Fla-2 抑制血管內皮生長因子引發之血管新生作用之探討

Abstract

血管新生(angiogenesis) 是一連串複雜的過程從既有的血管衍生出新生的微血管，其可供給腫瘤細胞所需的養分與氧氣，並且利於癌細胞沿著新生的血管爬行轉移至其他部位。於血管新生的進程當中，內皮細胞會進行增生、爬行、分化的作用，最終形成成熟的血管。抗血管新生的藥物可以減少腫瘤內部新生的血管以及限制腫瘤生長的大小，因此可當作一個治療癌症的新方針。我們以MTT assay 篩選了一系列flavonoids 的衍生物對人類臍帶靜脈內皮細胞(HUVECs) 存活率的影響，最後選擇效果較佳的fla-2 來繼續研究探討其抗血管新生作用與相關機轉。 Fla-2 會抑制內皮細胞的增生，且此抑制作用呈現劑量相關性，進一步的研究中發現fla-2 會增加細胞週期中sub-G1 相的分佈，顯示出fla-2 可能是藉由引發內皮細胞凋亡(apoptosis) 來抑制其增生的現象。Fla-2 可依劑量相關性顯著的抑制內皮生長因子(VEGF) 所誘發之內皮細胞移行現象。Fla-2 會抑制內皮細胞經由內皮生長因子所刺激的管狀生成，且於60 μM 即可完全抑制內皮生長因子的作用。內皮生長因子引誘內皮細胞從大鼠主動脈血管環中爬行出來並形成管狀結構的作用也會被fla-2 隨著劑量的增高而完全抑制。另一方面，fla-2 對內皮生長因子及其接受器結合所活化之訊息傳遞分子(Akt, ERK, JNK—MAPK) 也具有劑量相關性的抑制作用。除此之外，內皮細胞因為處於缺氧的環境下所產生的轉錄因子HIF-1α及其上游之訊息傳遞分子Akt 的活化也可被fla-2 所抑制。 總結來說，fla-2 藉由引起內皮細胞凋亡，抑制內皮生長因子所誘發之內皮細胞移行、管狀生成及其相關之訊息傳遞等作用來展現出fla-2 具有良好的抗血管新生能力，未來可依此方向發展成一嶄新的抗血管新生藥物。

Angiogenesis contributes to the progression of tumor and metastasis by forming new capillaries from the pre-existing blood vessels. Endothelial cells play an important role in the processes of angiogenesis including cell proliferation, migration, differentiation and tube formation. Anti-angiogenic agents reduce the tumor vasculature and growth, thereby being a therapeutic strategy for cancer. We investigated the action of fla-2, a synthetic flavonoids derivative compound, on human umbilical vein endothelial cells (HUVECs). Fla-2 inhibited HUVEC viability and proliferation in a concentration-dependent manner as measured by MTT assay and BrdU proliferation assay, respectively. Fla-2 inhibited cell proliferation by inducing cell apoptosis as evidenced by the increased sub-G1 phase examined by PI staining using flow cytometry. VEGF-induced cell migration and tube formation were concentration-dependently inhibited by fla-2. Moreover, fla-2 also inhibited endothelial cells sprouting from aortic ring stimulated by VEGF. Besides, fla-2 blocked the activation of Akt, ERK, JNK—MAPK stimulated by VEGF treatment. It also inhibited HIF-1α expression and Akt phosphorylation of endothelial cells induced by hypoxia in a concentration-dependent manner. In conclusion, fla-2 exhibited anti-angiogenic activity by inducing endothelial cell apoptosis and inhibiting cell migration, tube formation, and signaling involved in VEGF-VEGFR pathway. These results suggest that fla-2 could be a potential compound for developing as a anti-angiogenic agents.