自噬抑制剂氯喹通过线粒体ROS增加顺铂诱导QBC939细胞死亡的敏感性

发布时间：2018-08-15 14:27

【摘要】：背景胆管癌组织学起源于胆道被覆上皮细胞,与肝细胞癌分别居于肝胆系统最常见恶性肿瘤的前两位。相比于肝细胞癌,有较多研究指出,胆管癌具有较明显的原发多药耐药性,对顺铂等化疗药物普遍不敏感。对于两种不同组织来源的肿瘤细胞——肝细胞癌细胞和胆管癌细胞——可能因代谢差异而导致自身抗氧化能力不同,并因此对顺铂等抗肿瘤药物表现出不同的敏感性。由于糖代谢与细胞抗氧化能力的密切关系,而顺铂在诱导肿瘤细胞发生凋亡时会增加细胞内ROS水平,尤其是线粒体ROS,因此,推测胆管癌对顺铂耐药可能与其抗氧化能力增强有关。糖代谢为细胞提供了合成生物大分子所需的原料和各种生命活动所需的能量。磷酸戊糖途径作为糖代谢的重要分支之一,不仅能生成核苷酸的原料——磷酸核糖,还能产生大量NADPH。后者是细胞内重要的还原性底物,能够维持GSH的还原性,参与细胞内氧化还原平衡的调节。自噬是维持细胞代谢功能的必要条件,也可以被线粒体功能紊乱和氧化应激诱导激活。异常功能的蛋白和细胞器可以通过分子伴侣介导自噬或巨自噬降解,而自噬的降解产物可以参与代谢过程,重新用于生成生物大分子或提供能量。作为经典的自噬抑制剂CQ,能够抑制肺癌、结肠癌等肿瘤发展,具有治疗肿瘤的潜在可能。研究指出,自噬被抑制后细胞内ROS水平显著升高,由于自噬与代谢关系密切,推测自噬通过与代谢途径的相互作用发挥维持细胞内环境稳态的作用。与之相反的,通过抑制自噬是否能够抑制细胞代谢活性,进而降低其抗氧化能力,增加肿瘤细胞对化疗药物敏感性,成为我们所关注的重点。目的本实验选择肝脏不同组织来源的肿瘤,肝细胞癌Hep G2细胞和胆管细胞癌QBC939细胞,通过对比二者顺铂耐药性差异与代谢、抗氧化能力,探讨自噬抑制剂CQ通过抑制细胞PPP途径、促进细胞内ROS产生等多个途径增加细胞内mt ROS水平,促进顺铂诱导QBC939细胞死亡,进而为通过抑制细胞自噬-溶酶体途径降低细胞糖代谢活性,而增加肿瘤细胞对顺铂的敏感性机制的探索提供新的途径。方法(1)细胞活力和细胞凋亡率变化:利用MTT法分别检测药物作用后细胞活力变化;利用Annexin V/PI染色后流式细胞术检测药物作用后细胞凋亡率变化。(2)细胞内ROS水平和线粒体膜电势检测:应用荧光探针DCFH-DA对细胞进行染色后在倒置荧光显微镜下观察总体ROS水平变化;利用荧光探针Mito SOX对细胞进行染色后在倒置荧光显微镜下观察线粒体ROS水平变化;利用JC-1对细胞染色后通过流式细胞术检测细胞内荧光强度变化。(3)标准化培养基葡萄糖消耗及乳酸释放的检测:利用葡萄糖检测试剂盒和乳酸试剂盒分别检测培养基原液及培养细胞后所含葡萄糖及乳酸的量,通过计算二者差值得出细胞对培养基葡萄糖消耗和乳酸释放的量。利用Bradford法检测培养基内总细胞蛋白量后,将所测得的葡萄糖变化值及乳酸变化值与总蛋白数作比,得出标准化后的葡萄糖消耗量及乳酸释放量。(4)G6PDH活性检测:收集细胞并裂解后,裂解液利用G6PDH活性检测试剂盒处理并通过比色法检测G6PDH活性变化。(5)GSH/GSSG比值、NADPH/NADP比值及羟自由基含量检测:收集细胞并裂解,裂解液分别利用GSH/GSSG检测试剂盒、NADPH/NADP检测试剂盒和羟自由基检测试剂盒处理并通过比色法检测GSH、GSSG、NADPH、NADP和细胞内羟自由基的含量,通过分别作比,得出GSH/GSSG比值、NADPH/NADP比值和相对自由基。(6)自噬相关蛋白检测:收集细胞并裂解,裂解液利用免疫印迹法检测细胞内自噬相关蛋白LC3和p62的水平变化。结果(1)与肝细胞癌Hep G2相比,胆管细胞癌QBC939在CDDP处理后表现出细胞活力变化较小、凋亡发生率较低和细胞内线粒体ROS水平变化较小的特点。(2)与Hep G2相比,QBC939糖代谢方面表现出葡萄糖摄取及消耗能力明显增强、乳酸释放增多的特点,磷酸戊糖途径关键酶G6PDH的表达及活性、NADPH/NADP比值和GSH/GSSG比值也明显增高。(3)2-DG和DHEA均能使细胞内ROS水平升高,与Hep G2相比,QBC939对DHEA的敏感性较低,在DHEA作用下QBC939细胞内总ROS水平和线粒体ROS水平均升高程度较小。(4)CQ与3-MA作用下Hep G2与QBC939细胞活力被显著抑制,细胞内的LC3与p62水平也明显升高,其中QBC939细胞对CQ较为敏感。与Hep G2相比,在CQ作用下QBC939细胞内总ROS和线粒体ROS升高均较明显,同时对CDDP诱导凋亡敏感性也显著升高。(5)与Hep G2细胞相比,CQ处理后QBC939细胞表现出G6PDH活性、GSH/GSSG比值和NADPH/NADP比值均显著降低,线粒体膜电势也降低较明显,而细胞内羟自由基含量显著增加。结论(1)肝细胞癌细胞Hep G2和胆管癌细胞QBC939之间存在代谢模式差异。(2)磷酸戊糖途径活性增强可能是QBC939细胞对顺铂敏感性低的机制之一。(3)自噬抑制剂氯喹通过抑制细胞磷酸戊糖途径活性、降低抗氧化能力,可能是其增加QBC939细胞顺铂敏感性的机制之一。(4)氯喹通过抑制损伤线粒体清除和促进了芬顿反应发生,升高了细胞内ROS水平,促进了细胞对顺铂敏感性的增加。综上,我们以胆管细胞癌QBC939和肝细胞癌Hep G2为研究对象,通过对比研究细胞糖代谢相关抗氧化能力与顺铂耐药之间的关系,并发现自噬抑制剂氯喹通过抑制细胞磷酸戊糖途径等多种方式升高了细胞内ROS水平,增加了细胞对顺铂的敏感性。提示抑制自噬-溶酶体途径可能会成为抑制胆管癌细胞的新思路。
[Abstract]:BACKGROUND Histologically, cholangiocarcinoma originates from coated epithelial cells of the biliary tract and is the first two most common malignant tumors of the hepatobiliary system, respectively. Compared with hepatocellular carcinoma, many studies have shown that cholangiocarcinoma has obvious primary multidrug resistance and is generally insensitive to cisplatin and other therapeutic drugs. Tumor cells, hepatocellular carcinoma cells and cholangiocarcinoma cells, may have different antioxidant abilities due to metabolic differences, and