kB-dependent Tobacco components stimulate Akt-dependent proliferation and NF

Carcinogenesis vol.26 no.7 pp.1182--1195, 2005
doi:10.1093/carcin/bgi072
Advance Access publication March 24, 2005
Tobacco components stimulate Akt-dependent proliferation and NFkB-dependent
survival in lung cancer cells
Junji Tsurutani, S.Sianna Castillo, John Brognard,
Courtney A.Granville, Chunyu Zhang, Joell J.Gills,
Jacqueline Sayyah and Phillip A.Dennis
Cancer Therapeutics Branch, Center for Cancer Research, National Cancer
Institute, Bethesda, MD 20889, USA
To whom correspondence should be addressed at: Building 8, Room 5101,
8901 Wisconsin Avenue, Bethesda, MD 20889, USA. Tel: þ1 301 496 0929;
Fax: þ1 301 496 0047;
Email: [email protected]
Retrospective studies have shown that patients with
tobacco-related cancers who continue to smoke after their
diagnoses have lower response rates and shorter median
survival compared with patients who stop smoking. To
provide insight into the biologic basis for these clinical
observations, we tested whether two tobacco components,
nicotine or the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), could
activate the Akt pathway and increase lung cancer cell
proliferation and survival. Nicotine or NNK, rapidly and
potently, activated Akt in non-small cell lung cancer
(NSCLC) or small cell lung cancer (SCLC) cells. Nicotinic
activation of Akt increased phosphorylation of multiple
downstream substrates of Akt in a time-dependent manner, including GSK-3, FKHR, tuberin, mTOR and S6K1.
Since nicotine or NNK bind to cell surface nicotinic acetylcholine receptors (nAchR), we used RT--PCR to assess
expression of nine alpha and three beta nAchR subunits
in five NSCLC cell lines and two types of primary lung
epithelial cells. NSCLC cells express multiple nAchR subunits in a cell line-specific manner. Agonists of a3/a4 or a7
subunits activated Akt in a time-dependent manner,
suggesting that tobacco components utilize these subunits
to activate Akt. Cellular outcomes after nicotine or NNK
administration were also assessed. Nicotine or NNK
increased proliferation of NSCLC cells in an Akt-dependent manner that was closely linked with changes in cyclin
D1 expression. Despite similar induction of proliferation,
only nicotine decreased apoptosis caused by serum
deprivation and/or chemotherapy. Protection conferred
by nicotine was NFkB-dependent. Collectively, these results identify tobacco component-induced, Akt-dependent
proliferation and NFkB-dependent survival as cellular processes that could underlie the detrimental effects of smoking in cancer patients.
Abbreviations: DMXB, 3-[2,4-dimethoxybenzylidene] anabaseine; DnAkt,
dominant negative Akt; GSK, glycogen synthesis kinase; HA, hemagglutin;
MDM2, mouse double minute 2 homologue; mTOR, mammalian target of
rapamycin; nAchR, nicotinic acetylcholine receptors; NFkB, nuclear factor
kappa B; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NSCLC,
non-small cell lung cancer; PI3K, phosphatidylinositol 3-kinase; PARP, poly
(ADP ribose) polymerase; SCLC, small cell lung cancer.
Carcinogenesis vol.26 no.7 # Oxford University Press 2005; all rights reserved.
Introduction
Lung cancer is the leading cause of cancer death throughout the
world, causing ~1.2 million deaths annually (1). The development of lung cancer is associated with smoking in ~85--90% of
cases (2). The basis for marked lethality is probably on account
of the high frequency of advanced stage at diagnosis and the
inherent therapeutic resistance of lung cancer cells. Smokers
continue to have additional health risks, even after the diagnosis of a tobacco-related cancer. These risks include the
increased risk of developing a second tobacco-related malignancy, possibly because of field carcinogenesis effects (3,4).
In addition to the prospective risks, retrospective studies have
shown that continued smoking during therapy for tobaccorelated cancers, such as lung cancer is associated with lower
response rates to chemotherapy and/or radiation, and in some
cases, decreased survival (5--7). Thus, the identification of
molecular events associated with exposure to tobacco components that contribute to therapeutic resistance might provide a
basis for interventions aimed at ameliorating the effects of
smoking during therapy for the patients unable to quit smoking.
Although changes in oncogenes or tumor suppressor genes
are related to smoking and could contribute to therapeutic
resistance (8), activation of signal transduction pathways that
promote cellular survival such as the phosphotidylinositol
3-kinase (PI3K)/Akt pathway might also contribute to
tobacco-related therapeutic resistance. The PI3K/Akt pathway
is a critical pathway in cancer because it contributes to tumorigenesis, tumor growth and therapeutic resistance [reviewed in
(9)]. Akt serves at a nodal point in this pathway and is probably
important for the development and maintenance of lung cancer.
Active Akt has been detected in human lung cancer precursor
lesions and in established lung cancers (10). Non-small cell lung
cancer (NSCLC) cells frequently have constitutively active
Akt that promotes cellular survival and resistance to chemotherapy or radiation (11). In primary human lung epithelial
cells, tobacco components such as nicotine or the tobaccospecific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK), activate the Akt pathway which increases
cellular proliferation and confers protection against different
cellular stresses (12). Moreover, tobacco carcinogen-induced
transformation of bronchial epithelial cells increases activation
of the Akt pathway in vitro and in vivo (13). These studies
suggest that Akt activation is an early event that is related to
exposure to tobacco components, but no study has explored the
effects of this mechanism of Akt activation in established lung
cancer cells. This is highly relevant because smokers diagnosed
with tobacco-related cancers are often not encouraged to quit
smoking because of a perception that it is ‘too late’.
