EZH2抑制剂DZNEP影响人头颈部鳞癌增殖与凋亡的体内外研究

DZNEP inhibits cell proliferation and induces apoptosis by targeting EZH2 in human head and neck squamous cell carcinoma in vitro and vivo

目的:探讨Zeste同源序列2的增强子(EZH2)的小分子抑制剂DZNEP抑制人头颈部鳞状细胞癌细胞Tb3.1增殖、诱导凋亡的效果与机制。方法:甲基噻唑基四唑(MTT)法测细胞对DZNEP的半数致死量(IC50)、细胞的增殖能力;流式细胞术检测细胞凋亡;平板克隆法检测细胞形成克隆能力;JC-1荧光探针染色法检测细胞线粒体膜电位;蛋白质印迹法(WB)检测EZH2、细胞增殖核抗原(Ki-67)、B细胞淋巴瘤2(Bcl-2、BAX)、活化型含半胱氨酸的天冬氨酸蛋白水解酶3(CCASP-3)的表达。体内实验裸鼠皮下荷瘤模型,通过免疫组织化学染色及原位末端标记(TUNEL)法检测DZNEP对细胞增殖、凋亡的作用。结果:经DZNEP处理后,EZH2蛋白表达降低;MTT法检测DZNEP明显抑制鳞癌细胞Tb3.1的增殖,且具有浓度和时间依赖性;流式细胞术显示DZNEP可诱导细胞凋亡;平板克隆实验表明DZNEP能够明显抑制克隆形成能力,抑制细胞增殖;JC-1荧光探针染色法显示DZNEP可以改变细胞的线粒体膜电位;WB显示,DZNEP可增加促凋亡基因(CCASP-3、BAX)的表达,抑制Ki-67、Bcl-2的表达水平。体内实验观察结束时,DZNEP处理组肿瘤体积较对照组明显减小。免疫组织化学染色结果示,DZNEP组中BAX、CCASP-3表达增加,Ki-67、Bcl-2表达减少;TUNEL结果显示,DZNEP组比DMSO组肿瘤细胞凋亡指数上升。结论:DZNEP通过抑制EZH2表达在体外抑制Tb3.1细胞增殖并诱导凋亡;为进一步探讨EZH2参与人头颈部鳞状细胞癌发生发展的分子机制提供科学实验依据。

Objective：To investigate the anti-tumour molecular mechanism of DZNEP in human head and neck squamous cell carcinoma. Methods：Tb3.1 human head and neck squamous cell carcinoma cell lines were employed. DZNEP was used to suppress the expression of EZH2. Methyl thiazolyl tatrozolium （MTT） assay was applied to determine IC50 and cell survival. Flow cytometry（FCM） was used to measure cell apoptosis. JC-1 fluorescence was employed to determine MMP. The expression level of EZH2 , antigen 67（Ki-67）, B cell lymphoma 2 （Bcl-2, BAX） and cleaved cysteinyl aspartate specific proteinase-3（CCASP-3） were examined by Western blotting. Results：Compared with control and normal control cells, DZNEP inhibited EZH2 expression and affected cell proliferation. Apoptosis rate was elevated by DZNEP. Clone formation assay suggested that cell clone numbers were significantly reduced by DZNEP. BAX and CCASP-3 expression were enhanced by DZNEP treatment, while Ki-67 and Bcl-2 expression were suppressed. In vivo, the tumor volume after treatment with DZNEP was significantly smaller than control groups. Immunological histological chemistry suggested that BAX and CCASP-3 protein expression was up-regulated while Ki-67 and Bcl-2 protein of tumor tissue was down-regulated after treated DZNEP as compared to control groups. TUNEL assay indicated that apoptosis index was increased after treatment with DZNEP compared with control groups. Conclusion：DZNEP modulates proliferation and apoptosis of Tb3.1 cancer cell by inhibiting EZH2, which might promote further research in head and neck squamous cell carcinoma treatment.