氧化膽固醇於血管內皮細胞誘發COX-2蛋白表現:氧化膽固醇與癌症形成的關係

Cholesterol-3-beta,5-alpha,6-beta-triol induced cyclooxygenase-2 expression in endothelial cells:implication for carcinogenesis

羅翊伶(Yi-Ling Lo)

指導教授 ： 鄭幼文

臺北醫學大學/藥學院/藥學研究所/碩士(2006年)

摘要

氧化性的低密度脂蛋白(oxLDL)已被證實在粥狀動脈硬化的病理過程中扮演重要的角色。近年來的研究中，發現氧化性膽固醇(Oxysterols)為氧化性的低密度脂蛋白中主要的毒性成分。有關膽固醇氧化物會造成癌症的論點近年來也逐漸受到重視。然而，其確切的機轉到目前為止並不清楚。 氧化性膽固醇存在著許多不同的結構異構物以及擁有不同的生理活性。在許多不同形態的細胞上，許多氧化性膽固醇皆會產生顯著的毒性。我們已初步發現某些氧化性膽固醇cholesterol-3β,5α,6β-triol (α-Triol)會經由自由基的生成而誘導基因突變。許多證據顯示，致基因突變與致癌反應之間有著密不可分的關係。然而，有關於氧化性膽固醇與致基因突變與致癌作用相關性的研究，至今仍不明瞭。我們的初步研究發現，讓我們很有興趣地想接下去探討cholesterol-3β,5α,6β-triol (α-Triol)與致癌作用的相關性。 許多的證據支持了COX-2蛋白與癌化的相關性，多種惡性腫瘤細胞比正常細胞分泌過多的前列腺素(PGs)，而此前列腺素(PGs)的增加則來自於COX-2蛋白的過度表現。 在人類的疾病中，氧化性膽固醇存在著許多不同的生理活性。因此，在我們的研究中，嘗試著去探討α-Triol在內皮細胞是否會誘發COX-2蛋白表現，首先，我們同時利用人類臍靜脈內皮細胞(HUVECs)與血管內皮細胞株(ECV-304 cells)，來觀察α-Triol是否會誘發COX-2蛋白表現。在實驗結果中，我們發現α-Triol在人類臍靜脈內皮細胞(HUVECs)能誘發COX-2的蛋白表現，且呈現時間-效應與濃度-效應關係。在血管內皮細胞株(ECV-304 cells)上，同樣也發現COX-2蛋白表現呈現濃度-效應關係。除了會誘發COX-2蛋白表現量增加，我們發現α-Triol也會增加PGE2 的生成。而由α-Triol所造成PGE2 的生成增加，可藉由前處理專一性的COX-2抑制劑，而減少生成量。 α-Triol在血管內皮細胞株(ECV-304 cells)上，會同時活化p42/44，JNK，和p38 MAPK。使用p42/44，JNK，和p38 MAPK的特異性蛋白抑制劑皆可有效地降低COX-2蛋白的生成。證實了MAP kinase磷酸化反應，包括p-ERK、 p-JNK和p-p38三條路徑，皆會對COX-2蛋白表現有調控的作用。此外，我們也發現了α-Triol會活化NF-κB，藉由增加轉錄能力而調控COX-2蛋白表現。α-Triol在血管內皮細胞株(ECV-304 cells)上，會磷酸化p-eNOS 1177 蛋白而造成NO生成量呈現濃度-效應反應的增加。專一性的eNOS and AKT抑制劑可減緩由α-Triol在血管內皮細胞株(ECV-304 cells)上，所誘發COX-2蛋白的過度表現。 總結，在我們的實驗結果中發現，α-Triol在內皮細胞中所誘發COX-2蛋白的表現，需同時經由MAPK和 NF-κB的活化，以及牽涉到在訊息傳遞中扮演重要角色之NO的生成。這是過去對於氧化膽固醇的研究中，從來沒有被發現以及探討的部份。希望藉由我們的研究發現，可提供關於氧化性膽固醇在人類可能誘發癌化作用的一個新的論點，以及開啟另一條對於氧化膽固醇的研究領域。

