巴伐奇宁调节Akt/MDM2/p53信号通路影响肝癌细胞增殖、凋亡和细胞周期

Bavachinin regulats the Akt/MDM2/p53 signaling pathway and affects the proliferation,apoptosis and cell cycle of hepatoma cells

目的:探讨巴伐奇宁调节Akt/MDM2/p53信号通路影响肝癌细胞增殖、凋亡和细胞周期。方法:CCK-8检测巴伐奇宁对正常肝细胞和肝癌细胞的抑制作用;体外培养肝癌HepG2细胞,将HepG2细胞分为Control组,巴伐奇宁低剂量组(10μmol/L)、巴伐奇宁高剂量组(20μmol/L)、巴伐奇宁高剂量(20μmol/L)+SC79(8μg/mL)组;MTT和Edu实验检测细胞增殖;采用流式细胞术检测细胞凋亡和细胞周期;Western blot检测Bcl-2、Bax、p-Akt/Akt、p-MDM2/MDM2、p-p53/p53蛋白表达;建立肝癌肿瘤裸鼠模型,观察肿瘤生长情况,检测肿瘤组织中Bcl-2、Bax、p-Akt/Akt、p-MDM2/MDM2、p-p53/p53蛋白表达。结果:巴伐奇宁对几种肝癌细胞均具有抑制作用,不影响正常肝细胞增殖;体外实验表明,与control组相比,巴伐奇宁低、高剂量组HepG2细胞OD_(490)(24 h、48 h)值、细胞增殖率、S期和G_(2)/M期细胞比例、Bcl-2、p-Akt/Akt和p-MDM2/MDM2蛋白显著降低,凋亡率、G_(0)/G_(1)期细胞比例、p-p53/p53和Bax蛋白显著升高(P<0.05);与巴伐奇宁高剂量组相比,巴伐奇宁高剂量+SC79组HepG2细胞OD_(490)(24 h、48 h)值和细胞增殖率、S期和G_(2)/M期细胞比例、Bcl-2、p-Akt/Akt、p-MDM2/MDM2蛋白表达显著升高,凋亡率、G_(0)/G_(1)期细胞比例、p-p53/p53、Bax蛋白表达显著降低(P<0.05);裸鼠移植瘤实验结果表明,与对照组相比,巴伐奇宁组和Hu7691组裸鼠肿瘤质量和肿瘤体积、Bcl-2、p-Akt/Akt、p-MDM2/MDM2蛋白表达显著降低,裸鼠肿瘤组织中p-p53/p53、Bax蛋白表达显著升高(P<0.05);与巴伐奇宁组相比,Hu7691组裸鼠各检测指标均无统计学差异(P>0.05)。结论:巴伐奇宁可能通过调节Akt/MDM2/p53信号通路来抑制肝癌细胞增殖,阻滞细胞周期,促进细胞凋亡。

Objective:To investigate the effects of bavachinin on the proliferation,apoptosis and cell cycle of liver cancer cells by regulating the Akt/MDM2/p53 signaling pathway.Methods:CCK-8 was used to detect the inhibitory effect of bavachinin on normal hepatocytes and hepatocellular carcinoma cells.HepG2 cells from liver cancer were cultured in vitro and divided into control group,low dose Bavachinin group(10 μmol/L),high dose Bavachinin group(20 μmol/L),and high dose Bavachinin(20 μmol/L)+SC79(8 μg/mL) group.Cell proliferation was detected by MTT and Edu.The apoptosis and cell cycle was detected by flow cytometry.The expression of Bcl-2,Bax,p-Akt/Akt,p-MDM2/MDM2,and p-p53/p53 proteins was detected by Western blot.A nude mouse model of liver cancer was established,tumor growth was observed,and the expression of Bcl-2,Bax,p-Akt/Akt,p-MDM2/MDM2,and p-p53/p53 proteins in tumor tissue was detected.Results:Bavachinin inhibited several kinds of hepatocellular carcinoma cells,but did not affect normal hepatocyte proliferation.In vitro experiments showed that compared with the control group,the OD_(490)(24 h,48 h) value of HepG2 cells,cell proliferation rate,ratios of S phase and G_2/M phase cells,and the expression of Bcl-2,p-Akt/Akt,and p-MDM2/MDM2 proteins in the low and high dose Bavachinin groups were obviously reduced,the apoptosis rate,ratio of G_0/G_(1) phase cells,and the expression of p-p53/p53,and Bax proteins obviously increased(P < 0.05).Compared with the high-dose Bavachinin group,the OD_(490)(24 h,48h) value of HepG2 cells,cell proliferation rate,ratios of S phase and G_2/M phase cells,and the expression of Bcl-2,p-Akt/Akt,and p-MDM2/MDM2 proteins in the high dose Bavachinin + SC79 group were obviously increased,the apoptosis rate,ratio of G_0/G_(1) phase cells,and the expression of p-p53/p53,and Bax proteins obviously reduced(P < 0.05).The results of nude mouse tumor transplantation experiments showed that compared with the control group,the tumor mass and volume,and the expression of Bcl-2,p-Akt/Akt,p-MDM2/MDM2 proteins in the Bavachinin group and Hu7691 group were obviously reduced,the expression of p-p53/p53 and Bax proteins in nude mouse tumor tissue was obviously increased(P < 0.05).Compared with the Bavachinin group,there was no statistically obvious difference in all detection indicators of nude mice in the Hu7691 group(P > 0.05).Conclusion:Bavachinin may inhibit liver cancer cell proliferation,block cell cycle,and promote cell apoptosis by regulating the Akt/MDM2/p53 signaling pathway.