therefore exhibit different sensitivity to antitumor drugs such as cisplatin. Endochondrial ROS levels, especially mitochondrial ROS, suggest that resistance to cisplatin in cholangiocarcinoma may be associated with increased antioxidant capacity. Glucose metabolism provides cells with the raw materials needed to synthesize biological macromolecules and the energy needed for various life activities. Pentose phosphate pathway, as an important branch of glucose metabolism, can not only produce nucleotide progenitors. Phosphorus ribose also produces large amounts of NADPH. The latter is an important reductive substrate in cells, which maintains GSH reducibility and participates in the regulation of redox balance in cells. Autophagy is a necessary condition for maintaining cell metabolic function, and can also be activated by mitochondrial dysfunction and oxidative stress. Organelles can mediate autophagy or macrophagy degradation through molecular chaperones, and the degradation products of autophagy can participate in metabolic processes, and can be reused to produce biological macromolecules or provide energy. As a classical autophagy inhibitor, CQ can inhibit the development of lung cancer, colon cancer and other tumors, and has the potential to treat tumors. ROS levels were significantly elevated after treatment. Because of the close relationship between autophagy and metabolism, it is speculated that autophagy plays a role in maintaining homeostasis of cellular environment by interacting with metabolic pathways. Objective To compare the cisplatin resistance, metabolism and antioxidant capacity of hepatocellular carcinoma Hep G2 cells and cholangiocarcinoma QBC939 cells from different tissues of the liver, and to explore the effect of autophagy inhibitor CQ on the production of ROS by inhibiting PPP pathway. Methods (1) Changes of cell viability and apoptosis rate: MTT assay was used to detect the drug respectively. Cell viability was measured by Annexin V/PI staining and apoptosis rate was detected by flow cytometry. (2) ROS level and mitochondrial membrane potential were measured by fluorescent probe DCFH-DA staining, and ROS level was observed by inverted fluorescence microscope. Mitochondrial ROS level was observed under inverted fluorescence microscope after staining, and fluorescence intensity was detected by flow cytometry after JC-1 staining. (3) Detection of glucose consumption and lactic acid release in standardized medium: glucose detection kit and lactic acid kit were used to detect the original medium respectively. The amount of glucose and lactic acid in the culture medium was calculated by calculating the difference between them, and the amount of glucose consumption and lactic acid release in the culture medium was calculated. Consumption and lactic acid release. (4) G6PDH activity detection: after the cells were collected and lysed, the lysate was treated with G6PDH activity