ToxSci Advance Access originally published online on September 26, 2006
Toxicological Sciences 2007 96(1):21-29; doi:10.1093/toxsci/kfl118
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Functional Role of ß-Adrenergic Receptors in the Mitogenic Action of Nicotine on Gastric Cancer Cells
,1
* Department of Pharmacology
Department of Surgery
Research Centre of Infection and Immunology, Faculty of Medicine, The University of Hong Kong, Hong Kong, HKSAR, China
1 To whom correspondence should be addressed at Department of Pharmacology, Faculty of Medicine Building, 21 Sassoon Road, The University of Hong Kong, Hong Kong, HKSAR, China. Fax: +852-2817-0859. E-mail: chcho{at}hkusua.hku.hk.
Received July 12, 2006; accepted September 13, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We previously reported that nicotine promoted gastric cancer cell growth via upregulation of cyclooxygenase 2 (COX-2). In the present study, we further investigated whether ß-adrenoceptors, protein kinase C (PKC), and extracellular signal–regulated kinase-1/2 (ERK1/2) were involved in the modulation of COX-2 expression and cell proliferation by nicotine in AGS, a human gastric adenocarcinoma cell line. Results showed that nicotine dose dependently increased the phosphorylation of EKR1/2 and the expression of AP-1 subunits c-fos and c-jun. In this connection, the ERK1/2 inhibitor U0126 abrogated the upregulation of AP-1 and COX-2 as well as cell proliferation induced by nicotine. Moreover, nicotine induced the translocation of PKC-ßI from cytosol to membrane and increased the total levels of PKC expression. Inhibition of PKC by staurosporine attenuated nicotine-induced ERK1/2 phosphorylation and COX-2 expression. Furthermore, atenolol and ICI 118,551, a ß1- and ß2-adrenoceptor antagonist, respectively, reversed the stimulatory action of nicotine on the expression of PKC, ERK1/2 phosphorylation, and COX-2 together with cell proliferation. Collectively, these results suggest that nicotine stimulates gastric cancer cell growth through the activation of ß-adrenoceptors and the downstream PKC-ßI/ERK1/2/COX-2 pathway.
Key Words: nicotine; gastric cancer; ß-adrenergic receptor; protein kinase C; proliferation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Gastric cancer is the second most common cause of cancer mortalities in males and the fourth in females. Though the incidence of gastric cancer has been decreasing, it remains a common malignancy worldwide, especially in Asia. Cigarette smoking contributes to the increased risk of gastric cancer, with a 1.5- to 2.5-fold increase in the incidence among current smokers (Sasazuki et al., 2002
; Stadtlander and Waterbor, 1999). Cigarette smoking is therefore considered to be one of the major risk factors for gastric cancer. Cigarette smoke contains many carcinogens that have been shown to induce neoplastic lesions in humans or animals (Stadtlander and Waterbor, 1999). Any defined active component involved in the carcinogenesis of stomach, however, has yet to be identified.
Evolving evidence suggests that dysregulation of cyclooxygenase 2 (COX-2) expression may be centrally involved in the pathogenesis of gastric cancer. For instance, COX-2 expression is highly upregulated in cancerous tissues (> 70%) when compared with the normal surrounding tissues (Kawabe et al., 2002). Moreover, nonsteroidal anti-inflammatory drugs are used clinically to treat gastrointestinal cancers, owing to their ability to inhibit COX activity (Jiang and Wong, 2003; Koehne and Dubois, 2004). In this connection, the specific COX-2 inhibitor SC-236 has been reported to suppress gastric cancer cell growth through inhibition of c-jun N-terminal kinase (JNK) activity (Wong et al., 2004). Nimesulide, another selective COX-2 inhibitor, also induced G0/G1 cell cycle arrest and apoptosis in the gastric cancer cell line SGC7901 (Li et al., 2003). In relation to smoking-related malignancies, we previously reported that nicotine, a major component in cigarette, enhanced gastric cancer development by promoting cancer cell proliferation via upregulation of COX-2 (Shin et al., 2004). The mechanism by which nicotine regulates COX-2 expression in gastric cancer cells, however, remains poorly defined.
