Metabolomic alterations in human cancer cells by vitamin C-induced oxidative stress - PubMed (original) (raw)

Metabolomic alterations in human cancer cells by vitamin C-induced oxidative stress

Megumi Uetaki et al. Sci Rep. 2015.

Abstract

Intravenous administration of high-dose vitamin C has recently attracted attention as a cancer therapy. High-dose vitamin C induces pro-oxidant effects and selectively kills cancer cells. However, the anticancer mechanisms of vitamin C are not fully understood. Here, we analyzed metabolic changes induced by vitamin C in MCF7 human breast adenocarcinoma and HT29 human colon cancer cells using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The metabolomic profiles of both cell lines were dramatically altered after exposure to cytotoxic concentrations of vitamin C. Levels of upstream metabolites in the glycolysis pathway and tricarboxylic acid (TCA) cycle were increased in both cell lines following treatment with vitamin C, while adenosine triphosphate (ATP) levels and adenylate energy charges were decreased concentration-dependently. Treatment with N-acetyl cysteine (NAC) and reduced glutathione (GSH) significantly inhibited vitamin C-induced cytotoxicity in MCF7 cells. NAC also suppressed vitamin C-dependent metabolic changes, and NAD treatment prevented vitamin C-induced cell death. Collectively, our data suggests that vitamin C inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death.

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Figures

Figure 1

Figure 1. Effects of vitamin C-induced hydrogen peroxide (H2O2) on viability of cancer cells.

(a) Cancer cells were treated with vitamin C for 2 h, washed, and cultured for an additional 46 h in DMEM in the absence of vitamin C. Cell viability was determined using MTT assays. IC50 values indicate the concentration of vitamin C that inhibited survival by 50%, as determined by MTT assays. (b) Effects of vitamin C on HO-1 expression in MCF7 cells. Cells were treated with vitamin C (1 mM), NAC (10 mM), and H2O2 (1 mM) for 24 h. Expression levels of HO-1 mRNA were measured using qPCR. (c) Suppressive effects of antioxidants NAC and GSH on vitamin C-induced cytotoxicity in MCF7 cells. Cell viability was determined using MTT assays in MCF7 cells treated without or with vitamin C and antioxidants. Data are presented as means ± SDs from triplicate experiments, **P < 0.01.

Figure 2

Figure 2. Vitamin C-induced metabolic alterations in MCF7 cells.

(a) Metabolic alterations in glycolysis and the TCA cycle induced by vitamin C. MCF7 cells were incubated in DMEM without or with vitamin C, and metabolites levels were measured using CE-TOSMS. Colors of metabolites on heatmap indicate significant differences (red, upregulated; green, downregulated). Bar graphs indicate fold changes relative to control sample (None). (b) Effects of vitamin C on levels of AMP, ADP, ATP, GMP, GDP, GTP, and adenylate energy charge. Bar graphs indicate fold changes relative to control sample (None). Adenylate energy charge calculation: (ATP + 0.5 × ADP)/(ATP + ADP + AMP). (c) Effects of vitamin C on levels of GSH and GSSG and GSH:GSSG ratio. Bar graphs show relative metabolite levels compared to control (None). Data are presented as means ± SD of triplicate experiments, *P < 0.05, **P < 0.01. ND, not detected.

Figure 3

Figure 3. Effects of N-acetyl cysteine (NAC) on energy metabolism in MCF7 cells treated with vitamin C.

(a) Effects of NAC on metabolites of glycolysis, the TCA cycle, and the PPP in MCF7 cells stimulated with vitamin C. Heatmap depicts log2-transformed ratios of measured sample to control sample (None) concentrations. *P < 0.05, **P < 0.01 (comparing lanes 3 and 4). (b) Effects of NAC on levels of AMP, ADP, ATP, GMP, GDP, GTP, and adenylate energy charge. Bar graphs indicate fold changes relative to control sample (None). Adenylate energy charge calculation: (ATP + 0.5 × ADP)/(ATP + ADP + AMP). Data are presented as means ± SD of triplicate experiments. **P < 0.01.

Figure 4

Figure 4. Nicotinamide adenine dinucleotide (NAD) depletion induced by vitamin C-induced H2O2 in MCF7 cells.

(a) Metabolite map of glycolysis. Colors of metabolites indicate significant differences (red, upregulated; green, downregulated). Conversion of GAP to 1,3- BPG mediated by GAPDH. (b) NAD levels were decreased by vitamin C in MCF7 (left) and HT29 cells (right) and were determined using CE-TOFMS. Bar graphs indicate fold changes relative to control sample (None). (c) Effects of NAC on levels of NAD in MCF7 cells. Bar graphs show metabolite levels relative to those of control (None). (d) Effects of NAD on viability of MCF7 (left) and HT29 (right) cells determined by MTT assay in both cell lines treated without or with vitamin C and NAD. Data are presented as means ± SD of triplicate experiments, **P < 0.01.

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