- 學位論文
泡盛酒麴菌發酵代謝物中對口腔鱗狀癌細胞OEC-M1具TRAIL致敏性化合物之探討
TRAIL-sensitizing compounds from the fermentation metabolites of Aspergillus awamori against oral squamous carcinoma cell OEC-M1
摘要
根據我國衛生福利部統計，癌症近年來均蟬聯國人十大死因之首，其中尤以口腔癌發生率攀升迅速且治療困難，找尋安全且有效的治療方法已屬刻不容緩的課題。腫瘤壞死因子相關誘導配體 (tumor necrosis factor- related apoptosis inducing ligand, TRAIL)被證實是較安全且有效的癌症治療藥劑，因其能高選擇性地毒殺癌細胞，然而目前亦出現多種TRAIL抗藥性癌細胞株而成為目前TRAIL應用在癌症臨床治療上的一大阻礙。本研究著眼於從食用微生物菌種發酵物中，尋找能有效提升TRAIL誘導癌細胞凋亡之致敏因子(sensitizer)，特別鎖定天然物分子結構中含α,β共軛不飽和羰鍵(α,β-unsaturated carbonyl group)特定活性部位之TRAIL致敏活性物質，針對食用麴菌(edible Aspergillus)發酵代謝產物中對人類口腔鱗狀癌細胞OEC-M1具備提升TRAIL誘導細胞凋亡之標的活性成分，進行菌種活性代謝物之發酵生產、純化分離、TRAIL致敏效能評估及其活性作用機轉之探討。實驗中以所設計抗菌/拮抗模式做為TRAIL致敏性物質之篩選平台，而從日本沖繩傳統之泡盛酒麴菌(Aspergillus awamori)與 M-SDB培養基之發酵組合代謝物中發現符合我們篩選目的之特定活性成分。針對活性成分所在之菌種發酵濾液經乙酸乙酯酸鹼分層萃取 (acid-base fractionation)可得中性萃取層(NF)，再進行NF對OEC-M1癌細胞在增強TRAIL誘導細胞凋亡之致敏效果評估。結果顯示當NF添加濃度在62.5μg/mL合併TRAIL添加濃度在50ng/mL作用癌細胞時，可明顯降低OEC-M1癌細胞之存活率而達到協助TRAIL抑制OEC-M1癌細胞生長之致敏作用。由於所分離之活性成分源自食用菌種之發酵代謝產物，且合併TRAIL對OEC-M1癌細胞之致敏作用顯著，將來發展成為TRAIL治療口腔癌之致敏組合輔助製劑是可以期待的。
並列摘要
Cancer remains the top ten’s causes of death in Taiwan, especially the incidence of oral cancer rise quickly and is difficult to treat. Accordingly, in search of a novel and effective treatment for the disease is important to current issue. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induces apoptosis in cancer cells while exhibiting little or no toxicity in normal cells. It is considered as a suitable agent for cancer therapeutic. However, development of resistance to TRAIL in some tumor cells has become a major roadblock nowadays. A variety of natural products have been reported to inhibit tumor cell growth and sensitize TRAIL-induced tumor cell apoptosis. After carefully examining the activity-structure correlation of these compounds, we propose that the TRAIL sensitization activity are due to the presence of a reactive α,β-unsaturated carbonyl group in their molecules. Therefore, the main focus of our screening program was set to explore bioactive compounds containing a reactive α,β-unsaturated carbonyl group originally from the fermentation metabolites of edible Aspergillus microbial metabolites will enhance TRAIL-induced apoptosis in oral squamous carcinoma cell OEC-M1. The antibacterial/antagonistic test model against Staphylococcus aureus was used as the screening criteria for detecting the target microbial metabolites. The result showed that the strain of Aspergillus awamori, which was isolated from Okinawa, was able to produce the target bioactive metabolites when combined with M-SDB medium. The bioassay-directed fractionation by the EA extraction to the cultural filtrate of the strain, followed by a series of column and thin layer chromatographic separation, led to the isolation of one main bioactive principle namely AWM-NF02, which existing in the neutral fraction (NF) of the extract. Since the amount of AWM-NF02 obtained was too small to follow, the bioactive crude extract NF, in combination with TRAIL, was thereafter subjected to investigate its cytotoxic activity by using MTT and LDH assay against OEC-M1. When OEC-M1 was treated with NF to a dose range from 3.9 to 250μg/mL together with 50ng/mL of TRAIL, the survival rate of OEC-M1 significantly decreased as the dose of NF increased up to 62.5μg/mL. Overall, our results indicate that NF can show not only cytotoxic activity to OEC-M1 (dose≥125μg/mL) but sensitize the tumor cell line to TRAIL-induced apoptosis (dose≤62.5μg/mL) in a dose dependent manner. Therefore, we can conclude that the combinatory fermentation of Aspergillus awamori with M-SDB can produce the target bioactive metabolite that can act cytotoxic agent solely on OEC-M1 as well as a sensitizing agent for TRAIL-induced apoptosis. These finding can provide a clue that the microbial metabolite being isolated in this study may be a promising candidate for further development as a TRAIL sensitizer in treatment of oral squamous cell carcinoma.