Mitogens and growth factors bind to the cell-surface receptor to initiate cellular responses, such as cell proliferation and differentiation, which may contribute to cell transformation and cancer development. Protein kinase C (PKC), in this regard, has been shown to participate in various cellular signaling pathways that regulate cell proliferation, tumor promotion, differentiation, and apoptosis (Hug and Sarre, 1993). Tumor promoters like phorbol esters and some carcinogens in cigarette smoke (e.g., hydroquinone, catechol, and nitrosamines) are known to activate PKC (Gopalakrishna et al., 1994; Schuller, 1994). To date, 12 isoforms of PKC have been identified, namely, conventional PKC isoforms (
, ßI, ßII, and 
), novel PKC isoforms (
, 
, 
, and 
), atypical PKC isoforms (
, 
, and 
), and another subgroup PKC (µ). The localization and activation properties of these isoforms determine their differential functions. It has been shown that catechol and hydroquinone present in cigarette smoke can induce PKC translocation and increase tumor cell invasiveness (Gopalakrishna et al., 1994). Activation of PKC can also activate the Raf/MEK/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MAPK)–signaling cascade to promote cell growth (Debidda et al., 2003; Jull et al., 2001). AP-1, which consists of c-jun and c-fos as well as other subunits, is one of the major transcriptional targets of the MAPK family (Karin, 1995). An earlier study revealed that increased MAPK activity is associated with human gastric adenocarcinoma (Bang et al., 1998). The involvement of MAPK-signaling cascade in gastric carcinogenesis promoted by nicotine, however, remains to be elucidated.
Nicotine has been reported to promote the growth of various types of cancer through the engagement of signaling pathways mediated by two different receptors, namely, nicotinic acetylcholine receptors (nAChRs) and ß-adrenoceptors (Jin et al., 2004; Tsurutani et al., 2005). In this respect, nicotine and its derivatives, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), stimulate 7 subtype of nAChRs and activate the downstream Raf-1/MAPK signaling to promote lung cancer cell proliferation (Schuller et al., 2000a). A parallel line of evidence, nevertheless, demonstrates that adrenoceptors may also play a role in nicotine signaling. Antagonists for ß-adrenoceptors, in particular, inhibit the development of pulmonary adenocarcinoma in animal models (Schuller et al., 2000b). Thus, it is of great interest to investigate which specific receptors contribute to the carcinogenic effect of nicotine in the stomach. In the present study, we therefore aim to delineate the molecular mechanism underlying the promoting effect of nicotine on gastric cancer cell proliferation. The possible involvement of PKC and MAPK in nicotine-induced gastric cancer cell growth was investigated so as to better understand the pathogenesis of smoking-related gastric cancer.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Reagents and drugs.
U0126 (specific extracellular signal–regulated kinase-1/2 [ERK1/2] inhibitor), p-JNK–, and JNK-specific antibodies were purchased from Cell Signaling Technology (Beverley, MA). Antibodies specific to conventional PKC isoforms (
, ßI, ßII, and ), COX-2, p-ERK1/2, ERK1/2, c-fos, and c-jun were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). p38 and p-p38 antibodies were from BioLabs (Beverly, MA). SB203580 (specific p38 inhibitor) was obtained from Calbiochem (La Jolla, CA). Prostaglandin E2 (PGE2) enzyme immunoassay kit was bought from R&D Systems (Minneapolis, MN). (–)-Nicotine, propranolol (a nonselective ß-adrenergic receptor antagonist), atenolol (a specific ß1-adrenoceptor antagonist), ICI 118,551 (a specific ß2-adrenoceptor antagonist), -bungarotoxin (-BTX, 7-nAChR–specific inhibitor), staurosporine (PKC inhibitor), and other chemicals were purchased from Sigma (St Louis, MO) unless otherwise specified.
Cell culture and drug treatment.
Human gastric adenocarcinoma cell line AGS was purchased from American Type Culture Collection (Manassas, VA) (CRL-1739). Cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin G, and 100 µg/ml streptomycin and maintained at 37°C, 95% humidity, and 5% carbon dioxide. AGS cells were plated at a density of 8 x 104 cells per well in 24-well plates. At confluence, nicotine at a dose of 432µM was incubated with cells for 5 h (the optimal time for studying the nicotine action on cell proliferation in the present system) to study the mitogenic effect of nicotine on gastric cancer cells. In order to examine the effects of various inhibitors, cells were pretreated without or with staurosporine (100nM), U0126 (20µM), SB203580 (20µM), -BTX (200nM), propranolol (20µM), atenolol (10µM), or ICI 118,551 (10µM) for 45 min prior to nicotine treatment. Control cells were allowed to grow in the absence of any inhibitors for the same period of time as the treated cells. Experiments were performed in duplicate and repeated for three times (n = 6). The concentrations of nicotine currently chosen were used previously to study the mitogenic and motogenic effects of nicotine on gastric cancer cells (Shin et al., 2004, 2005).
Analysis of PKC isoform translocation.
