棉酚抑制子宮頸癌細胞轉移及相關機制的探討

Gossypol inhibits invasion potential of human cervical carcinoma cells in vitro and in vivo

林孟璇(MENG-HSUAN LIN)

指導教授 ： 謝易修 ； 陳霈霓

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2014年)

摘要

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。棉酚是從棉花植物中萃取的天然多酚類物質，可作為細胞內脫氫酶的抑制劑。過去棉酚被報導具有抗生育，抗氧化，抗癌，防病毒和抗菌功能。然而，棉酚對癌症侵及人宮頸癌腫瘤生長的影響仍不清楚。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酶（MMPs）與尿激酶型纖溶酶原激活物（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。另外棉酚也可以抑制TGF-β1所誘導的EMT，結果證實，棉酚可以減少侵襲力和回復子宮頸癌細胞 EMT、p-ERK1/2以及p-Smad3的表現同時還原TGF-β1所減少SiHa細胞的E-cadherin蛋白表現。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。最後，透過尾靜脈注射免疫缺陷小鼠的活體實驗來驗證棉酚可以抑制宮頸子宮頸癌細胞轉移到肺部的能力以及抑制腫瘤增生。從以上的實驗結果得知，棉酚可以減少子宮頸癌細胞侵襲同時也可以回復人子宮頸癌細胞的被TGF-β1所誘導的 EMT 。

關鍵字

棉酚 ； 轉移 ； 侵襲 ； 子宮頸癌

並列摘要

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. Gossypol is a natural phenol derived from the cotton plant and is a phenolic aldehyde that permeates cells and acts as an inhibitor for several dehydrogenase enzymes. Gossypol is reported to exhibit antifertility, antioxidant, anticancer, antivirus, and antimicrobial properties. However, the effects of gossypol on cancer invasion and tumor growth of the human cervical carcinoma remain unclear. 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 was sufficient to inhibit TGF-β1-induced p-ERK1/2 and p-Smad3 expression and increase TGF-β1-reduced E-cadherin (MET marker) expression in SiHa cells. Gossypol reverses the TGF-β1 induced conformation change (mesenchymal type) using immunofluorescence assay. Finally, gossypol was evidenced by its inhibition on the tumor growth and metastasis of SiHa cells via cancer cell xenografted nude mice and tail vein injection scid mice mode, respectively. Taken together, these results suggested that gossypol could reduce the invasion and reverse EMT in human cervical cancer cells.

並列關鍵字

Gossypol ； metastasis ； Invasion ； cervical cancer.

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