Combination of High-Dose Parenteral Ascorbate (Vitamin C) and Alpha-Lipoic Acid Failed to Enhance Tumor-Inhibitory Effect But Increased Toxicity in Preclinical Cancer Models

Abstract

Background: Intravenous vitamin C (IVC, ascorbate [Asc]) and alpha-lipoic acid (ALA) are frequently coadministered in integrative oncology clinics, with limited understanding of combination effects or drug-drug interactions. As high-dose IVC has anticancer activity through peroxide (H₂O₂), it is hypothesized that IV ALA, a thiol antioxidant, might have untoward effects when combined with IVC.

Methods: In vitro combination index (CI) was investigated in 6 types of human cancer cells, using clinically relevant concentrations of Asc (0.625-20 mM) and ALA (0.25, 0.5, and 1 mM) evaluated by nonconstant ratio metrics. Cellular H₂O₂ was measured using HeLa cells expressing a fluorescent probe HyPer. Mouse xenografts of the metastatic breast cancer MDA-MB-231 were treated with intraperitoneal injections of ALA (10, 20, and 50 mg/kg) and Asc (0.2, 0.5, and 4 g/kg) at various dose levels.

Results: Cancer cell lines were sensitive to Asc treatment but not to ALA. There is no evidence ALA becomes a prooxidant at higher doses. The CIs showed a mixture of synergistic and antagonistic effects with different ALA and Asc combination ratios, with a "U" shape response to Asc concentrations. The ALA concentrations did not influence the CIs or cellular H₂O₂ formation. Adding ALA to Asc dampened the increase of H₂O₂. Toxicity was observed in mice receiving prolonged treatment of ALA at all doses. The Asc at all doses was nontoxic. The combination of ALA and Asc increased toxicity. The ALA at all doses did not inhibit tumor growth. The Asc at 4 g/kg inhibited tumor growth. Adding ALA 50 mg/kg to Asc 4 g/kg did not enhance the effect, but lower doses of ALA (10 or 20 mg/kg) dampened the inhibitory effect of Asc.

Conclusions: These data do not support the concurrent or relative concurrent use of high-dose intravenous ALA with prooxidative high-dose IVC in clinical oncology care with potentially increased toxicity.

Keywords: Integrative oncology; alpha-lipoic acid; combination index; high-dose intravenous vitamin C; intravenous alpha-lipoic acid; vitamin C and cancer.

Figure 1.

Dose-response curves of the 6 tested cancer cell lines to ascorbate (A) or alpha-lipoic acid (B) treatment. Cells were treated for 24 hours, and cell viability was assessed by MTT assay and compared with untreated. Data represent mean ± SD of 2 to 3 independent experiments, each done in triplicates. HCT116 indicates colon cancer; MDA-MB231, metastatic breast cancer; Ovcar5, ovarian cancer; PANC-1, pancreatic cancer; PC3, prostate cancer; SK-Mel2, metastatic melanoma.

Figure 2.

CI values according to Asc concentrations (A) or ALA concentrations (B). (A) The CI values related to Asc concentration. The x-axis shows the 6 groups of Asc concentrations, and the y-axis shows the CI values. The CI values were averaged for all cell lines, and all 3 ALA concentrations were combined with that specific Asc concentration. The CIs showed a U-shape change as Asc concentration increased. (B) The CIs related to ALA concentration. The x-axis shows the 3 ALA concentration groups. No change in CIs was seen as ALA concentration increased.

Figure 3.

Normalized fluorescence of Hela-HyPer-Cyto cells under ALA or Asc treatment. (A) Cells were treated with ALA at 0, 0.25, 0.5, or 1 mM. (B) Cells were treated with Asc at 0.625, 1.25, 2.5, 5, 10 or 20 mM. Fluorescence was detected at 488/525 nm excitation/emission and then normalized to time 0 of the same treatment. The changes in fluorescence reflect concentrations of H₂O₂ in the cells.

Figure 4.

Normalized fluorescence of Hela-HyPer-Cyto cells under combination treatment of ALA and Asc. Cells were treated with Asc (0-20 mM) and ALA (0-1 mM). Fluorescence was detected at 24 hours of treatment at 488/525 nm excitation/emission and normalized to untreated control cells. The relative fluorescence intensity reflects H₂O₂ inside the cells. At lower Asc concentrations (0.625-2.5 mM), adding ALA inhibited H₂O₂ formation from Asc. At higher concentrations of Asc, ALA did not influence H₂O₂ formation.

Figure 5.

Average body weight of each group of mice under combination treatment of ALA and Asc. N = 9 to 20 per group.

Figure 6.

Final tumor weight at necropsy. Box-Whisker graph showing the distribution of tumor weight in each group. At the end of the treatment, mice were euthanized, and tumors were weighed. Data were combined from 2 independent experiments, each with 9 to 10 mice per group. The X mark in the box represents the average value, and the short line in the box represents the median. *P < .05 compared with the control group; ^&P < .05 compared with the ALA 50 mg/kg group. Asc 4 g/kg significantly inhibited tumor growth compared with control. The combination of ALA 50 mg/kg + Asc 4 g/kg treatment resulted in significantly smaller tumors than the control or ALA 50 mg/kg treatment. However, had no difference than Asc 4 g/kg. The combination of ALA 20 mg/kg or 10 mg/kg + Asc 4 g/kg had larger tumors than Asc 4 g/kg treatment alone but no difference than control.