chen rj nicotine exposure-induced chemoresistance is mediated by activated stat3 toxicological...
TRANSCRIPT
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
1/13
TOXICOLOGICAL SCIENCES 115(1), 118130 (2010)
doi:10.1093/toxsci/kfq028
Advance Access publication January 27, 2010
Long-term Nicotine ExposureInduced Chemoresistance Is Mediated byActivation of Stat3 and Downregulation of ERK1/2 via nAChR and
Beta-Adrenoceptors in Human Bladder Cancer Cells
Rong-Jane Chen,* Yuan-Soon Ho, How-Ran Guo,* and Ying-Jan Wang*,1
*Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan 70428; andSchool of Medical
Technology and Biotechnology, Taipei Medical University, Taipei 110, Taiwan
1To whom correspondence should be addressed at Department of Environmental and Occupational Health, National Cheng Kung University Medical College,
138 Sheng-Li Road, Tainan 70428, Taiwan. Fax: (886) 6-275-2484. E-mail: [email protected].
Received January 18, 2010; accepted January 22, 2010
Previous reports suggested that bladder cancer patients whocontinue to smoke while receiving chemotherapy have poorer
outcomes than their nonsmoking counterparts. Nicotine, the major
addictive compound in cigarette smoke, is known to induce che-
moresistance in some cancer cells. Chemoresistance has been linked
to the activation of Stat3 (signal transducer and activator of
transcription). The objective of this study was to identify the role of
Stat3 in chemoresistance induced by nicotine in human bladder
cancer cell line, T24 cells. Chemoresistant T24 cells were established
by persistent nicotine treatment. Apoptosis and cell cycle parameters
were analyzed by Annexin V staining, poly(ADP-ribose) polymerase
degradation, caspase activity, and propidium iodide staining. Signal
transduction mediating the chemoresistance was detected by
Western blotting and small interfering RNA (siRNA) transfection.
We provide evidence for the first time that nicotine strongly activatedStat3, leading to Cyclin D1 overexpression, cell cycle perturbations,
and chemoresistance. Furthermore, nicotine mobilized Stat3 signal-
ing, resulting in the loss of extracellular signal-regulated protein
kinase 1/2 (ERK 1/2) activation and reduced chemosensitivity via
nicotinic acetylcholine receptors and b-adrenoceptors. Inhibition of
Stat3 by siRNA or a specific inhibitor restored chemosensitivity in
T24 cells. Stat3 could be the major target for increasing chemo-
sensitivity in patients who develop chemoresistance during chemo-
therapy, and avoidance of cigarette smoking or nicotine-based
treatments may increase the efficacy of chemotherapy.
Key Words: nicotine; chemoresistance; Stat3; ERK1/2; nAChR;
b-AR.
Smoking is considered to be one of the most important risk
factor for urinary bladder cancer (UBC), the fifth most common
human neoplasm (Zeegers et al., 2000). Most of the deaths
from bladder cancer are due to advanced unresectable disease,
which is resistant to chemotherapy (Dreicer, 2001). A number
of studies have shown that bladder cancer patients who smoke
while receiving treatment for their malignancies have poorer
outcomes compared with their nonsmoking counterparts. For
instance, bladder cancer patients with superficial transitional
cell carcinoma who continue to smoke tend to have fasterrecurrences than those who quit smoking (median time to
recurrence of 8.9 vs. 13 months, respectively) (Fleshner et al.,
1999). In Chens study, the 3-year recurrence-free survival
(95% confidence interval) of smokers, nonsmokers, ex-
smokers, and quitters were 45% (3256%), 57% (4370%),
62% (4773%), and 70% (5381%), respectively, which
indicated a shorter recurrence-free survival in those who
continued to smoke. They concluded that continued smokers
have a 2.2-fold greater risk of bladder cancer recurrence than
quitters (Chen et al., 2007). These studies suggest that cigarette
smoking may exert a protective factor against drug-induced
cytotoxicity and further imply a survival mechanism in themaintenance of chemoresistance in these cells. However, very
little is known about the mechanism by which cigarette smoke
enhances chemoresistance.
It has been suggested that nicotine, the major component in
cigarette smoke originally thought to be only responsible for
tobacco addiction, also alters some cellular functions, such as
activation of mitogenic pathways, angiogenesis, and cell
growth in many cell types (Arredondo et al., 2006; Mousa
and Mousa, 2006). Nicotine acts its biological function mainly
through the nicotinic acetylcholine receptor (nAChR) (Schuller
et al., 2003), b-adrenoceptors (b-AR) (Shin et al., 2007), or
EGF receptor (Laag et al., 2006). Additionally, nicotine has
been shown to inhibit apoptosis induced by tumor necrosis
factor, UV (ultraviolet radiation) light, or chemotherapeutic
drugs such as cisplatin, vinblastine, paclitaxel, and doxorubicin
in a variety of cancer cells (Wrightet al., 1993; Xu et al., 2007).
The antiapoptotic activity of nicotine is known to be regulated
by multiple signaling proteins, such as Bcl-2, nuclear factor-jB,
Bax, Bad, active human protein kinases (AKT), and survivin
(Jin et al., 2004b; Tsurutani et al., 2005; Xu et al., 2007). These
studies indicate that exposure to nicotine may result in chemo-
resistance and decreased efficiency of cancer therapies.
The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: [email protected]
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
2/13
Tumor cells often respond to chemotherapy by engaging
protective mechanisms and survival signaling, which can
antagonize the chemotherapy (Mayo and Baldwin, 2000).
Furthermore, apoptosis inhibition is necessary to provide
cancer cells with the ability to survive in a stressful
environment; it has been proposed that oncogenes provide
cancer cells with intrinsic resistance. Chemoresistance inseveral types of cancer has been linked to the activation of
Stat3 (signal transducer and activator of transcription), and
upregulation of Stat3 directly confers a drug-resistant pheno-
type (Barre et al., 2007). For example, recent studies have
demonstrated that paclitaxel-resistant ovarian cancer cell lines
show an abnormal increase of Stat3 activity and that RNAi-
mediated downregulation of the transcription factor reduces
paclitaxel resistance (Duan et al., 2006). Stat3 can also inhibit
cell cycle arrest and senescence through upregulation of Cyclin
D1 and downregulation of p21WAF1 protein expression (Barre
et al., 2003). Taken together, these studies indicate that Stat3
confers on cancer cells an enhanced ability to survive from
genotoxic treatments and thus may be a predictive marker ofdrug resistance. Inhibition of the Stat3 pathway in several
models of human malignancies induces growth arrest,
apoptosis, and chemosensitivity (Duan et al., 2007).
Enhancement of the expression of survival proteins and
prevention of cell cycle arrest implied that Stat3 may be
involved in drug resistance in bladder cancer therapies and that
the chemoresistance induced by nicotine in bladder tumors
could trigger this process. Studying the mechanisms of
cigarette smokeinduced chemoresistance may explain how
cancer cells gain a survival advantage to combat chemother-
apeutic agentinduced cytotoxicity and could also be helpful
for the design of improved therapeutic strategies for enhancingchemosensitivity in bladder cancer patients who continue to
smoke. The following were the focus of this study: (1)
chemosensitivity of control cells and chemoresistance by long-
term nicotine treatment in bladder cancer cells, (2) constitutive
activation of Stat3 leading to Cyclin D1 overexpression was
correlated with chemoresistance induced by nicotine, (3) ef-
fects of Stat3 inhibitor AG490 and Stat3 small interfering RNA
(siRNA) on the reversal of chemoresistance, and (4) the role of
a7-, a4/b2-nAChR, and b-AR on Stat3 activation, extracellular
signal-regulated protein kinase 1/2 (ERK 1/2) downregulation,
and protective effects in response to anticancer agents.