Here, we report the rapid and potent activation of Akt by
tobacco components in lung cancer cells, and describe changes
in cellular proliferation and survival that occur after administration of these components. These studies identify signaling
mechanisms and cellular processes that could contribute to the
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Tobacco components, Akt, NFkB and lung cancer cells
poor outcomes associated with patients who smoke during
cancer therapy.
Materials and methods
Materials
Etoposide and paclitaxel were purchased from Calbiochem (La Jolla, CA). All
phospho-specific antibodies and poly (ADP ribose) polymerase (PARP)
antibodies were from Cell Signaling Technology (Beverly, MA). a7 nicotinic
acetylcholine receptors (nAchR) and a-tubulin antibodies were from SigmaAldrich (St Louis, MO). a4 nAchR, b2 nAchR, cyclin D1 antibodies and
hemagglutin (HA)-probed F7 were from Santa Cruz Biotechnology (Santa
Cruz, CA). Protease inhibitor cocktail was obtained from Sigma Chemical,
and protein assay materials were from Bio-Rad (Hercules, CA). All cell culture
reagents were purchased from Life Technologies (Rockville, MD). Protran
Pure nitrocellulose membranes were purchased from Schleicher & Schuel
(Dassel, Germany). Nicotine was from Sigma-Aldrich. NNK was from
ChemSyn Laboratories (Lenexa, KS). LY294002 and a-anatoxin (ATX) were
purchased from Sigma-Aldrich. 3-(2,4)-Dimethoxybenzylidine anabaseine
(DMXB) was a generous gift from Dr William Kem, University of Florida.
Cell culture
All lung cancer cell lines were established at the National Cancer Institute/
Naval Medical Oncology (Bethesda, MD) or were purchased from
ATCC (Manassas, VA). H157V and H157I cells were a generous gift from
Dr David Jones, University of Virginia. H157 and H1703 cells were maintained in 75 cm2 flasks in DMEM supplemented with 10% fetal bovine serum
(FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. H69, H157V
and H157I cells were maintained in RPMI 1640 supplemented with 10%
(FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were incubated
at 37 C in a 7.0% CO2 atmosphere incubator. The stock cultures were split on a
weekly basis at 1:5 or 1:10 ratio.
Pharmacological treatments
For time-dependent induction of Akt phosphorylation or assessing phosphorylation of downstream substrates, cells were serum deprived in 0.1%
FBS containing media for 24 h prior to addition of 10 mM nicotine or 100 nM
NNK. Dose--response curves were generated at 60 min. a-ATX (20 mM) or
DMXB (20 mM) was added to H157 cells after serum deprivation. For examination of cell proliferation, serum-deprived H157 or H1703 cells were treated
daily with nicotine or NNK. To assess apoptosis, H157 and H1703 cells were
treated with paclitaxel (100 nM) or etoposide (100 mM) for 48 h in 0.1% FBS
in the absence or presence of nicotine (10 mM) or NNK (100 nM). For
apoptosis of H157V or H157I cells, cells were plated in media containing
10% FBS. After attachment, cells were washed twice with PBS and incubated
in 0.1% FBS-containing medium for 48 h in the absence or presence of nicotine
or NNK.
Adenoviral infection
Adenoviruses containing b-galactosidase (b-gal) or dominant negative Akt
(DnAkt) were generous gifts from Dr Kenneth Walsh and have been described
previously (14). Cells were plated at a density of 3 104 cells per well in
12-well plates. After attachment, cells were washed with PBS, and infected
with media containing adenoviral constructs for b-gal or DnAkt [multiplicity
of Infection (MOI 50)] for 24 h. Infected cells were then treated with or without
nicotine or NNK and cells were harvested and analyzed for cell proliferation as
described below. Parallel samples were harvested for immunoblotting and
quantification of cell number in each experiment.
Immunoblotting
After the various pharmacologic treatments described above, cell extracts were
prepared as described previously (11). Protein yield was quantified using the
Bio-Rad DC Protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equivalent protein was loaded, and the lysates were separated by SDS--PAGE and
transferred to nitrocellulose membranes. Equivalent loading was confirmed by
staining of membranes with fast green. Membranes were blocked for 1 h in
blocking buffer [1 Tris buffered saline (TBS), 5% milk and 0.2% Tween-20]
and placed in primary antibody (1 TBS, 5% milk, 0.1% Tween-20 and
1:1000 antibody) overnight at 4 C. Membranes were washed three times in
wash buffer (0.1% NP40, 0.1% Tween-20 and 1 TBS). Primary antibody was
detected using horseradish peroxidase-linked goat anti-mouse or goat antirabbit IgG antibodies and visualized with the enhanced chemiluminescent
detection system ECL (Amersham Pharmacia Biotech, Amersham). All
phospho-specific antibodies were used at 1:1000 dilutions. Immunoblot
experiments were performed at least three times.
