褪黑激素對於檳榔誘導口腔表皮細胞毒性的預防作用

Abstract

實驗目的：檳榔嚼塊包括檳榔子，包覆檳榔的葉子和裡面的熟石灰等，盛產在世界許多地區，特別是東南亞地帶，使用比率分布在各個年齡層面都有，世界衛生組織（WHO）研究指出，全世界約有1/10的人口嚼食檳榔，僅次菸草、酒精和咖啡，第4種最常被使用於提神的飲食。台灣嚼食檳榔的人口甚至高達200多萬人，據衛生福利部於2012年的統計數據，台灣90％的口腔癌患者有嚼檳榔的習慣。目前有許多研究已經發現檳榔的致癌性，其最主要導致口腔癌的發生。本研究的目的著重在於探討因檳榔萃取物作用於口腔舌癌細胞上的毒性影響是否能被退黑激素預防或減輕，因褪黑激素為人體內部松果體的主要分泌產物，許多研究也證實褪黑激素，在抗發炎和抗癌上有顯著效果。 實驗方法：將SAS口腔舌癌細胞暴露在不同濃度的褪黑激素（100 & 250 μg/ ml）下一小時後，再加入檳榔萃取物（800 μg/ ml）後，培養24小時。於光學顯微鏡下觀察24小時後SAS細胞形態變化。並以細胞存活率分析(MTT assay)反轉錄聚合酶連鎖反應（RT-PCR）、西方墨點法（Western Blot）和免疫螢光法(Immunofluorescence) 、 流式細胞術 (flow cytometry) 探討褪黑激素對於檳榔萃取物在SAS舌癌細胞上，細胞週期和存活率，發炎反應和氧化壓力是否具有調節性。 實驗結果: 褪黑激素作用在SAS細胞上24小時後，在細胞形態上可以發現，加入褪黑激素500 μg/ ml濃度下細胞數量有所減少，在細胞調節相關基因例如Cyclin B1、P21、CDC2跟CDC25C，可以發現到Cyclin B1、CDC2跟CDC25C的表現量隨著褪黑激素濃度的上升而下降，對應到P21的表現量則是隨著濃度的變化而上升，而在發炎反應和ROS等的相關基因例如COX2、ICAM1、Nrf2、NQO1的表現量也會受到褪黑激素影響。此外，褪黑激素也會影響癌症的血管新生相關基因TF的表現。當加入檳榔萃取物（800 μg/ ml）後，觀察對於細胞調節基因的影響，發現到檳榔萃取物導致的Cyclin B1下降則會因加入褪黑激素100μg/ ml時有些微回升，在P21、CDC2跟CDC25C，褪黑激素似乎沒有影響，而在發炎反應和免疫反應等的相關基因，也發現到褪黑激素都有效調節因檳榔萃取物的影響而上升的趨勢讓其下降，褪黑激素沒有調節檳榔萃取物所導致的Nrf2、NQO1和HO1的上升趨勢，最後觀察檳榔萃取物在褪黑激素receptor上，MT1、MT2、NQO2(MT3)、RoRα的影響。 結論:褪黑激素單純作用在SAS細胞上，在到達一定濃度下，會影響細胞週期調節基因的表現，Cyclin B1的下降和P21的上升，對於SAS細胞的細胞週期有所調控，也會影響發炎反應和抗氧化路徑COX2、Nrf2、NQO1等。當加入檳榔萃取物時，褪黑激素會讓因其導致的發炎、細胞毒素等COX2、ICAM1、MMP9、IL6、IL8降低，這些初步結果，可以發現褪黑激素對於口腔癌的一些效果，如若臨床上能搭配手術等治療方式，進一步減緩口腔癌所造成的發炎、增生等反應，對於口腔癌的臨床應用和治療效果會有更多幫助。 關鍵字: SAS舌癌細胞、檳榔萃取物、褪黑激素、細胞週期、發炎反應、細胞毒素

Aim：Betel quid consist of areca nut (AN), betel leaves, and slaked lime, which are abundant in many places of the world, particularly in Southeast Asia. The betel quid (BQ)-chewers comes from all ages. According to a World Health Organization (WHO) study, the population of BQ-chewer are one-tenth as populous as world, followed by tobacco, alcohol and coffee, and the fourth most commonly-used for refresh oneself. The population in Taiwan is up to 2 million. According to the statistics of the Ministry of Health and Welfare in 2012, 90% of oral cancer patients in Taiwan have the habit of BQ chewing. Ｍany studies have found the carcinogenicity of AN, which is the main cause of oral cancer. The aim of this study is to investigate whether the toxic effects of AN extract on oral tongue cancer cells can be prevented or mitigated by melatonin which is the main secretory product of the pineal gland in human body. Many studies have also confirmed that melatonin have function as anti-inflammatory and anti-cancer. Materials and methods：The tongue cancer cells (SAS) were pretreated with melatonin in different concentration (100&250 µg/ml) for 1 hours, and adding AN Extract (ANE) (800 µg/ml) co-incubated for another 24 hours. The SAS cells’ morphology was observed under optical microscope. Cell viability assay (MTT assay), Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western Blot, Immunofluorescence and flow cytometry were used to detect whether melatonin can regulate ANE-induced cell cycle and survival, inflammatory response, and oxidative stress on SAS tongue cancer cells. Results：After 24 hours of exposure of SAS cells to melatonin, we first examined cell morphology. As the melatonin concentration up to 500 µg/ml, some of the cells were floated up and decreased. In addition, we also examined cell regulation related genes such as Cyclin B1, P21, CDC2 and CDC25C. We discovered that that the expression of Cyclin B1, CDC2 and CDC25C decreased with increasing concentrations, while the expression of P21 increased with the concentration. We discovered that melatonin also regulated inflammatory and ROS related genes, such as COX2, ICAM1, Nrf2, and NQO1. Moreover, melatonin also affects the expression of angiogenesis-related genes in cancer such as TF. When the ANE (800 µg/ml) was added, we discovered that ANE-induced the decreased expression of Cyclin B1 was slightly increased by the melatonin 100 µg/ml while ANE-induced expression of P21, CDC2 and CDC25C, melatonin seemed have no effect. We also examined inflammatory and immune response related gene and discovered that melatonin attenuated all of their ANE-induced expression. Moreover, we discovered melatonin did not regulate ANE-induced decreased expression of Nrf2, NQO1 and HO1. Finally, we examined the ANE effects of melatonin receptor such as MT1, MT2, NQO2 (MT3) and RoRα. Conclusions：When melatonin reached a certain concentration, it affected the expression of cell related genes on SAS cell. Melatonin regulated the cell cycle of SAS cells by decreased expression of Cyclin B1 and the increased expression of P21. Moreover, melatonin also affected inflammation and anti-oxidation such as COX2, Nrf2, NQO1, and so on. When ANE are added, melatonin could attenuate the ANE-induced expression of COX2, ICAM1, MMP9, IL6 and IL8. However, preliminary results could reveal some of the effects of melatonin in oral cancer. If the melatonin could be combined with other treatment such as surgery, it could further reduce the inflammation and cancer hyperplasia caused by oral cancer, for the clinical application and treatment of oral cancer will be more helpful. Key word：SAS tongue cancer cell, Areca Nut Extract, melatonin, cell cycle, inflammatory response, cytokines.