Tumor Biology

, Volume 37, Issue 7, pp 9493–9501 | Cite as

Silencing A7-nAChR levels increases the sensitivity of gastric cancer cells to ixabepilone treatment

  • Chao-Chiang Tu
  • Chien-Yu Huang
  • Wan-Li Cheng
  • Chin-Sheng Hung
  • Yu-Jia Chang
  • Po-Li Wei
Original Article


Gastric cancer is an important health issue worldwide. Currently, improving the therapeutic efficacy of chemotherapy drugs is an important goal of cancer research. Alpha-7 nicotine acetylcholine receptor (A7-nAChR) is the key molecule that mediates gastric cancer progression, metastasis, and therapy responses; however, the role of A7-nAChR in the therapeutic efficacy of ixabepilone remains unclear. A7-nAChR expression was silenced by small interfering RNA (siRNA) technology. The cytotoxicity of ixabepilone was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and ixabepilone-induced apoptosis was analyzed by flow cytometry and annexin V/propidium iodide (PI) apoptotic assay. The expression patterns of anti-apoptotic proteins (AKT, phospho-AKT, Mcl-1, and Bcl-2) and pro-apoptotic proteins (Bad and Bax) were determined by western blot. Our study found that A7-nAChR knockdown (A7-nAChR-KD) AGS cells were more sensitive to ixabepilone administration than scrambled control AGS cells. We found that A7-nAChR knockdown enhanced ixabepilone-induced cell death as evidenced by the increased number of annexin V-positive (apoptotic) cells. After scrambled control and A7-nAChR-KD cells were treated with ixabepilone, we found that pAKT and AKT levels were significantly reduced in both groups of cells. The levels of Bcl-2 and the anti-apoptotic Mcl-1 isoform increased dramatically after ixabepilone treatment in scrambled control cells but not in A7-nAChR-KD cells. Bad and Bax levels did not change between the treatment group and vehicle group in both A7-nAChR-KD and scrambled control cells, whereas cleaved PARP levels dramatically increased in ixabepilone-treated A7-nAChR-KD cells. Our results demonstrated that knockdown of A7-nAChR enhanced the sensitivity of gastric cancer cells to ixabepilone administration. Thus, the A7-nAChR expression level in patients with gastric cancer may be a good indicator of ixabepilone sensitivity.


Nicotine acetylcholine receptor Gastric cancer Ixabepilone 



Alpha-7 nicotine acetylcholine receptor



This work was supported by a grant from the National Science Council, Taiwan (Grant No. NSC101-2314-B-038-016-MY3).


