干擾素-α對肝癌細胞株之血管內皮生長因子與E-cadherin 蛋白質表現所造成的影響

目的： VEGF 與E-cadherin，分別被視之為血管新生作用與遠端轉移作用之觀察指標。由VEGF 與E-cadherin 表現量的增加或減少，我們期待找到腫瘤治療中相關的判定預後的指標。本研究嘗試評估干擾素-α於試管內對人類肝癌細胞株之腫瘤發生作用的影響效應。材料與方法：採用兩列分化程度良好的人類肝癌細胞株，Hep G2, Hep 3B，一列分化程度差的人類肝癌細胞株與SK-Hep I。細胞株經以干擾素-α處置培養，評估干擾素-α對其腫瘤遠端轉移能力的影響。我們用不同濃度的干擾素-α(5000 unit/m; 1000 unit/ml)，以不同時間(3;6天)處置Hep G與SK-Hep I。隨後，以ELISA test分析測試培養基中VEGF表現量。Hep 3B細胞株用對照組，以及分別含干擾素-α1000, 5000, 20000 unit/ml之新鮮培養基培養3天。之後，用西方墨點法與Immunocytochemistry（免疫細胞化學）確認細胞膜上E-cadherin 表現量的變化。除上述檢測外，這三株細胞株均經細胞計數與MTT測試以觀察其經干擾素-α處置後的存活率。結果：在血管新生作用的觀察，在A組(5000 unit/m; 3天)與B組(1000 unit/ml, 6天)，兩組均可見干擾素-α調降VEGF蛋白質表現量。A組：VEGF蛋白質表現量為，Hep G2 (無 IFN-α?恁G124.4，經IFN-α?捖B置：76.8)，SK-Hep-I (無IFN-α?恁G44.5，經IFN-α?捖B置：30.0). B組，VEGF蛋白質表現量為，Hep G2 (無IFN-α：225.5，經IFN-α?捖B置：151.4)，SK-Hep-I (無IFN-α?恁G74.1，經IFN-α?捖B置：53.4)。在細胞侵襲作用方面，西方墨點法與Immunocytochemistry（免疫細胞化學）顯示干擾素-α調升E-cadherin 表現量。細胞株經干擾素-α處置後均呈現細胞計數與MTT 測試之細胞存活率下降，顯示干擾素-α對肝癌細胞株有抗細胞增殖作用。結論：干擾素-α的抗腫瘤活性，至少部分來自於調降VEGF蛋白質表現量之抗血管新生作用，以及調升細胞膜上E-cadherin蛋白質表現量之抗細胞侵襲作用，細胞存活率下降之抗細胞增殖作用。藉本研究結果將來可再探討干擾素-α在治療計劃中適當的投與量，或在化學預防方面扮演起預防癌症復發的角色。

關鍵字

E-cadherin ； VEGF ；遠端轉移作用；血管新生作用；干擾素-α ；侵襲作用

並列摘要

Purpose: Vascular endothelial growth factor(VEGF) and E-cadherin have been examined and taken for indicators of angiogenesis and metastasis respectively. In our study, we evaluated the in vitro effects of IFN-α treatment on the tumorigenicity of liver cancer cell lines. From the changes of the expression of VEGF and E-cadherin, we expected to evaluate the prognostic markers. Materials and Methods: Two well differentiated (Hep G2, Hep 3B) and one poor differentiated (SK-Hep I) human liver cancer lines were used. Cells were incubated with Interferon-α to evaluate their metastatic potential. Hep G2, SK-Hep-I cells were incubated with IFN-α for different concentrations (5000 unit/ml; 1000 unit/ml) and different periods(3;6 days) of time. Then, the VEGF protein expression was assayed by ELISA test in those culture media. The Hep 3B cells were treated with fresh medium, that contained 1000, 5000, 20000 unit/ml INF-α. It were incubated for 3 days. After that, E-cadherin expression changes was confirmed by Western blotting and Immunocytochemistry. All three cell lines, treated with INF-α, their survival rates were measured by cell counting and MTT test . Results: In the angiogenesis study, both in group A(5000 unit/ml; 3days)and group B(1000 unit/ml, 6days), the VEGF protein expression was down-regulated by IFN-α. In Group A, the VEGF protein expression were, in Hep G2 (no IFN-α:124.4, with IFN-α: 76.8),and in SK-Hep-I (no IFN-α: 44.5, with IFN-α: 30.0). In Group B, VEGF protein expression were, in Hep G2 (no IFN-α: 225.5, with IFN-α:151.4) and SK-Hep-I (no IFN-α: 74.1, with IFN-α: 53.4). In the cell invasion study, Western blotting and Immunocytochemistry showed that E-cadherin expression were up-regulated by the increasing concentration of IFN-α. The declined cell number counts and survival rates of all three cell lines showed antiproliferative activity of IFN-α. Conclusion: IFN-α confers its antitumor activity, at least in part, by its antiangiogenic activity, which results from decreased VEGF expression, and by anti-invasive activity, which results from elevated E-cadherin expression and antiproliferative activity on cancer cell lines. The results may help us to design the treatment protocol and to define the chemopreventive role of IFN-α. Key words：Interferon-α，Angiogenesis，Invasion，Metastasis，VEGF，E-cadherin