MATERIALS AND METHODS
Materials
Nicotine, nonspecific nicotinic receptor inhibitor hexomethonium bromide,
nonselective antagonist for beta-adrenergic receptors, propranolol, ERK1/2
inhibitor U0126, JAK2/Stat3 inhibitor AG490, a7-subunit inhibitor methyl-
lycaconitine (MLA), a4/b2 subunit inhibitor a-lobeline (Lob), cisplatin, and
paclitaxel were purchased from Sigma-Aldrich, Inc. (St Louis, MO).
Antibodies against Cyclin D1, Cyclin A, Cyclin B, proliferation cell nuclear
antigen (PCNA), phospho-cdc2, Bcl-2, Bax, poly(ADP-ribose) polymerase
(PARP), ERK1/2, Stat3, phospho-ERK1/2, phospho-Stat3 Ser727, phospho-
Stat3 Tyr-705, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and
horseradish peroxidaseconjugated anti-mouse and anti-rabbit secondary
antibodies were purchased from Cell Signaling (Beverly, MA)
Cell Culture and Pharmacological Treatments
T24 bladder epithelial cancer cell line, immortalized human uroepithelial
cells SV-HUC-1, human lung cancer cell line A549, and immortalized
human lung epithelial cells Beas2B were purchased from ATCC. Human UBC
cell line UB47 was a kind gift of Dr Hsiao-Sheng Liu (National Cheng
Kung Medical College, Institute of Molecular Medicine, Tainan, Taiwan). T24
cells were maintained in 10-cm2
dishes in McCoy 5A medium (Sigma-Aldrich,
Inc), UB47 cells were grown in Roswell Park Memorial Institute medium 1640
(Life Technologies, Inc., Gaithersburg, MD), SV-HUC-1, A549, and Beas2B
cells were maintained in Dulbecco/Vogt Modified Eagles minimal essential
medium (Life Technologies, Inc.). All culture medium were supplemented with
100 U/ml penicillin, 100 lg/ml streptomycin (Life Technologies, Inc.), and
10% heat-inactivated fetal calf serum (HyClone, South Logan, UT). For the
generation of chemoresistance clones, T24 cells were grown in the presence of
1lM of nicotine for 20, 40, 60, and 80 passages. Control cells were cultured in
parallel with the treated cells and passaged for every 2 days. Control T24 cells and
p80 nicotinetreated T24 cell were used for chemosensitivity comparisons and for
studying the mechanism of nicotine-induced chemoresistance in this study.Determination of Cell Viability
Trypan blue exclusion assay. Cells were cultured in 96-well plates at
a density of 2 3 103 cells per well for 24 h for 5 days. Cell numbers were
counted every day after staining with 0.5% trypan blue using a cell counting
chamber.
MTT assay. Cells were seeded in a 96-well plate at a density of 8 3 103
cells per well for overnight. After removing the medium, 100 ll of serum-free
medium containing antitumor agents were added for 72 h. Then, 100 ll of MTT
was added to the wells, and the plate was incubated for 2 h at 37C. The
medium was removed, and 100 ll of dimethyl sulfoxide (DMSO) was added to
the wells. Absorbance was measured using an ELISA plate reader at 570 nm.
Assessment of Apoptosis
Annexin V staining as say. One of the early characteristics of apoptosis is
the rapid translocation and accumulation of the membrane phospholipids
phosphotidylserine from the cytoplasmic interface to the extracellular surface.
Cells were trypsinized, washed with 13 PBS, centrifugated, and resuspend in
13 Annexin V binding buffer (10mM Hepes, pH7.4; 0.14M NaCl; and 2.5 mN
CaCl2) containing 5 ll Annexin V-FITC (Becton Dickinson, San Jose, CA) at
room temperature for 15 min. Additional 400 ll of 13 binding buffer was
added to stop the reaction, and the percentage of Annexin Vpositive cells were
measured by FACScan (Becton Dickinson)
Flow cytometry analysis. The percentages of cells below the G1 peak
(subG0/G1 fraction) and the distribution of cell cycle were evaluated by
propidium iodide staining and analyzed by FACScan (Becton Dickinson) with
WinMDI software programs.
Caspase activity assay. The activity caspase-3 was quantified by means of
the Caspase Fluorometric Assay Kit (R&D Systems, Minneapolis, MN), ac-
cording to the manufacturers instructions. Briefly, cell extracts were incubated
with caspase substrate for 1 h at 37C. Caspase-specific peptides that are
conjugated to the fluorescent reporter molecule 7-amino-4-trifluromethyl
coumarin (AFC) were then added to the reaction and incubation for 1 h. The
cleavage of peptide by caspase released free AFC that can be quantified using
a fluorescence spectrophotometer (400-nm excitation and 505-nm emission).
Western Blot Analysis
The isolation of total cellular lysates, immunoprecipitation, gel electropho-
resis, and immunoblotting were performed, according to the methods described
previously (Lee et al., 2003). Immunoreactive proteins were visualized with the
ROLE OF NICOTINE INDUCES CHEMORESISTANCE 119
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
3/13
enhanced chemiluminescence detection system (PerkinElmer Life Science,
Inc., MA) and BioMax LightFilm (Eastman Kodak Company, New Heaven,
CT), according to the manufacturers instructions.
Electrophoretic Mobility Shift Assay
Nuclear extracts were performed according to Trombino et al. (2004). DNA
probes were biotin-labeled using a biotin 3# endlabeling kit (PIERCE
Biotechnology, Rockford, IL). Double-stranded labeled DNA oligonucleotides
encompassing the Stat3 consensus oligonucleotides (GATCCTTCTGG-
GAATTCCTAGATC) (Protech Technology Enterprise Co., Ltd, TaoYuan,
Taiwan) were used for a binding reaction. The DNA-binding activities of Stat3
were evaluated using an electrophoretic mobility shift assay kit (PIERCE
Biotechnology), according to the manufacturers instructions. Ten micrograms
of nuclear extracts was subjected to denaturing 4% polyacrylamide gel
electrophoresis and developed.
Transfection of the Constitutively Active Form Stat3C Plasmid and Stat3
siRNA
The constitutively active form of Stat3 (Stat3C) plasmid was kindly
provided by Dr Hsiao-Sheng Liu (National Cheng Kung Medical College,
Institute of Molecular Medicine, Tainan, Taiwan). Stat3C can bind DNA and
activate transcription without a specific stimulation. T24 cells were transfected
with 1.5 lg/ml of Stat3C plasmid and then cells were treated with cisplatin orpaclitaxel for 48 h. The siRNAs targeting Stat3 used in this study were
purchased from Ambion Inc. (Austin, TX). The siRNA sequences targeting
human Stat3 used in this study were as follows: sense: 5#-GGAUCUAGAA-
CAGAAAAUGtt-3# and antisense: 5#-CAUUUUCUGUUCUAGAUCCTG-3#.
