BJ-FA對於人類血管內皮細胞的生長抑制作用及分子機轉

Molecular Mechanisms of BJ-FA-Induced anti-proliferation effect in human vascular endothelial cells

蘇怡帆(Yi-Fan Su)

指導教授 ： 李文森

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2004年)

摘要

本篇研究計畫主要在探討化合物 (BJ-FA) 對人類臍靜脈內皮細胞 （Human umbilical vein endothelial cell，HUVEC）的生長抑制作用及其可能作用的分子機轉。近年來，有關藉由抑制血管增生 (Angiogenesis) 進而抑制腫瘤生長的研究議題，相當熱門。而在相關研究當中顯示，許多抑制血管增生的化合物不僅有抑制腫瘤生長的作用且相對有著較低的副作用，因此尋找有效且容易被人體吸收的化合物用以取代目前臨床對於癌症的治療藥物，似乎是比較妥當的方式。 本研究的發現，BJ-FA能使人類臍靜脈內皮細胞的生長產生抑制作用，且其抑制的效果和BJ-FA的劑量成正相關性。而利用3H-Thymidine incorporation和Flow cytometry的實驗結果，我們發現BJ-FA會抑制血管內皮細胞的DNA 合成，並使細胞週期停滯在G0/G1。此外，利用Western blot 的實驗分析，我們觀察到與細胞週期停滯相關的蛋白p21，在血管內皮細胞的表現可受BJ-FA的刺激而增加。我們進一步利用免疫沉澱法分析，發現BJ-FA會使細胞內與CDK2-cyclin complex 結合的p21有增加的現象。Kinase activity assay發現BJ-FA會抑制CDK2的活性。利用微血管生成實驗，我們發現 BJ-FA會抑制內皮細胞微小血管的生成。因此初步推論BJ-FA會干擾內皮細胞的細胞週期，進而達到抑制內皮細胞生長的目的。其作用可能是透過增加抑制細胞週期進行的調控蛋白所引起。因此，BJ-FA或許具有潛力能成為抗血管增生的藥物。

關鍵字

人類血管內皮細胞

並列摘要

The main purpose of this study is to investigate the antiproliferation effect of the compound, BJ-FA, on human umbilical vein endothelial cell (HUVEC), and its underlying molecular mechanisms. In these years, the topics about anti-angiogenesis have become an attractive theme for cancer therapy. It has been also demonstrated that lots of some chemical compounds not only have the utility of cancer therapy, but also can reduce the side effects of currently used drugs. Thus, to search for a succedaneum much more effective and easily absorbed by human-body seems to be an appropriate approach to substitute the presently used clinical remedy against cancer. Here we report that BJ-FA induced a dose-dependent inhibition of HUVEC growth. [3H]-thymidine incorporation and flow cytometry analyses demostrated that the cell cycle was arrested in G0/G1 phase and decreased the DNA synthesis. Western blot analysis revealed that the cell cycle inhibitor protein, p21, was significantly increased in the BJ-FA treated HUVEC as compared with the control. Accordingly, we hypothesize that BJ-FA may interrupt the progress of cell cycle of HUVEC and then inhibit the angiogenesis through increasing the levels of p21 protein which might inhibit the CDKs. Immuno-precipitation study showed that the CDK2-cyclin-linked p21 in HUVEC were significantly increased after BJ-FA treatment. Using matrix gel we further demonstrated that BJ-FA exerts the anti-capillary-like tube formation activity. Consequently, BJ-FA might have the potential to be an anti-angiogenesis drug.

並列關鍵字

human vascular endothelial cells

參考文獻

Bergers,G. and Benjamin,L.E. (2003b). Tumorigenesis and the angiogenic switch. Nat. Rev. Cancer 3, 401-410.

Bernatchez,P.N., Soker,S., and Sirois,M.G. (1999). Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent. J. Biol. Chem. 274, 31047-31054.

Budzynska,R. and Zakliczynska,H. (2004). [Methotrexate for treatment of rheumatoid arthritis]. Postepy Hig. Med. Dosw. (Online. ) 58, 108-117.

Bussolati,B., Dunk,C., Grohman,M., Kontos,C.D., Mason,J., and Ahmed,A. (2001). Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. Am. J. Pathol. 159, 993-1008.

Catsoulacos,P. and Camoutsis,C.H. (1979). Benzothiazinoone dioxides and their derivatives. J Heterocycl Chem 16, 1503-1524.