羥基白藜蘆醇藉由調節上皮-間質性轉化與微小核糖核酸進而抑制大腸癌細胞移行

Abstract

大腸直腸癌連續13年蟬聯國人癌症發生之冠，且死亡人數仍逐年上升。又大腸癌患者主要死因為癌症轉移，而上皮-間質性轉化 (epithelial-mesenchymal transition, EMT) 正為癌症轉移之起始步驟。EMT不僅受到乙型轉化生長因子 (transforming growth factor beta, TGF-β) 所誘發，若微小核糖核酸 (microRNA, miRNA) 失調也將導致癌細胞產生更強的移動與侵襲能力。因此大腸癌轉移之預防與治療刻不容緩。目前除了以手術、化療、放療等治療外，蔬果中的植化素 (phytochemical) 也具有預防或延緩癌症進程之功效，如紅酒、葡萄中的芪類化合物—白藜蘆醇 (resveratrol, RES) 能抑制大腸癌、乳癌、膀胱癌等癌細胞的增生、移行與侵襲。而RES衍生物羥基白藜蘆醇 (oxyresveratrol, OXY) —結構上僅比RES間位多一羥基，也具有抑制皮膚癌細胞移行之作用，然而OXY能否抑制大腸癌細胞移行仍尚待確認。有鑑於此，本研究會使用HCT116與HT29兩株人類大腸癌細胞，並以RES作為正對照組進行機制探討。此外，HT29細胞會額外予以TGF-β處理使之具較強的移行能力。首先由傷口癒合試驗與細胞遷移試驗結果顯示，OXY比起RES更能顯著抑制大腸癌細胞移行。進一步以西方墨點法及細胞免疫螢光染色法，分別分析了與EMT相關的轉錄因子Snail和抑制轉移的指標蛋白E-cadherin，亦發現高濃度OXY能顯著下調Snail並促進E-cadherin表現。然而於明膠酶譜試驗中，高濃度OXY下調HCT116細胞分泌至胞外的MMP-2與MMP-9表現不及RES，且HT29細胞之組間皆無顯著差異。最終本研究利用微小核糖核酸微陣列證實了高濃度OXY具調節TGF-β誘導HT29細胞中的miRNAs表現，且多種miRNAs與EMT相關，例如能顯著下調miR-3687、miR-301a-3p與上調miR-3612等miRNAs。綜合上述，OXY能藉由調節EMT與miRNAs表現進而有效抑制大腸癌細胞移行，且效果較RES佳。期以此結果提供OXY與受其調控的miRNAs於大腸癌轉移之應用。

Colorectal cancer (CRC) is the most commonly diagnosed cancer with increasing deaths in Taiwan. Moreover, metastasis is the major cause of death in CRC patients. A number of literatures has emphasized that epithelial-mesenchymal transition (EMT) is a pivotal step of metastasis. Not only transforming growth factor beta (TGF-β) but also dysregulation of microRNAs (miRNAs) can induce or regulate EMT, promoting cancer cells to lose intercellular adhesion and increase motility. Over the past several decades, resveratrol (RES), the most studied stilbenoid, has been a wealth of research on anti‑metastatic ability. Nevertheless, relatively little is known about oxyresveratrol (OXY), a natural derivative of RES found in white mulberry. Accordingly, the purpose of this study was to investigate the effect of OXY on colon cancer cell migration, and RES would be the positive control. First, we used HCT116 and TGF-β-induced HT29 cells to examine the inhibitory effects of OXY and RES by wound healing assay and transwell assay. Next, western blot, immunofluorescence and gelatin zymography were conducted to figure out the EMT-related mechanism of two compuonds. Lastly, the regulatory effects of OXY on miRNAs expression in TGF-β-induced HT29 cells were evaluated by miRNA microarray. Results showed that OXY, rather than RES, could markedly reduce both colon cancer cells migration via Snail/E-cadherin expression. However, OXY had less effect than RES to inhibit secreted MMP-2 and MMP-9 from HCT116 cells, and there was no significant difference in HT29 cells. Finally, OXY improved lots of EMT-related miRNAs expression, such as lowering the levels of miR-3687, miR-301a-3p while upregulating miR-3612. In conclusion, OXY inhibits human colon cancer cell migration through regulating EMT and miRNAs. Based on these findings, OXY has potential for anti‑metastatic properties in CRC.