紅麴山藥酒精萃取物化學防護效應對口腔癌預防與治療效果之研究

Abstract

紅麴發酵產物廣泛應用於亞洲，提供數種生理功效。本研究探究紅麴山藥酒精萃取物之化學防護效應對口腔癌預防與治療效果，以人類口腔癌細胞株為細胞模式，探討紅麴山藥酒精萃取物誘導癌細胞凋亡之效果及其分子機制；並透過倉鼠口腔頰囊癌變為動物模式，評估紅麴山藥酒精萃取物於口腔癌預防及治療之成效，並分析其作用機制。在細胞模式中，評估紅麴山藥酒精萃取物對於人類口腔癌細胞之細胞增殖、細胞週期及細胞凋亡之影響，並分析其分子機制。實驗結果顯示，紅麴山藥酒精萃取物對於口腔癌細胞較紅麴米酒精萃取物具有更強之細胞毒性，而紅麴山藥酒精萃取物對 SCC-25 細胞株具有最佳之毒殺能力 (IC50: 78.1 μg/mL)。在細胞週期分析中，紅麴山藥酒精萃取物促使 SCC-25 細胞停滯在 G2/M 期，此乃透過抑制 NF-κB 蛋白進而調控 G2/M 期的 Cyclin B1 及 CDK1 蛋白表現量下降，因而造成細胞週期停滯，此現象顯示紅麴山藥酒精萃取物對口腔癌細胞具有抑制活性。經由 Annexin V-FITC 雙染試驗發現，紅麴山藥酒精萃取物能促使口腔癌細胞凋亡，乃更進一步探討其作用機制。結果顯示，紅麴山藥酒精萃取物顯著地透過粒線體路徑來提升促凋亡蛋白 Bax 之表現量，因而活化了 caspase-9 酵素並進而活化 caspase-3 活性。此外，紅麴山藥酒精萃取物亦能增加 caspase-8 之活性，顯示死亡受體路徑亦參與紅麴山藥酒精萃取物所調控之口腔癌細胞進行細胞凋亡。在治療組動物模式中，研究目的乃在探討紅麴山藥酒精萃取物對於二甲基苯蔥 (7,12-dimethyl-1,2-benz[a]anthracene, DMBA) 誘導之倉鼠頰囊癌化之治療效果。以 0.5% DMBA，每週 3 次、連續 14 週塗抹黃金敘利亞倉鼠之頰囊，以誘導口腔鱗狀細胞瘤 (oral squamous cell carcinoma, OSCC) 生成。於第 9 週時以相同頻率加入塗抹抗發炎藥物 celecoxib 及 50、100、200 mg/kg 紅麴山藥酒精萃取物及 200 mg/kg 山藥酒精萃取物，並於 14 週後犧牲動物。結果顯示，DMBA 造成 nitric oxide (NO)、reactive oxygen species (ROS)、prostaglandin E2 (PGE2)、transforming growth factor-β (TGF-β) 之過度表現，而紅麴山藥酒精萃取物則減緩了這些因子的增加。此外，紅麴山藥提升血清中 tumor necrosis factor-α (TNF-α) 及interleukin-1β (IL-1β) 之含量，因而刺激了 caspase-8 及 caspase-3 之活性，顯示紅麴山藥酒精萃取物延緩 DMBA 所造成之氧化傷害並誘導口腔癌細胞凋亡，因而達到對 OSCC 之治療成效。以預防組動物模式探討紅麴山藥酒精萃取物於 DMBA 誘導之倉鼠頰囊癌化之預防作用，並評估其抗發炎及抗氧化能力。將 DMBA 每週 3 次，連續 14 週塗抹於倉鼠頰囊袋，並於每次塗抹 DMBA 的隔天進行抗發炎藥物 celecoxib 及 50、100、200 mg/kg 紅麴山藥酒精萃取物及 200 mg/kg 山藥酒精萃取物之塗抹，14 週後犧牲動物。結果證實，紅麴山藥酒精萃取物藉由提升抗氧化酵素之活性，抑制 ROS、NO、PGE2 以及促發炎細胞激素因 DMBA 誘導而造成之過度表現，進而延緩腫瘤形成，顯示紅麴山藥酒精萃取物展現良好之抗發炎及抗氧化活性而達預防口腔癌之效果。綜合以上結果得知，紅麴山藥代謝產物之化學防護功效在抵抗 OSCC 上具有相當良好之抑制能力，具潛力開發為預防口腔癌之功能性食品或於口腔癌之治療上作為輔助治療劑。

