with Gamma Knife irradiation.................................................. 124
Figure 1. Pharmacokinetic profiles of platinum concentration in whole blood,
serum, kidney, liver, muscle, and tumor after a single i.v. injection of cisplatin
or Lipoplatin™.....................................................................................
126-127
Figure 2. Time dependence of platinum concentration in cytoplasm, nucleus and
platinum-DNA adducts obtained after a single i.v. injection of cisplatin or Lipoplatin™......129
Figure 3. The tumor growth delay after initial treatment of nude mice bearing HCT116
colorectal tumor with cisplatin and Lipoplatin™ followed by radiotherapy........................... 131
Table 2. Efficacy of platinum-based chemotherapy alone and in combination with
radiation treatment........................................................................................................................133
Figure 4. Assessment of apoptosis after chemoradiotherapy of cisplatin and
Lipoplatin™ .................................................................................................................................134
CHAPTER VI - DISCUSSION
Table 1. The platinum-DNA binding after expousred HCT116 to cisplatin, oxaliplatin,
and their liposomal formulation for 4 h and 24 h of drug incubation...................................... 151
Figure 1. Diagram illustrated phases of cell cycle and their relative radiosensitivity or
radioresistance.............................................................................................................................. 156
Figure 2. Induction of the mitochondrial apoptotic pathway by cisplatin................................161
Figure 3. Histophatologic changes of HCT116 human colorectal cancer tumors
treated with platinum drugs and radiation.......................................................................... 163-166
Figure 4. Possible improvement for clinical chemoradiation treatment apply from our
preclinical studies.........................................................................................................................168
Table 2. Lists of other chemotherapeutic agents that are commonly combined with
platinum drugs....................................................
170
LIST OF ABBREVIATIONS/SYMBOLS
Pt
Platinum
DACH
Diaminocyclohexane
CDDP
Cisplatin
5-FU
5-Fluorouracil
FA
Folinic acid
DPPG
Dipalmitoyl phosphatidyl glycol
PEG
Polyethylene glycol
NER
Nucleotide excision repair
MMR
Mitmatch repair
HMG
High-mobility group
OCT
Organic cation transporter
CTR
Copper treansporter
GSH
Glutathione
MT
Metallothionein
DNA
Dioxyribonucleic acid
RNA
Ribonucleic acid
G
Gaunine
A
Adenine
SSB
Single strand break
DSB
Double strand break
ICP-MS
Inductively coupled plasma mass spectrometer
IR
Ionizing radiation
60c o
Cobalt-60
GK
GammaKnife
Gy
Gray
eV
Electronvolt
tl /2
Half-life
h
Hour
Micromolar
Hg
Microgram
ng
Nanogram
mg/kg
Milligram/kilogram
Td
Tumor growth delay
CHAPTER I
INTRODUCTION
Colorectal cancer
Colorectal cancer occurs when abnormal cells grow in the lining of both the colon and
rectum. Colorectal cancer is one of the most common types of cancer worldwide, accounting
for a significant percentage of cancer mortality (International Agency for Research on CancerWorld Health Organization, www.iarc.fr). In Canada, mortality rates continue to decline in
both sexes by 2.6% per year since 2003, however, colorectal cancer is still the second leading
cause of death from cancer, behind lung cancer in men and breast cancer in women. The
Canadian Cancer Society has estimated that there will be about 23,300 new cases of colorectal
cancer in 2012. On average, 64 Canadians will be diagnosed with colorectal cancer and 25
Canadians will die of this disease each day (Canadian Cancer Society, www.cancer.ca).
1
Table 1. Staging for colorectal cancer, percentage of occurrence and percentage of survival
(Greene and Page, 2002)
Stage
% Occurrence
% Survival
Tl-2, NO, MO
14
>90
IIA
T3, NO, MO
28
60-85
IIB
T4, NO, MO
IIIA
Tl-2, N I, MO
37
25-65
IIIB
T3-4, N I, MO
IIIC
T (any), N2, MO
21
5-7
I
IV
T (Primary tumor)
TX
Tis
T (any), N (any), M l
Primary tumor cannot be assessed
Carcinoma in situ
T1
Tumor invades submucosa
T2
T3
T4
Tumor invades muscularis propria
Tumor penetrates muscularis propria and invades subserosa
Tumor directly invades other organs or structures or perforates visceral
peritoneum
N (Nodal status)
NX
NO
N1
N2
Regional lymph nodes cannot be assessed
N metastases in regional lymph nodes
Metastases in one to three regional lymph nodes
Metastases in four or more regional lymph nodes
M (Distance metastases)
MX
Presence or absence cannot be determined
MO
No distant metastases detected
Ml
Distant metastases detected
2
Table 1 shows the colorectal cancer staging, the percentage of occurrence and
percentage of survival (Greene and Page, 2002). Surgical resection at an early stage is still the
only chance for a cure in patients with colorectal cancer (Canadian Cancer Society,
www.cancer.ca). The role o f adjuvant chemotherapy (5-fluorouracil) in stage II (T3/4, NO) is
debatable, there is a minimal benefit. For patients with stage III (TX, N I) rectal cancer,
adjuvant chemoradiotherapy was considered the standard treatment because it improved local
control and overall survival compared with surgery alone or surgery plus radiation (Krook et
al., 1991). Neoadjuvant chemoradiotheraapy protocols were mainly used for locally advanced
rectal cancers, aiming to achieve subsequent curative resection by decreasing tumor size and
decrease the risk of local recurrence (Kapiteijn et al., 2001). The radiotherapy is the use of
carefully measured dose of radiation (e.g. adjuvant radiotherapy is typically given 1.8 Gy per
day for 5 days per week for a number of weeks, total dose of 50.4 Gy). When the dose per day
is increased, the total number of days is correspondingly decreased to limit the risks for injury
to normal tissue from the increasing dose per fraction (Robertson, 2008). Fractionation
radiotherapy allows normal cells to recover and also allows tumor cells that were in a relatively
radio-resistant phase of the cell cycle during one treatment to cycle into a sensitive phase of the
cycle before the next fraction is given (Hall and Giaccia, 2005). Palliative therapy is
recommended for patients with stage IV (Andre and Schmiegel, 2005).
Three recent completed clinical phase III trials (ACCORD-0405, STAR-01 and
NSABPR-04) that added oxaliplatin to 5-FU or capecitabine treatment have failed to improve
results of chemoradiotherapy (Aschele et al., 2011; Gerard et al., 2010 and Roh et al., 2009),
with only the results from the German CAO/ARO/AIO-04 study (Rodel et al., 2012) that
showed an increase in the treatment efficacy when added oxaliplatin to 5-FU plus radiation
3
(Table 2). From the various treatment studies, non-optimal schedule for rectal cancer treatment
may be one of the important factors in clinical treatment failure.
Despite the fact that
approximately 70-80% of patients are eligible for curative surgical resection at the time of
diagnosis (Faivre-Finn et al., 2002), 5 years overall survival rate is only 50-60% et al., 2003).
Some
66%
of patients who undergo curative resection will experience local recurrence or
distant metastases. In 85% of people, relapse was diagnosed within the first 2.5 years after
surgery (Gill et al., 2004). Upon diagnosis of metastatic disease, patients have a median
survival rate of only
6
months and the 5-year overall survival rate was less than 10% (Greene
and Page, 2002).
Importantly, severe side effects of either chemotherapy or radiotherapy can limit the
potential for dose escalations. Chemotherapy is known to cause severe adverse reactions
including renal toxicity, gastrointestinal toxicity, peripheral neuropathy, asthenia and
ototoxicity (Howell, 1991; Kweekel et al., 2005 and O'Dwyer et al., 2000). Radiotherapy
potentially can cause skin irritation, nausea, diarrhea, trouble controlling of bowels, rectal or
bladder irritation, and tiredness (Hall and Giaccia, 2005). This will limit the anticancer
efficiency of both modalities. Moreover, the optimal schedule for adding platinum-based drugs
to the standard chemoradiotheapy for treating rectal cancer is not yet determined. Investigations
to improve the chemoradiation schedule for rectal adenocarcinoma to attain the maximum
concomitant effect, while minimizing the systemic toxicities is thus very important.
4
Table 2. Example of preclinical and clinical studies on oxaliplatin-based chemoradiotherapy of
colorectal cancer. (OXA = Oxaliplatin; 5FU = 5-Fluorouracil; Cape = Capecitabine; pCR =
Pathological complete response; RT = Radiation)
Treatment
Outcome
Blackstock et al,
1999
HT29 colorectal cancer; OXA
0.48 pM; 1 or 24 h incubation; 2
or 4Gy before or after drug
treatment
Oxaliplatin induced synergistic effect
with independent of the sequence of
the combination
Magn6 et al, 2003
SW403 colorectal cancer; OXA
6.5-100 pM; 1 or 4 Gy (60CO
gamma-rays, lGy/ min); RT 2h
before or 12 h after or 24 h after
the start of OXA
OXA induced the supra-additive
effect with no influenced of p53
Kjellstrom et al,
2005
WiDr colorectal cancer; OXA 14 pM; 2 h incubation; 0.5 - 4Gy
Oxaliplatin induced
radiosensitization
Folkwords et al,
HT29 colorectal cancer; OXA
10 mg/kg; dl weekly; 2Gy xlO
(60CO gamma-rays, 0.6Gy/ min)
RT 30 min after i.v.
Weak endpoints o tumor growth
delay after the combined treatment
Study
2008
Tippayamontri et
al, 2 0 1 2
HCT116 colorectal cancer; OXA Oxaliplatin induced
radiosensitization with influence of
2.5-10 pM: 8 h incubation;
the level of Pt-DNA adducts
0.025-0.5 pM: 48 h incubation;
2.3Gy after OXA (60CO gammarays, 1.64Gy/min)
Gerard et al, 2010
ACCORD-0405; n = 598; Cape
1600 mg/m2/day dl-5 weekly
x5; OXA 50 mg/m 2 dl weekly
x5; 50Gy for 5 weeks
Compared Cape- 45Gy versus CapeOXA-50Gy: pCR = 14% Vs 19%;
Grade 3 toxicity = 1% Vs 25%
Aschele et al, 2009
STAR-01, n = 747; 5FU 225
mg/m2/day dl-35; OXA 60
mg/m 2 dl weekly x 6 ; 50.4Gy in
28 daily fractions
Toxicity significantly increased
without affecting local tumor
response
Roh et al, 2011
NSABP R-04; n = 1608; 5FU
225 mg/m2/day or Cape 1650
mg/m2/day dl-5 weekly x5;
OXA 50 mg/m2 dl weekly x5;
45Gy in 25 fractions over 5
weeks
Compared group with OXA versus
without OXA: pCR = 19% Vs
20.9%; Grade 3 toxicity 6 .6 % Vs
15.4%
5
Rddel etal, 2012
The German CAO/ARO/AIO,04; n = 1265; 5FU 250
mg/m2/day d l-1 4 ,22-35; OXA
50 mg/m 2 d l, 8,22,29; 50.4Gy
Improve pCR
Platinum (Pt) compounds -Cisplatin and Oxaliplatin
History
In 1965, Rosenberg and his group discovered the potential of platinum-based drugs to
inhibit cell division (Rosenberg, 1980). They investigated the effect of electronic current on
bacterial mobility and, noticed that the cells under study formed a filamentous structure, which
is a characteristic of un-dividing cells. The chemical compound causing this structure was cisdiamminedichloroplatinum (II), or cisplatin, a compound known since the 1860’s as “Peryone’s
Chloride” (Rosenberg et al., 1969). After this finding, several studies of cisplatin for cancer
treatment were performed, and cisplatin was approved for clinical use in 1979 (Rosenberg et
al., 1969).
The treatment with cisplatin consists of a course of intravenous injections administered
every 3-4 weeks at a dose of 50-120 mg/m2 (Adelstein et al., 2003). Such treatment has a
reported high efficacy for testicular, ovarian and bladder cancers, and other solid tumors (Begg,
1990). Over the years, various platinum complexes have been studied in an attempt to
overcome the severe side effects of cisplatin
et al., 1998). Oxaliplatin or (trans-R,R)l,2-
diaminocyclohexaneoxalotoplatinum (II), is a third generation platinum-based compound in the
alkylating family of anticancer agents and the first clinically available diaminocyclohexane
(DACH) platinum compound. Oxaliplatin is distinctive from cisplatin and is non-cross-resistant
with some platinum-resistant tumors. Although oxaliplatin was discovered 34 years ago, it only
gained Food and Drug Administration (FDA) approval
8
years ago, specifically for the
management of advanced human colorectal cancer, for use in combination with 5-fluorouracil
(5FU) and folinic acid (FA) (National Cancer Institute at the national institute of health,
www.nih.gov). Unlike cisplatin, oxaliplatin is non-nephrotoxic with the main toxicity being
neuropathy. Neurotoxicity is dose-limiting. Oxaliplatin is commonly given at the dose of 85
mg/m i.v. injection every second week (Alcindor and Beauger, 2011).
Chemical structure
The chemical structure of cisplatin and oxaliplatin in 2D and 3D are shown in Figure 1.
Cisplatin has a square-planar structure with the central platinum (Pt) coordinated by two
chlorides (Cl) ion ligands and other two ammonia (NH3) ligands in cis to each other.
Oxaliplatin is a much bigger organic compound, with the two amine NH 3 ligands in cisplatin
substituted by the (trans-R,R)l,2-diaminocyclohexan (DACH) ligand to improve antitumor
activity; and the two chloride ion ligands replaced by the oxalatobidentate ligand derived from
oxalic acid to improve water solubility.
*'«•###/
Cisplatin
Oxaliplatin
Figure 1. The chemical structures of cisplatin and oxaliplatin
Chemistry
. Cisplatin of molecular formula H6Cl2N2Pt has molecular weight 301.1 g/mol. There is
evidence suggesting that due to a high concentration (about 100 mM) of chloride ions outside
of cell membranes, cisplatin remains in the dichloro form until it passes through the cell
membrane. Inside the cell membrane, the chloride ion concentration is about 4 mM which
allows the hydrolysis process to take place (Hall et al., 2008). In aqueous solution, the Cl' ion is
slowly lost and replaced by a water molecule (H20 ) to form the mono-aqua product, cis[Pt(NH3)2(OH2)Cl]+. Further substitution of the remaining chloroligand leads to the formation
of the diaquaplatinum complex, cis-[Pt(NH3)2(OH2)2]2+(Figure 2). At physiological pH
(pH=7.87), the equation shifts and the majority of molecules present are in the diaqua form, the
active form o f cisplatin (Dionisi et al., 2011; Esteban-Femandez et al., 2010).
first hydrolysis
h 3n '
x ci
cr
second hydrolysis
h 3n <" x o h 2
cr
h 3n ^
xoh2
cisplatin
pKa2
pK»i
third hydrolysis
H3N^
XI
/P tx
H3N
OH
cr
h3nx
n oh
P*a3
H3Nv
OH
h 3n x
oh
Figure 2. The hydrolysis reaction scheme of cisplatin (Reprinted with permission from Lau and
Deubel) (Lau and Deubel, 2006).
The oxaliplatin molecule has the formula CgHi4N 20 4Pt and a molecular weight o f 397.3
g/mol.
Oxaliplatin
slowly
hydrolyzes
in
water
according
to
following
reaction:
[(DACH)Pt(C20 4)] + 2H20 -> [(DACH)Pt(H20)]2+ + C20 42+(Esteban-Femandez et al., 2010).
The oxalate group can be shifted by nucleophile groups such as CT or HCO3' and H20 (Figure
3).
Upon entering the cell, cisplatin and oxaliplatin are biotransformed to their reactive
species. Through non-enzymatic reactions, the chloride atom of cisplatin and oxalate group of
oxaliplatin are displaced by H20 forming biaquated biotransfoimation products that readily
react with nucleophiles in the cells, including DNA, RNA and protein (Hall et al., 2008).
oxalate
Oxaliplatin
Dichloro intermediate
Diaquo intermediate
Figure 3. The hydrolysis reaction scheme of oxaliplatin. Permission license # 3034930431133
(Kweekel et al., 2005).
Mechanism of action
Interaction with the cell
Figure 4 illustrates the mechanisms affecting and controlling the cellular accumulation
of platinum drugs. In the extracellular environment, platinum drugs can be associated with
water molecules as explained previously. These may enter the cell or cross-react with
extracellular proteins such as serum albumin, reducing its bioavailablility. Platinum drugs can
enter the cell by passive diffusion across the lipid bilayer as well as by fluid phase endocytosis.
The main organic cation transporters such as OCT1-3, CTR1, and sodium-dependent process
have been identified.
Inside the cell, platinum drugs can be deactivated by binding to the thiol-rich
metallothionein (MT) or chelated by glutathione (GSH) and effluxed from the cell via the GSX pumps. Platinum drugs can also be entrapped in subcellular organelles such as vesicles or
melanosomes in melanoma cells and then subsequently exported out from the cells (Wang and
Lippard, 2005). The accumulation of platinum is a steady-state phenomenon which depends on
both the rates of influx and efflux of platinum drugs. When the shift of the balance between
influx and efflux occurs, an increase of the extrusion process leads to a reduction in cellular
accumulation of platinum. Drugs that evade the detoxification and efflux processes can enter
the nucleus and bind to DNA, eliciting apoptosis if the DNA lesion is not repaired.
Organic cation
transporters
Endocytosis
Passive diffusion
Pt
[Ct]= -100 mM
[COs2] = -24 mM
[P O t]~ 9 mM
Pt
[Ct]= 4-50 mM
[CO,2]= -10 mM
[P O j]= -8 0 mM
Pt
Pt
Pt
Pt detoxification
Pt
Pt
Pt accumulation
Nucleus
GSH, MT, Vesicles, Melanosome
i
efflux
Pt
Figure 4. Mechanism of platinum uptake and efflux.
10
(Pt-DNA
adducts)
j
j
Interaction with the DNA
The clinical efficacy o f platinum drugs against tumors is thought to be primarily due to
the formation of platinum-DNA adducts which interfere with cellular repair and DNA
replication and which therefore trigger a chain of cell regulatory events, ultimately leading to
cell death (Saris et al., 1996).
On DNA, cisplatin and oxaliplatin preferentially react with the highly nucleophilic N7
position on guanine or adenine, and form coordinated covalent bonds (Figure 5). Platinum
binds to DNA and causes a critical structure change in the DNA such as a bend of 45 degree,
unwinding of DNA and causing destacking of the purine bases (Sharma et al., 2007).
Cisplatin and oxaliplatin are bifiinctional agents. They can bind to two sites in DNA
with the following order of sequence preference: -GG- > -A G -» -GA. The resulting biadducts
composed o f approximately 60-65% 1,2-intrastrand GG, 25-30% 1,2-intrastrand AG, 5-10%
1,3-intrastrand GXG and 1-3% interstrand GG (Figure 6 ) (Eastman, 1987). Although cisplatin
and oxaliplatin form the same types of adduct at the same sites on DNA, their structures are
distinct (Sharma et al., 2007). The bulky DACH carrier ligand of oxaliplatin is thought to
contribute to a much bulkier adduct than cisplatin.
11
Adenine (A)
Guanine (G)
cis-diammine-Pt adducts
(Trans-RR) 1,2-diaminocyclohexane-Pt adducts
Figure 5. Formation of cisplatin- and oxaliplatin-DNA adducts. Permission license #
3034940510767 (Sharma et al., 2007).
12
> Oxaliplatin or Cisplatin-DNA monoadduct
N
Cl
C Y
" - V
Cl
»' x°
x
t
«*•'' x °
y
.
X
> Oxaliplatin or Cisplatin-DNA diadduct
K.
*
G
r
ck
/
G*
G
<
or
or
*
/ — * — >»,
kk
N H ,— _
A
' " '
y
—
6
/
N Hj
X
I
Intrastrand crosslink DNA
\
H
V
/ )
fy
xx
,
/
;
i
;<rx
Interstrand crosslink DNA
Figure 6. Interaction of cisplatin and oxaliplatin with the DNA.
or
The DNA repair system can detect many forms of damage. Repair of platinum-DNA
adducts is through the nucleotide excision repair pathway (NER), a complex process which is
essential in the repair of bulky covalent DNA lesions such as those attributed to platinum drugs
(Wozniak and Blasiak, 2002). Once a platinum adduct is detected, excision repair removes the
damaged DNA to form a gap. This gap is then filled by DNA polymerase. The order of DNA
repair efficiency is l,3-d(GTG) crosslink > AG > GG. This implies that l,2-d(GG) cross-links,
as formed by cisplatin or oxaliplatin are the most toxic. Cisplatin adducts, but not the bulky
oxaliplatin adducts, are recognized by a functional mismatch repair (MMR) complex. Binding
of the MMR complex to damage DNA is thought to mediate the apoptotic response (Sharma et
al., 2007).
Alterations to DNA structure prevent both cell replication and activation of cellular
repair mechanisms, and thus lead to cell death (Figure 7). The link between structure alteration
and cell death is due to the prevention of DNA transcription which proceeds by three
mechanisms o f action; i) Participation of high-mobility group (HMG) proteins, which have a
higher affinity to bind to platinum-modified DNA than to unmodified DNA. Possibly, the
HMG-Pt-DNA lesion prevents the binding of DNA-repair proteins, thus preventing replication
and causing cell death, ii) Irreversible platinum binding to transcription factors by replacing the
zinc ion with platinum, and iii) Abortive correlation repair will upregulate the p53 due to DNA
strand breaks, which leads to apoptosis (Zlatanova et al., 1998).
14
Cell death
t
t
Repair blocked
DNA-Pt
Cancer cell -> Binding by HMG-protein
(Note: It appears that HMG protein does
not occur in healthy cells in large amounts
and therefore cannot protect against
excision repair)
Healthy cell -> Excision DNA
repair
damage
I
T
Damage repaired
Cell lives
Figure 7. Mechanism of cell death after platinum chemotherapy. (Adapted with permission
from Zlatanova) (Zlatanova et al., 1998).
Liposomalformulation o f platinum compounds
Liposomes are spherical, self-enclosed structures consisting of one or several
concentrated lipid bilayers with an aqueous phase inside and between the lipid bilayers.
Attractive biological properties of liposomes include; i) liposomes are biocompatible, ii)
liposomes can entrap water-soluble (hydrophilic) pharmaceutical agents into the membrane, iii)
liposome-incorporated pharmaceuticals are protected from the inactivating effect of external
conditions, and iv) liposomes provide an unique opportunity to deliver drugs into cells or even
inside individual cellular compartments (Torchilin, 2005).
15
Lipoplatin™ and Lipoxal™
Cisplatin and oxaliplatin have been frequently used in therapy against cancer, despite
the strong side-effects often observed in patients. In this context, any improvements to platinum
complexes, related to their efficacy or reduction in toxicity, would be a substantial boost for
cancer therapy.
In recent years, liposomal encapsulations of cisplatin and oxaliplatin, Lipoplatin™ and
Lipoxal™, respectively, were achieved using Regulon’s platform technology (Boulikas, 2007).
They were developed to reduce the systemic toxicity of cisplatin and oxaliplatin while
attempting to improve the anticancer efficiency. Figure
8
depicts a liposomal formulation. The
lipid formulation is composed of soy phosphatidyl choline (SPC-3), cholesterol, dipalmitoyl
phosphatidyl
glycol
(DPPG),
and
methoxy-polythylene
glycoldisteraryl
phophatidylethanolamine (mPEG 2000-DSPE). The nanoparticles of liposomal formulation
have a 110 nm diameter. Lipoplatin™ is composed of 8.9% cisplatin and 91.1% lipid (w/w).
Concentrations of 3 mg/ml of cisplatin in Lipoplatin™, and 3 mg/ml of oxaliplatin in
TW
Lipoxal
were used in this study.
Lipoplatin™ and Lipoxal™ formulations were developed to have several therapeutic
advantages: i) the anionic lipid DPPG gives liposomal drugs their fusogenic properties
presumably acting at the level of entry of the drug through the cell membrane after reaching the
target tissue, ii) the total lipid to platinum compounds ratio is low in liposomal formulations
which means that less lipid is injected into the patient, and iii) the PEG polymer coating used
on liposomal formulations is meant to give the drug particles the ability to pass undetected by
the macrophages and immune-system cells to remain in the circulation of body fluids and
tissues for long periods (ti/2 = ~72 h for Lipoplatin™ and
16
~6
h for cisplatin), to extravasate
preferentially and to infiltrate solid tumors and metastatic tissue through the altered and often
compromised tumor vasculature (Boulikas and Vougiouka, 2003).
PEG
Free-Pt
Lipos
Figure 8. Depiction of a liposomal formulation. Free platinum compounds are showed as blue
spheres surrounded by the lipid bilayer with the PEGylated lipid sticking out like hair from the
outer surface (Reprinted with permission from Stathopoulos and Boulikas) (Stathopoulos and
Boulikas, 2012).
Mechanism of action
Drugs stably entrapped inside a liposome are not biologically active and must be
released to gain access to their intracellular target. Figure 9 shows the liposome-cell
interactions. Drug-loaded liposomes can specifically or non-specifically adsorb onto the cell
surface. Liposomes can also fuse with the cell membrane, and release their contents into the
cell cytoplasm, or can be destabilized by certain cell membrane components when adsorbed on
the surface, so that the released drug can enter the cell via micro pinocytosis. Liposomes can
undergo the direct or transfer-protein-mediated exchange of lipid components with the cell
membrane or can be subjected to a specific or non-specific endocytosis. In the case of
endocytosis, a liposome can be delivered by the endosome into the lysosome or, en route to the
17
lysosome, the liposome can provoke endosome destabilization, which results in drug liberation
into the cell cytoplasm. It has been suggested that Lipoplatin™ is mainly taken up into the cell
by phagocytosis, due to its 110 nm size, which is much greater than that of cisplatin (of
molecular dimensions < 1 nm).
,c?
✓/
.
// /
Liposome
I®'
/
& /
*
/
[Ct]= -100 mM
[CO,27= -24 mM
[PO/']= 9 mM
.
[C t]= 4-50 mM
[COs2]= -10 mM
[P O /]= -80 mM
Endosome
Lysosome
Nucleus
>
(Pt-DNA
adducts)
Figure 9. Liposome-cell interactions.
Preclinical and clinical studies of Lipoplatin
T IM
and Lipoxal
T IM
Devarajan and co-workers reported significantly less structural and functional evidence
of nephrotoxicity in mice and rats after treatment with Lipoplatin™ compared to equal doses
with cisplatin (Devarajan et al., 2004a). Phase I, II and III clinical trials have shown that
Lipoplatin™ has a similar efficacy to that of cisplatin in pancreatic, head and neck cancer,
breast cancer and non-small cell lung cancer, while reducing the severe side effects of cisplatin
18
(Boulikas et al., 2005; Boulikas, 2009; Ravaioli et al., 2009 and Stathopoulos et al., 2011).
