3-溴丙酮酸增强人鼻咽癌细胞对顺铂敏感性的作用及机制研究

时宗芬; 鲁星月; 张配; 赵素容

doi:10.13898/j.cnki.issn.1000-2200.2020.09.002

3-溴丙酮酸增强人鼻咽癌细胞对顺铂敏感性的作用及机制研究

Study on the effects of 3-bromopyruvate on the sensitivity of human nasopharyngeal carcinoma cells to cisplatin and its mechanism

摘要

摘要:
目的探讨3-溴丙酮酸（3-BrPA）增强人鼻咽癌细胞对顺铂敏感性的作用及作用机制。
方法MTT法检测3-BrPA和顺铂对鼻咽癌HNE1细胞增殖的影响。集落克隆形成实验观察3-BrPA和顺铂对HNE1细胞克隆形成能力的影响。线粒体膜电位检测试剂盒分析细胞早期凋亡情况。DAPI荧光染色法检测细胞核形态变化。AnnexinⅤ-FITC/PI双染法检测细胞凋亡率的变化。ATP检测试剂盒测定细胞内ATP水平的变化。Western blotting检测Bcl-2、Bax、Mcl-1、Bak蛋白的表达。
结果3-BrPA（20、40、80、160、320 μmol/L）、顺铂（2、4、8、16、32 μmol/L）以及80 μmol/L 3-BrPA联合不同浓度顺铂都能明显抑制HNE1细胞的体外增殖活性，与对照组比较，差异有统计学意义（P < 0.01）。8 μmol/L 3-BrPA+0.8 μmol/L顺铂组细胞集落数明显低于3-BrPA、顺铂单独处理组及对照组（P < 0.01）。80 μmol/L 3-BrPA+8 μmol/L顺铂组细胞红色荧光向绿色荧光转变明显；细胞核碎裂及核固缩明显增加；细胞凋亡率明显高于3-BrPA、顺铂单独处理组及对照组（P < 0.01）；细胞内ATP水平明显低于3-BrPA、顺铂单独处理组及对照组（P < 0.01）；Bcl-2、Mcl-1蛋白的表达降低，Bax、Bak蛋白的表达增高，与3-BrPA、顺铂单独处理组及对照组比较，差异有统计学意义（P < 0.05~P < 0.01）。
结论3-BrPA能诱导HNE1细胞凋亡，并能增强HNE1细胞对顺铂的敏感性，其机制可能与降低细胞内ATP水平以及下调Mcl-1和Bcl-2表达、上调Bak和Bax蛋白的表达有关。

Abstract:
ObjectiveTo investigate the effect of 3-bromopyruvate (3-BrPA) on enhancing the sensitivity of human nasopharyngeal carcinoma cells to cisplatin and its mechanism.
MethodsThe effects of 3-BrPA and cisplatin on the proliferation of nasopharyngeal carcinoma HNE1 cells were detected by MTT assay.The effects of 3-BrPA and cisplatin on the colony forming ability of HNE1 cells were observed using colony formation assay.The early apoptosis was analyzed by mitochondrial membrane potential detection kit.The nuclear morphological changes were detected by DAPI fluorescence staining.The apoptosis rate was detected by Annexin Ⅴ-FITC/PI double staining.The changes of intracellular ATP level were measured by ATP detection kit.The protein expressions of Bcl-2, Bax, Mcl-1 and Bak were analyzed by Western blotting.
Results3-BrPA (20, 40, 80, 160 and 320 μmol/L), cisplatin (2, 4, 8, 16 and 32 μmol/L) and 80 μmol/L 3-BrPA combined with different concentrations of cisplatin significantly inhibited the proliferation of HNE1 cells in vitro, which was statistically significant compared with control group (P < 0.01).The combination of 8 μmol/L 3-BrPA and 0.8 μmol/L cisplatin significantly decreased the colony formation of HNE1 cells compared with that of 3-BrPA or cisplatin alone and control group (P < 0.01).The transformation of red fluorescence to green fluorescence was obvious, and nuclear fragmentation and nuclear pyknosis increased significantly in 80 μmol/L 3-BrPA combined with 8 μmol/L cisplatin group.The apoptosis rate and intracellular ATP level in 80 μmol/L 3-BrPA combined with 8 μmol/L cisplatin group were significantly higher and lower than those in 3-BrPA or cisplatin alone group and control group, respectively (P < 0.01).The expression levels of Bcl-2 and Mcl-1 protein were significantly downregulated, and the expression levels of Bax and Bak protein were significantly upregulated in 80 μmol/L 3-BrPA combined with 8 μmol/L cisplatin group, which was statistically significant compared with 3-BrPA or cisplatin alone group and control group (P < 0.05 to P < 0.01).
Conclusions3-BrPA can induce apoptosis in HNE1 cells and enhance the sensitivity of HNE1 cells to cisplatin.The mechanism of which may be related to the decrease of intracellular ATP level, down-regulation of Mcl-1 and Bcl-2 protein, and up-regulation of Bak and Bax protein.

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