Nic-T24 cells were transiently transfected with Stat3 siRNA (50nM) by
electroporation using a MicroPorator (Digital Bio Technology, Suwon, Korea)
under condition of 1350 V and 20 ms, according to the manufactures instruction.
Determination of Noradrenaline Level
T24 and Nic-T24 cells were plated in a 12-well plate at a density of 2 3 104
per well. Cells were pretreated with thea7-subunit inhibitor methyllycaconitine
(MLA) at 200lM, the a4/b2-subunit inhibitora-lobeline (Lob) at 200lM, or
the b-adrenoceptor inhibitor propranolol (Prop) at 10lM for an hour. Nic-T24
cells were then treated with 1lM nicotine for 24 h. Supernatants were collected
to determine the concentration of noradrenaline. The noradrenaline level wasdetected using the Noradrenaline ELISA Kit (Immuno-Biological Laboratories,
Hamburg, Germany), according to the manufacturers instructions.
Statistical Analysis
Results are expressed as mean SEM. Experimental data were analyzed
using the Students t-test. Differences were considered to be statistically
significant when the p value was less than 0.05.
RESULTS
Growth Properties of T24 Cells with Long-term Nicotine
Exposure
In this study, we developed long-term nicotine exposure
models in T24 bladder cancer cells. T24 cells were exposed to
1lM nicotine for 20, 40, 60, and 80 passages (referred to as
p20, p40, p60, and p80 Nic-T24 cells). Figure 1A shows that
nicotine-treated T24 cells have a markedly higher time-
dependent proliferation rate compared with the control cells.
The percentage of subG0/G1 phase cells in control increased
significantly in a time-dependent manner under serum-free
condition. In contrast, at the same time point, nicotine-treated
T24 cells displayed a significantly lower increase in the
percentage of subG0/G1 phase cells (Fig. 1B) and a concomitant
increase in the percentage of G0/G1 phase cells (Fig. 1C).
Among the nicotine-treated T24 cells, p80 Nic-T24 cells,
which were exposed to nicotine for the longest time period,
exhibited the highest cell proliferation rate, the percentage of
cells in G0/G1 phase, and the lowest increased in the percentage
of subG0/G1 cells at 24, 48, and 72 h compared with the controland other nicotine-treated T24 cells. These results indicate that
long-term nicotine exposure disrupts serum withdrawal
mediated apoptosis, leading to continuous cell cycle pro-
gression. We then assessed expression of cell cycle regulatory
proteins. Figure 1D shows that Cyclin D1 and PCNA
expression was increased with increasing exposure periods of
T24 cells to nicotine. P80 Nic-T24 cells increased Cyclin D1
expression by 2.1-fold compared with control T24 cells.
Long-term Nicotine Treatment Induces Higher
Chemoresistance in Nic-T24 Cells
Cyclin D1 is associated with enhanced resistance toapoptosis induced by anticancer agents (Biliran et al., 2005).
To investigate the effect of long-term nicotine treatment on
apoptosis, control T24 cells (Con) and p80 Nic-T24 cells (Nic)
were treated with increasing concentrations of cisplatin (Cis) or
paclitaxel (Tax) for 72 h. Cell viability of the Con group was
significantly inhibited by 50% at 5lM cisplatin compared with
19% in the Nic group. Similarly, treated with paclitaxel
resulted in a dose-dependent growth inhibition in the Con
group but with decreased paclitaxel sensitivity in the Nic group
(Fig. 2A). In addition, Con groups treated with Cis and Tax
showed a significantly higher percentage of Annexin V
staining (22% by Cis treatment and 18.1% by Tax treatment)compared with Nic groups (13.5% by Cis treatment and 9.8%
by Tax treatment) (Fig. 2B). Caspase activity assay revealed
that the caspase-3 activity increased by about threefold in Con
groups, whereas only increased slightly in Nic groups after
antitumor agents application (Fig. 2C). Furthermore, treatment
of Con groups with antitumor agents showed cleavage of the
DNA repair enzyme PARP. Cleaved PARP was reduced in
cisplatin-treated Nic groups and was not observed in paclitaxel-
treated Nic groups. Moreover, a high Bax/Bcl-2 ratio can be
correlated with apoptotic cell death, while a low Bax/Bcl-2
ratio may represent a prosurvival profile (Perlman et al., 1999).
Figure 2D shows that the Bax/Bcl-2 ratio was increased in Con
groups (2.97-fold by Cis treatment and 1.4-fold by Tax
treatment), whereas a lower Bax/Bcl-2 ratio was observed in
Nic groups.
We then investigated whether the decreased chemosensitivity
in the Nic groups was associated with perturbations in the cell
cycle. Nic groups displayed a significant increase in percentage
of cells in G0/G1 phase in response to antitumor agents
compared to Con groups (Fig. 2E). Western blot analysis
confirmed that the Nic groups maintained an increased
expression of Cyclin D1, Cyclin A, and Cyclin B proteins after
120 CHEN ET AL.
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
4/13
treatment with antitumor agents, whereas the expression levels
of these cell cycle regulatory proteins decreased in the Congroups (Fig. 2F). These data indicate that persistent exposure to
nicotine may inhibit apoptosis then trigger chemoresistance in
bladder cancer cells.
Overactivation of Stat3 and Downregulation of ERK1/2 in
Chemoresistant Nic-p80 Cells
Our recent study indicated that Stat3 and ERK1/2 activation
was associated with Cyclin D1 overexpression and was linked
to nicotine exposure (Chen et al., 2008). The constitutive
activation of Stat3 may contribute to the survival advantage of
cancer cells and acquired drug resistance to chemotherapy.
Thus, we further examine the activation of Stat3 and ERK1/2
in response to long-term nicotine exposure. Exposure of Con
groups exposed to antitumor agents for 48 h resulted in ERK1/2
activation but Stat3 inhibition, by comparison, Stat3 phosphor-
ylation was increased, but ERK1/2 activation was inhibited in
Nic groups. Consistent with the Western blot results, the Stat3
DNAbinding activity was strongly induced in Nic groups by
antitumor agent treatment compared with Con groups (Fig.
3B). We conclude that chemoresistance in the Nic groups could
be mediated by inducing the Stat3 signaling pathway and
reducing ERK1/2 activity.
In order to clarify that nicotine-induced Sta3 activation is not
limited in T24 bladder cancer cells, normal human lungepithelial cells Beas2B, human lung cancer cells A549,
immortalized human uroepithelial cells SV-HUC-1, and human
bladder cancer cells UB47 were treated with 1lM nicotine for
15, 30, 60, and 120 min. Figures 3CF showed that nicotine
increased the phosphorylation of Stat3 in four cell lines, whereas
ERK1/2 activation was only observed in A549 and UB47 cells.
We suggested that Stat3 could be the major target for nicotine
exposure and may play important roles in proliferation, chemo-
resistance, or antiapoptosis in response to nicotine.
