PTEN在人類泌尿上皮癌中CEBPD參與順鉑介導作用的角色探討

The role of PTEN in CEBPD-mediated cisplatin response in human urothelial carcinoma

胡惠婷(Huei-Ting Hu)

指導教授 ： 黃阿梅

高雄醫學大學/醫學院/生物化學研究所/碩士(2012年)

摘要

順鉑（Cisplatin;cis-diaminedichloroplatinum，CDDP）為一種烷化劑，被廣泛的應用在各種癌症治療，包括人類泌尿上皮癌。順鉑是以鉑金屬為中心的順式化合物，進入細胞核中與DNA鍵結形成加合物（adducts）干擾細胞的複製與轉錄作用，且有研究指出順鉑會干擾癌細胞內的氧化壓力，進而使癌細胞走向凋亡。然而，在臨床治療中往往發現，病人長期使用順鉑後，會產生抗藥性導致治療成效大幅下降，因此找出藥物作用的分子機制與抗藥機轉是十分重要的。轉錄因子CCAAT/enhancer-binding protein delta（CEBPD）是與免疫發炎相關的基因，也參與在細胞增殖、分化、存活和腫瘤的生成等作用。在過去實驗室的研究中發現，CEBPD對於維持基因組的穩定和控制乳腺上皮細胞生長扮演重要的角色。此外，CEBPD也涉及腫瘤細胞發展出化療藥物抗藥性與轉移的相關機制；在人類泌尿上皮癌細胞中CEBPD會直接調控銅/鋅-超氧化物歧化酶（SOD1）的表現，進而抑制順鉑所誘導產生的自由基（ROS）含量與細胞凋亡，造成抗藥性的生成。因此我們推測，CEBPD在腫瘤發展的進程中扮演多重角色。然而至今藥物調控CEBPD表現之詳細機轉以及CEBPD是否還有其他的標的基因或訊息路徑影響腫瘤的發展仍然未明確。在本研究中，透過藥物作用時間及劑量的實驗，我首次發現PI3K/Akt訊號傳遞路徑會影響順鉑誘導CEBPD的表現。在順鉑的作用下，CEBPD直接調控PTEN的表現、活性（磷酸化PTEN）及其在核中的累積。另一方面，MTT細胞毒殺實驗也證實PTEN的表現及活性與順鉑對細胞的毒性有絕對相關性；其可能之作用機轉為影響細胞內自由基之含量。綜合以上結果顯示，在順鉑的作用下PTEN可做為CEBPD的一標靶基因，並因順鉑所導致的氧化壓力提高PTEN在核中的累積，造成細胞週期停滯於G1/S期，最終引發細胞凋亡。本研究提供了一個治療膀胱癌新的標的及方向。

關鍵字

順鉑 ； 泌尿上皮癌

並列摘要

Cisplatin (cis-diaminedichloroplatinum; CDDP) is a DNA alkylating agent and wildly used in treating cancers including human urothelial carcinoma. CDDP is a platinum-based anticancer drug. The cytotoxic effect of cisplatin is mainly mediated by DNA damage, primarily forming intrastrand crosslinking DNA adducts, and ROS production. The transcription factor CCAAT/enhancer binding protein delta (CEBPD) is originally identified as an inflammatory response gene and also implicated in cell proliferation, survival, differentiation, and tumorigenesis. Our previous studies had shown that CEBPD plays a pivotal role in maintaining genomic stability and mammary epithelial cell growth control. In addition, CEBPD also involves in the development of chemotherapeutic drug resistance and tumor metastasis. Thus, CEBPD may exhibit multiple roles in tumor progression. We have previously identified Cu/Zn-superoxide dismutase (SOD1) as a direct target of CEBPD to confer drug resistance by reducing CDDP-induced reactive oxygen species (ROS) and apoptosis in human urothelial carcinoma NTUB1 cells. However, the molecular mechanism by which CEBPD exerts these functions remaining elusive and very little is known about how the expression of the CEBPD is regulated. In this study, I first observed that PI3K/Akt pathways contributed to CDDP-induced CEBPD expression. In addition, CEBPD can not only regulate PTEN (phosphatase and tensin homolog) expression and its activity (phospho-Ser380- PTEN) directly but also its accumulation in the nucleus upon CDDP treatment. Furthermore, MTT cytotoxicity assay confirmed that the PTEN expresssion and activity are relevant to CDDP-induced cytotoxicity and may through regulating the intracellular content of free radicals. In conclusion, these data indicate that PTEN is a direct target gene of CEBPD in response to CDDP and accumulated in the nucleus to confer the cytotoxicity of CDDP by inducing G1/S arrest and apoptosis. This study provides a new direction for managing bladder urothelial carcinoma.

並列關鍵字

CEBPD ； CDDP ； PTEN

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