  1. 1.
    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.CrossRefPubMedGoogle Scholar
  2. 2.
    Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int J Cancer. 2006;118:3030–44.CrossRefPubMedGoogle Scholar
  3. 3.
    de la Torre BA, Kettenhofen Enriquez W, Roesch Dietlen F, Rodriguez Moguel L, Mejia Novelo A, Peniche BJ. Diagnosis and treatment guideline of gastric cancer. Epidemiology, risk factors, histologic varieties and natural history. Rev Gastroenterol Mex. 2010;75:237–9.Google Scholar
  4. 4.
    Macdonald JS, Smalley SR, Benedetti J, Hundahl SA, Estes NC, Stemmermann GN, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001;345:725–30.CrossRefPubMedGoogle Scholar
  5. 5.
    Graziano F, Catalano V, Baldelli AM, Giordani P, Testa E, Lai V, et al. A phase II study of weekly docetaxel as salvage chemotherapy for advanced gastric cancer. Ann Oncol. 2000;11:1263–6.CrossRefPubMedGoogle Scholar
  6. 6.
    Bang YJ, Kang WK, Kang YK, Kim HC, Jacques C, Zuber E, et al. Docetaxel 75 mg/m(2) is active and well tolerated in patients with metastatic or recurrent gastric cancer: a phase II trial. Jpn J Clin Oncol. 2002;32:248–54.CrossRefPubMedGoogle Scholar
  7. 7.
    Ohtsu A, Boku N, Tamura F, Muro K, Shimada Y, Saigenji K, et al. An early phase II study of a 3-hour infusion of paclitaxel for advanced gastric cancer. Am J Clin Oncol. 1998;21:416–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Bode CJ, Gupta Jr ML, Reiff EA, Suprenant KA, Georg GI, Himes RH. Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules. Biochemistry. 2002;41:3870–4.CrossRefPubMedGoogle Scholar
  9. 9.
    Vahdat L. Ixabepilone: a novel antineoplastic agent with low susceptibility to multiple tumor resistance mechanisms. Oncologist. 2008;13:214–21.CrossRefPubMedGoogle Scholar
  10. 10.
    Cheng KL, Bradley T, Budman DR. Novel microtubule-targeting agents—the epothilones. Biologics. 2008;2:789–811.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Moulder S, Li H, Wang M, Gradishar WJ, Perez EA, Sparano JA, et al. A phase II trial of trastuzumab plus weekly ixabepilone and carboplatin in patients with HER2-positive metastatic breast cancer: an Eastern Cooperative Oncology Group Trial. Breast Cancer Res Treat. 2010;119:663–71.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sparano JA, Vrdoljak E, Rixe O, Xu B, Manikhas A, Medina C, et al. Randomized phase III trial of ixabepilone plus capecitabine versus capecitabine in patients with metastatic breast cancer previously treated with an anthracycline and a taxane. J Clin Oncol. 2010;28:3256–63.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Thomas ES. Ixabepilone plus capecitabine for metastatic breast cancer progressing after anthracycline and taxane treatment. J Clin Oncol. 2008;26:2223.CrossRefPubMedGoogle Scholar
  14. 14.
    Liu G, Chen YH, Dipaola R, Carducci M, Wilding G. Phase II trial of weekly ixabepilone in men with metastatic castrate-resistant prostate cancer (E3803): a trial of the Eastern Cooperative Oncology Group. Clin Genitourin Cancer. 2012;10:99–105.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Harzstark AL, Rosenberg JE, Weinberg VK, Sharib J, Ryan CJ, Smith DC, et al. Ixabepilone, mitoxantrone, and prednisone for metastatic castration-resistant prostate cancer after docetaxel-based therapy: a phase 2 study of the Department of Defense Prostate Cancer Clinical Trials Consortium. Cancer. 2011;117:2419–25.CrossRefPubMedGoogle Scholar
  16. 16.
