Tumor Biology

, Volume 36, Issue 12, pp 9537–9544 | Cite as

Alpha 7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to 5-fluorouracil

  • Wei-Yu Chen
  • Chien-Yu Huang
  • Wan-Li Cheng
  • Chin-Sheng Hung
  • Ming-Te Huang
  • Cheng-Jeng Tai
  • Yen-Nien Liu
  • Chi-Long Chen
  • Yu-Jia Chang
Research Article


Gastric cancer is the second most common cause of cancer mortality worldwide. Most gastric cancer patients are asymptomatic until the advanced stages, for which current therapeutic treatments are suboptimal. 5-Fluorouracil (5-FU), an antimetabolite agent, is widely used in gastric cancer therapy. However, the presence of drug resistance in gastric cancer patients reduces the cytotoxic activity of 5-FU. In gastric cancer, no research has yet been conducted to analyze the effect of alpha 7-nicotinic acetylcholine receptor (A7-nAChR) on the therapeutic response to 5-FU. In this study, we generated A7-nAChR knockdown (A7-nAChR-KD) AGS cells by a small interfering RNA (siRNA) technique in gastric cancer cells. The anti-proliferative effects of 5-FU were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and cell cycle determination. We found that A7-nAChR-KD cells were more resistant to 5-FU treatment compared with the scrambled control cells according to the MTT assay. The apoptotic cell population was increased more in scrambled control cells treated with 5-FU than A7-nAChR-KD cells according to the cell cycle distribution and TUNEL assays. We analyzed expression levels of survival and apoptosis-associated proteins (pAkt, Akt, Mcl-1, Bcl-2, Bad, and Bax) altered by 5-FU treatment. Survival and antiapoptosis signaling (pAkt, Akt, Mcl-1 and Bcl-2) was downregulated, and the proapoptotic proteins (Bad and Bax) were upregulated in 5-FU-treated control cells but expression levels of Bcl-2, Bad, and Bad were not altered in 5-FU-treated A7-nAChR-KD cells. This is consistent with A7-nAChR-KD cells exhibiting more resistance to 5-FU treatment. In our study, we carried out an in vitro study on AGS gastric cancer cell line to elucidate the anticancer efficacy and molecular mechanisms of A7-nAChR silencing on 5-FU-induced cell death. The results clearly showed that depletion of A7-nAChR suppressed the drug sensitivity of gastric cancer cells to 5-FU treatment.


A7-nAChR Gastric cancer 5-Fluorouracil 5-FU 



Alpha 7-nicotinic acetylcholine receptor



This grant was supported by Ministry of Science and Technology, Taiwan (NSC101-2314-B-038-016-MY3).


