Vitamin C antagonizes the cytotoxic effects of antineoplastic drugs

Abstract

Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of reactive oxygen species-generating antineoplastic drugs. The therapeutic efficacy of the widely used antineoplastic drugs doxorubicin, cisplatin, vincristine, methotrexate, and imatinib were compared in leukemia (K562) and lymphoma (RL) cell lines with and without pretreatment with dehydroascorbic acid, the commonly transported form of vitamin C. The effect of vitamin C on viability, clonogenicity, apoptosis, P-glycoprotein, reactive oxygen species (ROS), and mitochondrial membrane potential was determined. Pretreatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by trypan blue exclusion and colony formation after treatment with all antineoplastic agents tested. Vitamin C given before doxorubicin treatment led to a substantial reduction of therapeutic efficacy in mice with RL cell-derived xenogeneic tumors. Vitamin C treatment led to a dose-dependent decrease in apoptosis in cells treated with the antineoplastic agents that was not due to up-regulation of P-glycoprotein or vitamin C retention modulated by antineoplastics. Vitamin C had only modest effects on intracellular ROS and a more general cytoprotective profile than N-acetylcysteine, suggesting a mechanism of action that is not mediated by ROS. All antineoplastic agents tested caused mitochondrial membrane depolarization that was inhibited by vitamin C. These findings indicate that vitamin C given before mechanistically dissimilar antineoplastic agents antagonizes therapeutic efficacy in a model of human hematopoietic cancers by preserving mitochondrial membrane potential. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

Figure 1. DHA, but not AA is taken up in malignant hematopoietic cells

K562 cells (A, B) and RL cells (C, D) were exposed to either AA or DHA. Dose-dependent uptake after 1 hour of exposure to varying concentrations of either AA or DHA (0–750 μM) (A, C). Time-dependent uptake after exposure to either AA or DHA (500 μM) for 0–240 seconds (B, D). Three separate experiments were conducted in triplicate and the results shown represent the mean values and the standard deviations of a single representative experiment.

Figure 2. Vitamin C attenuates the cytotoxicity of antineoplastic agents in K562 and RL cells without inhibiting normal cell growth

K562 (A) and RL (B) cells were loaded with DHA to varying concentrations of intracellular vitamin C. The cells were washed and treated with either vehicle or antineoplastic agents for 48 hours. Cell viability was measured by Trypan Blue exclusion. The results are plotted as the percentage of viable cells compared to cells not treated with either vitamin C or antineoplastic agents. The effect of vitamin C on colony formation after treatment with antineoplastic agents was determined (C). K562 cells, either treated with vehicle or with vitamin C to an internal concentration of 18 mM vitamin C, and RL cells either treated with vehicle or with vitamin C to an internal concentration of 8.5 mM vitamin C were washed and then treated with either vehicle (−) or antineoplastic agents for 48 hours. The cells were plated in methylcellulose and colony numbers were compared to, and expressed pair-wise, as a percentage of CFU’s resulting from treatment with antineoplastic agents without vitamin C pretreatment. Three separate experiments were conducted in triplicate and the results shown represent the mean values and the standard deviations of a single representative experiment.

Figure 3. Vitamin C attenuates the cytotoxicity of antineoplastic agents in vivo

ICR SCID mice were xenografted with RL cells in the right flank. Cohorts of 5 mice were treated with vehicle control, 1 mg/kg DOX, 250mg/kg DHA or DHA 2 hours prior to DOX on days as noted (▲), and tumor volumes were measured (A). Statistics were single factor ANOVA. Representative animals on day 28 from the left are DOX, DHA prior to DOX, and DHA. The background color was slightly modified to enhance contrast and the image was converted from color to black and white. (B). Two separate experiments were conducted in quintuplicate and the results shown represent the mean values and the standard deviations of a single representative experiment.

Figure 4. Vitamin C reduces the percentage of apoptotic cells

K562 and RL cells were treated with DHA to achieve varying concentrations of intracellular vitamin C. The cells were washed and treated with antineoplastic agents for up to 48 hours. Apoptosis was measured by TUNEL after 48 hours of treatment. Results are expressed as a percentage of TUNEL positive cells. Three separate experiments were conducted in triplicate and the results shown represent the mean values and the standard deviations of a single representative experiment.

Figure 5. Vitamin C has minimal effects on intracellular ROS and has a different effect on cytotoxicity than N-acetyl cysteine

K562 and RL cells were either untreated (−) or treated (+) to internal vitamin C (AA) concentrations of 18 mM and 8.5 mM respectively, and treated with antineoplastic agents for 6 hours. ROS was measured by incubation with 0.1μg/mL CM-H2DCFDA (A). Results are expressed as median fluorescence intensity (arbitrary units). K562 and RL cells were treated with antineoplastic agents for 48 hours in the presence (+) or absence (−) of 25 mM N-acetylcysteine (NAC) (B). Cell viability was measured by Trypan Blue exclusion and results are expressed as a percent of cells not concomitantly treated with NAC. Three separate experiments were conducted in triplicate and the results shown represent the mean values and the standard deviations of a representative experiment.

Figure 6. Vitamin C antagonizes mitochondrial membrane depolarization mediated by antineoplastic agents

Cells were stained with 10 μg/mL JC-1, washed, and equilibrated for 1 hour at 37°C. K562 and RL cells either untreated (−) or loaded (+) with 18 mM and 8.5 mM vitamin C (AA), respectively were exposed to antineoplastic agents for 6 hours and mitochondrial membrane potential was measured as the ratio of 530 nm to 585 nm fluorescence. Maximal depolarization was determined by exposure to 100 nM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The results are representative of at least 3 separate experiments. The differences in mitochondrial membrane potential between chemotherapy-treated cells with and without pretreatment with vitamin C were statistically significant (p<0.003 in RL cells, p<0.0005 in K562 cells, (student’s t-Test)). Three separate experiments were conducted in triplicate and the results shown represent the mean values and the standard deviations of a representative experiment.