Mechanism of Ascorbate-Induced Cell Death in Human Pancreatic Cancer Cells: Role of Bcl-2, Beclin 1 and Autophagy

Abstract

The present study investigates the anticancer effect of ascorbate in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. We showed that ascorbate suppresses the growth of human pancreatic cancer cells via the induction of oxidative stress and caspase-independent cell death. Ascorbate induces the formation of autophagosomes and the presence of autophagy inhibitors suppresses ascorbate-induced cell death. These data suggest that the induction of autophagosome formation contributes to ascorbate-induced pancreatic cancer cell death.

Key words

ascorbate - pancreatic cancer - cell death - autophagy

• References

• 1 Vainio H, Weiderpass E. Fruit and vegetables in cancer prevention. Nutr Cancer 2006; 54: 111-142
• 2 Bravi F, Polesel J, Bosetti C, Talamini R, Negri E, Dal Maso L, Serraino D, La Vecchia C. Dietary intake of selected micronutrients and the risk of pancreatic cancer: an Italian case-control study. Ann Oncol 2001; 22: 202-206
• 3 Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK, Levine M. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 2003; 22: 18-35
• 4 McGregor GP, Biesalski HK. Rationale and impact of vitamin C in clinical nutrition. Curr Opin Clin Nutr Metab Care 2006; 9: 697-703
• 5 Du J, Martin SM, Levine M, Wagner BA, Buettner GR, Wang SH, Taghiyev AF, Du C, Knudson CM, Cullen JJ. Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer. Clin Cancer Res 2010; 16: 509-520
• 6 Padayatty SJ, Sun H, Wang Y, Riordan HD, Hewitt SM, Katz A, Wesley RA, Levine M. Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann Intern Med 2004; 140: 533-537
• 7 Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, Park JB, Lazarev A, Graumlich JF, King J, Cantilena LR. Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci U S A 1996; 93: 3704-3709
• 8 Levine M, Wang Y, Padayatty SJ, Morrow J. A new recommended dietary allowance of vitamin C for healthy young women. Proc Natl Acad Sci U S A 2001; 98: 9842-9846
• 9 Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, Khosh DB, Drisko J, Levine M. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci U S A 2008; 105: 11105-11109
• 10 Chajes V, Sattler W, Stranzl A, Kostner GM. Influence of n-3 fatty acids on the growth of human breast cancer cells in vitro: relationship to peroxides and vitamin E. Breast Cancer Res Treat 1995; 34: 199-212
• 11 Colas S, Germain E, Arab K, Maheo K, Goupille C, Bougnoux P. Alpha-tocopherol suppresses mammary tumor sensitivity to anthracyclines in fish oil-fed rats. Nutr Cancer 2005; 51: 178-183
• 12 Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR, Shacter E, Levine M. Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci U S A 2005; 102: 13604-13609
• 13 Nagai H, Noguchi T, Takeda K, Ichijo H. Pathophysiological roles of ASK1-MAP kinase signaling pathways. J Biochem Mol Biol 2007; 40: 1-6
• 14 Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta 2010; 1802: 396-405
• 15 Ha YM, Park MK, Kim HJ, Seo HG, Lee JH, Chang KC. High concentrations of ascorbic acid induce apoptosis of human gastric cancer cell by p 38-MAP kinase-dependent up-regulation of transferrin receptor. Cancer Lett 2009; 277: 48-54
• 16 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479-489
• 17 Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 1997; 57: 1835-1840
• 18 Eguchi Y, Srinivasan A, Tomaselli KJ, Shimizu S, Tsujimoto Y. ATP-dependent steps in apoptotic signal transduction. Cancer Res 1999; 59: 2174-2181
• 19 Chen P, Yu J, Chalmers B, Drisko J, Yang J, Li B, Chen Q. Pharmacological ascorbate induces cytotoxicity in prostate cancer cells through ATP depletion and induction of autophagy. Anticancer Drugs 2012; 23: 437-444
