嶄新化合物PT-262造成細胞骨架改變及死亡的機轉

Abstract

癌症是國人死亡主因，研究有效對抗腫瘤的藥物，是當前迫切需要。最近我們發現一種嶄新的5,8-quinolinedione衍生物，稱為PT-262，它會嚴重地造成癌細胞骨架改變及細胞死亡，然而機轉仍不清楚，因此在本計畫中，我們主要研究PT-262如何誘發細胞骨架改變及細胞死亡的機制，建立人類肺癌細胞模式，並評估對正常肺細胞的影響。將探討PT-262是否直接結合肌動蛋白，或是間接經由影響RhoGTPases蛋白訊息傳遞的路徑，導致肌動蛋白異常的聚合作用，將以肌動蛋白聚合法、質譜儀分析及共軛焦原子力顯微鏡觀察，研究PT-262與肌動蛋白的結合反應，並以基因阻斷或抑制劑的方式，研究抑制RhoGTPases (包括RhoA、Rac1及Cdc42)與有絲抗原活化蛋白激酶(包括JNK、p38及ERK)路徑之後，對PT-262在細胞骨架改變及死亡的影響，並進一步利用RhoGTPases等基因表現載體，轉殖細胞後，觀察是否影響PT-262對細胞骨架及細胞死亡的作用，包括分析細胞的骨架改變、存活、凋亡及週期，並進行癌細胞移行及侵襲的分析，研究PT-262對抗腫瘤細胞轉移的能力。再者，研究RhoGTPases與有絲抗原活化蛋白激酶之間的相互調控角色，以西方墨點法、免疫沈澱法、免疫螢光染色和共軛焦顯微鏡，分析相關蛋白的表達、交互作用與細胞中的位置。本計畫最終目標為提供PT-262造成癌細胞骨架改變及死亡的機轉，並應用於癌症的治療。

Cancer has become the major mortality in Taiwan. Development of the more efficient anticancer drugs is highly desirable. Recently, we found a brand-new compound derived from 5,8-quinolinedione (designated as PT-262), which dramatically induced the cytoskeleton alteration and death in cancer cells. However, the mechanisms of PT-262 actions are still unclear. In this project, we will investigate the mechanisms of cytoskeleton alteration and cell death by PT-262. The model of human lung cancer cells will be established, and the effect of PT-262 on normal lung cells will be compared. We will determine whether PT-262 is through direct binding to actin filaments or indirect effect on RhoGTPase actin-remodeling signaling pathways for causing abnormal actin polymerization. Actin polymerization assay, MALDI-TOF MS analysis, and confocal bio-atomic force microscopy are adopted to examine PT-262 binding to actin. Furthermore, we will study the blockade of RhoGTPases (RhoA, Rac1, and Cdc42) and MAP kinases (JNK, p38, and ERK) by using gene knockdowns or inhibitors on the effects of PT-262-induced cytoskeleton alteration and cell death. Additionally, the cells will be transfected with expressed vectors of RhoGTPases for testing on the cytoskeleton alteration, survival, apoptosis, and cell cycle in the PT-262-exposed cells. The inhibition of migration and invasion will be evaluated for the anti-metastasis ability by PT-262. Finally, the interaction and regulation between RhoGTPases and MAP kinases will be investigated by Western blot, immunoprecipitation, immunofluorescence, and confocal microscopy for analysis of protein expression, interaction, and location in the cells. The final goal of this project is to delineate the mechanisms of PT-262-induced cytoskeleton alteration and cell death and offers applications on cancer therapy.