ERK1/2 Activation Mediates Apoptotic Cell Death Induced by
Antitumor Agents
Previous studies indicated that cell death induced by cisplatin
is ERK1/2 dependent; inhibition of ERK1/2 activation reduced
the chemosensitivity of cancer cells (Lu and Cederbaum, 2007).
ERK1/2-specific inhibitor U0126 was used to determine whether
ERK1/2 activity is needed for antitumor agentinduced
apoptosis. Pretreatment with U0126 in Con groups effectively
attenuated antitumor agentinduced ERK1/2 activation but did
not affect Stat3 phosphorylation (Fig. 4C) and resulted in
a decrease in the percentage of subG0/G1 cells in Cis- and Tax-
treated groups by 20 and 12%, respectively (Fig. 4A). Cell cycle
FIG. 1. Persistent exposure to nicotine increases cell proliferation, perturbs cell cycle progression, and upregulates Cyclin D1 and PCNA expression. (A)
Control (Con), p20, p40, p60, and p80 nicotine-treated T24 cells were seeded in 96-well plates in 10% serum medium for the indicated times. Cell proliferation
rates were measured by the trypan blue exclusion assay. Distribution of cells in subG0/G1 (B) and G0/G1 (C) phases were analyzed by flow cytometry after
propidium iodide staining. Data are represented as means SD of three independent experiments; *p < 0.05 compared with Con groups. (D) After the treatment,
cell lysates were isolated and immunoblotted with anti-Cyclin D1 and anti-PCNA antibodies. The membrane was probed with anti-a-tubulin to confirm equal
loading of proteins.
ROLE OF NICOTINE INDUCES CHEMORESISTANCE 121
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
5/13
distribution was also affected: most of the cells remained in
G0/G1 phase after ERK1/2 inhibition (Fig. 4B). The results
suggested that long-term nicotine-treated T24 cells inhibited
ERK1/2 activation could result in chemoresistance.
Role of Stat3 in Chemoresistance in T24 Cells
We further investigate whether inhibition of Stat3 activity
could reverse chemosensitivity in Nic groups. By transfection
with Stat3 siRNA in Nic-T24 cells (Nic si Stat3), the Stat3
protein levels were reduced by 30% and phosphorylated Stat3
was reduced by 50%, in comparison to Nic-T24 cells (Fig. 5A).
Consistently, the Nic si Stat3 groups exhibited higher levels
of apoptosis upon antitumor agent treatments compared with
Nic groups, as evidenced by the decrease of cell viability ( Fig.
5B) and increased number of cells in subG0/G1 phase (Fig. 5C).
To further confirm the role of Stat3 in chemoresistance, we
examined whether downregulation of Stat3 activity by the
FIG. 2. Effects of persistent nicotine exposure on chemoresistance. (A) Control (Con) and p80 nicotine-treated T24 cells (Nic) were seeded in 96-well plates
treated with 5lM cisplatin (Cis) or 10nM paclitaxel (Tax) under serum-free condition for 72 h. Cell viability was measured by the MTT assay. Following DMSO,
serum deprivation (SF), Cis, or Tax treatment, Con and Nic groups were collected and subjected to the following apoptotic assays: (B) Annexin V staining as
a specific apoptosis marker; (C) caspase-3 activities; and (D) Western blotting for PARP, Bax, and Bcl-2 expression by using specific antibodies. The intensities of
Bax and Bcl-2 bands were quantified by densitometry and expressed as Bax/Bcl-2 ratios. (E) Distribution of cells in G0/G1 phase was analyzed by a flow
cytometer. (F) Cell lysates were subjected to the SDS-polyacrylamide gel electrophoresis and probed with anti-Cyclin D1, -Cyclin A, and -Cyclin B antibodies.
Equal protein loading was determined by an anti-GAPDH antibody. Data are represented as means SD of three independent experiments; *p < 0.05 compared
with Con groups.
122 CHEN ET AL.
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
6/13
JAK2/Stat3 inhibitor AG490 enhanced susceptibility to
antitumor agentinduced apoptosis. Figure 5D shows that
pretreatment with AG490 followed by antitumor agents
induced a significant increase in apoptotic cells compared to
Nic groups without AG490 pretreatment. We also found that
Stat3 inhibition by Stat3 siRNA or AG490 reduced Cyclin D1
expression, indicating that Cyclin D1 expression requires the
Stat3 activation and is necessary for chemoresistance in Nic
groups (Figs. 5E and 5F). Interestingly, we found a correlation
may exist between Stat3 activation and ERK1/2 inhibition after
long-term nicotine treatment. Pretreatment with AG490 or
Stat3 siRNA restored the ERK1/2 activation, indicating that
nicotine-induced inhibition of ERK1/2 phosphorylation could
be mediated by Stat3 activation (Figs. 5E and 5F).
Previous findings prompted us to study the effect of
constitutively active Stat3 on chemoresistance in response to
chemotherapeutic agents. Transient transfection of Stat3C
plasmid into wild-type T24 cells resulted in increased
phosphorylation of Stat3 and reduced ERK1/2 phosphorylation
compared with control T24 cells (Fig. 5G). In addition,
increased expression of Cyclin D1 was observed in T24 cells
transfected with Stat3C. Cell viability increased about twofold
in Stat3C-transfected T24 cells compared with control T24
cells after treatment with cisplatin or paclitaxel (Fig. 5H).
These results confirmed that overexpression and/or constitutive
activation of Stat3 could desensitize T24 cells to apoptosis
induced by chemotherapeutic agents.
Activation of Stat3 and Deregulation of ERK1/2 after Long-
term Nicotine Exposure Are Mediated by nAChR andb-AR
We have previously indicated that nicotine activates Stat3,
leading to Cyclin D1 expression and cell proliferation through
FIG. 3. Stat3 but not ERK1/2 activity is increased by persistent exposure to nicotine. (A) Control (C) and nicotine-treated p80 T24 cells (N) were treated with
DMSO, serum deprivation (SF), 10lM cisplatin (Cis), or 2nM paclitaxel (Tax) for 48 h. Expressions of pStat3, Stat3, pERK1/2, and ERK1/2 were determined by
Western blotting. Equal protein loading was determined by anti-GAPDH antibody. (B) After the same treatment, Stat3 DNAbinding activity was assessed using
an electrophoretic mobility shift assay as described in Materials and Methods section. (C) SV-HUC-1 cells, (D) UB47 cells, (E) Beas2B cells, and (F) A549
cells were treated with nicotine 1lM under serum-free condition for 0, 15, 30, 60, and 120 min. Phosphorylated ERK1/2, ERK1/2, Stat3 Ser727, and Stat3 were
determined by immunoblotting with specific antibodies.
ROLE OF NICOTINE INDUCES CHEMORESISTANCE 123
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
7/13
a4/b2-, a7-nAChR, and b-AR (Chen et al., 2008). Chronic
exposure to nicotine has been reported to upregulate several
classes of neuronal nAChRs in a long-lasting manner (Kawai
and Berg, 2001). Our results also confirmed that a4- and a7-
nAChR subunits were upregulated after long-term exposure to
nicotine (Fig. 6A). It is not clear whether nAChRs upregulation
is involved in Stat3 activation and chemoresistance in Nic-T24
cells. The nonspecific nAChR antagonist hexamethonium
bromide (Hexa), a4/b2-specific inhibitor a-lobeline (L), a7-
selective antagonist methyllycaconitine (MLA), and nonspe-
cific b-AR antagonist propranolol (Prop) were used to confirm
which receptors upregulate Stat3 and induce chemoresistance.
