| Summary: | 碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 100 === Cancer is the top of ten causes of death in Taiwan; especially hepatocellular carcinoma threatens all of male. The metastasis (invasion) of cancer cells leads 80% patients died, and inhibition of cancer cells metastasis will markly decrease the death rate. However, at present, none of genes activation or loss has been demonstrated to be specific sufficient for the prognostic significance. lipocalin-2 (LCN2), also called Neutrophil gelatinase associated lipocalin (NGAL), belongs to the lipocalin protein family, it involved the cell inflammation, proliferation and invasion. Elevated NGAL expression has also been observed in multiple human cancers including breast, colorectal, and ovarian cancers; however, the biological roles of elevated LCN2 in human hepatocellular carcinoma is not yet clear.
We experiment data from western blotting, qRT-PCR and ELISA revealed the LCN2 was weak detected in the HCC cell lines, and LCN2 was found to be downregulated in tumor tissues in 16 HCC patients. In order to understand the biological function of LCN2, we set up the overexpression assay as study tool, and used the MTT, DAPI, TUNEL, and flow cytometry analyses revealed that LCN2 overexpression dramatically inhibited cell viability, induced apoptosis features of cell-cycle arrest in sub-G1 phase, induced DNA fragmentation, and induced condensation of chromatin in Huh-7 and SK-Hep-1 cells. In addition, western blots were used to detect the activation of caspase, pro-apoptosis, and anti-apoptosis protein expression in overexpress-LCN2 HCC cells. LCN2-induced apoptosis was characterized by cleavage of caspase-9, caspase-8, caspase-3, and PARP protein, and a reduction in the mitochondrial membrane potential (MMP). Furthermore, LCN2 also enhanced the down-regulated Bcl-2 and up-regulated the expression of Bax. Additional, our experiments with caspase inhibitors LEHD-FMK and IETD-FMK was prevent LCN2-induced apoptosis. These findings indicate that LCN2 overexpression can effectively induce apoptosis of HCC cells and may be used as a potent therapy against human HCC.
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