骨肉瘤是一種臨床常見的惡性骨腫瘤，約占所有骨腫瘤的百分之二十，好發於活動頻繁的關節部位，且具有很高的轉移能力，而骨肉瘤的肺轉移和肝轉移往往是導致患者死亡的主因。目前已知在癌症的轉移中，癌細胞會藉由產生蛋白?來分解細胞基質，而在這些蛋白?中以基質金屬蛋白? (matrix metalloproteinases; MMPs) 及尿激?型胞漿素原活化因子 (urokinase-type plasminogen activator; uPA) 較為常見。葉下珠也是俗稱的珍珠草，在過去研究中被認為具有抗氧化、抗高血壓及治療肝炎的作用，近幾年則有報告將它應用於癌症的治療上，但有關葉下珠在癌症轉移的研究並不多，因此我們想探討葉下珠與抑制骨癌轉移的相關性。首先我們利用人類骨肉瘤細胞株 Saos-2 處理葉下珠萃取物之後，發現並不會影響癌細胞的生長，而利用傷口癒合實驗則發現葉下珠萃取物能抑制 Saos-2 的移動能力，因此接著利用 modified Boyden chamber assay 也證實葉下珠萃取物確實能抑制癌細胞侵襲的能力。而在 gelatin zymography 與 casein zymography assay 中也發現葉下珠萃取物能抑制 MMP-2 及 u-PA 的活性。再利用 western blot 發現葉下珠萃取物能抑制細胞內 MMP-2 及 u-PA 的蛋白表現並增加內生性抑制劑 PAI-1 的表現，接著進一步利用 real-time PCR 發現 u-PA 的 mRNA 確實有下降的趨勢，但 MMP-2 則沒有顯著的變化。利用 luciferase reporter assay 也證實，葉下珠是透過抑制 u-PA 啟動子的活性來影響 mRNA 的表現。最後我們利用 western blot 去觀察葉下珠萃取物對訊息傳遞路徑的影響，發現葉下珠萃取物能抑制磷酸化 ERK1/2 與 Akt 的表現。總結實驗結果我們發現葉下珠萃取物能藉由抑制 MMP-2 及 u-PA 的表現，來達到抑制骨肉瘤細胞株 Saos-2 侵襲及轉移的能力。因此，葉下珠萃取物可能可以用來防止癌症的轉移，並期望能應用在未來癌症的治療上。
Osteosarcoma is one of the most common malignance of bone with an incidence of 20% in all bone tumor. This type of malignance often occurs in the joints with a high potential for metastasis to lung or liver, while metastasis is the most vital cause of death. From extensive studies for metastasis, it is well known that proteinases like MMPs and u-PA can destroy the ECM and promote cancer metastasis. Recently, Phyllanthus urinaria, used to be known with activities of antioxidants, anti- hypertensive and anti-hepatisis, was further proved to have various anti- carcinogenesis properties. However, the detailed effects and mechanisms of Phyllanthus urinaria on cancer cell invasion and metastasis were still unclear. In this study, the effect of ethanol extract of Phyllanthus urinaria on the metastasis ability of osteosarcoma Saos-2 cells was investigated with MTT assays. While results suggesting that Phyllanthus urinaria have no effect on cell viability, results from wound healing and modified Boyden chamber assays revealed that a treatment of Phyllanthus urinaria significantly inhibited the abilities of migration and invasion. Gelatin zymography and casein zymography assays also showed that MMP-2 and u-PA activities all were reduced by Phyllanthus urinaria. Furthermore, results from western blot showed that Phyllanthus urinaria extract may suppress the protein expression of MMP-2 and u-PA, but increase that of PAI-1, the u-PA inhibitor. Results from real-time PCR showed that Phyllanthus urinaria extract suppress the u-PA expression at the transcriptional level, while results from luciferase reporter assay further confirmed that Phyllanthus urinaria extract suppressed the transcription activity of u-PA promoter. Phosphorylation levels of ERK1/2 and Akt were also reduced in the presence of Phyllanthus urinaria extract. In conclusion, Phyllanthus urinaria may be a powerful candidate for a preventive agent against osteosarcoma invasion and metastasis.
