白藜蘆醇甲基衍生物3,5,4\'-Trimethoxystilbene及槲皮素調節乳癌細胞侵犯及反轉上皮-間質轉化效果之機制探討

乳癌是世界上婦女常見死亡原因之一。近期研究發現乳癌細胞侵襲力的獲得和晚期的轉移與乳癌細胞型態上的上皮細胞-間質細胞轉換(epithelial- mesenchymal transition，EMT) 有極為密切的關聯，因為EMT可以賦予乳癌細胞呈現腫瘤起始細胞與癌症幹細胞的特性，進而促進乳癌細胞的擴散與轉移。Wnt/β-catenin和Type I insulin-like growth factor receptor (IGF-1R)訊號路徑號的失調是EMT轉換的主要機制之一。因此，若能藉由白藜蘆醇甲基衍生物3,5,4’- trimethoxystilbene (MR-3) 及黃酮類化合物槲皮素 (quercertin) 抑制Wnt/β-catenin或IGF1R訊號路徑活化所造成的癌細胞EMT，或許能夠降低乳癌的轉移與致死率。對於研究乳癌轉移機制及現行的預後與治療策略也有重要的幫助，而且目前關於研究MR-3及槲皮素抑制乳癌轉移過程相關分子機制的也尚未明瞭 。 因此，本論文第一部份的研究，MR-3抑制乳癌細胞的上皮細胞-間質細胞轉換，以MR-3處理MCF-7人類乳癌細胞株，發現MR-3確實能夠增加MCF-7細胞中epithelial-like marker E-cadherin的表現，回復cobblestone-like細胞形態，並同時抑制mesenchymal-like markers如snail，slug與vimentin等之表現。此外，MR-3逆轉EMT的同時，亦抑制了MCF-7細胞在transwell assay下侵襲與轉移的能力。進一步探討MR-3逆轉MCF-7細胞EMT之作用機轉，發現MR-3會減少β-catenin在細胞核的堆積，降低β-catenin下游基因的表現，並同時增加membrane-bound β-catenin的出現。另一方面，MR-3亦透過抑制Phosphatidylinositol 3-kinase (PI3K) /Akt的活化，來增加Glycogen synthase kinase 3 beta (GSK-3β) 穩定性與活性，而GSK-3β會促成β-catenin經由ubiquitin proteasome pathway 被分解。因此，我們推論MR-3可能是先藉由調節PI3K/Akt/GSK-3β訊號路徑的活性，進而抑制β- catenin進入細胞核與減少其下游基因的表現，來促使MCF-7細胞EMT的逆轉與降低其侵襲力。 第二部分研究quercetin藉由調節IGF1R訊號路徑的活性，來減少三陰性乳癌細胞株MDA-MB-231的EMT轉換，藉由抑制insulin-like growth factor-1 (IGF-1)分泌及ADAM Metallopeptidase Domain 28 (ADAM28)表現，增加Insulin-like growth factor-binding protein 3 (IGFBP-3)在細胞培養液中濃度，抑制三陰性乳癌細胞的侵襲力亦影響了MDA-MB-231細胞Matrix Metalloproteinases- 2 (MMP-2), Matrix Metalloproteinases- 9 (MMP-9), urokinase plasminogen activator (uPA)與乳癌幹細胞分子標記Oct4、Sox2與Nanog等的表現。同時，quercetin亦抑制vimentin、fibronetin與vascular endothelial growth factor (VEGF) 等間質相關標記的表現，並促進keratin-18, keratin -19與ZO-1等上皮相關標記表現。Quercetin透過降低的IGF1R磷酸化程度，影響IGF1R激酶之受質Insulin receptor substrate 1 (IRS1) 的磷酸化和IGF1R下游Akt與 MAPK/ERK 之活性產生抑制作用。而如同quercetin對於IGF1R訊號傳遞路徑的活性的影響，quercetin亦明顯降低EMT轉換之相關轉錄因子，包含snail、slug與zeb1在蛋白質與mRNA的表現同時減少snail與slug啟動子的活性。最後以小鼠人類乳癌移植瘤動物實驗模式，證明quercetin確實能有效抑制ADAM28和IGF1R訊號傳遞路徑對MDA-MB-231乳癌移殖瘤的肺轉移與EMT轉換之影響。 總而言之，綜合以上研究，本論文指出白藜蘆醇甲基衍生物MR-3和槲皮素皆具有抑制上皮-間質轉化及侵襲性功效。分別透過不同分子機制來調控細胞上皮-間質轉化，MR-3主要透過抑制Wnt/β-catenin的訊號傳遞路徑而槲皮素是透過增加IGFBP-3分泌抑制IGF-1R傳遞路徑。

Breast cancer is the most common cancer in women worldwide. Epithelial- mesenchymal transition (EMT) was demonstrated to be one of the major mechanisms responsible for mediating invasiveness and metastasis of late-stage cancers. The molecular basis of EMT functions as a potential therapeutic target for breast cancer because EMT may endow breast tumor-initiating cells with stem-like characteristics and enable the dissemination of breast cancer cells. Understanding the mechanisms of 3,5,4’-trimethoxystilbene and quercetin in breast cancer metastasis will greatly improve diagnostic or interceptive strategies. In the part I, We have examined a series of resveratrol methoxylated derivative of to explore their cellular on EMT and the