探討CEBPD調控SOD1基因表現參與在人類泌尿上皮癌細胞的cisplatin抗藥作用

CEBPD-induced SOD1 expression mediates cisplatin resistance in human bladder urothelial carcinoma cell line NTUB1

賴彥良(Yan-Liang Lai)

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(2009年)

摘要

中文摘要
臨床抗癌用藥cisplatin (cis-diaminedichloroplatinum; CDDP)是一以鉑金屬為中心的化合物，可插入到DNA結構上而阻斷DNA的複製及轉錄，並可能透過影響細胞內的自由基(ROS)含量而抑制癌細胞生長，進而使腫瘤細胞凋亡，達到抗癌功效。然而癌症病患在使用cisplatin一段時間後，常產生抗藥性，致使治療無效。因此找出腫瘤細胞抗藥性的作用機轉是非常迫切需要的。CEBPD (CCAAT/ enhancer-binding protein delta)屬於轉錄因子C/EBP家族的一員。由本實驗室過去的研究已知CEPBD的mRNA及protein皆可在人類泌尿上皮癌細胞株NTUB1受cisplatin刺激被誘導出來；因此推論CEBPD可能參與在調控癌細胞對cisplatin的作用及其引發的抗藥性。故本研究目的是探討CEBPD如何參與在調控NTUB1對cisplatin之作用及抗藥性的分子機轉。為了探討此轉錄因子的表現是否與腫瘤細胞的抗藥性相關，進一步以NTUB1建立表達不同CEBPD的細胞株: N-V (empty vector pcDNA3); N-D#1 (WT); N-△DBD (CEBPD序列的C端basic leucine zipper domain發生缺失); N-R198A (CEBPD在胺基酸198位置上由晶胺酸置換為丙胺酸)和N-hd-pSi (為一降低CEBPD 表現的siRNA clone)做為材料，從其細胞存活率、細胞週期和自由基含量分析結果:發現CEBPD的DNA結合力及轉錄活性對於腫瘤細胞抵抗cisplatin是很重要的。同時透過reporter assay分析指出SOD1為CEBPD的一個目標基因，並且直接參與在CEBPD對cisplatin抗藥性上的調控。綜合以上結果顯示: NTUB1細胞經由cisplatin處理後可誘導CEBPD調控SOD1的表現，而致使腫瘤細胞提高對cisplatin的耐受性。轉錄因子CEBPD可作為一個具潛力的癌症治療標的基因，其更詳盡的分子機轉及應用則有待進一步研究。

關鍵字

人類泌尿上皮癌細胞 ； 抗藥

並列摘要

英文摘要
Cisplatin (cis-diaminedichloroplatinum;CDDP), a platinum- based anticancer drug, is one of the most effective chemo- therapeutic agents. Its configuration includes two amines, two chlorines, and the center of platinum. The cytotoxic effect of cisplatin is mainly mediated by DNA damage, primarily forming intrastrand crosslinking DNA adducts and ROS production. However, its clinical efficacy is largely limited to its toxic effects on normal cells and the development of drug resistance within tumor cells. Therefore, understanding the molecular and cellular events upon cisplatin treatment will improve the development of therapeutic options for the patients. CCAAT/enhancer-binding protein delta (CEBPD) belongs to C/EBP transcription factor family. We previously found CEBPD mRNA and protein were induced by cisplatin in human urothelial carcinoma cell line NTUB1. In order to explore the biological roles of CEBPD in cisplatin response and resistance, various CEBPD- expressing stable clones in NTUB1 were established: N-V (empty vector pcDNA3); N-D#1 (WT, CEBPD full length); N-△DBD (Mutant, C-terminal basic leucine zipper domain of CEBPD 197-269 amino acids was deleted); N-R198A (Mutant, CEBPD amino acid198 Arginine was replaced by Alanine) and N-hd-pSi (CEBPD siRNA knocking down). The results showed that C-terminal DNA binding domain activity of CEBPD is essential for the cisplatin response and resistance. Furthermore, SOD1 was identified as a direct target of CEBPD and contributed to CEBPD-mediated cisplatin resistance in NTUB1 cells. Thus, CEBPD might be a potential target for clinical treatment of cisplatin resistant bladder cancers. The underlying molecular mechanism of CEBPD-mediated SOD1 expression in cisplatin response/resistance needs to be further explored.

並列關鍵字

CEBPD ； SOD1 ； cisplatin ； human bladder urothelial carcinoma

參考文獻

1. Saunders Aioe. Campbell-Walsh Urology, 9th edition. 2007.

2. 行政院衛生署，民國97年主要死因統計. http://www.doh.gov.tw/statistic/eBAS/生命統計/主要死因統計/主要死因統計.xls.

3. Reuter V. The lower urinary tract. Sternberg S, editor Diagnostic Surgical Pathology New York, Raven 1994: 1767-805.

4. Soloway MS. Overview of treatment of superficial bladder cancer. Urology 1985; 26: 18-26.

5. Skinner DG, Lieskovsky G. 16 years experience in the management of patients with deeply invasive bladder cancer. Eur Urol 1988; 14 Suppl 1: 30-1.