Cytosolic and membrane fractions were isolated as described previously with modifications (Li et al., 1998). AGS cells were incubated without or with nicotine (432µM) for 0, 1, 3, or 5 h. Cells were scraped and homogenized in ice-cold homogenization buffer (10mM Tris-HCl [pH 7.4], 1mM EDTA, 1mM NaVO4, 0.5mM phenylmethylsulfonyl fluoride [PMSF], 1% aprotinin, and 1mM dithiothreitol) for 20 min, and then centrifuged at 800 x g for 10 min. The supernatant was centrifuged at 100,000 x g for 60 min. After centrifugation, the cytosolic fraction was collected in the supernatant and the pellets were resolubilized in solubilizing buffer (20mM HEPES, 400mM NaCl, 1mM EGTA, 1mM EDTA, 1mM NaF, 1mM NaVO4, 1mM PMSF, and 1% aprotinin). The solubilized pellets were centrifuged at 100,000 x g for 60 min to obtain the membrane fraction. Translocation of different PKC isoforms as determined by their differential levels in the cytosol and membrane was analyzed by Western blot.
Western blot.
AGS cells were harvested in radioimmunoprecipitation buffer containing proteinase and phosphatase inhibitors. Protein was quantified using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (80 µg per lane) were resolved by sodium dodecyl sulfate (SDS)–polyacrylamide gels electrophoresis and transferred to Hybond C nitrocellulose membranes (Amersham Corporation, Arlington Heights, IL). The membranes were probed with COX-2, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, c-fos, c-jun, or PKC antibodies overnight at 4°C and incubated for 1 h with secondary peroxidase–conjugated antibodies. They were developed with an enhanced chemiluminescence system (Amersham Corporation) and exposed to an x-ray film (Fuji Photo Film Co., Ltd, Tokyo, Japan). The correct molecular weight was confirmed with the molecular weight marker, and quantitation was carried out with a video densitometer (Scan Maker III, Microtek, Carson, CA).
Semiquantitative RT-PCR analysis.
The total cellular RNA was isolated from AGS cells using Trizol reagent. The RNA concentration was measured by GeneQuant II (Pharmacia, Uppsala, Sweden) at 260 nm. The same amount of total RNA (3 µg) was used to generate the first strand of cDNA by reverse transcription (Invitrogen) in accordance with the manufacturer's instructions. The PCR primers were as follows: COX-2, 5'-ACTGCGCCTTTTCAAGGATG-3' (sense), 5'-CATCACCCCATTCAGGATGC-3' (antisense); ß1-adrenergic receptor, 5'-CAAGTGCTGCGACTTCGTCACC-3' (sense), 5'-GCCGAGGAAACGGCGCTC-3' (antisense); ß2-adrenergic receptor, 5'-ACGCAGCAAAGGGACGAG-3' (sense), 5'-CACACCATCAGAATGATCAC-3' (antisense); and GAPDH, 5'-ACGGATTTGGTCGTATTGGG-3' (sense), 5'-TGATTTGGAGGGATCTCGC-3' (antisense). The amplification step includes 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. The correct molecular weight was confirmed by a molecular weight marker and normalized with GAPDH as the loading control.
COX-2 activity.
AGS cells were collected using a scraper and centrifuged at 1500 x g for 10 min at 4°C. Cell lysates were sonicated in ice-cold buffer containing 0.1M Tris-HCl (pH 7.8), 1mM EDTA, 250mM mannitol, and 0.3mM diethyldithiocarbamic acid and centrifuged at 10,000 x g for 15 min at 4°C. The supernatant was used to determine the peroxidase activity of COX using the COX activity assay kit (Cayman Chemical Company, Ann Arbor, MI). The peroxidase activity was measured spectrophotometrically by determining the amount of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine at 590 nm according to the manufacturer's instructions.
PGE2 assay.
Cell lysates were homogenized in enzyme immunoassay buffer (containing 50mM Tris-HCl at pH 7.4, 100mM NaCl, 1mM CaCl2, 1 mg/ml D-glucose, and 28µM indomethacin) for 30 s on ice. Samples were then centrifuged for 15 min at 9300 x g at 4°C. The supernatant was used for the determination of PGE2 using the PGE2 immunoassay kit (R&D Systems Inc.) according to the manufacturer's instructions.
3H-thymidine incorporation assay.
A modified 3H-thymidine incorporation assay was used to determine the amount of DNA synthesis as previously described (Wu et al., 2006). Cells were incubated in the absence or presence of nicotine (432µM) for 5 h and then incubated with 0.5 µCi/ml 3H-thymidine (Amersham Corporation) for another 5 h. Then it was washed with ice-cold 0.15M NaCl followed by 10% trichloroacetic acid and incubated for 15 min at room temperature. After several washings, 1% SDS was added and incubated for another 15 min at 37°C. Finally, hydrophilic scintillation fluid was added into the vial and the amount of DNA synthesized was measured using liquid scintillation spectrometry on a beta-counter (Beckman Instruments, Fullerton, CA).
