World Journal of Microbiology and Biotechnology ( IF 4.0 ) Pub Date : 2021-07-21 , DOI: 10.1007/s11274-021-03076-5 Kavitha Alapati 1 , Savithri S Handanahal 1
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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 µM) 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 (G1 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.
Graphic abstract
中文翻译：
链霉菌产生的胆固醇氧化酶的细胞毒活性。AKHSS 对抗癌细胞系：在 HeLa 细胞中的作用机制
癌症的复发是目前可用的抗癌疗法的主要缺点。因此，研究一种有效的酶，胆固醇氧化酶，由各种微生物产生，尤其是从未探索的海洋放线菌物种中获得的对抗人类癌细胞系的微生物，并了解其作用机制，有助于确定一种不可逆的强效抗癌剂。海洋链霉菌产生的胆固醇氧化酶的细胞毒性潜力sp. 通过 MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑] 测定证明了 AKHSS 对四种不同的人类癌细胞系的作用。进行了基于荧光共聚焦显微镜和流式细胞术的实验以了解酶促作用对 HeLa 细胞的效率。此外，通过蛋白质印迹检测凋亡相关蛋白。有趣的是，该酶在非常低的浓度 (0.093–0.327 µM) 下对所有测试的细胞都表现出有效的细胞毒性。荧光共聚焦显微镜揭示了酶在癌细胞系上诱导的形态变化，例如脂滴的形成和细胞核的凝聚。通过气相色谱质谱法分析的酶处理的 HeLa 细胞无细胞提取物显示膜胆固醇的消耗和取代酶氧化产物 cholest-4-ene-3-one 的存在。该酶对细胞活力产生了显着的抑制作用，例如细胞周期停滞（G1期）、细胞凋亡和活性氧的增加，通过流式细胞术很明显。此外，在酶处理的 HeLa 细胞中记录了线粒体膜的超极化、pAkt 磷酸化率的降低以及 Fas、Fas L、caspases（8 和 3）和 PARP-1 等凋亡死亡标志物的表达。因此，从海洋链霉菌属中纯化的胆固醇氧化酶。AKHSS 在非常低的浓度下对人类癌细胞系表现出有效的细胞毒性。所有离体实验都描绘了该酶对 HeLa 细胞的显着抑制作用，表明Streptomyces sp. 的胆固醇氧化酶。AKHSS 可能是一种突出的癌症化学治疗剂。
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