大麻素WIN55,212-2抑制SMMC-7721肝癌细胞增殖和侵袭迁移  

Cannabinoid WIN55,212-2 inhibits proliferation,invasion and migration of human SMMC-7721 hepatocellular carcinoma cells

作　　者：王江林[1] 邓远斐 谭茵[1] 李恪发 温博冠 赵青[1] 

机构地区：[1]广州医科大学生物化学教研室,广东广州511436 [2]广州医科大学,广东广州511436

出　　处：《细胞与分子免疫学杂志》2016年第5期619-624,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81372466);广东省研究生培养创新计划项目(2014JGXM-MS20);广东省高等教育教学改革项目(GDJG20141136);广州市教育科学规划课题(11A033)

摘　　要：目的研究大麻素WIN55,212-2(WIN)对SMMC-7721肝癌细胞增殖、侵袭和迁移能力的影响,并探讨其分子机制。方法应用CCK-8法分析(0、1、5、10、20)μmol/L WIN对SMMC-7721细胞活力的影响;Hoechst33258染色荧光显微镜下观察各组细胞形态改变;异硫氰酸荧光素标记的膜联蛋白Ⅴ/碘化丙啶(annexin V-FITC/PI)双标记结合流式细胞术检测细胞凋亡;Western blot法检测细胞凋亡相关蛋白P53、P21、Bcl-2、Bax和磷酸化蛋白激酶B(p-AKT)、磷酸化细胞外信号调节激酶(p-ERK)的表达;采用TranswellTM侵袭实验及划痕实验检测细胞侵袭、迁移的能力,Western blot法检测基质金属蛋白酶14(MMP-14)蛋白水平。结果 WIN抑制SMMC-7721细胞增殖并诱导其凋亡,两者都具有剂量依赖性;WIN处理后的细胞,经Hoechst33258染色,可发现细胞核浓缩、破碎,引起明显细胞凋亡;凋亡相关蛋白P53、P21、Bax激活,抗凋亡蛋白Bcl-2水平下降;AKT、p-AKT、p-ERK水平下降,而ERK总蛋白无明显变化;与对照组比较,WIN处理后的SMMC-7721细胞侵袭和迁移的能力均显著降低,MMP-14蛋白水平也下降。结论 WIN能抑制SMMC-7721细胞增殖和诱导其凋亡,与WIN激活P53,抑制AKT、p-AKT、p-ERK表达有关。WIN通过下调MMP-14蛋白水平,抑制SMMC-7721细胞侵袭和迁移。Objective To investigate the effects of WIN55, 212-2 （WIN） on the proliferation, invasion and migration of SMMC-7721 hepatocellular carcinoma ceils and its underlying mechanisms. Methods SMMC-7721 cells were treated with （0, 1, 5, 10, 20） μmol/L WIN, and cell viability was determined by CCK-8 assay. The morphological changes of the cells were observed under a fluorescence microscope with Hoechst33258 staining. Cell apoptosis was measured by flow cytometry combined with＇annexin V-FITC/PI staining. The expression levels of apoptosis-related proteins P53, P21, Bcl-2 and Bax, and the phosphorylated AKT （p-AKT） and phosphorylated extracellular signal-regulated kinase （p-ERK） were analyzed by Western blotting. TranswellTM invasion assay was used to detect cell invasion ability. Would healing assay was performed to test cell migration ability. The expression level of matrix metalloproteinase 14 （MMP-14） was evaluated by Western blotting. Results WIN inhibited the proliferation of SMMC-7721 cells and induced cell apoptosis in a dose-dependent manner. After treatment with WIN, the cell nucleus concentrated and broken, indicating obvious cell apoptosis. Western blotting exhibited an up-regulation in the protein expression of P53, P21 and Bax. And the anti-apoptotic protein Bcl-2 was repressed. The expression levels of AKT, p-AKT and p-ERK were down-regulated, whereas the expression of total ERK was not obviously changed. Compared with control group, there was a significant inhibition of cell invasion and migration abilities when SMMC-7721 cells were treated with WIN. The expression level of MMP-14 decreased as well. Conclusion WIN can inhibitthe proliferation of SMMC-7721 cells and induce cell apoptosis. The mechanism is associated with the activation of P53 and the inhibition of AKT, p-AKT and p-ERK. WIN can inhibit the invasion and migration of SMMC-7721 cells through down-regulating the protein expression of MMP-14.

关 键 词：WIN55 212-2 SMMC-7721细胞 细胞增殖 侵袭 迁移 

分 类 号：R735.7[医药卫生—肿瘤] R392-33[医药卫生—临床医学]