Cell proliferation assays
Cells were seeded in triplicate in 12-well plates at a density of 3 104 cells per
well and incubated overnight. Cells were then washed twice with PBS and
media were changed to serum-free medium. Where indicated, nicotine or NNK
was added daily. Cells were harvested at 72 h by trypsinization and either
counted with a Zeiss Coulter Counter (Beckman Coulter, Miami, FL), or fixed
in ice-cold 70% methanol and stained with crystal violet and the absorbance at
540 nm in each sample was measured using El 800 Universal Microplate
Reader (BIO-TEK Instruments, VT). Proliferation experiments were performed three times.
Apoptosis assays
Floating cells and adherent cells were harvested by trypsinization and then
centrifuged at 1000 g for 5 min. Cells were fixed in ice-cold 70% methanol
added dropwise and then incubated at 20 C for 30 min. Cells were centrifuged and incubated with propidium iodide (25 mg/ml) supplemented with
RNaseA for 30 min at room temperature. Quantification of sub-2N DNA was
determined by flow cytometry analysis using a Becton Dickinson FACSort and
by manual gating using CellQuest software. Gating was performed on blinded
samples. All apoptotic experiments were repeated three times.
Reverse transcription--PCR
Total RNA extraction and PCR reaction mixtures were as described previously
(11). Subunit specific primers for nAchR were synthesized by Sigma-Genosys,
TX, with the following sequences. a1: 50 -CGTCTGGTGGCAAAGCT-30
(sense), 50 -CCGCTCTCCATGAAGTT-30 (antisense); a2: 50 -CCGGTGGCTTCTGATGA-30 (sense), 50 -CAGATCATTCCAGCTAGG-30 (antisense) (15);
a3: 50 -CCATGTCTCAGCTGGTG-30 (sense), 50 -GTCCTTGAGGTTCATGGA-30 (antisense) (16); a4: 50 -CTCTCGAACACCCACTC-30 (sense), 50 AGCAGGCTCCCGGTCCCT-30 (antisense) (17); a5: 50 -TCATGTAGACAGGTACTTC-30 (sense), 50 -ATTTGCCCATTTATAAATAA-30 (antisense)
(18); a6: 50 -GGCCTCTGGACAAGACAA-30 (sense), 50 -AAGATTTTCCTGTGTTCCC-30 (antisense) (15); a7: 50 -CACAGTGGCCCTGCAGACCGATGGTACGGA-30 (sense), 50 -CTCAGTGGCCCTGCTGACCGATGGTACGGA-30 (antisense) (19); a9: 50 -GTCCAGGGTCTTGTTTGT-30 (sense),
50 -ATCCGCTCTTGCTATGAT-30 (antisense) (16); a10: 50 -CTGTTCCGTGACCTCTTT-30 (sense), 50 -GGAAGGCTGCTACATCCA-30 (antisense)
(16); b2: 50 -CAGCTCATCAGTGTGCA-30 (sense), 50 -GTGCGGTCGTAGGTCCA-30 (antisense) (15); b3: 50 -AGAGGCTCTTTCTGCAGA-30 (sense),
50 -GCCACATCTTCAAAGCAG-30 (antisense) (15); b4: 50 -CTGAAACAGGAATGGACT-30 (sense), 50 -CCATGTCTATCTCCGTGT-30 (antisense) (15);
and b-actin primers were 50 -GTGGGGCGCCCCAGGCACCA-30 (sense) and
50 -CTCCTTAAGTCACGCACGATTTC-30 (antisense). a9 and a10 cDNA
controls were generously provided by Dr L.Lustig, Johns Hopkins University.
nAchR primers yielded predicted products of 505 (a1), 466 (a2), 401 (a3), 371
(a4), 265 (a5), 413 (a6), 598 (a7), 403 (a9), 388 (a10), 347 (b2), 354 (b3)
and 310 (b4) bp.
Luciferase assays
H157V and H157I cells were plated at 3 104 cells per well in 24-well plates.
Cells were transiently transfected the next day with 1 mg DNA PathDetect
NFkB cis-reporting system vector (Stratagene, La Jolla, CA) using Superfect
transfection reagent (Qiagen, Valencia, CA), according to manufacturer’s
instructions. After incubation for 24 h, media were changed to fresh RPMI
containing 10% FBS or 0.1% FBS with or without nicotine (100 nM). Cells
were lysed 24 h later with 1 Reporter Lysis Buffer (Promega, Madison, WI)
and stored frozen at 20 C overnight. Luciferase activity was determined by
testing 20 ml of cell lysate and 100 ml of Luciferase Assay Reagent (Promega)
using a moonlight 2010 tube luminometer (Analytical Luminescence Laboratory, San Diego, CA). Relative luciferase units were normalized to total protein
levels from the cell lysates. Luciferase assays were performed three times.
Statistical analysis
Statistical comparison of mean values was performed using the Student t-test.
All P values are two tailed.
Results
Nicotine or NNK increases activation of Akt in a dosedependent and time-dependent manner
To investigate whether nicotine or NNK could stimulate the
Akt pathway in fully transformed lung cancer cells, we used
two NSCLC cell lines (H157 and H1703) and one small cell
lung cancer (SCLC) cell line (H69) to assess phosphorylation
of Akt at S473, which is indicative of Akt activation. Nicotine
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J.Tsurutani et al.