Lipoxal™ was a subject of a stability testing and a clinical study in 2003. As a single agent,
Lipoxal™ has produced significant cytotoxicity in human colorectal cancer cells and showed
T y
adequate effectiveness in pre-treated patients with colorectal cancer in phase I study. Lipoxal
is currently under study for phase II for gastric cancer and pancreatic cancer (Stathopoulos et
al., 2006a).
Chemoradiation treatment
Sometimes, chemotherapy or radiotherapy alone is not the most effective treatment. The
efficacy of cancer treatment using radiotherapy is often limited by the radiation tolerance of
normal healthy tissues. Chemoradiation treatment is the combined modality treatment using
chemotherapy and radiotherapy. Three clinical rationales support the use of combined
treatment (Seiwert et al., 2007), these include:
i) That concomitant chemoradiotherapy can be used with organ-preserving intent,
resulting in improved cosmiesis and function compared with surgical resection with or without
adjuvant treatment,
ii) The chemotherapeutic agent can act as radiosensitizer, by improving the locoregional
tumor control,
iii) Chemotherapy given as part of concurrent chemoradiotheapy may act systemically
and potentially eradicate distance metastases.
Figure 10 shows the classical framework for describing the possible interactions
between chemotherapy and radiation treatment (Steel and Peckham, 1979) including;
i)
Spatial cooperation: This concept is used to describe the scenario in which
radiotherapy is designed to target locoregionally primitive tumor sites, and chemotherapy acts
19
against distant micrometastases, without interaction between the agents. This operative effect
requires the agents to have non-overlapping toxicity profiles in order that both modalities can
be used at effective doses without increasing normal tissue effects.
ii) Additivity: Where it is intended that both modalities interact in a purely additive
mode with regards to sterilization of the target tumor.
iii) Supra-additivity: Synergism or radiosensitization, the use of combined modalities
leads to increased killing of cells more than expected from the summation of the effects for
each agent applied alone. The radiosensitizers should not have inherent cytotoxicity activity.
iv) Infra-additivity: Antagonism or radioprotective, where the chemotherapeutic agent
inhibits tumor regression by radiation.
The mechanisms o f chemotherapy and radiation interaction are summarized in the table
3. The interaction between chemotherapy agents and radiation may take place different leveal
o f organistaion (Hennequin and Favaudon, 2002) specifically;
i) The molecular level, altering DNA repair or modifying o f lesions induced by drugs
and radiation,
ii) The cellular level through cytokinetic cooperation from differential sensitivity of the
various compartments o f the cell cycle to drugs or radiation,
iii) The tissue level, including reoxygenation, increased drug uptake and inhibition of
repopulation or angiogenesis.
20
“Spatial coorporation”
Chemotherapy
Distal control
Chemoradiotherapy
Radiotherapy
Local regional
control
Local control
Wtmmm
•
•
•
Synergism (Radiosensitization)
Additive
Antagonism (Radioprotection)
Figure 10. The theoretical framework for the prediction of the outcome of combined treatments
with cytotoxic chemotherapy and radiotherapy.
Radiosensitizers are intended to modify tumor cells to become sensitive to radiation
therapy while having minimal toxicity on normal tissue (Tannock, 1996). Heidlberger’s
preclinical studies in 1958 were the first to establish the concept of giving drugs concomitantly
with radiation to enhance the effect of the radiation (Heidelberger et al., 1958).
21
Table 3. The mechanisms of chemotherapy and radiation interaction
Mechanism
Process affected
Increased radiation damage
Incorporation of chemotherapeutic agents into
DNA/RNA generate inter/intra- strand cross­
links with DNA/RNA
Inhibition of DNA repair process
Interfere with the DNA repair process after
radiation
Cell cycle interference (cytokinetic
Platinating agents are cell-cycle independent.
corporation and synchronization)
Radiation is cell-cycle specific.
Accumulation of cells in the G2 and M phases
are the most radiosensitive phases and in the S
phase is the most radioresistant phase.
Reoxygenation and tumor shrinkage
A reduction in tumor volume resulted in
improved blood supply to the tumor, leading to
reoxygenation and increase radiosensitivity
and drug sensitivity.
Platinum-based radiosensitizers induce additional damage or modification o f radiation
induced DNA damage
The biological impact of ionizing radiation results predominantly from the induction of
a variety of lesions in cellular DNA via energy deposition into the DNA itself (direct effect)
and its surrounding molecular environment, particularly water molecules (indirect effect) (Hall
and Giaccia, 2005). The intermediate species (e.g. DNA subunit radical cations, free electrons
and electronically excited DNA subunits) arising from the direct effect of radiation are nonthermal secondary electrons which carry most of the primary radiation energy. The majority of
secondary electrons have energies below 30 eV with the most probable energy at about 10 eV
(Pimblott and LaVeme, 2007). Even low energy electrons with energies less than that required
22
for ionization of DNA can induce DNA damage (M. Rezaee et al., 2013). In addition, low
energy electrons can also induce DNA damage via indirect action, which due to dissociative
electron transfer from water-interface electron traps to DNA bases, quenching of dissociative
electron attachment to DNA, and quenching of dissociative electronically excited states of H2O
in contact with DNA (Alizadeh et al., 2013).
Ionizing radiation results in DNA damage and exerts its therapeutic activity primarily
by inducing DNA strand breaks of two types: single strand break (SSB) and double strand
break (DSB). The DSB is more difficult to repair than the SSB and thus apoptosis typically
ensues following therapeutic application of chemoradiation treatment to a tumor. Radiation also
can induce DNA base damage, DNA to protein cross-links and install replication forks. In
addition, membrane damage leading to signal transduction may affect: i) gene expression of
cell cycle regulations, ii) growth factor production, and iii) oxidative stress pathway activation
(Hall and Giaccia, 2005).
Initial radiation induced cell death can be directly enhanced by the incorporation of a
radiosensitizer into DNA. Cisplatin and oxaliplatin can exert their cytotoxicity through the
formation of monofunctional adducts and bifunctional adducts, leading to the induction of
intra- and inter- strand DNA cross-links. Sanche and co-workers have demonstrated increased
yields of SSBs and DSBs in plasmid DNA after combining cisplatin with irradiation with 100
eV electrons (Figure 11). An enhancement in the electron cross section for damage to DNA at
the cisplatin binding site is thought to be the cause of this increase in SSBs and DSBs. When
cisplatin-DNA complexes are irradiated by high energy particles, secondary low energy
electrons can be captured at the cisplatin site, increasing the probability of rupture of the DNA
backbone, by an order of magnitude. Moreover the ionization cross section, which describes the
23
probability o f generating secondary electrons, is increased near cisplatin due to the presence of
high platinum atom (Z = 78), and this therefore also indirectly contributes to the huge increase
in local damage. Thus, strand breaks in the DNA of cancer cells, leading to cells death, might
be significantly increased if sufficient quantities of the drug were delivered or could
accumulate preferentially in the DNA of cancer cells (Zheng et al., 2008).
R a tio o f c is p la tin /p la s m id
Figure 11. Comparison of the yield of SSB (A) and DSB (B) as a function of different cisplatin
to plasmid DNA ratios. The yields were obtained from exposure curves of 5 molecule
cisplatin/plasmid mixture deposited on a tantalum substrate and bombarded with 100 ± 0.5 eV
electrons (Copyright from American Physical Society by author) (Zheng et al., 2008).
Platinum-based radiosensitizers inhibit or alter o f radiation damage repair
Radiation recovery is considered to be the main reason for the poor response o f tissue to
radiotherapy. Many studies have shown that sublethal DNA damage induced by radiation can
be repaired. This repair could occur through two major pathways; non-homologous end-joining
(NHEJ) and homologous recombination (HR) (Haveman et al., 2004; Myint and Raaphorst,
2002).
Platinum-based drugs can inhibit the sublethal damage repair that occurs after a cell
population is exposed to a radiation dose insufficient to cause cell death (Seiwert et al., 2007).
Repair involves enzymatic corrections of DNA breaks and aberrations caused by radiation and
can result in tumor cell recovery to full mitotic potential. The mechanism by which platinum
drugs inhibit DNA damage repair is related to the formation of platinum-DNA complexes that
lead to the transformation of DNA structure into forms that could not be recognized by the
DNA damage repair proteins (Hennequin and Favaudon, 2002). The integration of platinum
drugs into DNA in close proximity to a radiation-induced SSB would act synergistically to
make the DNA damages difficult to repair. If the cells are unable to successfully repair the
DNA damage induced by ionizing radiation, the cells undergo programed cell death or
apoptosis.
A summary of previous studies of combined treatment of cisplatin, oxaliplatin,
Lipoplatin™ and Lipoxal™ plus radiation is shown in table 4. The combination of
chemotherapy with radiation treatment has shown positive outcomes in preclinical and clinical
trials, both in terms of anticancer efficacy and toxicity, however, an optimal sequence for
administration, is still required. The principle aim of improving combined treatments is to have
the greatest impact on' current, solid tumor treatment practice. Any postulated protocol for an
optimal sequence for administration, should be first developed in the preclinical setting and be
well-grounded on a comprehensive, rational understanding of experimental results, before any
transition to clinical trials.
25
Table 4. Example of previous studies of combined treatment of cisplatin, oxaliplatin,
Lipoplatin™ and Lipoxal™ plus radiation.
Platinum
drug plus
radiation
Cisplatin
Oxaliplatin
Study
Treatment
Result
Zhang N, et al.
(Zhang et al., 2009)
HNSCC cell lins, 0.0380.330 pM + 2 Gy
Induce additive and supraadditive effect
Rave-Frank M, et al.
(Rave-Frank et al.,
2007)
CaSki cervical cancer,
2/24 h incubation + 0-6
Gy
Supra additive effect
HJ Groen, et al.
(Groen et al., 1995)
GLC4 HSCLC, 30 min,
4/
24 h incubation + 0-8
Gy
Synergism o f cisplatin plus
radiation
BC Boulmay, et al.
(Boulmay et al.,
2009)
Phase II,III,IV adjuvant
H&N cancer, 30
mg/m2/wk + 72 Gy
Improve clinical efficacy
Magne N, et al.
(Magne et al., 2003a)
SW403 colorectal
cancer; 6.5-1000 pM +
1 or 4 Gy
Oxaliplatin induced radio­
sensitization with no influence
of p53
HT29 colorectal cancer;
10 mg/kg repeated dose
fractionated radiation
Weak endpoint o f tumor
growth delay
Folkword S, et al.
(Folkvord et al.,
2008)
Kjellstrom J, et al.
(Kjellstrom, Kjellen
and Johnsson, 2005)
WiDr colorectal cancer;
2 h incubation 1-4 pM +
0.5-4 Gy
Koivusalo R, et al.
(Koivusaio et al.,
2002)
SiHa cervical cancer;
0.1-300 pM + 5 Gy 6h
incubation
Cividalli A, et al.
(Cividalli et al.,
2002)
Breast cancer; 6-14
mg/kg + fractionated IR
Phase I study rectal
26
Oxaliplatin induce radio­
sensitization
Increase apoptosis
Synergism of oxaliplatin plus
IR
Lipoplatin™
Freyer G, et al.
(Freyer et al., 2001)
cancer; oxaliplatin +
5FU +folinic acid +IR
Charest G, et al.
(Charest et al., 2010)
F98 Glioblastoma; 18.22
pM o f Lipoplatin + 2.21
Gy
Reduction of toxicity
Phase I/II study advance
gastric cancer; 120
mg/m2/ week + 5FU +
3.5 Gy
High complete response rate
F98 Glioplastoma; 12
mg/kg + 1.5-6.6 Gy
Prolong survival rate and
reduce toxicity
F98 Glioblastoma; 4.58
pM o f Lipoxal + 2.21
Gy
Improve the tumor uptake
F98 Glioplastoma; 12
mg/kg + 1.5-6.6 Gy
Prolong survival rate and
reduce toxicity
Koukourakis, et al.
(Koukourakis et al.,
2010)
Charest, et al.
(Charest et al., 2012)
Lipoxal™
Charest G, et al.
(Charest etal., 2010)
Charest G, et al.
(Charest et al., 2012)
27
Improve clinical efficiency
Objectives o f this study
Overall aim
To improve the chemoradiation treatment schedule for colorectal cancer based on “true”
concomitant chemoradiation treatment to attain the best synergic effect, while minimizing the
systemic toxicities. All studies were performed in vitro with the human colorectal cancer
HCT116 cells model, and in vivo with nu/nu nude mice model.
Specific aims
1) Study the cytotoxic effects o f platinum drugs, cisplatin and oxaliplatin and their liposomal
formulations (Lipoplatin™ and Lipoxal™) and elucidate their pharmacokinetic characteristic
such as difference tissues distribution, intracellular platinum accumulation, and DNA binding.
2) Study the effect of cell proliferation, cell cycle progression and cell death on the response to
chemoradiotherapy
at
different
treatment
schedules
to
better
understand
of how
chemotherapeutic agents act as radiosensitizers.
3) Determine the effect o f “true” concomitant chemoradiation treatment i.e. response when
radiation is delivered at such time that platinum-drugs have their highest concentration bound
to DNA.
28
CHAPTER II
ARTICLE NO. 1
In this chapter, we report our preliminary study regarding the cytotoxicity and
pharmacokinetics o f cisplatin, oxaliplatin and their liposomal formulation in human colorectal
cancer HCT116 cell.
This work is presented in the following article, entitled: “Cellular uptake and cytoplasm
/ DNA distribution of cisplatin and oxaliplatin and their liposomal formulation in human
colorectal cancer cell HCT116”, by Thititip Tippayamontri, Rami Kotb, Benoit Paquette and
L6on Sanche.
This article is published in Investigational New Drugs, 2011,29(6): 1321-1327.
In.csi
l)ru£. iJO II) M -IK I I K ’
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PRECLINICAL STUDIES
Cellular uptake and cytoplasm / DNA distribution
of cisplatin and oxaliplatin and their liposomal formulation
in human colorectal cancer cell HCT116
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29
Resume
Les formulations liposomales de cisplatine et d'oxalipiatine (Lipoplatin™ et Lipoxal™,
respectivement) ont ete recemment proposees pour leur capacity a rdduire la toxicity
systemique, tout en optimisant l'efficacite anti-cancereuse de ces composes. Comme l'activite
anti-tumorale oul’action radio-sensibilisante de ces medicaments est attribue a leur liaison k
l'ADN, nous avons dvalu6 1'impact de ces formulations de liposomes en fonction du temps
d'accumulation de ces composes de platine dans les lignees humaines de cellules de cancer
colorectal HCT116 et leur distribution entre le cytoplasme et l'ADN a ete mesurd. Leur
cytotoxicite a ete determinee par un test de formation de colonies. La quantite de platine
intracellulaire et de platine lie k l’ADN a ete mesuree par plasma induit couple a la
spectrometrie de masse. Bien que, comme agent chimiotherapeutique, le cisplatine etait aussi
efficace que l'oxaliplatine apres une exposition pendant une courte periode, l'oxaliplatine et
Lipoxal ™ est devenu plus actif que le cisplatine contre les cellules HCT116 apres 24 h
d'incubation. Le Lipoxal ™ a fait preuve d'une plus grande accumulation dans le cytoplasme
des cellules HCT116 par rapport a l'oxaliplatine libre, en accord avec le m6canisme propose de
la fusion des liposomes avec la membrane cellulaire. La distribution cytoplasme / ADN du
cisplatine libre et du Lipoplatin™ etaient similaires. A l'inverse, la distribution cytoplasme /
ADN du Lipoxal ™ et de Poxaliplatin a ete significativement differente : plus de 95% de
l'oxaliplatine transportes par les liposomes a ete pris au piege dans le cytoplasme, meme apres
48 heures d'incubation. Notre etude indique que le Lipoxal ™ peut ameiiorer grandement
1'absoiption cellulaire de l'oxaliplatine, mais cela n'a pas mene a une augmentation similaire des
bris k l’ADN.
Mots cies: liposomes, le cisplatine, l'oxaliplatine, Lipoplatin ™ et Lipoxal ™, cancer colorectal
30
Abstract
Liposomal formulations of cisplatin and oxaliplatin (Lipoplatin™ and Lipoxal™,
respectively) were recently proposed to reduce systemic toxicity, while optimizing the anti­
cancer effectiveness of these compounds. As the anti-neoplastic or radio-sensitizing activity of
these drugs is attributed to their binding to DNA, we assessed the impact of the liposomal
formulations on the time course of accumulation of these platinum compounds in the human
colorectal cancer HCT116 cell lines and their distribution between cytoplasm and DNA. Their
cytotoxicity was determined by colony formation assay. Intracellular platinum and platinum
bound to DNA was measured by inductively coupled plasma mass spectrometry. Although, as a
chemotherapeutic agent, cisplatin was as efficient as oxaliplatin after exposure for a short time,
oxaliplatin and Lipoxal™ became more active than cisplatin against HCT116 cells after 24 h
incubation. Lipoxal™ displayed a higher accumulation in the cytoplasm of HCT116 cells
compared to free oxaliplatin, consistent with its proposed mechanism of fusion with the cell
membrane. The distribution cytoplasm/DNA of free cisplatin and Lipoplatin™ were similar.
Conversely, Lipoxal™ had a significantly different cytoplasm/DNA distribution from
oxaliplatin: more than 95% o f oxaliplatin transported by the liposome was trapped in the
cytoplasm, even after 48 h incubation. Our study indicates that Lipoxal™ can largely improve
the cellular uptake of oxaliplatin, but this was not followed by a similar increase in the DNA
bound fraction.
Key words: Liposome, cisplatin, oxaliplatin, Lipoplatin™ and Lipoxal™, colorectal cancer
31
Introduction
Despite its nephrotoxicity and bone marrow toxicity, cisplatin (CDDP) has shown
significant antineoplastic and radiosensitising activity for various solid tumours [1]. Oxaliplatin
has shown a much better tolerance and similar or superior activity in different tumors compared
to CDDP [2,3].
Combined with 5-fluorouracil and folinic acid, oxaliplatin improved the
response rate, progression free and overall survival of patients with advanced colorectal cancer.
Although no nephrotoxicity has been observed, the administration dose was still limited by the
development o f a neurotoxicity [4].
Lipoplatin™ and Lipoxal™, the liposomal formulation of CDDP and oxaliplatin, have
been designed with the aim of reducing systemic toxicity, while simultaneously improving the
targeting of drugs to primary tumor and metastases [5,6]. Preclinical studies in animal models
supported an important reduction of nephrotoxicity when administrating Lipoplatin™
compared to CDDP [7]. In a phase I study, no nephrotoxicity was observed in the patients, even
at a dose of 300 mg/m2 [8], Same trend was observed with Lipoxal™, which was better
tolerated with fewer side effects than in patients receiving oxaliplatin [6]. Reduction of the
systemic toxicity with Lipoplatin™ and Lipoxal™ did not seem to decrease their therapeutic
potential, as suggested by preclinical and few clinical reports [9,10].
As radiosensitisers, only CDDP is routinely included in radio-chemotherapy protocols
for different cancers [1,11]. Preliminary data on the added benefit of oxaliplatin to 5-flurouracil
and radiotherapy for rectal cancer are inconsistent, and requires further evaluation to determine
the best combination and schedule [12].
For all these drugs, especially the liposomal formulations, important information
regarding their ability to improve the accumulation of platinum in DNA is still missing,
although it is of paramount importance to schedule radiotherapy in combination with any of
these compounds.
In the present study, we assess the time course of intracellular platinum accumulation
and DNA binding for each of these compounds in the human colorectal cancer HCT116 cells.
Materials and methods
Cell culture and drugs
The HCT116 human colorectal carcinoma cell line obtained from ATCC were routinely
cultures in modified Eagle’s medium (MEM) (Sigma-Aldrich, Oakville, Canada) supplemented
with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml
penicillin and 100 pM streptomycin in a folly humidified incubator at 37°C in an atmosphere
containing 5% CO2. Cisplatin was purchased from Sigma-Aldrich, while oxaliplatin was
obtained from Sanofi-Aventis Canada through the pharmacy of the Centre Hospitalier
Universitaire de Sherbrooke. Lipoplatin™ and Lipoxal™, the liposomal formulation of
cisplatin and oxaliplatin respectively, were generously provided by Regulon Inc. (Athena,
Greece). Their chemical structures are illustrated on figure 1. All platinum solutions were
freshly prepared before usage in FBS-free MEM.
33
Cisplatin
Oxaliplatin
Liposome..
Schematic of a liposome
Figure 1. Chemical structures of platinum-based drugs studied and schematic of a liposome.
Clonogenic assay
The anticancer potential of platinum compounds was assessed by using a colony
formation assay. Briefly, 10 cells from a single-cell suspension were seeded into 100-mm cell
culture dishes containing 10 ml of culture medium and incubated at 37°C in a humidified
atmosphere containing 5% CO2 for 24 h. After removal of the culture medium, the cells were
washed with PBS before treatment with platinum compounds at appropriate concentrations and
further incubated for the desired period. Thereafter, the drugs were removed and the cells were
incubated for another 7 days to allow the formation of colonies. Cells were fixed and stained
with 0.1% crystal violet, after washing with tap water. Colonies containing more than 50 cells
were counted manually to calculate the clonogenic survival fractions. For each treatment, three
parallel samples were scored and the assays were repeated for 3 independent experiments. The
relative colony formation (% clonogenic survival) was plotted against drug concentrations, and
the concentration of platinum compound leading to a 50% reduction of colony formation (IC50)
was calculated.
Cellular uptake of platinum compounds
HCT116 cells (6 x 104/plate) were incubated for 48 h in 100-mm tissue culture plates.
Cells were then incubated at 37°C for 1,4, 8, 24 or 48 h with a platinum compound at the IC50
concentration previously measured after 4 h incubation with the drug. Cells were then washed
twice with PBS, harvested by trypsinization, resuspended in PBS and counted. The quantity of
platinum accumulated in HCT116 cells was determined with an inductively coupled plasma
mass spectrometer (ICP-MS, ELAN DRC-II, PerkinElmer). Briefly, cell suspensions were
treated with 23% of nitric acid, 8% hydrogenperoxide and wet autoclaved for 1 h. The solutions
were then injected as is in the ICP-MS to quantify the intracellular platinum. Cell-uptake
platinum was expressed as ng of platinum per 1 x 106 cells. As internal controls, platinum and
thallium (m/z 195 and 205, respectively) were quantified in untreated cells.
Binding of platinum to DNA
Cells (25 x 104/plate) were incubated with platinum compounds at their respective IC50
for 1, 4, 8, 24 or 48 h. DNA was extracted according to a salting-out procedure [13]. Briefly,
cells were washed twice with warm PBS, harvested after trypsinisation, and centrifuged at 1300
x g for 3 min. The cell pellets were cleaned one time with PBS and centrifuged another time,
followed by resuspended in 3 ml of a lysing buffer containing 10 mM Tris-HCl, 400 mM
sodium chloride and 2 mM EDTA. To this cell suspension, 0.1 ml sodium dodecylsulfate (SDS
20%) and 0.5 ml proteinase K (10 mg/ml, DNase free) were added and incubated overnight at
37°C. The DNA was precipitated by adding of 1.2 ml of 5 M sodium chloride. The tube was
35
agitated for 1 min, centrifuged at 2500 x g for 15 min and then the supernatant was transferred
to another tube. The DNA was precipitated with 2.5 vol. of 95% ethanol, the tube was gently
inverted for 30 s, and the DNA was spooled out and air-dried briefly. The DNA was dissolved
in TE buffer (lOmM Tris-HCl, ImM EDTA, pH 8.0) and RNase A (0.05 ml of a 10 mg/ml
solution) was added and incubated for 1 h at 37°C. The DNA was precipitated a second time
with ethanol as described above and redissolved in TE buffer. The amount of DNA extracted
was evaluated by measuring the absorbance of the DNA solution at 260 nm (A260) with a
spectrophotometer (Synergy HT, BIO-TEX) and calculated using the following equation: A260
x 50 (pg/ml). Concentration of platinum was expressed as ng of platinum per pg DNA. No
platinum was detected in the untreated control cells.
Statistical analysis
The mean ± SD were calculated. P < 0.05 was considered statistically significant (twotailed paired Student’s t-test).
Results
Cytotoxicity potential
The IC50 values for the platinum compounds tested are reported in Table 1. After 4 h
incubation with the drugs, higher concentrations of Lipoplatin™ and Lipoxal™ were required
to reduce the cell survival by 50% compared to their free analogs cisplatin and oxaliplatin.
Since the rate of platinum accumulation in cancer cells in vitro could be reduced when the
drugs were transported into liposomes, the incubation time with the drugs Was extended to 24 h
(Table 1). In this case, lower concentrations for all the platinum drugs were required to reach
the IC50. The free cisplatin and oxaliplatin still led to a better anticancer effect than their
36
respective liposomal formulation, the most effective platinum drug being oxaliplatin for
treating the HCT116 cells.
Table 1. ICso® values of the platinum compounds for the HCT116 cells
Incubation time
Platinum compounds
Cisplatin
Oxaliplatin
4h
24 h
7.5 ± 0.7
1.25 ±0.1
7 ±2.2
0.15 ± 0.02
Lipoplatin™
70 ±10.5
5.5 ± 3
Lipoxal™
21 ± 2.7
0.5 ± 0.2
aICso in pmol/1 ± S.D.
Time course of cellular accumulation
The time course o f platinum compounds accumulated in HCT116 cells is shown in Fig.
TW
2. After 4 h incubation, cisplatin, oxaliplatin and Lipoxal
accumulated at similar levels in the
HCT116 cells (~ 1 ng Pt / 106 cells). Although incubation with Lipoplatin™ led to the same
level of cytoxicity (IC50), the concentration of platinum measured in the HCT116 cells using
this liposomal formulation was about 3-fold higher than the other platinum compounds.
Incorporation of oxaliplatin into liposomes considerably improved cellular uptake.
While the cellular concentration of free oxaliplatin has reached a plateau after 24 h incubation,
its liposomal formulation Lipoxal™ continued to accumulate in HCT116 cells even after 48 h
incubation.
37
<riu|
Regarding cisplatin and Lipoplatin
, the uptake was initially accelerated with the
liposomal preparation. However, a similar accumulation in HCT116 cells was measured after
48 h incubation.
30
o
a
smm
(0) cisplatin
(□) oxaliplatin
(♦) Lipoplatm™
(■) Lipoxal™
25
§1
5 o
S
« ©
"S.
