Antiangiogenic Effect Of 4-isothiocyanate-2,2,6,6-tetramethyl Piperidinooxyl And Its Mechanisms

Objective:For the growth and metastasis of tumor depending on angiogenesis, inhibition of angiogenesis has been recognized as a promising therapeutic approach for the control of tumor growth and metastasis. The angiogenic process is regulated by various angiogenic and antiangiogenic factors. Reactive oxygen species (ROS) is involved in the signalling pathway trigered by angiogenic factors. Thus, seeking antiangiogenic agents from antioxidants is becoming an important diretion for the present study.4-isothiocyanate-2,2,6,6-tetramethyl piperidinooxyl (4-ISO-Tempo) exhibits potent antioxidant acticity. However, the antiangiogenic effect of 4-ISO-Tempo has yet not been reported. The aim of this study was to investigate the effect of 4-ISO-Tempo on antiangiogenesis in vitro and in vivo and its mechanisms.Methods:SRB assay was performed to test the effect of 4-ISO-Tempo on cell proliferation of SW620, A549 and HMEC-1 cells. The effect of 4-ISO-Tempo on the expression of MMP-2 and MMP-9 in HMEC-1 was assayed by Gelatin Zymography. Transwell chamber was used to test the effect of 4-ISO-Tempo on the migration of HMEC-1. Tube Formation assay was performed to test the effect of 4-ISO-Tempo on tube formation. Western blot was used to investigate the effect of 4-ISO-Tempo on the phosphorylation of VEGFR2 stimulated by VEGF, phosphorylation of Tie2 stimulated by Ang-1 and ERK1/2 activation. The effect of 4-ISO-Tempo on ROS generation in HMEC-1 induced by VEGF was examined using DCFH-DA as fluorescence probe. Chicken chorioallantoic membrane model was performed to test the effect of 4-ISO-Tempo on the angiogenesis in vivo.Results:(1) The IC50 of 4-ISO-Tempo is 58 times on SW620 cells and 27 times on A549 cells than that of 4-OH-Tempo after 72h. The inhibitory effect of 4-ISO-Tempo is stronger than that of 4-OH-Tempo. (2) 4-ISO-Tempo can inhibited the growth of HMEC-1 cells significantly in a dose-dependent manner. The IC50 of 4-ISO-Tempo is 4.668μg/ml after 24h and 1.386μg/ml after 72h. The inhibitory effect is stronger than that on tumor cells. (3) There is obvious inhibitory effect of 4-ISO-Tempo on expression MMP-2 and MMP-9 expression by HMEC-1 cells. 10μg/ml of 4-ISO-Tempo inhibited completely. (5) 4-ISO-Tempo suppressed significantly the migration of HMEC-1. The inhibition rate was 34.8%,44.6% and 66.5% with 0.625,1.25 and 2.5μg/ml 4-ISO-Tempo respectively. (5) The inhibitory effect of 4-ISO-Tempo on tube formation is the most significant among these experiments.2.5μg/ml of 4-ISO-Tempo inhibited completely. The tube formation was inhibited by 4-ISO-Tempo with inhibition rate of 27%,41.2%,76.5%,88.2% at the concentrations of 0.156,0.312,0.625 and 1.25μg/ml. (6) 4-ISO-Tempo inhibited ROS generation stimulated by VEGF. (7) 4-ISO-Tempo inhibited the phosphorylation of VEGFR2 stimulated by VEGF as well as phosphorylation of Tie2 induced by Ang-1 and had little effect on ERK1/2 activation. (8) There is no effect on angiogenesis with CAM model in vivo.Conclusion:4-ISO-Tempo exhibits more potent antitumor and antiangiogenic activity. The antiangenic effect is related to blocking ROS production and further inhibiting VEGFR2 and Tie2 phosphorylation.