Abstract

MHY1485 is an mTOR activator that inhibits the autophagy process by inhibiting the fusion between autophagosomes and lysosomes. This study aimed to explore the role and mechanism of MHY1485 in hepatocellular carcinoma (HCC) and to provide an in-depth understanding of the mechanisms of autophagy regulation in relation to adriamycin (ADM) resistance, as well as the development of a molecularly targeted autophagy-modulating approach. Here, ADM was used to treat HepG2 cells and construct an ADM-resistant cell model. The HepG2/ADM cell line and HepG2 cells were treated with MHY1485 and ADM, respectively, and the proliferation and apoptosis of HCC cells were detected using CCK8, clone formation, flow cytometry, and 5-ethynyl-2′-deoxyuridine staining assays. Ki-67, mTOR phosphorylation, and LC3A expression were detected by IF staining; the expression or phosphorylation levels of autophagy-related proteins (i.e., GLUT1, PGI, PFK, END, and MTHFD2) and apoptosis-related proteins (caspase-3, caspase-8, and caspase-9) were detected by qPCR and western blotting. The number of autophagosomes was determined by monodansylcadaverine staining. Our results showed that MHY1485 can inhibit the proliferation and growth of liver cancer cells, and that MHY1485 combined with ADM can effectively inhibit the tolerance of HepG2/ADM cells to ADM and enhance the efficacy of ADM. The results of the detection of the autophagy-related protein LC3A also indicated that MHY1485 activates mTOR and can affect the phosphorylation level of ULK1, inhibit autophagy, and enhance the sensitivity of liver cancer cells to adriamycin. In summary, MHY1485 can enhance the sensitivity of adriamycin-resistant cells to adriamycin by activating mTOR and blocking the autophagy process in cells; therefore, mTOR may become a potential target for the treatment of liver cancer.

中文翻译：

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MHY1485通过抑制自噬促进HepG2细胞对阿霉素的敏感性

摘要

MHY1485 是一种 mTOR 激活剂，通过抑制自噬体和溶酶体之间的融合来抑制自噬过程。本研究旨在探讨MHY1485在肝细胞癌（HCC）中的作用和机制，深入了解与阿霉素（ADM）耐药相关的自噬调控机制，以及分子靶向自噬的发展。 -调节方法。在这里，ADM被用来处理HepG2细胞并构建ADM耐药细胞模型。分别用MHY1485和ADM处理HepG2/ADM细胞系和HepG2细胞，并使用CCK8、克隆形成、流式细胞术和5-乙炔基-2'-脱氧尿苷染色法检测HCC细胞的增殖和凋亡。 IF染色检测Ki-67、mTOR磷酸化、LC3A表达；通过qPCR和western blotting检测自噬相关蛋白（即GLUT1、PGI、PFK、END和MTHFD2）和凋亡相关蛋白（caspase-3、caspase-8和caspase-9）的表达或磷酸化水平。通过单丹磺酰尸胺染色测定自噬体的数量。我们的结果表明MHY1485能够抑制肝癌细胞的增殖和生长，并且MHY1485与ADM联用能够有效抑制HepG2/ADM细胞对ADM的耐受，增强ADM的疗效。自噬相关蛋白LC3A的检测结果也表明MHY1485激活mTOR并能影响ULK1的磷酸化水平，抑制自噬，增强肝癌细胞对阿霉素的敏感性。综上所述，MHY1485可以通过激活mTOR、阻断细胞内自噬过程，增强阿霉素耐药细胞对阿霉素的敏感性；因此，mTOR可能成为肝癌治疗的潜在靶点。