© m
4> O
«
20
15
10
0
0
10
20
30
Incubation time (h)
40
50
Figure 2. Time course o f platinum derivatives accumulation in HCT116 cells. The cells were
incubated at the IC50 concentrations previously measured after 4 h incubation. The amount of
platinum accumulated in the cells was measured by ICP-MS. Each point represents the mean ±
SD (n=3).
Accumulation of the platinum compounds in HCT116 cells induces DNA damages that
could result in cell death, and consequently a reduction of cell number. Cisplatih, oxaliplatin
and Lipoxal
TXX
accumulated initially in the HCT116 cells at a similar level and they led to an
equivalent reduction of cell number as shown in Fig. 3. Regarding Lipoplatin™, an initial
reduction of cell number was followed 24 h later by a significant recovery o f the cell
proliferation capacity. This result contrasts with the higher ability of this liposomal formulation
38
to accumulate platinum in the cells during the first 24 h incubation, compared to the other
platinum compounds.
(0) cisplatin
(□) oxaliplatin
(♦) Lipoplatin™
(■) Lipoxal™
140
120
100
8
o
H
60
40
20
0
10
20
30
40
50
Incubation time (10
Figure 3. Cell proliferations in presence of the platinum derivatives. Cells were incubated at
the IC50 concentration previously measured after 4 h incubation. Each point represents the
mean ± SD (n=3).
DNA accumulation
Binding of the four platinum compounds to DNA are compared in Fig. 4. For the same
level of toxicity (IC50) after 4 h incubation, cisplatin and oxaliplatin accumulated in DNA at a
similar level. However, when included into liposomes, much lower quantities of cisplatin and
oxaliplatin were linked to DNA for the same cytotoxicity level.
39
E
0
1
cisplatin
1
oxaliplatin Lipoplatin
Lipoxal
Drugs
Figure 4. Amount of platinum bound to DNA after exposing the HCT116 cells to platinum
drugs for 4 and 24 h. Cells were incubated at the IC50 concentrations previously measured after
4 h ( ■ ) and 24h ( ■ ) . The amount of platinum bound to DNA was measured by ICP-MS. Each
point represents the mean ± SD (n=3).
After 24 h incubation, lower concentrations were required to reach the IC50 for all
platinum compounds (Table 1). A similar reduction (4-5 folds) of cisplatin and oxaliplatin
accumulated in DNA was also measured (Fig. 4). Conversely, their respective liposomal
formulation resulted in better accumulation in DNA than recorded with IC50 after 4 h
incubation. DNA binding measured for Lipoplatin™ was even higher than that measured with
cisplatin.
The kinetic o f platinum uptake in DNA was then followed up to 48 h, with
concentrations of platinum compounds leading to the IC50 measured after 4 h incubation. For
the free cisplatin and oxaliplatin, the rapid initial accumulation in DNA was followed by a
progressive reduction which seem to slowly stabilized at about 0.14 ng Pt/pg DNA (Fig. 5).
T X jI
When included in their liposomal formulation Lipoplatin
and Lipoxal
t w
•
, their kinetics of
accumulation in DNA were much slower starting at a level more than 10 times lower. After 48
h incubation, Lipoplatin™ led to a level of DNA accumulation approaching that of the free
cisplatin. Lipoxal™ bound about 4 times less platinum to DNA compared to the other
compounds (Fig. 5).
0.5 n
<
2
Q
o
2
4* Q
M>
vs
>
«
>
r*
mm
0.2
-
es
E
0.0
0
10
20
30
Incubation time (h)
40
50
Figure 5. Time course of platinum binding to DNA after exposing the HCT116 cells for 4 h.
Cells were incubated at the IC50 concentrations previously measured after 4 h. The amount of
platinum accumulated in the cells was measured by ICP-MS. Each point represents the mean ±
SD (n=3).
41
After entering the cancer cell, the distribution of platinum compounds between the
cytoplasm and the nucleus could affect their anti-cancer potential. The kinetics of their
distribution was therefore assessed to determine the impact of the liposomal formulation (Fig.
6 ).
A) Cisplatin
B) Oxaliplatin
100 -i
I
•a
ce
•a
60
3
A
bm
G
•mm
a
4
8
24
lacabatioa time (b)
48
8
24
lacabatioa time (h)
48
8
24
lacabatioa tiam (b)
48
D) Lipoxal TM
C) Lipoplatin TM
se
‘■O
£
c
o
•a
A
A
bm
bm
a
3
3
a
4
8
24
lacabatioa tnae (b)
48
Figure 6. Distribution o f platinum compounds in cytoplasm ( ■ ) or bound to DNA ( ■ ) . Each
point represents the mean ± SD (n=3).
42
After 4 h incubation with the HCT116 cells, the majority of the cisplatin was found in
DNA. This equilibrium then moved to the cytoplasm where more than 90% of the cisplatin was
measured after 48 h incubation with the cells (Fig. 6A). For oxaliplatin, the maximal
accumulation and distribution in DNA was measured after 8 h incubation. As observed with
cisplatin, most of the oxaliplatin accumulated after longer exposure to the cells was mainly
found in the cytoplasm (Fig. 6B). Lipoplatin™ resulted in a generally lower distribution of
platinum in DNA compared to the free cisplatin (Fig. 6C). Again, the platinum was mainly
measured in the cytoplasm after 48 h incubation.
A completely different distribution compared to free oxaliplatin was observed (Fig. 6D)
with Lipoxal™: despite the higher cell uptake reached (Fig. 2), most of the Lipoxal™ remained
in the cytoplasm and the fraction bound to DNA never exceeded 5% (Fig. 6D).
Discussion
Liposomal formulations of cisplatin and oxaliplatin (Lipoplatin™ and Lipoxal™) have
been proposed to reduce the systemic toxicity of these platinum compounds, while optimizing
their anti-cancer effectiveness [5,6]. In the present study, effects of the liposomal formulations
in the time course of the cellular accumulation of these platinum compounds and their
distribution between cytoplasm and DNA were evaluated.
The relative cytotoxicity of cisplatin, oxaliplatin and their respective liposomal
formulations Lipoplatin™ and Lipoxal™ varied according to the incubation time with the
colorectal carcinoma cells HCT116. As expected, lower concentration of all these platinum
compounds were required to reach the IC50 after 24 h incubation, than measured after 4 h
incubation. Although cisplatin was as efficient as oxaliplatin after a short exposure time,
43
oxaliplatin either under its free form or transported in the liposome Lipoxal™ became more
efficient than cisplatin for treating the HCT116 cells after 24 h incubation. The highest
effectiveness of oxaliplatin corresponds to clinical observations showing that DNA mismatch
repair (MMR) deficient colorectal cancer cells, such as the HCT116 cells, are more sensitive to
oxaliplatin than cisplatin. The oxaliplatin DNA-adducts are apparently not recognized or
processed by the MMR system in the same way as cisplatin DNA-adducts. The structural basis
for the lack o f oxaliplatin recognition is unclear and could involve a distortion in DNA that is
distinct from that produced by cisplatin [14].
In our study, the concentration of free cisplatin has to be about 8-fold higher than that of
oxaliplatin to reach similar accumulations in DNA and lead to the same level of cancer cell
toxicity. Similar results were reported by Kitada et al. [15]. These results further supported the
hypothesis that oxaliplatin adducts are not efficiently processed by the MMR deficient HCT116
cells as it is the case with cisplatin adducts.
Mechanisms responsible for saturation of the cellular uptake of free oxaliplatin remain
unclear. Platinum compounds accumulation was reported to occur through some copper
transporters such as the uptake transporter hCtrl and the polyspecific organic cation transporter
of hOCTl [16]. Conversely, platinum compounds can be pumped out through the efflux
transporters ATP7A and ATP7B, suggesting a contribution of these transporters to the
sensitivity o f cells [17-19]. A decrease in the accumulation of platinum is reported to be the
most common mechanism of resistance [20]. Our study supports that incorporation of
oxaliplatin in the liposome Lipoxal™ largely improved its accumulation in the HCT116 cells.
This result suggests that liposomes can bypass the uptake and efflux transporters, and
potentially could increase the anti-cancer activity of oxaliplatin. These data further support the
44
proposal [10] that liposomal encapsulation promotes fusion of the liposome nanoparticle with
the cell membrane resulting in higher cellular accumulation compared with the free drug. Thus,
liposomal platinum drugs could find clinical application as second-line therapy treatments after
front-line platinum drug-treatment against tumors in patients with resistance developed at the
level of cell membrane rather than at the level of faster repair of DNA lesions or increased
levels of glutathione.
Regarding cisplatin, it continued to accumulate in a time-dependent manner. Its
incorporation into the liposome Lipoplatin™ accelerated its cellular uptake during the first 24 h
of exposure; but no significant improvement was measured when the exposure time was
extended to 48h. Therefore, the uptake and efflux transporters seemed to be less limiting for
cisplatin in HCT116 cells.
The major target of platinum-based drug is DNA. Thus, when a platinum complex
enters a tumor cells, cytotoxicity is not yet assured until drug enters the nucleus and reacts with
DNA [21]. It was suggested that Lipoplatin™ and Lipoxal™ directly fusion with the
membrane of the tumor cell to deliver their contents [22]. On the other hand, liposomes can
also delivery their content by endocytosis. The resulting endosome ferry the platinum
throughout the cytoplasm and fuse with a lysosome to form an endolysosome, followed by the
release of the drugs. The rate of these events varies typically from half to several hours [23].
We have determined whether delivery of cisplatin and oxaliplatin through the liposome
Lipoplatin™ and Lipoxal™ affected their cytoplasm/DNA distribution. Using isotoxic doses,
Tw
Lipoplatin
initially reduced the distribution of cisplatin to DNA, but a similar distribution
cytoplasm/DNA was measured after 48 h incubation. On the other hand, Lipoxal™ has largely
modified the distribution cytoplasm/DNA of oxaliplatin. More than 95% of oxaliplatin
45
accumulated in cancer cells was trapped in the cytoplasm, even after 48 h incubation. These
results suggest that different pathways for delivering cisplatin and oxaliplatin occurred when in
T 't /
their liposomal formulation. The cell uptake study showed that Lipoxal
•■ p w
and Lipoplatin
led
to similar platinum accumulations, although Lipoplatin™ initially accelerated the accumulation
of cisplatin. However, the lower accumulation of oxaliplatin in DNA indicates that oxaliplatin
seems to be entrapped in the cytoplasm when delivered by Lipoxal
TT1VX
. Conversely,
Lipoplatin™ behaved quite differently, leading instead of an improvement of cisplatin
accumulation in DNA. These results suggest that cispaltin transported by its liposomal
formulation may be released at a faster rate from the endolysosome. Another hypothesis is that
the delivery pathway through the direct fusion of the liposome with the membrane of the tumor
cell could be more efficient for Lipoplatin™ than Lipoxal™.
In general, the degree of cytotoxicity correlated with the amount of platinum bound to
DNA [18]. In our study, Lipoxal™ was a more efficient anti-cancer agent than Lipoplatin™,
although much less platinum was accumulated into DNA using this liposome. These results
suggest that either oxaliplatin is distributed in DNA following a different and more efficient
pathway than cisplatin when carried under their liposomal formulation, or oxaliplatin reacts
with different sensitive targets in the cytoplasm. A third hypothesis could be that cisplatin
DNA-adducts are removed less efficiently in the MMR deficient HCT116 cells than the
oxaliplatin DNA-adducts resulting in a higher accumulation of cisplatin in DNA. One
additional suggestion is that platinum drugs kill cancer cells by modulating signalling pathways
that are different than those o f DNA damage-induced apoptotic pathways [10]; in this case,
•
Lipoxal
TU
could activate a more potent signal transduction regulatory pathway leading to
apoptosis in cancer cells than Lipoplatin™.
46
In conclusion, incorporation of oxaliplatin into the liposome Lipoxal
T * \v t
largely
improved its accumulation in the HCT116 cells. However, for the same cytotoxic effect (IC50),
less platinum was bound to DNA when using Lipoxal
. Animal studies are required to
determine the anti-cancer effect of Lipoxal™ and its potential advantage in reducing the
systemic toxicity of oxaliplatin.
Acknowledgments
Canadian Institutes of Health Research (grant # MOP 81356). Ldon Sanche, Benoit
Paquette and Rami Kotb are members of the Centre de recherche Clinique-Etienne Lebel
supported by the Fonds de la Recherche en Sante du Quebec. Sanofi-Aventis Canada has
offered a partial unrestricted grant to support this project.
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20.
Akiyama S, Chen ZS, Sumizawa T, Furukawa T (1999) Resistance to cisplatin. Anti-
Cancer Drug Design 14:143-151
21.
Desoize B, Madoulet C (2002) Particular aspects of platinum compounds used at
present in cancer treatment. Critical Reviews in Oncology-Hematology 42:317-325
22.
Boulikas T, Stathopoulos GP, Volakakis N, Vougiouka M (2005) Systemic Lipoplatin
infusion results in preferential tumor uptake in human studies. Anticancer Research 25:30313039
23.
Zlatanova J, Yaneva J, Leuba SH (1998) Proteins that specifically recognize cisplatin-
damaged DNA: a clue to anticancer activity of cisplatin. FASEB Journal 12:791-799
50
CHAPTER III
ARTICLE NO. 2
To investigate the role of radiation sensitizing of platinum drugs, induction of DNA
damage and cell cycle accumulation, we performed our study presented in article no.2.
The complete work is reported in the following article, entitled: “Synergism in
concomitant chemoradiotherapy of cisplatin and oxaliplatin and their liposomal formulation in
human colorectal cancer HCT116 model”, by Thititip Tippayamontri, Rami Kotb, Benoit
Paquette and Ldon Sanche.
This article has been accepted to publish in Anticancer Research, vol. 32(10), 2012.
A n t ic a n c e r R e s e a r c h .*’ : 4 .« 5 - uo 4 i : o i : i
Synergism in Concomitant Chem oradiotherapy o f
Cisplatin and Oxaliplatin and their Liposomal Formulation
in the Human Colorectal Cancer HCT116 Model
T H ITm PTIPPA Y A M O N TR l'
RAMI KOTB3. B EN O rr PAQUETTE1- and LEON SANCHE1 :
'Department o f Nuclear Medicine and Radiobtology, *Center fo r Research in Radiotherapy.
Faculty o f Medicine and Health Sciences, I'nnersite de Sherbrooke,
■'Department o f Medicine. Centre Hospttalier Untversitatre dc Sherbrooke. Sherbrooke. QC. Canada
51
R6sum6
Contexte: Nous avons choisi de tester l'effet de Passiciation chimio-radiotherapie a 8 h
(le plus haut niveau de l'ADN-platine) et 48 h (le plus bas niveau de l'ADN-platine) afin de
verifier si l'irradiation au maximum de concentration ADN-platine pourrait ameiiorer la
synergie. Methodes: Inhibition de la croissance de la lignee humaine de cellules HCT116
t u
TM
(cancer colorectal) traitds avec le cisplatine, l'oxaliplatine, le Lipoplatin
et le Lipoxal
combing avec le rayonnements gamma a ete determine par un essai de formation de colonies.
La synergie a ete 6valuee par l'indice de combinaison. Resultats: Pour les 8 h et 48 h
d'exposition au cisplatine ou Lipoplatin™ suivie d'une irradiation, les concentrations de
medicament superieures a PCI50 a ete jugee synergique, tandis qu'une plus faible concentration
que PICso etait antagoniste. Pour l'oxaliplatine, l'exposition a une concentration superieure k
PIC50
pendant 8 h etait synergique, tandis que l'exposition a l'oxaliplatine (k n'importe quelle
«T*w
concentration) pendant 48 heures etait antagoniste. Le Lipoxal
ameiiore de fason
significative la synergie par rapport a 1’oxaliplatin fibre. Tous les composes platines testes
sensibilisaient les cellules HCT116 soumises a l’irradiation en recrutant les cellules en phase
G2. Conclusion: Les differentes concentrations de composes platines et l'intervalle de temps
entre l'administration du medicament et la radiotherapie pourrait donner des resultats differents
dans la radiochimiotherapie.
52
Abstract
Background: We choose to test the effect of associating chemoradiotherapy at 8 h (the
highest level of DNA-platinum) and 48 h (the lower level of DNA-platinum) to clarify if
irradiation at the maximum DNA-platinum concentration could improve the synergism.
Methods: Growth inhibition of the human colorectal cancer cell line HCT116 treated with
cisplatin, oxaliplatin, Lipoplatin™ and Lipoxal™ plus gamma-radiations was determined by a
colony formation assay. The synergism was evaluated using the combination index method.
Results: For 8 h and 48 h exposure to cisplatin or Lipoplatin™ followed by irradiation, drug
concentrations higher than IC50 was found to be synergic, while a lower than IC50
concentrations was antagonistic. For oxaliplatin, exposure to a concentration above IC50 for 8 h
was synergistic, while the exposure to oxaliplatin (at any concentrations) for 48 h was
antagonistic. Lipoxal™ significantly improve synergism compared to its parent drugs. All
platinum drugs tested sensitize radiation-treated HCT116 cells by inducing G2 phase fraction.
Conclusion: The difference of drug concentrations and time interval between drug
administration and radiotherapy could give different results in chemoradiation therapy.
53
Introduction
The addition of chemotherapy to radiotherapy is expected to improve treatment
outcome of numerous malignant diseases. This is evident in head and neck, lung, anal and
rectal cancers (1-3), but for some other cancers the benefit of such association is less clear (ex.
pancreatic cancer) (4). Some chemotherapeutic agents (as well as other drugs) have also been
proposed to enhance the efficacy of radiotherapy, in addition to their potential anti-neoplastic
effect.
In vitro, in vivo and clinical studies revealed the complexity of the interactions and
i
showed promising results when platinum chemotherapeutic agents were combined with
radiation (5, 6). Platinum salts, cisplatin and oxaliplatin have been shown to possess
radiosensitizing properties (7-9) by means of increasing apoptotic cells death. In addition, the
integration of platinum derivatives into DNA in close proximity to a radiation-induced SSB can
act synergistically to make the defect significant more difficult to repair (10). Cisplatin and
oxaliplatin appeared to be a particularly plausible choice for combination with radiation, since
they are not considered to be radio-mimetic drugs (11).
However, the mechanism underlying such radiosensitisation of platinum drugs is not
fully understood in most cases. Moreover, the effect of the relative time sequence of the
combined treatment of chemotherapy and radiation has not been systematically studied. We
recently observed the relationship between exposure time to various concentrations of different
platinum salts and the platinum level in different intracellular compartments (cytoplasmic,
nuclear and DNA-bound platinum) of HCT116 cells as a function of incubation time (12).
Preclinical investigation on the concomitant interaction of platinum and radiation at a specific
54
time point might case some light on the clinical relevance of the optimal synergism and the
reduction of systemic toxic effect for colorectal cancer treatment.
Although the wide range of applications as anticancer agent of cisplatin and oxaliplatin,
severe side effects have been reported such as neurotoxicity, nephrotoxicity, ototoxicity and
retinopathy (13). In addition, colorectal cancers have been reported for the intrinsic resistance
to cisplatin and chronic resistance to oxaliplatin (14, 15), which has been observed in the
reduction of the intracellular accumulation of free platinum (16). These severe side effects and
cancer cells resistance to cisplatin and oxaliplatin provoke a limitation of their therapeutic
efficiency in colorectal cancer treatment. In order to enhance its efficacy and reduce systemic
toxicity, the drug is encapsulated in liposomal formulation.
•
TXil
Lipoplatin
and Lipoxal
TM
, the liposomal formulation of cisplatin and oxaliplatin,
have been designed with the aim of reducing systemic toxicity, while simultaneously
improving the circulation half-life time of the encapsulated drug that is slowly released as
liposomes degrade (17, 18). In addition, the liposomal formulation has been suggested to
bypass the uptake and efflux transporters, and potentially increase the delivery o f free-platinum
drugs to the tumor cells. Liposomal drugs have a better tolerance profile and are highly
accumulated in the tumor, properties that promise an optimal radiosensitzation. A Phase I/II
study in advanced gastric cancer showed that Lipoplatin™ radio-chemotherapy is feasible, with
minor hematological and nonhematological toxicity (19).
In the current work, we describe the effect of radiotherapy at different times of exposure
to these platinum salts (which implies different concentrations of platinum drugs). Another
purpose was to compare the synergistic effects between free-platinum formulation, cisplatin
55
>rw
and oxaliplatin, with their liposomal formulation, Lipoplatin
and Lipoxal
TKyf
. The interaction
of platinum and radiation was investigated with respect to colonies formation, combination
index and apoptosis. From these data, we assessed the impact of these timings on the synergism
between chemo-radiotherapies.
Materials and Methods
Cell lines and drugs
The HCT116 colorectal carcinoma cell line obtained from American Type Culture
Collection is a p53 wild type. Cells were routinely cultures in modified Eagle’s medium
(MEM) (Sigma, Oakville, Canada) supplemented with 10% fetal bovine serum (FBS), 2 mM
glutamine, 1 mM Sodium-Pyruvate, 100 units/ml penicillin and 100 pM streptomycin in a fully
humidified incubator at 37°C in an atmosphere containing 5% CO2. Cisplatin and oxaliplatin
were purchased from Sigma-AJdrich. Lipoplatin™ and Lipoxal™, the liposomal formulation of
cisplatin and oxaliplatin, respectively, were generously provided from Regulon Inc. (Athena,
Greece). All drugs were diluted to the given concentrations in culture medium immediately
before to use in FBS-free MEM.
Clonogenic assay
The cytotoxicity of platinum and ionizing radiation against HCT116 cell line was
assessed by colony formation assay. For chemoradiation combination therapeutic studies on
HCT116 cells, we performed with three groups of treatment: 1) the control group treated with
four representative platinum-based drugs only at five concentrations, 2) the group treated with
ionizing radiation at four doses of radiation, 3) the group treated with a combination of drugs
and radiation, at a constant of the IC50 and LD50 values (IC50 referred to, and LD50 referred to
56
the dose ofdrugs or radiation causing a 50% reduction of cell growth compared with untreated
cells). All the experiments began by growing the 1000 cells from a single-cell suspension in
100-mm cell culture dishes containing 10 ml of culture medium and incubated at 37°C in a
humidified atmosphere containing 5% CO2 for 24 h. After the culture medium was removed,
the cells were washed twice with PBS before following treatments.
For chemotherapy alone, cells were treated with platinum compounds at appropriate
concentrations and further incubated for the desired period of 8 h and 48 h. Thereafter, the
drugs were removed and the cells were incubated for another 7 days to allow the formation of
colonies. Cells were fixed and stained with 0.1% crystal violet, after washing with tap water.
Colonies containing more than 50 cells were counted manually to calculate the clonogenic
survival fractions. Each experiment was performed at least of 3 times using triplicate cultures
for each drug concentration. In addition, the cytotoxicity of platinum drugs alone was examined
in order to determine the IC50 values, i.e. the concentrations of drugs causing a 50% reduction
of cell growth compared with untreated cells.
For radiation treatment, cells were irradiated with gamma-rays (y-rays) during
exponential cell growth using a 60Co unit at a dose rate of 1.64 Gy/min. Cells were maintained
in MEM supplemented with 10% FBS during all the radiation exposures, which were
performed at room temperature. Dose response curves were established for the HCT116 cells
using a total with 4 single doses of 1 ,2 ,3 and 5 Gy.
For concomitant chemoradiotherapy, cells were treated with the platinum compound at
appropriate concentrations, which were the same concentrations as used to study the
cytotoxicity of chemotherapy alone. Thereafter, the drug was removed and the cells were
57
further incubated for the desired periods. We selected exposure time of 8 and 48 h as they were
previously shown to correlate with the highest and lowest levels of DNA-platinum adducts,
concentration in the nucleus j respectively (12). Then, the cells were irradiated with y-rays at the
LD 50 value (i.e., at a single dose of 2.3 Gy, obtained from the results of Fig. 1). This figure
shows the cell survival fraction decreased as a function of radiation dose. The 2.3 Gy value
was obtained on platinum-free HCT116 cells and corresponds to the radiation dose which
resulted in a reduction of 50% o f the colonies formation.
After treatment with either radiation alone or concomitant chemoradiotherapy, the cells
were incubated for additional 7 days (to allow the formation of colonies) before cells fixation
and counting colonies formation were performed as described above. Each experiment was
performed at least of 3 times using triplicate cultures for each drug concentration.
The result of colonies formation assays from different groups (chemotherapy alone,
radiation alone and combined treatments) were used to calculate the combination index (Cl) in
order to assess the level o f synergy as described in the following section.
Determination of combination index: Antagonism/Synergism/Additivity
Evaluation of the nature of activity of platinum complexes in combination with
radiation is mutually non-exclusive modalities, since the plots for drug, radiation and their
combination are not parallel to each others. This evaluation and the Cl calculation with
respected to cell survival were performed according to the method described by Chou and
Talalay (20). The Cl was evaluated for each drug at 8 and 48 h of incubation. We also sought to
determine whether the synergism would be reached at platinum drug dose equivalent, lower
and higher than that of respective IC50. A lower concentration was defined as the average of
58
minimum tested concentration to the concentration at IC50 values, and while a higher
concentration defined as the average of concentration at IC50 values to maximum tested
concentration.
Mathematical evaluation of the data was carried out by computerized analysis of the
median dose effect (IC50) and the combination index as well as linear correlation coefficient (r).
According to the CALCUSYN software of BIOSOFT (Ferguson, MO) developed by Chou
(21), the Cl is expressed by the relationship where each term represents,
CJ
( Pt) i ( IR) |
( Ptx)
( IRx)
(P tjlR )
(Ptx\lRx) -
the denominator, (Ptx) is for the concentration of platinum compound “alone” that inhibits
colonies formation at x%, and (IRx) is for the dose of radiation alone that inhibits colonies
formation at the same x%. In the numerator, (Pt) + (IR) “in combination” also inhibit the
colonies formation also at the same x%. Cl values <1.0, and = 1.0, and >1.0 indicate
synergistic, additive, and antagonistic effects, respectively.
