Effects of Bombesin on Growth of Human Small Cell Lung...

(CANCER RESEARCH 48, 1439-1441, March 15, 1988]
Effects of Bombesin on Growth of Human Small Cell Lung Carcinoma in Vivo1
Robert W. Alexander, James R. Upp, Jr., Graeme J. Poston,2 Vicram Gupta, Courtney M. Townsend, Jr.,3 and
James C. Thompson
Department of Surgery [R. W. A., J. R. V., G. J. P., C. M. T., J. C. T.] and Department of Internal Medicine [V. G.J, The University of Texas Medical Branch,
Galveston, Texas 77550
ABSTRACT
Bombesin-like peptides are found in many different human tumors and
are thought to function as an autocrine growth factor for small cell lung
cancer in humans. In this study, a human small cell lung carcinoma (NCIH69) was s.c. implanted bilaterally into the flanks of 12 nude mice. The
mice were randomized and divided into two groups and given either
bombesin (20 Mg/kg) or saline i.p. 3 times a day. Tumor areas were
measured twice weekly for 6 wk. At sacrifice, the tumors and normal
pancreas were excised, weighed, and assayed for DNA, KN'A,and protein
content. Significant stimulation of rumor growth was observed at weeks
4, 5, and 6. Tumor weight at sacrifice was significantly elevated (77%)
above the control, as was DNA content (78%). Bombesin significantly
increased the weight (42%), DNA (48%), and protein (61%) contents of
the normal mouse pancreas. We conclude that bombesin may act as an
autocrine growth factor, or indirectly through the release of other growth
factors, on human small cell lung carcinoma.
INTRODUCTION
It is estimated that lung cancer will cause approximately
136,000 deaths in the United States in 1987 (1). SCLC4 or oat
léñate
(30 HM)], HITES supplemented with bombesin or argi
nine vasopressin, as well as in serum-supplemented media,
suggesting that SCLC secrete an autocrine growth factor (15).
Cuttitta and colleagues (16) developed a monoclonal antibody
against a synthetic analogue of amphibian bombesin. This
antibody inhibited the growth of a xenografted SCLC (NCIN592) in nude mice and inhibited cloning of SCLC cell lines
in soft agarose in vitro. Analysis of membrane preparations of
both rat brain and SCLC revealed the antibody blocked a
specific bombesin receptor.
Specific receptors for bombesin have been demonstrated on
the Swiss 3T3 fibroblasts (17) as well as in the rat brain (18),
guinea pig pancreatic acini (19), rat pituitary (20), and human
small cell tumors and cell lines (5, 16, 21).
The purpose of our study was to determine whether exoge
nous administration of bombesin would affect the growth of a
human SCLC growing in nude mice.
MATERIALS
AND METHODS
cell carcinoma accounts for approximately 20% of the classified
lung tumors (2). Many SCLC have been found to contain
bombesin-like immunoreactivity (3-10). Bombesin is a 14amino acid peptide initially isolated from two European frogs,
Bombina bombino and Bombina veriegata veriegata (10). Bom
besin-like immunoreactivity is not specific for SCLC; other
tumors of neuroendocrine (bronchial and gastrointestinal carcinoids) and nonneuroendocrine origin (squamous, large cell,
and adenocarcinoma of the lung) have been found to contain
bombesin-like immunoreactivity (7-10). However, bombesinlike immunoreactivity has been found in elevated concentra
tions in SCLC when compared with other tumors (3-6, 9).
Bombesin stimulates in vitro proliferation of 3T3 mouse
fibroblasts (11) and of epithelial cells from the normal human
bronchus (12). GRP, the mammalian counterpart of bombesin,
has recently been shown to serve as a mitogen for SCLC cell
lines in vitro (13). The carboxyterminal fragment (GRPI4~27),
Animals. Male athymic nude BALB/c mice (21-24 g) were purchased
from Life Sciences, St. Petersburg, FL. The mice were housed in a
specific pathogen-free, temperature-controlled isolation compound and
were exposed to a light-controled day (lights on, 0700-1900 h). Diet
was composed of standard chow (Autoclavable Rodent Chow No. 5010;
Ralston Purina, St. Louis, MO) and water given ad libitum.
Tumor. A human SCLC (NCI-H69) (American Type Culture Collec
tion, Rockville, MD), which contains bombesin-like immunoreactivity
(3, 4), was xenografted as 2-mm2 pieces s.c. through an interscapular
which exhibits significant homology to bombesin, appears to
be the active site, since GRP1"16 does not act as a mitogen (13).
