棉酚抑制肺癌及子宮頸癌細胞之上皮-間質轉化、侵襲轉移與血管新生作用表現機制之相關研究-1

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 研究計劃 > Item 310902500/15563

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/15563

Title:	棉酚抑制肺癌及子宮頸癌細胞之上皮-間質轉化、侵襲轉移與血管新生作用表現機制之相關研究-1 Studies of the Mechanism of Action of Gossypol on Epithelial-Mesenchymal Transition, Cancer Metastasis and Angiogenesis in Lung and Cervical Cancer Cells-1
Authors:	謝易修陳霈霓許立松 Hsieh Yih-Shou Pei-NiChen LI-SUNGHSU
Contributors:	中山醫學大學生化暨生物科技研究所
Keywords:	基礎醫學
Date:	2015
Issue Date:	2016-07-25T08:02:21Z (UTC)
Abstract:	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。子宮頸癌的轉移是患者死亡的最常見原因，因此我們可以針對預防子宮頸癌轉移的發生來來當做治療子宮頸癌的其中一種策略。在本研究中使用棉酚對兩株子宮頸癌細胞進行研究（ SiHa細胞和HeLa細胞），觀察棉酚是否具有抗轉移，抗血管生成，抗腫瘤以及回復 EMT的作用。將SiHa與HeLa細胞分別處理棉酚，利用MTT分析觀察棉酚對子宮頸癌細胞增生的影響。為了研究棉酚在子宮頸癌細胞侵襲和移動的抑制作用，利用Boyden chamber侵襲實驗和傷口癒合實驗進行分析。我們使用Western blot檢測棉酚對子宮頸癌轉移和上皮與間質轉化(EMT)的影響。吸取培養過細胞的培養液，分別針對基質金屬蛋白?(MMPs)與尿激?型纖溶?原激活物(u-PA)進行酵素活性的測試。進一步利用Western blot來研究棉酚透過何種分子機制包括MAPK、 FAK/Src和PI3K/Akt訊號傳遞路徑影響的腫瘤轉移和EMT。隨著劑量和時間的增加，證實棉酚可以抑制人類宮頸癌細胞的細胞存活率。透過細胞侵襲實驗，隨著棉酚濃度增加，可以降低SiHa細胞和HeLa細胞的侵襲的能力。酵素活性分析也顯示出棉酚可以降低的u-PA和MMP- 2的活性。此外利用免疫螢光染色分析，可觀察到棉酚可以回復TGF-β1處理下子宮頸癌細胞型態的改變。 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, theeffects molecular mechanism of gossypol in human lung cancer or cervical carcinoma are presently unknown. The metastasis of cervical cancer is the most prevalent cause of patient death, and various treatment strategies have targeted the prevention of the occurrence of metastasis. In this study, anti-metastasis, anti-angiogenesis, anti-tumor effects and reversion of EMT were investigated using gossypol on two cervical cancer call lines (SiHa and HeLa). SiHa and HeLa cells were treated with gossypol to determine the effect on cell proliferation by MTT assay. To examine the inhibitory effect on the cell invasion and migration, Boyden chamber invasion assay and wound healing assay were performed. We examined the effect of gossypol on factors of cancer metastasis and epithelial to mesenchymal transition (EMT) by Western Blot. Condition media of cells were subjected to gelatin zymography and casein zymography to investigate the expression of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA). The molecular mechanisms of gossypol mediated cancer metastasis and EMT were further investigated by Western blotting analysis including MAPK, FAK/Src and PI3K/Akt signaling pathways. Gossypol inhibits the viability of human cervical carcinoma cells in a dose- and time-dependent manner. By cell invasion assay, gossypol reduces the invasion of SiHa and HeLa cells in a concentration-dependent manner. Zymography assay has shown gossypol reduces the activities of u-PA and MMP-2. Gossypol inhibits cell migration of SiHa and HeLa cells. Gossypol reverses the TGF-β1 induced conformation change (mesenchymal type) using immuno?uorescence assay.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/15563
Appears in Collections:	[生化微生物免疫研究所] 研究計劃

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