探討脂質載運蛋白-2 誘發人類肝癌細胞凋亡之分子機制

癌症居國內十大死因之首位，其中肝癌是男性的十大惡性腫瘤之一，平均約有百分之八十的癌症病人因癌細胞之轉移而致命，但是至今仍然無法得知何種專一性基因活化或缺失會造成人類肝癌形成。脂質載運蛋白-2 (LCN2)又稱嗜中性白血球明膠酶相關性脂質載運蛋白(NGAL)，它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方式測試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、mRNA和蛋白分泌量都不表現在SK-Hep-1和Huh-7細胞。同時我們收集16組肝癌病人檢體證明LCN2在人類肝癌鄰近正常組織表現量比肝癌組織還高。為了證明LCN2在肝癌細胞的生物功能，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，同時未來希望可以有效抑制肝癌的形成。

關鍵字

脂質載運蛋白-2 ；人類肝癌細胞；凋亡

並列摘要

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.

並列關鍵字

lipocalin-2 ； Neutrophil gelatinase associated lipocalin ； LCN2 ； apoptosis ； hepatocellular carcinoma

參考文獻

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探討脂質載運蛋白-2 誘發人類肝癌細胞凋亡之分子機制

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