薑黃素及其結構類似物誘導人類乳癌細胞(MCF-7和MDA-MB-231)計畫性死亡之相關研究

Studies on curcumin and its analogs on apoptotic activities in human breast adenocarcinoma cells (MCF-7 and MDA-MB-231)

江庭萱(Ting-Hsuan Chiang)

指導教授 ： 侯文琪

臺北醫學大學/藥學院/生藥學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00052

摘要

薑黃素 (curcumin)是植物薑黃 (turmeric)內主要具活性的成分，包含抗發炎、抗氧化、抗癌症轉移、誘導癌細胞執行細胞計畫性死亡等活性。本篇研究探討薑黃素及其二十三個結構類似物抑制兩株人類乳癌細胞 (MCF-7和MDA-MB-231)增生和誘導細胞計畫性死亡的相關機制。經由細胞存活率測試 (SRB assay)，發現結構上具有多個甲氧基的結構類似物二號、六號、八十五號對於兩株乳癌細胞具有顯著毒性，且有濃度依存性、時間依存性的現象，不具細胞毒性之結構類似物九號被選做結構-活性探討之用。在5 μM濃度下，以雷射共軛焦顯微鏡觀察Annexin-V-FITC對於兩株乳癌細胞螢光染色，發現結構類似物六號螢光強度最強，顯示其phosphatidylserine (PS)外翻程度最高，表現出細胞計畫性死亡的特徵。以15 μM濃度處理乳癌細胞24小時，再以流式細胞儀分析Annexin-V-FITC/PI雙染之螢光比例，發現結構類似物六號對於兩株乳癌細胞誘導細胞計畫性死亡的比例將近100%﹔結構類似物二號誘導MDA-MB-231細胞株計畫性死亡達80%﹔結構類似物八十五號誘導MCF-7細胞株計畫性死亡達60%。在15 μM濃度下，探討薑黃素及結構類似物對於細胞週期的影響，發現薑黃素與結構類似物二號、六號、八十五號造成MCF-7細胞株G2/M期的停滯﹔薑黃素與結構類似物二號、八十五號造成MDA-MB-231細胞株G2/M期的停滯，結構類似物六號則造成G0/G1的停滯。結構類似物九號對於兩株乳癌細胞皆不造成細胞周期停滯。探討結構類似物六號對於細胞內活性氧化物 (reactive oxygen species; ROS)之影響，以濃度15 μM處理MDA-MB-231細胞株30分鐘，可顯著誘導細胞內ROS的表現；處理24小時後，細胞內Heme oxygenase-1 (HO-1)蛋白質表現顯著增加。結構類似物六號誘導caspase-9及caspase-3活化，經由細胞內途徑使MDA-MB-231細胞進行細胞計畫性死亡。此外，本研究中發現結構類似物六號有抑制癌症轉移的潛力，經由明膠酵素電泳法及畫痕試驗，結構類似物六號可顯著抑制MMP-9的活性及抑制MDA¬-MB-231細胞遷移。

關鍵字

薑黃素 ； 薑黃素結構類似物, ； 乳癌 ； 細胞計畫性死亡

並列摘要

Curcumin is the primary bioactive constituent of turmeric, which is an herbal medicine used in Asia for a long time. It has been shown to possess anti-inflammatory, antioxidant, anti-metastatic, and apoptosis- induced properties. In this study, curcumin and 23 different kinds of curcumin analogs were evaluated for cytotoxicity effects on two breast cancer cell lines—MCF-7 and MDA-MB-231. According to the results of cell viability assay, analog 2, 6 and 85 showed stronger growth inhibition activities in both MCF-7 and MDA-MB-231 cell lines than curcumin under concentration of 5 μM and were selected as test candidates. The analog 2, 6, and 85 together with negative control of analog 9 with four to six methoxyl substituents in the phenyl groups were chosen to compare structure-activity relationships. Apoptosis activity was examined by Annexin-V-FITC fluorescence stain by a confocal microscopy under 5 μM treatments. Among them, analog 6 was demonstrated to effectively induce apoptosis activity. By flow cytometry analysis for Annexin-V-FITC/PI fluorescence under concentration of 15 μM treatment for 24 hours, analog 6 showed almost 100% apoptosis in both MCF-7 and MDA-MB-231 cell lines; analog 2 induced 80% apoptosis in MDA-MB-231 cell line; analog 85 exhibited 80% apoptosis in MCF-7 cell line. Cell cycle distribution was arrested by curcumin, analog 2, 6, and 85 in G2/M phase in MCF-7 cells. In MDA-MB-231 cells, curcumin, analog 2, and 85 caused cell cycle arrest in G2/M phase; analog 6 caused cell cycle arrest in G0/G1 phase. Analog 9 showed no influence on cell cycle distribution in both cell lines. To study the mechanism of apoptosis induction by analog 6 and curcumin, cellular reactive oxygen species (ROS) produced levels were detected. It was found that curcumin and analog 6 elevated ROS levels in MDA-MB-231 cells after being treated with 30 minutes. Furthermore, curcumin and analog 6 for 24-h treatments induced heme oxygenase-1 (HO-1) protein expressions in both cell lines. Analog 6 increased MDA-MB-231 cells caspase-9 and caspase-3 actived form expression. We concluded that analog 6 induce MDA-MB-231 cells undergo apoptosis through intrinsic pathway. In addition, according to gelatin zymograhy and wound healing assay, analog 6 inhibits MDA-MB-231 cells migration through suppressing MMP-9 activity.

並列關鍵字

curcumin ； curcumin analog ； breast cancer ； apoptosis

參考文獻

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