Cell viability.
Cell viability was measured by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method as previously described (Ozkan and Fiskin, 2006). In brief, after incubation with nicotine at different concentrations, cells were incubated with 2.5% MTT solution (5 mg/ml) for another 4 h at 37°C. Thereafter, 0.04M HCl-isopropanol was added into the mixture and mixed thoroughly. The color change was recorded using spectrophotometry with the microplate reader (MRX, Dynex Technologies Inc., Sullyfield Circle Chantilly, VA) at 570 nm. The same test was repeated three times, and the optical density was calculated for statistical analysis.
Statistical analysis.
Results were expressed as the mean ± SEM. Statistical analysis was performed with an ANOVA followed by the Tukey's t-test. Those p values that were less than 0.05 were considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nicotine Increased COX-2 mRNA Expression and Activity
As upregulation of COX-2 expression is involved in the pathogenesis of gastric cancer, the mRNA expression and activity of COX-2 in response to nicotine treatment were determined. Results showed that nicotine (22–432µM) markedly increased the COX-2 mRNA expression (Fig. 1A) and activity (Fig. 1B) in AGS cells after a 5-h incubation (22–432µM). These results were in line with our previous finding that nicotine dose dependently stimulated gastric cancer cell proliferation, accompanied by a marked increase in COX-2 protein expression and PGE2 release (Shin et al., 2004
).
|
U0126 Abolished Nicotine-Induced ERK1/2 Phosphorylation, COX-2 Expression, and Cell Proliferation
As MAPKs might be involved in the signal transduction pathway activated by nicotine, the alternation of phosphorylation levels of ERK1/2, JNK, and p38 was determined. Results showed that treating AGS cells with nicotine for 5 h dose dependently increased ERK1/2 phosphorylation. The maximal increase in ERK1/2 phosphorylation was observed at the dose of 432µM (Fig. 2A). In contrast, the phosphorylation levels of JNK and p38 were not altered by nicotine treatment. As ERK1/2 signaling is generally involved in cell proliferation, the above finding implicated that nicotine might promote gastric cancer growth through phosphorylation of ERK1/2. To this end, pretreating AGS cells with U0126, a specific ERK1/2 inhibitor, at the doses of 5–20µM effectively abolished nicotine-induced ERK1/2 phosphorylation (Fig. 2B), COX-2 protein expression (Fig. 2C), and cell proliferation (Fig. 2D).
|
Staurosporine Inhibited Nicotine-Induced ERK1/2/COX-2 Signaling
Activation of PKC/MAPK cascade is known to be involved in cancer growth and progression (Stewart and O'Brian, 2005
). To determine whether there is a possible linkage between PKC and ERK1/2 in the mitogenic signaling pathway activated by nicotine in gastric cancer cells, staurosporine (a potent PKC inhibitor) was used to determine the effect of PKC inhibition on the activation of ERK1/2/COX-2 cascade. Pretreating the cells with staurosporine (100–400nM) strongly attenuated ERK1/2 phosphorylation induced by nicotine (Fig. 2B). In addition, staurosporine blocked the nicotine-induced COX-2 expression (Fig. 2C), with a concomitant decrease in cell proliferation (Fig. 2D). These findings suggest that nicotine-induced gastric cancer cell proliferation depends on the activation of PKC and the downstream ERK1/2/COX-2 pathway.
Nicotine Induced the Translocation of PKC-ßI and Upregulated Total PKC Expression
To delineate which specific isoformss of PKC were involved in the nicotine-induced gastric cancer cell proliferation, the expression of conventional PKC isoforms in the cell membrane and cytosol was determined. Exposure of cells to nicotine (432µM) for 0, 1, 3, and 5 h resulted in a time-dependent increase in PKC-ßI protein in the membrane fraction, accompanied by a parallel reduction in the cytosolic fraction, suggesting that PKC-ßI translocated from the cytosol to cell membrane upon stimulation with nicotine. The translocation of other PKC isoforms, however, was not affected by nicotine treatment (Fig. 3). Moreover, an upregulation of total PKC expression was also observed in nicotine-treated AGS cells (Fig. 5C).
|
|
Nicotine Upregulated AP-1 Subunits c-fos and c-jun Which Was Reversed by U0126
AP-1, which consists of subunits c-fos and c-jun, is the downstream transcriptional target of ERK1/2 and is involved in induction of COX-2 and tumor promotion (Karin, 1995
; Uenoyama et al., 2006). The expression of c-fos and c-jun in relation to ERK1/2 phosphorylation in nicotine-induced gastric cancer cell proliferation, however, remains unclear. Results showed that nicotine dose dependently upregulated the expression of AP-1 subunits c-fos and c-jun, with a maximum increase in expression starting from 108µM and 216µM, respectively (Fig. 4A). To determine whether MAPKs activation contributes to c-fos and c-jun upregulation, the effect of U0126 (ERK1/2 inhibitor) and SB203580 (p38 inhibitor) on AP-1 expression induced by nicotine was determined. Results showed that U0126 but not SB203580 blocked the nicotine-induced upregulation of c-fos and c-jun (Fig. 4B), implicating that the mitogenic effect of ERK1/2 signaling evoked by nicotine was AP-1 dependent.