    Rocha Lima CM, Lin EH, Kim GP, Giguere JK, Marshall J, Zalupski M, et al. A phase 2 trial of ixabepilone plus cetuximab in first-line treatment of metastatic pancreatic cancer. Gastrointest Cancer Res. 2012;5:155–60.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Edelman MJ, Schneider CP, Tsai CM, Kim HT, Quoix E, Luft AV, et al. Randomized phase II study of ixabepilone or paclitaxel plus carboplatin in patients with non-small-cell lung cancer prospectively stratified by beta-3 tubulin status. J Clin Oncol. 2013;31:1990–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Dizon DS, Blessing JA, McMeekin DS, Sharma SK, Disilvestro P, Alvarez RD. Phase II trial of ixabepilone as second-line treatment in advanced endometrial cancer: gynecologic oncology group trial 129-P. J Clin Oncol. 2009;27:3104–8.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Vishnu P, Colon-Otero G, Kennedy GT, Marlow LA, Kennedy WP, Wu KJ, et al. RhoB mediates antitumor synergy of combined ixabepilone and sunitinib in human ovarian serous cancer. Gynecol Oncol. 2012;124:589–97.CrossRefPubMedGoogle Scholar
  20. 20.
    Huang H, Menefee M, Edgerly M, Zhuang S, Kotz H, Poruchynsky M, et al. A phase II clinical trial of ixabepilone (Ixempra; BMS-247550; NSC 710428), an epothilone B analog, in patients with metastatic renal cell carcinoma. Clin Cancer Res. 2010;16:1634–41.CrossRefPubMedGoogle Scholar
  21. 21.
    Ho J, Zhang L, Todorova L, Whillans F, Corey-Lisle P, Yuan Y. Budget impact analysis of ixabepilone used according to FDA approved labeling in treatment-resistant metastatic breast cancer. J Manag Care Pharm. 2009;15:467–75.PubMedGoogle Scholar
  22. 22.
    Guo JZ, Tredway TL, Chiappinelli VA. Glutamate and GABA release are enhanced by different subtypes of presynaptic nicotinic receptors in the lateral geniculate nucleus. J Neurosci. 1998;18:1963–9.PubMedGoogle Scholar
  23. 23.
    Wonnacott S. Presynaptic nicotinic ACh receptors. Trends Neurosci. 1997;20:92–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Dujic Z, Roerig DL, Schedewie HK, Kampine JP, Bosnjak ZJ. Presynaptic modulation of ganglionic ACh release by muscarinic and nicotinic receptors. Am J Physiol. 1990;259:R288–93.PubMedGoogle Scholar
  25. 25.
    Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 2001;411:269–76.CrossRefPubMedGoogle Scholar
  26. 26.
    Dasgupta P, Rizwani W, Pillai S, Kinkade R, Kovacs M, Rastogi S, et al. Nicotine induces cell proliferation, invasion and epithelial-mesenchymal transition in a variety of human cancer cell lines. Int J Cancer. 2009;124:36–45.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Heeschen C, Weis M, Aicher A, Dimmeler S, Cooke JP. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. J Clin Invest. 2002;110:527–36.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Pavlov VA, Wang H, Czura CJ, Friedman SG, Tracey KJ. The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Mol Med. 2003;9:125–34.PubMedPubMedCentralGoogle Scholar
  29. 29.
    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
  30. 30.
    Tu CC, Huang CY, Cheng WL, Hung CS, Uyanga B, Wei PL, Chang YJ. The alpha7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to taxanes. Tumour Biol. 2015Google Scholar
  31. 31.
    Wozniak KM, Nomoto K, Lapidus RG, Wu Y, Carozzi V, Cavaletti G, et al. Comparison of neuropathy-inducing effects of eribulin mesylate, paclitaxel, and ixabepilone in mice. Cancer Res. 2011;71:3952–62.CrossRefPubMedGoogle Scholar
  32. 32.
    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
  33. 33.
    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.CrossRefPubMedGoogle Scholar
  34. 34.
    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
  35. 35.
    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
  36. 36.
    Chen WY, Huang CY, Cheng WL, Hung CS, Huang MT, Tai CJ, et al. Alpha 7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to 5-fluorouracil. Tumour Biol. 2015;36:9537–44.CrossRefPubMedGoogle Scholar
  37. 37.
    Wang W, Chin-Sheng H, Kuo LJ, Wei PL, Lien YC, Lin FY, et al. NNK enhances cell migration through alpha7-nicotinic acetylcholine receptor accompanied by increased of fibronectin expression in gastric cancer. Ann Surg Oncol. 2012;19 Suppl 3:S580–8.CrossRefPubMedGoogle Scholar
  38. 38.