A
B
Fig. 1. Time- and dose-dependent induction of Akt activation by nicotine or NNK. (A) Nicotine. Lung cancer cell lines were plated on 6-well plates in DMEM
with 10% FBS overnight. Media were then changed to DMEM with 0.1% FBS, with or without addition of nicotine (10 mM) for indicated times. Cells were
harvested and processed for immunoblotting using phospho-specific antibodies against S473 and antibodies against total Akt. Scanned images of immunoblots
were analyzed using NIH Image 1.62. Each pAkt signal was normalized to native Akt, and the bars indicate the fold induction of the pAkt signals compared with
the control. (B) NNK. Cell extracts were prepared as in (A), except that NNK (100 nM) was administered for the indicated times. (C) Dose dependence. Cells were
plated, treated, and processed as in (A), except that cells were harvested at 45 min.
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Tobacco components, Akt, NFkB and lung cancer cells
Fig. 1. Continued.
or NNK rapidly increased Akt activation in all three cell lines
(Figure 1A and B). Nicotine increased Akt activation 2- to
3-fold within 15--30 min (Figure 1A). Similar induction of Akt
activation was observed with NNK, with 2- to 3-fold induction
observed by 30 min (Figure 1B). Nicotine or NNK did not
affect total levels of Akt expression. In experiments designed
to test dose-dependent responses to nicotine, nicotine
increased Akt phosphorylation in H157 cells with doses as
low as 10 nM, but maximum Akt phosphorylation in H157
or H1703 cells was observed at 1--10 mM (Figure 1C). These
concentrations might be achievable in smokers, because average steady-state serum concentrations of nicotine have been
reported at ~200 nM, and acute increases to 10--100 mM in
serum or to 1 mM at the mucosal surface immediately after
smoking have been reported (20--22). NNK was more potent
than nicotine, because increased phosphorylation of Akt was
observed with doses as low as 1 nM in both cell lines. These
studies demonstrate that nicotine, the addictive component of
tobacco, and NNK, an important tobacco-specific carcinogen,
activate Akt within minutes at doses that could be physiologically relevant.
Nicotine or NNK increases phosphorylation of downstream
substrates of Akt
To demonstrate that activation of Akt by nicotine or NNK
propagated signaling cascades within lung cancer cells, we
initially assessed phosphorylation of downstream substrates
using an antibody raised against the consensus sequence identified in all known Akt substrates (RXRXXS/T). Nicotine or
NNK induced a time-dependent increase in phosphorylation
of numerous downstream substrates in H157 (Figure 2A) and
H1703 cells (Figure 2B) that reflected the rapid onset of
increased Akt phosphorylation. The pattern of induction for
H157 cells was different from H1703 cells, possibly reflecting
differences between these cell lines in the status of molecules
that control the Akt pathway such as PTEN or K-Ras (11). To
identify which known Akt substrates were altered by nicotine
or NNK treatment and to rule out contribution of kinases such
as SGK that have similar consensus sequences to that of Akt,
H157 cell extracts were probed with phospho-specific antibodies against glycogen synthase kinase (GSK-3), mouse double
minute 2 homologue (MDM2), ASK1, FKHR, tuberin, and
mammalian target of rapamycin (mTOR), which are direct
Akt substrates, and eukaryotic initiating factor 4 binding protein 1 (4EBP-1) and S6 kinase 1 (S6K1), which are not direct
Akt substrates but are phosphorylated in response to Akt
activation (Figure 2C). Nicotine increased phosphorylation of
all proteins tested within minutes (Figure 2C, left panels),
although the time course varied in a substrate-specific manner.
The greatest induction of phosphorylation was observed in
substrates that control transcription and protein translation.
NNK induced similar effects on phosphorylation of these proteins (Figure 2C, right panels), except that NNK did not induce
MDM2 or mTOR phosphorylation. Variability in phosphorylation of Akt substrates could reflect differential sensitivity to
regulatory mechanisms such as cellular phosphatases or the
contribution of other kinases. Collectively, these data show
that nicotine- or NNK-mediated stimulation of Akt results in
broad propagation of the pathway, but that nicotine or NNK
propagates the Akt signal differently.
Identification of nAchR in lung cancer cells
Nicotine or NNK exerts its biological effects through binding
to nAchR. nAchR were originally described and are predominantly expressed in neural tissue, but nAchR have recently
been reported to be expressed in other cell types, including
small cell lung cancer (SCLC) cells (23--25). Functional
nAchR are composed of homopentamers of a7--a10 subunits
or heteropentamers derived from 5 a (a2--a6) and 3 b (b2--b4)
subunits. a3- or a4-containing nAchR are most abundant in
neural tissue (26), and a7-containing nAchR have been
described in normal human bronchial epithelial and
endothelial cells (16). To evaluate expression of individual
nAchR subunits in NSCLC cell lines and to compare
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J.Tsurutani et al.
expression with normal human airway epithelial cells, we
performed nAchR subunit-specific RT--PCR analysis of
a1--a10 and b2--b4 subunits (Table I). In the five NSCLC
cell lines, a4, a5, a7 and b2 subunits were ubiquitously
expressed. a1, a2, a10 and b3 subunits were not expressed
in any NSCLC cell line tested, and a3, a6, a9 and b4 subunits
were expressed in an NSCLC cell line specific manner. Differences in nAchR subunit expression were also observed
Fig. 2. Time-dependent induction of Akt substrate phosphorylation by nicotine or NNK. (A) H157 cells. Cells were plated on 6-well plates and incubated
in DMEM with 10% FBS overnight. Media were then changed to 0.1% FBS and cells treated with nicotine or NNK for the indicated time periods.