|
Blockade of ß-Adrenoceptors Abrogated Mitogenic Signals Evoked by Nicotine
In order to elucidate which receptor was involved in nicotine-induced cell growth,
-BTX (7-nAChR antagonist) and propranolol (nonspecific ß-adrenoceptor antagonist) were used to block the respective receptor. Pretreatment of cells with propranolol for 45 min attenuated the nicotine-induced gastric cancer cell proliferation. In contrast, -BTX had no effect on nicotine-induced gastric cancer cell proliferation (Fig. 5A). To further examine which specific ß-adrenoceptor subtype was involved in mediating the mitogenic signal evoked by nicotine, atenolol (ß1-adrenoceptor antagonist) and ICI 118,551 (ß2-adrenoceptor antagonist) were selected. Results showed that both atenolol and ICI 118,551 blocked nicotine-induced AGS cell proliferation. ICI 118,551 (10µM), however, exhibited a stronger inhibitory action with a significant reduction of cell proliferation by more than 50% (Fig. 5B). Both atenolol and ICI 118,551 also markedly reduced the expressions of total PKC, phosphorylation of ERK1/2, and upregulation of COX-2, in which ICI 118,551 produced a more prominent response (Fig. 5C). A similar reduction of PGE2 levels by atenolol and ICI 118,551 in nicotine-induced AGS cells was also observed (Fig. 5D). These results indicate that the stimulatory action of nicotine on gastric cancer cell proliferation possibly involves the activation of ß-adrenoceptors and the downstream PKC/ERK1/2/COX-2/PGE2 pathway (Fig. 6).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cigarette smoking is a major risk factor for gastric cancer in which COX-2 is known to play a critical role in the modulation of inflammation and tumorigenesis (Li et al., 2006
; Saukkonen et al., 2003). We recently reported that nicotine in drinking water increased the growth of orthotopically implanted gastric cancer in association with upregulation of COX-2 in athymic mice (Shin et al., 2004). The molecular mechanism involving the regulation of COX-2 expression by nicotine, however, is poorly understood. In the present study, we further demonstrated that nicotine induced gastric cancer growth through the PKC/ERK1/2/COX-2 pathway. All these evidences not only support the notion that nicotine may contribute to the carcinogenesis of smoking-related gastric cancer but also partially delineate the regulatory mechanism of COX-2 expression in gastric cancer cells.
PKC has been implicated in processes related to cancer development, such as modulation of tumor cell metastasis and apoptosis. The contributory role of PKC in the carcinogenesis of gastrointestinal tumor is further substantiated by the fact that chemoprophylactic drugs such as resveratrol suppressed gastric cancer cells through the PKC-–mediated pathway (Atten et al., 2001). An earlier study also demonstrated that safingol, a PKC inhibitor, suppressed gastric cancer growth by inducing apoptosis (Schwartz et al., 1995). In the colon, PKC-ßII is known to promote carcinogenesis through the activation of COX-2 and Raf/MAPK pathway (Gokmen-Polar et al., 2001; Zhang et al., 2004). All these studies highlight the significance of PKC isoforms in gastrointestinal cancers. Here we also provide evidence that nicotine-mediated PKC-ßI translocation is responsible for ERK1/2 phosphorylation and COX-2 upregulation, resulting in increased gastric cancer cell proliferation (Fig. 2). However, the mechanism by which PKC is activated remained elusive.
MAPKs are serine/threonine kinases that are present in most cell types and have been reported to regulate COX-2 gene expression (Chun and Surh, 2004). Our results clearly demonstrated that nicotine stimulated gastric cancer cell proliferation, along with ERK1/2 phosphorylation, but had no effect on JNK and p38 activities (Fig. 2A). These findings indicate that JNK and p38 are not involved in gastric cancer growth promoted by nicotine. U0126 (ERK1/2 inhibitor) not only blocked the nicotine-induced ERK1/2 phosphorylation but also the COX-2 induction (Fig. 2B and 2C). In another study, lipopolysaccharides-induced COX-2 expression in macrophages was also abrogated by U0126 (Scherle et al., 1998). These findings further support the notion that COX-2 expression is regulated by ERK1/2. In this connection, we hypothesized that AP-1 might be involved in the upregulation of COX-2 in relation to ERK1/2 phosphorylation during gastric carcinogenesis. In this regard, it was known that AP-1 activity was determined by the abundance of AP-1 subunits c-fos and c-jun and is involved in the induction of COX-2 and tumor growth (Karin, 1995; Uenoyama et al., 2006). The present data showed that there was a marked increase in the levels of c-fos and c-jun after a 5-h nicotine treatment (Fig. 4A). Furthermore, pretreatment with U0126 significantly reduced the upregulation of c-fos and c-jun (Fig. 4B), implicating that nicotine-mediated ERK1/2 phosphorylation could lead to an upregulation of these AP-1 subunits and subsequently stimulate COX-2 expression.
