自噬通过促进MGMT的表达介导胃癌烷化剂化疗耐药的分子机制研究

The chemoresistance of tumor cells to alkylating agents is one of the main reasons which cause the treatment failure of patients with gastric cancer. Autophagy, an irritable reaction to protect cells and organisms from external stimuli, plays an important role in the chemoresistance of tumor cells to alkylating agents. However, the possible mechanism of autophagy promotes the chemoresistance of tumor cell to alkylating agents is not clear until now. Some studies have reported that autophagy could lead to the elevated chemoresistance of tumor cells to DNA-damaging agents via enhancing the capacity of DNA damage repair. O6-methylguanine-DNA methyltransferase (MGMT), as an important DNA repair protein, plays an important role in repairing DNA damage caused by alkylating agents. We found that，in gastric cancer cells，inhibition of autophagy could significantly reduce the expression of MGMT，and sensitized cells to alkylating agents. Therefore, we hypothesized that autophagy may participate in the process of the chemoresistance of gastric cancer cells to alkylating agents by regulating the expression of MGMT. This study is planning to explore the possible mechanism of autophagy regulates MGMT and its role in the chemoresistance of gastric cancer cells to alkylating agents, by the neutral comet assay, dual luciferase experiments and animal experiments, which will provide a new theoretical basis for the treatment of gastric cancer.

烷化剂耐药是胃癌患者治疗失败的主要原因之一。自噬作为细胞及机体应对外界不良刺激的一种应激性保护反应，在肿瘤烷化剂耐药中发挥重要作用。然而，目前有关自噬促进肿瘤细胞烷化剂耐药的机制尚不清楚。文献报道，自噬可通过增强细胞DNA损伤修复能力从而促进肿瘤化疗耐药。O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）作为一种重要的DNA损伤修复蛋白，主要修复烷化剂造成的DNA损伤。我们前期预实验发现，在胃癌细胞中，抑制自噬后可明显降低MGMT的表达，并且明显增强细胞对烷化剂的敏感性。因此，我们推测自噬可能通过调控MGMT表达从而导致胃癌烷化剂耐药。据此，本课题拟采用中性彗星实验、双荧光素酶实验及动物实验等，通过自噬对DNA损伤修复能力的影响、自噬对MGMT表达调控及其调控机制等内容的研究，从影响DNA损伤修复的角度来探讨自噬促进胃癌烷化剂耐药的分子机制，以期为胃癌的治疗提供新的实验基础和理论依据。

研究背景和目的： 胃癌（gastric cancer）是我国发病率最高的恶性肿瘤之一，我国男性和女性中发病率分列第二位和第五位。但胃癌患者的5年总体生存率仍不令人满意。顺铂是治疗胃癌的一线化疗药物。但胃癌细胞顺铂化疗敏感性降低在临床上较为常见。成为制约胃癌患者预后的重要因素之一。因此，揭示胃癌细胞顺铂化疗敏感性降低的具体分子机制，有望促进顺铂抗肿瘤效果、改善胃癌患者预后。主要研究内容： 本课题以自噬为切入点，利用细胞生物学和分子生物学技术，探讨了自噬在胃癌细胞顺铂化疗敏感性降低中的作用及机制。重要结果：①顺铂通过诱导胃癌细胞凋亡杀伤胃癌细胞；②顺铂可剂量及时间依赖性地激活胃癌细胞自噬；而抑制胃癌细胞自噬，能促进顺铂诱导的胃癌细胞凋亡、促进胃癌细胞顺铂化疗敏感性；③顺铂激活自噬的具体机制是顺铂通过抑制DNA损伤反应过程中的转甲基酶---O-6-甲基鸟嘌呤-DNA甲基转移酶（O-6-methylguanine-DNA methyltransferase，MGMT），从而上调自噬相关基因（autophagy-related gene）ATG4B，最终激活了保护性的细胞自噬，使顺铂化疗敏感性降低。