Arecoline誘導口腔上皮細胞OCT4表現及其機轉

Abstract

衛生福利部今年公佈的 103 年癌症登記報告指出，口腔癌為臺灣十大癌症發生率的第五位，也是台灣年增率最高癌症。臺灣口腔癌發生與嚼檳榔有密切關係，但檳榔致癌機制仍未詳細闡明。文獻指出檳榔中的主要成分檳榔鹼（arecoline）是主要導致口腔癌發生的致病因子。八聚體結合轉錄因子4（Oct4）是口腔癌中幹細胞基因，研究發現OCT4的表現與口腔癌病人的預後效果呈高度負相關性，但Arecoline是否可誘導口腔上皮細胞OCT4表現及其機轉並不清楚。在本篇研究，我們發現Arecoline會誘導牙齦正常上皮SG細胞、口腔癌CA9-22及SAS細胞株中OCT4的表現。競爭性乙醯膽鹼蕈毒接受器(muscarinic acetylcholine receptor)抑制劑atropine及M4 muscarinic acetylcholine receptor拮抗劑Tropicamide可降低由Arecoline所誘導的OCT4的表現。TGF-β1中和抗體、ALK5抑制劑（SB431542）以及Smad3抑制劑（SIS3）可降低Arecoline誘導SG及Ca9-22細胞中OCT4的表現，顯示Arecoline 經由TGF-β1訊息傳遞路徑誘導口腔上皮細胞OCT4的表現。進一步發現Arecoline可以增加SG及Ca9-22細胞培養液中活化態TGF-β1的表現量。前處理抗氧化劑NAC能夠明顯降低Arecoline誘導SG及Ca9-22細胞的活化態TGF-β1。抗氧化劑NAC、PEG-catalase、mitochondria-targeted O2-抑制劑（Mito-TEMPO）、NO抑制劑（L-NAME）以及MnTBAP能夠明顯抑制Arecoline誘導SG及Ca9-22細胞活化態TGF-β1的表現量。EGCG可抑制Arecoline誘導SG及Ca9-22細胞活化態TGF-β1的表現量。 EGFR抑制劑（AG1478）、PI3K抑制劑（LY294002）、P38/ MAPK抑制劑（SB203580）、JNK抑制劑（SP600125）能夠明顯降低TGF-β1誘導SG及Ca9-22細胞中OCT4的表現。此外我們發現兒茶素（EGCG）對於檳榔鹼誘導誘導SG及CA9-22細胞中OCT4的表現皆有明顯的抑制效果。這些結果顯示，Arecoline經由Muscarinic receptor、ROS以及TGF-β1路徑誘導OCT4的表現，並得知此誘導路徑可藉由EGCG抑制Arecoline誘導細胞產生OCT4。

In Taiwan, oral cancer is the forth leading cause of death in males 2011. Areca nut (AN) chewing is the most important risk factor of oral cancer. Octamer-binding transcription factor 4 (OCT4) has been shown to play a central role in the pathogenesis of many human cancers. Chiou et al. reported that patients with higher OCT4 expression have worse overall survival. We observed Arecoline, a main alkaloid found in AN, increased OCT4 synthesis in a dose- and time- dependent manner in SG, Ca9-22 and SAS cell. Pretreatment with muscarinic receptor inhibitor (Atropine and Tropicamide) significantly reduced Arecoline induced OCT4 synthesis. We aimed to examine the possible signal transduction pathways involved in TGF-β1-induced OCT4 expression in SG and Ca9-22 cell, we found the effect that Arecoline induces OCT4 activation was inhibited by TGF-β1 neutralizing antibody, ALK5 inhibitor(SB431542) and Smad3 inhibitor(SIS3). We have also shown that EGFR inhibitor(AG1478), PI3K inhibitor(LY294002), P38/MAPK inhibitor(SB203580), and JNK inhibitor(SP600125) significantly abrogate the TGF-β1-induced levels of OCT4 protein, indicating ALK5/Smad3, EGFR, PI3K, P38/MAPK, and JNK are involved in the TGF-β1-induced OCT4 in SG and Ca9-22 cells. Furthermore, epigallocatechin-3-gallate (EGCG) completely inhibited Arecoline-induced OCT4 synthesis and inhibition is dose-dependent. We found Arecoline increased activated TGF-β1 level in SG and Ca9-22 cells. Pretreatment with antioxidant NAC significantly reduced Arecoline-induced activated TGF-β1 level in SG and Ca9-22 cells. We further found antioxidant NAC, PEG-catalase, mitochondria-targeted O2- inhibitor(Mito-TEMPO), NO inhibitor(L-NAME) and MnTBAP significantly inhibited Arecoline-induced activated TGF-β1 level in SG and Ca9-22 cells. In addition, EGCG significantly inhibited Arecoline-induced activated TGF-β1 level in SG and Ca9-22 cells. Taken together, our results indicate the Arecoline enhances cancer stem cell marker by increasing OCT4 expression via muscarinic receptor, ROS and TGF-β1 pathways, and EGCG potentially qualifies as a useful reagent for the prevention and therapy of oral cancer.