Cell cycle and apoptosis analysis
Cells were treated with platinum drugs at concentrations corresponding to their specific
IC 50, as determined by the clonogenic assay. After
8
h and 48 h of platinum drug exposure with
or without radiation treatment, both adherent and floating cells were washed and allowed to
grow for an additional 48 h in culture medium. Cells were harvested by trypsinization, pooled
with the culture medium that contained floating cells, and collected by centrifugation (3 min,
1300 rpm). The cell pellets were resuspended in 0.5 ml of PBS and fixed with 1.5 ml of icecold 95% ethanol. Before analysis, 100 pi of FBS was added. The cell pellets were re­
59
suspended in 2 ml PBS and collected the pellet by centrifugation (3 min, 1300 rpm). The 200 pi
of staining solution containing o f 0.5 mg/ml of RNase A and 5 pg/ml of propidium iodide was
added, and the suspension was incubated at 37°C for 30 min. The samples were analyzed using
FACScan (Becton Dickinson). The cells in the subdiploid (sub-Gj/Go) region o f the histogram
are classified as death cell in apoptosis assay. For each sample 10,000 events were analyzed.
These analyses were performed at least three separate cultures.
Statistical analysis
Results were expressed as the mean ± SD of three experiments performed in triplicate.
P < 0.05 was considered statistically significant (two-tailed paired Student’s t-test).
Results
Cytotoxicity potential:
Clonogenic assay fo r platinum-drugs chemotherapy
The survival of HCT116 clonogenic cells as a function of platinum-based drug
concentration for four tested drugs on cells at 8 and 48 h incubation are shown in Figure 2 and
3, respectively, for the four drugs tested. The IC50 values for all of the drugs tested in HCT116
cells for 8 and 48 h are summarized in Figure 4. After 8 h incubation of cisplatin, oxaliplatin,
Lipoplatin™ and Lipoxal™, the IC50 values were observed at 4.65 pM, 2.29 pM, 41.62 pM
and 5.61 pM, respectively. These IC50 values of four tested drugs were reduced about 6.4-,
35.8-, 9.95- and 13.7-times, respectively, when the incubation time was increased to 48 h.
60
Clonogenic assay for radiation treatment
According to the clonogenic assay, the cell survival fraction decreased as a function of
radiation dose (Figure 1). The LD50 for the irradiated HCT116 cells was 2.30 ± 0.98 Gy. This
value was further used in combination with platinum drugs at different concentrations to
measure the CL
100
o'
98
>
an
w
p•m
4)
10
CL
3mm
©
0
1
0
1
2
3
4
5
Dose (Gy)
Figure 1. Dose response curve for radiotherapy alone on HCT116 cells. Cells were irradiated
with gamma-rays (y-rays) during exponential cell growth using a 60Co unit at a dose rate of
1.64 Gy/min. Each point represents mean of 3 independent experiments and the bars indicate
the standard deviation (S.D).
Clonogenic assay fo r chemoradiation combination
Similar plot of clonogenic cell survival were constructed for all chemoradiation
combinations. The results are shown in the same graph as those of the platinum-drug treatment
alone at 8 h and 48 h incubation in Figure 2 and 3. The IC50 values for all of the drugs tested
combination with radiation at LD50 dose in H C T 116 cells for 8 and 48 h are summarized in
Figure 4. The platinum concentration needed to obtain the IC50 was considerably reduced
61
where the tested drugs were combined with radiation. Theses IC50 values were reduced about
11.4-, 7.3-, 44.2- and 10.89-times for combination of radiation and cisplatin, oxaliplatin,
Lipoplatin™ and Lipoxal™, respectively, when increased the time of incubation to 48 h.
B)
A)
- Q - D i u g alone
D rug-R ad
100
100
8>
>
•9
W
10
■a
&
u
1
0
2
8
6
4
0
10
Cisplatin (pM)
3
1
2
Oxaliplatin (pM)
4
D)
C)
100
100
5 10
w
0
20
40
60
0
80
5
10
15
Lipoxal (pM)
Lipoplatin (pM)
Figure 2. Response curves for chemoradiotherapy on HCT116 cells at 8 h incubation time. (□)
Cells were exposed to various concentrations of each platinum derivative alone, or (■) in
combination with gamma radiation (2.3 Gy). The concentration of the drugs is based on the
IC50 values, which were previously obtained from the clonogenic assay at 8 h incubation. Each
point represents mean o f 3 independent experiments and the bars indicate the S.D.
62
- O - D r u g alone
—■ —D m g-R ad
A)
B)
100
100
r
>
IWW
10
■a
&
§o
0
1
0
0.25
0.5
0.75
1
0
Cisplatin(pM )
0.1
0.2
0.3
0.4
0.5
Oxaliplatin (pIM)
C)
D)
100 q-
100
R
R
>
>
i
M
■S
&
w
■s
&
ge
9
u
10
■Ml
0
o
1
0
1
1.5
Lipoplatin (pM)
0.5
0.25
0.5
0.75
1
Llpoxal (pINI)
Figure 3. Response curves for chemoradiotherapy on HCT116 cells at 48 h incubation time.
(□) Cells were exposed to various concentrations of each platinum derivative alone, or (■) in
combination with gamma radiation (2.3 Gy). The concentration of the drugs is based on the
IC50
values, which were previously obtained from the clonogenic assay at 48 h incubation.
Each point represents mean of 3 independent experiments and the bars indicate the S.D.
63
A)
5 ° -I
□ D ru g alone
,4 0 -
Sh
■ D rug 8 h-radiation
53
«
C. ‘ 30 *■ 3
M
fi
3 §."0 "1
J
e
w 10 J
I
n
Cisplatin Oxaliplatin Lipoplatin
B)
£ L Lipoxal
1.5
□ D ru g alone 48 h
■D rug 48 h-radiation
53
ss
c.
3
e
a 0.5
IL
I
Cisplatin Oxaliplatin Lipoplatin
Lipoxal
Figure 4. The IC50 values o f cisplatin, oxaliplatin and their liposomal formulation
(Lipoplatin™ and Lipoxal™) in combination with radiation. A for 8 h of drug incubation with
or without radiation, and B for 48 h o f drug incubation with or without radiation.
64
Concomitance effect o f platinum compounds with radiation
The effect of adding radiation to cells treated at platinum drug concentration equivalent
to, lower or higher than their respective IC50, in the time interval of 8 h and 48 h platinum drug
incubation are shown in Figure 5A and 5B.
Cisplatin: With a similar trend after exposure for 8 h or 48 h, the treatment with
cisplatin at IC50 showed only an additive effects (Cl = 1.02 and 1.05). With a dose lower than
IC50,
the combination with radiation showed an antagonistic (less than additive) effect (Cl =
1.22 and 1.22), while a synergism was only observed with a concentration above IC50 (Cl =
0.74 and 0.70).
Oxaliplatin: 8 h exposure to oxaliplatin at or below IC50 showed only an additive effect
(Cl = 0.99 and 1.05), but a synergism was noted when a dose above IC50 was used (Cl = 0.87).
Exposure to oxaliplatin for 48 h (at, above or below IC50) resulted in an antagonistic effect (Cl
= 1.22,1.14 and 1.4, respectively).
Lipoplatin: The treatment with Lipoplatin at IC 50, for 8 h or 48 h, showed only an
additive effect (Cl = 0.98 and 0.94). Exposure to a concentration below IC 50 for 8 h showed an
additive effect (Cl = 1.06), while a similar concentration for 48 h -exposure resulted in an
antagonistic effect (Cl = 1.19). Treatment with a concentration higher than IC50 resulted in a
synergism after 8 h or 48 h o f exposure (Cl = 0.77 and 0.77).
Lipoxal: The treatment with Lipoxal at, above or below IC50 value for 8 h showed a
synergism (Cl = 0.88, 0.89 and 0.77, respectively); but this gave only an additive effect with 48
h (Cl = 1.04,1.09 and 1.01, respectively).
65
A)
n<IC50
■-IC50
a>IC50
Antagonistic
t
. A dditive
Synergistic
Cisplatin Lipopaltin Oxaliplatin Lipoxal
B)
15541554
262
.
Cisplatin Lipopaltin Oxaliplatin
Lipoxal
Figure 5. The combination index (Cl) of chemoradiotherapy on HCT116 cells at A)
48 h incubation time. Column
( 3
8
h and B)
), ( ■ ) and ( B ) indicate Cl values at the concentration of
platinum drugs lower, higher and equal to the IC50 value.
66
Cell cycle analysis
The cell cycle progression of HCT116 cell exposed to four platinum drugs for 8 h and
48 h of incubation time with and without radiation treatment were observed as shown in the
Figure 6A and 6B. Radiation treatment increased the accumulation of the cells in the Go/Gj and
G2/M (1.15- and 1.22-time), while decreased the S phase fraction (1.63-time) compared to the
control group (57.33 ± 3.26%, 29.77 ± 3.18%, 13.03 ± 1.02% for G0/G 1, S and G2/M phase,
respectively).
Platinum chemotherapy: After 8 h incubation, the cell accumulation o f the G0/G 1, S and
G2/M phase were observed for cisplatin, oxaliplatin, Lipoplatin™ and Lipoxal™, respectively,
at 56.88 ± 1.58%, 50.57 ± 1.06%, 47.32 ± 3.00% and 57.14 ± 5.86%, respectively; 22.45 ±
0.81%, 23.09 ± 1.98%, 35.10 ± 1.31% and 28.32 ± 3.86% respectively; and 20.67 ± 1.01%,
26.38 ± 1.48%, 17.58 ± 1.73% and 14.29 ± 2.36%, respectively. These values of cell
accumulation in the G0/G 1 were increased about 1.4-, 1.46-, 1.68- and 1.29-times, respectively,
when the incubation time was increased to 48 h. For the cell accumulation in the S and G2/M
phase were reduced about 2.30-, 1.48-, 3.88- and 1.47-times, respectively; and 1.97-, 2.56-,
1.70- and 1.32-times, respectively, when the incubation time was increased to 48 h.
The chemoradiation combination: After combined radiation treatment with cisplatin,
oxaliplatin, Lipoplatin™ and Lipoxal™ administration, the cells accumulation in the G0/G 1
phase for 8 h was increased, while the slightly reduced of cell accumulation was observed after
48 h incubation. The reduction of the cells accumulation in the S phase after combined drug
and radiation, no substantial change was observed after the combination at 48 h of drug
incubation. The similar trend of the increasing of the cells in the G2/M phase for 8 h and 48 h
drug incubation was observed.
67
Induction o f apoptotic cells
To investigate whether the induction of apoptosis cells after the combined treatment, the
cells in the subdiploid (sub-Gi/Go) phase were analysed (Figure 7A and 7B).
Platinum-drug chemotherapy: After 8 h incubation of cisplatin, oxaliplatin,
Lipoplatin™ and Lipoxal™, the percentage of the apoptotic cells were observed at 1.54%,
1.68%, 0.76% and 1.70%, respectively. These percentages of four tested drugs were reduced
about 10.3, 8.0, 1.0 and 1.2-times of magnitude, respectively, when the incubation time was
increased to 48 h.
Chemoradiation combination: The percentage of apoptotic cells were considerably
induced where HCT 1.16 cells were incubated to the tested drugs for 8 h prior the radiation
treatment. The apoptotic cells were reduced about 1.1-, 2.2-, 1.6- and 1.3-times for combination
of radiation and cisplatin, oxaliplatin, Lipoplatin™ and Lipoxal™, respectively, when
increased the time of incubation to 48 h.
68
^
Lipoxal 8h-IR
Lipoxal 8h
Lipoplatin Sh-ER.
Lipoplatin 8h
Oxaliplatin 8h-IR
Oxaliplatin 8h
Cisplatin Sh-IR
■G0G1 (%)
□S (%)
b G2M(%)
Cisplatin Sh
IR alone
Control
0
20
40
60
80
Cell accumulation (%)
100
B)
Lipoxal 4 8h-IR
Lipoxal 4 8h
Lipoplatin 48h-IR
Lipoplatin 48h
Oxaliplatin 4Sh-lR
■G0/G1 (%)
□s (%)
Oxaliplatin 48h
Cisplatin 48h-IR
b
G 2 M (% )
Cisplatin 48h
IR alone
Control
0
20
40
60
80
Cell accumulation (%)
100
Figure 6. Flow cytometric measurement of cell-cycle distribution of the HCT116 cell after
-p w
cisplatin, oxaliplatin, Lipoplatin
< P |L l
and Lipoxal
administration for A) 8 h and B) 48 h
incubation, followed by irradiation at 2.3 Gy. The concentration of the drugs is based on the
IC 50 values, which were previously obtained from the clonogenic assay at 8 h or 48 h
incubation. Mean of three experiments is shown.
69
A)
Lipoxal 8h-IR
Lipoxal 8h
Lipoplatin 8h-IR
Lipoplatin 8h
Oxaliplatin 8h-IR
Oxaliplatin 8h
Cisplatin 8h-ER
Cisplatin 8h
IR alone
Control
0
1
2
3
4
5
2
3
4
Sub-Gl phase (%)
5
Sub-Gl phase (%)
^
Lipoxal 48h-IR
Lipoxal 48h
Lipoplatin 48h-IR
Lipoplatin 48h
Oxaliplatin 48h-IR
Oxaliplatin 48h
Cisplatin 48h-IR
Cisplatin 48h
IR alone
Control
0
1
Figure 7. Induction of apoptotic cell death after treatment with platinum drugs and radiation.
The number of apoptotic cells was quantified by PI staining and flow cytometric analysis in
HCT116 cells treated with A) 8 h and B) 48 h incubation of platinum drugs alone or in
combination with radiation at 2.3 Gy. The concentration of the drugs is based on the IC50
values, which were previously obtained from the clonogenic assay at 8 h or 48 h incubation.
Mean of 3 independent experiments is shown and the bars indicate the S.D.
70
Discussion
Despite their narrow therapeutic index and cumulative toxicity, platinum compounds
are widely used as chemotherapeutic agents. Almost all current cisplatin-based chemoradiotherapy protocols consist of daily administration of radiotherapy, with intermittent
administration of the agent. This implies that the tumor and healthy tissues are exposed to
radiotherapy after different periods of administration of cisplatin, and that the drug level varies
overtime. The same scheme is also found in most protocols associating oxaliplatin and 5-FU
regimens in the chemoradiotherapy treatment of rectal cancer.
The cytotoxicity o f platinum-based drugs is dose and time-dependent (5, 22). An
effective combination o f radiation and chemotherapy is expected to lead to synergism or at
least yield an additive effect on tumor cells. Bentzen and co-worker have been suggested that
for maximal cytotoxic enhancement the drug must be present at the time of irradiation to
modify the initial stage o f radiation-induced cell killing or repair (23). Adequate information
concerning the pharmacokinetics of each drug used as well as the intra-tumor drug distribution
could therefore help optimizing the treatment schedule.
We previously studied the kinetic of cisplatin and oxaliplatin uptake and that o f their
liposomal formulation uptake in human colorectal cancer cell line HCT116 (12) to determine
the amount of platinum in different tumor cell compartments and DNA-bound platinum at
different time intervals after administration. For the free cisplatin and oxaliplatin, the rapid
initial accumulation in DNA reached respectively 0.16 ng Pt/pg DNA and 0.32 ng Pt/pg DNA
after 8 h incubation. The levels of platinum DNA adducts were progressively reduced as a
function of time down to a minimum at 48 h. More than 90% of the drugs were accumulated in
the cytoplasm after 48 h incubation with the cells. Lipoplatin™ and Lipoxal™ resulted in a
71
generally more than 10-fold lower distribution of platinum in DNA compared to their free
formulation. The results of cellular uptake and cytoplasm / DNA distribution of cisplatin and
oxaliplatin and their liposomal formulation in HCT116 suggested the point of maximum DNAplatinum adducts formation at 8 h.
In addition, the radiosensitivity is a function of the cell cycle. The radiosensitive is
pronounced when the cells are in the Go/Gi and G2/M phase, while cells are radioresistent when
the cells are in the S phase. The Gi arrest was a majority of the cell cycle analysis showed that
the majority o f the cells after drug treatment for both 8 h and 48 h incubation with and without
radiation were accumulated in the Gi phase. Our results are similar to the previous studied (2426). Moreover, the cell accumulation in the G2/M phase which is the most radiosensitive phase,
were mostly observed after 8 h of drug incubation with and without radiation, while no
significantly change was observed after 48 h incubation. These results accompanied by a higher
proportion of apoptotic cells after 8 h incubation than after drug exposure for 48 h incubation.
Thus, we chose to test the effect o f associating radiotherapy and chemotherapy at 8 h and 48 h
to clarify if the maximum DNA-Pt could improve synergism.
Clonogenic assays after 8 h of incubation indicated that the concentration of cisplatin
and oxaliplatin needed to obtain the same level of cytotoxicity was approximately 3-10 times
lower than their liposomal formulation. When the incubation period is increased to 48 h, there
were still about a factor o f two differences in the IC 50 values between cisplatin and oxaliplatip
compared to their liposomal formulations. This confirms our initial results on the
pharmacokinetics of platinum drugs (12) where the cells were less sensitive to the liposomalplatinum formulation than free-platinum formulation. For all tested drugs, whether after 8 h or
72
48 h of incubation, the association of radiotherapy reduced the IC 50 and increased the efficiency
of HCT116 cells killing by factors of 3 to 7.
As each of these drugs remain for long time in the target tissues, while the patient is
given radiotherapy daily, difference of drug concentrations and time interval between drug
administration and radiation treatment could cause a difference in the combined effect. Our
results confirm our hypothesis that radiotherapy at various times after injection of the platinum
drugs at different concentrations could lead to different outcomes. For cisplatin, a drug
concentration higher than IC50 is synergic, while a lower than IC50 concentration is
antagonistic. Although, cisplatin showed a great improvement in synergism at high
concentrations, its severe toxicity is of considerable concern. For oxaliplatin, exposure to a
concentration above IC50 for 8 h is synergic, while the exposure to oxaliplatin (at any
concentrations) for 48 h is antagonistic. Lipoplatin™ showed a similar trend as observed with
cisplatin, but Lipoxal™ differed significantly from its parent drugs. These results also
suggested that time-dependence on irradiation are more important to reach the synergism in the
case of oxaliplatin and Lipoxal™ than for cisplatin and Lipoplatin™.
Several potential mechanisms of platinum-mediated radiation sensitization have been
reported (5,10,27) to rationalize the synergic and additive effects of chemoradiation treatment.
Zheng and co-worker clearly demonstrated that there is a significant increase of single and
double strand breaks of DNA when DNA is irradiated in the presence of cisplatin (10). In
addition, it has been proposed that ionizing radiation caused higher uptake rate, probably due to
an increased permeability of the cells membrane (28, 29). Some authors suggested that a
sufficient number o f platinum atoms must accumulate in the cancer cell to reach a
radiosensitizing effect. Gabriel and co-workers showed the relationship between cell uptake
73
and radiosensitizing potential (30). Liposomal formulation showed promising results in order to
increase the level of cell uptake, compared to their free platinum form. High accumulation Of
liposomes in the cancer cells could be considerable value to increase the benefit of
chemotherapy when it is combined with radiotherapy.
An antagonistic effect was proposed to be associated with the efficiency of DNA repair,
as well as, alterations in cell oxygenation and the cellular levels of thiol groups (31, 32). The
prolonged time o f drug incubation could lead to the reduction of platinum accumulation and PtDNA adducts. An antagonistic effect was clearly observed when HCT116 cells were treated
with oxaliplatin and Lipoxal™ at time interval of 48 h before irradiation. Importantly in the
clinical treatment, typical radiation treatment schedule is 2 Gy irradiations each day for 5 days
a week (33), irradiation at the time point that displayed low level of platinum concentrations in
the cell would lead to antagonistic effect.
Conclusion
The present study provides information about the schedule dependence of the synergism
of platinum-based drugs and radiation. Confirmation of these data and their validation in
animal models can have a tremendous impact on designing future platinum based chemoradiotherapy protocols. Associating radiotherapy to the time intervals of maximum synergism
could improve efficacy and limit toxicity of chemotherapeutic agents.
Acknowledgments
This research was financed by the Canadian Institute of Health Canada (grant # 81356),
Leon Sanche, Benoit Paquette and Rami Kotb are members of the Centre de recherche
74
Clinique-fitienne Lebel supported by the Fonds de la Recherche en Sant6 du Quebec. SanofiAventis Canada has offered a partial unrestricted grant to support this project.
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79
CHAPTER IV
ARTICLE NO. 3
In this chapter, we investigated the synergism of combined oxaliplatin and its liposomal
formulation plus radiation treatment using an animal model. This work was performed to
determine radiosensitizing activity of platinum drugs in an animal model.
The complete work is reported in the following article, entitled: “New therapeutic
possibilities of combined treatment of radiotherapy with oxaliplatin and its liposomal
formulations (Lipoxal™) in colorectal cancer using nude mouse xenograft”, by Thititip
Tippayamontri, Rami Kotb, Benoit Paquette and Leon Sanche.
This article has been submitted to Radiation Research, 2012.
80
New therapeutic possibilities of combined treatment of radiotherapy with oxaliplatin and
its liposomal formulations (Lipoxal™) in colorectal cancer using nude mouse xenograft
Running title: Chemoradiotherapy for colorectal cancer
Thititip Tippayamontri8’b. Rami Kotb0, Benoit Paquette81b and Leon Sanche8, b
“Department of Nuclear Medicine and Radiobiology, bCenter for research in radiotherapy,
Faculty of Medicine and Health Sciences, Universite de Sherbrooke, departm ent of Medicine,
Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Quebec, Canada.
Corresponding author:
Prof. Benoit Paquette, Faculty of Medicine and Health Science, Universite de Sherbrooke,
3 0 01,12e Avenue North, Sherbrooke, Quebec, Canada, J1H 5N4.
Tel. (1) (819) 820-6868, ext. 14767
Fax: (1) (819) 564-5442
E-mail address: [email protected]
ACKNOWLEDMENTS: This research was financed by the Canadian Institute of Health
Research (grant # 81356). Ldon Sanche, Benoit Paquette and Rami Kotb are membres of the
Centre de recherche Clinique-Etienne Lebel supported by the Fonds de la Recherche en Santd
du Qudbec. Sanofi-Aventis Canada has also offered a partial unrestricted grant to support this
project.
81
Tippayamontri, T., Kotb, R., Paquette, B. and Sanche, L. New therapeutic possibilities
of combined treatment of radiotherapy with oxaliplatin and its liposomal formulations
(Lipoxal™) in colorectal cancer using nude mouse xenograft. Radiat. Res.
Meme si l’ADN est consider^ comme la cible principale pour la radiosensibilisation
menant a la mort cellulaire, la periode d’incubation suivant 1’administration d’un compose
plating menant a une accumulation maximum a l’ADN et un meilleur effet concomitant n’est
pour le moment pas bien determinee pour une application clinique. Dans cet ouvrage, nous
explorons le lien entre le niveau d’adduits platine-ADN menant a un effet concomitant
potentiel et different temps d’attente entre 1’administration de compost plating et la
radiotherapie. Pour augmenter l’efficacife anticancereuse tout en surmontant ces effets
secondaires, nous avons egalement evalue une formulation liposomale de l'oxaliplatine, le
Lipoxal™. Des Etudes ont ete effectuees dans le carcinome colorectal humain HCT116
implanfe chez la souris nu/nu. La quantife de platine dans la tumeur, dans l'ADN tumoral, dans
des tissus normaux et dans le sang a ete mesuree par un spectrometre de masse coupfe au
plasma inductit. . L’effet concomitant avec le rayonnement a efe evaluee a 4, 24, et 48 h apfes
l'injection de drogue et lorsque les medicaments ont ete administres immediatement apres la
radiotherapie. Le retard de croissance tumorale obtenu est rapporfe et corrdfe a l'analyse
d ’apoptose. Le retard de croissance tumorale obtenu est rapporte et correfe a l'analyse
d’apoptose. Tandis que la quantite de platine dans le tissu tumoral atteint un pic initial 4h
suivant 1’administration et diminue par la suite avec le temps, le niveau d’adduits platine-ADN
oscille en deux pics apres 1’administration. En effet, le niveau d’adduit platine-ADN atteint un
premier pic a 4 h suivi d’une diminution progressive pour atteindre un premier nadir k 24 h
suivi d’une nouvelle augmentation pour atteindre un pic plus eieve a 48 h avant de rebaisser,
sauf pour Lipoxal™. Un important potentiel radiosensibilisant est observe lorsque la
radiotherapie est effechfee 4 et 48 h apfes l'injection de medicaments, ce qui correspond k la
pointe de concentration d’adduits platine-ADN. Ces resultats ont ete assocfes a un plus grand
nombre de cellules apoptotiques. A l'inverse, un effet antagoniste a
efe
observe lorsque la
efe deiivree 24 heures apres l'injection d'oxaliplatine, ce qui correspond k des
niveaux inferieurs d’adduit platine-ADN. Un effet concomitant a efe obtenu avec le Lipoxal™.
radiotherapie a
82
Tippayamontri, T., Kotb, R., Paquette, B. and Sanche, L. New therapeutic possibilities
of combined treatment of radiotherapy with oxaliplatin and its liposomal formulations
(Lipoxal™) in colorectal cancer using nude mouse xenograft, Radiat. Res.
Although DNA is considered the main target of radiosensitizing activity leading to
cancer cell death, there are limited number of publications that studied on the beneficial of
adding radiotherapy at the optimal time at which the platinum is bound to the DNA of tumor
cells. Here, we explored relationships between different levels of platinum-DNA adducts and
the potential o f the concomitant effect after different schedules of combined treatment. To
further increase the anticancer effectiveness while overcoming the side effects of oxaliplatin,
we also evaluated its liposomal formulation, named Lipoxal™. Studies were performed with
the human colorectal carcinoma HCT116 implanted in nude mice. The amount of platinum in
the tumor, tumoral DNA, and in normal tissues and blood was measured in function of time
using an Inductively Goupled Plasma Mass Spectrometer. The concomitant effect with
radiation was assessed at 4, 24, and 48 h after drug exposure. The tumor growth delay was
reported and correlated to the apoptosis analysis. While the amount of platinum in tumor
tissues reached an initial peak at 4 h after drug exposure and declined over time, the
concentration of platinum-DNA adducts was more varied and two maxima were observed at 4
h and 48 h after drug administration, excepted for Lipoxal™. The concomitant effect was
observed when radiotherapy was performed at 4 h and 48 h after oxaliplatin administration,
which were related to maximum levels of oxaliplatin-DNA adducts. These results were
associated with an increase of apoptotic cell death. Conversely, no advanced improvement of
the concomitant effect was observed when radiotherapy was delivered at 24 h after oxaliplatin
administration, which was related to the lower level of platinum-DNA adducts. The
concomitant effect was obtained with Lipoxal™ for each of combined treatment schedules.
