苦参碱联合硫蒽酮调控自噬靶向逆转胃癌干细胞耐药的作用及机制研究

Chemoresistance is a major cause of treatment failure in gastric cancer. Residual cells after chemotherapy contain a high proportion of cancer stem cells which are responsible for cancer recurrence and metastasis. So it is very important for effectively eradicating the remaining cancer cells to reverse chemoresistance in gastric cancer stem cells (GCSCs). Our previous study showed that autophagy is activated as a protective mechanism during the matrine-induced apoptosis of gastric cancer cells and inhibition of autophagy enhances the antitumor potential of matrine in gastric cancer. In the study, we use experimental methods and technologies including establishment of chemoresistant gastric cancer cell lines, sorting of GCSCs, immune coprecipitation, immunofluorescence, transmission electron microscopy, gene overexpression, gene knockdown and knockout. We would for the first time clarify the chemoresistant mechanisms in GCSCs from the perspective of autophagy, and investigate effectiveness that matrine combined lucanthone reverses chemoresistance in GCSCs by regulating autophagy which is regarded as a new therapeutic target for overcoming drug resistance. We further study the roles of lysosome, DRAM1 and cathepsin D in the regulation of autophagy and apoptosis and reversion of chemoresistance. This study would be expected to provide new insights into improving the therapeutic effect for gastric cancer, and provide experimental data about combinations of monomer extracted from traditional Chinese medicine with autophagy inhibitor in the treatment of gastric cancer.

耐药性是胃癌化疗失败的主要原因，肿瘤干细胞在化疗后的残留细胞中比例较高，被认为是肿瘤复发和转移的根源，因此只有靶向逆转胃癌干细胞（GCSCs）耐药才能有效杀灭清除胃癌细胞。我们前期研究发现苦参碱在诱导胃癌细胞凋亡的同时产生保护性自噬，抑制自噬能增强其抗肿瘤效应。本项目拟通过构建耐药胃癌细胞株、胃癌干细胞分选、免疫共沉淀、免疫荧光、透射电镜、基因过表达、基因敲减和基因敲除等方法，从分子、细胞、组织以及动物整体水平，首次从自噬角度阐述GCSCs的耐药机制，并将自噬作为逆转GCSCs 耐药的新靶点，研究苦参碱联合新型自噬抑制剂硫蒽酮通过调控自噬在靶向逆转GCSCs耐药中的作用，并深入探讨溶酶体、自噬基因DRAM1、组织蛋白酶D在调控自噬和凋亡转换、逆转耐药中扮演的重要作用。本课题的实施可为进一步提高胃癌治疗效果提供新的思路，为中药单体及联合自噬抑制剂治疗胃癌提供实验依据。

胃癌是严重危害人类健康的恶性肿瘤。尽管近年来胃癌的化疗取得了一定的进展，但耐药性依然是化疗失败的主要原因，只有靶向逆转胃癌细胞耐药才能有效杀灭胃癌细胞。本项目研究了耐药胃癌细胞SGC7901/VCR和SGC7901/ADR的耐药倍数、苦参碱对耐药胃癌细胞的逆转作用，并深入研究了苦参碱逆转胃癌耐药的机制，主要从细胞增殖、细胞周期、凋亡、自噬、蛋白组学方面进行探讨。.研究结果表明胃癌细胞SGC7901/VCR和SGC7901/ADR为多药耐药细胞株，SGC7901/VCR对ADR、VCR的耐药倍数分别为370倍、23.2倍，SGC7901/ADR对ADR、VCR的耐药倍数分别为123.3倍、15.1倍。苦参碱具有逆转胃癌细胞耐药的作用，对耐药细胞SGC7901/VCR和SGC7901/ADR的逆转倍数分别为23.2和123.3。同时苦参碱具有抑制耐药胃癌细胞增殖、细胞周期阻滞、诱导凋亡的作用，能明显抑制细胞周期蛋白及激酶的表达水平，并能降低耐药蛋白P-gp表达水平。研究还发现耐药细胞SGC7901/VCR用苦参碱干预后，Atg5和Atg7表达量减少，同时LC3-II表达量增加，p62表达无明显变化，说明苦参碱对耐药胃癌细胞自噬的早期阶段和晚期阶段具有抑制作用。蛋白组学结果表明苦参碱能显著影响涉及溶酶体、自噬、凋亡等关键分子的表达，并能明显影响涉及溶酶体、自噬、凋亡等的信号转导通路，初步结果表明苦参碱能通过自噬溶酶体途径逆转胃癌细胞耐药，自噬可作为逆转胃癌耐药的新靶点，苦参碱可作为逆转胃癌耐药的有效药物。本项目可为传统中药提取物治疗胃癌提供新的思路和理论依据。