Liu Yan, Li Jihong, Xu Hui, Zhang Yuwen, Liu Yuanli, Liu Xinghan
Department of Biochemistry and Molecular Biology, Harbin Medical University, The Key Laboratory of Biomedical Engineering of Heilongjiang Province, Harbin 150081, PR China.
Int J Mol Med. 2009 Nov;24(5):653-9. doi: 10.3892/ijmm_00000276.
The effect of a 19-amino-acid C-terminal peptide of tumstatin (aa 185-203, peptide 19) on human hepatoma cell (HepG2) proliferation was studied, as well as the mechanism by which it induces tumor cell apoptosis. Recombinant peptide 19 was purified by chitin affinity chromatography and identified by Tricine-SDS-PAGE. The DTT was removed with sephadex G-10. MTT colorimetry was used to evaluate the proliferation of tumor cells. Hematoxylin and eosin staining (H&E staining) and AO/EB double staining were used to view morphological changes during apoptosis. Mitochondrial potential was measured via flow cytometer. Western blot analysis was performed to detect the transfer of cytochrome C from mitochondria to the cytoplasm and to monitor the expression levels of caspase-8, caspase-9, Fas, p53, Bcl-2, Bax and Bid in human hepatoma cells. Recombinant peptide 19 effectively suppressed the proliferation of HepG2 cells and induced apoptosis. Each of the two effects had a dose-dependent relationship with recombinant peptide 19. Peptide 19 upregulated the expression of caspase-9, Fas, p53, Bax and Bid, downregulated the expression of Bcl-2 and had little effect on the expression of caspase 8. Peptide 19 decreased the mitochondrial membrane potential and induced the release of cytochrome C from mitochondria to the cytoplasm. In conclusion, peptide 19 induced HepG2 cell apoptosis through the mitochondrial apoptosis pathway.
研究了肿瘤抑素19个氨基酸的C末端肽（aa 185 - 203，肽19）对人肝癌细胞（HepG2）增殖的影响及其诱导肿瘤细胞凋亡的机制。重组肽19通过几丁质亲和层析纯化，并通过Tricine - SDS - PAGE鉴定。用葡聚糖凝胶G - 10去除二硫苏糖醇（DTT）。采用MTT比色法评估肿瘤细胞的增殖。苏木精和伊红染色（H&E染色）以及AO/EB双重染色用于观察凋亡过程中的形态变化。通过流式细胞仪测量线粒体膜电位。进行蛋白质免疫印迹分析以检测细胞色素C从线粒体向细胞质的转移，并监测人肝癌细胞中半胱天冬酶 - 8、半胱天冬酶 - 9、Fas、p53、Bcl - 2、Bax和Bid的表达水平。重组肽19有效抑制HepG2细胞的增殖并诱导凋亡。这两种作用均与重组肽19呈剂量依赖性关系。肽19上调半胱天冬酶 - 9、Fas、p53、Bax和Bid的表达，下调Bcl - 2的表达，对半胱天冬酶8的表达影响不大。肽19降低线粒体膜电位并诱导细胞色素C从线粒体释放到细胞质中。总之，肽19通过线粒体凋亡途径诱导HepG2细胞凋亡。
