| 摘要: | 臨床數據顯示，癌細胞的轉移往往是造成癌症病人病灶復發致死的最主要原因，故 若能有效抑制癌細胞的轉移及侵襲作用，將可大幅降低癌症的死亡率。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。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。棉酚(gossypol)已知的生物 活性包括：抗攝護腺癌轉移、誘導癌細胞死亡、抗氧化和抑制病毒等多重功用。但是， 棉酚針對於人類子宮頸細胞癌與肺癌的作用機制、EMT現象與血管新生作用，至今仍然 不清楚。本實驗室初步分析研究結果發現低濃度的棉酚能明顯抑制人類子宮頸癌或肺癌 細胞的侵襲及移動能力，及VEGF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-2的活性與侵襲移動的能力，棉酚能促進MET marker E-cadherin的蛋白表達，並有效降低u-PA的蛋白及基因轉錄活性。在肺癌細胞中可降低 p-Src的蛋白表現。在高濃度作用下則是會誘導癌細胞死亡，此外，在動物實驗中證實棉 酚可抑制裸鼠(nude mice)皮下異種移植SiHa cells的腫瘤生長能力。綜合上述，在抑制子 宮頸癌細瘤細胞及肺癌侵襲轉移能力、EMT、腫瘤血管新生作用及誘導癌研包死亡研究 方面應該會有所貢獻。針對這些課題我們將以三年計畫分述各個階段的目標與策略: 第一年目標:(1) gossypol對於癌細胞in vitro表型特性[例如:影響細胞增生、移動、侵襲、 血管新生、細胞凋亡(apoptosis)與細胞自噬(autophagy)的能力]；(2) gossypol作用是否影 響水解蛋白酶及相關轉錄因子等蛋白及mRNA的表現並探討其相關結合位置與轉錄活 性；(3) 進一步利用訊息傳遞路徑的抑制劑和siRNA(例如: siRNA-FAK/Src、E-cadherin、 vimentin)及overexpression (例如: p-FAK/p-Src、E-cadherin、vimentin)方式，分別探討 gossypol對子宮頸癌與肺癌細胞的相關訊息傳遞路徑及其蛋白變化。 第二年目標:(1)闡釋TGF-對子宮頸癌鱗狀上皮及肺癌細胞誘發EMT現象及其相關的基 因表現，證實gossypol調控TGF-誘導癌上皮細胞與EMT有關基因的表現。釐清gossypol 抑制TGF-啟動EMT之可能途徑及其訊息傳遞，分別以抑制劑或拮抗劑、過度表現或 siRNA方式逐一探討；(2)探討gossypol抑制由子宮頸癌或肺癌細胞condition media與 VEGF或b-FGF所誘導血管上皮細胞形成tube及增生之能力;分析與血管新生之相關之基 因表現；(3)釐清gossypol 誘導細胞凋亡及自噬之間的關係及分子機轉；(4)以細胞模式 中使用一些臨床抗癌藥物(如：cisplatin、doxorubicin)或抑制劑搭配gossypol，藉以減緩 癌細胞生長、侵襲轉移及能力。 第三年目標:: 主要目標為gossypol對於癌細胞in vivo 表型特性影響: (1) 以裸鼠(nude mice)分別於皮下異種移植子宮頸癌或肺癌細胞，以口服gossypol的方式，觀察腫瘤生長， 評估裸鼠存活率，及對腫瘤內血管新生的影響。(2)利用SCID mice以尾靜脈注射子宮頸 癌或肺癌細胞，以口服投藥的方式，觀察細胞轉移到肺部或其他臟器的能力; (3)利用組 織化學染色、Western Blot及real-time RT-PCR並驗證gossypol對於EMT、侵襲或血管新 生相關蛋白在in vivo改變(4) 利用動物模式中使用一些臨床抗癌藥物(如：cisplatin、 doxorubicin)、抑制劑或化療方式搭配gossypol，藉以減緩肺癌細胞生長、侵襲轉移及能 力。
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. Epithelial-to-mesenchymal transition (EMT) in cancer cells is considered pre-requisite for acquiring invasive/migratory phenotype, and subsequent metastasis. It has been showed gossypol that may have potentially beneficial effects, including anti-metastasis of prostate cancer, induction of cancer cell death, anti-oxidant and anti-bacterial effects. However, theeffects molecular mechanism of gossypol in human lung cancer or cervical carcinoma are presently unknown. In this study, anti-metastasis, anti-angiogenesis, anti-tumor effects and reversion of EMT were investigated using gossypol on various cervical cancer call lines (SiHa, HeLa and Caski) and lung cancer cell lines (A549 and H1299). In our preliminary study, we demonstrated that gossypol inhibited cell invasion of SiHa, HeLa and A549 cells. Gossypol was sufficient to inhibit TGF--induced MMP-2 expression and increase TGF--reduced E-cadherin (MET marker) expression in SiHa human cervical cancer cells. Gossypol could reduce VEGF-induced tube formation of HUVECs. Gossypol inhibited migration and the secretion and transcription activity of u-PA, and was sufficient to inhibit TGF--induced MMP-2 expression in SiHa cells. Gossypol also could reduce the expression of p-Src and MMP-2 in lung cancer cells. Treatment of high concentration of gossypol could induce cell death of lung and cervical cancer cells. Gossypol was evidenced by its inhibition on the tumor growth of SiHa cells via cancer cell xenografted nude mice mode. Therefore, three years grant proposal aims to study whether treatment of gossypol would inhibit cell invasion and induced cell death via mechanism(s) involved in vitro and in vivo models. In the first year of this study, we propose to (1) investigate the effect of gossypol on invasion, migration in various cervical and lung cancer cells; (2) analyze the protein and mRNA levels of invasion relevance genes, including MMP-2, u-PA and claudin-1…etc. (2) investigate the signal pathways (such as FAK/Src, PI3K, Rho) for gossypol affect human cerviacl aor lung cancer cell invasion and migration; (3) analyze the effects of gossypol for VEGF, b-FGF or cancer induced-angiogenesis; (4) analyze the effects of gossypol on cancer cell death. In the second year of this study, (1) we will investigate whether gossypol could reverse TGF--induced epithelial-to-mesenchymal transition phenotype and related gene expression; (2) to determine gossypol 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 gossypol inhibit TGF- induced-EMT signaling pathways ; (4) using some specific inhibitors, blockers and RNA interference to alleviate TGF--induced EMT pathway in human cervical and lung 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; (6) analyze the effects of gossypol for VEGF, b-FGF or cancer induced-angiogenesis. (7) analyze the mechanisms of the relationship between apoptosis and autophagy upon treatment of gossypol. In the third year of this study, we aim to (1) via an scid mice model determine the effect of gossypol on metastasis; (2) investigate tumor growth of different cervical and lung cancer cells in vivo via cancer cell xenografted nude mice model and BALB/c-bearing mice model; (3) investigate the effects of gossypol 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 gossypol on the development of intersegmental vessel and blood cells. These studies support that gossypol may have potential as anti-cancer and anti-invasion agents in human cervical or lung cancer cells. |