  1. 1.
    Park SC, Chun HJ. Chemotherapy for advanced gastric cancer: review and update of current practices. Gut Liver. 2013;7:385–93.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wagner AD, Unverzagt S, Grothe W, Kleber G, Grothey A, Haerting J, et al. Chemotherapy for advanced gastric cancer. Cochrane Database Syst Rev. CD004064.Google Scholar
  3. 3.
    Mizuarai S, Yamanaka K, Kotani H. Mutant p53 induces the gef-h1 oncogene, a guanine nucleotide exchange factor-h1 for rhoa, resulting in accelerated cell proliferation in tumor cells. Cancer Res. 2006;66:6319–26.CrossRefPubMedGoogle Scholar
  4. 4.
    Longley DB, Harkin DP, Johnston PG. 5-Fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003;3:330–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Chu E, Callender MA, Farrell MP, Schmitz JC. Thymidylate synthase inhibitors as anticancer agents: from bench to bedside. Cancer Chemother Pharmacol. 2003;52 Suppl 1:S80–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Vinod BS, Antony J, Nair HH, Puliyappadamba VT, Saikia M, Narayanan SS, et al. Mechanistic evaluation of the signaling events regulating curcumin-mediated chemosensitization of breast cancer cells to 5-fluorouracil. Cell Death Dis. 2013;4:e505.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Grando SA. Connections of nicotine to cancer. Nat Rev Cancer. 2014;14:419–29.CrossRefPubMedGoogle Scholar
  8. 8.
    Tabuchi T, Ito Y, Ioka A, Nakayama T, Miyashiro I, Tsukuma H. Tobacco smoking and the risk of subsequent primary cancer among cancer survivors: a retrospective cohort study. Ann Oncol. 2013;24:2699–704.CrossRefPubMedGoogle Scholar
  9. 9.
    Shen T, Le W, Yee A, Kamdar O, Hwang PH, Upadhyay D. Nicotine induces resistance to chemotherapy in nasal epithelial cancer. Am J Rhinol Allergy. 2010;24:e73–7.CrossRefPubMedGoogle Scholar
  10. 10.
    Cucina A, Dinicola S, Coluccia P, Proietti S, D'Anselmi F, Pasqualato A, et al. Nicotine stimulates proliferation and inhibits apoptosis in colon cancer cell lines through activation of survival pathways. J Surg Res. 2012;178:233–41.CrossRefPubMedGoogle Scholar
  11. 11.
    Dinicola S, Morini V, Coluccia P, Proietti S, D'Anselmi F, Pasqualato A, et al. Nicotine increases survival in human colon cancer cells treated with chemotherapeutic drugs. Toxicol In Vitro. 2013;27:2256–63.CrossRefPubMedGoogle Scholar
  12. 12.
    Chang YJ, Chiu CC, Wu CH, An J, Wu CC, Liu TZ, et al. Glucose-regulated protein 78 (grp78) silencing enhances cell migration but does not influence cell proliferation in hepatocellular carcinoma. Ann Surg Oncol. 2010;17:1703–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Chiou JF, Tai CJ, Huang MT, Wei PL, Wang YH, An J, et al. Glucose-regulated protein 78 is a novel contributor to acquisition of resistance to sorafenib in hepatocellular carcinoma. Ann Surg Oncol. 2010;17:603–12.CrossRefPubMedGoogle Scholar
  14. 14.
    Lien YC, Wang W, Kuo LJ, Liu JJ, Wei PL, Ho YS, et al. Nicotine promotes cell migration through alpha7 nicotinic acetylcholine receptor in gastric cancer cells. Ann Surg Oncol. 2011;18:2671–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Wei PL, Chang YJ, Ho YS, Lee CH, Yang YY, An J, et al. Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor. Ann Surg. 2009;249:978–85.CrossRefPubMedGoogle Scholar
  16. 16.
    Wei PL, Kuo LJ, Huang MT, Ting WC, Ho YS, Wang W, et al. Nicotine enhances colon cancer cell migration by induction of fibronectin. Ann Surg Oncol. 2011;18-1782-90.Google Scholar
  17. 17.
    Wang SK, Liang PH, Astronomo RD, Hsu TL, Hsieh SL, Burton DR, et al. Targeting the carbohydrates on hiv-1: interaction of oligomannose dendrons with human monoclonal antibody 2g12 and dc-sign. Proc Natl Acad Sci U S A. 2008;105:3690–5.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Kohler K, et al. Membrane nanotubes physically connect t cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol. 2008;10:211–9.CrossRefPubMedGoogle Scholar
  19. 19.
    McLean MH, El-Omar EM. Genetics of gastric cancer. Nat Rev Gastroenterol Hepatol. 2014;11:664–74.CrossRefPubMedGoogle Scholar
  20. 20.
    Ladeiras-Lopes R, Pereira AK, Nogueira A, Pinheiro-Torres T, Pinto I, Santos-Pereira R, et al. Smoking and gastric cancer: systematic review and meta-analysis of cohort studies. Cancer Causes Control. 2008;19:689–701.CrossRefPubMedGoogle Scholar
  21. 21.
    Hecht SS, Carmella SG, Chen M, Dor Koch JF, Miller AT, Murphy SE, et al. Quantitation of urinary metabolites of a tobacco-specific lung carcinogen after smoking cessation. Cancer Res. 1999;59:590–6.PubMedGoogle Scholar
  22. 22.
    Wolfgang CL, Herman JM, Laheru DA, Klein AP, Erdek MA, Fishman EK, et al. Recent progress in pancreatic cancer. CA Cancer J Clin. 2013;63:318–48.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Smedlund K, Tano JY, Margiotta J, Vazquez G. Evidence for operation of nicotinic and muscarinic acetylcholine receptor-dependent survival pathways in human coronary artery endothelial cells. J Cell Biochem. 2011;112:1978–84.CrossRefPubMedGoogle Scholar
  24. 24.