83
INTRODUCTION
Concomitant chemoradiotherapy is proposed to improve the treatment effectiveness in
several malignant diseases, including rectal cancer, advance head and neck squamous cell
carcinoma and cervical cancer (1-4). The efficacy of concurrent chemotherapy with radiation
treatment is often increased by using chemotherapeutic drugs as radiosensitizers (5). The
common chemoradiation treatment for colorectal cancer is the combination of 5-fluorouracil
(5-FU), leucovarin, oxaliplatin and radiation (6).
While the increased treatment efficacy of combined treatment, recent completed clinical
phase III trials (ACCORD-0405, STAR-01 and NSABPR-04) that added oxaliplatin to 5-FU
backbone or capcitabine have failed to improve results of combined treatment (7-9), with the
exception of the German CAO/ARO/AIO-04 study (10) when added oxaliplatin to 5-FU plus
radiation. In addition to increase efficacy of combined treatment, some difficulties in managing
side effects from this combined treatment are still commonly found in clinical practices, such
as hematologic and gastrointestinal toxicity (11). Since limited number of publications on the
use of oxaliplatin in combination with radiation, Flatmark and Ree have suggested the need of
initial testing radiosensitizing activity of oxaliplatin alone in precliriical setting prior the clinical
use as a local radiosensitizer for radiation (12). Moreover, the radiosensitizing activity, and an
accurate scheduling of combined treatment to achieving the benefit of drug-radiation
interactions while maximizing tumor responses have to be determined.
The role of oxaliplatin as radiosensitizers has been recently described (6, 13). Likeother platinum compounds, the main mechanism of antineoplastic and radiosensitizer activity
of oxaliplatin is thought to occur mostly via the formation of platinum-DNA adducts (14, 15).
The oxalate bidentate ligand of oxaliplatin preferentially reacting with the highly nucleophilic
84
N7 position on guanine or adenine, and form coordinated covalent bonds. Oxaliplatin can bind
to two sites in DNA with the following order of preference: -GG- > -AG- »
-GA. The
resulting biadducts composed of approximately 60-65% 1,2-intrastrand GG, 25-30% 1,2intrastrand AG, 5-10% 1,3-intrastrand GXG and 1-3% interstrand GG (16). Platinum binds to
DNA and causes a critical structure change in the DNA such as a bend of 45 degree, unwinding
o f DNA and causing disrupting of the purine bases. The formation of platinum-DNA adducts
which interfere with cellular repair and DNA replication, and which therefore trigger a chain of
cell regulatory events, ultimately leading to cell death (17).
Although the
most favorable concomitant effect would be occurred when
radiosensitizers reach the DNA, the activity of radiosensitizers is often referred to the
concentration of drugs accumulation in the tumor tissue (18-20). To date very few number of
preclinical publications stutied on combined treatment of oxaliplatin and radiation for
colorectal cancer. Also the information underlying the assessment of radiotherapy at the
maximum oxaliplatin-DNA adducts is not yet clarified in both preclinical and clinical setting.
In this study we hypothesized that the potential of combined treatment possibly depend on the
interaction of radiation at different times after drug administration, which were referred to
difference levels of platinum-DNA adducts.
Previous studies demonstrated efficacy of oxaliplatin in the treatment of colorectal
cancer, however the antitumor activity of oxaliplatin may be limited by its severe systemic side
effects, such as neurotoxicity, haematological and gastrointestinal toxicity (21). In recent years,
liposomal oxaliplatin (Lipoxal™) was developed to reduce the systemic toxicity of oxaliplatin,
while attempting to improve the anticancer efficiency (22). As a single agent, Lipoxal™ has
produced significant cytotoxicity in human colorectal cancer cells and showed adequate
85
effectiveness in pretreated patients with colorectal cancer in phase I study (23). Lipoxal™ is
currently under study for phases II for colorectal cancer (22). The combination of radiation and
TW
Lipoxal
is an interesting preclinical research subject that might shed some light on new
clinical treatment strategies. To date, there is not sufficient data concerning the
pharmacokinetic, anticancer activity, and combined treatment efficacy of Lipoxal™
comparative to its parent compound (oxaliplatin).
In the present work, variations of platinum accumulation with incubation time in blood,
different normal and tumor tissues compartments, as well as platinum-DNA were measured.
Therefore, we explore relationships between different levels of platinum-DNA adducts and the
potential of the concomitant effect after combined treatment of oxaliplatin and Lipoxal™ plus
radiation. The concomitant effects were evaluated with respect to tumor growth delay and
apoptosis analysis.
86
MATERIALS AND METHODS
Cell line
The HCT116 colorectal carcinoma cell line was obtained from the American Type
Culture Collection. Cells were routinely cultured in modified Eagle’s medium (MEM) (Sigma,
Oakville, Canada) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine,ImM
sodium pyruvate, 100 units/mL penicillin and 100 pM streptomycin in a fully humidified
incubator at 37°C and 5% CO2.
Animal xenograft model
All experiments were performed with outbred male nu/nu nude mice at 4-6 weeks of
age (Charles River, USA). The animals were maintained in our conventional animal facility,
under specific pathogen-free conditions. Housing and all procedures involving animals
manipulation were performed according to the protocol approved by Universite de Sherbrooke
Animal Care and Use Committee. HCT 116 tumor cells were inoculated by subcutaneous (s.c.)
injection into each rear flank with 0.1 mL of cell suspension containing 2 x 106 cells. Tissue
culture medium without serum was used as injection vehicle. Tumor measurement began 1
week post-injection and continued twice weekly through the course of the experiment. Tumor
volumes were measured with a caliper and calculated by the formula: V (mm3) = 7t/6 x a (mm)
x b (mm ), where a and b referred to the largest and smallest perpendicular tumor diameters,
respectively. The pharmacokinetic study began when tumor volumes reached the range o f 100300 mm , with an average tumor volume of approximately 200 mm , The tumor-bearing
animals were randomized into different groups of three to five animals. For all procedures
87
(implantation, chemotherapy, radiotherapy and euthanasia) nu/nu nude mice were anesthetised
with an intraperitoneal injection of ketamine/xylazine (87/13 mg/mL) at 1 mL/kg.
Drug preparation
Oxaliplatin was obtained through the pharmacy of the Centre Hospitalier Universitaire
de Sherbrooke. Lipoxal
, the liposomal formulation of oxaliplatin, was generously provided
by Regulon Inc. (Athens, Greece).
Pharmacokinetics studies
Three groups of mice were studied, including: 1) oxaliplatin, 2) Lipoxal™ and 3)
control group. The tested drugs were dissolved in 5% dextrose for a final concentration of 1
mg/mL to be given as a single dose of 10 mg/kg intravenously (i.v.) via the tail vain. Animals
in the control group received an injection of only 5% dextrose. Three to five animals from each
group were sacrificed at 4, 24, 48, 72, and 96 h post-injection. A sufficient blood sample was
obtained via cardiac puncture for later whole blood and serum platinum assessments. Tumor
and different normal tissues were taken to determine platinum accumulation in the cytoplasm,
the nucleus as well as in DNA extractions.
Determination of cytoplasmic and nuclear platinum accumulation
Samples were processed with the nuclear and cytoplasm extraction kit Activemotif
(ActiveMotife North America). Briefly, tumors were weighed and diced into very small pieces
using a razor blade. Three mL of ice cold IX hypotonic buffer supplemented with 3 pL of 1 M
dithiothreitol and 3 pL o f detergent were added per gram of tissue, homogenized, incubated on
ice for 15 min. and then centrifuged for 10 min. at 850 x g at 4°C. The hypotonic buffer and
88
detergent were supplied in the kit. Subsequently, the supernatants corresponding to the
cytoplasmic fraction were transferred into pre-chilled microcentrifuge tubes, while the pellets
were gently resuspended in 500 pL IX hypotonic buffer, transferred to a pre-chilled
microcentrifuge tube, and incubated on ice for 15 min. The detergent (25 pL) was added and
vortex for 10 sec at the highest setting. The samples were centrifuged for 30 sec at 14,000 x g
in a microcentrifuge pre-cooled at 4°C and these second supernatants were combined with the
first ones. The resulting pellets corresponded to the nuclear fraction.
Quantification of DNA-bound platinum
DNA was extracted according to a salting-out procedure (24). Briefly, the tumor was
weighed and diced into very small pieces, and homogenized with a pre-chilled Dounce
homogenizer. On ice, 3 mL of a lysing buffer containing 10 mM Tris-HCl, 400 mM sodium
chloride and 2 mM EDTA were added. Then, 0.1 mL sodium dodecylsulfate (SDS 20%) and
0.5 mL proteinase K (10 mg/mL, DNase free) were added and incubated overnight at 37oC.
DNA was precipitated by adding 1.2 mL of 5 M sodium chloride. The tube was gently agitated
for 1 min, centrifuged at 2500 x g for 15 min and then the supernatant was transferred to
another tube. DNA was precipitated with 2.5 vol. of 95% ethanol, the tube was gently inverted
for 30 s, and DNA was spooled out and air-dried briefly. DNA was dissolved in TE buffer
(lOmM Tris-HCl, lmM EDTA, pH 8.0) and RNase A (0.05 mL of a 10 mg/mL solution) was
added and incubated for 1 h at 37oC. DNA was precipitated a second time with ethanol as
described above and re-dissolved in TE buffer. The amount of DNA extracted was evaluated by
measuring the absorbance of the DNA solution at 260 nm (A260) with a spectrophotometer
(Synergy HT, BIO-TEX) and calculated using the following equation: A260 x 50 (pg/mL).
89
Platinum quantification using an Inductively Coupled Plasma Mass Spectrometer
The quantities of platinum accumulated in the cytoplasm, nucleus and DNA of tumor
tissue samples were determined with an inductively coupled plasma mass spectrometer (ICPMS) (ELAN DRC-II, PerkinElmer). Briefly, samples were treated with 23% nitric acid, arid 8%
H2O2. The solutions were then injected in the ICP-MS to quantify the platinum concentration.
As internal controls, platinum and thallium (m/z 195 and 205, respectively) were quantified in
untreated animals. Cytoplasm and nucleus-uptake of platinum of tumor tissue samples were
expressed as ng of platinum per g of cytoplasm or nucleus. Concentration of platinum in whole
blood and in serum was expressed as pg of platinum per mL of whole blood and serum. DNAPt adducts were expressed as ng of platinum per pg of DNA. Muscle, liver and kidney uptake
was expressed as ng of platinum per g of tissue.
Chemotherapy treatment
The tested drugs were dissolved in 5% dextrose for a final concentration at 1 mg/mL to
be given as a single dose of 10 mg/kg intravenously (i.v.) via the tail vain. Then, platinum
drugs were further incubated for 4, 24 and 48 h prior the radiation treatment, as shown in
Figure 3. The selected drug concentration of oxaliplatin and Lipoxal™ did not induce the
appearance of any side effects when combined with radiation treatment, as no significant
decrease in body weight was observed between time of initiation and termination of treatment
(Table 1). Animals in the control group received an injection of only 5% dextrose.
90
Tablel. Change in body weight of male nu/nu nude mice treated with i.v.° oxaliplatin or
Lipoxal™ in combination with GK6 irradiation
Platinum drugs
Control
Oxaliplatin (48 h)-IR
Lipoxal™ (48 h)-IR
Body weight (g) (n = 3-5)
Before the treatment
21.57±1.00
27.37±1.14
27.60±0.95
After the treatment
27.07±2.25
31.03±1.18
30.00±0.56
p-value
0.04
0.02
0.03
a i.v. = intravemeous injection of single concentration 10 mg/kg of platinum drug
aGK = Gamma Knife irradiation of single dose 15 Gy
^Control group = no chemotherapy and radiation treatment
Chemotherapy was given 48 h initially followed by radiotherapy
Tumor irradiation
To prevent the damage repair that may occur after fractionated radiation treatment (25),
a single dose of radiation treatment was used in this study. Mice were anestbietised and
positioned in our home made stereotactic frame designed for irradiation with a 4C Gamma
Knife (Elekta Instruments AB). The radiation treatment (15 Gy with a dose rate of 3.6 Gy/min)
with 8 mm collimators was delivered at predetermined coordinates targeting the tumor (Fig.3).
The radiation dose o f 15 Gy was taken from a previous study (26) as the dose that induced a
temporary tumor growth delay and not led to a complete tumor cure. The tumors were
irradiated with a single dose of radiation at one side of the rear flank, whereas the other side
was kept as control for non-irradiated tumor.
91
A) Pharmacokinetics study
—2 weeks
(~125mmJ)
£
T
4/24/48/72/96h
s.c. implantation
HCT116 cells
Blood, Serum, Normal and Tumoral tissues
phancnacokinetics
i.v. injection
oxaliplatin/Lipoxal™
10mg4cg
B) Chemoradiation treatment
—2 weeks
(~ 125mm3)
s.c. implantation
HCT116 cells
T
4/24/48 h
&E, Apoptosis
i
F ^ 4 8 h
i.v. injection
15Gy GK irradiation
oxaliplatin/Lipoxal™
10 mg/kg
C) Gamma Knife irradiation
£
Determination synergistic effect
k -^n
a) Homemade stereotactic frame
b) Coordinate for Gamma Knife
Figure 1. The experiment setup for chemoradiotherapy in nu/nu nude mice model. A) represent
the pharmacokinetics study scheme, B) represent the chemotherapy was given initially 4, 24,
and 48 h followed by radiotherapy and C) represent a) the homemade stereotactic frame and b)
the coordinate taking from program GKElecta. GK = Gamma Knife irradiation; s.c. =
subcutaneous implantation; i.v. = intravenous injection. The dose of chemotherapy is 10 mg/kg
of oxaliplatin and Lipoxal™; the single dose of radiation at 15Gy.
92
Determination of combination indices: Synergistic/Additive/Antagonistic
In each group of animals, the relative tumor volume was expressed as Vt/Vo ratio where
Vt is the mean tumor volume on a given day during the treatment and Vo is the mean tumor
volume at the beginning of the treatment. The details for antitumor activity after
chemoradiotherapy have been modified from a previous study (27). Briefly, the combined
treatment activity was evaluated by the time to reach a tumor volume that was five times
greater than the initial volume [5Td]. The expected 5Td of combined treatment was calculated
according to the following formula: Expected 5Td = Mean control + (mean drug alone - mean
control) + (mean radiation alone - mean control). The effect of combined treatment
(synergistic/additive/antagonistic) was assessed by calculating the ratio of the observed 5Td
[05Td] divided by that of the expected 5Td [E5Td]. Previous study on the effect of combined
treatment reported an additivity of combined treatment at the range of 0.9-1.1 (28). Then in this
study, the ratio values of [05Td]/ [E5Td] is > 1.1, the combination is indicate a synergistic; 0.9
to 1.1 the combination is additive; and < 0.9 the combination is antagonistic.
Apoptosis analysis
Groups of three nu/nu nude mice bearing HCT116 tumors were treated with platinum
drugs for 24 or 48 h before radiation treatment, as described. Two days after irradiation, mice
were euthanized and tumors were immediately removed, fixed in 10% buffered formalin and
embedded in paraffin. Tumor sections of 4 pm were taken at three different levels. Analysis of
apoptotic cells within tumor tissue was done by using a commercially available ApopTag®
Peroxidase In Situ Apoptosis Detection Kit (Millipore), following the manufacturer's protocol.
Briefly, formalin-fixed, paraffin-embedded tissue slides were rehydrated using xylene to
alcohol washings, followed by a hydrogen peroxide-methanol quench. The samples were
93
treated with 20 pg/mL of proteinase K for 15 min at room temperature. After washing and
incubation with equilibration buffer for 5 min, TdT (33 pL of TdT enzyme in 77 pL of reaction
buffer) was incubated on the slides for 1 h at 37° C. After applying stop solution for 10 min and
washing, the samples were incubated with anti-digoxigenin peroxidase conjugate (65 pL/cm
of specimen surface area) for 30 min at.room temperature. Slides were developed with a 1:20
dilution of diaminobenzidine (3,3'-diaminobenzidine) substrate, counterstained with methyl
green, dehydrated, and coverslipped. TUNEL-positive apoptotic nuclei were detected with a
fluorescent microscope. Positive control slides were prepared by nicking DNA with DNasel
and treated similarly to the sample (data not shown). Samples treated similarly but without
enzyme treatment served as negative controls. To determine the amount of cellular area stained
with 3,3'-diaminobenzidine (positive for apoptosis) and the amount of tumor area
counterstained with methyl green (negative for apoptosis), the tumor sections were scanned
with the Olympus FSX100 microscope and the values were quantified using the Adobe
Photoshop CS5 analysis software. Briefly, the percentage of the apoptotic cells was calculated
as the quotient o f 3,3'-diaminobenzidine-positive
area over the total area (3,3'-
diaminobenzidine positive + methyl green negative), times 100 (29).
Statistical analysis
The mean ± SD were calculated. P< 0.05 was considered statistically significant (twotailed paired Student’s t-test).
94
RESULTS
Platinum concentration in whole blood, serum, tumor and normal tissues
After i.v. administration, the total blood levels of oxaliplatin or Lipoxal™ declined
steadily, with a tendency for later stabilisation or even slight increased levels. These changes
were more pronounced for oxaliplatin than Lipoxal™, and for whole blood than serum levels.
Evolutions of total blood and serum levels are given in Figure 2A, B. Serum levels were about
10% of the corresponding whole blood levels. Figure 2C shows the accumulation of platinum
in tumor tissues after oxaliplatin and Lipoxal™ administration. At 4 h, the concentration of
platinum in tumor tissues after treatment with oxaliplatin was about two times higher compared
to the Lipoxal
TK>f
group. For both drugs, the total tumour platinum content declined gradually
over time. Similar results were obtained in muscle and kidney tissues (Figure 2D and E).
Oxaliplatin and Lipoxal
TTUf•
exhibited similar levels of platinum accumulation in liver tissues
Platinum derivative
accumulated in w hole
blood (ng Pt/mL blood)
(Figure 2F).
0.05
0.3
Oxaliplatin
Lipoxal™
0.2
Oxaliplatin
• I
2
Lipoxal™
S® <»
•M fl
-
g 0.025
0.1
1 1
eg 3
JT U
u
Pi
eg
0
0
0
20
40
60
80
100
Time after i.v. injection (h)
95
20
40
60
80
100
Time after i.v. injection (h)
Platinum derivative
accumulated in tum or
(ng Pt/g tissue)
0.5
0.8
Oxaliplatin
Oxaliplatin
Lipoxal™
Lipoxal™
0.6
0.4
0.2
0
0
20
40
60
80
Time after i.v. injection (h)
0
100
3
5
Platinum derivative
accumulated in kidney
(jig Pt/g tissue)
20
40
60
80
100
Time after i.v. injection (h)
4
-a-O xaliplatin
Oxaliplatin
-♦-Lipoxal™
Lipoxal™
2
3
2
1
1
0
0
0
20
40
60
80
Time after i.v. injection (h)
0
100
20
40
60
80
100
Time after i.v. injection (h)
Figure 2. Pharmacokinetic profiles of platinum concentration presented in blood, tumor tissues
and normal tissues. The platinum accumulation in A) whole blood, B) serum, C) muscle, D)
tumor, E) liver, and F) kidney, was obtained after a single i.v. injection at 10 mg/kg oxaliplatin
or Lipoxal™ in colorectal cancer HCT116 tumor nude mice. The amount of platinum was
measured by ICP-MS. Each point represents the mean ± SD of the concentration in 3-5 mice.
96
Platinum accumulation in cytoplasm, nucleus of tumor cells and tumoral DNA-bound
platinum
The time profiles of platinum accumulation in the cytoplasm and nucleus of tumor cells
are shown in Figure 3A and 3B. After peaking initially at 4 h, the amount of platinum in the
cytoplasm and the nucleus declined gradually over time for both oxaliplatin and Lipoxal™. The
level of tumoral DNA-bound platinum showed similar kinetics only in the case of Lipoxal
TKyf
.
For oxaliplatin, the level of tumoral DNA-bound platinum reached a first peak at 4 h, and then
gradually declined to reach a first nadir at 24 h, then rose again to reach a second and higher
peak at 48 h before re-declining. Even at 72 h and 96 h, levels of tumoral DNA-bound platinum
were found to be closer to those at 4 h (Figure 3C).
T um or grow th delay
The tumor growth delay after nu/nu nude mice were treated initially with chemotherapy
for 4, 24 and 48 h and followed by radiotherapy is shown in Figure 4. Efficiency of combined
treatment is reported in Table 1. A single oxaliplatin or Lipoxal
administration induced a
significant tumor growth delay compared to non-treatment animals (p < 0.05). Treatment with
radiation alone had considerable effects compared to the control group (p < 0.01). When
oxaliplatin or Lipoxal™ were combined with the radiation treatment, the tumor growth delay
increased significantly compared to the control group {p < 0.01). In addition, no significant
decreased in body weight of animals was observed between time of initiation and termination
of treatment (Table 2), suggesting the absence of additive toxicity of these combined treatment.
Figure 4A, combined treatment of oxaliplatin administration for 4, 24 and 48 h prior
radiation treatment induced a dramatic reduction in the tumor growth (5TdoX4h = 25 ± 1.41 days;
5Tdox24h - 23 ± 2.5 days; 5TdoX48h = 39 ± 2 days, respectively) compared to oxaliplatin alone
97
(5TdoXaione= .16 ± 0.71, p < 0.01). Similar enhancement of tumor growth delays was observed
after combined treatment with Lipoxal
for 4, 24 and 48 h of drug incubation plus radiation
(Figure 4B), (5Tdiip0x4h = 39 ± 1.41 days; 5Tdiip0x24h = 32 ± 1 days; 5Tdiip0x48h = 35 ± 0.5 days,
respectively) compared to Lipoxal™ alone (5Tdiipox'alone = 14.5 ± 0.71, p < 0.01). The
significant different of tumor growth delay between the group of drug treatment alone and
combined treatment was observed after 7 days and 5 days for oxliplatin and Lipoxal™,
respectively.
Oxaliplatin and Lipoxal
TK/I
, in combination with radiation showed a greater efficacy than
with radiation treatment alone (5TiRaione:= 19 ± 1.00,/? < 0.01), excepted for the combination of
radiation at 24 h after oxaliplatin administration (p = 0.08). Significant differences of tumor
growth delay were observed 20 days after combined treatment between 48 h of oxaliplatin
administration and 4 h or 24 h of oxaliplatin administration. For Lipoxal™, the tumor growth
delay showed no significant different among each treatment schedules.
98
200
200
Platinum derivative
accumulated in cytoplasm
(ng Pt/g tissue)
Oxaliplatin
B
v 2
5 « ~
Lipoxal™
-o-O xaliplatin
-•-L ipoxal™
I § S
_
C/i
a tg
2
«k. —
^ ’g
100
s J3 Bu
B 3 Ot
'B S d
.2 a w
S
"l uv
0
20
40
60
80
100
Time after i.v. injection (h)
0
20
40
60
80
10(
Time after i.v. injection (h)
10
Platinum derivative bound
to DNA (pg Pt/jig DNA)
Oxaliplatin
8
Lipoxal™
6
4
2
0
0
20
40
60
80
Time after i.v. injection (h)
100
Figure 3. Time dependence of platinum concentration in A) cytoplasm, B) nucleus and C)
platinum-DNA adducts obtained after a single i.v. injection at 10 mg/kg oxaliplatin or
•
Lipoxal
T lu |
.
m colorectal cancer HCT116 tumor nude mice. The amount of platinum bound to
DNA was measured by ICP-MS. Each point represents the mean ± SD of the concentration in
3-5 mice.
99
20
1
"o
>
ha
©£
n
<u
«
J5
■ - * — Control
— * • - Oxaliplatin (48h) alone
---A — 1R-GK alone
— A — Oxaliplatin (4h) plus GK
— B— Oxaliplatin (24h) plus GK
— ©— Oxaliplatin (48h) plus GK
>5
10
-
5 ^
&
i i i i | ) i i i | ) i i 1m
0
5
10
m
15
i i | i i i i i i i i i i i i ) i | i i r v r 1 1"1
20 25
Days
20
"©
>
i. ^
0 kP
35
40
45
>
Control
— *■ — Lipoxal™ (48h) alone
— a— IR-GK alone
— A— Lipoxal™ (4h) plus GK
S — Lipoxal™ (24h) plus GK
O — Lipoxal™ (48h) plus GK
B
1
30
15
1 ^ 10 H
3 >
<U w
s"35
H
94
i
0
i
i
i
I
5
t
i
i
i
1
i
10
i
i " T " )' i
15
i
i
i
|
i ' i " ) 'i
|
i
20 25
Days
i
i
i
|
i
30
i 'r
r "| " r
35
i i
i
i
i
i
i
40 45
Figure 4. The tumor response after nu/nu nude mice were treated initially with chemotherapy
and followed by radiotherapy. A and B show the combination of radiation with oxaliplatin and
Lipoxal
TW
, respectively. Treatments were administered with a single dose of platinum drugs of
10 mg/kg; and a single dose o f radiation of 15Gy. Each symbol represents the mean of the
results obtained with 5 mice and the bars ± S.D. (p < 0.05).
100
Table 2. In vivo efficacy of platinum-based chemotherapy alone and in combination with
radiation treatment in the HCT116 human colorectal cancer s.c. flank xenografts grown in male
nu/nu nude mice
Treatment
Control
IR alone
Oxaliplatin alone
Lipoxal™ alone
Oxaliplatin (4h)-IR
Oxaliplatin (24h)-IR
Oxaliplatin (48h)-IR
Lipoxal™ (4h)-IR
Lipoxal™ (24h)-IR
Lipoxal™ (48h)-IR
5Td° (days)
10.5 ±0.71
19 ±1.00
16 ±0.71
14.5 ±0.71
25 ±1.41
23 ±2.50
39 ± 2.00
39 ±1.41
32 ±1.00
35 ±0.50
TGD4
8.5 ± 1.00*
5.5 ±0.71*
4.0 ±0.71*
14.5 ±1.41**
12.5 ±2.50**
28.5 ±2.00**
28.5 ±1.41**
21.5 ± 1.00**
24.5 ±0.71**
%TGDC
Ratio of 5Td/ETdrf
80.95
52.38
38.10
138.10
119.05
271.43
271.43
204.76
233.33
1.07 ±0.03*
0.92 ± 0.06
1.49 ±0.08*
1.76 ±0.09
1.33 ±0.07
1.54 ±0.07*
a 5Td; Time necessary to reach a tumor volume five times greater than the initial volume
4 TGD is 5Td minus Control group (C) ( Control group = no chemotherapy and radiation
treatment)
c %TGD = (5Td - C)/C x 100%
d Ratio of 5Td/ETd obtained by dividing the 5Td by the expected 5Td of the combination.