detection kit and the activity of G6PDH was detected by colorimetry. (5) GSH / GSSG ratio, NADPH / NADP ratio and hydroxyl radical content detection: cells were collected and lysed, and the lysate was detected by GSH / GSSG detection kit, NADPH / NADP respectively. The contents of GSH, GSSG, NADPH, NADP and intracellular hydroxyl radicals were detected by colorimetric method. The ratios of GSH/GSSG, NADPH/NADP and relative free radicals were obtained by comparing them. 6. Autophagy-associated protein detection: cells were collected and lysed, lysate was detected by immunoblotting. Results (1) Compared with hepatocellular carcinoma Hep G2, cholangiocarcinoma QBC939 showed less changes in cell viability, lower apoptosis rate and less changes in mitochondrial ROS level after CDDP treatment. (2) Compared with Hep G2, QBC939 showed glucose uptake and consumption in glucose metabolism. The expression and activity of G6PDH, NADPH/NADP ratio and GSH/GSSG ratio were also significantly increased. (3) Both 2-DG and DHEA increased the intracellular ROS level. Compared with Hep G2, QBC939 was less sensitive to DHEA, and the total ROS level and mitochondrial R in QBC939 cells were increased by DHEA. The activity of Hep G2 and QBC939 cells was significantly inhibited by CQ and 3-MA, and the levels of LC3 and p62 were also significantly increased. QBC939 cells were more sensitive to CQ. Compared with Hep G2, the total ROS and mitochondrial ROS in QBC939 cells were significantly increased by CQ and 3-MA, and the sensitivity to CDDP-induced apoptosis was also obvious. (5) Compared with Hep G2 cells, QBC939 cells treated with CQ showed G6PDH activity, GSH / GSSG ratio and NADPH / NADP ratio decreased significantly, mitochondrial membrane potential decreased significantly, and intracellular hydroxyl free radical content increased significantly. Conclusion (1) There was a metabolic pattern difference between hepatocellular carcinoma cells Hep G2 and cholangiocarcinoma cells QBC939. Increased activity of pentose phosphate pathway may be one of the mechanisms underlying the low sensitivity of QBC939 cells to cisplatin. (3) Chloroquine, an autophagy inhibitor, inhibits the activity of pentose phosphate pathway and decreases the antioxidant capacity of QBC939 cells, which may be one of the mechanisms by which it increases the sensitivity of QBC939 cells to cisplatin. (4) Chloroquine enhances the clearance of damaged mitochondria In summary, we studied the relationship between antioxidant capacity related to glucose metabolism and cisplatin resistance in cholangiocarcinoma QBC939 and hepatocellular carcinoma Hep G2, and found that chloroquine, an autophagy inhibitor, inhibited cell phosphoric acid by inhibiting cell phosphoric acid. Inhibition of autophagy-lysosome pathway may be a new way to inhibit cholangiocarcinoma cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.8

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