α-生育醇醋酸醚對人類臍靜脈內皮細胞之血管增生作用的影響及其機制探討

Angiogenesis is an important process for tumor growth and metastasis. New blood vessel formation provides oxygen, nutrients, hormone, and removes CO2 and metabolites for cancer cells. Angiogenesis is regulated by several angiogenic factors including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and so on. Vascular endothelial growth factor (VEGF) is a powerful angiogenic activator, which is produced by many types of tumor cells. VEGF influences angiogenesis by inducing a variety of protein production of endothelial cells, including matrix metalloproteinases (MMPs), plasminogen activator and others. MMPs lead to the invasion and migration of endothelial cells by destroying the extracellular matrix. Focal adhesion kinase (FAK) and p38 cause endothelial cell migration. α-Tocopherol ether-linked acetic acid (α-TEA) is one of vitamin E analog and elicits apoptosis in a variety of human cancer cells. However, there are no reports to demonstrate the inhibition ofα-TEA on angiogenesis. In this study, we investigated that the effect of α-TEA inhibits VEGF-induced angiogenesis on human umbilical vein endothelial cells (HUVECs). We measured the effect of α-TEA on tube formation, invasion, migration, adhesion, MMP-2 activity and a diversity of proteins expression of HUVECs. The results show that α-TEA significantly prevented angiogenesis, invasion and migration on VEGF-induced HUVECs. Furthermore, we found thatα-TEA decreased MMP-2 activity and the expression of MMP-2, FAK and p38 proteins in a dose-dependent manner. By contrast, α-TEA dose-dependently increased the expression of nm23-H1 and tissue inhibitors of metalloproteinases (TIMP-2) proteins . Thus, α-TEA may be useful an anti-angiogenic and an anti-metastatic agent for tumor therapy.血管增生對腫瘤生長和癌轉移是一個重要的過程。新的血管形成會提供癌細胞氧氣、營養、荷爾蒙、並且移除二氧化碳和代謝廢物。血管增生受到多種血管增生因子所調控，包含血管內皮生長因子(vascular endothelial growth factor, VEGF)、介白素-8(Interleukin-8, IL-8) 等。血管內皮生長因子是一種有效的血管增生活化物，許多種癌細胞均可產生。血管內皮生長因子可藉由誘導內皮細胞產生多種蛋白質，包含基質金屬蛋白酶 (matrix metalloproteinases, MMPs)、纖維溶原活化物(plasminogen activator)等。基質金屬蛋白酶會破壞胞外基質導致內皮細胞侵入和移行，而粘著斑激酶(Focal adhesion kinase, FAK)和p38會影響內皮細胞的移行。α-生育醇醋酸醚 (α-tocopherol ether-linked acetic acid, α-TEA)是維生素E的衍生物，研究指出其能夠誘導多種癌細胞凋亡。然而，並無任何研究報導指出α-TEA對於VEGF所誘發血管增生的影響。在本研究中，我們以人類臍靜脈內皮細胞(human umbilical vein endothelial cells, HUVECs)為模式，探討抑制VEGF所誘發的血管增生之效果。我們測試α-TEA對於人類臍靜脈內皮細胞之脈管生成 (tube formation)、侵入能力 (invasion)、移行能力 (migration)、黏附能力 (adhesion)、基質金屬蛋白酶-2 (MMP-2) 活性和多種蛋白的表現。從結果顯示，α-TEA能顯著抑制VEGF誘發的人類臍靜脈內皮細胞之血管增生、侵入和移行能力。更進一步，我們發現α-TEA能降低VEGF誘導的人類臍靜脈內皮細胞之MMP-2活性，並抑制MMP-2、FAK和p38蛋白質表現，且呈劑量關係。另外，α-TEA亦能增加nm23-H1 和 TIMP-2蛋白質的表現。因此，對腫瘤治療而言，α-TEA可能可作為抗血管增生的抑制劑。中文摘要 I 英文摘要 II 圖目次 VII 縮寫表 IX 實驗架構 X 文獻整理 1. 癌症與轉移 (metastasis) 1 2. 癌症轉移與血管增生(angiogenesis) 3 3. 血管增生與血管增生因子 5 4. 血管內皮生長因子(vascular endothelial growth factor, VEGF) 7 5. 血管內皮生長因子受體(vascular endothelial growth factor receptor, VEGFR) 9 6. 內皮細胞與VEGF 11 7. 血管增生與基質金屬蛋白酶(matrix metalloproteinases或matrixins, MMPs) 13 8. 基質金屬蛋白酶與非轉移蛋白23 (nonmetastatic protein 23, nm23) 16 9. 血管增生與基質金屬蛋白酶組織抑制物(tissue inhibitors of metalloproteinases, TIMPs) 18 10. 血管增生與粘著斑激酶 (focal adhesion kinase, FAK) 20 11. 血管增生與p38 21 12. 薑黃素 (curcumin) 25 13. 生育醇醋酸醚(α-tocopherol ether-linked acetic acid, α-TEA) 27 14. 研究動機與假說 29 材料與方法 1. 實驗試劑 30 2. 細胞解凍 30 3. 細胞保存 31 4. 細胞培養 31 5. α-Tocopherol ether-linked acetic acid合成 31 6. MTT assay 32 7. Chicken chorioallantoic membrane (CAM) assay 32 8. In vitro angiogenesis assay 33 9. VEGF-induced angiogenesis assay 33 10. Cell invasion assay 33 11. Cell migration assay 34 12. Cell adhesion assay 34 13. 培養基中MMP-2 activity 之測定 35 14. 胞内蛋白質測定 36 15. 統計分析 39 結果 1. α-TEA對於細胞存活率之影響 40 2. α-TEA處理對於雞胚胎血管增生之影響 40 3. α-TEA對於體外脈管生成之影響 40 4. α-TEA對於VEGF誘發脈管生成之影響 41 5. α-TEA對於HUVECs細胞侵入能力(invasion)之影響 42 6. α-TEA對於HUVECs細胞移行能力(migration)之影響 42 7. α-TEA對於HUVECs細胞黏附能力(adhesion)之影響 43 8. α-TEA對於HUVECs細胞所分泌MMP-2活性之影響 43 9. α-TEA對於HUVECs細胞MMP-2蛋白質表現之影響 44 10. α-TEA對於HUVECs細胞nm23-H1蛋白質表現之影響 44 11. α-TEA對於HUVECs細胞TIMP-2蛋白質表現之影響 45 12. α-TEA對於HUVECs細胞FAK蛋白質表現之影響 46 13. α-TEA對於HUVECs細胞p38和p-p38蛋白質表現之影響 46 14. α-TEA對於HUVECs細胞VEGFR-2蛋白質表現之影響 46 圖表 48 討論 66 參考文獻 71 附錄 8