甘草酚和5-FU联合处理对结肠癌细胞增殖、凋亡及化疗敏感性的影响

Effect of glycyrol combined with 5-FU on proliferation,apoptosis and chemosensitivity of colon cancer cells

目的探讨甘草酚(GC)和5-FU联合处理对结肠癌细胞的增殖、凋亡及化疗敏感性的影响及机制。方法以人结肠癌细胞系HCT116为研究对象,细胞分别以0,2.5,5,10,15,20μmol/L 5-FU以及0,5,10,20,30,40μmol/L GC处理细胞48 h,用结晶紫染色法计算细胞存活率以确定最佳处理浓度。HCT116细胞分别以10μmol/L 5-FU、20μmol/L GC及10μmol/L 5-FU+20μmol/L GC处理12,24,36,48,60 h,用结晶紫染色法计算细胞存活率以确定最佳处理时间。HCT116细胞分别常规培养(对照组)、10μmol/L 5-FU、20μmol/L GC及10μmol/L 5-FU+20μmol/L GC处理48 h,采用结晶紫染色法测定细胞存活率,流式细胞术测定细胞凋亡率,蛋白免疫印迹法检测裂解多聚腺苷二磷酸核糖聚合酶(cleaved-PARP)、程序性死亡配体1(PD-L1)、磷酸化组蛋白H3(p-Histone H3)和磷酸化蛋白酪氨酸激酶2(p-JAK2)的表达水平。固定5-FU与GC的浓度比例为1∶2,细胞分别以不同浓度的5-FU、GC单独或联合处理细胞48 h,采用Chou-Talalay方程计算协同指数(combination index,CI)评估二者之间的协同效应。结果5-FU和GC抑制细胞增殖的最佳浓度分别为10μmol/L和20μmol/L,5-FU和GC联合处理HCT116细胞抑制增殖的最佳时间为48 h。结晶紫染色结果显示,与对照组相比,5-FU+GC组细胞存活率显著降低(P<0.01);与5-FU组和GC组相比,5-FU+GC组细胞存活率降低,但差异无统计学意义。流式细胞术结果显示,与对照组相比,5-FU+GC组细胞凋亡率显著升高(P<0.01)。协同指数计算结果显示,所有处理组合的CI值均小于1,GC和5-FU的联合处理有协同效应。蛋白免疫印迹结果表明,与对照组相比,5-FU+GC组HCT116细胞中cleaved-PARP表达上调(P<0.05),PD-L1和p-JAK2表达显著下调(P<0.05),p-Histone H3表达显著下调(P<0.01);与5-FU组相比,5-FU+GC组HCT116细胞中cleaved-PARP表达显著上调(P<0.05),p-Histone H3表达显著下调(P<0.01)。结论GC和5-FU联合处理通过下调JAK2的磷酸化水平抑制PD-L1、p-Histone H3的表达,抑制HCT116细胞增殖,诱导细胞凋亡,并增加其化疗敏感性。

Objective To investigate the effects and mechanism of glycyrol(GC)plus 5-FU on the proliferation,apoptosis and chemo-sensitivity of colon cancer cells.Methods The human colon cancer cell line HCT116 cells were treated with 0,2.5,5,10,15,20μmol/L 5-FU and 0,5,10,20,30,40μmol/L GC for 48 h,respectively,and the cell survival was calculated by crystal violet staining method to determine the optimal treatment concentration.The HCT116 cells were treated with 10μmol/L 5-FU,20μmol/L GC and 10μmol/L 5-FU+20μmol/L GC for 12,24,36,48,60 h,respectively,and the cell survival rate was calculated by crystal violet staining to determine the optimal treatment time.The HCT116 cells were routinely cultured(control group)or treated with 10μmol/L 5-FU,20μmol/L GC and 10μmol/L 5-FU+20μmol/L GC for 48 h,respectively.The cell viability was determined by crystal violet staining,the apoptosis was determined by flow cytometry,and the expression levels of cleaved polyadenosine diphosphate ribose polymerase(cleaved-PARP),programmed death ligand 1(PD-L1),phosphorylated histone H3(p-Histone H3)and phosphorylated protein tyrosine kinase 2(p-JAK2)were detected by Western blot.The HCT116 cells were treated with different concentrations of 5-FU,GC,or in combination at the concentration ratio of 1∶2 for 48 h,and the Chou-Talalay equation was used to calculate the combination index(CI)to assess the synergistic effect of 5-FU and GC.Results The optimal concentrations of 5-FU and GC to inhibit cell proliferation were 10μmol/L and 20μmol/L,respectively,and the optimal time for cell proliferation inhibition by the combined treatment of 5-FU and GC was 48 h.The results of crystal violet staining showed that compared with control group,the cell viability was significantly reduced in 5-FU+GC group(P<0.01).Compared with 5-FU group and GC group,the cell survival rate was reduced in 5-FU+GC group,but there was no statistically significant difference.Flow cytometry results showed a significanly higher apoptosis rate in 5-FU+GC group than in control group(P<0.01).The combination index(CI)values of all combinations were less than 1,and the combined treatment of GC and 5-FU had a synergistic effect.Western blot results showed that compared with control group,cleaved-PARP was upregulated in HCT116 cells in 5-FU+GC group(P<0.05),PD-L1 and p-JAK2 expression were downregulated(P<0.05),and p-Histone H3 expression was downregulated(P<0.01).Compared with 5-FU group,cleaved-PARP expression was significantly upregulated in HCT116 cells in 5-FU+GC group(P<0.05),while p-Histone H3 expression was significantly downregulated(P<0.01).Conclusion Combination of glycyrol and 5-FU can inhibit the expressions of PD-L1 and p-Histone H3 by downregulating the phosphorylation level of JAK2,inhibiting the proliferation,inducing apoptosis,and increasing the chemosensitivity of HCT116 cells.