探討脂質載運蛋白-2誘發人類肝癌細胞凋亡之分子機制

Study on Molecular Mechanism of Lipocalin-2 Inducing Apoptosis in Human Hepatocellular Carcinoma Cells

脂質載運蛋白-2（LCN2）是屬於脂質載運蛋白家族成員之一，主要參與細胞發炎、增殖和侵襲作用。許多報導指出大部分的癌症都有大量LCN2的表現，包含乳癌、大腸癌和卵巢癌等等。但是，目前LCN2在人類肝癌細胞的角色，至今仍然未知。本實驗結果以Western blotting、qRT-PCR和ELISA方式來測定人類肝癌細胞SK-Hep-1和Huh-7內LCN2蛋白質、mRNA和蛋白分泌的表現量。實驗結果證實LCN2蛋白表現、mRNA和蛋白分泌量都在SK-Hep-1和Huh-7細胞中極低。我們建立過度表現LCN2的細胞模式，利用MTT、DAPI、TUNEL和流式細胞儀方式證實過度表現LCN2會抑制肝癌細胞生長、誘導肝癌細胞產生細胞核濃染、細胞週期停留在Sub G1和DNA斷裂等現象，同時我們也採用JC-1染色來證實過度表現LCN2會造成粒線體膜電位的改變，也會增加Bax/Bcl-2的比例。另一方面，西方墨點法證實過度表現LCN2會造成caspase-9、caspase-8、caspase-3和PARP蛋白的活化，同時利用caspase-9和caspase-8抑制劑加入過度表現LCN2的肝癌細胞證實會減緩細胞凋亡的現象。綜合以上結果說明過度表現LCN2會誘導人類肝癌細胞凋亡，未來希望可以調控LCN2表現來有效抑制肝癌的形成。

lipocalin-2 (LCN2) belonging to the lipocalin protein family is involved in the cell inflammation, proliferation and invasion. Elevated LCN2 expression has also been observed in multiple human cancers including breast, colorectal, and ovarian cancers; however, the biological role of increased LCN2 expression in human hepatocellular carcinoma is not yet clear. Our experimental data from Western blotting, qRT-PCR and ELISA revealed the LCN2 was weakly 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 overexpressed LCN2 in cancer cells, and characterized the results by the MTT, DAPI, TUNEL, and flow cytometry analyses. LCN2 overexpression dramatically inhibited cell viability, induced apoptosis features including 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, in overexpressing, the activation of caspase, pro-apoptosis, and anti-apoptosis protein expression in -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, experiments with caspase inhibitors LEHD-FMK and IETD-FMK prevented LCN2-induced apoptosis. These findings indicate that LCN2 overexpression can effectively induce apoptosis of HCC cells and may be developed as a potent therapy against human HCC.