• 20 Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122: 927-939
• 21 Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, Tsujimoto Y. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol 2004; 6: 1221-1228
• 22 Elgendy M, Sheridan C, Brumatti G, Martin SJ. Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol Cell 2011; 42: 23-35
• 23 Carosio R, Zuccari G, Orienti I, Mangraviti S, Montaldo PG. Sodium ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake. Mol Cancer 2007; 6: 55
• 24 Hong SW, Jin DH, Hahm ES, Yim SH, Lim JS, Kim KI, Yang Y, Lee SS, Kang JS, Lee WJ, Lee WK, Lee MS. Ascorbate (vitamin C) induces cell death through the apoptosis-inducing factor in human breast cancer cells. Oncol Rep 2007; 18: 811-815
• 25 Lin SY, Lai WW, Chou CC, Kuo HM, Li TM, Chung JG, Yang JH. Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells. Melanoma Res 2006; 16: 509-519
• 26 Kang JS, Cho D, Kim YI, Hahm E, Yang Y, Kim D, Hur D, Park H, Bang S, Hwang YI, Lee WJ. L-Ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway. Cancer Immunol Immunother 2003; 52: 693-698
• 27 Frömberg A, Gutsch D, Schulze D, Vollbracht C, Weiss G, Czubayko F, Aigner A. Ascorbate exerts anti-proliferative effects through cell cycle inhibition and sensitizes tumor cells towards cytostatic drugs. Cancer Chemother Pharmacol 2011; 67: 1157-1166
• 28 Verrax J, Delvaux M, Beghein N, Taper H, Gallez B, Buc Calderon P. Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study. Free Radic Res 2005; 39: 649-657
• 29 Verrax J, Cadrobbi J, Marques C, Taper H, Habraken Y, Piette J, Calderon PB. Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death. Apoptosis 2004; 9: 223-233
• 30 Jamison JM, Gilloteaux J, Taper HS, Summers JL. Evaluation of the in vitro and in vivo antitumor activities of vitamin C and K-3 combinations against human prostate cancer. J Nutr 2001; 131: 158S-160S
• 31 Gilloteaux J, Jamison JM, Arnold D, Taper HS, Summers JL. Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells. Ultrastruct Pathol 2001; 25: 183-192
• 32 Taper HS, Jamison JM, Gilloteaux J, Gwin CA, Gordon T, Summers JL. In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K₃ combination. J Histochem Cytochem 2001; 49: 109-120
• 33 Hong SW, Jin DH, Hahm ES, Yim SH, Lim JS, Kim KI, Yang Y, Lee SS, Kang JS, Lee WJ, Lee WK, Lee MS. Ascorbate (vitamin C) induces cell death through the apoptosis-inducing factor in human breast cancer cells. Oncol Rep 2007; 18: 811-815
• 34 Cullen JJ. Ascorbate induces autophagy in pancreatic cancer. Autophagy 2010; 6: 421-422
• 35 Yo YT, Shieh GS, Hsu KF, Wu CL, Shiau AL. Licorice and licochalcone-A induce autophagy in LNCaP prostate cancer cells by suppression of Bcl-2 expression and the mTOR pathway. J Agric Food Chem 2009; 57: 8266-8273
• 36 Chen Y, Klionsky DJ. The regulation of autophagy – unanswered questions. J Cell Sci 2011; 124: 161-170
• 37 Giansanti V, Torriglia A, Scovassi AI. A conversation between apoptosis and autophagy: “Is it your turn or mine?”. Apoptosis 2011; 16: 321-333
• 38 Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 2008; 9: 1004-1010
• 39 Fukui M, Kang KS, Okada K, Zhu BT. EPA, an omega-3 fatty acid, induces apoptosis in human pancreatic cancer cells in vitro and in vivo: role of ROS accumulation, caspase 8 activation, and autophagy induction. J Cell Biochem 2013; 114: 192-203
• 40 Fukui M, Choi HJ, Zhu BT. Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death. Free Radic Biol Med 2010; 49: 800-813
• 41 Hanaichi T, Sato T, Iwamoto T, Malavasi-Yamashiro J, Hoshino M, Mizuno N. A stable lead by modification of Satoʼs method. J Electron Microsc (Tokyo) 1986; 35: 304-306