| ObjectsAngiogenesis, the formation of new blood vessels from preexisting vessels, has been demonstrated to be essential for tumor growth and metastasis. Tumor cells can secrete kinds of angiogenic factors which can disrupt the balance between angiogenic factors and inhibitors of vascularization which has existed in normal and this leads to the concentration of angiogenic factors increasing and/or that of inhibitors declining and stimulates the proliferation, division and capillarogenesis of endothelial cells. Angiogenesis can increase the blood flow, accelerate tumor progression, inhibit the apoptosis of tumor cells and make tumor cells apt to progress. The inhibition of tumor angiogenesis may directly obstruct the nutrients and angiogenic factors emerging as a promising strategy for treating tumors.TNP - 470 , an analog of fumagillin, is a specific angiogenesis inhibitor. It has been undergoing in vivo and clinical trials for treating a variety of cancers. However, the molecular mechanisn of TNP -470 has not been completely elucidated. At present, many reports have explained the mechanism of TNP - 470 from multiple levels and the explanation of the effect of TNP - 470 is mainly focused on the inhibition of tumor angiogenesis. In this experiment, we discuss the influence of TNP - 470 on the expression of proliferating cell nuclur antigen (PCNA) , vascular endothelial growth factor receptor -2 (VEG-FR - 2/Flk -1) and fibroblast growth factor receptor ( FGFR) and on the cell cycle of cultured human umbilicial vascular endothelial cell (HUVEC) induced by tumor cells (AGZY82A). Moreover, we detect the combinative efficacy of TNP -470 and IL -2.MethodsHUVECs were cultured in 24 or 6 - well plates by modified Jaffe method and tumor cells were cultured, also. The HUVECs were divided into groups; normal control group (only added normal medium) ; conditional control group (only added conditional medium or crushed medium of tumor cells ) ; TNP - 470 group ( respectively the concentration is divided into 10-12,10-10,10-8,10-6g/ml); IL - 2 group (1000IU/ml) ; TNP - 470 + IL - 2 group (10-8 g/ml TNP - 470 + 1000IU/ml IL -2). The conditional medium or the crushed medium of tumor cells were made , then added to endothelial cells when they were subconfluent. The expression of PCNA,Flk - 1,FGFR of endothelial cells were tested by immunocytochemical analysis and positive cell -count and image - analysis were used . The influence of TNP -470 on cell cycle of endothelial cells induced by tumor cells was analyzed by FACScan flow cytometer.ResultsCompared with the conditional control group, TNP - 470 inhibited the expression of PCNA of endothelial cells induced by tumor cells dose - dependently at the range of 10-12 ~ 10 -6g/ml ( P < 0.05 ). The expression of PCNA of endothelial cells decreased after TNP -470 or IL - 2 was added. But the efficacy was not stronger than that of TNP -470 or IL - 2 alone when they were combined ( P < 0. 05 ). Com-pared with the conditional control group, after TNP - 470 was added to the subconfluent HUVECs, the percentage of G0/G1 phase of cell cycle of endothelial cells increased and that of S phase decreased; the percentage of S phase decreased with the treatment of IL-2; the percentage of G0/G1 phase increased and that of S phase decreased with the combination of TNP -470 and IL -2 ( P <0.05). TNP -470 inhibited the expression of Flk - 1 and FGFR of HUVECs induced by tumor cells dose - dependently after conditional medium of tumor cells or crushed medium of tumor cells was added, respectively ( P < 0. 05).DiscussionTumor growth and metastasis are angiogenesis - dependent. In vivo, tumor angiogenic factors are secreted by tumor cells in which VEGF and FGFs are the main positive regulating factors. VEGF is a kind of secreting glucoprotein cytokine. It can be secreted by tumor cells and specially stimulate the proliferation of vascular endothelial cells. In cultured tumor cells, VEGF exists in the conditional medium generally. FGFs are a kind of non - secreting growth...
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