木樨草素抑制乳癌細胞惡化及調控訊息路徑之關聯性研究

Luteolin inhibits invasion of breast cancer by regulating multiple signal pathways

張安琪(An-Chi Chang)

指導教授 ： 胡超群 ； 曾翠華

中山醫學大學/健康管理學院/應用化學系碩士班/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00197

摘要

類黃酮是一種天然的多酚類化合物，廣泛存在於日常食用的蔬菜水果中，而諸多文獻證實類黃酮具有許多特殊活性，近年來文獻指出類黃酮具有抗發炎、抑制癌症轉移、抑制血管新生及誘導癌細胞凋亡之能力等。為了瞭解類黃酮在抑制乳癌細胞生長及轉移的機制，本研究選用惡性乳癌細胞株 (MDA-MB-231及4T1細胞)進行實驗，利用boyden chamber assay篩選四種類黃酮化合物 (Catechin, Genistein, Quercetin, Luteolin)，結果發現經木樨草素處理後對4T1的抑制較MDA-MB-231的效果佳，而過去已做過木樨草素能夠抑制血管新生及降低腫瘤生長速度，但在抑制乳癌轉移的機制上不是很清楚，因此後續以探討木樨草素抑制4T1惡性乳癌入侵轉移作用之影響及調控分子機制為研究主題。首先利用細胞毒性分析、細胞存活率分析、傷口癒合試驗、入侵試驗、貼附試驗發現及凝膠酶譜法觀察luteolin對4T1惡性乳癌細胞的存活率、移動、侵襲、貼附及分泌基質金屬蛋白酶能力的影響，結果顯示luteolin能有效的抑制4T1惡性乳癌細胞移行、運動及附著。因此進一步以西方點墨法分析其相關蛋白表現，發現木樨草素會抑制基質金屬蛋白酶的活性及表現，並抑制腫瘤轉移前的上皮-間質轉化的過程，使細胞與細胞間的標記蛋白E-cadherin增加、間質型標記蛋白vimentin和N-cadherin減少。
此外文獻中提到高度侵襲性的癌細胞，骨橋蛋白會過度表現，因此進一步探討發現木樨草素會抑制骨橋蛋白OPN，而在過去文獻提到骨橋蛋白OPN會受到ERK、Akt、stat3、NF-κB這些訊息傳遞路徑所調控，結果顯示木樨草素在4T1惡性乳癌細胞中會對訊息路徑相關調控分子ERK、Akt、stat3、NF-κB磷酸化產生抑制作用。接著使用四種抑制劑PD98059 (ERK inhibitor)、Wortmannin (PI3K/Akt inhibitor)、S3I-201 (stat3 inhibitor)、PDTC (NF-κB inhibitor)分別處裡，發現加入ERK、Akt、NF-κB、Stat3的抑制劑會抑制OPN、vimentin、MMP-2及促進E-cadherin的表現。最後更進一步探討用si-OPN 轉染作用，骨橋蛋白與上皮-間質轉化間之關聯性，結果證實抑制OPN會抑制MMP-2、vimentin及促進E-cadherin的蛋白表現。綜合以上結果，木樨草素可能是透過抑制ERK、Akt、NF-κB、Stat3 pathway 中蛋白的磷酸化及轉錄因子的活性而抑制OPN進而反轉EMT達到抑制癌細胞轉移的能力。

關鍵字

乳癌 ； 木樨草素 ； 上皮 ； 間質轉化 ； 骨橋蛋白 ； 金屬基質蛋白酶

並列摘要

Flavonoids, a large group of natural polyphenols existing in a wide range of daily fruits and vegetables, have been evidenced to progress various prominent bio-activities. Recently, members of flavonoids have found a variety of anticancer effect such as cell growth, apoptosis induction, suppression of the secretion of matrix metalloproteinases and tumor invasive behavior in various studies. First, our experiments chose two high malignant breast cancer cells (MDA-MB-231 and 4T1), and we used boyden chamber assay to investigate that the inhibitory effects of four flavonoids in two breast cancer cells. It showed that luteolin exhibited the highest anti-invasive capacity. However, the molecular mechanism regulation of inhibition of metastatic in breast cancer is not well clarified. We assayed the cell viability, cell invasion/motility, matrix metalloproteinases (MMPs) activity of 4T1 cells with luteolin treatment by MTT assay, trypan blue dye exclusion assay, wound healing, invasion assay, adhesion assay and gelatin zymography. The result showed that luteolin can inhibit 4T1 cell migration, movement, and adhesion. In addition, luteolin inhibited matrix metalloproteinases activity and expression. Luteolin also inhibits tumor metastasis in front of epithelial-mesenchymal transition process, so that increased cell-cell-marker protein :E-cadhetin and reduced the mesenchymal marker: vimentin and N-cadherin.Some research also demonstrated that OPN have high expression in malignant breast cancer cells. Therefore, further to explore that luteolin inhibited OPN, whereas in the past that literature mentioned OPN is regulated by ERK, Akt, stat3 and NF-κB signaling pathway. Our results showed that luteolin inhibited ERK, Akt, Stat3, NF-κB phosphorylation inhibition. Then using four inhibitor PD98059 (ERK inhibitor), Wortmannin (PI3K/Akt inhibitor), S3I-201 (stat3 inhibitor), PDTC (NF-κB inhibitor), respectively, discovered that adding ERK, Akt, NF-κB, Stat3 inhibitors will inhibit OPN, vimentin, MMP-2 and promote of E-cadherin expression.
Finally explore further transfected with si-OPN and epithelial - mesenchymal conversion between correlation results confirmed inhibition of OPN may inhibit MMP-2, vimentin and promotion of E-cadherin protein expression. Above of all, these results demonstrated anti-invasion effect of luteolin likely through inhibition of ERK, Akt, NF-κB, Stat3 signaling pathway resulting in inhibition of the activity of transcription factors of OPN then reversing EMT's ability to inhibit cancer metastasis.

並列關鍵字

Breast cancer ； Luteolin ； EMT ； OPN ； MMPs

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