蛇莓萃取物抑制人類口腔癌細胞轉移和侵襲之機制探討

Study of the inhibitory mechanisms of Duchesnea indica extracts on metastasis and invasion of human oral cancer cells

賴芝庭(Chih-Ting Lai)

指導教授 ： 楊順發

中山醫學大學/醫學院/醫學研究所/碩士(2019年)

https://doi.org/10.6834/CSMU201900078

摘要

口腔癌在十大惡性腫瘤死因中排名第五，且病患以男性居多。在臨床統計中，以鱗狀細胞癌為主，其好發的部位以頰黏膜最多。造成口腔癌的主因大多是長期慢性的刺激，例如抽菸、喝酒、嚼食檳榔以及病毒的感染。在癌症轉移中，當腫瘤釋放出基質金屬蛋白水解酶 (Matrix metalloproteinases, MMPs) 後，能夠分解細胞外基質 (extracellular matrix, ECM)，接著藉由血液或淋巴系統轉移至其他器官。在過去的文獻指出，具轉移性的鱗狀細胞癌中，MMP-2及MMP-9的活性較高，在癌細胞的轉移過程中扮演了重要的角色。蛇莓 (Duchesnea indica) 屬於薔薇科的植物，目前已知具有抗發炎、抗氧化壓力的能力，以及在某些癌症當中能夠抑制癌細胞的生長，並且有研究發現蛇莓萃取物對於肺癌具有抑制癌細胞轉移的機制，然而蛇莓萃取物對於口腔癌的影響尚未釐清。因此在本篇研究中，將探討蛇莓萃取物在口腔癌中是否有抑制其轉移及侵襲的能力。首先，以細胞毒性實驗觀察到蛇莓萃取物 (0, 10, 20, 40 g/mL) 在口腔癌細胞 (SCC-9, TW2.6) 中並無細胞毒性，接著以wound healing assay、migration assay及invasion assay來觀察，發現蛇莓萃取物確實能抑制口腔癌細胞的爬行和侵襲能力。接著進一步利用gelatin zymography assay觀察口腔癌細胞分泌出的MMP-2表現量，結果顯示MMP-2的分泌量會隨著蛇莓萃取物的增加而顯著地下降，另外在MMP-2 mRNA及蛋白也觀察到有相同抑制效果。Western blot結果也顯示，蛇莓萃取物有效地抑制磷酸化FAK、Src及Raf/MEK/ERK的表現。接著使用ERK上游蛋白MEK抑制劑U0126後，觀察到蛇莓萃取物對於口腔癌細胞爬行能力有協同抑制的現象，且對分泌到細胞外的MMP-2表現也有相同抑制效果。綜合以上結果，蛇莓萃取物在未來或許能夠應用於口腔癌轉移之預防或作為口腔癌之輔助治療。

關鍵字

口腔癌 ； 基質金屬蛋白水解酶蛇莓萃取物

並列摘要

Oral cancer ranked fifth in malignant tumor deaths, and the major of patients are male. In clinical statistics, over 90% of oral cancer patients diagnosed as oral squamous cell carcinoma (OSCC). The main causes of oral cancer are long-term chronic irritation, such as smoking, alcohol consumption, betel nut chewing and viral infections. Numbers of studies indicated that suppress cancer cell metastasis is a key stage in the inhibition of cell progression. Metastasis is the major cause of cancer death and involves a variety of processes including the extracellular matrix (ECM) remodeling and degradation. Some of MMPs, MMP-2 and MMP-9, are involved in degradation of ECM, and found to have high activity in metastatic oral cancer. Recently reports documented that extract of Duchesnea indica (Andr.) Focke (DIE) has anti-inflammatory, antioxidant and anti-metastasis activities in various cancer cells. However, the effects and underlying mechanism of DIE on oral cancer metastasis are still unclear. The present study aimed to examine the anti-metastatic properties of DIE in oral cancer cell lines. The results of wound healing assays, migration assays and invasion assay revealed that DIE significantly reduced oral cancer cells motility, migration, and invasion. Gelatin zymography, real-time PCR, and Western blot analyses showed that DIE reducd MMP-2 enzyme activity as well as the expression of MMP-2 mRNA and protein in a dose-dependent manner. In addition, DIE significantly decreased Focal adhesion kinase (FAK), steroid receptor coactivator (Src), and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 levels. Furthermore, blocking of the ERK1/2 pathway also enhanced the anti-metastatic ability of DIE. In conclusion, DIE possesses the anti‐metastatic activity of oral cancer cells by suppressing MMP‐2 activity via MEK/ERK signaling pathway. Therefore, DIE may be used in the future for the prevention of oral cancer metastasis and in secondary oral cancer treatment.

並列關鍵字

oral cancer ； matrix metalloproteinase ； extracts of Duchesnea indica

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