Hypoxia-inducible factor-1α inhibition by a pyrrolopyrazine metabolite of oltipraz as a consequence of microRNAs 199a-5p and 20a induction

Oltipraz, a cancer chemopreventive agent, has antitumor and antiangiogenic effects. In animal models and clinical studies, a considerable amount of oltipraz is metabolized to pyrrolopyrazines, including M2, 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine; M3, 7-methyl-8-(methylsulfinyl)-6-(methylthio)pyrrolo[1,2-a]pyrazine and M4, 7-methyl-6,8-bis(methylsulfinyl)pyrrolo[1,2-a]pyrazine. In view of the role of hypoxia-inducible factor-1 (HIF-1) alpha in tumor growth and angiogenesis, this study investigated whether pyrrolopyrazine metabolites of oltipraz inhibit HIF-1 alpha induction, and if so, what the molecular basis is. M2 treatment inhibited the induction of HIF-1 alpha by a variety of stimuli including insulin, hypoxia, CoCl2 and hydrogen peroxide in HCT116 cells, whereas M3 or M4 failed to do so. Consistently, M2 prevented HIF-1 alpha target gene induction. Moreover, it inhibited cancer cell invasion and migration. M2 caused no change in the expression of HIF-1 alpha transcript but increased the levels of precursor forms of microRNAs (miRNAs) 199a-5p and 20a, but not those of primary forms, indicating facilitation of the maturation process of the miRNAs by M2. Increased levels of the miRNAs contributed to HIF-1 alpha repression, as shown by the results of experiments using mimics. Consistently, M2 treatment inhibited de novo synthesis of HIF-1 alpha, as supported by decreased incorporation of [S-35]-methionine into HIF-1 alpha with no changes in its ubiquitination or degradation. 7-Ethyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine, a synthetic analog of M2, had a similar inhibitory effect. In conclusion, M2 with pyrrolopyrazine structure and its 7-ethyl congenor have the ability to prevent the induction of HIF-1 alpha, which may result from the inhibition of HIF-1 alpha de novo synthesis, as mediated by the induction of miR-199a-5p and miR-20a.