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dc.contributor.advisorHungchen E. Yenen_US
dc.contributor.authorHo, Tsing-Fenen_US
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Carcinogenesis, 2007. 28(7): p. 1533-42.zh_TW
dc.description.abstractProdiginines 是一群化學構造上具有三個砒咯環的衍生物,多由鏈黴菌屬 (Streptomyces) 及鋸桿菌屬 (Serratia) 這類微生物所產生的紅色色素。Prodigiosin (PG) 與undecylprodigiosin (UP) 皆屬於prodiginines 這一類結構物。已有文獻證實 PG 確有抑癌作用,但 UP 的抑癌活性尚未揭示。本研究主要是探討 PG 與 UP 的抑癌活性與作用機制,我們以乳癌細胞株為生物研究模式,包括 BT-20、MCF-7、 MBA-MD-231、T47-D 四株乳癌細胞株,及一株正常的乳腺細胞株 (MCF-10A)。針對 UP 抑癌活性的研究,首先以 MTS assay 評測 UP 對乳癌細胞株活性之抑制現象。結果發現 UP 對乳癌細胞株有顯著的細胞毒殺作用,但在正常乳腺細胞株並無明顯作用,証實 UP 的細胞毒殺作用具有選擇性,而一般臨床常用之抗癌藥 cisplatin,卻無此選擇性。顯示 UP 是一極具潛力之抗癌藥。進一步由 TUNEL assay 及 Annexin V 染色,以流式細胞儀偵測,UP 確能導致細胞凋亡效應。另輔以 z-VAD.fmk 可證實, UP 所引起的細胞凋亡效應與 caspase 活化有關。由 Western blotting 的蛋白表現量可得, UP 處理後會抑制 BCL-xL、BCL-2、survivin、XIAP 表現,促進 BIM、Mcl-1s 表現。又以 RNA 干擾技術減少 MCF-7 細胞株之 p53 蛋白表現量,進行 MTS assay,獲知 UP 抑癌作用與 p53 無關。依目前研究結果推論,UP 可能透過抑制 XIAP、survivin 蛋白表現,達到活化 caspase 所致細胞凋亡作用。Prodiginines 成員中,關於 PG 的抗癌機制的研究最多,但IAP (inhibitor of apoptosis) 在 PG 所誘發的細胞凋亡效應所扮演的角色,尚未揭示。本篇研究證實 PG 可誘發人類乳癌細胞株的細胞凋亡作用,由 Western blotting 的蛋白表現量及即時定量 RT-PCR 結果,顯示 PG 抑制 survivin 及 XIAP 蛋白表現,不僅調控蛋白質轉譯的層次,而且也調控基因轉錄的層次。我們建立可以持續穩定表現 survivin 基因或 XIAP 基因的乳癌細胞株,會對PG 產生抗性,証實 survivin 與 XIAP 蛋白在 PG 對乳癌細胞的毒殺作用中的重要性。紫杉醇會抑制 XIAP 蛋白表現,卻促使 survivin 蛋白表現量增加,可能與抗性的產生有關。藉由螢火蟲冷光酶報導系統顯示 PG 會抑制 survivin 啟動子活性而抑制 survivin 基因表現。藉由 PG 抑制 survivin 基因表現的特性,期能發展有效的治療新方法。PG 與紫杉醇合併處理乳癌細胞後,由 MTS 檢測法及細胞群落形成能力檢測法,都可發現 PG 的確可增效紫杉醇對乳癌細胞的毒殺作用,但這種合併效應在穩定表現 survivin 基因的細胞株卻消失,證實 survivin 在 PG 與紫杉醇合併效應所扮演的重要性。zh_TW
dc.description.abstractProdiginine is a family of tripyrrole red pigments produced by a restricted group ofmicroorganisms, including some Streptomyces and Serratia strains, and characterized by a common pyrrolydipyrrolylmethene skeleton. Prodigiosin (PG) and undecylpridigiosin (UP) are the members of prodiginine. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47-D and one nonmalignant human breast epithelial cell line, MCF-10A, were to study on the anticancer effect and the underlying mechanisms of prodiginines. On the anticancer effect of UP, we found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP''s cytotoxic effect is selective for malignant cells. UP''s cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase-9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-xL, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47-D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP''s cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status. On the other hand, we try to elucidate the role of IAP in PG's apoptotic effect. We found that PG down-regulates the levels of survivin and XIAP at both transcriptional and translational levels. Furthermore, breast cancer carcinoma cells gain resistance to PG-induced apoptosis via expression of the XIAP or survivin. The role of IAP including XIAP and survivin is important in PG-induced apoptosis. This information unveils molecular targets allowing efficient induction of apoptosis by PG, but also provides valuable insight to facilitate the design of combination effect of PG with other anticancer agents. Paclitaxel, a chemotherapeutic agent, down-regulates XIAP but up-regulates survivin