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目的研究白杨素联合喜树碱对鼻咽癌细胞CNE2凋亡的影响,并对其分子机制做初步探讨。方法白杨素以不同浓度(10、20、40μmol/L)单独/联合喜树碱(1μg/mL)处理CNE2细胞后,于普通及荧光倒置显微镜下观察细胞形态变化,获得细胞死亡的定性资料;MTT法分析细胞活性,获得细胞死亡的定量资料;并以Western blotting方法检测凋亡标志蛋白caspase-3和PARP的变化情况及凋亡抑制蛋白Bcl-xL随时间的变化规律。结果形态学观察发现,与对照组比较,白杨素联合喜树碱处理CNE2细胞后,细胞出现明显的死亡量增加,而单独白杨素、喜树碱和未处理的对照组则未观察到细胞的明显减少;MTT法分析的定量资料也支持这一结果,联合处理组细胞存活率随着白杨素剂量增加而下降,与未处理的对照组比较均有统计学意义(P<0.05),而且与单独白杨素及单独喜树碱组比较均有统计学意义(P<0.05);Hochest 33342荧光染色在联合处理组可观察到明显的核固缩细胞增加;Western blotting检测到凋亡标志蛋白caspase-3和PARP原蛋白减少、相应的活化裂解片段出现;全caspase酶抑制剂z-VAD-fmk可有效抑制联合处理引起的CNE2细胞凋亡,阻止凋亡标志蛋白caspase-3和PARP的活化降解;喜树碱引起的凋亡抑制蛋白Bcl-xL表达量增加,随联合处理时间延长而明显下调。结论白杨素联合喜树碱具有促进CNE2细胞凋亡的能力,凋亡抑制蛋白Bcl-xL表达下调是其重要的分子机制。
Objective To investigate the effect of chrysin and camptothecin on the apoptosis of nasopharyngeal carcinoma cell line CNE2 and to explore its molecular mechanism. Methods Cinobuycin was used to treat CNE2 cells with different concentrations (10, 20 and 40μmol / L) of CaCl2 alone or in combination with camptothecin (1μg / mL). The morphological changes of CNE2 cells were observed under ordinary and inverted fluorescence microscope. Cell viability was assayed by MTT assay, and quantitative data of cell death were obtained. The changes of apoptotic marker protein caspase-3 and PARP and the change of apoptosis-inhibiting protein Bcl-xL with time were detected by Western blotting. Results Morphological observation showed that, compared with the control group, significant decrement of cell death occurred after CNE2 cells were treated with chrysin and camptothecin, and no significant difference was found between the cells treated with chrysin, camptothecin and untreated control (P <0.05). The quantitative data by MTT method also support this result. The cell viability of the combined treatment group decreased with the increase of the dosage of the chrysin, which was statistically significant compared with the untreated control group (P <0.05) (P <0.05). Hochest 33342 fluorescence staining showed significant increase of nuclear condensation cells in the combined treatment group. Western blotting detected the expression of caspase- 3 and PARP protein decreased, and the corresponding activated fragment appeared. The caspase inhibitor z-VAD-fmk could effectively inhibit the apoptosis of CNE2 cells induced by the combined treatment and prevent the activation and degradation of the apoptotic markers caspase-3 and PARP. Camptothecin-induced apoptotic protein Bcl-xL expression increased, with the combined treatment time significantly reduced. Conclusion Chrysin combined with CPT can promote the apoptosis of CNE2 cells, and the down-regulation of Bcl-xL is an important molecular mechanism.