三氧化二砷对人肝癌SMMC-7721细胞中P27kip1、JAB1表达的影响及其作用机制

背景与目的:三氧化二砷(arsenic trioxide,As2O3)作为治疗实体瘤的新药已应用于临床,但其作用机理尚不清楚.本研究拟探讨As2O3对肝癌SMMC-7721细胞增殖的影响,及其对细胞周期素依赖性激酶抑制剂(cyclin-dependent kinase inhibitors,CDKIs)P27kip1和P27kip1相关蛋白c-Jun结合蛋白-1(c-Jun activation domain-binding protein 1,JAB1)表达的调控作用.方法:体外培养人肝癌细胞株SMMC-7721,用0～8 μmol/L As2O3处理96 h,用WST-8法检测细胞的存活率.用2 μmol/L As2O3作用72 h,在指点时间点收集细胞,用Western blot技术检测P27kip1、JAB1在SMMC-7721细胞中的表达,同时采用核浆分离方法及细胞免疫荧光技术检测P27kip1、JAB1表达和亚细胞定位的改变.结果:与对照组比较,As2O3可明显抑制SMMC-7721细胞的增殖,96 h时IC50为(1.81±0.41)μmol/L.2μmol/LAs2O3处理12 h后,SMMC-7721细胞中P27kip1蛋白表达增加,而JAB1蛋白表达减少.在As2O3处理后12 h、24 h,P27kip1与JAB1均发生从胞浆向胞核的易位.免疫荧光检测P27kip1和JAB1的亚细胞定位情况,结果也显示2 μmol/L As2O3可诱导两者在胞核的积聚.结论:As2O3可下调JAB1的表达,从而影响P27kip1的核内外分布及表达,并影响P27kip1的功能状态,进而参与调控肝癌细胞的增殖.

Inhibitory effect of arsenic trioxide on proliferation of human hepatocellular carcinoma cell line SMMC-7721 and the mechanism

BACKGROUND & OBJECTIVE: Arsenic trioxide (As(2)O(3)) is a new drug used to treat solid tumors. However, the mechanism is still unclear. This study was to investigate the effects of As(2)O(3) on the proliferation of human hepatocellular carcinoma (HCC) cell line SMMC-7721, and to explore the mechanisms. METHODS: When treated with 0-8 micromol/L As(2)O(3) for 96 h, the survival rate of SMMC-7721 cells was determined by WST-8 assay. When treated with 2 micromol/L As(2)O(3) for 72 h, the expression of P27(kip1) and c-Jun activation domain-binding protein 1 (JAB1) in SMMC-7721 cells were detected at different time points by Western blot, the subcellular localization of P27(kip1) and JAB1 was detected by subcellular fractionation and immunofluorescent staining. RESULTS: As(2)O(3) significantly inhibited the proliferation of SMMC-7721 cells. The 50% inhibition concentration (IC50) of As(2)O(3) to SMMC-7721 cells was (1.81+/-0.41) micromol/L at 96 h. When SMMC-7721 cells were treated with 2 mumol/L As(2)O(3) for 12 h, the expression of JAB1 was down-regulated and that of P27kip1 was up-regulated. Furthermore, P27(kip1) and JAB1 proteins were translocated from the cytoplasm into nuclei when cells were exposed to 2 micromol/L As(2)O(3) for 12 h and 24 h, respectively. The nuclear accumulation of both proteins was also observed under fluorescence microscope after treatment of 2 micromol/L As(2)O(3). CONCLUSION: As(2)O(3) attenuates JAB1 expression, thereby disturbs the location and expression of P27(kip1), and may participate in regulating the proliferation of SMMC-7721 cells through interfering with the function of P27(kip1).