薯蓣皂苷的次皂苷元B(DRG)体外诱导人大肠癌HCT-15细胞凋亡的机制研究

Mechanism Study on Induction of Apoptosis by Diosgenin-3-O-α-L-Rhamnopyranosyl-(1→4)-β-D-Glucopyranoside (DRG) in HCT-15 Cell in Vitro

目的探讨中药单体薯蓣皂苷的次皂苷元B(DRG)体外诱导HCT-15细胞凋亡的分子作用机制。方法采用Westernblotting、体外Bcl-xL蛋白竞争结合检测实验及逆转录PCR(RT-PCR)对DRG诱导细胞凋亡的机制进行了研究。结果在HCT-15细胞中,DRG促发了线粒体调控的凋亡途径,细胞色素c呈时效性地从线粒体中释放到胞质中,同时Bax与Bcl-2蛋白表达的相对比例上调,caspase-9、caspase-3和PARP等被剪切成具有活性的片段,但是caspase-7却未见剪切,同时还不影响P53和Bcl-xL的表达;而与死亡受体途径相关的FADD表达无明显变化,caspase-8酶原也未见剪切;另外,DRG也不抑制BH3短肽与Bcl-xL的结合;RT-PCR检测结果表明,DRG诱导HCT-15细胞凋亡相关基因Bcl-2和Bax的mRNA水平的改变,进而导致Bcl-2蛋白表达水平下降,而Bax蛋白表达水平则明显增加,说明DRG诱发的细胞凋亡中涉及到Bcl-2和Bax基因水平的变化。结论 DRG诱发HCT-15细胞凋亡的分子机制在于从基因水平影响Bax和Bcl-2靶基因的转录表达,上调Bax/Bcl-2蛋白表达水平比例,并最终通过激活经典的线粒体途径而不是死亡受体途径来发挥其HCT-15细胞凋亡诱导作用,且对P53呈非依赖型。

OBJECTIVE To investigate the molecular mechanism of the induction of apoptosis by Chinese medicine monomer diosgenin-3-O-a-L-rhamnopyranosyl-（1→4）-β-D-glucopyranoside （DRG） in HCT-15 cell in vitro. METHODS The apoptotic mechanism induced by DRG was investigated by Western blotting, in vitro Bcl-xL competitive binding assay and reverse transcription-PCR （ RT- PCR） assay. RESULTS It was proved that DRG triggered a mitochondria-controlled apoptotic pathway to induce apoptosis in HCT-15 cells, which involved the release of cytochrome C from mitochondria into the cytosol in a time-dependent manner, the up-regulation of the ratio of Bax/Bcl-2 expression, and the cleavage of caspase-9, caspase-3 and PARP, without effect on the expression of P53, Bcl-xL and the cleavage of caspase-7. Whereas, the changes of proteins related to death receptor pathway such as FADD and caspase-8 were not detected. In addition, DRG was not found to inhibit the BH3 peptide from combining with Bcl-xL protein. Semi-quantitative RT-PCR indi- cated the changes of mRNA levels of Bcl-2 and Bax genes, which might contribute to the decreased protein levels of Bcl-2 and the increased protein levels of Bax caused by DRG, revealing that both Bcl-2 and Bax gene were involved in the apoptosis induced by DRG. CONCLUSION The molecular mechanism of DRG inducing HCT-15 cells apoptosis involves in such processes as affecting the mRNA transcription level of Bax and Bcl-2 genes, up-regulating the ratio of Bax/Bcl-2 protein expression level and at last activating classical mitochondrial pathway but not death receptor pathway and P53-independent manner to induce cell apoptosis.