The results indicate that the a4/b2-specific inhibitor and b-AR
antagonist inhibited Stat3 activation, BCl-2 expression (Figs.6BD), and cell viability (Figs. 6E and 6F), whereas the a7-
selective antagonist methyllycaconitine (MLA) did not. These
results indicate that Stat3 activation and chemoresistance by
long-term nicotine stimulation involves the action of a4/b2
nAChR, and b-AR in Nic-T24 cells.
Nicotine was found to evoke noradrenaline or adrenaline
release through nAChR (Al-Wadei and Schuller, 2009), and in
turn, these hormones stimulate human cancer cells growth and
metastasis (Shin et al., 2007; Sood et al., 2006). Our previous
study showed that nicotine did not induce the release of
adrenaline (Chen et al., 2008). However, it is possible that
nicotine could induce noradrenaline to stimulate tumor cell
growth. Figure 6G shows that nicotine induced the release ofnoradrenaline, which was inhibited by a4/b2-nAChR and
b-AR antagonist but was not affected by a7-nAChR
antagonist. Our results further confirm that a4/b2-nAChR
and b-AR act upstream of Stat3, cell growth, noradrenaline
release, and chemoresistance in long-term nicotinestimulated
human bladder cancer cells.
DISCUSSION
Bladder cancer patients who continue smoking tend to develop
chemoresistance compared with patients quit smoking beforetreatment (Fleshner et al., 1999). The molecular mechanisms of
cigarette smokeinduced chemoresistance in bladder cancer
remain to be identified. Nicotine is the major component in
cigarette smoke and can be detected in the urine of smokers.
Previous studies have shown that nicotine inhibits apoptosis
and induces chemoresistance in many cancer cells. Thus, we
hypothesize that long-term exposure to nicotine in bladder cancer
cells could be the leading cause of chemoresistance. To fully
understand the potential chemoresistant properties of nicotine, we
analyzed various signaling pathways in response to persistent
nicotine exposure and its subsequent effects on apoptosis in-
hibition and cell cycle regulatory events in T24 cells.
Herein, we provide evidence for the first time that over-
activation of Stat3 but fails to activate ERK1/2 activity (and
downregulation of ERK1/2) contribute to chemoresistance in
response to long-term nicotine exposure in bladder cancer cells.
Previous studies indicated that ERK1/2 activation is generally
considered a survival signaling pathway; however, many evi-
dences exist that the ERK1/2 pathway mediates apoptosis
induced by different stimuli in different tissues. Wang et al.
(2000) showed that ERK1/2 activation is the single most
important factor for cisplatin-induced apoptosis. In human
FIG. 4. Effects of U0126 pretreatment on the percentage of subG0/G1 (A)
and G0/G1 (B) observed following 48-h exposure to 5lM Cis or 2nM Tax
analyzed by flow cytometry and the expression of pERK1/2, ERK1/2, pStat3,
Stat3, and Cyclin D1 levels determined by Western blotting (C). Means SDof three independent experiments; *p < 0.05 compared with Con groups.
124 CHEN ET AL.
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
8/13
FIG. 5. Effects of Stat3 inhibition on cell viability and apoptosis induced by antitumor agents. (A) Nic-T24 cells were transfected with Stat3 siRNA (50nM);
incubated for 24-h posttransfection; and Stat3, ERK1/2, pStat3, pERK1/2, and Cyclin D1 expression were determined by Western blotting. (B) Cell viability of
Con, Nic, and Nic si Stat3 groups after treated with 5lM Cis or 10nM Tax were measured by the MTT assay. (C) The percentage of subG0/G1 were detected
either transfected with Stat3 siRNA or (D) pretreatment with 15lM AG490 followed by treated with antitumor agents for 48 h. *p < 0.05 compared with Con
groups; #p < 0.05 compared with Nic groups. (E) ERK1/2, Stat3, pERK1/2, pStat3, and Cyclin D1 expressions were determined by Western blotting in Nic-T24
cells transfected with 50nM Stat3 siRNA for 24 h or (F) pretreated with 15lM AG490 for 1 h and then treated with 10lM cisplatin or 2nM paclitaxel for a further
48 h under serum-free condition. (G) T24 cells were transfected with Stat3C plasmid (1.5 lg) (Stat3C), incubated for 24-h posttransfection, and Stat3, ERK1/2,
pStat3, pERK1/2, and Cyclin D1 expression were determined by Western blotting. Equal protein loading was determined by an anti-GAPDH antibody. (H) Cell
viability of Con, Nic, and Con Stat3C groups after treated with 5lM Cis or 10nM Tax were measured by the MTT assay. *p < 0.05 compared with Con groups.
ROLE OF NICOTINE INDUCES CHEMORESISTANCE 125
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
9/13
cervical carcinoma SiHa and hepatoblastoma HepG2 cells,
suppression of ERK1/2 signal pathway by mitogen-activated
protein kinase/extracellular signal regulated kinase kinase
(MEK) inhibitor PD98059 resulted in an increase in cisplatin
resistance (Yeh et al., 2002). Consistent with these findings, we
found that ERK1/2 activation and subsequent apoptosis were
observed in control T24 cells but not in chemoresistant Nic-
T24 cells after antitumor agent treatment. Inhibited ERK1/2
phosphorylation by U0126 effectively reduced anticancer
agentinduced apoptosis and increased G0/G1 arrest (Fig. 4).
The results provide the evidence that Nic-T24 cells with lower
ERK1/2 activity were prone to stay in G0/G1 phase rather than
undergo apoptosis in response to antitumor agents.
Stat3 pathway is one of the major prosurvival signal
transduction pathways linked to chemoresistance in various
cancer cell lines. We provide evidence that nicotine-treated
cancer cells exhibit stronger prosurvival signaling through
Stat3 activation, which leads to cell survival in response to
antitumor agents. We also found that Stat3C-transfected cells
had elevated cell viability compared with T24 cells after
treatment with cisplatin or paclitaxel (Fig. 5H). Thus, over-
activation of Stat3 may stimulate cell cycle progression and
provide protection against apoptosis. Indeed, Stat3 promotes
uncontrolled cell growth and survival through deregulation of
the expression of cell cycle genes, including Cyclin D1
(Kobayashi et al., 2006). Overexpression of Cyclin D1 in
FIG. 6. Involvement of nAChR and b-AR in the Stat3 activation and ERK1/2 downregulation in Nic-T24 cells. (A) The expression ofa4-, b2-, and a7-nAChRs
in T24 and Nic-T24 cells was detected by specific antibodies. (B) Nic-T24 cells were starved and treated with inhibitors nAChR antagonist; hexomethonium bromide
(Hexa) 0.1, 0.2, and 0.4mM (MLA, M); or (C) nonspecific b-AR antagonist, propranolol (Prop) 10, 25, and 50lM for 120 min. (D) Nic-T24 cells were pretreated
with a7-nAChR inhibitor MLA (M) 200lM ora4-nAChR antagonist Lobeline (L) 200lM for 1 h followed by cisplatin (Cis 10lM) or paclitaxel (Tax 10nM) for 48
h. Protein expression was detected by Western blotting using anti-ERK1/2, pERK1/2, pStat3, Stat3, or BCl-2 antibodies, and (E) Cell viability was then measured by
the MTT assay. *p < 0.05 compared with Con groups; #p < 0.05 compared with Nic groups. (F) Pretreatment with propranolol (Prop) 10lM increased drug
sensitivity in both T24 and Nic-t24 cells. *p < 0.05 compared with groups in the absence of Prop; (G) pretreatment with propranolol (P) 10lM ora4-nAChR
antagonist Lobeline (L) 200lM reduced the release of noradrenaline. *p < 0.05 compared with Con groups; #p < 0.05 compared with Nic groups.
