骨肉瘤是骨癌中惡性程度很高的一種，約占百分之二十，其常好發於孩童跟青少年，臨床上一旦從良性轉成惡性腫瘤後，常容易轉移到肺臟跟肝臟，也是癌症病人死亡的主要原因。類黃酮是一種天然多酚類，廣泛存在我們日常食用的蔬菜水果中，近幾年類黃酮被醫界廣泛地研究，目前已證實能抑制乳癌及前列腺癌細胞的增生及誘導其凋亡，而黃酮醇(flavonols)為類黃酮中的一種，3-hydroxyflavone為黃酮醇的結構骨架，因此希望能藉由研究3-hydroxyflavone，來了解其是否能抑制人類骨肉瘤細胞U2OS的侵襲與轉移，並探討機制期望能應用在癌症的臨床治療上。首先，我們使用3-hydroxyflavone處理人類骨肉瘤細胞U2OS，利用傷口癒合試驗確認其明顯能夠抑制細胞的移動，因此我們再利用MTT assay確認3-hydroxyflavone對U2OS並無細胞毒性。接著使用Boyden chamber assay觀察3-hydroxyflavone處理U2OS細胞時期侵襲和轉移的能力被抑制。用Zymography assay看MMP-2的活性也明顯下降。Westernblot的結果發現磷酸化的FAK表現量下降，再以Western blot看其他相關蛋白量的變化，p-Src、p-ERK1/2和vimentin等蛋白明顯被抑制，證實3-hydroxyflavone會透過Src、ERK1/2的路徑來影響細胞的侵襲與轉移。綜合上述結果可發現3-hydroxyflavone確實能抑制細胞的轉移與侵襲能力，在未來或許可以應用在臨床治療與預防骨肉瘤的轉移。
Osteosarcoma is a highly malignant bone tumor and is the most commonly encountered malignant bone tumor in children and adolescents. Osteosarcoma is about 20% in all bone tumor. Osteosarcoma patients often die owing to tumors metastasis to liver and lung. Flavonols(3-hydroxyflavone) is one of flavonoids, and flavonoids has been proposed as a potential drug for cancer chemoprevention and treatment because it is a natural polyphenol contained in plant-based foods. Recent studies have demonstrated that flavonoids protects against breast cancer and prostate cancer. Based on this finding, we investigated the mechanisms by which flavonols produces the anti-metastatic effect in human osteosarcoma cell U2OS. First, 3-hydroxyflavone significantly inhibited migration and invasion of U2OS cells based on results from the wound healing assay. The cytotoxic effect analysis indicated that 3-hydroxyflavone at the range of 0 to 50μM did not alter U2OS cells viability. 3-hydroxyflavone significantly also inhibited migration and invasion of U2OS cell in Boyden chamber invasion and migration assay. The protein levels of MMP-2 were decreased as shown by zymography assay. Then, the effect of 3-hydroxyflavone on cellular morphology-related proteins was slso examined by western blot, the 3-hydroxyflavone can inhibit p-FAK expression. To futher explore the detailed molecular mechanisms, we found the expression of phosph-Src、phospho-ERK1/2、and vimentin were suppressed by 3-hydroxyflavone through a western blot, it demonstrated that 3-hydroxyflavone could inhibit cell invasion、migration and EMT via Src、ERk1/2 pathway. These findings provide new insights into the molecular mechanisms involved in the anti-metastatic effect of 3-hydroxyflavone, and are valuable in the prevention of human osteosarcoma cell U2OS metastasis.
