抗氧化蛋白在嚼食檳榔引發口腔鱗狀細胞癌之研究

口腔鱗狀細胞癌是最常見的口腔癌，嚼食檳榔嚼塊被認為是造成口腔癌的主要原因之一。文獻指出，抗氧化蛋白如金屬硫蛋白 (metallothioneins; MTs)、第一型血基質氧化酵素(heme oxygenase-1; HO-1)及熱休克蛋白70 (heat shock protein 70; HSP70)等，在許多癌症有明顯表現的現象，但是鮮少有研究探討抗氧化蛋白在嚼食檳榔嚼塊所引發之口腔鱗狀細胞癌中所扮演的角色。在本研究中，我們利用免疫組織化學染色法來分析正常口腔上皮組織與嚼食檳榔嚼塊引發之口腔鱗狀細胞癌組織切片，並探討其表現強度與臨床特徵及TNM stage之相關性。另培養口腔上皮細胞株GNM細胞以反轉錄聚合脢連鎖反應(reverse transcription polymerase chain reaction, RT-PCR)及西方點墨法(western blotting)探討檳榔素(arecoline)引發口腔鱗狀細胞癌可能的致病機轉。實驗結果發現在口腔鱗狀細胞癌的組織中MT-1、HO-1及HSP70的表現明顯高於正常的口腔上皮組織(p<0.05)，MT-1及HO-1的表現強度與淋巴結轉移成正相關(p<0.05)，而HSP70表現強度與淋巴結轉移成負相關(p=0.005)。香菸中主要的致癌物benzo[a]pyrene則可以增加arecoline對GNM細胞誘發MT-1及HO-1的表現(p<0.05)。抗氧化劑N-acetyl-L-cysteine則可以抑制MT-1、HO-1及HSP70的表現(p<0.05)。curcumin、PD98059及staurosporine可以抑制arecoline對GNM細胞誘發HSP70蛋白的表現(p<0.05)。由實驗結果顯示檳榔嚼塊誘發MT-1、HO-1及HSP70的表現可能是造成口腔鱗狀細胞癌的致病機轉之一，benzo[a]pyrene對於造成口腔鱗狀細胞癌有加成作用，這可說明臨床上嚼食檳榔且吸菸者會有較多機會得到口腔癌，且檳榔誘發HSP70的表現可能是經由extracellular signal-regulated kinase inhibitor、activating protein-1及protein kinase C的傳導路徑來調控。

Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer. Previous studies have shown that antioxidant proteins such as metallothionein-1 (MT-1), heme oxygenase-1 (HO-1), and heat shock protein 70 (HSP70) were overexpressed in various cancers. Little is known about the role of antioxidant proteins in the pathogenesis of areca quid chewing-associated OSCC. In the present study, we examined MT-1, HO-1, and HSP70 expression in normal oral epithelium specimens from non-areca quid chewers and OSCC specimens from areca quid chewers by immunohistochemistry. To verify whether arecoline could affect the MT-1, HO-1, and HSP70 production by oral epithelial cell line GNM cells, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were used. Furthermore, glutathione precursor N-acetyl-L-cysteine (NAC), tobacco smoke carcinogen benzo[a]pyrene (BaP), extracellular signal-regulated kinase (ERK) inhibitor PD98059, activating protein-1 (AP-1) inhibitor curcumin, and protein kinase C (PKC) inhibitor staurosporine were added to find the possible regulatory mechanism. MT-1, HO-1 and HSP70 expression were higher in OSCC specimens than normal epithelium specimens (p<0.05). NAC was found to reduce MT-1, HO-1, and HSP70 expression induced by arecoline (p<0.05). BaP was found to enhance MT-1 and HO-1 expression induced by arecoline (p<0.05). Curcumin, PD98059, and staurosporine were found to markly inhibit the HSP70 protein expression induced by arecoline (p<0.05). These results suggest that areca quid chewing may activate MT-1, HO-1, and HSP70 expression that may play an important role on pathogensis of OSCC. BaP may act synergistically in the pathogenesis of OSCC in areca quid chewers. HSP70 expression induced by arecoline in GNM cells may be mediated by ERK inhibitor activation, AP-1, and PKC inhibitor signal transduction pathway.