野薑花化合物誘導頭頸癌細胞株產生細胞凋亡之機制

研究目的：一個理想的抗癌藥物應該是對於惡性癌症細胞具有毒殺性，但是對正常細胞的毒殺性為最小。然而，具有此種特性並可用於臨床使用的試劑仍然有限，因此新穎的癌症治療藥物篩選開發是一個重要的和具有挑戰性的任務。Coronarin D (CD)是提煉自薑花中，又稱為穗花山奈，對於癌症細胞有抑制細胞增生的療效。但Coronarin D 對於5FU抗藥性之口腔癌細胞的療效目前尚未明確。因此我們希望藉由此研究計畫內容的設計與執行，能夠進一步的深入研究Coronarin D (CD)對於誘發5FU抗藥性口腔癌細胞細胞凋亡的影響及探討所影響的相關分子機制。研究方法及資料： 本研究利用薑花素D作用於口腔癌5-FU抗藥性細胞株上，分析其促進凋亡之路徑。分析薑花素D作用在口腔癌抗藥性細胞株上，我們進行了細胞週期分析、細胞毒性試驗、DAPI染色進行細胞凋亡之定量，同時也利用Annexin細胞染色法、粒線體膜電位分析、西方墨點法測定蛋白質表現。研究結果： 研究結果顯示，薑花素D作用於5FU抗藥性口腔癌細胞株上，會顯著地降低口腔癌細胞株的活性。其中. 薑花素D的細胞毒性致使5FU抗藥性口腔癌細胞株引起細胞週期在G2/M期呈現停滯 ，並同時經由內始途徑及外始途徑引起細胞凋亡。其中發現薑花素D在5FU抗藥性口腔癌細胞株上透過磷酸化，藉由加入JNK 抑制劑 SP600125會降低 JNK路徑磷酸化的表現，推測其為細胞凋亡的分子路徑。結論與建議：薑花素D透過影響 JNK 1/2 的途徑進而造成促凋亡蛋白（Bim 和 Bak）表現上升及抗凋亡蛋白（Bcl-2 和 Bcl-xL）表現下降，進而影響 cleaved caspases-3, cleaved caspases-8, cleaved caspases-9 及 cleaved PARP 表現增加後引起凋亡現象。這樣的作用機轉是第一次有研究在5FU人類抗藥性口腔癌細胞上，未來可以運用在5FU人類抗藥性口腔癌細胞的治療上。

Objective：Coronarin D (CD) is one of the main components of Hedychium coronarium rhizome , which has therapeutic potential by reducing cell proliferation in cancer cells. However, the mechanism of CD to 5-FU oral cancer cell remain unclearly. This study discusses the CD to 5FU chemoresistance oral squamous cell carcinoma (OSCC) biochemical mechanisms and possibly pathways to inhibit multiplication in oral cancer.Methods and Materials：We demonstrated the study of CD-treated 5FU-chemoresistance humanoral cancer cell lines were analysis by MTT assay, cell cycle assay, DAPI assay, annexin-V/PI double staining assay and mitochondrial membranepotential measurement. Besides, western blotting was performed to realized effect of CD on the expression levels of apoptosis related proteinand MAPK signaling pathway.Results：The results of the study evidenced that CD reduced viability of 5FU cancer cells in a dose- and time-dependent manner compared with control. The cytotoxic effect of CD lead to cell cycle arrest in the G2/M phase and induced apoptosis in both internal and external pathways. CD induces apoptosis by enhancing phosphorylation of JNK, further exploring the combination of CD and SP600125 reduced the overexpression of phosphate JNK levels.Conclusion and Suggestion：This study results indicated that CD potentially inhibits the proliferation of 5FU-resistant human oral cancer cells by regulating the MAPK pathway, followed by inducing cell cycle arrest and apoptosis. Our study is the first study to report the mechanism of effect of CD in 5FU on oral cancer cells. The study may be potential therapeutic agent for 5FU against human oral cancer cells. The current combination therapy of 5FU and CD can be demonstrate in animal models. In the future, human trials will be conducted to improve drug efficacy in oral cancer and reduce the recurrence rate.