| Abstract: | 棉黃素(gossypetin, GTIN)為一種類黃酮(flavonoid)物質，源自於錦葵科洛神和棉花植株中的成份。過去研究指出GTIN具有抗氧化、抗菌、抗動脈粥狀硬化以及抗癌的活性，然而其對於抗乳癌(breast cancer)之功效仍尚未釐清。因此，本文擬探討GTIN抑制乳癌細胞生長之作用及釐清其分子機制。首先，以GTIN處理正常人類乳腺細胞株M10及乳癌細胞株MCF-7並進行細胞毒性測試。實驗結果發現GTIN對於MCF-7細胞具有抑制生長之作用且增加細胞週期(cell cycle) subG1期的表現；而GTIN處理之M10細胞則無明顯毒性。接著，以DAPI染色和Annexin V雙染試驗結果發現GTIN可促使MCF-7細胞產生凋亡(apoptosis)，GTIN主要藉由抑制抗凋亡蛋白Bcl-2和p-Bad表現，達到增加細胞凋亡的反應。另外，從AVO染色及LC3-II螢光分析發現GTIN抑制MCF-7細胞自噬(autophagy)的表現，並進一步指出GTIN抑制class III PI3K/Beclin-1訊息傳遞，進而阻斷自噬路徑的活化。後續實驗發現以預先處理細胞自噬抑制劑3-methyladenine (3-MA)具有加成GTIN抑制MCF-7細胞生長及自噬的效果，確認GTIN在自噬啟始路徑的調控，以及提出此自噬作用為一種癌細胞自我保護性的存活方式。最後，以MCF-7細胞同時處理GTIN與臨床治療乳癌藥物tamoxifen (Tam)也發現兩者合併使用對於MCF-7細胞生長抑制更為明顯。因此，我們提出GTIN具有誘導乳癌細胞凋亡及抑制保護性自噬之作用，可作為一個有潛力發展為治療乳癌的試劑或輔助用藥。 Gossypetin (GTIN), a naturally occurring hexahydroxy flavone, has been shown to possess antimutagenic, antioxidant, antimicrobial, and antiatherosclerotic effects. Here, we investigated the mechanism(s) underlying the anticancer potential of GTIN. Trypan blue exclusion test of cell viability showed that GTIN exhibited the anti-proliferation effect on human breast cancer MCF-7 cells in a dose-dependent manner, but not in normal human mammary epithelial M10 cells. GTIN was evaluated for its apoptotic activity in MCF-7 cells as demonstrated by morphological and biochemical features, including apoptotic bodies formation, distribution of hypodiploid phase of cell cycle, and caspase-3 activation. Molecular data showed the apoptotic effect of GTIN in the cells might be mediated via downregulation of antiapoptotic proteins Bcl-2 and p-Bad. Next, the GTIN showed potential in reducing the formation of acidic vesicular organelles and the up regulation of the autophagy-related genes (LC3 and Atg5/12 conjugate). The protective autophagy inhibition of GTIN enhances MCF-7 cell death, and is dependent on class III PI3K/Baclin-1 pathway, as demonstrated by the usage of the autophagy inhibitor 3-methyladenine (3-MA). Finally, the combination therapy of GTIN with tamoxifen (Tam), a medicine to treat breast cancer, was evidenced by its synergistic inhibition on the growth of MCF-7 cells. Taken together, this is the first report to demonstrate that GTIN exhibited anti-proliferative effect in breast cancer cells by inducing apoptosis and inhibiting autophagy, and provide a new mechanism for its anticancer activity. |
