葉酸調控人類臍靜脈內皮細胞p21蛋白表現之分子機轉

Abstract

本篇研究計畫主要探討水溶性維生素葉酸（Folic Acid）抑制人類臍靜脈內皮細胞（Human umbilical vein endothelial cell, HUVEC）生長作用的可能分子機轉。近年來，在癌症的相關研究當中，藉由抑制血管新生（Angiogenesis）進而抑制腫瘤生長的研究相當受到重視。研究發現，許多抑制血管新生的化合物不但能有效抑制癌細胞的生長，且具有相對較低的副作用。在本實驗室先前的研究證實，葉酸對於人類臍靜脈內皮細胞生長具有抑制的作用，且此抑制作用是透過影響細胞的細胞週期而達成。葉酸能促使人類臍靜脈內皮細胞的細胞週期停滯於G0/G1期，且主要是透過增加細胞週期停滯相關蛋白P21及P27的表現。本研究延續先前實驗室的發現，進一步釐清葉酸抑制人類臍靜脈內皮細胞生長作用的分子機轉。在本研究中，利用Western Blot（WB）的實驗分析證實，葉酸能夠去刺激P21的蛋白表現，且增加的效果和葉酸的劑量呈現正相關。而利用Luciferase Activity Assay以及RT-PCR的分析也分別發現葉酸能夠去促進p21 promoter的活性和p21 mRNA的表現量。更深入觀察細胞內的訊息傳遞路徑，在給予葉酸刺激的早期便會促進ERK蛋白的磷酸化。進一步利用給予ERK抑制劑U0126和Transfect dominant negative ERK2（DN-ERK2）兩種方式阻斷ERK的訊息傳遞路徑，並利用WB和3H-Thymidine Incorporation觀察阻斷ERK的訊息傳遞路徑後p21蛋白的表現量和細胞DNA 合成量的多寡。實驗證實，經由兩種方式抑制阻斷ERK的訊息傳遞路徑都會使葉酸促進P21蛋白的表現量增加以及抑制DNA合成的作用消失。最後，我們也發現，利用Transfect DN-ERK2方式阻斷ERK的訊息傳遞路徑，會使得葉酸促進p21 promoter活性的作用消失。經由以上實驗結果可以初步的推估，葉酸對人類臍靜脈內皮細胞生長的影響是經由ERK調控的途徑去促進p21 promoter活性，進一步促進p21 mRNA和P21蛋白的表現量，而導致細胞增生的抑制作用。

The main purpose of this study is to investigate the underlying molecule mechanisms folic Acid–induced anti-proliferation effect on human umbilical vein endothelial cells (HUVEC). Recently, anti-angiogenesis has been considered to be an alternative strategy on cancer therapy. Seveal compounds with anti-angiogenic activity have been demonstrated to exert an anti-cancer activity with fewer side effects. In our lab, we have previously found that folic acid inhibits angiogenesis through arresting the cell cycle of human umbilical vein endothelial cell. We demonstrated that folic acid increased the levels of p21 and p27 protein, which caused a decrease of CDK2 activity, and finally arrested the cell cycle at the G0/G1 phase. Here, we reported that folic acid dose-dependently increased the levels of p21 protein. Luciferase activity assay and RT-PCR demonstrated that folic acid induced the p21 promoter activity and mRNA level. Moreover, the levels of phosphorylated ERK protein were increased at 5 min after folic acid treatment. Blockade of the ERK pathway by ERK inhibitor (U0126) or dominant ERK2 (DN-ERK2) transfection prevented the folic acid-induced increases of p21 protein and promoter activity, and inhibition of DNA synthesis. The present study demonstrated that folic acid increased the p21 transcription though an ERK-dependent pathway.