invasiveness of human MCF-7 breast adenocarcinoma cell line were also explored for further development of new chemotherapeutic agents. We found that MR-3 significantly increased epithelial marker E-cadherin expression and decreasing of mesenchymal markers, such as snail, slug, and vimentin. In parallel with EMT reversal, MR-3 downregulated the invasion and migration of MCF-7 cells. Exploring the action mechanism of MR-3 on the suppression of EMT and invasion indicates that MR-3 markedly reduced the expression and nuclear translocation of β-catenin, accompanied with the downregulation of β-catenin target genes and the increment of membrane-bound β-catenin. MR-3 restored glycogen synthase kinase-3β activity by inhibiting the phosphorylation of Akt, the event required for β-catenin destruction via a proteasome-mediated system. These findings indicate that the anti-invasive activity of MR-3 on MCF-7 cells may result from the suppression of EMT via down-regulating phosphoinositide 3-kinase (PI3K)-AKT signaling, and consequently, β-catenin nuclear translocation. In the part II, we investigated quercetin can suppress the Type I insulin-like growth factor (IGF-1) growth axis may be of preventive or therapeutic value. We have demonstrated quercetin modulates Type I insulin-like growth factor receptor (IGF1R) signaling through upregulation of Insulin-like growth factor-binding protein 3 (IGFBP-3) and downregulation IGF-1 secretion and ADAM Metallopeptidase Domain 28 (ADAM28) expression . In this study, we investigated the mechanism of quercetin can induce the reversion of EMT . Quercetin down-regulation mesenchymal- like markers vimentin、fibronetin and vascular endothelial growth factor (VEGF) and up-regulated epithelial-like markers keratin-18, -19 and ZO-1 proteins expression。Quercetin also inhibition of IGF-1R and IRS-1 phosphorylation and decrease IGF-1R downstream signaling molecules AKT and ERK1/2 phosphorylation. The transcript levels of transcription factors involved in EMT control, such as snail, slug, and zeb1 were reduced in protein and mRNA levels and downregulation of snail and slug activation in MDA-MB-231 cells after quercetin treatment. In vivo administration of quercetin to MDA- MB-231 tumor xenografts inhibited tumor growth and lung metastasis, resulted in IGF-IR inactivation and regulation ADAM28. These results suggest that inhibition of cell invasive activity and metastasis induction of IGFBP-3 and by Quercetin are mediated inhibition of IGF-IR signaling pathway. Taken together, these findings indicate that resveratrol methoxylated analog MR-3 and quercetin lead to the blockage of EMT and the invasion of breast cells. We found that MR-3 inhibitor of Wnt/β-catenin signaling and quercetin increas level of IGFBP- 3 through aberrant regulation of IGF-1R signaling.