Immunoblotting was performed with phospho-specific antibodies that recognize phosphorylated Akt substrates (RXRXXS/ T) (panels A and B). Equivalent
loading is shown by immunoblotting for a-tubulin. Extracts of NIH3T3 cells were included as controls. Asterisks indicate bands whose intensity increased or
decreased after exposure to tobacco components. (B) H1703 cells. Same as (A) except that H1703 cells were used. (C) Effects of nicotine or NNK on
phosphorylation of individual Akt substrates. H157 cells were treated similarly with nicotine or NNK for immunoblotting experiments performed with
phospho-specific antibodies directed against individual downstream substrates at the indicated sites or with other antibodies. Immunoblots from
nicotine- (left panels) or NNK-treated (right panels) cells are shown for a representative experiment done three times. Direct Akt substrates are shown
by labels shaded in gray. Equivalent loading was confirmed by staining membranes with fast green.
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Tobacco components, Akt, NFkB and lung cancer cells
Fig. 2. Continued.
Table I. RT--PCR expression of nAchR subunits
Total RNA was isolated from primary human airway epithelial cells (NHBE,
normal human bronchial epithelial cells; SAEC, small airway epithelial cells)
or NSCLC cell lines as described, and RT--PCR was performed to analyze
individual nAchR subunits.
between normal airway epithelial cells and NSCLC cells,
such as loss of a10 subunits in all five NSCLC cell lines,
but the significance of these differences is unknown. To confirm the results of the RT--PCR analysis, we performed
immunoblotting with antibodies against a4, a7 and b2 nAchR
subunits (Figure 3A). Each of these subunits was expressed at
a protein level in H157, H1703 and A549 cells. These studies
suggest that functional a4b2 and a7 nAchR could be
expressed in NSCLC cells.
To identify nAchR that might be capable of activating Akt in
NSCLC cells, we tested if subunit-specific nicotinic agonists
could stimulate Akt activation (Figure 3B). a-ATX, an a3/a4
agonist (27), increased Akt activation in H157 and H1703 cells
within minutes. The onset of a-ATX-induced Akt activation
was slower in H157 cells than in H1703 cells, where
Akt activation increased ~3-fold within 5 min (densitometry
not shown). DMXB, an a7 agonist (28), also increased Akt
activation within minutes, and this response was prolonged
in the H157 cells. Together, these data show that a3/a4 or
a7 nAchR agonists can activate Akt in NSCLC cells.
Nicotine and NNK are Akt-dependent mitogens for NSCLC
cells
Activation of the PI3K/Akt/mTOR pathway promotes cellular
proliferation in multiple cell types. To determine if nicotinic
activation of Akt induced NSCLC cell proliferation, different
doses of nicotine were added daily to H157 cells in 0.1% FBS
and cell number was determined after 5 days (Figure 4A).
At doses as low as 100 nM, nicotine increased proliferation 2- to 4-fold. With higher doses, increased proliferation
was observed compared with sham-treated cells, but dosedependent increases in proliferation were not consistently
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Fig. 3. Role of nAchR in Akt activation. (A) Immunoblotting of a4, a7 and b2 nAchR subunits. Lysates of H157, H1703 and A549 cells were examined for
expression of a4, a7 and b2 proteins. Brain lysates were used as positive controls. Molecular sizes of a4, a7 and b2 are shown (70 kD, 56 kD and 60 kD,
respectively). (B) Nicotinic agonists. H157 or H1703 cells were serum-deprived in media containing 0.1% FBS and treated with a-ATX or DMXB (20 mM) for the
indicated times. Cells were harvested for immunoblotting with antibodies against total or activated Akt.
observed, perhaps due to the cumulative toxicity of daily
dosing of 1 or 10 mM nicotine. Increased proliferation of
H157 cells after nicotine treatment (10 mM) was associated
with a 3.5-fold increase in Akt activation and increased cyclin
D1 levels (Figure 4A, right panel).
To extend these studies and minimize the contribution of
cumulative toxicity, the ability of lower doses of daily nicotine
or NNK to induce proliferation was compared in H157 or
H1703 cells after 3 days of exposure (Figure 4B). Nicotine or
NNK (100 nM) increased proliferation ~2-fold in both cell
lines. Immunoblotting analysis revealed that after daily administration of nicotine or NNK, Akt activation increased 41.5fold in both cell lines. The reasons for the less robust increase
in Akt activation on day 3 is unclear, but might be related to
the higher dose (10 mM) or longer time course (5 days) used in
Figure 4A. Increased NSCLC cell proliferation was associated
with increased cyclin D1 expression, but was not accompanied
by decreased levels of the cdk inhibitor, p27 (data not shown).
(Expression of the cdk inhibitor p21 was not detectable in
these cells.) These studies show that nicotine or NNK are
mitogenic for NSCLC cells, which was associated with
increased expression of cyclin D1.
To determine the relative contribution of Akt activation to
tobacco component-mediated NSCLC cell proliferation, we
used adenoviruses expressing mutant Akt (Figure 4C).
