Mitochondria-mediated tumstatin peptide-induced HepG2 cell apoptosis

Abstract

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.