ETd (Expected o f combination) = Mean Control + (Mean drug alone- Mean Control) +
(Mean radiation - Mean Control)
Ratio of Td/ETd obtained by dividing the observed Td by the expected Td of the combination.
A ratio =0.9-1.1,>1.1, <0.9 indicates additivity, synergism and antagonism
Statistically significant (P<0.05), compared to the control group, ** compared to both either
chemotherapy or radiation treatment alone group, and * compared to combined treatment
groupat 24 h of drug administration before irradiaiton.
101
Combination index
Figure 5 shows a combination index after chemoradiation treatment. When combined
radiation at 4 h, 24 h and 48 hof oxaliplatin administration, the combination index (Cl) was
1.07 ± 0.03,0.92 ± 0.06,1.49 ± 0.08, respectively.
In case of Lipoxal™ combined with radiation, the Cl was 1.76 ± 0.09, 1.33 ± 0.07 and
1.54 ± 0.07 at time intervals of 4, 24 and 48 h of drug incubation, respectively, suggesting a
synergic effect for every time schedules.
Induction of apoptosis
Induction of apoptosis at different time intervals between drug injection and radiation
treatment is shown in Figure 6A. The percentage of apoptotic cells (Figure 6B) was 0.94 ± 0.38
% and 2.86 ± 0.89 % in the untreated group and in the radiotherapy alone group, respectively.
Compared to treatment with oxaliplatin alone (1.59 ± 0.31 %) (p < 0.05), combined treatment
of oxaliplatin with radiation significant increased the percentage of apoptotic cells to 3.89 ±
1.05 % and 12.53 ± 2.35 % for 24 h and 48 h drug administration prior radiation treatment,
respectively. For Lipoxal™, the percentage of apoptosis cells was of 1.78 ± 0.69 %, 6.41 ±
1.72 % and 16.32 ± 4.81 % for Lipoxal™ alone and when combined with radiation at 24 and
48 h of drug incubation, respectively.
102
w
jS
"eS
►
Oxahplatin-IR
□ Lipoxal-IR
,3 2
a
o
v
«
a
aS 1
S
o
U
-
0
4h
24 h
48 h
Times between drug administration
and radiotherapy
Figure 5. The combination index of chemoradiotherapy in nude mice model. Animals were
treated initially with oxaliplatin (■) or Lipoxal™ (□) chemotherapy followed by radiotherapy.
*, statistically significant (P<0.05) from the combined treatment at 24 h of drug administration
group.
103
A)
IR
Control
OxaliDlatin alone
Oxaliplatin+IR
24 h
Linoxal™
Lipoxal™ +IR
48 h
24 h
48 h
B)
Lipoxal(48h)-IR
Lipoxal (24h)-IR
Lipoxal alone
Qxaiiplalin(48h) -IR
Oxaliplatm(24h) -IR
Oxaliplatin alone
IR alone
Control
0
5
10
15
% Apoptotic cells
20
25
Figure 6. A) TUNEL assay after chemoradiotherapy. Animals were irradiated at 24 and 48 h of
drug administration. Treatment was administered with a single dose of oxaliplatin or Lipoxal™
of 10 mg/kg; and a single dose of radiation of 15Gy. B) Percentage of apoptosis cells after
chemoradiotherapy. Apoptotic Fig.s were counted in 10 random fields per tumor (n=3) (Mean
± S.D.). Magnification, X40. Statistically significant (P<0.05) * compared to the control group;
** compared to both radiation treatment and the control sample; *** compared to both from
104
DISCUSSION
The optimal chemoradiotherapy schedules for colorectal cancer using oxaliplatin-based
therapy are being actively investigated. However, preclinical evidences of oxaliplatin as a
radiosensitizer for colorectal cancer treatment are limited and remain undetermined. The
radiosensibilizing activity o f platinum-based drugs has been previously suggested involving
two successful mechanisms of interaction between drug and radiation; firstly, by inducing
additional DNA damage or modifying radiation induced DNA damage, and secondly, by
inhibiting post-irradiation DNA damage repair (30).
After a single dose injection of chemotherapeutic agents, the platinum concentration in
tumor tissues is declined gradually over time after drug administration. However, the amount of
platinum drugs binding to tumoral DNA was varied with the time. Additionally, Pieck and co­
workers have reported a characteristic time course of oxaliplatin-DNA adducts in white blood
cells of cancer patients, which was not correlated to platinum pharmacokinetics (31).
Therefore, this raises the concern of whether or not the binding of drug to DNA during
irradiation interferes with the degree of inhibiting post-irradiation DNA damage repair, and
subsequent to an efficacy o f the concomitant effect.
The variation o f platinum-DNA adducts may be explained by different types of binding
to DNA. The initial peak at 4 h may suggest the formation of platinum-DNA mono-adducts (PtdG or Pt-dA), which rapidly removed by the nucleotide excision repair system, leading to the
reduction of the amount of platinum-DNA adducts. At 24 h of oxaliplatin administration, most
of DNA mono-adducts have possibly been repaired, and thus minimizing the amount of
platinum binding to DNA. Therefore, the exposure of oxaliplatin at 4 h and 24 h followed by
105
radiotherapy had no impact on the HCT116 xenografts, as only a lower degree of the
concomitant effect was observed. The time interval between these two modalities appeared to
be important since a profound concomitant effect was observed at 48 h of drug incubation. This
may explain by the formation of platinum-DNA adducts at the later time after drug
administration. The platinum-DNA adducts which are formed directly as bi-functional adducts
or rearranged from mono-functional adducts (Previous measurement of the rate constant for
oxaliplatin mono-adduct to di-adduct conversion was about 0.00000576 s-1 (32)), kept
increasing upto 48 h after drug administration. The interstrand cross links are known to
contribute a significant drug activity and are more vulnerable to DNA repair systems (33-35).
Therefore, a maxima formation of platinum-DNA adduct especially as interstrand cross links
might be occurred after 48 h of oxaliplatin administration, leading to a maximum effect of
radiosensitivity o f oxaliplatin.
This may contribute to a better understanding of
radiosensitizing activity of oxaliplatin, and explain the wide variation of different clinical
reports which describing the efficacy of oxaliplatin as a radiosensitizer (13, 35-37). In addition,
the amount of platinum-adducts after drug administration may be considered as an excellent
indicator to evaluate the concomitant effect of combined treatment.
Moreover, the induction of apoptotic cell death after combined treatment was similar to
the previous study (38), the number of apoptotic cells was significant increased after combined
treatment of oxaliplatin or Lipoxal™ with radiation treatment compared to untreated mice.
Chater and co-workers reported the reduction of the inhibitor of apoptosis protein after
combined treatment, which was not observed for either chemotherapy or radiation treatment
alone (38). In addition, Soini and co-workers suggested the improvment of platinum
106
distribution to DNA after radiotherapy may contribute to a higher DNA fragmentation,
subsequent to inducing high apoptosis and cell shrinkage (39).
The ability of liposomes to extravasate through leaky tumor vessels was suggested to
contribute to selective localization of liposomal oxaliplatin in tumor tissues (40). However our
results shown that the encapsulation of oxaliplatin in liposome did not improve its
accumulation in tumor cells, neither its fraction bound to DNA. In this reason,, the higher
concentration of Lipoxal™ can be used compared to oxaliplatin. In addition, low severe side
effects observed with Lipoxal™ may suitable for the combination with radiation to produce a
high potential o f the concomitant effect.
The radiosensitzer role of Lipoxal™ was previously evaluated in terms of a cellular
response for systemic cytotoxicity and the potential of a synergistic effect with radiation
treatment (41). Previous in vivo studies of glioblastoma treated with combined Lipoxal™ and
radiation showed promising results of animal survival (42), however, little is known about the
mechanisms of this improved outcome. In our experimental studies, applied radiation treatment
at different time intervals of Lipoxal™ administration, a slight difference in antitumor
efficiency was observed. However, the lowest concomitant effect was observed when radiation
administered 24 h after Lipoxal™ treatment.
Although the encapsulation of oxaliplatin in liposome gave a smaller amount of
platinum-DNA adducts compared to its free form, a high level of the concomitant effect of
combined treatment was observed. As shown in our results, Lipoxal™ administration alone or
combined with radiation induced a larger number of apoptotic cells than oxaliplatin. The
different mechanisms of interaction between Lipoxal™ and radiation remain to be clarified.
107
Previous study has suggested the binding of platinum drugs to cellular proteins via sulfur atoms
in the cysteine and/or methionine residues may affect the activity of enzymes, receptors, and
*
other proteins (43). Moreover, platinum drugs can induce a high level of mitochondrial reactive
oxygen species which would further interfere with vital cellular functions, leading to cell death
(44). Therefore, these data may apply to further investigations on the concomitant effect after
combined treatment between Lipoxal™ and radiation both in experimental systems and in the
clinical setting.
With respect to improving combined treatment outcomes of oxaliplatin in clinical
radiotherapy, our results demonstrated that the potential of concomitant effects between
radiation and oxaliplatin depended on the exposure schedule of both modalities. This correlates
considerably with platinum levels in different cellular compartments, mainly with platinumDNA adducts. The high degree of concomitant effects after Lipoxal™ plus radiation treatment
was observed, further evaluation of the radiosensitizing activity of Lipoxal™ for clinical trials
should be determined.. A better understanding of the mechanisms of synergism between
radiotherapy and oxaliplatin could lead to the design of new and more efficient colorectal
cancer chemoradiation treatment protocols.
108
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114
CHAPTER V
ARTICLE N 0.4
In this chapter, we investigated the concomitant effect of combined cisplatin and its
liposomal formulation plus radiation treatment using animal model. This work was performed
to confirm our previous results from in vitro studies.
This work is presented in the following article, entitled: “Efficiency of cisplatin and
T»l
Lipoplatin
for the concomitant treatment with radiation of a colorectal tumor in nu/nu nude
mouse”, by Thititip Tippayamontri, Rami Kotb, Benoit Paquette and Leon Sanche.
This article has been accepted to publish in Anticancer Research, 2013.
115
Efficiency of cisplatin and Lipoplatin
TM
for the concomitant treatm ent with radiation of a
colorectal tum or in nu/nu nude mouse
Short title: Synergism of cisplatin and Lipoplatin™ with radiation
Thititip Tippavamontri^b. Rami Kotb°, Benoit Paquette4’b and Leon Sanche8, b
/
J
Department o f Nuclear Medicine and Radiobiology, Center o f Radiotherapy Research,
Faculty o f Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, QC, Canada
3Department o f Systemic Therapy, BC Cancer Agency's Vancouver Island Centre, Victoria,
BC, Canada.
Correspondence to: Professor Leon Sanche, Department of Nuclear Medicine and
Radiobiology, Faculty o f Medicine and Health Sciences, University de Sherbrooke, 3001,12th
Avenue North, Sherbrooke, QC, Canada, J1H 5N4
Tel. 1-819-820-6868, ext. 14601, Fax: 1-819-564-5442, e-mail: Leon.Sanche@,USherbrooke.ca
Key Words: Radiotherapy, chemotherapy, concomitant therapy, colorectal cancer, cisplatin,
Lipoplatin™.
116
Rdsumd
Dans le but de ddvelopper de plus efficaces protocole de traitement utilisant la
concomitance chimio-radiothdrapeutique, nous avons cherchd les conditions de cddule
temporelle menant a une radiosensibilisation optimale. Nous avons proposd que la
concomitance soit maximale lorsque le niveau maximal de liaison de platine a l'ADN serait
atteint. D'autre part, les effets toxiques causes par le cisplatine aux tissus sains peuvent
empecher l'atteinte de son accumulation optimale a l'ADN. Pour pallier a cet inconvdnient, le
Lipoplatin™, la formulation liposomale du cisplatine, a dtd inclus dans cette dtude. Nous avons
ddfini la "fenetre Platinum" en dtudiant la pharmacocindtique et la distribution intracellulaire
temps-dependante du cisplatine et du Lipoplatin™ dans le carcinome colorectal humain
HCT116 implantdes chez des souris nu/nu. L'effet concomitant a dtd evalude lorsque le
rayonnement (15 Gy) a dtd donne 4, 24, et 48 h apres l'administration du mddicament, temps
correspondant aux variations maximum d’adduits platine-ADN. Le retard de croissance
tumorale rdsultant est reporte et corrdle a des analyses d’apoptose. La meilleure concomitance
et le plus haut taux d’apoptose ont dtd observes lorsque le rayonnement a dtd donne k 4 h ou 48
h apres l'injection de drogues. Ces temps correspondent aux delais pour une liaison maximale
de platine a l'ADN de la tumeur et sont probablement dus a une augmentation immddiate de
produits ldtaux gdndres par les radiations ionisantes. Ces donnees ameliorent notre
comprehension des mdcanismes de radiosensibilisation induite par le platine et peuvent avoir
un impact significatif sur la conception de protocoles de traitement plus efficaces.
Mots-clds: cisplatine; Lipoplatin™; pharmacocinetiques; radiation; tumeur colorectale
117
Abstract
Background: Optimal conditions for efficient concomitant chemoradiation treatment of
colorectal cancer with cisplatin still need to be better defined. In addition, intolerance of
healthy tissue to cisplatin prevents the full exploitation of its radiosensitizing potential. A
liposomal formulation of cisplatin, Lipoplatin™, was proposed to overcome its toxicity. Using
an animal model of colorectal cancer, we determine the platinum window, defined by studying
the pharmacokinetics and time-dependent intracellular distribution of cisplatin and
Lipoplatin™. Materials and Methods: In nude mice bearing HCT116 human colorectal
carcinoma treated with cisplatin or Lipoplatin™, the platinum accumulation in blood, serum,
different normal tissues, tumor and different tumor cell compartments was measured by
inductively coupled plasma mass spectrometry. Radiation treatment (15 Gy) was given 4, 24,
and 48 h after drug administration and was correlated to the amount of platinum-DNA adducts
in the cancer cells. The resulting tumor growth delay is reported and correlated to apoptosis
analysis. Results: The greatest effects and highest apoptosis were observed when radiation was
given at 4 h or 48 h after drug injection. These times correspond to the times of maximal
platinum binding to tumor DNA. An enhancement factor (ratio of group treated by combined
treatment compared to chemotherapy alone) of 13.00 was obtained with Lipoplatin™, and 4.09
for cisplatin when tumor irradiation was performed 48 h after drug administration. Conclusion:
The most efficient combination treatment of radiation with cisplatin or Lipoplatin™ was
observed when binding of platinum to DNA was highest. These results improve our
understanding of the mechanisms of platinum-induced radiosensitization and should have
significant impact on the design of more efficient treatment protocols.
118
Introduction
Cisplatin is a chemotherapeutic agent, which is also widely used as radiosensitizer (16). Addition of cisplatin to radiotherapy has been approved as a standard procedure because
improved treatment has been achieved in a number of randomized trials compared with
radiotherapy alone. However, in some other studies, concurrent radiation treatment with
cisplatin administration did not result in better efficacy (3, 6-9). The explanation for the latter
results is mainly associated with severe side-effects of either chemotherapy or radiotherapy,
\vhich may limit the escalation of anticancer drug concentrations, as well as radiation doses. In
addition, there is still no general consensus for the optimal timing between drug administration
and radiation treatment. Therefore, the precise determination of this timing appears a promising
objective for improving the efficacy of the combined treatment without added toxicity.
Optimal timing requires determination of platinum drug concentration in the tumor during the
course of radiation treatment in order to assess the time evolution of radiosensitization (10,11).
DNA damage induced by cisplatin-based radiosensitization has been proposed to be
mainly responsible for cytotoxicity (12). This toxicity is related to the formation of cisplatinDNA adducts, which include intra- and interstrand cross links of two guanosine nucleotides
(GG) or adenosine guanosine nucleotides (AG), and monobifunctional binding to guanosine
(12). Different mechanisms underlying the antitumor effect of concurrent radiation and
cisplatin treatments have been proposed (13). Radiation induces single-and-double-strand DNA
breaks and DNA base damage, while cisplatin forms adducts with DNA. The presence of
cisplatin-DNA adducts can reduce the repair of sublethal and potentially lethal DNA damage
(14). Moreover, ionizing radiation preferentially induces DNA damage where cisplatin is
119
located (15). In a previous in vitro study, we showed that a high level of cisplatin-DNA adduct
formation appeared to be associated with better treatment effect in human HCT116 colorectal
cancer cells (16). However, there are still some inconsistencies in the literature regarding the
relationship between the efficacy of anticancer platinum therapy and cisplatin-DNA adduct
formation (17), and it is not yet clear how to assess, in the clinic, the information underlying the
efficacy of radiotherapy at maximum cisplatin-DNA adduct concentration in the cancer cells.
Lipoplatin™ is a new liposomal formulation of cisplatin. It is currently being
developed, and aims to reduce the systemic toxicity of cisplatin, while improving its
accumulation in primary tumor tissue (18). A phase I/II study in advanced gastric cancer shows
that chemoradiotherapy with Lipoplatin™ is feasible, with minor toxicity (19). This suggests
that the therapeutic index of Lipoplatin™ could be larger than that of cisplatin. In our previous
report (20), we showed that distribution of cisplatin in different tumor cell compartments was
affected by its encapsulation in liposomes. Therefore, studying the mechanisms of its
combination with radiotherapy is important in defining the role of Lipoplatin™ as a
radiosensitizer and also to confirm our hypothesis for this formulation.
In the present study, we determined the correlation between different cellular levels of
DNA-platinum adduct as a function of time after administration of the drug and the efficacy of
combined treatment with ionizing radiation. The investigations were performed using nude
mice implanted with the human colorectal xenografts of HCT116. We also assessed the
variation of platinum concentrations in blood and in different normal tissues. The results of this
study should provide useful information enabling suitable scheduling of chemoradiotherapy
treatment of colorectal cancer.
120
Materials and methods
Cell line. The HCT116 colorectal carcinoma cell line was obtained from the American Type
Culture Collection (Manassas, VA, USA). Cells were routinely cultured in modified Eagle’s
medium (MEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum
(FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin and 100 pM
streptomycin in a fully humidified incubator at 37°C and with 5% CO2.
Animal xenograft model. All experiments were performed with outbred male nude mice at 4-6
weeks of age (Charles River Laboratories, Saint-Constant, QC, Canada). The animals were
maintained in our animal facility, under specific pathogen-free conditions. Housing and all
procedures involving animals were performed according to the protocol approved by the
University de Sherbrooke Animal Care and Use Committee (protocol number: 235-10B).
HCT116 tumor cells (2X106, 0.1 ml) were inoculated subcutaneously (s.c.) into each rear flank.
Tumor measurements began one week post-injection and continued biweekly. Tumor volumes
were calculated with the formula: V (mm3) = tc/6 X a (mm) X b2 (mm2), where a and b were
the largest and smallest perpendicular tumor diameters, respectively. The pharmacokinetic
studies began when tumor volumes reached the range of 30-60 mm . The tumor-bearing
animals were randomized into different groups of three to five animals each. For all procedures
(implantation, chemotherapy and radiotherapy), nude mice were anesthetised with an
intraperitoneal injection of ketamine/xylazine (87/13 mg/ml) at 1 ml/kg.
Drug preparation. Cisplatin was obtained through the pharmacy of the Centre Hospitalier
Universitaire de Sherbrooke. Lipoplatin™ was generously provided by Regulon Inc. (Athens,
Greece). All drugs were diluted to the given concentrations in a solution of 5% dextrose
immediately before use.
121
Pharmacokinetic studies. Drugs were dissolved in 5% dextrose for a final concentration of 1
mg/ml to be given as a single dose of 10 mg/kg into the mice tail vein. Three groups of mice
were studied with the following treatment: i) cisplatin, ii) Lipoplatin™, and iii) 5% dextrose
alone, as a control group. Three to five animals from each group were sacrificed at 4, 24, 48,
72, and 96 h post-injection. Blood samples were obtained via cardiac puncture for whole-blood
and serum platinum assessments. Tumors and samples of different tissues were taken to
determine platinum accumulation in the cytoplasm, and the nucleus, as well as in DNA
extractions.
Determination o f cytoplasmic and nuclear platinum accumulation. Samples were processed
with the nuclear and cytoplasm extraction kit Activemotif (ActiveMotif, Carlsbad, CA, USA).
Briefly, tumors were weighed and diced into very small pieces using a razor blade. Three
milliliters of ice-cold hypotonic buffer supplemented with 3 pi of 1 M dithiothreitol and 3 pi of
detergent were added per gram of tissue and incubated on ice for 15 min, and then centrifuged
for 10 min at 850 X g and 4°C. The hypotonic buffer and detergent were supplied in the kit.
Thereafter, the supernatants corresponding to the cytoplasmic fraction were transferred into
pre-chilled microcentrifuge tubes, while the pellets were gently resuspended in 500 pi of
hypotonic buffer, and incubated on ice for 15 min. The detergent (25 pi) was added and then
tubes vortexed. Samples were centrifuged for 30 s at 14,000 X g in a microcentrifuge pre­
cooled at 4°C and these second supernatants were combined with the first ones. The resulting
pellets corresponded to the nuclear fraction.
Quantification o f DNA-bound platinum. DNA was extracted according to a salting-out
procedure (21). Briefly, the tumors were weighed and diced into very small pieces, and
homogenized with a pre-chilled Dounce homogenizer. On ice, 3 ml of a lysing buffer
containing 10 mM Tris-HCl, 400 mM sodium chloride and 2 mM EDTA were added.
Subsequently, 0.1 ml of 20% sodium dodecylsulfate and 0.5 ml of proteinase K (10 mg/ml)
were added and the samples incubated overnight at 37°C. DNA was precipitated by adding 1.2
ml of 5 M sodium chloride. The tube was gently agitated for 1 min, centrifuged at 2,500 X g for
15 min and then the supernatant was transferred to another tube. The DNA was precipitated
with 2.5 vol. of 95% ethanol, spooled out and air-dried briefly. The DNA was then dissolved in
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and RNase A (0,05 ml of a 10 mg/ml
solution) was added and incubated for 1 h at 37°C. The DNA was precipitated a second time
with ethanol and re-dissolved in TE buffer. The amount of DNA extracted was evaluated by
measuring its absorbance in the solution at 260 nm (A260) with a spectrophotometer (Synergy
HT, BIO-TEX, Winooski, VT, USA) and calculated using the following equation: A260 X 50
(pg/ml).
Platinum quantification using an inductively coupled plasma mass spectrometer (ICP-MS). The
quantity of platinum accumulated in tissue samples and in the cytoplasm, nucleus and DNA of
tumor were determined with ICP-MS (ELAN DRC-II; PerkinElmer, Waltham, MA, USA) (22).
Briefly, samples were treated with 23% nitric acid and 8% H202. The solutions were then
injected in the ICP-MS to quantify the platinum concentration. As internal controls, platinum
and thallium (m/z 195 and 205, respectively) were quantified in untreated animals.
Chemotherapy treatment. The tested drugs were dissolved in 5% dextrose for a final
concentration of 1 mg/ml to be given as a single dose of 10 mg/kg into the tail vein. The
platinum drugs were injected at 4, 24 and 48 h prior to radiation treatment. The selected drug
concentration of Cisplatin and LipoplatinTM did not induce the appearance of any side-effects
123
after combined treatment, as the body weight of all groups of animals significantly increased
between initial and final time of combined treatment (Table I).
Table 1. Change in body weight of male nu/nu nude mice after combined
treatment of
cisplatin or Lipoplatin™ and Gamma Knife irradiation
Platinum drugs
Control0
Cisplatin (48 h)-IR
Lipoplatin™ (48 h)-IR
Body weight (g) (n=3-5)
Before the treatment
21.57±1.00
27.20±1.27
22.50±2.29
After the treatment
27.07±2.25
31.25±0.35
25.43±1.46
p-value
0.04
0.12
0.08
a Control group = no chemotherapy and radiation treatment
i.v. = intravemeous injection of single concentration 10 mg/kg of platinum drug
GK = Gamma Knife irradiation of single dose 15 Gy
p<0.05 is considered as statistically significant between body weight before and after the
treatment
Tumor irradiation. Mice were anaesthetized and positioned in our in-house constructed
stereotactic frame designed for the 4C Gamma Knife (Elekta Instruments AB, Stockholm,
Sweden) (23, 24). The radiation treatment (15 Gy, dose rate of 3.6 Gy/min) using 8 mm
collimators was delivered at predetermined coordinates targeting the tumor, which had a
diameter of about 7 mm. Radiation was applied to one side of the rear flank, whereas the other
side was kept as the non-irradiated control tumor.
Apoptosis analysis. Groups o f three mice bearing HCT116 tumors were treated with platinum
drugs at 24 or 48 h before the radiation treatment, as previously described. Two days after
irradiation, tumors were removed, fixed in 10% buffered formalin and embedded in paraffin.
Tumor sections o f 4 pm were taken at three different levels. Apoptotic cells within tumor
tissues were analyzed with a commercially available ApopTag® Peroxidase In Situ Apoptosis
Detection Kit (Millipore, Temecula, CA, USA), following the manufacturer's protocol. Briefly,
124
formalin-fixed, paraffin-embedded tissue slides were rehydrated using xylene, rinsed with
ethanol, and then incubated with a solution of 3% hydrogen peroxide. The samples were treated
with 20 pg/ml of proteinase K for 15 min at room temperature. After washing and incubation
with equilibration buffer included in the kit for 5 min, then 33 pi of terminal deoxynucleotidyl
transferase (TdT) enzyme in 77 pi of reaction buffer included in the kit was incubated on the
slides for 1 h at 37°C. After applying stop solution included in the kit for 10 min and washing,
the samples were incubated with anti-digoxigenin peroxidase conjugate (65 pl/cm2 of specimen
surface area) for 30 min at room temperature. Slides were developed with a 1:20 dilution of
diaminobenzidine (3,3'-diaminobenzidine) substrate, counterstained with methyl green,
dehydrated, and coverslipped. Terminal deoxynucleotidyl transferase-mediated dUPT nick end
labeling (TUNEL)-positive apoptotic nuclei were detected with a fluorescence microscope. A
positive control slide was prepared by nicking DNA with DNasel and treated similarly (data
not shown). Samples treated similarly but without enzyme treatment served as negative
controls. To determine the amount of cellular area stained with diaminobenzidine (positive for
apoptosis) and the amount of tumor area counterstained with methyl green (negative for
apoptosis), the tumor sections were scanned with an Olympus FSX100 microscope (Olympus,
Center Valley, PA, USA) and the values were quantified using Adobe Photoshop CS5 (Adobe
Systems Canada, Ottawa, Ontario, Canada) analysis software. Briefly, the percentage of
apoptotic cells was calculated as the quotient of the diaminobenzidine-positive area over the
total area (diaminobenzidine positive + methyl green negative), multiplied by 100.
