- 學位論文
抗組織胺藥物Cyproheptadine 對膀胱癌的抑癌機制與ANGPTL4基因在膀胱癌中所扮演的角色與功能之探討
Investigation of The Antitumor Mechanism of Cyproheptadine and The Role and Function of ANGPTL4 in Bladder Urothelial Carcinoma
摘要
抗組織胺藥物佩你安是第一代抗組織胺藥物，近年來的研究指出它在癌症中可用來當作新的抗癌藥物，但是對於泌尿上皮癌的治療效果至今從未被探討過。在本研究中，我們利用人類膀胱癌細胞株及異種移植的動物試驗去檢測佩你安對癌細胞生長之影響。在試管及活體試驗中結果皆顯示，佩你安會導致細胞週期停滯在G0/G1階段，具有抑制膀胱癌細胞生長之功效，接著導致細胞凋亡及壞死。導致細胞週期停滯的潛在機制主要是與p21, p27蛋白的增加、c-Myc蛋白的減少及Rb蛋白穩定的表現有關。除此之外，同時也觀察到在佩你安處理過的膀胱癌細胞株中，其GSK3β/β-catenin信號及GSK3β/TSC2/mTOR訊息傳遞路徑會被抑制。而且，佩你安引起的細胞凋亡現象與PPARγ及血管生成素樣4的大量表現有關，它會活化泌尿上皮癌細胞中的caspase3 及PARP蛋白。我們的結果證據顯示: 佩你安是一個有潛力可用在治療泌尿上皮癌的藥物。 過去的研究報導指出: 血管生成素樣4在某些癌症會高度表現，但是在另一些癌症中會受到DNA甲基化調控作用而低度表現。因此，由於在不同的癌症中, 它顯然有不同的功能，而血管生成素樣4在泌尿上皮癌的病因學中，確切的角色目前仍不清楚。本篇研究結果顯示: 血管生成素樣4的轉錄作用在膀胱癌細胞株及臨床檢體組織中被抑制，是受到DNA甲基化作用影響。功能性研究進一步指出：在試管試驗中異位的表現血管生成素樣4會抑制膀胱癌細胞的增殖及單層細胞集落形成；在活體試驗中會抑制異種移植癌細胞的生成。有趣的是，在泌尿上皮癌病人的血清中可偵測到循環的血管生成素樣4 (cANGPTL4) 蛋白存在，故認為它有可能用來當作侵犯程度最低限度的預測標記。有趣的是，當我們外加cANGPTL4會促進膀胱癌細胞的增殖及透過活化Erk/FAK axis訊息傳遞而促使細胞的移動。我們進一步利用老鼠的異位移殖活體試驗去證實，當注射cANGPTL4蛋白會促進異位腫瘤生長。總而言之，在膀胱癌的癌化生成過程當中，我們的實驗數據顯示，血管生成素樣4不是單一的腫瘤抑制基因或是腫瘤促進基因，在癌細胞微環境中它具有雙重角色。 關鍵字: 佩你安、mTOR信號、血管生成素樣4、泌尿上皮癌
並列摘要
Cyproheptadine (CPH), a first-generation antihistamine, has recently been reported to function as a novel therapeutic agent in cancers, but never been explored in urothelial carcinoma (UC). In our study, we determined the effect of CPH on the growth of human bladder cell lines and an in vivo xenograft model. The results showed that CPH induced cell cycle arrest in the G0/G1 phase and exerted an inhibitory effect on cancer cells proliferation both in vitro and in vivo, subsequently followed by apoptosis and necrosis. The underlying mechanisms of cell cycle arrest in CPH-treated bladder cells were associated with the induction of p21 and p27, reduction of c-Myc and the stabilization of Rb expression. In addition, deregulation of the GSK3β/β-catenin signaling and the GSK3β/TSC2/mTOR pathway were observed. Furthermore, CPH-induced apoptosis was associated with overexpressed PPARγ and ANGPTL4 followed by activation of caspase3 and PARP in UC cells. Our results provide evidence that CPH is a potential therapeutic agent for the treatment of UC. Previous studies had demonstrated that ANGPTL4 is highly expressed in some cancers, but downregulated, by DNA methylation, in others. Consequently, due these seemingly conflicting functions in distinct cancers, the precise role of ANGPTL4 in the etiology of UC remains unclear. In this study, we show that ANGPTL4 is transcriptionally repressed by DNA methylation in UC cell lines and primary tumor samples. Functional studies further demonstrated that ectopic expression of ANGPTL4 potently suppressed UC cell proliferation, monolayer colony formation in vitro, and xenograft formation in vivo. Interestingly, circulating ANGPTL4 (cANGPTL4) was detectable in plasma samples from UC patients, suggesting it could be used as a minimally invasive diagnostic marker. Interestingly, our data also indicated that exogenous cANGPTL4 could promote cell proliferation and cell migration via activation of signaling through the Erk/FAK axis. We further confirmed that mouse xenograft tumor growth could be promoted by administration of exogenous cANGPTL4. In conclusion, our data supports dual roles for ANGPTL4 in UC progression, either as a tumor suppressor or oncogene, in response to microenvironmental context. Keywords: Cyproheptadine; mTOR signaling; ANGPTL4; Urothelial Carcinoma