檳榔鹼誘導口腔上皮細胞中Cyr61表現之機轉

Abstract

根據行政院衛生署民國 95 年統計，口腔癌已成為台灣男性十大癌症發生率的第四位。嚼檳榔已早被證實為導致台灣口腔癌發生最主要的危險因子。檳榔鹼arecoline 是檳榔中最主要的興奮成分，在唾液中的濃度可達 0.9 mM。我們先前發現在口腔癌組織中有較高量的 Cyr61 表現。 將過度表現 Cyr61 的口腔癌上皮細胞打入 SCID 鼠會增大腫瘤體積與看到明顯的血管新生。本研究以 0.6 mMarecoline 處理人類牙齦上皮 S-G 細胞株，口腔癌 Ca9-22 細胞株與 KB 細胞株發現都會誘導 Cyr61 蛋白的表現。Cyr61 蛋白在刺激後 4 小時開始上升，12 小時達到高峰，而且 mRNA 的表現也在 0.5 小時開始上升，4 小時達到高峰。並發現0.6 mM arecoline 處理可以誘導含氧自由基（ROS）的產生。加入抗氧化劑N-acetylcysteine (NAC)後可降低 arecoline 誘導的 Cyr61 蛋白表現，說明了在口腔細胞中，arecoline 經由 ROS 而誘導 Cyr61 蛋白的表現。 此外，arecoline 也會促使 ras-related small GTPase Rac1 的活化。以 Rac1、P38 MAPK 與 MSK1 之抑制劑(分別為 NSC23766、SB203580 與 H-89)前處理可阻斷 arecoline 誘導的 Cyr61蛋白表現，但是 Rac1 與 p38 MAPK 之抑制劑無法阻斷 arecoline 誘導的 ROS 的產生。西方墨點分析顯示了arecoline 可以活化 CREB。另外 arecoline 亦可以活化 Egr-1 不論是蛋白或是 mRNA 的表現。而加入 AP-1 的抑制劑時也會抑制掉arecoline 對於 Cyr61 的誘導。當細胞加入 HDAC 抑制劑時可加強 arecoline 對於Cyr61 的誘導現象。本研究首次指出arecoline 可誘導口腔細胞Cyr61 蛋白之表現，可能經由 ROS-Rac1-p38-MSK-CREB 訊息傳遞路徑。組蛋白乙醯化(acetylation)也在 arecoline 對 Cyr61 誘導的過程中扮演了重要的角色。另外Ｅgr-1 與 AP-1 在其中所扮演的角色有待更進一步的證實。冀望未來可以藉由抑制這些訊息傳遞路 徑成員來達到抑制嚼食檳榔導致的口腔癌。

According to the 2006 census of department of health, Executive Yuan, ROC, oral cancer ranks fourth in the mortality rate of male cancer patient in Taiwan. Areca chewing has been affirmed as the major risk factor of oral caner. Arecoline is the major ingredient of areca nut and the concentration can reach 0.9 mM in saliva. Recent studies show that Cyr61 is expressed at high level in oral cancer. Cyr61-expressed clones could promote in vivo tumor formation and angiogenesis in SCID mice. We identify 0.6mM arecoline can induce the expression of Cyr61 in oral epithelial cell SG, Ca9-22 and KB lines. Arecoline also can induce generation of ROS . ROS inhibitor, N-acetylcysteine can inhibite the expression of Cyr61 in KB cell. Arecoline can activate ras-related small GTPase Rac1. Rac1 inhibitor, NSC23766 and p38 MAPK inhibitor, SB203580 and MSK1 inhibitor, H89 also inhibited the expression of Cyr61 induced by arecoline whereas NSC23766 and SB203580 did not inhibit the generation of ROS. Western blotting showed that arecoline activated CREB. Arecoline also induced the expression of Egr-1. Furthermore, curcumin, an AP-1inhibitor, inhibited the arecoline-induced Cyr61 expression. Thus, arecoline may induce the expression of Cyr61 through ROS-Rac1-p38 MAPK-MSK-CREB signaling pathway. HDAC inhibitor, SAHA or TSA increased the induction of Cyr61 by arecoline. Histone acetylation may play an important role in the expression of Cyr61 induced by arecoline. Blocking the arecoline-induced Cyr61 signaling may provide an alternative therapeutic approach for the treatment of oral cancer in Taiwan.