Previous reports showed that neuronal AChRs (7-nAChR) and ß-adrenoceptors might be involved in the mediation of the effect of nicotinic stimulation on cancer growth (Park et al., 1995; Tsurutani et al., 2005). The involvement of these receptors in gastric cancer cell proliferation promoted by nicotine remains undefined. To this end, the present study revealed that propranolol (nonselective ß-adrenoceptor antagonist), but not -BTX (7-nAChR antagonist), suppressed the stimulatory action of nicotine on AGS cell proliferation (Fig. 5A). Selective antagonists of ß1- and ß2-adrenoceptors also completely blocked the effect of nicotine on gastric cancer cell growth, implicating that the stimulatory effect of nicotine was solely mediated through the ß-adrenoceptors. Indeed, it has been reported that nicotine can function as a ß-adrenergic receptor agonist and the effect is abrogated by a nonspecific ß-adrenergic receptor blocker propranolol but not by -BTX, the 7-nAChR–specific inhibitor, on nicotine-induced Bad phosphorylation in a pulmonary adenocarcinoma cell line (Jin et al., 2004). More noteworthy was that the blockade of ß-adrenoceptors also reduced cell proliferation far below the basal control level, suggesting that activation of the same type of receptors could be involved in control of basal cancer growth (Fig. 5B). Furthermore, atenolol and ICI 118,551 blocked the regulatory action of nicotine on PKC/ERK1/2/COX-2 pathway, suggesting that ß-adrenoceptors are closely associated with this signal pathway in the promotion of gastric cancer development. Nevertheless, it is worthwhile to notice that the concentration of nicotine we used in the current study is on the high side, when compared with the neuroscience literature in which nicotine binds to nAChRs but not ß-adrenoceptors (Messi et al., 1997). Nicotine at such a high concentration could activate ß-adrenoceptors, and whether the ß-adrenergic activity induced by nicotine was mediated specifically through the direct ligand-receptor interaction or through a yet undefined signaling pathway leading to transactivation of ß-adrenoceptors requires further investigation.
Here we report a unique picture of smoking-related gastric tumorigenesis. Nicotine can promote gastric cancer cell growth through the ß-adrenoceptors followed by the activation of PKC-ßI/ERK1/2/COX-2/PGE2–signaling pathway (Fig. 6). Indeed, there are reports showing that the derivative of nicotine, NNK, could induce cancer growth also via the ß-adrenoceptor activation (Schuller et al., 1999; Wu et al., 2005). Moreover, the ß-adrenergic agonist isoproterenol has been shown to exert cancer-promoting effects in human pulmonary and colon adenocarcinoma cell lines (Schuller and Cole, 1989; Wu et al., 2005). All these findings along with the present experimental data suggest that ß-adrenergic antagonism may be an effective therapeutic and chemopreventive approach to treat and prevent smoking-related gastrointestinal cancers.
|
ACKNOWLEDGMENTS |
|---|
This work is supported by The Committee on Research and Conference Grants of the University of Hong Kong and the Hong Kong Research Grants Council (HKU 7499/05M).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Atten MJ, Attar BM, Milson T, Holian O. (2001) Resveratrol-induced inactivation of human gastric adenocarcinoma cells through a protein kinase C-mediated mechanism. Biochem. Pharmacol. 62:1423–1432.[CrossRef][ISI][Medline]
Bang YJ, Kwon JH, Kang SH, Kim JW, Yang YC. (1998) Increased MAPK activity and MKP-1 overexpression in human gastric adenocarcinoma. Biochem. Biophys. Res. Commun. 250:43–47.[CrossRef][ISI][Medline]
Chun KS and Surh YJ. (2004) Signal transduction pathways regulating cyclooxygenase-2 expression: Potential molecular targets for chemoprevention. Biochem. Pharmacol. 68:1089–1100.[CrossRef][ISI][Medline]
Debidda M, Sanna B, Cossu A, Posadino AM, Tadolini B, Ventura C, Pintus G. (2003) NAMI-A inhibits the PMA-induced ODC gene expression in ECV304 cells: Involvement of PKC/Raf/Mek/ERK signalling pathway. Int. J. Oncol. 23:477–482.[ISI][Medline]
Gokmen-Polar Y, Murray NR, Velasco MA, Gatalica Z, Fields AP. (2001) Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis. Cancer Res. 61:1375–1381.