Drugs. Mice with implanted tumors were randomized and divided
into two groups, each receiving 0.1 ml i.p. injections 3 times per day.
The control mice received saline with 0.1% bovine serum albumin
(Calbiochem, La Jolla, CA), and the mice in the treatment group
received bombesin tetradecapeptide in 0.1% bovine serum albumin (20
Mg/kg) (Sigma Chemical Co., St. Louis, MO).
Biochemical Assays. Tissues were extracted for measurement of
DNA, RNA, and protein (22). DNA was measured by the Burton
modification of the diphenylamine procedure, with calf thymus DNA
as a standard (23). RNA was measured using the orcinol procedure and
yeast RNA as a standard (24). Protein was determined by the method
of Lowry and colleagues (25).
Statistical Analysis. Statistical significance was determined by the
Kruskal-Wallis analysis of variance. A value of P< 0.05 was considered
to be significant.
Bombesin, when added to cultures of SCLC, stimulates colony
formation in soft agarose (14). Media taken from established
cultures of SCLC stimulated the soft agarose cloning of SCLC
in serum-free HITES medium [RPMI Medium 1640 supple
mented with hydrocortisone (10 nM), bovine insulin (5 fig/ml),
transferrin (10 ^g/rnl), 17/î-estradiol(10 nM), and sodium seReceived 8/31/87; revised 11/30/87; accepted 12/11/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by grants from N1H (ROÕDK 15241, POI DK 35608, and RCDA
CA 00854), from the American Cancer Society (PDT-220), and from the National
Cancer Institute (CA 17701).
'Visiting Scientist from the Department of Surgery, Royal Postgraduate
Medical School, University of London, London, United Kingdom. Supported by
the Wellcome Foundation, Ethicon Foundation, and British Digestive Founda
tion.
3 To whom requests for reprints should be addressed.
4 The abbreviations used are: SCLC, small cell lung cancers; GRP, gastrinreleasing peptide.
incision bilaterally into the flanks (two tumors per mouse) of 12
anesthetized mice. Mice were weighed weekly, tumors were measured
twice weekly with calipers, and tumor surface areas were calculated
using the product of the two greatest perpendicular tumor diameters.
Tumor doubling times were calculated from semilogarithmic graphs of
tumor area versus day from implantation. At termination of the exper
iment, mice were sacrificed by cervical dislocation followed by excision
of tumors and pancreas. Tissues were frozen in liquid nitrogen and
stored at —70°C
until assay.
RESULTS
The body weights in both groups increased by approximately
87% over the 6-wk study period and were not significantly
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BOMBESIN AND HUMAN SMALL CELL LUNG CANCER
different from each other. Tumor growth was seen in 8 of 12
tumors implanted in each group.
Bombesin reduced the tumor cell doubling time by 24% from
7.5 days in the control to 5.75 days in the treated group.
Bombesin produced significant stimulation of tumor growth;
the areas increased from weeks 4-6 (Fig. 1). At sacrifice, both
tumor weight (77%) (Fig. 2) and tumor DNA content (78%)
(Fig. 3) were significantly stimulated above the control group.
Tumor RNA and protein content were also increased (67 and
32%), although they were not significantly different from con
trol (Table 1).
Bombesin significantly increased the weight (42%), protein
(61%), and DNA content (48%) of the normal mouse pancreas
(Table 1).
150^120¿
:<
90K
CO§\1
«0¡
30*rira
lu1234WEEKS.Li*I_ä8iÈ¡LU
reu
Fig. 1. Tumor area in relation to time from implantation. Values, mean ±
SEM (n = 8 in each group; *, P < 0.05).
We found that bombesin produced significant stimulation of
growth of a human SCLC and the normal mouse pancreas. The
tumor cell line that we studied has been reported to contain
bombesin-like immunoreactivity (3, 4). The importance of our
study is that, to our knowledge, this is the first demonstration
that exogenous bombesin stimulates growth of human SCLC
in vivo. Our findings support and extend the work of Cuttitta
and colleagues (16) who suggested that bombesin-like peptides
may act as autocrine growth factors in SCLC.
We and others have previously shown that bombesin stimu
lates growth of the normal rat pancreas (26-29). In the present
study, bombesin (20 ¿tg/kgi-p- 3 times a day) produced hyperplasia of the normal mouse pancreas which was characterized
by significant increases in weight, DNA, and protein content.
The mechanism by which exogenous bombesin induces growth
may involve both a direct effect of bombesin and an indirect
effect through bombesin-mediated release of cholecystokinin
(27, 29).