in breast carcinoma cancer cell lines. We next addressed whether PG can enhanced the cytotoxicity effect of paclitaxel. In such a combination treatment, PG enhanced cell cytotoxicity compared to inducement by taxol alone was determined by MTS assay and colony-forming ability assay. Overexpression of survivin abrogated the combination effect with prodigiosin and paclitaxel in breast carcinoma cell lines. Therefore, we postulate that down-regulation of survivin by prodigiosin may be one of the key events involved in the combination effect.en_US
dc.description.tableofcontents壹、序論 一、 腫瘤的發生 (p.1) 二、 乳癌 (p.2) 三、 細胞凋亡 (Apoptosis) (p.4) 四、 BCL-2 家族蛋白 (The Bcl-2 family) (p.6) 五、 IAP 家族蛋白 (The IAP family) (p.8) 六、 Prodiginine 藥物發展史 (p.11) 七、 Prodiginine 藥物作用機制 (p.13) 貳、研究目的與策略 一、 天然抗癌藥物 UP 對於人類乳癌細胞株的抑癌作用之探討 (p.16) 二、 探討 IAP 在抗癌藥物 PG 誘發人類乳癌細胞株之細胞凋亡的重要性 (p.18) 参、實驗材料與方法 一、 細胞培養 (Cell culture) (p.20) 二、 藥物處理 (Drug treatment) (p.21) 三、 細胞增殖試驗 (Cell proliferation) (p.21) 四、 細胞存活試驗 (Cell viability assay) (p.22) 五、 蛋白質電泳與西方墨點法 (Western blotting) 1. 總蛋白之萃取 (Total protein extraction) (p.22) 2. 蛋白濃度定量分析 (protein quantification) (p.23) 3. 西方墨點法 (Western Blot) 與 SDS-PAGE (p.23) 六、 即時定量 RT-PCR法 (Real time RT-PCR) (p.25) 七、 冷光測定法 (Chemiluminescent reporter gene assasystem) 1. survivin 啟動子基因之選殖 (p.26) 2. survivin 啟動子基因之重組 (p.26) 八、 穩定表現標靶基因的細胞株 1. 基因選殖 (p.26) 2. 基因重組 (p.27) 3. 轉型作用 (Transformation) (p.27) 4. 煮沸法 (p.27) 5. G418 篩選劑量 (G418 killing curve) (p.28) 6. 基因轉化入乳癌細胞株 (p.29) 九、 流式細胞儀分析 1. 細胞週期檢測 (p.29) 2. 磷脂醯絲胺酸外翻偵測 (Annexin V-FITC) (p.30) 3. 去氧核糖核苷酸末端轉移酶介導的缺口末端標記法 (TUNEL) (p.31) 十、 細胞群落形成能力檢測法 (Clonogenic assay) (p.31) 十一、 統計 (Statistics) (p.32) 肆、結果 一、 天然抗癌藥物 UP 對於人類乳癌細胞株的抑癌作用之探討 1. UP 對於人類乳癌細胞株的選擇性抑癌作用之劑量效應 (p.33) 2. UP 對於人類乳癌細胞株的選擇性抑癌作用之時間效應 (p.33) 3. UP 誘發細胞凋亡作用需 caspase 參與 (p.34) 4. UP 改變 BCL-2 及 IAP 蛋白表現引發細胞凋亡 (p.34) 5. UP 誘發細胞凋亡作用與 p53 無關 (p.35) 二、 探討 IAP 在抗癌藥物 PG 誘發人類乳癌細胞株之細胞凋亡的重要性 1. PG 對於人類乳癌細胞株的選擇性凋亡作用之劑量效應 (p.36) 2. PG 抑制 survivin 及 XIAP 表現量 (p.36) 3. Survivin 或 XIAP 大量表現之乳癌細胞株建立及其 PG 感受性 (p.36) 4. 抗癌藥物---紫杉醇在有效劑量作用下會增加 IAP 蛋白表現量 (p.37) 5. 抗癌藥物---紫杉醇與 PG 合併使用的加成現象 (p.37) 6. Survivin 過量表現的乳癌細胞失去紫杉醇與 PG 合併使用的加成現象 (p.37) 伍、討論 一、 天然抗癌藥物 UP 對於人類乳癌細胞株的抑癌作用之探討 (p.39) 二、 探討 IAP 在抗癌藥物 PG 誘發人類乳癌細胞株之細胞凋亡的重要性 (p.40) 陸、圖表 圖一、比較 UP 與 Cisplatin 對於人類乳癌細胞株的選擇性抑癌作用之劑量效應 (p.43) 圖二、UP 對於人類乳癌細胞株的選擇性抑癌作用之時間效應 (p.44) 圖三、UP 誘發細胞凋亡作用需 caspase 參與 (p.45) 圖四、UP 改變 BCL-2 蛋白群及 IAP 蛋白群之蛋白表現量 (p.46) 圖五、UP 調控 BCL-2 蛋白推論圖 (p.47) 圖六、UP 誘發細胞凋亡作用與 p53 無關 (p.48) 圖七、綜合本篇研究所繪製之 UP 抑癌機制圖 (p.49) 圖八、PG 對於人類乳癌細胞株的凋亡作用之劑量效應 (p.50) 圖九、PG 抑制 survivin 及 XIAP 轉譯及轉錄作用 (p.51) 圖十、Survivin 或 XIAP 穩定表現之乳癌細胞株其 PG 感受性 (p.52) 圖十一、抗癌藥物---紫杉醇在有效劑量作用下會抑制 X IAP 卻增加 survivin 蛋白表現量 (p.53) 圖十二、抗癌藥物---紫杉醇與 PG 合併使用的加成現象 (p.54) 圖十三、Survivin 過量表現的乳癌細胞株失去紫杉醇與 PG 合併使用的加成現象 (p.55) 圖十四、紫杉醇與 PG 合併使用的加成現象之推論圖 (p.56) 柒、參考文獻 (p.57) 捌、附錄 一、研討會 1. 2007 年度生物醫學聯合學術年會 --- 海報展示 (p.63) 2. 2008 年度生物醫學聯合學術年會 --- 口頭論文報告 (p.64) 二、期刊論文 1. Toxicology and Applied Pharmacology 期刊 (p.65)zh_TW
dc.subjectBCL-2 proteinen_US
dc.subjectIAP proteinen_US
dc.subjectBCL-2 家族蛋白zh_TW
dc.subjectIAP 家族蛋白zh_TW
dc.titleProdiginines 抑癌活性與機制之探討zh_TW
dc.titleProdiginines: the anticancer effect and the underlying mechanismsen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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