吉九里香碱体外诱导人大肠癌HCT-15细胞凋亡的机理研究

目的: 探讨中药单体吉九里香碱体外诱导HCT-15细胞凋亡的分子作用机理。 方法: 采用Western Blotting、体外Bcl-xL蛋白竞争结合检测实验及RT-PCR对吉九里香碱诱导细胞凋亡的机制进行研究。 结果: 结果显示50 μmol·L^-1吉九里香碱分别作用HCT-15细胞6 h、12 h及24 h,P53蛋白表达水平基本没有变化;Cyto c呈时效性的从线粒体释放到胞质中,Bcl-2蛋白表达基本没有变化,Bcl-xL的表达被抑制在较低水平,吉九里香碱处理细胞6 h后Bax表达开始增加,Bax与Bcl-xL蛋白表达的相对比例上调;与死亡受体通路中相关的FADD表达未见异常,同时线粒体通路中的蛋白包括Caspase-7、Caspase-8、Caspase-9、Caspase-3等均没有被剪切,但相应的凋亡标志物PARP和Rb等以时间依赖形式被剪切;RT-PCR检测结果显示Bcl-2的mRNA水平基本不变,而Bax的mRNA水平随时间的增加而略有增加,说明吉九里香碱从基因水平影响Bax靶基因的转录表达;另外,吉九里香碱能明显竞争BH3与Bcl-xL蛋白的结合,强度甚至强于阳性药HA14-1。 结论: 吉九里香碱诱发HCT-15细胞凋亡的分子机制在于从基因水平影响Bax靶基因的转录表达,同时通过与Bcl-xL蛋白的BH3结构域特异性结合置换出Bax蛋白以增加Bax同源二聚体的浓度,进而影响二者的蛋白表达水平比例,最终导致线粒体功能紊乱来发挥其诱导HCT-15细胞凋亡的效应,且为P53和Caspases非依赖型。

Objective: To investigate the molecular mechanism study on induction of apoptosis by Chinese medicine monomer girinimbrine in HCT-15 cell in vitro. Methods: The mechanism of apoptosis of HCT-15 cells induced by girinimbrine was investigated by Western Blotting, in vitro Bcl-xL competitive binding assay and reverse transcription-PCR(RT-PCR) assay. Results: The results showed that the expression of P53 protein did not change when HCT-15 cells was treated with 50 μmol·L^-1 girinimbrine for 6, 12, 24 h, respectively; Moreover, the Cyto c was released from mitochondria into the cytosol in a time-dependent manner. The expression of Bcl-2 protein kept unchangable and the expression of Bcl-xL has been suppressed to a lower level, therefore while Bax began to increase at 6 h after the treatment of girinimbrine, the ratio of Bax/Bcl-xL expression was up-regulated. But the expression of proteins related to death receptor such as FADD, Caspase-8 maintained invariable, and the proteins of mitochondrial pathways including Caspase-7, Caspase-9 and Caspase-3 were not cleaved, whereas PARP and Rb which are relevant key substrates for apoptosis were cleaved in a time-dependent manner. The result of RT-PCR assay indicated that the transcription level of mRNA for Bcl-2 gene was not altered and Bax gene increased slightly in HCT-15 cells treated with 50 μmol·L^-1 girinimbrine for different times, revealing that Bax gene was involved in the apoptosis induced by girinimbrine but Bcl-2 was not. In addition, girinimbrine could competitively bind with Bcl-xL protein structure domain and showed stronger binding affinity than that of the positive control HA14-1. Conclusion: The molecular mechanism of girinimbrine inducing HCT-15 cells apoptosis involves in such processes as affecting the transcription level of Bax gene, binding with the Bcl-xL protein structure domain and exchanging the Bax protein by specific binding with the BH3 structural domain of Bcl-xL protein to increase the concentration of the Bax homodimers which further affect the ratio of Bax/Bcl-xL expression level and at last leading to mitochondrial dysfunction and thus to induce cell apoptosis, but not depending on P53 and Caspases of the death receptor and mitochondrial pathways.