Although we have demonstrated stimulation of human SCLC
growth In vivo, we have not used a bombesin receptor antagonist
to show inhibition of bombesin stimulated growth. The syn
thetic peptide, spantide [(o-Arg1, o-Pro2, o-Trp7-9, Leu") sub
stance P], inhibits the in vitro effects of bombesin (17, 30, 31);
however, the nonspecificity of action and lack of potency limit
the use of spantide in vivo (32). We have found that bombesin
does not stimulate the growth of a human colon cancer growing
in nude mice,5 suggesting it may be specific for tumors with
bombesin receptors.
Bombesin has been implicated as a mitogen or as an autocrine
growth factor for normal and malignant cells (11-17). The term
"autocrine secretion" has been proposed for self-stimulation
0.6-
0.4
DISCUSSION
Õ
0.2-
Fig. 2. Tumor weight at sacrifice. Values, mean ±SEM (n = 8 in each group;
*,P<0.05).
4-•2
n -JL_j8Iw/mi
Fig. 3. Tumor DNA content. Values, mean ±SEM (n = 8 in each group; :
P < 0.05).
Table 1 Tumor and pancreas weight and content of DNA, RNA, and
protein ±SEM
TumorWeight
±0.10°
±0.03"
(g)
±0.10
±0.04
DNA (mg)
2.42 ±0.66 4.30 ±0.72° 0.69 ±0.10 1.02 ±0.04°
RNA (mg)
1.41 ±0.38 2.35 ±0.42
3.20 ±0.47
3.46 ±0.28
5.7°'F<
70.5 ±
Protein (mg)Control0.38
67.7 ±18.4Bombesin0.67
89.1 ±15.4PancreasControl0.25
43.8 ±6.1Bombesin0.35
whereby a cell produces endogenous factors for which they have
receptors and to which they are able to respond (33). Bombesin
has been found in human SCLC as well as neuroendocrine and
other pulmonary tumors (3-10). Moreover, specific receptors
for bombesin have been identified in SCLC tissue culture cell
lines (5, 16, 21). Bombesin, when added to cultures of SCLC,
stimulates colony formation in soft agarose (14). One of the
most convincing studies that supports the autocrine hypothesis
is that of Cuttitta and colleagues (16). They found that a
monoclonal antibody, raised against a synthetic analogue of
amphibian bombesin, blocked the binding of bombesin to bom
besin receptors and inhibited the clonal growth of SCLC in
vitro and the growth of SCLC xenografts in vivo (16).
The amphibian peptide, bombesin, and the mammalian coun
terpart, GRP, have striking homology, the C-terminal heptapeptides are identical. Sausville and colleagues (34) have re
cently described the presence of prepro-GRP gene products in
5 human SCLC. It then appears that the bombesin-like immu
noreactivity (and probably the growth-stimulatory agent) is due
to GRP.
The finding of bombesin-like immunoreactivity in pulmonary
tumors of all types (including squamous and adenocarcinoma)
may be of major significance. The presence or absence of
bombesin-like immunoreactivity in the lung may be related to
the developmental stage of cellular growth. Yesner (35) postu
lated that all lung cancers reflect a spectrum of differentiation
and the degree of ectopie hormone secretion rather than the
type of secretion may be associated with stage of cellular mat
uration. In Yesner's growth concept, SCLC is more primitive
(35); bombesin immunoreactivity appears to diminish as cellu-
0.05.
9 Unpublished data.
1440
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BOMBESIN AND HUMAN SMALL CELL LUNG CANCER
lar differentiation occurs. Further evidence to support this
hypothesis includes the finding of elevated levels of bombesinlike immunoreactivity in fetal or neonatal lungs compared to
adult lung tissue (36) and differences in endocrine cell distri
bution during varying stages of pulmonary development (37).
Cellular maturation may explain the differences in bombesin
immunoreactivity observed between fetal-neonatal and adult
lung tissue, as well as the differences seen between the subtypes
of pulmonary carcinoma. The significance of these findings is
the idea that malignant cells may indeed be related to their
earlier embryonic states and that malignant transformation
occurred due to inappropriate expression of the autocrine mech
anism.
Our study demonstrates the action of bombesin as an exog
enous growth factor for SCLC in vivo. These data support the
possibility that GRP is indeed an autocrine growth factor for
SCLC in vivo. As specific autocrine growth factors are identi
fied, specific antagonists may be synthesized to combat or
control the process of malignant transformation.
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Effects of Bombesin on Growth of Human Small Cell Lung
Carcinoma in Vivo
Robert W. Alexander, James R. Upp, Jr., Graeme J. Poston, et al.
Cancer Res 1988;48:1439-1441.
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