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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 cannabinoid WIN55 and 212-2 (WIN) on the proliferation, invasion and migration of SMMC-7721 hepatocellular carcinoma cells and to explore its molecular mechanism. Methods The effects of (0, 1, 5, 10, 20) μmol / L WIN on the viability of SMMC-7721 cells were analyzed by CCK-8 method. The morphological changes of cells were observed under Hoechst33258 staining and fluorescein isothiocyanate The apoptotic rate was detected by double labeling of annexin V-FITC / PI and flow cytometry. The expressions of apoptosis-related proteins P53, P21, Bcl-2, Bax and phosphoric acid (P-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK). The invasion and migration of cells were detected by Transwell invasion assay and scratch assay. The expression of MMP-14 MMP-14) protein levels. RESULTS: WIN inhibited the proliferation and induced apoptosis of SMMC-7721 cells in a dose-dependent manner. After treated with WIN, Hoechst33258 staining showed that the nuclei were concentrated and broken, resulting in obvious apoptosis. The apoptosis-related protein P53 , P21 and Bax, the level of anti-apoptotic protein Bcl-2 decreased; the levels of AKT, p-AKT and p-ERK decreased but the total protein of ERK did not change; compared with the control group, the invasion of SMMC- And migration ability were significantly reduced, MMP-14 protein levels also decreased. Conclusion WIN can inhibit the proliferation and induce the apoptosis of SMMC-7721 cells, which is related to the activation of P53 by WIN and the inhibition of the expression of AKT, p-AKT and p-ERK. WIN can inhibit the invasion and migration of SMMC-7721 cells by down-regulating the protein level of MMP-14.