Gopalakrishna R, Chen ZH, Gundimeda U. (1994) Tobacco smoke tumor promoters, catechol and hydroquinone, induce oxidative regulation of protein kinase C and influence invasion and metastasis of lung carcinoma cells. Proc. Natl. Acad. Sci. U.S.A. 91:12233–12237.
Hug H and Sarre TF. (1993) Protein kinase C isoenzymes: Divergence in signal transduction? Biochem. J. 291:329–343.
Jiang XH and Wong BC. (2003) Cyclooxygenase-2 inhibition and gastric cancer. Curr. Pharm. Des. 9:2281–2288.[CrossRef][ISI][Medline]
Jin Z, Gao F, Flagg T, Deng X. (2004) Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis. J. Biol. Chem. 279:23837–23844.
Jull BA, Plummer HK III, Schuller HM. (2001) Nicotinic receptor-mediated activation by the tobacco-specific nitrosamine NNK of a Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc in human small cell lung carcinoma cells and pulmonary neuroendocrine cells. J. Cancer Res. Clin. Oncol. 127:707–717.[ISI][Medline]
Karin M. (1995) The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270:16483–16486.
Kawabe A, Shimada Y, Uchida S, Maeda M, Yamasaki S, Kato M, Hashimoto Y, Ohshio G, Matsumoto M, Imamura M. (2002) Expression of cyclooxygenase-2 in primary and remnant gastric carcinoma: Comparing it with p53 accumulation, Helicobacter pylori infection, and vascular endothelial growth factor expression. J. Surg. Oncol. 80:79–88.[CrossRef][ISI][Medline]
Koehne CH and Dubois RN. (2004) COX-2 inhibition and colorectal cancer. Semin. Oncol. 31:12–21.[ISI][Medline]
Li GQ, Xia HH, Chen MH, Gu Q, Wang JD, Peng JZ, Chan AO, Cho CH, So HL, Lam SK, et al. (2006) Effects of cyclooxygenase-1 and -2 gene disruption on Helicobacter pylori-induced gastric inflammation. J. Infect. Dis. 193:1037–1046.[CrossRef][ISI][Medline]
Li H, Oehrlein SA, Wallerath T, Ihrig-Biedert I, Wohlfart P, Ulshofer T, Jessen T, Herget T, Forstermann U, Kleinert H. (1998) Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene. Mol. Pharmacol. 53:630–637.
Li JY, Wang XZ, Chen FL, Yu JP, Luo HS. (2003) Nimesulide inhibits proliferation via induction of apoptosis and cell cycle arrest in human gastric adenocarcinoma cell line. World J. Gastroenterol. 9:915–920.[ISI][Medline]
Messi ML, Renganathan M, Grigorenko E, Delbono O. (1997) Activation of alpha7 nicotinic acetylcholine receptor promotes survival of spinal cord motoneurons. FEBS Lett. 411:322–328.[CrossRef][ISI][Medline]
Ozkan A and Fiskin K. (2006) Protective effect of antioxidant enzymes against drug cytotoxicity in MCF-7 cells. Exp. Oncol. 28:86–88.[Medline]
Park PG, Merryman J, Orloff M, Schuller HM. (1995) Beta-adrenergic mitogenic signal transduction in peripheral lung adenocarcinoma: Implications for individuals with preexisting chronic lung disease. Cancer Res. 55:3504–3508.
Sasazuki S, Sasaki S, Tsugane S. (2002) Japan Public Health Center Study Group. Cigarette smoking, alcohol consumption and subsequent gastric cancer risk by subsite and histologic type. Int. J. Cancer 101:560–566.[CrossRef][ISI][Medline]
Saukkonen K, Rintahaka J, Sivula A, Buskens CJ, Van Rees BP, Rio MC, Haglund C, Van Lanschot JJ, Offerhaus GJ, Ristimaki A. (2003) Cyclooxygenase-2 and gastric carcinogenesis. Acta Pathol. Microbiol. Immunol. Scand. 111:915–925.
Scherle PA, Jones EA, Favata MF, Daulerio AJ, Covington MB, Nurnberg SA, Magolda RL, Trzaskos JM. (1998) Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes. J. Immunol. 161:5681–5686.
