一、CHM-1藉由p38介導上調死亡接受器5之表現而誘發人類卵巢癌SKOV3細胞凋亡

二、大苞雪蓮抑制人類荷爾蒙抗性前列腺癌PC-3細胞之表皮生長因子受體訊息路徑

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题名:	一、CHM-1藉由p38介導上調死亡接受器5之表現而誘發人類卵巢癌SKOV3細胞凋亡二、大苞雪蓮抑制人類荷爾蒙抗性前列腺癌PC-3細胞之表皮生長因子受體訊息路徑 Part I.CHM-1 induces apoptosis via p38-mediated up-regulation of DR5 expression in human ovarian cancer SKOV3 cells. Part II.Inhibition of epidermal growth factor receptor signaling by Saussurea involucrata in human hormone-resistant prostate cancer PC-3 cells.
作者:	李正常
贡献者:	藥物化學研究所博士班
关键词:	Part I. CHM-1;卵巢癌;死亡受體5;p38;細胞凋亡。Part II. 大苞雪蓮;前列腺癌;PI3K/AKT;STAT3;表皮生長因子受體。 Part I. apoptosis;CHM-1;death receptor 5;ovarian cancer;p38. Part II. Saussurea involucrata;EGFR;AKT;STAT3;prostate cancer.
日期:	2011-07-30
上传时间:	2011-10-17 16:58:03 (UTC+8)
出版者:	中國醫藥大學
摘要:	PartI. 卵巢癌是全世界婦女常見癌症，佔所有女性癌症4%，估計全球每年約有204,000新病例，125,000名婦女死於卵巢癌。卵巢癌有著極高之發病率和死亡率，目前臨床有效治療卵巢癌並遏止卵巢癌復發之藥物依然有限，因此，開發安全有效治療卵巢癌之藥物實為當前迫切需要之課題。本研究主要探討新穎的2-芳香基-6, 7-亞甲二氧基-4-喹啉酮衍生物對卵巢癌SKOV3細胞之抗癌活性及其抑癌機轉，藉以評估此類化合物能否發展成為安全有效治療卵巢癌之藥物。利用細胞存活率分析評估2-芳香基-6, 7-亞甲二氧基-4-喹啉酮衍生物抑制人類卵巢癌SKOV3細胞的生長作用；以細胞週期分析、免疫墨點測定法和流式細胞計數法探討誘發卵巢癌SKOV3細胞凋亡之分子機制；利用皮下及原位移植腫瘤模式評估在活體之抗腫瘤活性。細胞存活率分析研究發現，在10個受測試2-芳香基-6, 7-亞甲二氧基-4-喹啉酮衍生物當中，2-(2-fluorophenyl)-6,7-methylenedioxoyquinolin- 4-one (CHM-1)能以濃度依賴性抑制卵巢癌SKOV3細胞生長並誘發細胞凋亡，但對人類正常二倍體皮膚纖維原Detroit-551細胞之毒性相對較低。西方墨點實驗顯示，CHM-1能誘發死亡受體5 (Death receptor 5) 和腫瘤壞死因子相關誘導凋亡配體 (tumor necrosis factor-related apoptosis-inducing ligand; TRAIL)，同時CHM-1所誘導之細胞凋亡和p38介導上調DR5有密切關聯。在裸鼠皮下移植SKOV3腫瘤模式，CHM-1及其親水性磷酸鹽 (CHM-1-P) 皆明顯的以依賴劑量和時間之方式抑制卵巢癌腫瘤生長。經腹腔注射CHM-1治療原位移植卵巢癌SKOV3 ip1/luc細胞之裸鼠，CHM-1除能抑制腫瘤生長外，15或30 mg/kg (i.p×11) 劑量之CHM-1分別延長卵巢癌裸鼠存活期達137% 及161%。除此，以靜脈注射CHM-1-P (10 or 20 mg/kg, i.v×10)治療原位移植SKOV3腫瘤細胞之ICR-Foxn1nu，相較於控制組，實驗組之存活期亦分別延長142% 及150%。由上述結果顯示CHM-1及其親水性磷酸鹽(CHM-1-P)具有新穎的抑癌機制及強效的抗腫瘤活性，值得進一步臨床試驗評估，以發展成為治療人類卵巢癌之藥物。 PartII.前列腺癌(prostate cancer)是臨床男性最常被診斷的惡性腫瘤，同時也是2009年美國男性因罹癌致死的第二位。截至目前，尚無有效的治療方法足以遏止前列腺癌細胞轉化發展成具侵襲性(invasive)之型式。大苞雪蓮(Saussurea involucrata Kar. et Kir.)習慣簡稱為雪蓮(Snow lotus)，是中國珍貴稀有的藥草，本研究確認雪蓮能有效抑制雄激素非依賴性(androgen- independent)前列腺癌PC-3細胞之增殖，進而探討其生化機制，及評估能否發展成為有效的抗腫瘤藥物。細胞存活率分析之結果顯示，雪蓮甲醇抽出物(SI-1)以濃度和時間依賴性抑制多種人類癌症細胞株之增殖，尤其是對荷爾蒙抗性(hormone-resistant)前列腺癌PC-3細胞之抑制效果最強。為探討雪蓮所含之生物活性成分，及雪蓮抑制腫瘤細胞生長之分子機制，進一步將雪蓮甲醇粗抽出物以不同極性之溶劑萃取，而分配得到甲醇抽出物(SI-1)、乙酸乙酯抽出物(SI-2)、正丁醇抽出物(SI-3)及水抽出物(SI-4)。經細胞存活率分析結果顯示，雪蓮乙酸乙酯抽出物(以下簡稱SI-2)對PC-3細胞增殖之抑制效果最為顯著。SI-2能明顯的以依賴濃度之方式抑制前列腺癌PC-3細胞之生長，導致細胞週期停滯於G1-phase，並誘發PC-3細胞凋亡。西方墨點法檢測結果顯示SI-2可不依賴p53訊息途徑而誘發p21WAF1/CIP 和 p27KIP1，並向下調節Cyclin D1 和CDK4之表現。除此之外，PC-3細胞經SI-2處理後，Bax, cytochrome c, activated caspase-3,和caspase-9的表現量會增加，而Bcl-2表現量則下降。表皮生長因子受體(EGFR)是治療前列腺癌的一個重要標的，SI-2能明顯降低EGFR的磷酸化，並抑制PI3K/AKT和STAT3的活化。經口餵食 (p.o) SI-2之動物(in vivo)實驗結果指出，SI-2能以依賴劑量之方式抑制前列腺癌PC-3腫瘤之生長。歸納本研究之結果，證實雪蓮是荷爾蒙抗性前列腺癌PC-3細胞EGFR信息的有效抑制劑，可發展並應用於治療人類EGFR陽性癌症。 PartI. Ovarian cancer is the sixth most commonly diagnosed cancer among women in the world, accounting for nearly 4% of all female cancers. On a worldwide basis, an estimated 204,000 new cases are diagnosed and 125,000 women die of ovarian cancer annually. Although most ovarian carcinomas initially respond to first-line therapy, recurrence with drug-resistant disease is the majority of patients eventually succumb to their diseases, and the overall 5-year relative survival rate is 45%. Treatment for advanced-stage and recurrent disease remains limited, consequently, an effective drug for ovarian cancer is urgently needed today. As part of our continuing investigation of 2-phenylquinolin-4-ones derivatives as potential anticancer drug candidates, a