穿山龍抑制口腔癌細胞其轉移和侵襲之機制探討

口腔癌(oral cancer)在台灣之盛行率與致死率與日俱增，為台灣十大癌症之第六名。口腔癌是發生在口腔部位之惡性腫瘤的總稱，可出現在口腔的任何部位，而造成口腔癌的死亡原因主要在於癌症的轉移(metastasis)。穿山龍(Dioscorea nipponnica)是坊間廣為使用的中草藥，具有降血壓及鎮咳功效。有許多文獻指出，穿山龍具有抗氧化、抗過敏、抗發炎及抗腫瘤等功效，對於穿山龍抑制癌細胞轉移與侵襲其詳細機轉仍不清楚。因此，在此實驗中主要探討中草藥對於癌細胞侵襲轉移及其調控路徑。在本實驗中，我們使用 50% 酒精萃取的穿山龍之萃取物處理 HSC-3 細胞株，首先利用 MTT assays 分析方法顯示穿山龍萃取物並不會影響 HSC-3 細胞的存活率，接著我們利用modified Boyden chamber assay發現穿山龍萃取物對於 HSC-3 細胞的移動與侵襲能力有抑制的作用。在 gelatin zymography assay 中也發現到穿山龍萃取物可以抑制 HSC-3 細胞中 MMP-2、MMP-9 的表現。再利用 western blot 發現穿山龍萃取物會抑制蛋白酶 MMP-2 及其內生性抑制劑 TIMP-2 的蛋白表現，也會抑制訊息傳遞蛋白 PI3K 與磷酸化的 ERK、Akt 之表現，利用 Real-time PCR 發現穿山龍萃取物可以抑制 HSC-3 細胞中 MMP-2 及 u-PA mRNA 的表現，最後我們利用 luciferase reporter assay 發現穿山龍萃取物可以抑制 HSC-3 的 MMP-2 啟動子活性進而影響 HSC-3 細胞中 MMP-2 表現。有文獻指出許多不同的訊息傳遞蛋白以及轉錄因子會調控 MMPs 啟動子的活性，進而影響腫瘤的轉移。因此我們利用染色質免疫沉澱法發現穿山龍萃取物可以抑制 c-Jun 、c-Fos 及 CREB-1 等轉錄因子與 DNA 的結合。在本實驗中，我們發現穿山龍萃取物透過PI3K/Akt 及ERK訊息傳遞路徑影響下游轉錄因子 AP-1 及 CREB-1 調控MMP-2啟動子活性使 MMP-2 表現下降，進而抑制口腔癌的轉移。綜合以上結果，穿山龍萃取物或許可以應用在預防口腔癌的轉移或輔助口腔癌的治療上。

關鍵字

穿山龍；口腔癌；轉移；基質金屬蛋白水解酶

並列摘要

In Taiwan, the incidence and mortality of oral cancer have increased gradually. Oral cancer may affect every part of the oral cavity, including the lips, tongue, mouth, and throat and the main mortality cause is metastasis. Dioscorea nipponica was a popular folk medicine with several proven biological benefits including reducing blood pressure and anti-cough activities, together with abilities of oxidation resistance, anti-allergy, anti-inflammation and anti-tumor. However, the detailed effects and mechanisms of this medicine on cancer cell invasion and metastasis were still unclear. First of all, HSC-3 cells were treated with ethanol extracts of Dioscorea nipponica and then subjected to MTT assays. Results suggested that Dioscorea nipponica have no effect on cell viability of HSC-3 cells. The modified Boyden chamber assays revealed that a treatment of Dioscorea nipponica significantly inhibited the cell motility/invasion capacities of HSC-3 cells. Since that tumor metastasis is accompanied with proteolytic degradation of the extracellular matrix and changed cell motility, MMP-2 and MMP-9 activity were also studied via gelatin zymography assay to show that they all were inhibited by Dioscorea nipponica. Furthermore, results from western blot showed that Dioscorea nipponica extract may suppress the protein expression of proteinase MMP-2 and their proteinase inhibitor TIMP-2. Dioscorea nipponica extract also inhibited the signal transduction protein expression of PI3K and phosphorylation of ERK and Akt while the expression of nuclear protein CREB-1, c-Jun and c-Fos were inhibited as well. The CHIP assay also revealed that the DNA binding activity with AP-1 and CREB-1 was also decreased by Dioscorea nipponica extract. In conclusion, Dioscorea nipponica may be a powerful candidate for a preventive agent against oral cancer development and metastasis.

並列關鍵字

Dioscorea nipponnica ； oral cancer ； metastasis ； MMP

參考文獻

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穿山龍抑制口腔癌細胞其轉移和侵襲之機制探討

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