氯通道参与姜黄素诱导的乳腺癌MCF-7凋亡

目的： 探讨姜黄素（curcumin）诱导的乳腺癌细胞MCF-7凋亡性途径中氯通道是否参与。方法： MTT检测姜黄素对MCF-7细胞的增殖抑制作用，及用氯通道阻断剂5-nitro-2-（3-phenylpropyl-amino）-benzoate （NPPB）预孵2 h后再加入姜黄素的增殖抑制作用；流式细胞术验证姜黄素诱导细胞的凋亡；采用全细胞膜片钳技术记录加入姜黄素后，细胞氯电流的变化。结果： （1）姜黄素对MCF-7细胞的增殖抑制作用呈现剂量依赖性和时间依赖性。NPPB组与单加入姜黄素组相比，细胞生存率存在明显差异，提示阻滞氯通道可导致姜黄素抑制MCF-7细胞增殖的作用减弱。（2）流式结果表示，加入姜黄素（25 μmol·L^-1）24 h后凋亡率33.16%；而姜黄素（25 μmol·L^-1）+NPPB （100 μmol·L^-1）组凋亡率仅为9.16%，相比对照组（6.26%）、NPPB组（7.31%），氯通道阻断剂明显抑制姜黄素诱导的MCF-7乳腺癌细胞的凋亡，且对早期凋亡的抑制作用明显。（3）全细胞膜片钳技术结果，细胞外灌流姜黄素（25 μmol·L^-1）可激活乳腺癌细胞MCF-7氯通道的开放，产生氯电流，翻转电位（－3.18±1.30） mV，外向电流密度（3.16±1.42） pA·pF^-1，内向电流密度为（－2.76±1.38） pA·pF^-1，该电流可被氯通道阻断剂NPPB、DIDS完全抑制，细胞外灌流高渗溶液亦可完全抑制该电流。结论： 姜黄素可能通过激活容积敏感性氯通道而诱导细胞的凋亡，氯通道的激活可能在姜黄素诱导肿瘤凋亡通路中起着重要的作用。

Curcumin induced apoptosis by activiting chloride channels in MCF-7 cells

OBJECTIVE To investigate the role of chloride channels in curcumin-induced apoptotic pathway in MCF-7 breast cancer cells. METHODS The inhibitory effect of curcumin on the proliferation of MCF-7 cells and the inhibitory effect of curcumin on the proliferation of MCF-7 cells after incubation with 5-nitro-2-(3-phenylpropylamino) -benzoate(NPPB), a chloride channel blocker, was detected by MTT assay. The apoptosis induced by curcumin was verified by flow cytometry. RESULTS (1) Curcumin inhibited the proliferation of MCF-7 cells in a dose-dependent and time-dependent manner.There was a significant difference in cell survival rate between the NPPB group and the curcumin alone group, suggesting that blocking the chloride channel could lead to a decrease in the effect of curcumin on inhibiting the proliferation of MCF-7 cells. (2) Flow results indicated that the apoptotic rate was 33.16% after the addition of curcumin(25 μmol·L^-1) for 24 h; while the apoptotic rate was only 9.16% in the curcumin(25 μmol·L^-1) + NPPB(100 μmol·L^-1) group, compared with the control group(6.26%) and NPPB group(7.31%), the chloride channel blocker significantly inhibited curcumin-induced apoptosis of MCF-7 breast cancer cells, and the inhibition of early apoptosis was significant. The results of whole cell patch clamp technique showed that extracellular perfusion of curcumin(25 μmol·L^-1) could activate the opening of MCF-7 chloride channels in breast cancer cells, resulting in a chloride current, with a reversal potential of (-3.18±1.30) mV, an outward current density of (3.16±1.42) pA·pF^-1, and an inward current density of (-2.76±1.38) pA·pF^-1, which was completely inhibited by the chloride channel blockers NPPB and DIDS, and also by the extracellular perfusion of hypertonic solution. CONCLUSION Curcumin induced the cells apoptosis possibly by activating volume-sensitive chloride channels, and the activation of chloride channel may play an important role in curcumin-induced tumor apoptosis.