    Chang YJ, Tai CJ, Kuo LJ, Wei PL, Liang HH, Liu TZ, et al. Glucose-regulated protein 78 (GRP78) mediated the efficacy to curcumin treatment on hepatocellular carcinoma. Ann Surg Oncol. 2011;18:2395–403.CrossRefPubMedGoogle Scholar
  39. 39.
    Chang YJ, Huang CY, Hung CS, Liu HH, Wei PL. Glucose-regulated protein 78 mediates the therapeutic efficacy of 17-DMAG in colon cancer cells. Tumour Biol. 2015;36:4367–76.CrossRefPubMedGoogle Scholar
  40. 40.
    Chang YJ, Huang CY, Hung CS, Chen WY, Wei PL. GRP78 mediates the therapeutic efficacy of curcumin on colon cancer. Tumour Biol. 2015;36:633–41.CrossRefPubMedGoogle Scholar
  41. 41.
    Kim D, Dan HC, Park S, Yang L, Liu Q, Kaneko S, et al. AKT/PKB signaling mechanisms in cancer and chemoresistance. Front Biosci. 2005;10:975–87.CrossRefPubMedGoogle Scholar
  42. 42.
    Nam SY, Lee HS, Jung GA, Choi J, Cho SJ, Kim MK, et al. Akt/PKB activation in gastric carcinomas correlates with clinicopathologic variables and prognosis. APMIS. 2003;111:1105–13.CrossRefPubMedGoogle Scholar
  43. 43.
    Sasaki T, Kuniyasu H. Significance of AKT in gastric cancer (Review). Int J Oncol. 2014;45:2187–92.PubMedGoogle Scholar
  44. 44.
    Wong HP, Yu L, Lam EK, Tai EK, Wu WK, Cho CH. Nicotine promotes cell proliferation via alpha7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells. Toxicol Appl Pharmacol. 2007;221:261–7.CrossRefPubMedGoogle Scholar
  45. 45.
    Trevino JG, Pillai S, Kunigal 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 pancreatic adenocarcinoma. Neoplasia. 2012;14:1102–14.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Yuge K, Kikuchi E, Hagiwara M, Yasumizu Y, Tanaka N, Kosaka T, et al. Nicotine induces tumor growth and chemoresistance through activation of the PI3K/Akt/mTOR pathway in bladder cancer. Mol Cancer Ther. 2015;14:2112–20.CrossRefPubMedGoogle Scholar
  47. 47.
    De Rosa MJ, Esandi Mdel C, Garelli A, Rayes D, Bouzat C. Relationship between alpha 7 nAChR and apoptosis in human lymphocytes. J Neuroimmunol. 2005;160:154–61.CrossRefPubMedGoogle Scholar
  48. 48.
    Giannakakou P, Gussio R, Nogales E, Downing KH, Zaharevitz D, Bollbuck B, et al. A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc Natl Acad Sci U S A. 2000;97:2904–9.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Dumontet C, Sikic BI. Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death. J Clin Oncol. 1999;17:1061–70.CrossRefPubMedGoogle Scholar
  50. 50.
    Horwitz SB, Cohen D, Rao S, Ringel I, Shen HJ, Yang CP. Taxol: mechanisms of action and resistance. J Natl Cancer Inst Monogr. 1993:55–61Google Scholar
  51. 51.
    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
  52. 52.
    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
  53. 53.
    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
  54. 54.
    Benowitz NL. Nicotine addiction. N Engl J Med. 2010;362:2295–303.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Shao XM, Feldman JL. Central cholinergic regulation of respiration: nicotinic receptors. Acta Pharmacol Sin. 2009;30:761–70.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Solinas M, Scherma M, Fattore L, Stroik J, Wertheim C, Tanda G, et al. Nicotinic alpha 7 receptors as a new target for treatment of cannabis abuse. J Neurosci. 2007;27:5615–20.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  1. 1.Graduate Institute of Clinical Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  2. 2.Division of General Surgery, Department of SurgeryNew Taipei HospitalTaipeiTaiwan
  3. 3.Division of General Surgery, Department of Surgery, Shuang Ho HospitalTaipei Medical UniversityTaipeiTaiwan
  4. 4.Department of Surgery, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  5. 5.Division of General Surgery, Department of SurgeryTaipei Medical University Hospital, Taipei Medical UniversityTaipeiTaiwan
  6. 6.Cancer Research CenterTaipei Medical University Hospital, Taipei Medical UniversityTaipei CityTaiwan

Personalised recommendations