虎杖苷调节Akt/MDM2/p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响

Polydatin regulates the proliferation,migration and cell cycle progression of gallbladder cancer cells by regulating the Akt/MDM2/p53 signaling pathway

目的探讨虎杖苷(PD)调节蛋白激酶B/原癌基因MDM2/抑癌基因p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响。方法以人胆囊癌细胞株(GBC-SD)为研究对象,体外培养人胆囊癌细胞株(GBC-SD),使用浓度为10~160 mmol/L的虎杖苷处理细胞24、48、72 h,采用CCK-8法检测细胞的增殖能力,确定最佳实验浓度。将GBC-SD细胞分为对照组(Control组)、虎杖苷低、中、高浓度组(PD-L组、PD-M组、PD-H组)、虎杖苷+Akt激活剂组(PD+SC79组),Transwell小室法评价细胞的迁移能力,Hoechst染色观察细胞的凋亡,流式细胞术检测细胞周期与细胞凋亡,Western blot检测Akt、MDM2、p53磷酸化水平,建立荷瘤小鼠模型评价虎杖苷对胆囊癌肿瘤生长的影响。结果浓度为10~160 mmol/L的虎杖苷处理细胞24 h,可显著抑制GBC-SD细胞的增殖活性,选择10、20、40 mmol/L的虎杖苷进行后续实验;与Control组比较,PD-L组、PD-M组、PD-H组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著降低,G0/G1期细胞比例、P-p53蛋白表达显著升高,且呈浓度依赖性(P<0.05);与PD-H组比较,PD+SC79组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著升高,G0/G1期细胞比例、P-p53蛋白表达显著降低(P<0.05);虎杖苷干预治疗后,小鼠移植瘤的生长速度显著降低(P<0.05)。结论虎杖苷可以通过调节Akt/MDM2/p53信号通路使细胞周期阻滞,抑制胆囊癌细胞增殖、迁移。

Objective To investigate the effects of polydatin(PD)on the proliferation,migration and cell cycle progression of gallbladder cancer cells by regulating the protein kinase B(PKB/Akt)/oncogene MDM2/p53 tumor suppressor gene signaling pathway.Methods The human gallbladder cancer cell line GBC-SD was cultured in vitro,and treated with polydatin at the concentrations of 10-160mmol/L for 24 hours,48 hours and 72 hours.Cell proliferation was detected by CCK-8 assay to determine the optimal experimental concentration.GBC-SD cells were induced with blank control,low-dose,medium-dose and high-dose polydatin,and polydatin+Akt activator SC79.Cell migration was detected by Transwell assay.Apotpsis was detected by Hoechst staining.Flow cytometry was performed to detect apoptosis and cell cycle progression.Phosphorylation levels of Akt,MDM2 and p53 were detected by Western blot.A tumor-bearing mouse model was established to evaluate the effect of polydatin on the growth of gallbladder cancer.Results Treatment of polydatin at 10-160mmol/L for 24h significantly inhibited the proliferation of GBC-SD cells.We selected the concentrations of 10,20 and 40mmol/L as the low-dose,medium-dose and high-dose concentration.Compared with those of blank control,GBC-SD cells induced with low-dose,medium-dose and high-dose polydatin presented significantly lower migratory cell number,apoptotic rate,proportions of cells in G2/M and S phases and protein levels of p-Akt and p-MDM2,and significantly higher ratio of cells in G0/G1 phase and protein level of p-p53 in a dose-dependent manner(P<0.05).Compared with those induced with high-dose polydatin,GBC-SD cells induced with polydatin+SC79 presented significantly higher migratory cell number,apoptotic rate,proportions of cells in G2/M and S phases and protein levels of p-Akt and p-MDM2,and significantly lower ratio of cells in G0/G1 phase and protein level of p-p53(P<0.05).Treatment of polydatin significantly slowed down the growth of gallbladder cancer in mice(P<0.05).Conclusion Polydatin can block cell cycle and inhibit the proliferation and migration of gallbladder cancer cells by regulating the Akt/MDM2/p53 signaling pathway.