Perforin Expression Driven by Survivin Promoter Inhibits Tumor Growth

Abstract

[[abstract]]生存蛋白在成熟的正常組織表現低落，但卻廣泛地、大量地表現於癌細胞中，為著名的IAP (Inhibitor of Apoptosis Protein) 家族成員之一。功能歸納出有三類，分別是抑制細胞凋亡、參與細胞分裂和抗藥性的產生。因為生存蛋白幾乎不在正常細胞表現，已有許多研究利用位於生存蛋白基因上游的啟動子進行基因治療。在生存素啟動子下游接上可對癌細胞產生細胞毒性的蛋白之基因，然後將此載體送入癌細胞內，使細胞大量表現此一蛋白質，而達到抑癌的結果。 穿孔素為細胞內重要的毒殺蛋白，參與CTL、NK cell…等毒殺細胞所執行的細胞毒殺作用。毒殺作用途徑有二，穿孔素參與的為穿孔素/顆粒?途徑，首先利用穿孔素在目標細胞膜上形成類似膜攻擊複合體的孔道，顆粒?再經由此孔道進入到細胞內，活化天冬胺酸特?性半胱胺酸蛋白脢，進而造成細胞凋亡。已發表的文獻多探討穿孔素基因剔除老鼠所產生的生理變化，或著重於分析穿孔素蛋白的功能區。 在本篇研究中結合上述兩個角色，構築穿孔素表現載體，利用生存素啟動子攜帶穿孔素序列在癌細胞裡表現。除了觀察腫瘤生長的抑制效果外，還欲研究如何在腫瘤引發癌細胞的毒殺效果。目前的實驗結果已初步證實穿孔素表現載體確實可在癌細胞裡表現，且具有功能性，可抑制癌細胞的生長。最後我們實際地將穿孔素表現載體運用至一個抗癌藥物的模式上，由結果發現，如果癌細胞內轉染有此載體時，可大幅提高抗癌藥物毒殺癌細胞的效果。日後如果可將此模式應用於癌症的治療，就可以較低的抗癌藥物劑量，達到和以往相當的治療效果，進而降低藥物對於患者的危害。[[abstract]]Survivin was found to express in many different cancer cells and rarely detected in normal cells or tissues. It belongs to IAP (Inhibitor of Apoptosis protein) family that is relateded to the suppression of apoptosis, cell division, and drug resistance. Survivin promoter has been reportedly applied in gene therapy against cancer by introducing a cytotoxicity-induced protein. Perforin is a cytolytic protein produced in CTL, NK cells,….etc. Its major function is involved in cell killing through the synergistic effect of granzymes. Once degranulated from T cells, perforin inserts into the plasma membrane of target cell, and form MAC-like structures, which allow granzymes to pass through the membrane. The caspase signaling pathway is then activated, resulting in the final apoptosis. Studies have addressed on the discussion on physiological condition in perforin- nocked out mice, and the functional domains of perofrin protein. In this study, we combined survivin promoter and perforin codeing sequence to construct a perforin expression vector. The construct is then transfected to colon cancer cell line, CT26, to determined its anti-cancer effect. Results indicated that perforin were able to exert an inhistory effect on CT26 proliferation. Furthermore, if combined with anti-tumor drug, Doxorubicin, a synergistic inhibitory effect was observed. Above all, the perofrin-expressing vector was proven to show a suppressive effect on tumor growth when introducting to them. Moreover, lower does of anti-tumor drug can be used to reach a similar inhibitory effect on tumor growth, and probably further to decrease the side effect on patients.[[note]]碩