series of 2-(substituted phenyl) 6, 7-methylenedioxyquinolin-4-one derivatives was evaluated for developing new anti-ovarian cancer drugs. MTT assay indicated that, 2-(2-fluorophenyl)-6, 7-methylenedioxy quinolin-4-one (CHM-1), was the most active compound. CHM-1 inhibited the growth of SKOV3 cells and induced apoptosis in a concentration -dependent manner, but it was less cytotoxic to human diploid skin fibroblast Detroit-551 cells. The Western blot experiments showed that CHM-1 caused the upregulation of death receptor (DR) 5 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, CHM-1-mediated cellular apoptosis was found to be closely involved with the p38-mediated upregulation of DR5 expression. In SKOV3 subcutaneous xenograft model, both CHM-1 and its hydrophilic phosphate (CHM-1-P) caused significant dose- and time-dependent tumor regression. Furthermore, CHM-1 inhibited tumor growth and prolonged the lifespan in the SKOV3 ip1/luc orthotopic xenograft model. The average lifespan was prolonged by 137% and 161% in animals treated with 15 mg/kg or 30 mg/kg (i.p×11) of CHM-1, respectively. Intravenous (i.v) administration of CHM-1-P also significantly prolonged the survival time in the SKOV3/ICR-Foxn1nu orthotopic xenograft model. The average lifespan was prolonged by 142% and 150% in mice administered with 10 mg/kg CHM-1-P (i.v×10) and 20 mg/kg CHM-1-P (i.v×10), respectively. Based on the excellent antitumor activity with interesting mechanism of action, CHM-1 was considered as a new anti-ovarian cancer drug candidate. PartII. Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. In this study, we identified Saussurea involucrata Kar. et Kir., a rare traditional Chinese medicinal herb, as a potential agent for androgen independent prostate cancer patients and investigated its biological mechanism as an anti-neoplasia agent. S. involucrata (SI-1) caused a concentration- and time-dependent inhibition of cell proliferation in human hormone-resistant prostate cancer PC-3 cells. Moreover, in vitro studies in a panel of several types of human cancer cell lines revealed that S. involucrata inhibited cell proliferation with high potency. To evaluate the bioactive compounds, we successively extracted the S. involucrata with fractions of methanol (SI-1), ethyl acetate (SI-2), n-butanol (SI-3), and water (SI-4). Among these extracts, SI-2 contains the most effective bioactivity. SI-2 treatment resulted in significant time-dependent growth inhibition together with G1-phase cell-cycle arrest and apoptosis in PC-3 cells. In addition, SI-2 treatment strongly induced p21WAF1/CIP and p27KIP1 expression, independent of the p53 pathway, and down-regulated expression of cyclin D1 and cyclin dependent kinase 4 (CDK4). SI-2 treatment increased levels of Bax, cytochrome c, activated caspase-3, active caspase-9, and decreased Bcl-2 expression level. One of the major targets for the therapy in prostate cancer can be epidermal growth factor receptor (EGFR). SI-2 markedly reduced phosphorylation of EGFR and inhibited activation of AKT and STAT3. Moreover, p.o. administration of SI-2 induced a dose-dependent inhibition of PC-3 tumor growth in vivo. In summary, our study identifies S. involucrata as an effective inhibitor of EGFR signaling in human hormone-resistant prostate cancer PC-3 cells. We suggest that S. involucrata could be developed as an agent for the management of EGFR positive human cancers.
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