Epigenetic Impacts of Ascorbate on Human Metastatic Melanoma Cells

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.

Gene expression response to ascorbic acid in mice implanted with sarcoma S180 cells

“…Compared to untreated tumor-bearing mice, we found a statistically significant increase in expression of gene p53 (p<0.02) in tumor-bearing mice treated with ascorbate. This is consistent with reports in the literature of ascorbate's effect on the p53 gene [30, 64,80]. Our data showed that p53 gene expression was elevated in ascorbate treated groups of mice with tumors, tended to decrease as tumors became larger and was reduced in animals with metastatic tumors.…”

“…reverse transcriptase (TERT), which regulates telomere length, can promote tumor development, increases the anti-apoptotic capacity of cells, enhances DNA repair [77] and nuclear factor erythroid derived 2 (Nrf2), which has anti-apoptotic effects and may therefore protect cancer cells [78][79]. Ascorbate has been shown to target the expression of these genes (promoting p53 while inhibition expression of the others) in tumor cells [57][58][59]80], which in turn shifts cells toward the sub-G1 fraction.…”

The Cure from Nature: The Extraordinary Anticancer Properties of Ascorbate (Vitamin C)

“…An epochal event is taking place in cancer treatment, but the misunderstanding is always in ambush! The Authors of a related article [84] conclude: "… Pauling may have been correct on the use of high doses of Vitamin C for cancer therapy, but for wrong reasons -not as an antioxidant, but as a pro oxidant anticancer agent"; knowing the real story of Vitamin C, one can easily realize that this is simply not true! Pauling never considered the anti-or pro-oxidant properties of ascorbate; he rather pointed out its effects as a booster of the immune response!…”

“…• It improves the quality of life of cancer patients [72] • It prolongs their survival [26,27,73] • It potentiates the cytotoxic effects and reduces the side effects of conventional chemotherapy [74] • It is remarkably safe in both physiologic, and pharmacologic concentrations [75] • It boosts the immune response, in cancer patients [76][77][78][79] • It increases the host resistance to neoplasia [80,81] • It protects against the development of neoplasia [82,83] • It is an epigenetic modulator of genes with a key role in cancer development [84,85] • It is highly cytotoxic in vitro against a number of different human tumor cell lines [30,31,[33][34][35][36] • It exert a powerful cytotoxic effect against colorectal cancer cells refractory to targeted therapies [86,87].…”

Silencing DNA methyltransferase 1 leads to the activation of the esophageal suppressor gene p16 in vitro and in vivo

“…The role of DNMT1 is to ensure the inheritance of the DNA methylation pattern through cell division. Since aberrant expression of DNMT1 occurs in the majority of cancer types, DNA methylation-based biomarkers mediated by DNMT1 have been used as diagnostic tools, with modification typically occurring in the CpG-rich island of the gene promoter region (19)(20)(21). Therefore, it has been suggested that DNA methylation patterns may be used to distinguish cancer cells from normal cells (22).…”