| Abstract: | 上皮間質的轉化(epithelial-mesenchymal transition；EMT) 與腫瘤血管新生(angiogenesis)作用是上皮細胞腫瘤癌化時造成侵襲(invasion)和轉移(metastasis)的關鍵。有許多文獻指出在具有高度惡性轉移能力的腫瘤細胞中，發現其皆具有EMT特性的現象。在臨床上顯示，癌細胞的轉移擴散往往是造成癌症病人病灶復發致死及最難根治的最主要原因，故若能有效抑制癌細胞的轉移及入侵，將可大幅減低癌症的死亡率。因此，發展新穎天然藥物來當作癌症預防或是治療藥物是目前最重要的課題。目前檜木醇(-thujaplicin)與黃連素(berberine)被發現的生物功能中包括：抗轉移、抗癌、抗發炎、抗氧化和細胞凋亡等多重功用。但是，-thujaplicin與berberine對於人類子宮頸細胞癌的作用機制、血管新生作用與EMT現象，至今仍然不清楚。本實驗室初步分析研究結果發現檜木醇(-thujaplicin)與黃連素(berberine)能明顯抑制人類子宮頸癌細胞的侵襲及移動能力，及VEGF或子宮頸癌細胞培養液所誘導血管內皮細胞管柱形成(tube formation)的能力，並降低由TGF-所誘導上升MMP-9的活性。此外，-thujaplicin能促進EMT marker E-cadherin的蛋白表達。並在動物實驗中證實berberine可抑制裸鼠皮下異種移植SiHa cells的腫瘤生長能力。綜合上述，在抑制子宮頸癌細瘤細胞侵襲轉移能力、EMT及腫瘤血管新生作用研究方面應該會有所貢獻，因此本計畫擬分為三年進行，第一年擬進行: (1)利用invasion assay、傷口癒合實驗、細胞貼附實驗，探討-thujaplicin與berberine抑制多種不同子宮頸癌細胞轉移之能力，並分析-thujaplicin與berberine對子宮頸癌細胞其轉移相關基因之蛋白層次與mRNA層次的影響，包含MMP-2, MMP-9, u-PA and claudin-1…等；(2) 利用免疫螢光染色、Western blot assay、CHIP、EMSA、RT-PCR，探討-thujaplicin與berberine調控癌細胞轉移之細胞骨架蛋白在細胞中分布與相關基因之蛋白層次與mRNA層次的影響，FAK, MAPK, PI3K, Rho…等;(3) 探討-thujaplicin與berberine抑制由腫瘤細胞、VEGF或FGF所誘導血管新生作用及相關訊息傳遞路徑。計畫第二年擬進行: (1) 闡釋TGF-對子宮頸癌鱗狀上皮細胞誘發EMT 現象、侵襲轉移及其相關蛋白酶的基因蛋白表現及mRNA表現; (2) 利用IF、Western blot、EMSA、ChIP assay，探討-thujaplicin與berberine抑制由TGF-啟動EMT之能力其詳細機轉與訊息傳遞路徑，包含: E-cadherin、fibronectin、slug 和snail…等；(3) 建立相關基因之Luciferase gene，分析其抑制上皮-間質轉化、細胞侵襲相關機轉是否影響於mRNA層次; (4) 以專一性抑制劑、siRNA及overexpression直接驗證相關基因的路徑是否正確。計畫第三年擬進行: (1) 免疫缺陷的小鼠(SCID mice)由尾靜脈分別接種子宮頸癌細胞，探討-thujaplicin與berberine抑制癌細胞轉移之能力；(2) 以免疫缺陷的小鼠(BALB/c nu/nu mice)皮下接種各種不同之子宮頸癌細胞，觀察-thujaplicin與berberine抑制腫瘤生長的能力分析; (3) 以小鼠實驗、血管螢光基因轉殖斑馬魚TG及雞胚絨毛膜血管生成試驗觀察-thujaplicin與berberine在動物體內抑制血管新生的能力。
Epithelial to mesenchymal transition (EMT) and angiogenesis have been considered essential for cancer metastasis, a multistep complicated process including local invasion, intravasation, extravasation, and proliferation at distant sites. Moreover, metastasis, the major cause of cancer death and various treatment strategies have targeted on preventing the occurrence of metastasis, is a multi-step process involving change of cytoskeleton, cell adhesion and proteolytic degradation of the extracellular matrix (ECM), essential to achieving cell motility. It has been showed that -thujaplicin and berberine may have potentially beneficial effects, including anti-metastatic, anti-cacinogenic, anti-inflammatory, anti-oxidant and apoptotic effects. However, theeffects molecular mechanism of -thujaplicin and berberine in human cervical carcinoma are presently unknown. In this study, anti-metastasis, anti-angiogenesis, anti-tumor agents and reversion of EMT were investigated using -thujaplicin and berberine on various cervical cancer call lines (SiHa, HeLa and Caski). In our preliminary study, we demonstrated that -thujaplicin inhibited cell invasion in SiHa cervical cancer cells. -thujaplicin was sufficient to inhibit TGF--induced MMP-9 expression and increase TGF--reduced E-cadherin (MET marker) expression in SiHa human cervical cancer cells. -thujaplicin could reduce VEGF-induced tube formation of HUVECs. Berberine inhibited migration, the secretion of MMP-2 and u-PA, and was sufficient to inhibit TGF--induced MMP-9 expression in SiHa cells. Berberine also could reduce cervical cancer induced tube formation and angiogenesis of transgenic zebrafish Tg (fli1:EGFP)×Tg (gata1:RFP). Therefore, in the first year of this study, we propose to (1) investigate the effect of -thujaplicin and berberine on invasion, migration in various cervical cancer cells and analyze the protein and mRNA levels of invasion relevance genes, including MMP-2, MMP-9, u-PA and claudin-1…etc. (2) investigate the signal pathways (such as FAK, MAPK, PI3K, Rho) for -thujaplicin and berberine affect human cerviacl cancer cell invasion and migration; (3) analyze the effects of -thujaplicin and berberine for VEGF, FGF or cervical cancer induced-angiogenesis. In the second year of this study, (1) we will investigate whether -thujaplicin and berberine could reverse TGF--induced epithelial-to-mesenchymal transition phenotype and related gene expression; (2) to determine -thujaplicin and berberine inhibit or reduce TGF- induced-EMT gene (including E-cadherin, -catenin, fibronectin, slug and snail) in cervical cancer cells by real-time PCR, western blot and confocal microscope.; (3) to evaluate that -thujaplicin and berberine inhibit TGF- induced-EMT signaling pathways ; (4) using some specific inhibitors, blockers and RNA interference to alleviate TGF--induced EMT pathway in human cervical cancer cells; (5) using EMSA and Luciferase assay to confirm what kinds of transcription factor binding, and whether through transcription factor to regulate of specific regions of E-cadherin promoter. In the third year of this study, we aim to (1) via an scid mice model determine the effect of -thujaplicin and berberine on metastasis; (2) investigate tumor growth of different cervical cancer cells in vivo via cancer cell xenografted nude mice model and BALB/c-bearing mice model; (3) investigate the effects of -thujaplicin and berberine on VEGF or tumor-induced angiogenesis by Matrigel plug assay via C57BL/6-bearing mice model; (4) finally using transgenic zebrafish Tg (fli1:EGFP)×Tg (gata1:RFP) and CAM assay as animal models to observe the effects of -thujaplicin and berberine on the development of intersegmental vessel and blood cells. These studies support that -thujaplicin and berberine may have potential as anti-cancer and anti-invasion agents in human cervical cancr cells. |
