Abstract
l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.
Similar content being viewed by others
Abbreviations
- NAC:
-
N-acetyl-l-cysteine
- ROS:
-
reactive oxygen species
- Δψm :
-
mitochondrial membrane potential
References
Afansev VN, Korol BA, Mantsygin YA (1986) Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death. FEBS Letter 194:347
Bram S, Froussard P, Guichard M, Jasmin C, Augery Y, Sinoussi-Barre F, Wray W (1980) Vitamin C preferential toxicity for malignant melanoma cells. Nature 284:629
De Pauw-Gillet MC, Siwek B, Pozzi G, Sabbioni E, Bassleer RJ (1990) Control of B16F10 melanoma cells differentiation and proliferation by CuSO4 and vitamin C. Anticancer Res 10:391
De Pauw-Gillet MC, Siwek B, Pozzi G, Sabbioni E, Bassleer RJ (1990) Effects of Fe SO4 on B16F10 melanoma cells differentiation and proliferation. Anticancer Res 10:1029
Du C, Fang M, Li Y, Li L, Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102:33
Ferrone S, Marincola FM (1995) Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance. Immunol Today 16:487
Gardiner NS, Duncan JR (1988) Enhanced prostaglandin synthesis as a mechanism for inhibition of melanoma cell growth by ascorbic acid. Prostaglandins Leukot Essent Fatty Acids 34:119
Gardiner NS, Duncan JR (1989) Inhibition of murine melanoma growth by sodium ascorbate. J Nutr 119:586
Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309
Green D, Kroemer G (1998) The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol 8:267
Hahne M, Rimoldi D, Schroter M, Romero P, Schreiter M, French LE (1996) Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science 274:1363
Huber D, Philipp J, Fontana A (1998) Protease inhibitors interfere with the transforming growth factor-beta-dependent but not the transforming growth factor beta-independent pathway of tumor cell-mediated immunosuppression. J Immunol 148:277
Krammer PH (2000) CD95's deadly mission in the immune system. Nature 407:789
Kroemer, G., N. Zamzami, S. A. Susin (1997) Mitochondrial control of apoptosis. Immunol Today 18:44
Li LY, Luo X, Wang X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412:95
Liu X., Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell free extracts: requirement for dATP and cytochrome c. Cell 86:147
Marczewska J, Koziorowska JH, Anuszewska EL (2000) Influence of ascorbic acid on cytotoxic activity of copper and iron ions in vitro. Acta Pol Pharm 57:415
Mukherji B, Chakraborty NG (1995) Immunobiology and immunotherapy of melanoma. Curr Opin Oncol 7:175
Nicholson DW, Thornberry NA (1997) Caspases: killer proteases. Trends Biochem Sci 22:299
Nungester WJ, Ames AM (1948) The relationship between ascorbic acid and phagocytic activity. J Infect Dis 83:50
Peter ME, Krammer PH (1998) Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis. Curr Opin Immunol 10:545
Savill J, Fadok V, Henson P (1993) Phagocyte recognition of cells undergoing apoptosis. Immunol Today 14:131
Siegel BV (1975) Enhancement of interferon production by poly(rl)poly(rC) in mouse cell cultures by ascorbic acid. Nature 254:531
Stockinger HE (1977) The case for carcinogen TLVs continues strong. Int J Occup Health Saf 46:54
Stone I (1980) The possible role of mega-ascorbate in the endogenous synthesis of interferon. Med Hypotheses 6:309
Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 8:613
Thornberry NA, Lazebnik Y (1998) Caspases, enemies within. Science 281:1312
Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM, Day CL, Tikoo A, Burke R, Wrobel C, Moritz RL, Simpson RJ, Vaux DL (2002) HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem 277:445
Victor VV, Guayerbas N, Puerto M, Medina S, De la Fuente M (2000) Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock. Immunopharmacology 46:89
Wang J, Chun HJ, Wong W, Spencer DM, Lenardo MJ (2001) Caspase-10 is an initiator caspase in death receptor signaling. Proc Natl Acad Sci USA 98:13884
Wyllie AH, Morrid RG, Smith AL (1998) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142:67
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Pen T-I, Jones DP, Wang X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked, Science 275:1129–1132
Zoratti M, Szabo I (1995) The mitochondrial permeability transition. Biochim Biophys Acta 1241:139
Acknowledgment
This study is supported by the grant from Korea Eundan Company
Author information
Authors and Affiliations
Corresponding author
Additional information
Jae Seung Kang and Daeho Cho contributed equally to this work.
Rights and permissions
About this article
Cite this article
Kang, J.S., Cho, D., Kim, YI. et al. l-Ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8–independent pathway. Cancer Immunol Immunother 52, 693–698 (2003). https://doi.org/10.1007/s00262-003-0407-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00262-003-0407-6