H157 cells were infected with adenoviruses expressing b-gal
or DnAkt, and nicotine- or NNK-induced NSCLC cell
proliferation was assessed on day 3. When nicotine or NNK
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was added to cells infected with adenoviruses expressing
b-gal, proliferation was increased ~2-fold. In contrast, when
nicotine or NNK was added to cells expressing DnAkt, no
increase in proliferation was observed. The inhibition of
nicotine- or NNK-induced proliferation by mutant Akt was
statistically significant (P 5 0.05). In cells that were infected
with DnAkt, expression of the HA epitope tag did not change
with nicotine or NNK administration (top right panel). Inhibition of nicotine-induced proliferation correlated with an
inhibition of cyclin D1 induction by nicotine or NNK, and
was not due to an increase in apoptosis because nicotine or
NNK did not induce PARP cleavage (Figure 4C, right panel).
Thus, tobacco component-induced proliferation of NSCLC
cells is dependent upon Akt activation.
Nicotine increases NSCLC cellular survival and activation of
NFkB
To determine if nicotine or NNK could affect responsiveness
to chemotherapy, NSCLC cells were treated with chemotherapeutic agents commonly used in the treatment of NSCLC, in
the absence or the presence of nicotine or NNK (Figure 5A).
Nicotine decreased paclitaxel-induced apoptosis in H157 cells
by 40% (upper left panel), but NNK did not inhibit paclitaxelinduced apoptosis (upper right panel), even when higher doses
were used (data not shown). Similar effects were observed in
the H1703 cells, where nicotine decreased etoposide-induced
apoptosis by 75% (lower left panel). NNK was again ineffective (lower right panel). To determine the contribution of the
Tobacco components, Akt, NFkB and lung cancer cells
Fig. 4. Effects of nicotine or NNK on NSCLC cell proliferation. (A) H157 cells were plated on 6-well plates in triplicate. Nicotine was added daily to the
existing DMEM with 0.1% FBS. On day 5, the cells were harvested and counted with Beckman Coulter Counter or immunoblotting was performed for
phospho-Akt, total Akt and cyclin D1 (right panels). Tubulin was used as a loading control. Asterisks indicate statistically significant differences between
nicotine treatment and no treatment (P 5 0.05). The fold increase in pAkt: total Akt is shown in the right lower panel. (B) H157 or H1703 cells were treated as in
(A) except that lower doses of nicotine and NNK were used (100 nM), and the experiment was conducted for 3 days. Immunoblotting for phospho-Akt, total Akt
and cyclin D1 is shown. Asterisks indicate statistically significant differences between treatment with tobacco components and no treatment (P 5 0.05). The fold
increases in pAkt: total Akt are shown in graphs below the corresponding immunoblots. (C) H157 cells were infected with adenoviruses expressing b-gal or
DnAkt. After recovery for 24 h, cells were serum-deprived and nicotine or NNK was added as indicated for 3 days. Cells were harvested for assessment of
proliferation using crystal violet staining and for immunoblotting for HA, cyclin D1 and PARP. Tubulin is shown as a loading control. Asterisks indicate
statistically significant differences in proliferation between cells infected with b-gal or DnAkt and treated with nicotine or NNK (P 5 0.05).
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Fig. 4. Continued.
PI3K/Akt pathway to nicotine-mediated protection of H157
cells, we used the PI3K inhibitor, LY294002, in the absence or
presence of nicotine and/or paclitaxel. As shown in Figure 5B,
combining LY294002 with paclitaxel increased apoptosis
above that observed with paclitaxel alone, consistent with
prior studies from our laboratory (11). Although nicotine
protected the cells from paclitaxel alone, the protective effect
of nicotine was lost in the presence of LY294002, suggesting
that the anti-apoptotic effects of nicotine are dependent upon
the activation of the PI3K/Akt pathway.
Chemotherapeutic resistance can be associated with
increased NFkB activity (29). To determine if nicotineinduced cellular survival was dependent upon NFkB activity,
we utilized H157 cells that stably overexpress IkBa, which
inhibits NFkB (H157I cells), or H157 cells that stably overexpress vector alone (H157V cells) (30). H157I cells have
decreased NFkB activity and increased sensitivity to chemotherapy. Relative expression of IkBa in H157V or H157I cells
is shown in Figure 6A (insets, upper right). When these cells
were grown in the presence of 10% FBS and apoptosis was
measured after 2 days, there was no change in basal levels of
apoptosis in H157V or H157I cells, in the absence or presence
of nicotine (left columns, Figure 6A). When these stably transfected cells were serum deprived, however, levels of apoptosis
in H157I cells increased ~2.5-fold above that observed in
H157V cells. Nicotine inhibited serum deprivation-induced
apoptosis by 48% in H157V cells. The protection conferred
by nicotine in H157V cells was not observed in H157I cells. To
address whether the high levels of apoptosis in H157I cells
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caused by serum starvation precluded nicotine from protecting
these cells from apoptosis, we performed a time course experiment with serum starved H157V and I cells with or without
nicotine over 24, 48 and 72 h (data not shown). Basal levels of
apoptosis increased progressively with time, but regardless of
levels of apoptosis over 50% in H157V cells, nicotine was still
able to protect these cells, indicating that the high levels of
serum starvation-induced apoptosis do not preclude protection
by nicotine. Interestingly, Akt activation was induced by ~1.5fold in both H157I and H157V cells, indicating that pathways
leading to Akt activation are intact in both cell types, suggesting that the ability of nicotine to promote cell survival is
dependent on differences in signal propagation downstream
of Akt (Figure 6A, insets, lower right). These experiments
were repeated using NNK. Similar to the results obtained
with parental H157 cells treated with chemotherapy, NNK
did not protect H157V or H157I cells from serum
deprivation-induced apoptosis (data not shown). These experiments showed that nicotine promoted survival of serum
deprived NSCLC cells in an NFkB-dependent manner.
