Abstract
In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of ascorbate for melanoma therapy.
Highlights
In recent years, a large number of studies demonstrated that in pharmacological doses, ascorbic acid in the low micromolar-range exerts cytotoxic effects on cancer cells in vitro and in vivo [1,2,3] via pro-oxidative mechanisms [4]
PHARMACOLOGICAL ASCORBATE INHIBITS DNA METHYLTRANSFERASES Due to the negative results of the histone deacetylase (HDAC) inhibition assays, we investigated if ascorbate had a DNA methyltransferase (DNMT) inhibitory activity in the human metastatic MeWo and BLM melanoma cells
We mainly investigated the role of epigenetic mechanisms accompanying ascorbate-induced cytotoxicity, we did not focus on miRNA expression changes induced by physiological ascorbate (200 μM), which bears no cytotoxic effect on melanoma cells
Summary
A large number of studies demonstrated that in pharmacological doses, ascorbic acid (ascorbate, vitamin C) in the low micromolar-range exerts cytotoxic effects on cancer cells in vitro and in vivo [1,2,3] via pro-oxidative mechanisms [4]. This cytotoxicity is conducted by ascorbyl radicals and H2O2 being catalyzed by serum components [5]. Oral ascorbate supplementation modulates B16FO melanoma growth, metastasis, and inflammatory cytokine secretion as well as enhanced encapsulation of tumors in scorbutic (l-gulono-gamma lactone oxidase −/−) mice [12, 13]
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