    Schaal C, Chellappan SP. Nicotine-mediated cell proliferation and tumor progression in smoking-related cancers. Mol Cancer Res. 2014;12:14–23.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Shin VY, Wu WK, Chu KM, Wong HP, Lam EK, Tai EK, et al. 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. 2005;3:607–15.CrossRefPubMedGoogle Scholar
  26. 26.
    Wang YY, Liu Y, Ni XY, Bai ZH, Chen QY, Zhang Y, et al. Nicotine promotes cell proliferation and induces resistance to cisplatin by α7 nicotinic acetylcholine receptor-mediated activation in Raw264.7 and E14 cells. Oncol Rep. 2014;31:1480–8.PubMedGoogle Scholar
  27. 27.
    Guha P, Bandyopadhyaya G, Polumuri SK, Chumsri S, Gade P, Kalvakolanu DV, et al. Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin, α9 nicotinic acetylcholine receptor and STAT3. Breast Cancer Res Treat. 2014;145:5–22.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Chen RJ, Ho YS, Guo HR, Wang YJ. Long-term nicotine exposure-induced chemoresistance is mediated by activation of Stat3 and downregulation of ERK1/2 via nAChR and beta-adrenoceptors in human bladder cancer cells. Toxicol Sci. 2010;115:118–30.CrossRefPubMedGoogle Scholar
  29. 29.
    Trevino JG, Pillai S, Kunigai S, Singh S, Fulp WJ, Centeno BA, et al. Nicotine induces inhibitor of differentiation-1 in a Src-dependent pathway promoting metastasis and chemoresistance in pancreastic adenocarcinoma. Neoplasia. 2012;14:1102–14.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Xian XS, Park H, Choi MG, Park JM. Cannabinoid receptor agonist as an alternative drug in 5-fluorouracil-resistant gastric cancer cells. Anticancer Res. 2013;33:2541–7.PubMedGoogle Scholar
  31. 31.
    Florou AN, Gkiozos IC, Tsagouli SK, Souliotis KN, Syrigos KN. Clinical significance of smoking cessation in subjects with cancer: a 30-year review. Respir Care. 2014;59:1924–36.CrossRefPubMedGoogle Scholar
  32. 32.
    Singh S, Pillai S, Chellappan S. Nicotinic acetylcholine receptor signaling in tumor growth and metastasis. J Oncol. 2011;456743.Google Scholar
  33. 33.
    Warren GW, Sobus S, Gritz ER. The biological and clinical effects of smoking by patients with cancer and strategies to implement evidence-based tobacco cessation support. Lancet Oncol. 2014;15:e568–80.CrossRefPubMedGoogle Scholar
  34. 34.
    Wessler I, Kirkpatrick CJ, Racke K. Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in human. Pharmacol Ther. 1998;77:59–79.CrossRefPubMedGoogle Scholar
  35. 35.
    Valiante S, Capaldo A, Virgilio F, Sciarrillo R, De Falco M, Gay F, et al. Distribution of α7 and α4 nicotinic acetylcholine receptor subunits in several tissues of Triturus carnifex (Amphibia, Urodela). Tissue Cell. 2004;36:391–8.CrossRefPubMedGoogle Scholar
  36. 36.
    Wu CH, Lee CH, Ho YS. Nicotinic acetylcholine receptor-based blockade: applications of molecular targets for cancer therapy. Clin Cancer Res. 2011;17:3533–41.CrossRefPubMedGoogle Scholar
  37. 37.
    Al-Wadei MH, Al-Wadei HA, Schuller HM. Pancreatic cancer cells and normal pancreatic duct epithelial cells express an autocrine catecholamine loop that is activated by nicotinic acetylcholine receptors alpha3, alpha5, and alpha7. Mol Cancer Res. 2012;10:239–49.CrossRefPubMedGoogle Scholar
  38. 38.
    Murakami D, Tsujitani S, Osaki T, Saito H, Katano K, Tatebe S, et al. Expression of phosphorylated Akt (pAkt) in gastric carcinoma predicts prognosis and efficacy of chemotherapy. Gastric Cancer. 2007;10:45–51.CrossRefPubMedGoogle Scholar
  39. 39.
    Oki E, Baba H, Tokunaga E, Nakamura T, Ueda N, Futatsugi M, et al. Akt phosphorylation associates with LOH of PTEN and leads to chemoresistance for gastric cancer. Int J Cancer. 2005;117:376–80.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Wei-Yu Chen
    • 1
    • 2
    • 3
  • Chien-Yu Huang
    • 4
    • 5
  • Wan-Li Cheng
    • 6
  • Chin-Sheng Hung
    • 1
    • 7
    • 8
    • 9
  • Ming-Te Huang
    • 4
    • 7
    • 8
  • Cheng-Jeng Tai
    • 6
    • 12
  • Yen-Nien Liu
    • 10
  • Chi-Long Chen
    • 1
    • 2
    • 11
  • Yu-Jia Chang
    • 1
    • 7
    • 8
    • 9
  1. 1.Graduate Institute of Clinical Medicine, College of MedicineTaipei Medical UniversityTaipei CityTaiwan
  2. 2.Department of Pathology, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  3. 3.Department of Pathology, Wan Fang HospitalTaipei Medical UniversityTaipeiTaiwan
  4. 4.Division of General Surgery, Department of Surgery, Shuang Ho HospitalTaipei Medical UniversityTaipeiTaiwan
  5. 5.Department of Neurosurgery, Shuang Ho HospitalTaipei Medical UniversityTaipeiTaiwan
  6. 6.Department of Internal Medicine, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  7. 7.Department of Surgery, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  8. 8.Division of General Surgery, Department of Surgery, Taipei Medical University HospitalTaipei Medical UniversityTaipeiTaiwan
  9. 9.Cancer Research Center, Taipei Medical University HospitalTaipei Medical UniversityTaipeiTaiwan
  10. 10.Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and TechnologyTaipei Medical UniversityTaipeiTaiwan
  11. 11.Department of Pathology, Taipei Medical University HospitalTaipei Medical UniversityTaipeiTaiwan
  12. 12.Division of Hematology and Oncology, Department of Internal MedicineTaipei Medical University HospitalTaipeiTaiwan

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