- 學位論文
探討Sunitinib引起人類泌尿上皮癌細胞毒性之分子特性
Molecular characterization of sunitinib mediated cytotoxicity in human urothelial carcinoma cells
摘要
中文摘要 膀胱癌是少數幾種在台灣發病率逐漸增加的癌症。最常見的膀胱癌是泌尿上皮癌(urothelial carcinoma; UC)，其主要是源自於膀胱黏膜內側表皮細胞癌化所造成。而癌症病人在進行化學治療時常會衍發出抗藥性問題而導致治療失敗。因此，找出對具抗藥性膀胱癌毒殺效果的治療為首要課題。抒癌特(Sunitinib，SU11248)是一種可以選擇性針對多種蛋白質受體(受體酪胺酸激酶，receptor tyrosine kinase，RTK)的口服多激酶抑制劑，同時具有抗增生與抗血管生成活性。在本篇我們藉由MTT assay分析膀胱癌細胞對於sunitinib的藥物敏感性，發現其對N/P(14)具有高度的藥物敏感性，再經由西方墨點法、real-time PCR與RT-PCR探討其分子機制，以及流式細胞儀(flow cytometry)分析reactive oxygen species(ROS)含量和結合處理藥物。發現當處理sunitinib後，對於膀胱癌細胞株的作用，主要是經由抑制蛋白質受體酪胺酸激酶，包含了：EGFR、PDGFRβ與FGFR1影響其蛋白質層次的下游傳遞路徑，(1) MAPK和PI3K/Akt的訊息傳遞分子受到抑制，降低細胞增生(proliferation)，減少細胞生長。(2)另外也會誘發細胞凋亡(apoptosis)相關分子的產生，包含了：p21、Bid 、Bcl-2、cytchrome C、caspase 3與caspase 8。(3)減少血管新生相關分子：STAT3與VEGF的表現。(4)降低抗藥性分子的表現，包含了：XPF、GSTP1、GSTT2 、GSTM4與MRP1。最後再經由結合處理sunitinib與cisplatin，發現對N/P(14)有顯著的藥物協同作用，以及得到N/P(14)具有高量ROS的表現。因此我們證明sunitinib對於抗藥性的膀胱癌細胞株呈現有意義的毒殺作用，未來sunitinib或許可以做為膀胱癌臨床用藥。
並列摘要
Abstract Urinary bladder cancer is one of the few cancers whose incidence has been increasing in Taiwan. Urinary bladder cancer arises from the cell lining in the interior surface of the bladder, and it is named urothelial carcinoma (UC). However, drug-resistance problems during chemotherapy lead to the failure of the therapy for many cancer patients. Therefore, an important clinical objective is to find an effective cytotoxic drug for drug-resistant bladder cancer cells.Sunitinib (known as SU11248) is an orally medicated multi-targeted receptor tyrosine kinase (RTK) selective inhibitor, and it also has anti-proliferation and anti-angiogenesis efficacy.In this study, we examined the sensitivity of the bladder cancer cell lines in response to the treatment of sunitinib by MTT assay, and found that N/P(14) cell line has the highest sensitivity to sunitinib. Further assessments using Western blotting, reali-time PCR, RT-PCR, flow cytometry and two-drug combination MTT assay show that the cytotoxicity of sunitinib toward UC cells is mainly through inhibition of receptor tyrosine kinase, including EGFR, PDGFRβ, and FGFR1, in which the following signal pathways MAPK and PI3K/Akt were inhibited. Our results showed resulted the following phenomenons: (1) down-regulated cell proliferation and cell growth, (2) induced cell pro-apoptosis related factors, such as p21, Bid, Bcl-2, cytchrome C, caspase 3 and caspase 8, (3) decreased angiogenesis molecules, such as STAT3 and VEGF, and (4) decreased drug-resistant molecules, such as XPF,GSTP1,GSTT2, GSTM4 andMRP1. Finally, combination of sunitinib with cisplatin showed synergistic effect on N/P(14) UC cell line and flow cytometry show high expression of ROS on N/P(14) UC cell line. In this study, sunitinib has been shown to have an excellent anti-cancer drug on UC cells, and it’s potential as a clinical drug is strongly suggested.