Christie Michaud and Dr. Byron Murray, Microbiology
Colorectal cancer is the second leading cause of cancer deaths in the United States and doxorubicin, is one of the more widely chemotherapeutic treatments for this disease. This study investigated the possibility of exposing adenocarcinoma cells to vitamin C/vitamin E (VC/VE) combinations as a means to reduce toxicity associated with doxorubicin (DXR) treatment.
Vitamins and their derivatives have been shown to increase the sensitivity of cancer cells to various chemotherapeutic agents both in vitro and in vivo (1, 2). This is especially interesting in light of the observation that multiple vitamins may actually protect normal cells from DNA damage and apoptosis (3). DXR has been reported to induce apoptosis (programmed cell death) through inhibition of topoisomerase II. DXR has also been shown to generate harmful free radicals. One attractive hypothesis is to use antioxidants to improve treatment efficacy through not only increasing tumor response, but through reducing the toxicity of common chemotherapeutic agents (4). However, oxidation reactions, which are frequently destructive to biological molecules, also appear to initiate apoptosis. A second hypothesis is that antioxidants might interfere with oxidation reactions, interfering with apoptosis and leaving cancer cells alive (5). Data obtained in this study support the first hypothesis.
Data obtained from cell proliferation assays in cultures seeded at high density 2 and 4 days after vitamin exposure were at control levels while cultures exposed to DXR were reduced to 54% of control values. However, when VE, VC, or VE+VC were added in combination with DXR, cell populations returned to control levels. In low density studies, cultures exposed to DXR for 8 days only obtained 48% of controls. The addition of VC and/or VE with DXR had no effect on increasing the population density when compared to DXR alone treated cultures.
Caspases, proteins that activate during apoptosis, were then analyzed. Caspase 3 levels in high density populations increased 2-fold in DXR treated cultures, while in low density cultures, caspase 3 activity was elevated 5-fold. When VE was added in with DXR, caspase 3 levels in high density cultures remained at control levels. In contrast, when VE was added in combination with DXR in low density cultures, caspase 3 levels were decreased but were still 3-fold higher than controls.
Caspase 9 levels in high density populations increased 2-fold in DXR treated cultures, while in low density cultures, caspase 9 activity was elevated 3-fold. When VC was added in combination with DXR, caspase 9 levels in high density cultures decreased by 30% and in low density cultures by 50%. When VE was added in combination with DXR, no significant change occurred in caspase 9 activity in either high or low density cultures. Addition of VC/VE collectively with DXR, resulted in 50% reduction of caspase 9 activity in high density cultures and 20% reduction in low density cultures.
Alterations in mitochondrial membrane potential, often resulting from free radical damage, was determined using JC-1 in a flow cytometry assay. In high density populations, no significant differences were observed in mitochondrial membrane potential after DXR treatment while in low density populations, mitochondrial membrane potential was decreased by DXR treatment but increased 25% by VC, VE, and VC/VE when added in combination with DXR. Together, this data suggests that while induction of apoptosis by DXR was not inhibited, toxicity was reduced by VC and/or VE treatment, which may improve chemotherapeutic treatment of human cancer.
References
- Cole, W. C., and K. N. Prasad. 1997. Contrasting effects of vitamins as modulators of apoptosis in cancer cells and normal cells: a review. Nutr. Cancer, 29: 97-103.
- Qiao, L., R. Hanif, E. Sphicas, S. J. Shiff, and B. Rigas. 1998. Effect of aspirin on induction of apoptosis in HT-29 human colon adenocarcinoma cells. Biochem. Pharmacol., 55: 53-64.
- Prasad, K.N., Kumar, A., Kochupillai, V., and Cole, W.C. 1999. High doses of multiple antioxidant vitamins: essential ingredients in improving the efficacy of standard cancer therapy. J. Am. Coll. Nutr., 18:13-25.
- Prasad, K.N., Cole, W., Kumar, B., and Prasad, K. 2001. Scientific rationale for using high-dose multiple micronutrients as an adjunct to standard and experimental cancer therapies. J. Am. Coll. Nutr., 20: 450S-463S.
- Salganik, R. 2001. The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. J. Am. Coll. Nutr., 20: 464S-472S.