探討人類泌尿上皮癌細胞對Cisplatin化學抗藥性機制之研究

Characterization of Cisplatin-Resistant Mechanism in Human Urothelial Carcinoma Cells

官靜儀(Ching-I Kuan)

指導教授 ： 侯自銓

高雄醫學大學/醫學院/生物化學研究所/碩士(2007年)

摘要

Cisplatin 是癌症化療的重要用藥，對於卵巢癌，小細胞肺癌及膀胱癌均有良好的治療效果。不過有部分泌尿上皮癌病人使用 cisplatin 治療一段時間後會產生抗藥性，進而影響治療效果。因此了解泌尿上皮癌細胞對於 cisplatin 抗藥性的作用機轉是當今重要的課題。我們利用泌尿上皮癌細胞株 NTUB1，培養出具 cisplatin 抗藥性之細胞株 NTUB1/P(14)。以西方墨點法發現 CCAAT/enhancer binding protein (C/EBP) 家族成員− CEBPD 在四株不同藥物抗藥性細胞株中，以 NTUB1/P(14) 有較高表現量，而其他三株抗藥株則表現量較低。接著以 NTUB1 利用 stable transfection 建立過度表現 CEBPD 細胞株 (N-CEBPD)，並在 MTT assay 中發現N-CEBPD 對
cisplatin 之 IC50 值為 NTUB1 的 2.9 倍 (P<0.001) ； 但是在 N-CEBPD-ΔDBD 因為不具 CEBPD binding domain 細胞株則對於cisplatin 的抗藥性降低，近似於 NTUB1。在先前研究中發現，細胞在 cisplatin 處理下，會誘導 extracellular signal-regulated kinase (ERK) 磷酸化 (p-ERK)，亦有研究發現 cisplatin 會誘導細胞 P53 之 ser15 磷酸化 (P53-p-ser15)。因此為了探討 CEBPD 及 cisplatin 抗藥性相關蛋白分子的關係，利用西方墨點法，發現 N-CEBPD 其 P53 表現量較對照組為低，SOD (superoxide dismutase) 表現量較對照組為高，而 N-CEBPD-ΔDBD 則相反；在處理cisplatin 10 μM 時 P53-p-ser15、p-ERK 活性被抑制，以上蛋白分子的表現均與 NTUB1/P(14) 相似，同時亦發現，N-CEBPD-ΔDBD 在處理 cisplatin 10 μM 時 P53-p-ser15、p-ERK 活性被抑制的現象卻被逆轉，因此 CEBPD 可透過抑制 P53 及 ERK 活化而增加細胞對 cisplatin 的抗藥性。另一方面，使用 DAPI 免疫染色發現 N-CEBPD 在 cisplatin 20 μM 處理下細胞發生 DNA fragmentation 的情形明顯比對照組少， 而 N-CEBPD-ΔDBD 卻是和對照組相似；同時利用流式細胞儀分析細胞凋亡 (apoptosis) 比例，亦發現 N-CEBPD 在 cisplatin 20 μM 處理下細胞週期之 G1 前期 (sub-G1) 分布比例明顯比對照組少，而 N-CEBPD-ΔDBD 易發生和對照組相似的情形。由此可知，N-CEBPD 對於 cisplatin 的抗性較 N-vector 及 N-CEBPD-ΔDBD 高，可見 CEBPD 確實與 cisplatin 抗藥性有明顯相關性，未來希望藉此能對於臨床上 cisplatin 治療膀胱癌有所幫助。

關鍵字

人類泌尿上皮癌細胞 ； 化學抗藥性機制

並列摘要

Cisplatin is one of the most effective anticancer drugs used for treatment of solid tumors, including ovarian, small cell lung and bladder cancers. During the cisplatin treatment of urothelial cancer, some patients will become to be cisplatin resistance, and then it will influence the outcome of treatment. Therefore, it is an important topic how to characterize the cisplatin-resistant mechanism in human urothelial carcinoma (UC) cells. In this study, we discovered that CEBPD (CCAAT/enhancer binding protein delta), a member of the transcription regulator protein, C/EBP, was higher expressed in cisplatin-resistant cells than in As2O3, paclitaxel and gemcitabine-resistant cells. Then we established the CEBPD transfectant subclones, N-vector and N-CEBPD by stable transfection. In MTT assay,
the IC50 value of N-CEBPD in response to cisplatin was 2.9-fold than NTUB1 (P<0.001). It indicated two cell lines were significant different in cisplatin resistance. Our results showed the lower P53 protein level in N-CEBPD than N-vector when cells were treated with 10 μM 24hr, and P53-p-ser15, p-ERK were inactivated. The expression of these proteins
were similar with NTUB1/P(14) in basal level or in cisplatin-treatment. We also found higher protein level of superoxide dismutase (SOD) in N-CEBPD than in N-vector and N-CEBPD-ΔDBD. Based on the DAPI staining and flowcytometry assays, N-CEBPD had less DNA fragmentation and sub-G1
fraction than in control cells when cells were treated with 20 μM 24hr. Therfore, in overexpressed CEBPD cells, they can increase cisplatin resistance through deactivated p-ERK and P53-p-ser15 when cells are treated with cisplatin. We also find that the transcriptional activity of CEBPD is very important in cisplatin-resistance. Briefly, cisplatin-resistant mechanism is associated with the CEBPD expression in UC.

並列關鍵字

Human Urothelial Carcinoma Cells ； Cisplatin-Resistant

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