Schuller HM. (1994) Carbon dioxide potentiates the mitogenic effects of nicotine and its carcinogenic derivative, NNK, in normal and neoplastic neuroendocrine lung cells via stimulation of autocrine and protein kinase C-dependent mitogenic pathways. Neurotoxicology 15:877–886.[ISI][Medline]
Schuller HM and Cole B. (1989) Regulation of cell proliferation by beta-adrenergic receptors in a human lung adenocarcinoma cell line. Carcinogenesis 10:1753–1755.
Schuller HM, Jull BA, Sheppard BJ, Plummer HK. (2000a) Interaction of tobacco-specific toxicants with the neuronal alpha(7) nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: Potential role in lung carcinogenesis and pediatric lung disorders. Eur. J. Pharmacol. 393:265–277.[CrossRef][ISI][Medline]
Schuller HM, Porter B, Riechert A. (2000b) Beta-adrenergic modulation of NNK-induced lung carcinogenesis in hamsters. J. Cancer Res. Clin. Oncol. 126:624–630.[CrossRef][ISI][Medline]
Schuller HM, Tithof PK, Williams M, Plummer H III. (1999) The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a beta-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via beta-adrenergic receptor-mediated release of arachidonic acid. Cancer Res. 59:4510–4515.
Schwartz GK, Haimovitz-Friedman A, Dhupar SK, Ehleiter D, Maslak P, Lai L, Loganzo F Jr, Kelsen DP, Fuks Z, Albino AP. (1995) Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells. J. Natl Cancer Inst. 87:1394–1399.
Shin VY, Wu WK, Chu KM, Wong HP, Lam EK, Tai EK, Koo MW, Cho CH. (2005) Nicotine induces cyclooxygenase-2 and vascular endothelial growth factor receptor-2 in association with tumor-associated invasion and angiogenesis in gastric cancer. Mol. Cancer Res. 3:607–615.
Shin VY, Wu WK, Ye YN, So WH, Koo MW, Liu ES, Luo JC, Cho CH. (2004) Nicotine promotes gastric tumor growth and neovascularization by activating extracellular signal-regulated kinase and cyclooxygenase-2. Carcinogenesis 25:2487–2495.
Stadtlander CT and Waterbor JW. (1999) Molecular epidemiology, pathogenesis and prevention of gastric cancer. Carcinogenesis 20:2195–2208.
Stewart JR and O'Brian CA. (2005) Protein kinase C-{alpha} mediates epidermal growth factor receptor transactivation in human prostate cancer cells. Mol. Cancer Ther. 4:726–732.
Tsurutani J, Castillo SS, Brognard J, Granville CA, Zhang C, Gills JJ, Sayyah J, Dennis PA. (2005) Tobacco components stimulate Akt-dependent proliferation and NFkappaB-dependent survival in lung cancer cells. Carcinogenesis 26:1182–1195.
Uenoyama Y, Seno H, Fukuda A, Sekikawa A, Nanakin A, Sawabu T, Kawada M, Kanda N, Suzuki K, Yada N, et al. (2006) Hypoxia induced by benign intestinal epithelial cells is associated with cyclooxygenase-2 expression in stromal cells through AP-1-dependent pathway. Oncogene 25:3277–3285.[CrossRef][ISI][Medline]
Wong BC, Jiang XH, Lin MC, Tu SP, Cui JT, Jiang SH, Wong WM, Yuen MF, Lam SK, Kung HF. (2004) Cyclooxygenase-2 inhibitor (SC-236) suppresses activator protein-1 through c-Jun NH2-terminal kinase. Gastroenterology 126:136–147.[CrossRef][ISI][Medline]
Wu WK, Li GR, Wong HP, Hui MK, Tai EK, Lam EK, Shin VY, Ye YN, Li P, Yang YH, et al. (2006) Involvement of Kv1.1 and Nav1.5 in proliferation of gastric epithelial cells. J. Cell. Physiol. 207:437–444.[CrossRef][ISI][Medline]
Wu WK, Wong HP, Luo SW, Chan K, Huang FY, Hui MK, Lam EK, Shin VY, Ye YN, Yang YH, et al. (2005) 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via ß-adrencoceptors. Cancer Res. 65:5272–5277.
Zhang J, Anastasiadis PZ, Liu Y, Thompson EA, Fields AP. (2004) Protein kinase C (PKC) betaII induces cell invasion through a Ras/Mek-, PKC iota/Rac 1-dependent signaling pathway. J. Biol. Chem. 279:22118–22123.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R.-J. Chen, Y.-S. Ho, H.-R. Guo, and Y.-J. Wang Rapid Activation of Stat3 and ERK1/2 by Nicotine Modulates Cell Proliferation in Human Bladder Cancer Cells Toxicol. Sci., August 1, 2008; 104(2): 283 - 293. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||