To show that nicotine directly stimulates NFkB activity, we
transiently transfected H157V or H157I cells with an NFkB
reporter and measured luciferase activity without or with
exposure to nicotine (Figure 6B). When cells were grown in
media with 10% FBS without nicotine, H157I cells had 65%
less basal luciferase activity than H157V cells. In H157V cells
grown in 10% FBS or 0.1% FBS, nicotine increased luciferase
activity 2- to 3-fold. H157I cells did not increase luciferase
activity in response to nicotine in either growth condition.
Tobacco components, Akt, NFkB and lung cancer cells
Fig. 5. Nicotine but not NNK is an NFkB-dependent survival factor for NSCLC cells. (A) Nicotine but not NNK decreases chemotherapy-induced apoptosis.
H157 cells (upper panels) or H1703 cells (lower panels) were serum deprived in DMEM with 0.1% FBS and exposed to chemotherapeutic agents with or without
nicotine or NNK for 48 h. Apoptosis was assessed morphologically and was measured using flow cytometry as described. Asterisks indicate statistically
significant differences in apoptosis induced by chemotherapy in the presence of nicotine and chemotherapy alone (P 5 0.05). (B) The PI3K inhibitor LY294002
inhibits nicotine-mediated survival. H157 cells were serum deprived in DMEM with 0.1% FBS overnight, and exposed to 10 mM taxol with or without 10 mM
nicotine or 10 mM LY294002 for 24 h. Apoptosis was measured using flow cytometry as described. Asterisk indicates statistically significant differences in
survival between paclitaxel-treated cells exposed to nicotine or not (P 5 0.05).
Thus, nicotine stimulates NFkB activity in NSCLC cells. Collectively, these studies show that nicotine is a survival factor
for NSCLC cells under conditions of exposure to chemotherapy or serum deprivation, and that nicotine-mediated survival
is dependent upon induction of NFkB activity.
Discussion
The activation of Akt and the promotion of lung cancer cell
proliferation and survival by tobacco components recapitulates
biologic processes that may underlie the poor clinical
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J.Tsurutani et al.
Fig. 6. Role of NFkB in nicotine-mediated survival of NSCLC cells. (A) H157 cells overexpressing IkB are resistant to nicotine protection. H157V or H157I cells
were grown in media containing 10% FBS or 0.1% FBS, in the absence or presence of daily nicotine (10 mM) for 48 h, and apoptosis was assessed. Expression of
IkBa, phosphorylated Akt and total Akt are shown. Tubulin and total levels of Akt are used as loading controls. Asterisks indicate statistically significant
differences in apoptosis between addition of nicotine or no treatment (P 5 0.05). (B) Nicotine activates NFkB activity in H157V but not H157I cells. H157V or
H157I cells were transiently transfected with a luciferase reporter containing an NFkB promoter. Cells were grown in 10% FBS or 0.1% FBS for 48 h in the
absence or presence of nicotine (10 mM) and luciferase activity was measured. Asterisks indicate statistically significant differences in apoptosis between addition
of nicotine or no treatment (P 5 0.05).
outcomes for patients with tobacco-related cancers who
continue to smoke. Moreover, these studies advance prior
studies of nicotine and NNK by identifying Akt and NFkB
as the key cellular targets of these tobacco components in
established lung cancer cells. Activation of Akt by tobacco
1192
components has been observed in many epithelial cell systems
relevant to lung cancer. The dose- and time-dependent
activation of Akt by nicotine or NNK in lung cancer cells is
similar to that we observed in primary cultures of human
bronchial or small airway epithelial cells (12). Likewise,
Tobacco components, Akt, NFkB and lung cancer cells
activation of Akt by nicotine or NNK has also been observed
with human lung epithelial cells at intermediate steps in
the transformation process (data not shown). Taken together,
human lung epithelial cells throughout the phenotypic
spectrum activate Akt in response to tobacco components
in vitro. Inhibiting the induction of Akt activity by tobacco
components might therefore be a valuable approach to mitigate
the effects of smoking in smokers at risk for the development
of lung cancer, and/or in lung cancer patients who continue to
smoke.
Such approaches would need to consider the consequences
of inhibiting nicotinic induction of Akt in other cell types,
especially in neuronal tissues. In neuronal tissues, activation
of Akt by tobacco components might ironically confer health
benefits, which could hypothetically complicate interventions
suggested above. Many epidemiologic studies have shown
that smokers are at decreased risk for the development of
neurodegenerative diseases such as Alzheimer’s disease and
Parkinson’s disease [reviewed in (31)]. In vitro model systems
of Alzheimer’s disease have shown that nicotine-mediated
protection of neuronal cells against b-amyloid-induced cytotoxicity is dependent upon Akt activation (32--34). Thus,
would a therapeutic strategy against Akt tailored for lung
cancer increase neuronal degeneration? Animal models may
be useful to address this question.
