Chk2於檳榔成分誘發細胞毒性過程中所辦演之角色

Abstract

嚼食檳榔是一項普遍風行於亞洲、非洲、歐洲部分地區各個社會階層的流行嗜好，尤其是泰國、馬來西亞、印尼、印度、中國、台灣等地區，根據統計，全世界有嚼食檳榔習慣的人口達到六億人之譜。嚼食檳榔也被證明是多種口腔粘膜病變：口腔白斑、口腔黏膜下纖維化甚至口腔癌的主要致病因子之一，國際癌症研究中心也發表專論指出，檳榔子本身即為一級致癌物，許多研究指出檳榔萃取物的確存在著具細胞與基因毒性物質，能夠造成DNA斷裂、DNA與蛋白質交互連結、非正常程序的DNA合成、細胞週期改變等異常現象。目前已經有研究指出，當細胞發生DNA傷害時，會有ATM/ATR-Chk1/Chk2路徑的活化，使細胞週期停滯，並活化其他參與DNA修補蛋白的表現。但由於檳榔成分過於複雜，對於檳榔成分究竟是如何造成各種細胞變異甚至口腔疾病的詳細致病機轉目前並不清楚，因此本篇研究的主要目的即是針對細胞內兩種細胞週期檢查點Chk1/Chk2在檳榔成分所造成的基因與細胞毒性中所扮演的角色進行探討。在dose dependent的實驗中發現，隨著檳榔子萃取物與檳榔素濃度的提高，SAS舌癌細胞不只外型出現了明顯變圓，較不貼附於培養皿的現象，細胞內也出現大量空泡。在細胞的存活性實驗也發現，隨著檳榔子萃取物與檳榔素濃度提高，分別在800 ug/ml與0.8 mM濃度時，SAS細胞存活率會有明顯下降的情況。利用流式細胞儀進行細胞週期分析也發現隨著檳榔子萃取物濃度提高，SAS細胞會有G2/M phase arrest的現象。進一步使用RT-PCR與西方點墨法研究檳榔子萃取物與檳榔素是否能有效活化各個與細胞週期調控以及細胞凋亡有關之指標分子表現，發現檳榔子萃取物與檳榔素能夠活化細胞內Chk2訊息傳遞路徑表現，藉由Chk2的磷酸化，進一步促進下游分子如p-Cdc25C、p-Cdc2表現，而Chk2的活化同時也影響了與細胞週期有關的cyclin B1、cyclin D表現，檳榔子萃取物與檳榔素也能調控和細胞凋亡有關的p-p53、Bcl-2、Bax在細胞內的表現。而ATM/ATR抑制劑咖啡因與Chk2抑制劑的使用則更進一步的釐清了Chk2訊息傳遞路徑中各分子的上下游關係，令人意外的是，使用Chk2抑制劑降低Chk2表現後，SAS細胞的G2/M phase arrest現象消失了，取而代之的是細胞大量堆積在G1時期。總結來說，我們提供了檳榔子萃取物與檳榔素對於細胞變異的可能分子機制，檳榔子萃取物與檳榔素能夠活化SAS細胞Chk2訊息傳遞路徑，證實了Chk2的表現在檳榔成分引起的細胞變異中的確扮演著舉足輕重的角色，而在未來臨床應用上，Chk2也許可以做為檳榔引起的口腔疾病治療中一個新的標的。

Betel quid (BQ) chewing is a very common habit in Asia, Africa, and a portion of Europe. It enjoys complete social acceptance in many societies especially in Thailand, Malaysia, Indonesia, India, China and Taiwan. It has been estimated that there are about 6 million BQ-chewers living in different regions of the world. BQ chewing is demonstrated to be one of the major risk factors leading to leukoplakia, oral submucous fibrosis, and oral cancer. In a monograph published by the International Agency for Research on Cancer（IARC）for the evaluation of cancer risks (IARC, 2004), areca nut was ranked as a group I carcinogen to humans. There are many reports indicating the components of areca nuts have genotoxicity and cytotoxicity leading to DNA strands breaks, DNA-proteins crosslink, unschedualed DNA synthesis and cell cycle aberration. When DNA damage occurs, the ATM/ATR-Chk1/Chk2 pathway is activated, cell cycle arrested, and induces the activation of other DNA-repair proteins. So far, because of the components of AN are very complex, the mechanisms of AN-induced cytotoxicity to cause cell aberrations and oral diseases are not clearly understood. The purpose of this study is trying to investigate the roles of two check point kineses: Chk1/Chk2 in the ANE-induced geno- and cytotoxicity. In the ANE-dose dependent experiments, the morphology of SAS cells became much roundly, lost the connection to the plate and many vacuoles appeared in the cells due to the increased ANE-concentration. The viability of SAS cells decreased obviously because of the raised of the ANE and arecoline, especially in the concentration of 800 ug/ml and 0.8 mM respectively. By using flow-cytometry assay, we found that the SAS cells arrested in G2/M phase because of the effects of the components of areca nuts. We further analysed whether the ANE and the arecoline can regulate the cell cycle- and apoptosis-related molecules by RT-PCR and western blot. ANE and arecoline activated the Chk2 pathway and induced the downstream p-Cdc2, p-Cdc25C expression through the Chk2 phosphorylation. The ANE and arecoline also regulated the cell cycle-related cyclin B1, cyclin D1 and the apoptosis-related p-p53, Bax and bcl-2. By using caffeine, an ATM/ATR inhibitor and Chk2 inhibitor, we clarified the correlation between the molecules of the ATM-Chk2 signaling transduction pathway. Unexpectedly, Chk2 inhibition not only blocked the ANE-induced G2/M phase arrest of SAS cells but also resulted in the G1 phase arrest. In conclusion, we provide the possible molecular mechanism of Chk2 pathway, which is induced by ANE and arecoline to cause aberration in SAS cells. Chk2 activation indeed plays a very important role in the cell mutagenesis induced by areca nuts components. Maybe Chk2 can be the new target in the prevention and clinical treatment of the areca nuts-induced oral mucosa diseases in the future.