穿心蓮內酯標靶口腔癌癌幹細胞之研究

目的：近年來研究已顯示口腔癌癌症幹細胞（oral cancer stem cells , OCSCs）具有高度致腫瘤特性、化療及放療抗藥性、及轉移性。CD44或醛脫氫酶(Aldehyde dehydrogenase, ALDH1)可作為口腔癌癌症幹細胞的確認標記。發展能標靶這些癌症幹細胞的有效治療可能改善目前有關口腔癌相關疾病的治療方式。穿心蓮內酯（Andrographolide, Andro）是由植物穿心蓮萃取出的二帖內酯，本研究的目的是探討穿心蓮內酯對口腔癌癌幹細胞的治療效果及其調控分子機制。方法：我們檢測穿心蓮內酯對人類正常角化細胞（normal human oral keratinocyte, NHOK）原代細胞及具ALDH1+CD44+特性的口腔癌癌幹細胞之細胞毒性效應。經穿心蓮內酯處理之口腔癌癌幹細胞其醛脫氫酶活性以Aldefluor assay分析，並將口腔癌幹細胞處以不同濃度之穿心蓮內酯，評估其自我更新能力、侵襲力、微核糖核酸microRNA-218表現量、體外致癌特性。結果：穿心蓮內酯可抑制ALDH1+CD44+特性的口腔癌癌幹細胞之增生速率，呈現濃度依賴效應，而對人類正常角化細胞的抑制則有限。首先發現穿心蓮內酯顯著地下調口腔癌癌幹細胞之醛脫氫酶活性、球體形成能力、侵襲能力及爬行能力，並呈現濃度依賴性。由微核糖核酸反轉錄聚合酵素鏈鎖反應分析，穿心蓮內酯可顯著地增加microRNA-218的表現，並確認Bmi1在口腔癌幹細胞中為microRNA-218之直接標靶基因。更重要的是，活體裸鼠實驗顯示口服餵食穿心蓮內酯可顯著抑制裸鼠皮下腫瘤生成。結論：穿心蓮內酯藉由上調microRNA-218的表現量而抑制口腔癌幹細胞的腫瘤侵襲性。

Scope: Recent reports have demonstrated that oral cancer stem cells (OCSCs) presented high tumorigenic, chemo-radioresistant, and metastatic properties. CD44 or ALDH1 could be the markers to identify OCSCs. An effective therapeutic approach targeting these OCSCs may help to improve current treatment regimens for oral cancer-related malignancies. The aim of this study was to investigate the chemo-therapeutic effect and regulatory mechanisms of Andrographolide (Andro), a diterpenoid lactone isolated from a plant called Andrographis paniculata, on OCSCs. Methods: We examined the cytotoxic effect of Andro on normal human oral keratinocyte (NHOK) primary cells and ALDH1+CD44+- OCSCs. ALDH1 activity of OCSCs with Andro treatment was assessed by the Aldefluor assay analysis. Self-renewal, migration, invasiveness, miR-218 expression, and in vivo tumorigenicity of OCSCs with different doses of Andro was presented.Results: Andro inhibited the proliferation rate of ALDH1+CD44+-OCSCs in a dose-dependent manner, whereas the inhibition on NHOK cells proliferation was limited. We first observed that the treatment of Andro significantly down-regulated the ALDH1 activity, spheres-forming ability, and migration/invasion properties of OCSCs in a dose dependent manner. Using microRNA real-time RT-PCR analysis, Andro significantly increased expression of tumor suppressive miR-218 and identified Bmi1 as the targets of miR-218 in OCSCs. Importantly, in vivo nude mice model showed that Ando treatment by oral gavage to xenograft tumors reduced tumor growth. Conclusion: From these results, we conclude that the inhibition of tumor aggressiveness in OCSCs by Andro was mediated by up-regulation of miR-218, suggesting that Andro would be a valuable therapeutics clinically in treatment modalities for malignant oral cancer by elimination of properties cancer stem cells properties.