Statistical analysis. The mean±SD were calculated. p<0.05 was considered statistically
significant (two-tailed paired Student’s t-test).
125
Results
Platinum concentration in whole blood, serum, tumor and normal tissues. The timeconcentration profiles of platinum in the blood and serum are shown in Figures la and b. After
i.v. administration (10 mg/kg) of cisplatin or Lipoplatin™, the initial maximum of platinum
concentration in whole blood and serum was reached at the first assessment time point (4 h),
followed by a rapid decline over the next 24 h. Thereafter, the decline was very slow. The
platinum concentration in whole blood was about three times higher than that in the serum.
While free cisplatin reached a higher peak level in kidney tissues than observed with the
liposomal formulation (Figure lc), both drugs exhibit similar levels of platinum accumulation
in liver tissues at different time points, with no well-defined initial peak (Figure Id). A higher
level of platinum accumulation was observed in muscle tissues with cisplatin than with
Lipoplatin™ (Figure le). Accumulation of platinum in the tumor tissue after administration of
cisplatin, and Lipoplatin™ are shown in the Figure If. Over a sampling period of 96 h, the
platinum concentration in tumor was about five times higher in the cisplatin-treated group than
in the Lipoplatin™- treated group. For both drugs, the total tumor platinum content declined
gradually over time.
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126
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Figure 1. Pharmacokinetic profiles of platinum concentration in a) whole blood, b) serum, c)
kidney, d) liver, e) muscle, and f) tumor obtained after a single i.v. injection of 10 mg/kg
cisplatin or Lipoplatin™, in nude mice bearing HCT116 colorectal tumor. The amount of
platinum was measured by an inductively coupled plasma mass spectrometer. Each point
represents the mean±SD of the concentration in 3-5 mice.
127
Platinum accumulation in cytoplasm and nucleus, and.platinum-DNA adducts in tumor tissue.
The time profiles of platinum accumulation in the cytoplasm and nucleus of HCT116 tumor
cells are shown in Figures 2a and b. The amount of platinum in the cytoplasm and the nucleus
declined gradually over time for both cisplatin and Lipoplatin™. Cisplatin administration led to
about 3- to 5-fold more platinum accumulation in the cytoplasm and the nuclei of cells of tumor
tissues, respectively, than did Lipoplatin™. Regarding the level of DNA-bound platinum in the
nuclei of tumor cells, for both drugs, the maximum was measured at 4 h, declining to reach a
first nadir at 24 h, increasing again to reach a second peak at 48 h before declining again
(Figure 2c). While the initial and later peak levels of DNA-bound platinum were much higher
in the case of cisplatin, for both drugs, the level of DNA-bound platinum declined to a similar
nadir level at 24 h.
128
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Figure 2. Time dependence of platinum concentration in a) cytoplasm, b) nucleus and c)
platinum-DNA adducts obtained after a single i.v. injection of 10 mg/kg cisplatin, or
Lipoplatin™ , in nude mice bearing HCT116 colorectal tumor. The amount of platinum bound
was measured by inductively coupled plasma mass spectrometer. Each point represents the
mean ±SD o f the concentration in 3-5 mice.
129
Tumor growth delay. Figure 3 shows the tumor response when the drugs were injected before
radiation treatment. Results in term of the time necessary to reach five times the initial tumor
volume (5Td) and the tumor growth delays (TGD = 5Td value for a treated group minus 5Td
for the control group) are reported in the Table II. A single cisplatin administration
significantly induced a tumor growth delay compared to non-treated animals (p<0.05), whereas
no significant improvement was observed when Lipoplatin™ was given alone. Treatment with
radiation alone (15 Gy) led to a good tumor response, with a 5Td of 19±1.0 days compared to
10.5±0.71 days for the non-treated control group (p<0.01). The tumor response was further
improved when cisplatin or Lipoplatin™ were combined with tumor irradiation (Figures 3a and
b). A significant difference in TGD between the groups of single drug treatments and combined
modalities was initially observed after eight and six days for cisplatin and Lipoplatin™,
respectively. With both drugs, the TGD was better when the irradiation was delivered at 4 h or
48 h after drug administration compared to 24 h after drug administration (p<0.01). It is
noteworthy that no improvement was observed when cisplatin was administered 24 h prior the
radiation treatment, compared to the irradiated control. Indeed, the TGDs were similar for the
group treated with radiation alone (8.5±1.00 days), and the group treated with cisplatin 24 h
prior the radiation treatment (10.5±1.41 days, p=0.19) (Table II).
130
a Cisplatin + IR
Control
*■ - Drug alone
■A— IR alone
■A— Drug plus IR at 4 h
■B— Drug plus IR at 24 h
■&— Drug plus IR at 48 h
0
5
10 15 20 25 30 35 40 45
Days
b Lipoplatin™ + IR
20
%
15
10
5
0
0
5
10 15 20 25 30 35 40 45
Days
Figure 3. The tumor growth delay after initial treatment of nude mice bearing HCT116
colorectal tumor with a) cisplatin and b) Lipoplatin™ followed by radiotherapy. Tumor growth
delay is reported as Vt/Vo ratio where Vt is the mean tumor volume on a given day during the
treatment and Vo is the mean tumor volume at the beginning of the treatment. A single dose of
platinum drug o f 10 mg/kg and a single dose of radiation of 15 Gy were administered. Each
symbol represents the meancfcS.D of the results obtained with five mice.
131
Enhancement factor (EF). The EF, which is the ratio of TGD measured with a group treated by
radiation and chemotherapy compared to TGD obtained with chemotherapy alone, was
calculated to better assess the effect obtained by radiation with cisplatin, and Lipoplatin™ at 4,
24 and 48 h after drug administration (Table II). When radiation was given at 4 h and 48 h after
cisplatin administration, the EF was 3.73 and 4.09 respectively, suggesting an important
improvement, while a modest EF of 1.91 was measured when radiation treatment was given 24
TXvl
h after cisplatin administration. The encapsulation of cisplatin in a liposome, i.e. Lipoplatin ,
resulted in a much more important effect when combined with tumor irradiation^ the EF was
13.67,10.33 and 13.00 at time intervals of 4,24 and 48 h when tumor irradiation was combined
TXjI
with Lipoplatin
after drug administration, respectively. As for cisplatin, the lowest EF with
Lipoplatin™ was measured at 24 h post drug administration.
Induction o f apoptosis. The number of apoptotic cells was scored in tumor sections after
treatment (Figure 4). The percentage of apoptotic cells was very low in the untreated control
group (0.94±0.38%) and the group treated with radiation alone (2.86±0.89%). When the tumor
was irradiated 24 h after cisplatin administration, a modest but significant increase of apoptotic
cells (3.48±0.69%) was measured compared to tumors treated with cisplatin alone
(1.97±0.77%) (p<0.05). Incubation with cisplatin alone required 48 h before a significant
increase in the number o f apoptotic cells (13.40±2.81%) was observed compared to treatment
with cisplatin alone or radiation alone (p<0.01). With a similar trend, a higher percentage
(p<0.05) o f apoptotic cells was observed when radiation was given 48 h after Lipoplatin™
administration (15.29±2.90%) compared to when given 24 h after drug administration
(5.72±1.23%).
132
Table II. Efficacy o f platinum-based chemotherapy alone and in combination with radiation
treatment (IR).
Treatment
5Td (days)
Control
IR alone
Cisplatin alone
Lipoplatin™ alone
Cisplatin + IR at 4h
Cisplatin + IR at 24h
Cisplatin + IR at 48h
Lipoplatin™ + IR at 4h
Lipoplatin™ + IR at 24h
Lipoplatin™ + IR at 48h
10.5±0.71
19±1.00
16±0.71
12±1.29
31 ±1.25
21±1.41
33±2.00
31±2.12
26±1.52
30±1.29
TGD (days)
EF
...
8.5±1.00*
5.5±0.71*
1.5±1.29
20.5±1.25*,#
10.5il.41*
22.5±2.00*’*
20.5±2.12*,#
15.5±1.52*,#
19.5±1.29*’#
—
—
—
3.73
1.91
4.09
13.67
10.33
13.00
5Td, Time necessary to reach a tumor volume five times greater than the initial volume; TGD,
tumor growth delay, is the 5Td value minus that for the control group (no chemotherapy or
radiation treatment); EF, enhancement factor, calculated by dividing the TGD of the group
treated by combined treatment with that o f the group treated by chemotherapy alone.
M
Statistically significant at /K0.05 *compared to the control group, compared to chemotherapy
•
•
•
alone and radiation treatment alone.
133
Control
a
Cisplatin alone
IR
Cisplatin + IR
24 h
48 h
Lipoplatin™ alone
Lipoplatin™ + IR
24 h
48 h
Control
IR alone
Cisplatin alone
Cisplatin +IR 24 h
Cisplatin + IR 48 h
Lipoplatin™ alone
Lipoplatin™ +IR 24 h
Lipoplatin™ + IR 48 h
0
5
10
15
20
Apoptotic cells (%)
Figure 4. Assessment of apoptosis after chemoradiotherapy of cisplatin and Lipoplatin
TW
. a)
Terminal deoxynucleotidyl transferase-mediated dUPT nick end labeling (TUNEL)-positive
apoptotic nuclei assay after chemoradiotherapy. Animals were irradiated at 24 or 48 h after
drug administration. A single dose of cisplatin or Lipoplatin™ of 10 mg/kg and a single dose of
radiation (15 Gy) were administered, b) Percentage of apoptotic cells after chemoradiotherapy.
Apoptotic cells were counted in 10 random fields per tumor (n=3) (mean±S.D.). Magnification,
X40. Statistically significant differences: p<0.05 *from the control group; **ffom both
radiation-treated and the control group; ***from radiation alone, chemotherapy alone and the
control group.
134
Discussion
For most clinical chemoradiotherapy protocols of weekly cisplatin plus five fractionated
doses of radiation, time between the administration of cisplatin and tumor irradiation can
change the anti-tumor response obtained by combining them. The present experiments were
performed with the chemotherapeutic agents cisplatin and Lipoplatin™ to investigate the
maximum concomitant effect between these agents and radiation at different times after drug
administration. We also assessed the level of platinum in blood, serum, and different normal
tissues with the aim to facilitate the transition to human trials where extraction of cancerous
tissue samples is more difficult. To correlate the efficacy of combined treatments, we defined
the platinum window of the maximum concomitant effect, which was associated with the
maximum level of platinum- DNA adducts in tumor cells. Supporting the importance of
platinum-DNA adducts, in clinical study with cisplatin chemotherapy Los and co-workers
reported that cisplatin-induced DNA modifications were observed in human tumor biopsies,
and were positively correlated with tumor remission (25).
It was clear from the EF values that cisplatin and its liposomal formulation
(Lipoplatin™) can act as radiosensitizers. The EFs, the results of apoptotic analysis and the
pharmacokinetics data showed that cisplatin and Lipoplatin™ were most efficient
radiosensitizers when their binding to tumoral DNA was maximized. In contrast, we found less
or even nearly no improvement in the combined effect of cisplatin and radiotherapy, when
binding of cisplatin to tumoral DNA was minimal (i.e. 24 h after drug injection).
While the initial high DNA-platinum peak at 4 h is explained by drug influx into tumor
cells, the reasons for the low level of adduct at .24 h and the second DNA-platinum peak at 48 h
are unclear. However, this observation leads to the following hypothesis. At 4 h, the majority
135
of DNA-platinum adducts are intrastrand cross-links binding as monoadducts. These
monoadducts are rapidly excised by the nucleotide excision repair system, leading to the
reduction of DNA-platinum binding (26). Twenty-four hours after administration of cisplatin,
most of the DNA monoadducts disappear, while the repair system becomes more saturated with
repair-resistant DNA-platinum interstrand cross links, which would maximize at 48 h.
There are many mechanisms that can enhance the effects of radiation (27, 28). Among
these, essentially two mechanisms may explain the radiosensitizing properties of cisplatin.
Previous in vitro studies (29-31) and some experiments with tumor-bearing mice (32-34)
suggested the inhibition of repair of radiation damage to DNA. Whereas from other
investigations, it was postulated that owing to the binding of cisplatin to DNA, there is an
increase in the immediate species created by the primary ionizing events in cells which causes
the additional damage (35). Zheng et al. (15) measured DNA single-strand breaks (SSBs) and
DNA double-strand breaks (DSBs) induced by the impact of 1, 10, 100 and 60,000 eV
electrons on thin solid films of DNA with and without cisplatin bonded to two adjacent guanine
bases. From a comparison of their results obtained at low energy (1-100 eV) with those
obtained at 60 keV, they found that production of SSBs and DSBs induced by low-energy
secondary electrons, created by the primary radiation, was substantially enhanced when
cisplatin was covalently bonded to DNA. Such damage is known to promote cell death. The
latter hypothesis implies that the effect of platinum drugs and radiation should be maximal
when binding of platinum to DNA of cancer cells is maximized. This is precisely the result
obtained in the present study. Determination of such fundamental mechanisms of the
radiosensitizing action of platinum drugs may have implications in the design of new
chemotherapeutic agents, as well as in the development of more efficient protocols for
136
chemoradiation therapy. In addition, since there was no correlation in the effect of combined
therapies and cisplatin found either in blood or in normal tissues, this support the notion that
the best combined effect can only be determined by quantifying the level of cisplatin-DNA
adducts in tumor cells.
We reported previously that the intracellular platinum distribution in tumor from human
HCT116 colorectal cancer cells for encapsulated cisplatin may be different than that of
cisplatin alone (20). In this in vivo study, identical concentrations of platinum drugs (10 mg/kg)
j
were administered to the animals. LipoplatinTM administration alone led to similar results in
TGD to that of untreated animals; the liposomal formulation was therefore less effective than
cisplatin administered alone. Indeed, most previous animal and human studies have
administered 3-6 times higher doses of Lipoplatin™ than cisplatin (36). For example, Ravaioli
and co-workers used Lipoplatin™ at 100 mg/m2 every two weeks as second-line chemotherapy
in heavily pre-treated patients with advanced non-small cell lung cancer (NSCLC) and obtained
5% partial remission and 16% stable disease (36). On the other hand, Stathopoulos and co­
workers used 400 mg/m2 every two weeks (200 mg/m2 D1 and 2 every 14 days) on a similar
group of patients, and obtained 38% partial remission and 43% stable disease, with only minor
toxicities of grade I (37). The dose of cisplatin in 14-day schedules was 75 mg/m2 compared to
200 mg/m2 for LipoplatinTM in randomized phase III study in combination with paclitaxel in
nonsquamous NSCLC had shown a much lower toxicity profile in the Lipoplatin™ arm (38).
Furthermore, the study of nonsquamous NSCLC demonstrated a statistically significantly
higher response rate with the Lipoplatin™ treatment. Consequently, Lipoplatin™ should
achieve a higher level of tumor accumulation of platinum and DNA-platinum adducts.
137
Although the radiosensitizing role of Lipoplatin™ has not yet been determined in
clinical practice, the wider therapeutic index of this formulation suggests such potential (3638). In addition, our previous study on the concurrent administration of Lipoplatin™ and
radiation showed the high potential of concomitant effect in the HCT116 cells when radiation is
applied at maximum formation of DNA-platinum adducts (16). In the present study, combined
treatment of radiation and Lipoplatin™ led to the highest increase in antitumoral activity. An
enhancement factor of 13.00 was obtained with Lipoplatin™, compared to only 4.09 for
cisplatin, when tumor irradiation was performed 48 h after drug administration. The effect after
treatment with Lipoplatin™ and radiation still correlated with maximum tumoral DNAplatinum levels (4 h and 48 h after drug administration). Administration of Lipoplatin™ also
led to lower accumulation of platinum in the normal tissues tested, except for the liver.
TW
Considering the lower toxicity of Lipoplatin
compared to that of cisplatin reported in clinical
trials (37- 38), the therapeutic index should be larger for Lipoplatin™ than for cisplatin. It
remains to be determined whether a larger dose of Lipoplatin™ administered in this preclinical
model of cancer would result in an improvement of the antitumoral effect while maintaining an
acceptable level of toxicity. Nevertheless, these data are valuable to highlight the potential of
Lipoplatin™ as a radiosensitizer.
Although similar level of Lipoplatin™ and cisplatin bound to the DNA 24 h after drug
administration was observed, superior treatment occurred with combined administration of
Lipoplatin™ and radiation. Only a weak correlation between the cytotoxicity of liposomal
platinum drug and the degree of DNA platination was found. This observation might imply that
platinum-based drugs have important targets other than nuclear DNA (e.g. cytoplasmic
compartments) (39). For example, it has been proposed that Lipoplatin™ can release cisplatin
138
molecules into the cytoplasm of the tumor cell, which then activate the mitochondrial apoptotic
cascade (40). In addition, any released cisplatin molecules may i) undergo reaction with
phospholipids; ii) inhibit amino acid transport, protein synthesis and ATPases; and iii)
uncouple oxidative phosphorylation (41-43). However, the importance of targets other than
DNA in relation to cytotoxicity is still unclear and remains a subject of future study.
It has been proposed that the binding affinity of drugs to the mitochondria is regulated
by lipid solubility (44). Hori and co-workers reported that highly lipophilic drugs are more
rapidly accumulated in the mitochondria than are non- or poorly lipophilic drugs (45). The
improvement in terms of lipophilicity for Lipoplatin™ compared to ffee-platinum forms could
indicate a higher efficacy for binding to the mitochondrial membrane that would lead to a
greater accumulation o f drug within the mitochondria. Thus, promotion of liposomal platinum
drug accumulation in the mitochondria may contribute to the enhancement of apoptotic cancer
cell death. Although this mechanism may explain in part the anticancer effect of Lipoplatin™
for some cancer types, we observed similar induction of apoptosis with cisplatin and
*
TU
Lipoplatin
in the HCT116 colorectal carcinoma model used in this study. However, the fact
remains that Lipoplatin™ was also associated with a large improvement of the radiosensitizing
effect.
In conclusion, the narrow therapeutic index of cisplatin excludes dose escalation to
improve its radiosensitizing action in the treatment of various types of cancer. There exists a
general impression that the maximum potential of this drug may have been reached. However,
our current report shows that the radiosensitizing effect of platinum drugs varies considerably
according to the time after exposure to the drugs and to the platinum level in different tumor
cellular compartments. The efficacy of combined treatments essentially correlates with the
139
amount of DNA-bound platinum in tumor cells:
it reaches a maximum at the times
corresponding to the highest DNA-platinum concentration in the nucleus (4 h and 48 h);
furthermore, efficacy reaches a minimum at low DNA-platinum levels (24 h). The best
T W
combined effect was observed with Lipoplatin
and was also correlated with the maximum
DNA-platinum levels in the tumor cells.
Acknowledgements
This research was financed by the Canadian Institute of Health Research (grant
#81356). Leon Sanche, Benoit Paquette and Rami Kotb are membres of the Centre de
recherche Clinique-Etienne Lebel supported by the Fonds de la Recherche en Sante du Quebec.
Sanofi-Aventis Canada also provided a partial unrestricted grant to support this project.
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146
CHAPTER VI
DISCUSSION
The scope o f this thesis
Concomitant chemoradiotherapy consists of administering chemotherapeutic agents,
such as radiosensitizers, during the course of radiotherapy. Concomitant chemoradiotherapy is
proposed to locally improve primary tumor control and eliminate metastatic foci. However,
some difficulties in managing the side effects from this combined treatment are still commonly
encountered in clinical practice. Therefore, the scheduling of radiosensitizing drugs in relation
to radiotherapy is crucial to gain the benefit of drug-radiation interactions for maximizing
tumor response. The aim of this study was to develop an optimal schedule for
chemoradiotherapy to assess the best concomitant effect while minimizing the level of
toxicities.
The articles presented in this thesis emphasize preclinical in vitro and in vivo studies
including; studies on the cytotoxicity, pharmacokinetic characteristics of platinum-based drugs
(e.g. intracellular accumulation and platinum-DNA adducts) and key information on cell
proliferation, cell cycle progression and cell death in the response to chemoradiotherapy. We
also investigated the optimal chemoradiation schedule regarding the hypothesis of the “true”
concomitant chemoradiotherapy that the maximum therapeutic effect of concomitance is
obtained if radiation is administered at the time at which the maximum quantity ,of platinum is
bound to the DNA in tumor cells. We first performed experiments in vitro to verify this
hypothesis. Since vascularization is not present in experiments with the cells, an animal model
was additionally investigated to model human tumors and be more predictive of the therapeutic
effects that might be observed in humans.
147
Cytotoxicity potential o f cisplatin and oxaliplatin
DNA damage is mainly responsible for the cytotoxic properties of platinum drugs (Saris
et al., 1996). Thus differences in the cytotoxicity of oxaliplatin and cisplatin likely derive from
differences in their interactions at the DNA level. Our results and existing evidences (Saris et
al., 1996; Woynarowski et al., 2000), showed that oxaliplatin forms fewer platinum-DNA
adducts, but inhibits DNA synthesis more efficiently than equimolar concentrations of
cisplatin.
That cells were more sensitive to oxaliplatin than to cisplatin may be because
oxaliplatin affects DNA with different and more efficient pathways than does cisplatin. This
was attributed to the fact that oxaliplatin-DNA adducts have different biological properties,
specifically they are bulkier and more hydrophobic than are cisplatin-DNA adducts, and can
therefore cause different damage to DNA by altering the conformation of the molecule
(Floltinova et al., 2008). Moreover, the formation of a bifunctional adduct with oxaliplatin was
reported to be greater, and this could also represent a mechanism for the increased activity of
oxliaplain (Jennerwein et al., 1989; Kim et al., 2010). The formation of bifunctional adducts of
oxaliplatin may associated with more lethal DNA damage than cisplatin-DNA adduct (Vaisman
et al., 1998; Ferry et al., 1999).
The difference of cell sensitivity to the two platinum drugs may also be explained by
differences in the cell’s ability to repair platinum-induced DNA damage lesions after cisplatin
and oxaliplatin treatment. Scheeff and coworkers have shown that oxaliplatin-induced damage
was more difficult to repair than cisplatin-induced damage (Scheeff et al., 1999). Nehme and
co-workers have suggested that cisplatin activates particular components of damage-response
pathways, such as JNK and c-Abl kinases. (Nehme et al., 1999). Consequently, cisplatin
148
depends on an intact mismatch repair (MMR) system for its minimal cytotoxicity for signaling
apoptosis via the JNK-mediated pathway. In contrast, the DACH ligand which is present in
oxaliplatin, but not in cisplatin, inhibit recognition by the MMR protein complex and so not
activate JNK and c-Abl (Vaisman et al., 1999), thus providing oxaliplatin with a means to
maintain its cytotoxicity in cells.
Pharmacokinetics o f platinum drugs
Our studies on the pharmacokinetic characteristics of platinum drugs were performed to
obtain a better understanding of their cytotoxicity and to better identify differences between the
drugs.
Previously, Boulikas has reported that the cisplatin concentration can reach 0.4 cisplatin
molecules/ 1000 b.p. in DNA (Boulikas et al., 2005). Zheng and co-workers demonstrated the
considerable sensitivity o f DNA/cisplatin complexes to low energy electrons that resulted in an
increase of SSB and DSB with an estimated 0.625 cisplatin molecules/ 1000 b.p. covalently
bound to DNA, when samples were irradiated by a 60 keV primary electron beam (Zheng et al.,
2008). By conducting in vitro studies using an inductively coupled plasma mass spectrometer
(ICP-MS), we found the fraction of platinum drugs bound to DNA. The pharmacokinetic
characteristics of the various platinum drugs at the DNA level are shown in Table 1. The
cisplatin and oxaliplatin concentration reached values of respectively 0.795 and 0.726 platinum
molecules / 1000 base pair (b.p.) in DNA, which is consistent with previous studies. These
observations suggest an adequate distribution of these platinum drugs into the DNA of the
cancer cells, which could lead to improvements in efficiency in combination with radiation
treatment.
149
Table 1 shows that while the quantities of cisplatin- or oxaliplatin-DNA adducts
decreases by a factor 3 as incubation periods increase from 4 h to 24 h, there is an
augmentation by a factor 10 in the concentration of platinum-DNA adducts from treatment with
liposomal platinum formulations. This suggests that the comparatively slow release of platinum
compounds into the cell by liposomal-platinum drug administration may prolong exposure of
tumor cell to the cytotoxic drugs and consequently promote their binding to DNA.
In our study (Table 1) and as discussed in Chapter 2, the cellular uptake and
cytoplasmic/DNA distribution of platinum-based drugs in HCT116 cells showed similar levels
for cisplatin and oxaliplatin accumulation after 4 h of incubation, subsequently, the level for
oxaliplatin attained a plateau; whereas cisplatin continued to accumulate. These results are
consistant with a recent study by Virag and co-workers (Virag et al., 2012). They observed that
the cellular uptake o f oxaliplatin gradually increased with time, while cisplatin entered rapidly
into the cells. Thus, the cellular accumulation of cisplatin was several times that of oxaliplatin.
In our study, the. concentration of cisplatin in DNA has to be eight times higher than that of
oxaliplatin to reach the same level of both platinum-DNA adducts and cancer cell toxicity. A
higher concentration of cisplatin was accumulated in cells compared to the oxaliplatin,
probably because of the less limiting efflux transporters (e.g. some copper transporters) for
cisplatin versus oxaliplatin. The plateau level of oxaliplatin in the cells after 24 h of drug
incubation may be related to the reaction of oxaliplatin with other targets in the cytoplasm, such
as proteins, with higher frequency than cisplatin. Cisplatin-DNA or cisplatin-protein adducts
may be removed less efficiently than oxaliplatin-DNA adducts, resulting in a higher
accumulation of cisplatin in the cells (Virag et al., 2012).
150
Table 1. The platinum-DNA binding after HCT116 cells exposured to cisplatin, oxaliplatin,
and their liposomal formulation for 4 h and 24 h o f drug incubation
DNA-Pt (Molecule Pt/1000 b.p.)
