辛二酰苯胺异羟肟酸对人肝癌SMMC-7721细胞增殖和凋亡的影响

Effects of suberoylanilide hydroxamic acid on proliferation and apoptosis of human hepatocellular carci-noma cell line SMMC-7721 cells

目的：探讨辛二酰苯胺异羟肟酸（SAHA）对人肝癌细胞株SMMC-7721细胞增殖和凋亡的影响。方法体外培养SMMC-7721细胞，给予不同浓度（2.5、5.0和7.5μmol/L）SAHA处理12~72h，采用噻唑蓝（MTT）比色法检测细胞增殖；加入SAHA（5.0和7.5μmol/L）处理SMMC-7721细胞24h或48h，使用流式细胞仪检测细胞凋亡和细胞周期的变化；采用RT-PCR法检测p53、bcl-2及bax基因mRNA水平；采用分光光度法检测Caspase-3蛋白表达。结果经5.0μmol/L SAHA处理细胞24h和48h时，细胞增殖率较对照下降了25.8%和28.8%，经7.5μmol/L SAHA处理细胞24h和48h时，下降了30.6%和48.6%；经5.0μmol/L或7.5μmol/L SAHA处理细胞24 h后，S期细胞从对照水平（24.33±0.17）%分别显著上升至（32.08±0.160）%和（33.96±0.20）%，（P=0.00），早期凋亡率均由（0.19±0.04）%显著上升至（1.67±0.59）%和（8.92±0.94）%，（P=0.03），而在48h后，S期细胞由（24.33±1.18）%分别显著上升至（32.25±0.53）%和（34.61±0.08）%，早期凋亡率由（0.19±0.04）%分别显著上升至（14.49±2.26）%和（26.23±0.55）%，（P=0.00）；SAHA能够上调p53、bax基因mRNA水平，下调bcl-2基因mRNA水平；经5.0μmol/L和7.5μmol/L SAHA处理细胞24h后，Caspase-3蛋白活性由对照水平（0.41±0.07）分别上升至（0.81±0.02），（P=0.01）和（1.09±0.21），（P=0.00），而在处理48 h后，Caspase-3蛋白活性由对照水平分别上升至（1.43±0.23）和（2.01±0.01），（P均=0.00）。结论 SAHA通过影响p53、bcl-2及bax凋亡相关基因水平及Caspase-3蛋白的活性，对人肝癌细胞株SMMC-7721细胞具有抑制增殖和促进凋亡的作用。

Objective To investigate the effects of suberoylanilide hydroxamic acid （SAHA）on the prolifera-tion and apoptosis of human hepatocellular carcinoma （HCC）cell line SMMC-7721 cells. Methods SMMC-7721 cells cultured in vitro were subjected to different concentrations of SAHA（2.5,5.0 and 7.5μmol/L）for 12 to 72h, and cell viability was determined by MTT assay;SMMC-7721 cells were incubated with SAHA （5.0 and 7.5μmol/L）for 24 or 48 hours,and flow cytometry was used to detect cell apoptosis and cell cycle turnover;RT-PCR was used to detect the mRNA levels of p53,bcl-2 and bax,while caspase-3 activity was detected by spectropho-tometric. Results After 24 or 48 hours of incubation with SAHA at concentration of 5.0μmol/L,the prolifera-tion of SMMC-7721 cells decreased by 25.8% and 28.8%,respectively,and with SAHA at 7.0μmol/L decreased by 30.6% and 48.6%,respectively;After 24 hours of incubation with SAHA at 5.0 and 7.5μmol/L,the percentage of cells in S-stage increased from the control level of（24.33±0.17）% to（32.08±0.160）% and （33.96±0.20）%,re-spectively（P=0.00）,and the apoptosis rate increased from （0.19±0.04） to （1.67±0.59） and （8.92±0.94）,respec-tively,P=0.03）,while after 48 hours of incubation,cells in S-stage increased from（24.33±1.18）%to（32.25±0.53） and （34.61±0.08）%,respectively,and the apoptosis rate increased from （0.19±0.04） to （14.49±2.26） and（26.23±0.55）,（P=0.00）,respectively （P=0.03）;SAHA increased the p53 and bax mRNA and reduced the bcl-2 mRNA levels;After incubation with SAHA at 5.0μmol/L and 7.5μmol/L for 24h,the caspase-3 activity increased from the control level of（0.41±0.07）to（0.81±0.02）,（P=0.01）and（1.09±0.21）,（P=0.00）,respectively,while after 48 hours,the caspase-3 activity increased from （0.41±0.07）to（1.43±0.23） and （2.01±0.01）,respectively（P=0.00）. Conclusions SAHA inhibits proliferation of and induces apoptosis in SMMC-7721 cells,and this effect is associ-ated with an impact on the apoptosis-related gene of p53,bcl-2 and bax and caspase-3 pathway.