126 CHEN ET AL.
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
10/13
human cancer cell lines have been shown to perturb cell cycle
progression leading to resistance to chemotherapy (Kornmann
et al., 1999) Our results also demonstrated that bladder cancer
cells can gain a survival advantage after chronic exposure to
nicotine. For instance, upon serum starvation, p80 Nic-T24
with Cyclin D1 overexpression consistently displayed shorter
doubling times and a higher proliferation rate, suggesting thatnicotine treatment renders T24 cells less dependent on growth
factors. Through activation of Stat3 and Cyclin D1, Nic-T24
cells were prone to enter into G0/G1 phase rather than apoptosis
after treatment with antitumor agents compared with control
groups. In the present study, disrupting the Stat3 oncogenic
pathway with either a JAK2/Stat3 inhibitor or an Stat3 siRNA
resulted in inhibition of Cyclin D1 expression, cell pro-
liferation, and restored the sensitivity antitumor agents. These
finding suggest that elevated Stat3 activation from long-term
nicotine treatment could trigger subsequent Cyclin D1 over-
expression and contribute to chemoresistance in Nic-T24
bladder cancer cells by maintaining cell cycle progression
and attenuating drug-induced apoptosis.Duan et al. (2006) reported that the Stat3 pathway is often
overexpressed and activated in many paclitaxel-resistant
ovarian cancer cells compared to cell lines that are paclitaxel
nave. Stat3 inhibition increased paclitaxel-induced apoptosis
even in the paclitaxel-resistant ovarian cancer cells (Duan
et al., 2006). Introduction of antisense Stat3, Stat3 decoy DNA,
or a dominant-negative Stat3 into human tumor cells with
constitutively activated Stat3 leads to apoptosis (Boehm et al.,
2008; Leong et al., 2003). These results reveal that Stat3
inhibition is an effective strategy for enhancing chemo-
sensitivity. However, both Stat3 siRNA and AG490 could
not completely reverse chemosensitivity in response tochemotherapeutic agents (Fig. 5C), indicating that other
mechanisms may also be involved in chemoresistance in
nicotine-treated T24 cell. For instance, the AKT pathway (Xu
et al., 2007) or AKT/protein kinase C (PKC) pathways (Jin
et al., 2004a) may be required for the antiapoptotic effects of
nicotine. Further work is necessary to show that the disruption
of Stat3 alone or in combination with other pathways such as
AKT pathway could be a potential strategy for increasing
chemosensitivity in bladder cancer therapy.
We found that treatment with Stat3 siRNA or AG490 induced
cell death and ERK1/2 activation in response to antitumor agents
in Nic-treated T24 cells, indicating the essential role of Stat3 in
the suppression of ERK1/2 activity. Several studies suggested
a negative regulation between the Stat3 and Src-homology 2
domain-containing tyrosine phosphatase/ERK pathways (Ernst
and Jenkins, 2004). For example, Arany et al. found that
pretreatment with AG490 or direct inhibition of Stat3 via
a dominant-negative mutant restored ERK1/2 activation. They
suggested that Stat3 may compete with the binding site of
growth factors and thus terminate ERK1/2 activation. The other
possibility is that Stat3-mediated activation of SOCS3 can inhibit
growth factor receptor activation leading to ERK inactivation
(Xiaet al., 2002). Based on these studies, it is possible that Stat3
overactivation in Nic-T24 cells increases SOCS3 activation,
leading to the suppression of ERK1/2 activation. Impaired
induction of the ERK1/2 pathway in Nic-T24 cells becomes
highly susceptible to Stat3-dependent suppression of apoptosis
and concomitant induction of proliferation. Thus, inhibition of
Stat3 activation by AG490 or siRNA restored ERK1/2 activationand chemosensitivity in Nic-T24 cells.
In this study, we found that a4- and a7-nAChR subunits
were upregulated in response to chronic exposure to nicotine.
A previous study indicated that a7-nAChR is the primary
receptor that mediates proliferation and antiapoptosis effects of
nicotine in cancer cells. Nevertheless, a3/b4- ora4/b2-nAChR
might also be important for these processes (Chen et al., 2008;
Marrero and Bencherif, 2009; West et al., 2003). Our results
show that long-term nicotine stimulationinduced Stat3
activation and ERK1/2 downregulation were effectively
inhibited by a-lobeline (a4/b2-specific inhibitor) (Fig. 6),
indicating that the chemoresistance induced by nicotine could
be mediated by a4/b2-nAChR and not by a7-nAChR. Thus,we suggest that chronic nicotine exposure results in a a4/b2-
nAChR-Stat3-Cyclin D1 prosurvival and antiapoptosis cascade
in bladder cancer cells.
In addition to nAChRs, nicotine has been reported to promote
the growth of cancer cells through the engagement of signaling
pathways mediated by another receptor, b-adrenoceptor. Jin et al.
(2004a) indicated that low-dose nicotine (1lM) is able to induce
cell survival through Bad phosphorylation mediated by b-adre-
noceptor. Antagonists for b-AR inhibit the development of
pulmonary adenocarcinoma induced by 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone (NNK) (Schuller et al., 2000), reversed
the stimulatory action of nicotine on PKC, ERK1/2 activation,and COX-2 expression together with gastric cancer cell
proliferation (Shin et al., 2007). These studies demonstrate that
adrenoceptors may play a role in nicotine-mediated signaling.
Consistently, we found that nicotine induced activation of Stat3,
and the release of noradrenaline was blocked by pretreatment
with the b-adrenoceptor antagonist propranolol (Figs. 6D and
6E), indicating a direct role of nicotine on b-adrenoceptor.
Nicotine is also reported to transactivate b-adrenoceptor by
releasing adrenaline or noradrenaline to stimulate the growth of
colon cancer cells (Al-Wadei and Schuller, 2009). Some
studies have indicated that synthesis of noradrenaline is
mediated by nAChRs, such as a7-nAChR, b2-nAChR, or
a2-nAChR (Al-Wadei and Schuller, 2009; Wong et al., 2007).
These reports are in accord with our study that nicotine may
also transactivate b-adrenoceptor through the release of
noradrenaline. Our results suggest that a4/b2-nAChR plays
a more important role in chemoresistance and regulation of
noradrenaline levels than a7-nAChR (Fig. 6).
