藤黃酚衍生物,MCPP,誘導人類大腸癌細胞凋亡

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Title:	藤黃酚衍生物,MCPP,誘導人類大腸癌細胞凋亡 MCPP, a phloroglucinol derivative, induces cell apoptosis in human colon cancer
Authors:	黃思銘;Ssu-Ming Huang
Contributors:	醫學院臨床醫學研究所碩士班
Keywords:	apoptosis;phloroglucinol;colon cancer;ER stress;GSK3α/β;apoptosis;phloroglucinol;colon cancer;ER stress;GSK3α/β
Date:	2010
Issue Date:	2010-09-29 12:17:08 (UTC+8)
Abstract:	背景：大腸直腸癌是世界上第三常見的惡性腫瘤，每年世界新增病患約100 萬人，其中，在已開發國家，此病之死亡率約有33%。許許多多的天然化合物已被證實是抗氧化物質、是癌症預防物質、甚至具有抗腫瘤特質。藤黃酚(phloroglucinol)在過去的文獻中也被報告過，透過許多不同的機轉表現出抗腫瘤(anti-tumor)的效果。然而，藤黃酚在人類大腸癌細胞的抗腫瘤效果還是一個未曾完全研究的嶺域。於本次實驗，我們研究一種藤黃酚衍生物MCPP(3-monochlorophenyl phloroglucinol)對於人類大腸癌細胞的抗癌機轉。方法：藉由官能基的置換，合成數種藤黃酚衍生物。先以MTT法與SRB法篩選對於人類大腸癌細胞有明顯抑癌作用的衍生物。接著，以抑癌效果最明顯的MCPP進行更深入的研究。以PI染DNA含量，檢測sub-G1期之細胞數所佔百分比的變化。以Annexin V-Propidium iodide 雙染法，測定細胞凋亡比例的變化。以TUNEL法，測定細胞凋亡比例的變化。以西方墨點法，分析cleaved PARP量之改變。以西方墨點法，分析Bcl-2蛋白質家族與內質網壓力相關蛋白質(GRP78, GRP94, p-eIF2α, CHOP與GSK3α/β)之變化。以反轉錄聚合酶連鎖反應(RT-PCR) 分析目標基因(GRP78與CHOP)的mRNA轉錄表現量。最後，利用siRNA技術，將人類大腸癌細胞HCT-116轉染所須之siRNA。結果：藤黃酚衍生物MCPP會造成人類大腸癌細胞HCT-116的細胞活性下降，且此現象具有時間依賴性與藥物濃度依賴性。MCPP對於人類大腸癌細胞HCT-116的細胞存活率抑制是經由”細胞凋亡”機轉造成。在MCPP處理之下，caspase-3、caspase-7、caspase-9被活化，而且，有時間依賴性。MCPP會造成粒線體凋亡訊息路徑的啟動。MCPP會增加內質網壓力訊息路徑相關蛋白質的表現增加(GRP78、p-eIF2α與CHOP)。MCPP會造成GRP78與CHOP的mRNA表現量增加。給予人類大腸癌細胞HCT-116轉染GRP78 siRNA或CHOP siRNA會減緩MCPP所造成的細胞凋亡。將人類大腸癌細胞HCT-116給予GSK3βinhibitor處置，會減緩MCPP所造成的細胞凋亡。結論：由本實驗，證明了內質網壓力反應路徑在藤黃酚衍生物MCPP所造成的人類大腸癌細胞凋亡，扮演重要的角色。所以，MCPP可能是未來新的、有潛力的對抗人類大腸癌之標靶治療藥物。 Background: Colorectal cancer is one of the most prevalent solid organ cancers in developed countries. Approximately 1 million cases are recorded every year worldwide, and over half a million patients die of this disease yearly. The natural phloroglucinol has been reported to exhibit multiple functions resulting in anti-tumoral effects. However, its effect on human colon carcinoma cells has never been scrutinized. In this study, the mechanisms underlying the anti-cancer effect of a new phloroglucinol derivative, 3-monochlorophenyl phloroglucinol (MCPP) in human colon carcinoma cells were studied. Methods: Several derivatives of phloroglucinol were synthesized. MTT and SRB assays (for cell viability) were used to screen the synthesized phloroglucinol derivatives. MCPP was found to have the most obvious anti-tumor effect. The cell cycle and sub-G1 accumulation were evaluated by PI flowcytometry. The percentage of apoptotic cells was quantified by Annexin V-PI double labeling. Also, the percentage of apoptotic cells was determined by TUNEL method. The expression level of cleaved PARP was assessed by Western blotting. Bcl-2 family proteins and the associated endoplasmic recticulum stress response proteins (GRP78, GRP94, p-eIF2α, CHOP and GSK3α/β) were evaluated by Western blotting. With reverse transcription polymerase chain reaction (RT-PCR), the mRNA expression of target genes (GRP78 and CHOP) was quantified. Finally, with siRNA technology, HCT-116 cells were transfecetd with indicated siRNA (siRNA against GRP78 or CHOP) for further evaluation. Results: MCPP inhibited HCT-116 cell growth in a concentration- and time-dependent manner. The inhibitory effects of HCT-116 cells were correlated with the arresting cell cycle at G0/G1 phase. Annexin V-PI double labeling also demonstrated the induction of cell apoptosis by MCPP treatment. With MCPP treatment, TUNEL method also revealed the induction of cell apoptosis. Cleaved-PARP level was also elevated after MCPP treatment. Treatment of HCT-116 human colon carcinoma cells with MCPP was found to induce pro-apoptotic member of Bcl-2 family proteins: Bax and Bak. Treatment of HCT-116 human colon carcinoma cells with MCPP was found to decrease the anti-apoptotic member of Bcl-2 family proteins: Bcl-xL and Bcl-2. The changed ratio of Bcl-2 family of proteins shifted the cells from pro-survival to pro-apoptotic pathway. Treatment of HCT-116 human colon carcinoma cells with MCPP was also found to induce a number of signature ER stress markers: phosphorylation of eukaryotic initiation factor-2α(eIF-2α), up-regulation of glucose-regulated protein (GRP)-78, and up-regulation of CCAAT / enhancer-binding protein homologous protein (CHOP). MCPP also induced activation of caspase-3, caspase-7, and caspase-9. Furthermore, transfection of cells with GRP78 or CHOP small interfering RNA (siRNA) attenuated MCPP-mediated cell death. GSK3βinhibitor also reduced MCPP-mediated cell apoptosis. Conclusion: Taken together, the present study thus provides evidence to support an important role of ER stress response in mediating the MCPP-induced human colon cancer cell apoptosis. Thus, our findings reveal that MCPP might be a potential, new chemopreventive agent targeting human colon cancer cells.
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