Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological...

7/30/2019 Chen RJ Nicotine Exposure-Induced Chemoresistance is Mediated by Activated STAT3 Toxicological Scniences 2010

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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]


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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

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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

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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.



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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.

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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.



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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.

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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.



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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.


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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;



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(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.


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



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.

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