In conclusion, as shown in Figure 7, persistent nicotine-
induced chemoresistance in bladder cancer cells could occur
via three processes: (1) increased release of noradrenaline
through a4/b2-nAChR and may transactivate b-adrenoceptor;
ROLE OF NICOTINE INDUCES CHEMORESISTANCE 127
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
11/13
(2) activation of Stat3 via a4/b2-nAChR and b-adrenoceptor,
leading to Cyclin D1 overexpression, perturbation of cell cycle
progression, and inhibition of apoptosis induced by antitumor
agents; and (3) inhibition of ERK1/2 activation and sub-
sequently reduction in sensitivity to chemotherapeutic agents.
To the best of our knowledge, this is the first evidence of long-
term nicotine treatment inducing chemoresistance through
overactivation of Stat3 leading to inhibition of ERK1/2
activation via a4/b2-nAChR and b-AR. It is noteworthy that
nicotine-mediated inhibition of cell death may not only occur
in the failure of chemotherapy but may also help to explain the
poor prognostic value of bladder cancer patients who continued
cigarette smoking during chemotherapy. Most importantly, we
also provide evidence that Stat3 aberrant activation could be
due to long-term exposure to environmental toxicants, such as
nicotine. Our study had given rise to these considerations for
clinical bladder cancer therapy: (1) Avoidance of cigarette
smoking or nicotine-based treatment may increase the efficacy
of chemotherapy. (2) a4/b2-nAChR, b-AR, and their down-
stream Stat3 could be the target for increasing chemosensitivity
in bladder cancer patients who develop chemoresistance during
chemotherapy.
FUNDING
National Science Council (NSC 95-2314-B-006-095-MY3).
REFERENCES
Al-Wadei, H. A., and Schuller, H. M. (2009). Nicotinic receptor-associated
modulation of stimulatory and inhibitory neurotransmitters in NNK-
induced adenocarcinoma of the lungs and pancreas. J. Pathol. 218,
437445.
Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., and
Grando, S. A. (2006). Receptor-mediated tobacco toxicity: cooperation of
FIG. 7. A model of chemoresistance by persistent nicotine exposure in T24 bladder cancer cells. Antitumor agents cause moderate ERK1/2 activation,
apoptotic cell death, caspase-3 activation, and low activation of Stat3 in T24 cells. However, persistent nicotine exposure induces chemoresistance through three
processes: (1) increased release of noradrenaline through a4/b2-nAChR and may transactivate b-adrenoceptor; (2) activation of Stat3 viaa4/b2-nAChR and
b-adrenoceptor leading to Cyclin D1 overexpression, perturbation of cell cycle progression, and inhibition of apoptosis induced by antitumor agents; and (3)
inhibition of ERK1/2 activation and subsequent reduction in sensitivity to chemotherapeutic agents. Inhibition ofa4/b2-nAChR and b-AR by antagonists reduced
Stat3 activity and reversed ERK1/2 activation. Inhibition of Stat3 activation by siRNA or the specific inhibitor AG490 restored chemosensitivity in T24 cells.
128 CHEN ET AL.
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
12/13
the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of
alpha7 nicotinic receptor in oral keratinocytes. FASEB J. 20, 20932101.
Barre, B., Avril, S., and Coqueret, O. (2003). Opposite regulation of myc and
p21waf1 transcription by STAT3 proteins. J. Biol. Chem. 278, 29902996.
Barre, B., Vigneron, A., Perkins, N., Roninson, I. B., Gamelin, E., and
Coqueret, O. (2007). The STAT3 oncogene as a predictive marker of drug
resistance. Trends Mol. Med. 13, 411.
Biliran, H., Jr, Wang, Y., Banerjee, S., Xu, H., Heng, H., Thakur, A.,Bollig, A., Sarkar, F. H., and Liao, J. D. (2005). Overexpression of cyclin D1
promotes tumor cell growth and confers resistance to cisplatin-mediated
apoptosis in an elastase-myc transgene-expressing pancreatic tumor cell line.
Clin. Cancer Res. 11, 60756086.
Boehm, A. L., Sen, M., Seethala, R., Gooding, W. E., Freilino, M.,
Wong, S. M. Y., Wang, S., Johnson, D. E., and Grandis, J. R. (2008).
Combined targeting of epidermal growth factor receptor, signal transducer
and activator of transcription-3, and Bcl-X(L) enhances antitumor effects in
squamous cell carcinoma of the head and neck. Mol. Pharmacol. 73,
16321642.
Chen, C.-H., Shun, C.-T., Huang, K.-H., Huang, C.-Y., Tsai, Y.-C., Yu, H.-J.,
and Pu, Y.-S. (2007). Stopping smoking might reduce tumour recurrence in
nonmuscle-invasive bladder cancer. BJU Int. 100, 281 286; discussion 286.
Chen, R. J., Ho, Y. S., Guo, H. R., and Wang, Y. J. (2008). Rapid activation ofStat3 and ERK1/2 by nicotine modulates cell proliferation in human bladder
cancer cells. Toxicol. Sci. 104, 283293.
Dreicer, R. (2001). Locally advanced and metastatic bladder cancer. Curr.
Treat. Options Oncol. 2, 431436.
Duan, Z., Bradner, J., Greenberg, E., Mazitschek, R., Foster, R., Mahoney, J.,
and Seiden, M. V. (2007). 8-benzyl-4-oxo-8-azabicyclo[3.2.1]oct-2-ene-6,7-
dicarboxylic acid (SD-1008), a novel janus kinase 2 inhibitor, increases
chemotherapy sensitivity in human ovarian cancer cells. Mol. Pharmacol.
72, 11371145.
Duan, Z., Foster, R., Bell, D. A., Mahoney, J., Wolak, K., Vaidya, A.,
Hampel, C., Lee, H., and Seiden, M. V. (2006). Signal transducers and
activators of transcription 3 pathway activation in drug-resistant ovarian
cancer. Clin. Cancer Res. 12, 50555063.
Ernst, M., and Jenkins, B. J. (2004). Acquiring signalling specificity from thecytokine receptor gp130. Trends Genet. 20, 2332.
Fleshner, N., Garland, J., Moadel, A., Herr, H., Ostroff, J., Trambert, R.,
OSullivan, M., and Russo, P. (1999). Influence of smoking status on the
disease-related outcomes of patients with tobacco-associated superficial
transitional cell carcinoma of the bladder. Cancer 86, 23372345.
Jin, Z., Gao, F., Flagg, T., and Deng, X. (2004a). Nicotine induces multi-site
phosphorylation of Bad in association with suppression of apoptosis. J. Biol.
Chem. 279, 2383723844.
Jin, Z., Gao, F., Flagg, T., and Deng, X. (2004b). Tobacco-specific nitrosamine
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional coop-
eration of Bcl2 and c-Myc through phosphorylation in regulating cell
survival and proliferation. J. Biol. Chem. 279, 4020940219.
Kawai, H., and Berg, D. K. (2001). Nicotinic acetylcholine receptors containing
alpha 7 subunits on rat cortical neurons do not undergo long-lastinginactivation even when up-regulated by chronic nicotine exposure.
J. Neurochem. 78, 13671378.
Kobayashi, S., Shimamura, T., Monti, S., Steidl, U., Hetherington, C. J.,
Lowell, A. M., Golub, T., Meyerson, M., Tenen, D. G., Shapiro, G. I., et al.
