目的

探讨蛋白酶体抑制剂Velcade 诱导人肝癌细胞株HepG2 凋亡的作用和机制。

方法

以不同浓度Velcade 处理HepG2细胞24 h 和48 h。四甲基偶氮唑蓝比色法评价细胞生长情况；流式细胞术检测细胞凋亡；Western blot 测定caspase 3 改变；RT-PCR 检测Bcl-2家族、Cyclin A 和Cyclin D mRNA 的表达。

结果

不同Velcade 浓度（32、64、128、256、512 nmol/L）处理HepG2 细胞48 h 后，细胞活性较对照组明显减少（分别是91.4%±2.1%、75.0%±3.7%、57.3%±1.6%、52.7%±1.6%和31.4%±2.6%），与对照组（100.0%±1.7%）比较具有显著性差异（P＜0.05）。128 nmol/L 的Velcade 处理HepG2 细胞，48 h 流式细胞仪检测结果显示SubG1 细胞百分数明显升高，SubG1 细胞百分数（27.3%±5.3%），与对照组（4.3%±0.5%）差别显著（P＜0.05），同时，Pro-caspase 3 明显下降；但Velcade 对Bcl-2 家族表达无明显影响；Velcade 诱导HepG2 细胞G2/M 阻滞，抑制Cyclin A 和Cyclin D 表达。

结论

Velcade 诱导肝癌细胞凋亡不通过改变Bcl-2 家族表达，可能存在其它途径；Velcade 诱导肝癌细胞G2/M 周期阻滞与调节通过下调Cyclin A 和Cyclin D 表达相关。

Objective

To investigate the effects of Velcade,a proteasome inhibitor,on HepG2 cells,and to explore the mechanism.

Methods

HepG2 cells were treated with various concentrations of Velcade for 24 h and 48 h. The cell viability was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)assay.Cell apoptosis was investigated by flow cytometry.Western blot assayed the change of caspase 3.The expressions of Bcl-2 family,Cyclin A and Cyclin D were detected by reverse transcription-polymerase chain reaction (RT-PCR).

Results

Viabilities of HepG2 cells treated with various concentrations of Velcade(32,64,128,256,512 nmol/L)for 48 h were significantly decreased compared to control group.Viabilities of HepG2 cells gradually reduced to 91.4%±2.1%, 75.0%±3.7%, 57.3%±1.6%, 52.7%±1.6% and 31.4%±2.6%, respectively. The differences compared with control group (100.0%±1.7%)were significant (P<0.05).The percent of SubG1 of HepG2 cells (27.3%±5.3%)treated with 128 nmol/L Velcade 48 h was significantly higher than that in control group(4.3%±0.5%,P<0.05).After treatment with Velcade(128 nmol/L)for 24 h,the expression of caspase 3 markedly decreased compared with control. Velcade greatly increased the G2/M phase in HepG2 cells. Velcade resulted in no significant changes of Bcl-2 family member.However,Velcade down regulated the expressions of Cyclin A and Cyclin D.

Conclusion

The mechanism of Velcade to induced apoptosis in HepG2 is not associated with change of Bcl-2 family member.Velcade induced G2/M phase arrest via down regulation of Cyclin A and D.