| Abstract: | 根據行政院衛生福利部公布民國101年國人十大癌症死因中，口腔癌排名第五位，文獻指出，其五年存活率為百分之六十，比起乳癌及大腸癌具有較差的存活比率，因此對於提高口腔癌存活率也一直是重要的研究方向。其中口腔癌的轉移是造成低存活率的主因，癌細胞容易藉由基質金屬蛋白?-2 (matrix metalloproteinases-2, MMP-2) 的分泌來降解細胞外基質，並可以透過MAPK (mitogen activated protein kinase) pathway以及細胞核轉錄因子 (Nuclear Factor - Kappa B, 簡稱NF-kB) 的活化來調控MMP-2的表現。銀合歡萃取物 (Leucaena leucocephala extract,簡稱LLE) 來源為含羞草科銀合歡屬植物，原產於中南美洲，其種子療效具有抗氧化、抗胃炎、抗憂鬱、解毒消炎等功效，並在過去文獻指出，銀合歡具有抗肝癌細胞增生的效果，同時誘導癌細胞走向細胞凋亡。但在銀合歡對於口腔癌的相關研究機制則尚未明確。因此本實驗目的擬探討LLE與口腔癌之間的相關性，進一步觀察LLE是否會對口腔癌細胞的轉移與生長造成影響。本研究採用口腔癌細胞株SCC-9進行實驗，首先利用MTT assay偵測LLE在不同濃度下對細胞存活率的影響(0、10、20、40、80 µg/mL)，結果顯示LLE對SCC-9的細胞存活率並沒有顯著的影響，並可以藉由抑制MMP-2的分泌來降低癌細胞的轉移與侵襲。而利用western blot檢測蛋白的表現方面，結果也如預期顯示隨著LLE濃度增加可以有效抑制MMP-2的蛋白分泌，且MMP-2的內生性抑制劑 (TIMP-2) 也有明顯的上升效果。另外在訊息傳遞路徑部分也發現LLE對於ERK1/2及p38都具有明顯的抑制效果，並且能抑制活化的NF-kB進入細胞核內。進一步我們加入ERK inhibitor (U0126)及p38 inhibitor (SB202190)來進行反證，結果也顯示對於細胞的爬行能力皆具有加乘抑制的效果。在MMP-2 mRNA的表現方面，實驗結果顯示LLE對於MMP-2的mRNA表現沒有顯著影響。另外，微核醣核酸 (microRNA)是新發現ㄧ種?會轉譯成蛋白質的小分子核醣核酸，由於mRNA的表現不受影響，我們進一步利用Real-time PCR偵測microRNA-21，已知對MMP-2的表現具調控能力，隨著LLE濃度增高，microRNA-21的表現也隨之增加。實驗也發現LLE可以透過增加microRNA-21的表現並具有調控SCC-9細胞MMP-2的分泌以及migration的效果。綜合以上結果，銀合歡萃取物在未來或許可以應用在預防口腔癌的轉移或輔助口腔癌的治?。
Oral cancer ranked fifth in a list of the 10 most fatal cancers issued by the Ministry of Health and Welfare. The literature indicates that its 5-year survival rate of 60% is poor, to compared to breast cancer and colorectal cancer; thus, improving the survival rate for oral cancer is a critical research objective. Metastasis of oral cancer is mainly caused by the low survival rate of cancer cells that easily secrete MMP-2, degrading the extracellular matrix, and can occur via an MAPK pathway and nuclear transcription factor (Nuclear Factor-Kappa B, NF-kB) activation, regulating the expression of MMP-2. Leucaena leucocephala extract (LLE), from the mimosa branch Leucaena genus, is native to Central and South America. The seeds exert antioxidant, anti-gastritis, anti-depressant, anti-inflammatory, and detoxification effects. The literature has reported that Leucaena causes antihepatoma cell proliferation and induces apoptosis in cancer cells. However, the mechanism of LLE in oral cancer remains unclear. The purpose of the experiment in the current study was to investigate the correlation between LLE and oral cancer. SCC-9 oral cancer cell lines were used in this study. First, we employed an MTT assay for detecting the various concentrations of LLE (0, 10,20,40,80 µg/mL) to determine cell viability. The results revealed that LLE did not significantly influence the cell viability of SCC-9 and reduced the transfer and invasion of cancer cells by suppressing the secretion of MMP-2. We then used a western blot for detecting protein expression, and the results indicated that increasing concentrations of LLE effectively suppressed the protein expression of MMP-2, and endogenous inhibitors of MMP-2 (TIMP-2) exhibited a clear upward effect. In addition, part of the message transfer path of LLE detected for ERK1/2 and p38 exerted a significant inhibitory effect and can inhibit the activation of NF-kB in the nucleus. Furthermore, we added an ERK inhibitor (U0126) and p38 inhibitor (SB202190) for validation. The results showed that the cells exert a substantial synergistic inhibition effect. In MMP-2 mRNA expression, the experimental results revealed no significant effect on the LLE mRNA expression of MMP-2. In addition, micro ribonucleic acid (microRNA) is not be translated into a protein of small RNA molecules because mRNA expression is not affected. We further use real-time PCR to detect microRNA-21, which is known for MMP-2 expression with a regulatory capacity. The microRNA-21 performance increased as the LLE concentration increased. The experiment revealed that LLE increased the expression of microRNA-21 to modulate SCC-9 cells through the secretion of MMP-2 regulation and migration effects. Based on the aforementioned results, Leucaena leucocephala can be applied in the future to prevent oral cancer metastasis and in secondary oral cancer treatment. |