(2006). Transcriptional profiling identifies cyclin D1 as a critical downstream
effector of mutant epidermal growth factor receptor signaling. Cancer Res.
66, 1138911398.
Kornmann, M., Danenberg, K. D., Arber, N., Beger, H. G., Danenberg, P. V.,
and Korc, M. (1999). Inhibition of cyclin D1 expression in human pancreatic
cancer cells is associated with increased chemosensitivity and decreased
expression of multiple chemoresistance genes. Cancer Res. 59, 35053511.
Laag, E., Majidi, M., Cekanova, M., Masi, T., Takahashi, T., and Schuller, H.
(2006). NNK activates ERK1/2 and CREB/ATF-1 via beta-1-AR and EGFR
signaling in human lung adenocarcinoma and small airway epithelial cells.
Int. J. Cancer 119, 15471552.
Lee, W. S., Chen, R. J., Wang, Y. J., Tseng, H., Jeng, J. H., Lin, S. Y.,
Liang, Y. C., Chen, C. H., Lin, C. H., Lin, J. K., et al. (2003). In vitro
and in vivo studies of the anticancer action of terbinafine in human cancer
cell lines: G0/G1 p53-associated cell cycle arrest. Int. J. Cancer106,
125137.
Leong, P. L., Andrews, G. A., Johnson, D. E., Dyer, K. F., Xi, S., Mai, J. C.,
Robbins, P. D., Gadiparthi, S., Burke, N. A., Watkins, S. F., et al. (2003).
Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates
head and neck cancer cell growth. Proc. Natl. Acad. Sci. U.S.A. 100,
41384143.
Lu, Y., and Cederbaum, A. (2007). The mode of cisplatin-induced cell death in
CYP2E1-overexpressing HepG2 cells: modulation by ERK, ROS, glutathi-
one, and thioredoxin. Free Radic. Biol. Med. 43, 10611075.
Marrero, M. B., and Bencherif, M. (2009). Convergence of alpha 7 nicotinic
acetylcholine receptor-activated pathways for anti-apoptosis and anti-
inflammation: central role for JAK2 activation of STAT3 and NF-kappaB.
Brain Res. 1256, 17.
Mayo, M. W., and Baldwin, A. S. (2000). The transcription factor NF-kappaB:control of oncogenesis and cancer therapy resistance. Biochim. Biophys.
Acta. 1470, M55M62.
Mousa, S., and Mousa, S. A. (2006). Cellular and molecular mechanisms of
nicotines pro-angiogenesis activity and its potential impact on cancer.
J. Cell. Biochem. 97, 13701378.
Perlman, H., Zhang, X., Chen, M. W., Walsh, K., and Buttyan, R. (1999). An
elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell
apoptosis. Cell Death Differ. 6, 4854.
Schuller, H. M., Plummer, H. K., 3rd, and Jull, B. A. (2003). Receptor-
mediated effects of nicotine and its nitrosated derivative NNK on pulmonary
neuroendocrine cells. Anat. Rec. 270, 5158.
Schuller, H. M., Porter, B., and Riechert, A. (2000). Beta-adrenergic
modulation of NNK-induced lung carcinogenesis in hamsters. J. Cancer
Res. Clin. Oncol. 126, 624630.Shin, V. Y., Wu, W. K., Chu, K. M., Koo, M. W., Wong, H. P., Lam, E. K.,
Tai, E. K., and Cho, C. H. (2007). Functional role of beta-adrenergic
receptors in the mitogenic action of nicotine on gastric cancer cells. Toxicol.
Sci. 96, 2129.
Sood, A. K., Bhatty, R., Kamat, A. A., Landen, C. N., Han, L., Thaker, P. H.,
Li, Y., Gershenson, D. M., Lutgendorf, S., and Cole, S. W. (2006). Stress
hormone-mediated invasion of ovarian cancer cells. Clin. Cancer Res. 12,
369375.
Trombino, S., Cesario, A., Margaritora, S., Granone, P., Motta, G., Falugi, C.,
and Russo, P. (2004). Alpha7-nicotinic acetylcholine receptors affect growth
regulation of human mesothelioma cells: role of mitogen-activated protein
kinase pathway. Cancer Res. 64, 135145.
Tsurutani, J., Castillo, S. S., Brognard, J., Granville, C. A., Zhang, C.,
Gills, J. J., Sayyah, J., and Dennis, P. A. (2005). Tobacco componentsstimulate Akt-dependent proliferation and NFkappaB-dependent survival in
lung cancer cells. Carcinogenesis 26, 11821195.
Wang, X., Martindale, J. L., and Holbrook, N. J. (2000). Requirement for ERK
activation in cisplatin-induced apoptosis. J. Biol. Chem. 275, 3943539443.
West, K. A., Brognard, J., Clark, A. S., Linnoila, I. R., Yang, X.,
Swain, S. M., Harris, C., Belinsky, S., and Dennis, P. A. (2003). Rapid
Akt activation by nicotine and a tobacco carcinogen modulates the
phenotype of normal human airway epithelial cells [see comment]. J. Clin.
Invest. 111, 8190.
Wong, H. P., Yu, L., Lam, E. K., Tai, E. K., Wu, W. K., and Cho, C. H. (2007).
Nicotine promotes cell proliferation via alpha7-nicotinic acetylcholine receptor
ROLE OF NICOTINE INDUCES CHEMORESISTANCE 129
-
7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010
13/13
and catecholamine-synthesizing enzymes-mediated pathway in human colon
adenocarcinoma HT-29 cells. Toxicol. Appl. Pharmacol. 221, 261267.
Wright, S. C., Zhong, J., Zheng, H., and Larrick, J. W. (1993). Nicotine
inhibition of apoptosis suggests a role in tumor promotion. FASEB J. 7,
10451051.
Xia, L., Wang, L., Chung, A. S., Ivanov, S. S., Ling, M. Y., Dragoi, A. M.,
Platt, A., Gilmer, T. M., Fu, X. Y., and Chin, Y. E. (2002). Identification
of both positive and negative domains within the epidermal growthfactor receptor COOH-terminal region for signal transducer and
activator of transcription (STAT) activation. J. Biol. Chem. 277,
3071630723.
Xu, J., Huang, H., Pan, C., Zhang, B., Liu, X., and Zhang, L. (2007). Nicotine
inhibits apoptosis induced by cisplatin in human oral cancer cells. Int. J. Oral
Maxillofac. Surg. 36, 739744.
Yeh, P. Y., Chuang, S. E., Yeh, K. H., Song, Y. C., Ea, C. K., and Cheng, A. L.
(2002). Increase of the resistance of human cervical carcinoma cells to
cisplatin by inhibition of the MEK to ERK signaling pathway partly via
enhancement of anticancer drug-induced NF kappa B activation. Biochem.
Pharmacol. 63, 14231430.
Zeegers, M. P., Tan, F. E., Dorant, E., and van Den Brandt, P. A. (2000). The
impact of characteristics of cigarette smoking on urinary tract cancer risk:
a meta-analysis of epidemiologic studies. Cancer 89, 630639.
130 CHEN ET AL.