Cytotoxic activity of cholesterol oxidase produced by Streptomyces sp. AKHSS against cancerous cell lines: mechanism of action in HeLa cells

Re-occurrence of cancer is the major drawback for the currently available anticancer therapies. Therefore, study of an efficient enzyme, cholesterol oxidase produced by various kinds of microbes especially obtained from unexplored marine actinobacterial species against human cancer cell lines and understanding its mechanism of action helps to identify an irreversible and potent anticancer agent. The cytotoxic potential of cholesterol oxidase produced by a marine Streptomyces sp. AKHSS against four different human cancer cell lines was demonstrated through MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Fluorescent confocal microscopy and flow cytometry based experiments were performed to understand the efficiency of the enzymatic action on HeLa cells. Further, the apoptotic related proteins were detected through western blotting. Interestingly, the enzyme exhibited potent cytotoxicity at very low concentrations (0.093-0.327 mu M) against all the cells tested. Fluorescent confocal microscopy revealed the morphological variations induced by the enzyme on cancer cell lines such as the formation of lipid droplets and condensation of nuclei. The enzyme treated cell-free extracts of HeLa cells analyzed through gas chromatography mass spectrometry showed the depletion of membrane cholesterol and the presence of substituted enzyme oxidized product, cholest-4-ene-3-one. The enzyme had induced significant inhibitory effects on the cell viability such as cell cycle arrest (G(1) phase), apoptosis and rise of reactive oxygen species as evident through flow cytometry. Besides, hyperpolarization of mitochondrial membrane, reduced rates of phosphorylation of pAkt and the expression of apoptotic death markers like Fas, Fas L, caspases (8 and 3) and PARP-1 were recorded in the enzyme treated HeLa cells. Thus, cholesterol oxidase purified from a marine Streptomyces sp. AKHSS exhibits potent cytotoxicity at very low concentrations against human cancer cell lines. All the ex vivo experiments portrayed the substantial inhibitory effect of the enzyme on HeLa cells suggesting that cholesterol oxidase of Streptomyces sp. AKHSS could be a prominent cancer chemotherapeutic agent.