關鍵字

血管內皮細胞 ； 氧化膽固醇 ； 癌症

並列摘要

Oxidized low-density lipoproteins (oxLDL) are now believed to play an important role in the pathogenesis of atherosclerosis. Oxysterols, found in oxLDL particularly in LDL-,appear to account for most of the cytotoxicity produced by oxLDL in recent investigations. The contribution of cholesterol oxidation products to carcinogensis has been noticed for the past few years,however,the precise mechanisms still remain unclear. Oxysterols have many different structures and possess diverse biological properties. Many oxysterols alone produce significant toxicity in a variety of cell types. In our preliminary study,we found that reactive oxygen species (ROS) were involved in cholesterol-3β,5α,6β-triol (α-Triol)-induced genotoxicity. It was known that many mutagens also exert the effect of carcinogenesis. Studies on the role of oxygenated derivatives of cholesterol (oxysterols) in carcinogenesis and mutagenesis, which listed, are largely inconclusive. Our findings have raised the interest to further investigate the effect ofα-Triol on carcinogenesis. Multiple lines of compelling evidence supports that the enzyme of cyclooxygenase-2 (COX-2) plays a crucial role in carcinogenesis. Many human malignancies produce more prostaglandin (PGs) than normal tissues from which they arise. Increased synthesis of PGs in transformed cells and tumors can be a consequence of enhanced expression of cyclooxygenase-2. In human diseases, oxysterols possess diverse biological properties. Therefore，in our study, we aimed to investigate if α-Triol can modulate COX-2 expression in endothelial cells. First，the effect of α-Triol on COX-2 expression in both human umbilical vein endothelial cells (HUVECs) and ECV-304 cell line was examined. We found α-Triol dose- and time-dependently enhanced COX-2 protein expression in HUVECs and ECV-304 cells. In addition,α-Triol increased the production of PGE2. Theα-Triol-dependent accumulation of PGE2 was attenuated by preincubation with COX-2 inhibitor(SC-791). The oxysterol activated p42/44，JNK，and p38 MAPK in ECV-304 cells . The inhibitor of p42/44，JNK，and p38 MAPK could effectively attenuate COX-2 induction, suggesting that MAPK pathway was involved inα-Triol -induced COX-2 expression. We also found thatα-Triol may participate in NF-κB activation，which in turn activates transcription of COX-2 production，α-Triol upregulated p-eNOS 1177 protein phosphorylation and dose-dependently increase nitric oxide expression in ECV-304 cells. The eNOS and AKT inhibitors abrogated theα-Triol-induced COX-2 expression in ECV304 cells. In conclusion,the results demonstrate that α-Triol can increase COX-2 protein expression in endothelial cells, this effect requires co-operation with MAPK and NF-κB, and NO might play an important role in transducing the signal. Our findings also provide a new insight of oxysterols induced carcinogenesis in humans.

並列關鍵字

COX-2 ； Oxysterol ； Carcinogenesis

參考文獻

1. Gutteridge JM and Halliwell B, The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem Sci 15(4): 129-35, 1990.

2. Dean RT, Thomas SM and Garner A, Free-radical-mediated fragmentation of monoamine oxidase in the mitochondrial membrane. Roles for lipid radicals. Biochem J 240(2): 489-94, 1986.

3. Vaca CE, Wilhelm J and Harms-Ringdahl M, Interaction of lipid peroxidation products with DNA. A review. Mutat Res 195(2): 137-49, 1988.

4. Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD and Lusis AJ, Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 91(9): 2488-96, 1995.

5. Salonen JT, Nyyssonen K, Salonen R, Porkkala-Sarataho E, Tuomainen TP, Diczfalusy U and Bjorkhem I, Lipoprotein oxidation and progression of carotid atherosclerosis. Circulation 95(4): 840-5, 1997.