地尔硫卓通过下调钙激活氯离子通道抑制肝癌细胞增殖和运动

Diltiazem inhibits proliferation and motility of hepatocellular cells in vitro by down-regulating calcium-activated chloride channel TMEM16A

目的观察钙离子通道抑制剂地尔硫卓对肝癌MHCC97H、7402细胞增殖和运动侵袭能力的作用,探讨其相应机制。方法采用不同浓度地尔硫卓(0、25、50、100、200、400μmol/L)干预肝癌MHCC97H、7402细胞不同时间(12、24、48 h)后,用MTT法测定地尔硫卓对肝癌细胞增殖的影响,体外细胞划痕实验检测处理后肝癌细胞运动侵袭能力;通过RT-PCR和免疫细胞化学检测地尔硫卓对肝癌细胞中TMEM16A mRNA和蛋白表达水平的影响。结果地尔硫卓能够有效抑制肝癌MHCC97H、7402细胞的增殖和运动侵袭能力,且具有一定的时间和浓度依赖性特点(P<0.05)。其中,100μmol/L浓度的地尔硫卓作用24 h对MHCC97H肝癌细胞抑制较明显,TMEM16A mRNA和蛋白表达减少(P<0.05),而50μmol/L浓度的地尔硫卓作用48 h对7402肝癌细胞抑制较明显,TMEM16A mRNA和蛋白表达减少(P<0.05)。结论地尔硫卓能一过性抑制MHCC97H、7402肝癌细胞侵袭,这种作用可能与其通过调节钙离子通道TMEM16A的mRNA和蛋白表达而发挥肿瘤抑制作用有关。

Objective To assess the inhibitory effect of diltiazem, a calcium channel inhibitor, on the proliferation and mobility of human hepatocellular carcinoma cells in vitro and explore the possible mechanism. Methods Two human hepatocellular carcinoma cell lines, MHCC97 H and 7402, were treated with different concentrations（0-400 μmol/L） of diltiazem for 12, 24, or48 h, and the changes in the cell proliferation and mobility were observed with MTT assay and wound healing assay,respectively. The changes in the expressions of calcium-activated chloride channel TMEM16 A at mRNA and protein levels in the treated cells were detected using RT-PCR and immunocytochemistry. Results Treatment with diltiazem obviously inhibited the proliferation and suppressed the mobility of MHCC97 H and 7402 cells in a time-and concentration-dependent manner（P〈0.05）. Treatment with 100 μmol/L diltiazem for 24 h significantly inhibited the proliferation of MHCC97 H cells and down-regulated the mRNA and protein levels of TMEM16 A. In 7402 cells, diltiazem treatment at 50 μmol/L for 48 h resulted in the most significant inhibitory effect on the cell proliferation and TMEM16 A expressions. Conclusion Diltiazem can transiently inhibit the invasion of hepatocellular carcinoma cells in vitro possibly by down-regulating the expression of TMEM16 A at both the mRNA and protein levels.