尼古丁誘導口腔上皮細胞中Cyr61之表現及其機轉

Nicotine induced the expression of Cyr61 in oral epithelial cells

謝佳穎(Chia-Ying Hsieh)

指導教授 ： 郭彥彬

共同指導教授 ： 林思洸(Sze-Kwan Lin)

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02878

摘要

根據衛生署公佈之國人「十大死亡原因」，癌症已蟬聯第一位達二十一年之久；其中口腔癌的發生率及死亡率位居國內男性十大癌症之第四位及第五位，且又以鱗狀上皮細胞癌(squamous cell carcinoma, SCC)最為常見。許多研究已證實菸草內之致癌物質為促使口腔癌發生的重要因素之一，而尼古丁(nicotine)為菸草中最主要的成分。尼古丁可改變細胞的功能，如生長、附著，及matrix protein之合成等。Cyr61為CCN family中的成員，參與調控細胞之附著、移動、增生、分化、凋亡及血管新生等，在許多癌症中被指出與癌症生成有關。本實驗室先前的研究中發現，口腔癌病人癌化組織中有較正常組織高的Cyr61表現。轉殖過量表現Cyr61之細胞株至SCID小鼠體內則可增加腫瘤之體積及增進血管新生之情形，顯示Cyr61和口腔癌之間有著密不可分的關係。本研究主旨即為了解尼古丁在口腔上皮細胞中是否會活化Cyr61的表現，並探討其訊息傳導路徑。我們發現10μM 尼古丁可誘導正常人類口腔上皮細胞株S-G及口腔癌細胞株Ca-922與KB之Cyr61蛋白表現。KB之Cyr61蛋白和mRNA在尼古丁刺激0.5小時後開始上升，分別於2小時及0.5小時達到高峰之後開始下降，8小時及2小時後降回正常值。當加入α3、α7與α9 nicotinic acetylcholine receptor (nAchR)之抑制劑d-tubocurarine及α7 nAchR之抑制劑MG 624時可反轉Cyr61被活化之情形，但α3 nAchR抑制劑macamylamine和α9抑制劑strychnine則無此效果，且單獨以α7 nAchR之促進劑choline處理KB亦使Cyr61表現量提升，顯示尼古丁係經含有α7但不含α3與α9 subtype之nAchR誘導Cyr61表現。muscarinic acetylcholine receptor (mAchR) M2及M3 mAchR抑制劑4-DAMP與gallamine並無此影響，顯示尼古丁非經由mAchR刺激Cyr61表現。以 p38 MAPK之抑制劑SB203580前處理可阻斷尼古丁所誘導之Cyr61蛋白表現，顯示此活化路徑極可能經由p38 MAPK達成；但MSK1、PKA與Rho A GTPase抑制劑無此影響，顯示尼古丁並非經由這些路徑誘導Cyr61蛋白表現。本論文首次指出尼古丁可經由p38 MAPK訊息傳遞路徑誘導Cyr61蛋白之表現。冀望未來可藉由抑制此訊息傳遞路徑之成員來達到抑制抽菸導致的口腔癌之目的。

關鍵字

口腔癌 ； 口腔上皮細胞 ； 尼古丁

並列摘要

According to the top 10 causes of death in Taiwan published by the department of health, cancer has been the top 1 for 21 years. The occurrence rate and mortality rate of oral cancer is the top four and top five of the ten most common cancers of Taiwanese male, respectively. Among oral caner, squamous cell carcinoma (SCC) is the most common one. Many researches had proved that the carcinogen in tobacco is one of the most important causes of oral cancer, and nicotine is one of the main components of tobacco. Nicotine can alter the cell functions, such like the growth and adhesion of the cells, and the production of matrix protein. Cyr61 is a member of the CCN family, which involves the regulation of cell adhesion, migration, proliferation, differentiation, apoptosis and angiogenesis, and was reported to be related to the occurrence of cancer. Previous study of our laboratory found that there was much higher expression of Cyr61 in the cancerous tissue of oral cancer patients than that of normal tissue; Cyr61 overexpressing transgenic SCID mice had larger tumor and improvement of angiogenesis. These data indicated that Cyr61 is closely related to oral cancer. The main purpose of this research is to find out whether nicotine can induce the expression of Cyr61 in the oral epithelial cells and to investigate the signaling pathway. We found that 10μM nicotine could induce the protein expression of the normal epithelial cell line S-G cells and the cancerous cell lines Ca9-22 and KB. The expression of protein and mRNA of Cyr61 in KB was elevated after treatment of 0.5 hour, and reached a high after 2 and 0.5 hours, and returned to the origin after 8 and 2 hours, respectively. Pre-treatment of KB cells with the inhibitor of α3、α7 and α9 nicotinic acetylcholine receptor (nAchR) d-tubocurarine and the inhibitor of α7 nAchR MG 624 but not the inhibitor of α3 nAchR macamylamine and that of α9 nAchR strychnine reversed the activation of Cyr61 expression; treatment of α7 nAchR agonist choline alone also stimulated Cyr61 expression, indicated that nicotine induced Cyr61 expression through α7-containing but not α3- and α9- containing nAchR. The inhibitor of cholinergic receptor atropine but not the inhibitor of M2 and M3 muscarinic acetylcholine receptor (mAchR) 4-DAMP and gallamine could suppress Cyr61 expression, proved that the activation was not through the mAchR. The expression of Cyr61 could be blocked by pre-treatment of p38 MAPK inhibitor SB203580 showed that p38 probably participated in the activation pathway, and other specific inhibitors had ruled out the possibility of involvement of MSK1, PKA and the Rho A GTPase family. This article showed that nicotine could induce the expression of Cyr61 through p38 MAPK pathway for the first time, and the signaling pathway can be the target of treating the oral cancer caused by smoking.

並列關鍵字

oral cancer ； oral epithelial cells ； nicotine

參考文獻

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