GHSC-73对人肝癌细胞株HepG2细胞增殖及细胞周期的影响

Effects of GHSC-73 on proliferation and cell cycle of human hepatocellular carcinoma HepG2 cells

目的:观察来源于海芒果种子的强心苷化合物β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin(GHSC-73)对人肝癌细胞株HepG2细胞增殖和细胞周期的影响,并初步探讨其作用机制。方法:采用MTT法检测不同浓度GHSC-73对人肝癌细胞株HepG2增殖的影响;采用流式细胞仪Propidium Iodide(PI)单标法检测GHSC-73对HepG2细胞周期进程的影响;Real-time RT-PCR检测S期相关基因在GHSC-73处理前后表达的变化。结果:GHSC-73可抑制HepG2细胞增殖,其作用具有时间和浓度依赖性,作用HepG2细胞24、48、72h后,IC50分别为(5.18±0.21)、(0.37±0.08)、(1.66±0.16)μmol/L。与对照组相比,随着作用时间的延长,S期细胞百分比逐渐增多,而G0/G1期细胞百分比逐渐减少(P<0.05),G2/M期细胞百分比基本保持不变,表明GHSC-73阻滞HepG2细胞于S期。Real-timeRT-PCR检测S期相关基因的结果显示:GHSC-73诱导后HepG2细胞GADD153、cyclin D1、p21基因表达上调,cyclin A2、DHFR、TYMS基因表达下调。结论:GHSC-73可通过S期阻滞来抑制HepG2细胞增殖,其阻滞作用可能与GADD153、cyclin D1、p21、cyclinA2、DHFR和TYMS基因表达变化有关。

Objective：To investigate the effects of β-D-glucosyl-（1-4）-α-L-thevetosides of 17β-digitoxigenin （GHSC-73）,an isolate from the seeds of Cerbera manghas L.,on cell growth and cell cycle regulation of human hepatocellular carcinoma cell line HepG2,and to discuss the related mechanism.Methods：HepG2 cells were treated with different concentrations （0-80 μmol/L） of GHSC-73.Cell viability was determined using MTT assay at 24,48,and 72 h after treatment.Cell cycle distribution was assessed by flow cytometry after propidium iodide （PI） staining.Expression of the S phase associated genes GADD153,cyclin D1,cyclin A2,DHFR,TYMS,and p21 was determined by real-time RT-PCR before and after GHSC-73 treatment.Results：GHSC-73 inhibited the cell proliferation of HepG2 cells in a dose-and time-dependent manner.The values of IC50 were （5.18±0.21）,（0.37±0.08）,and （1.66±0.16） μmol/L at 24,48,and 72 h,respectively.Compared with control group,the proportion of cells in S phase increased in the treatment group with the prolongation of treatment,the cells in G0/G1 phase gradually decreased （P〈0.05）,and cells in G2/M phase remained unchanged,indicating that GHSC-73 blocked HepG2 cell in S phase.Real-time RT-PCR showed that cyclin A2,DHFR,and TYMS were down-regulated,and p21,GADD153,and cyclin D1 were up-regulated,which might be associated with the GHSC-73-induced S phase arrest in HepG2 cells.Conclusion：GHSC-73 can inhibit growth of HepG2 cells by inducing S phase arrest; down-regulation of cyclin A2,DHFR,and TYMS genes and up-regulation of p21,GADD153,and cyclin D1 genes might participate in the induction of arrest.