Activation of Akt by nicotine or NNK clearly propagated the
Akt pathway in NSCLC cells because increased phosphorylation of multiple identified and unidentified downstream substrates of Akt was observed in a time-dependent manner.
Which substrate(s) might be most important for determining
cellular responses to tobacco components? The patterns of
phosphorylation of downstream substrates were mostly similar
for nicotine or NNK, but two potentially important differences
were that nicotine, but not NNK, increased phosphorylation of
MDM2 and mTOR. Phosphorylation of MDM2 by Akt can
promote nuclear translocation of MDM2 and degradation of
p53, which can clearly affect apoptosis (35,36). mTOR can
also promote cellular proliferation and cellular survival, especially in cells with high levels of Akt activity [reviewed in
(37)]. Thus, propagation of the Akt signal to MDM2 or mTOR
might be responsible for nicotine-induced survival. The availability of inhibitors of mTOR such as rapamycin will facilitate
studies to determine the relative roles of these Akt substrates
in nicotine-mediated survival.
The identification of nAchR subunits in NSCLC cell lines
provides a mechanistic basis for Akt activation in response to
tobacco components, and fills a gap in the analysis of
nAchR subunit expression in cells derived from lung tissues.
Once thought to be restricted to neuronal cells, nAchR
subunit expression has now been demonstrated in
human lung epithelial cells (12,16), SCLC cells (38), and
NSCLC cells. In addition, nAchR have been described in
keratinocytes (24) and endothelial cells. This wide distribution
of nAchR may underlie the systemic physiological responses
to smoking.
Although other studies have shown that nicotine or other
agonists of nAchR such as acetylcholine can promote the
growth of SCLC cells (25,39), our study is the first to demonstrate that nicotine or NNK stimulates NSCLC proliferation
that is dependent upon Akt. The fact that expression of mutant
Akt inhibited nicotine- or NNK-induced proliferation of H157
cells, suggests that other signaling pathways that might be
increased by nicotine stimulation such as the MEK / ERK
pathway (40) would not contribute greatly to proliferation in
these cells unless they were directly upstream or downstream
of Akt.
In addition to nAchR, NNK might also exert its biologic
activity through activation of other cell surface receptors.
NNK can indirectly stimulate DNA synthesis in NSCLC
cells through stimulation of b-adrenergic receptors and the
release of arachidonic acid (41), but the role of Akt was not
assessed by these investigators. It may be noted that
b-adrenergic antagonists did not block NNK-induced Akt
activation in H157 or H1703 cells (data not shown). Thus,
NNK-induced Akt activation and proliferation of H157 cells
is probably due to stimulation of nAchR rather than badrenergic receptors. Regardless of the relative roles of different receptors at the cell surface, nicotine- or NNK-induced
signaling converged on Akt, and proliferation was dependent
upon Akt activation.
Although NNK served as a mitogen for H157 and H1703
cells, only nicotine served as a mitogen and an NFkBdependent survival factor. In addition to the neuronal cell
systems discussed above, nicotine can promote survival of
many cell types including head and neck cancer cells (42,43).
Nicotine can inhibit apoptosis induced by diverse stimuli such
as administration of opioids, tumor necrosis factor, UV light or
g-radiation (42,44). In our studies, nicotine protected NSCLC
cells against chemotherapy-induced apoptosis and serum
deprivation-induced apoptosis. One mechanism by which
nicotine protected NSCLC cells was through NFkB, because
NSCLC cells expressing an inhibitor of NFkB, IkBa, were
resistant to nicotine-mediated protection, and NFkB activity
was directly stimulated by nicotine. These data are consistent
with prior observations that cigarette smoke condensate (CSC)
can increase NFkB activity (45). Given that cigarette smoke
condensate is a complex mixture of ~4000 chemicals (46), we
have identified a single component in this mixture, nicotine, as
an NFkB-dependent survival factor. This may be relevant
because increased expression of p50, a subunit of the NFkB
complex, is observed in NSCLC tumors compared with normal
lung tissues (47). Other mechanisms that could also contribute
to nicotine-mediated survival include increased bcl-2 phosphorylation (48), but we were unable to detect phosphorylated
bcl-2 in H157 cells (data not shown). The fact that NFkB
mediates nicotine-induced NSCLC cellular survival suggests
that new drugs that target the proteosome and NFkB such as
bortezomib might mitigate possible detrimental effects of
nicotine on chemotherapeutic responsiveness (49). Future
studies will test this hypothesis.
Finally, these studies highlight the complex biology of
nicotine and tobacco. Nicotine, originally thought only to be
responsible for tobacco addiction, is now recognized for
modulation of key cellular proteins, such as Akt, cyclin D1,
NFkB and bcl-2, and key cellular processes, such as increased
proliferation and survival, two components of the transformed
phenotype (50). If the conditions used in these studies were to
be replicated in vivo, it is hypothetically possible that increased
growth of nascent, undiagnosed cancers and/or increased resistance to cytotoxic therapy could occur with exposure to
nicotine. Because of the widespread availability and use of
nicotine supplements in current or former smokers, further
investigation on the sustained biological activities of nicotine
is warranted.
Conflict of Interest Statement: None declared.
1193
J.Tsurutani et al.
Supplementary material
Supplementary material can be found at: http://www.
carcin.oupjournals.org/.
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Received July 9, 2004; revised March 10, 2005; accepted March 12, 2005
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