Platinum derivatives
4 h drug incubation
24 h drug incubation
Cisplatin
0.795 ±0.09
0.173 ±0.01
Oxaliplatin
0.726 ±0.04
0.207 ±0.01
Lipoplatin
0.069 ± 0.02
0.588 ± 0.04
Lipoxal
0.007 ± 0.002
0.069 ±0.01
In the in vivo studies following the administration of a fixed intravenous dose (10
mg/kg) of cisplatin, oxaliplatin or their liposomal formulations, only a small fraction of
platinum found in the serum, suggesting that the most of the platinum drug molecules are free
to diffuse out from blood vessels into tumor and/or normal tissues (Hall et al., 2008; Wang and
Lippard, 2005). While there is a gradual decrease of platinum drug concentration in the serum
over time, the total concentration of platinum drugs in the blood tended to stabilize or to
increase, probably via redistribution from other tissues. While the design of this study consists
of a single i.v. injection of each of the tested drugs, these particular results underline the
possible long term blood accumulation of platinum drugs especially with repetitive
administrations, which may have an impact on drug pharmacokinetics and tolerability. This
result also suggests that the haemoglobin level may have an effect on the efficacy and toxicity
of the platinum drugs (Mandal et al., 2004; Mantovani et al., 1999).
Our results agree with previous observations that the binding of oxaliplatin to red blood
cells (RJBCs) is rapid (ti /2 RBCs = 0.58 h, reaching equilibrium by 4 hours) (Luo et al., 1999)
151
and its clearance was subsequently slow (Kim and Erlichman, 2007). These data indicate the
ratio of haemoglobin (Hb) level / packed cell volume (PCV) might affect oxaliplatin
metabolism. Since many patients treated with oxaliplatin are anemic from digestive bleeding
and/or bone marrow toxicity (Gundling et al., 2008; Noronha et al., 2005), the effect of the
variation in Hb level on the efficacy and toxicity of oxaliplatin should be clarified. Fortunately,
the relative contribution of Hb level on the anticancer efficacy in the clinical practice may be
small since the drugs are captured mainly in DNA in tissues throughout the body (Culy et al.,
2000).
Cisplatin and oxaliplatin exhibited levels of platinum drug accumulation in liver tissues
similar to those of their liposomal formulations. The free platinum forms however showed a
higher peak levels in kidney tissues than those observed with their liposomal formulations.
Indeed, the maximum level of platinum drug accumulation in kidney tissues was 3-5 times
higher with cisplatin and oxaliplatin than with to Lipoplatin™ or Lipoxal™. This observation
is in agreement with previous studies (Boulikas, 2004; Devarajan et al., 2004b). These
pharmacokinetic data may account for the low renal toxicity of liposomal platinum
formulations. A low concentration of platinum was accumulated in muscle tissues after
treatment with Lipoplatin™ and Lipoxal™ as it was with the free formulations, suggesting
slow clearance of all drugs.
The accumulation of platinum drugs in the tumor tissue after cisplatin and oxaliplatin
administration was higher relative to their liposomal forms in the animal model. However, this
is the opposite of what was observed in the in vitro study with HCT116 cells, where higher
TTKX
levels of platinum accumulation were observed after treatment with Lipoplatin
and
T (U [
Lipoxal
relative to the free-platinum forms. This difference in accumulation between in vitro
152
and in vivo studies is likely explained by our experimental setup. To determine the platinum
accumulation in the HCT116 cells, we incubated all platinum drugs at the same equitoxic dose
(or their IC50 concentration). With in vivo studies, the identical concentrations of platinum
drugs at 10 mg/kg were administered to the animals because of the difficulty to determine the
exact equitoxic dose among these platinum drugs. Indeed, most animal and human studies have
used 3-6 times higher doses of Lipoplatin™ compared to cisplatin. For example, Ravaioli and
coworkers (Ravaioli et al., 2009) used Lipoplatin™ 100 mg/m2 every two weeks, as second line
chemotherapy in heavily pre-treated patients with advanced non-small-cell lung cancer and
obtained 5% partial remission and 16% stable disease. On the other hand, Stathopoulos and
coworkers (Stathopoulos, 2010) used 400 mg/m2 every two weeks (actually 200 mg/m2 Dl,2
every 14 days) on a similar group of patients, and obtained 38% partial remission and 43%
stable disease with only minor toxicities of Grade I. The dose of cisplatin in 14-day schedules
is 75 mg/m2 compared to 200 mg/m2 for Lipoplatin™ in two randomized Phase III studies
(Stathopoulos et al., 2011) in combination with paclitaxel one in NSCLC and the other in nonsquamous-NSCLC patients; both studies have shown a much lower toxicity profile in the
Lipoplatin™ arm. Furthermore, the non-squamous-NSCLC study demonstrated a statisticallysignificant higher response rate in the Lipoplatin™ treatment. Consequently, Lipoplatin™
should achieve a higher level of tumor accumulation and also in platinum-DNA adducts.
Despite the fact that the amount of platinum accumulated in the tumoral tissue gradually
decreased with the time (Chapter 4 and 5), the concentration of platinum-DNA adducts showed
a more complicated behaviour and two maxima were observed at 4 h and 48 h after drug
administration. The rational for measuring the number of platinum-DNA adducts as a
predictive assay is based on the assumption that higher levels of platinum adducts predicts
153
favourable treatment outcomes (van de Vaart et al., 2000). Furthermore, it seems plausible that
the maximum combination effects with radiation treatment might be observed if radiation were
administered when platinum-DNA adducts levels are highest. Indeed, we observed a high
concomitant effect under such conditions (Chapter 4 and 5).
The poor accessibility of tumor tissues is a major obstacle for the routine measurement
of platinum-DNA adducts formation, but it is known that platinum-DNA adducts form in both
tumors and in normal tissues (Hoebers et al., 2008). Might levels in normal tissue be useful
markers to levels in tumors? The measurement of platinum-DNA adducts in normal tissues and
tumor response was performed previously (Blommaert et al., 1993; Schellens et al., 1996; Van
der Vaart et al., 2000). The data on correlations between the amounts of platinum-DNA adducts
and tumor responses are contradictory. In one experimental study, no significant correlations
were found between adduct levels in normal tissues and primary tumor biopsies, nor between
white blood cells and buccal cells (Hoebers et al., 2008). However, various other studies
(Blommaert et al., 1993; Redd et al., 1987, Schellens et al., 1996; Van der Vaart et al., 2000)
showed a positive correlation between the numbers of platinum-DNA adducts in normal tissues
and the response to therapy in patients. They suggested that the higher levels of platinum-DNA
adducts in normal tissue could predict a favourable treatment outcome. In future study, the
evaluation of the concentration of platinum-DNA adducts in normal tissue as well as in
leucocytes could be performed as a potential predictive assay, with the assumption that normal
tissues may be used as a surrogate marker for the tumor. Additionally, such a study might allow
for the assessment o f the concomitant effects in a clinical setting, where repetitively obtaining
cancerous tissue samples to determine platinum-adduct concentration would be problematic.
154
Such a study would look to identify correlations between in platinum-DNA adduct levels in
normal tissue and patient response to chemoradiotherapy in clinical trials.
Platinum-based radiosensitizers and cell cycle progression to chemoradiotherapy in human
colorectal cancer HCT116 cell
To determine the radiosensitivity of cisplatin and oxaliplatin in colorectal cancer in
vitro and in vivo studies were performed. For in vitro study (Chapter 3), the colony formation
assay demonstrated that cisplatin and oxaliplatin act as radiosensitizers in human colorectal
cancer HCT116 cells. It should be noted that the concentration range used in this study
(cisplatin = 1 - 1 0
pM; oxaliplatin = 0.5 - 3.5 pM) is close to clinically obtainable
concentrations, e.g. oxaliplatin at 2 h i.v. injection of a normal patient dose (85 mg/m ) reaches
a peak plasma concentration of 3.6 pM (Boulikas and Vougiouka, 2003).
While cisplatin and oxaliplatin belong to, the alkylating agent group of chemotherapies
(idweekel et al., 2005), they are not cell cycle specific. However, their radiosensitizing activity
is a function of the cell cycle (Hall and Giaccia, 2005). The diagram in Figure 1 illustrates the
cell cycle phases and their relative radiosensitivity or radioresistance. Cells appear to be
maximally sensitive to platinum drugs and radiation in Go/Gi and G2/M phases and minimally
sensitive at peak DNA synthesis in the S phase. Unlike G2/M arrest, the S phase delay lacks the
sustained maintenance phase of the cell cycle arrest (Shiloh, 2003). Moreover, DNA damage
induction by platinum drug and radiation has been shown to cause G2/M arrest prior to
inducing apoptosis (Sorenson et al., 1990).
The cell cycle progression after chemoradiotherapy was investigated in HCT116 human
colorectal cancer cells to better understand of how chemotherapeutic agents cisplatin,
oxaliplatin and their liposomal formulations (Lipoplatin™ and Lipoxal™) can act as
155
radiosensitizers in vitro, and to determine the effect of platinum drugs on cell cycle
progression. To examine cell distribution in the different phases of the cell cycle, we used flow
cytometry at 8 h and 48 h after drug exposure, with and without radiation treatment.
R adiation
Cisplatin
Oxaliplatin
Figure 1. Diagram illustrated phases of cell cycle and their relative radiosensitivity or
radioresistance (Adapted with permission from Hill) (Hill et al., 2012).
A similar characteristic of cell progression at both 8 h and 48 h after drug incubation,
with and without radiation treatment, in that the majority of cells were accumulated in the
Go/Gi phases. The combined treatment (i.e., with radiation) yielded a higher level of cell
accumulated in the G2/M phase than with platinum drug treatment alone. However, we
observed a difference in cell accumulation in the G2/M phase, (which is the most radiosensitive
phase) between 8 h and 48 h of drug incubation. Samples incubated for 8 hours and then
irradiated showed a higher level of cell blocking at G2/M phase than the untreated controls,
156
while a lower level o f cell accumulation at G2/M phase was measured in sample irradiated after
48 h of drug incubation. Moreover, a higher level of apoptotic cell death was observed with the
combined radiation treatment at 8 h than was the case when radiation was administered after 48
h of drug incubation. Consideration of time-dependence in cell cycle progressions may
contribute to the variation o f the concomitant effect of combined treatment, and consequently
have implications for the enhancement for cancer cell death in chemoradiotherapy involving
platinum-based drugs.
As described in chapter 3, the concentration of platinum drugs used to incubate the
HCT116 human colorectal cancer cells (e.g. at concentrations lower, equal and higher than
their IC50 values) may affect the cell progression through the cell cycle, affecting the potential
for a concomitant effect with combined treatment. It has been suggested that a high level of cell
accumulation at G2/M phase, combined with a delay in the S phase, resulting from a reduction
in DNA synthesis, is correlated with the augmentation of drug concentrations (William-Faltaos
et al., 2006). Such an increase of cell accumulation at G2/M phase could lead to an
enhancement in apoptotic cell death (Zheng et al., 2007). We clearly observed a synergetic
effect as well as an additive effect when assessing radiation treatment at high or median
concentration of the drug (> IC50 or = IC50 values) for both 8 h and 48 h of drug incubation,
except in the the case of oxaliplatin after 48 h, where a reduction of cell accumulation at G2/M
phase of more than 60% was measured. Similarly previous studies have demonstrated a
decrease in G2/M accumulation when HCT116 cells were exposed to oxaliplatin for a long
incubation period (Amould et al., 2002). Such a decrease suggests that a reduction in the
radiosensitizing activity of combined treatment at long incubation times with this drug. The
treatment with all drugs, excluding Lipoxal™, at low concentration (i.e., below the IC50 values)
157
for 48 h, showed a significantly antagonistic effect, indicating no pronounced G2/M arrest in
these cases.
Radiosensitizing activity o f Lipoplatin™ and Lipoxal™
•
The radiosensitizing activity of liposomal platinum formulations, Lipoplatin
tw
and
Lipoxal™, has been evaluated for cellular response, for systemic cytotoxicity and for the
potential for synergetic effects with radiation treatment. In previous a phase I/II study
concerning concurrent liposomal cisplatin (Lipoplatin), 5-fluorouracil and radiotherapy for the
treatment of locally advanced gastric cancer (Koukourakis et al., 2010), Lipoplatin™ was given
at a dose o f 120 mg/m2/week, 5-fluorouracil at 400mg/m2/week (Day 1), and radiotherapy was
given as 3.5-Gy fractions on Days 2, 3, and 4. Two groups of 6 patients received four and five
consecutive cycles, respectively. The concurrent administration of Lipoplatin™ and radiation
showed a high potential for improving complete response rates to chemoradiation treatment in
locally advanced gastric cancer with acceptable toxicities. Such results appear consistent with
those of this study (e.g., chapters 4 and 5), where we observed a radiosensitizing enhancement
after combined radiation treatment with Lipoplatin™ and Lipoxal™ in both cell cultures and
mouse xenografts.
While in chapter 4 and 5, Lipoplatin™ and Lipoxal™ were found to produce fewer
platinum-DNA adducts than did their free platinum formulations, a superior concomitant effect
was observed with concurrent radiation treatment. Only a weak correlation between the
cytotoxicity of liposomal platinum drugs and the degree of DNA platination could be found.
This might imply that the platinum-based drugs have important targets other than from nuclear
DNA (e.g. the cytoplasmic compartments) (Yang et al., 2000). For example it has been
proposed that Lipoplatin™ and Lipoxal™ can release cisplatin and oxaliplatin molecules into'
158
the cytoplasm of the tumor cell which then activate the mitochondrial apoptotic cascade
(Stathopoulos and Boulikas, 2012). In addition, any released cisplatin and oxaliplatin molecules
may i) undergo reaction with phospholipids; ii) inhibit amino acid transport, protein synthesis
and ATPases; and iii) uncouple oxidative phosphorylation (Gourdier et al., 2004; Mandic et
al., 2003; Wang et al., 1996). However, the importance of targets other than DNA in relation to
cytotoxicity is still unclear and remains the subject of future study.
Due to the abundance o f mitochondria in almost all cells, platinum drugs may interact
with components of the mitochondrial membrane (Weissig et al., 2006). It has been suggested
that platinum drugs can induce high levels of mitochondrial reactive oxygen species (ROS)
such as the superoxide radical anion (O2 *), hydroxyl radical ( OH) and hydrogen peroxide
(H2O2) (Mukhopadhyay et al., 2012). ROS can interact with cellular macromolecules to
interfere with vital cellular functions and so lead to cell death ( Conklin, 2004). Yakes and Van
Houten have been demonstrated oxidative stress-induced mitochondrial DNA damage (Yakes
and Van Houten, 1997). Figure 2 shows a schematic illustrating the induction of the
mitochondrial apoptotic pathway by cisplatin. Cisplatin-induced mitochondrial ROS trigger the
release of cytochrome c from the mitochondrial intermembrane space onto the cytosol, which
induces the formation of the apoptosome. The apoptosome is an Apaf-1 cytochrome c complex
which activates procaspase-9. Procaspase-9 molecules can bind to the inner "hub" region of the
apoptosome. This complex promotes the efficient activation of procaspase-3. Therefore, the
cleavage of procaspase-9 is not required to form an active cell death complex (Boulikas, 2007).
Cisplatin can activate the proapoptotic protein Bax, resulting in cytochrome c release, caspase
activation, and apoptosis (Wei et al., 2007). Bcl-2 also plays an important role in the
mitochondrial apoptotic pathway.
159
It has been proposed that the binding affinity of drugs to the mitochondria is regulated
by its lipid solubility (Boddapati et al., 2005). Hori and co-workers have reported that high
lipophilicity drugs are more rapidly accumulated in the mitochondria than are non- or lowT
»i
lipophilic drugs (Hori et al., 1987). The improvement in terms of lipophilicity for Lipoplatin
and Lipoxal™ above that of their free-platinum forms could indicate a higher efficacy for
binding to the mitochondria membrane, that would lead to a greater accumulation of the drugs
within the mitochondria. Thus, induction of the liposomal platinum drug accumulation in the
mitochondria may contribute to the enhancement of apoptotic cancer cell death. This
characteristic o f Lipoplatin™ and Lipoxal™ may be further associated with the improvement
of the radiosensitizing effect after the chemoradiation treatment.
160
Figure 2. Induction of the mitochondrial apoptotic pathway by cisplatin (Adapted with
permission from Boulikas) (Boulikas, 2007).
Histopathological analysis
Part of the work of this thesis was to evaluate the efficiency of various combinations of
radiation with platinum-based drugs on the basis of their effect on tumor growth delay (Chapter
4 and 5). Additionally, histopathologic changes were also studied to determine the response of
161
HCT116 colorectal tumor xenografts to chemoradiation treatments. Examples are shown in the
Figure 3A. Mitotic cells were not counted in necrotic areas. Figure 3B shows a mitotic cell
count after chemoradiotherapy. The tumor sections treated with chemotherapy alone remained
mitotically active compared to the control group. In contrast, HCT116 tumor sections with
combined platinum-based drugs and radiotherapy,, display a significant decrease in the number
of mitotic cells. In addition, the combined treatments demonstrate a shrinking nuclear
pleomorphism in relation to form and size; one or two nucleoli observed in the nuclei. Islands
of geographic necrosis are frequent between the sheets of tumor cells. Vascularisation is poor.
The tumoral mass seems contained in a very thin collagen capsule-like structure.
The relationship between mitotic activity and apoptotic cell death has been reported
(Brustmann, 2004). In the present study, an intense reduction in mitotic activity was observed
when radiation was applied 48 h after drug administration. In addition, we also observed an
increased number of cells dying following the combined treatment when compared to the
control group. After apoptotic analysis using TUNEL assay, we observed a higher percentage
o f apoptotic cells at 48 h of drug administration combined with radiation treatment, compared
to the control group or to both chemotherapy and radiotherapy alone. These results on mitotic
activity and the fraction o f apoptotic cells are remarkably consistent with idea that a strong
concomitant effect is obtained when radiotherapy is performed 48 h after drug administration
(i.e., when there is a high level of platinum-DNA adducts).
162
i) Control
ii) Irradiation alone
iii) Cisplatin
iv) Lipoplatin
163
v) Oxaliplatin
vi) Lipoxal™
vii) Cisplatin (24h)-IR
viii) Lipoplatin™ (24h)-IR
ix) Oxaliplatin (24h)-IR
x) Lipoxal™ (24h)-IR
xi) Cisplatin (48h)-IR
xii) Lipoplatin™ (48h)-IR
xiii) Oxaliplatin (48h)-IR
xiv) Lipoxal™ (48h)-IR
165
B)
Lipoxal(48h)-IR
Lipoxal(24h)-IR
Lipoxal alone
Oxaliplatin(48h)-IR
Oxaliplatin(24h)-IR
Oxaliplatin alone
Lipoplatin(48h)-IR
Lipoplatin(24h)-IR
Lipoplatin alone
Cisplatin(48h)-IR
Cisplatin(24h)-IR
Cisplatin alone
IR alone
Control
Mitotic cells
Figure 3. Histophatologic changes of HCT116 human colorectal cancer tumors treated with
platinum drugs and radiation. A) H&E staining after chemoradiotherapy. Animals were
irradiated at 24 and 48 h of drug administration. Treatments were administered with a single
dose of cisplatin, oxaliplatin, Lipoplatin™, Lipoxal™ of 10 mg/kg; and a single dose of
radiation of 15 Gy. Arrow indicated mitotic figures and arrow head indicated cells undergoing
death. Mitotic cells were counted in 10 random fields per tumor (n=3) (Mean ± S.D.).
Magnification, X40. B) Mitotic cells count after chemoradiotherapy.
Emerging strategies for improvement in chemoradiotherapy: Clinical aspects
The efficacy of radiation treatment is often increased by using chemotherapeutic agents
such as radiosensitizers. Concurrent chemoradiotherapy has become the standard treatment of
colorectal cancer (Prestwich et al., 2007; Glimelius et al., 2003; Minsky et al., 1992). Bosset
and co-workers reported downsizing and down-staging of tumors, as well as changes in
histologic characteristics of tumors after chemoradiotherapy. These effects were associated
166
with a significant increase in local control, but no improvement in progression-free or overall
survival (Bosset et al., 2005). The combined therapy is still much restricted by its narrow
therapeutic index. The chemotherapeutic drugs scheduling in relation to radiation fractions is
one of the important. Optimal scheduling is essential in concomitant chemoradiotherapy, not
only to maximize tumor radioresponse, but also minimize toxicity to critical normal tissues.
Before novel clinical treatment strategies are attempted in the field of chemoradiation,
there is a need for adequate preclinical data that establishes whether the addition of drugs to
radiation is likely to be of clinical benefit. The selection of optimal timing of drug
administration must be based on multiple factors including; mechanisms of tumor
radioenhancement by a given drug, the drug’s normal tissues toxicity, and conditions under
which the highest enhancement is achieved. The successes in our preclinical studies offer the
possibility to develop an optimal concomitant chemoradiotherapy schedule, and this should
encourage further clinical implementation.
The hypothesis for further development of an
optimal clinical treatment schedule that follows the observation in vitro and in vivo studies is
shown in the Figure 4.
For most current chemoradiotherapy protocols, the radiation is given at equal doses on a
daily basis (five sessions per week, for about five weeks) while the addition of cisplatin or
oxaliplatin is given intermittently (Bosset et al., 2006; Marcu, van Doom and Olver, 2003).
Assessment of radiotherapy at the time at which the amount of platinum accumulated in the
tumor or bound to DNA is low, likely to be clinically relevant. This may reduce the anticancer
effectiveness of the combined treatment.
167
Synergistic
Additive
0
QnfeMfo
oncumi*
20
40
60
80
Antagonistic
Time after injection®
Figure 4. Possible improvement for clinical chemoradiation treatment based on our preclinical
studies
for radiation treatment.
The improvement of antitumor effectiveness after fixed sessions of radiotherapy at
which the maximum level of platinum bound to DNA, so far not tested yet in the clinical
practice. Cividalli and co-workers reported the improvement of tumor growth delay in mouse
adenocarcinoma after combined treatment of oxaliplatin (10 mg/kg) with a single dose of 10
Gy X-rays better relative to a treatment with 10 daily X-rays sessions (Choi and Deasy, 2002).
The modifying of the conventional chemoradiation treatment schedule from daily 5 x 2 Gy per
week to a few sessions radiotherapy which correspond to the amount of platinum-DNA adducts
(e.g. 2 x 5 Gy per week) should be further explored.
Furthermore, considering that the majority of platinum radiosensitizers are not
administered alone, but combined with other drugs. Table 2 shows the lists of other
chemotherapeutic agents that are commonly combined with platinum drugs (Nieder and
Lordick, 2006). Here, we used platinum-based radiosensitizers as a monotherapy, but
concurrent administration of 5-FU or other drugs could impact the distribution of those drugs,
and vice versa. Kjellstrom and co-workers reported the importance of the period of incubation
with oxaliplatin in a.colorectal cancer cell line exposed to ionizing radiation and 5-FU [33]. It
has shown also the integration of drugs such as 5-fluorouracil, folinic acid, capecitabine and
irinotecan to oxaliplatin chemoradiotherapy protocols (Andre and Schmiegel, 2005; WilliamFaltaos et al., 2006). For cisplatin combined with 5-fluorouracil, folinic acid and gemcitabine
has been purposed (Chang et al., 2007). Thus, the use of cisplatin, oxaliplatin and their
liposomal formulations in combination with other drugs plus radiation could be further
explored.
169
T able 2 Lists of other chemotherapeutic agents that are commonly combined with platinum
drugs (Nieder and Lordick, 2006).
Mode of action
Compounds
Irinotecan
Topoisomerase-I inhibitor
Etoposide
Topoisomerase-II inhibitor
Taxanes (Paclitaxel, Docetaxel)
Microtubule inhibitors, disruption of the
centrosome network, inhibition of mitotic spindle
formation
Vinblastine, Vincristine, Vinorelbine
Tubuling-binding vinca alkaloids, inhibition of
mitosis
5-Fluorouracil, Capecitabine,
Affect DNA synthesis, nucleoside, and nucleotide
Gemcitabine
metabolism deplete the deoxynucleoside
triphosphate pool (5-FU: inhibition of thymidilate
synthase; Capecitabine: oral prodrug, converted to
FU by thymidine phosphorylase; Gemcitabine
diphosphate inhibits ribonucleotide reductase
inducing a depletion of cellular deoxynucleotides
(dNTP)
Mitomycin C
Affects DNA synthesis in hypoxic cells through
formation of adducts and crosslinks
Tirapazamine
Affects hypoxic cells through intracellular
reduction resulting in a highly reactive radical
capable of causing DNA strand breaks
170
CHAPTER VII
CONCLUSIONS
Aiming to enhance the synergism and increase the efficiency of concomitant
chemoradiotherapy in patients with rectal cancers, we tried to identify the optimal conditions
associated with higher synergistic anticancer effect with no or minimal added toxicity.
The in vitro experiments confirm the sensitization of the colorectal cell line HCT116 to
radiation therapy after exposure to cisplatin, oxaliplatin and their liposomal formulations
Lipoplatin
T\jf
and Lipoxal
T»i
. Such radiosensitization was shown to depend on the drug
concentration (that should exceed IC50), and the duration of exposure. The timing of radiation
in relation to drug exposure, which implies variation of drug concentration in tumor cells and
its different intra-cellular compartments, was shown to be a major element impacting
synergism. A maximal synergism appears to correlate to the time of highest platinum-DNA
adducts.
These data was confirmed in animal model, where the outcome of combined
chemoradiotherapy varied in relation to the timing of drug exposure and radiotherapy. The
maximal synergism and antitumor effect was observed when platinum drug was administered
48 h prior to radiation, which mainly correlated to the highest level of platinum bound to the
DNA.
These observations goes with the hypothesis of “true” or “physical” synergism between
radiation and platinum molecules bound to the DNA, rather than a concomitant or
complimentary biologic effects.
171
These studies show that the time-dependent accumulation of platinum in tumor cells
and in its different cellular compartments is not linear, and is not completely similar to other
healthy tissue cells. This opened the way to define a time-window where radiation has the
highest synergism with platinum.
Given that most clinical platinum based chemoradiotherapy protocols in rectal cancers
or other neoplasia consist of daily hyperffactionated radiation and intermittent chemotherapy, it
appears clear that this window of maximal synergism correlates only to a small part of the
radiation treatment, and most of radiation is given at times of sub-optimal or minimal
synergism. There is clearly a wide room to improve these clinical protocols for better patient
outcomes
The liposomal platinum formulations (Lipoplatin™ and Lipoxal™) have been
investigated to overcome side effects of cisplatin and oxaliplatin. Our in vitro and in vivo data
confirm their potential roles as radiosensitizers. While the general concepts of maximal
synergism correlating with highest DNA platinum still applies, there were some kinetic
different between each o f these drugs, as described, and these should be taken in consideration
in defining the “platinum window” of each drug.
Collectively, these studies allow a better understanding of the pharmacokinetics and the
synergistic mechanisms at the base of platinum-based chemoradiotherapy, and open the door to
a better patient outcome with minimal or no added toxicity. This should be confirmed in
clinical trials
172
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