Monascus-fermented products offer valuable therapeutic benefits and have been extensively used in East Asia. The aim of this study was to investigate the chemopreventive effect of ethanol extract of red mold dioscorea (RMDE) on oral cancer prevention and therapy. In the cell model, we used the human oral cancer cell line to realize the effect of RMDE on apoptosis and the molecular mechanism. In the animal model, we evaluated the effect of RMDE on DMBA-induced hamster buccal pouch carcinogenesis to understand the preventive and therapeutic effect of RMDE against oral cancer. In the cell model, RMDE was selected for investigating the effects on cell proliferation, cell cycle and the mechanism of apoptosis in human oral cancer cells. The results showed that the growth inhibitory effects of RMDE was maximally decreased on the SCC-25 cells (IC50: 78.1 μg/mL), and RMDE displayed greater cytotoxicity than ethanol extract of red mold rice (RMRE). In the cell cycle analysis, RMDE-mediated G2/M phase arrest was associated with down-regulation of NF-κBto inhibit Cyclin B1 and CDK1 expression, suggesting that RMDE apparently displayed the inhibitory activity on cancer cells. Furthermore, RMDE showed the activity of pro-apoptosis in the result of Annexin V-FITC double staining assay. To further investigate the apoptotic mechanism, RMDE effectively affected the expression of Bax in the mitochondria to activate caspase-9 and caspase-3, and subsequently triggering the mitochondrial apoptotic pathway. In addition, caspase-8 activity was been promoted when treating RMDE, indicating that death receptor pathway was also involved in RMDE-mediated SCC-25 cells apoptosis. The aim of treating animal model is to investigate the anti-tumor ability of the RMDE on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. We induced oral squamous cell carcinoma (OSCC) in the buccal pouch of male Syrian golden hamsters by painting with 0.5% DMBA three times a week for 14 weeks. After 8 weeks, celecoxib and a dose of 50, 100, and 200 mg RMDE per kg body weight were fed in the hamsters for 6 weeks, and extract of dioscorea (DE) group provided 200 mg per kg body weight. The results demonstrated that RMDE decreased nitric oxide (NO), reactive oxygen species (ROS), prostaglandin E2 (PGE2) and transforming growth factor-β (TGF-β) overexpression in hamster buccal pouches in the DMBA treatment group and increased serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) to significantly stimulate caspase-8 and caspase-3 activities, indicating that RMDE reduced oxidative damage causing by DMBA and induced apoptosis in oral cancer cells. The prevention animal model investigated the prevention of oral tumor formation, anti-inflammatory and antioxidative ability of RMDE on DMBA-induced HBP carcinogenesis. The HBP was painted with DMBA for 3-time/week for 14 consecutive weeks, and animals were painted with celecoxib, RMDE (50, 100 and 200 mg/kg bw) and DE (200 mg/kg bw) on days alternate to the DMBA application. The results demonstrated that RMDE attenuated tumor formation by elevating the antioxidase activity and suppressing the overproduction in ROS, NO, PGE2 and proinflammatory cytokines in the HBP caused by DMBA induction. These results indicated that RMDE exerted anti-inflammatory and anti-oxidative activity to prevent oral cancer. To integrate above results obtained a conclusion that the metabolite from Monascus-fermentated dioscorea has chemopreventive effect to against OSCC and may serve as a possible functional food for the prevention of oral cancer or adjuvant agent for oral cancer therapy.