The Structure-activity Relationship Of Nitroxides And Their Reduced Agents In Antiangiogenesis

Objective:The occurrence and development of tumor are related to the angiogenesis which is a complex process regulated by angiogenic and antiangiogenic factors.Vascular endothelial growth factor(VEGF)is one of the most important angiogenic factors which induce angiogenesis.And reactive oxygen species(ROS)is determined to one of the factors inducing the angiogenic effects of VEGF.Therefore,the investigation of novel antiantiogenesis agents from antioxidants is a new way for tumor therapy.As antioxidants,nitroxides has unique antioxidant.In present study,we investigated the antitumor and antiangiogenesis activities and the mechanisms of nitroxides and their reduced agents,and analysis the structure-activity,further investigated the effect of ROS on angiogenesis.Methods:Sulforhodamine B(SRB)assay was used to test the proliferation of A549 cells,SW620 cells and human microvascular endothelial cells(HMEC-1).Transwell chamber migration assay and tube structure formation assay were used in HMEC-1 cells.The expression of MMP-2 of HMEC-1 was assesssed by Gelatin Zymography.Weston blot was used to detect the VEGE-induced activation of VEGFR2(KDR)and ERK1/2.Intracellular ROS level was measured by 2’,7’-dichlorofluorescein diacetate(DCFH-DA)as a fluorescent dye probe.Cell cycle of HMEC-1 was analysed by Flow Cytometer.Results:Our results showed that the IC50 of 4-OH-Tempo of A549cells,SW620 cells and HMEC-1 were lower than that of 4-OH-TempOH.4-O-Tempo inhibited the proliferation of A549,SW620 and HMEC-1 cells while 4-O-TempOH did not affect their proliferation.Meanwhile,4-OH-TempH and 4-O-TempH have no effects on proliferations of A549,SW620 and HMEC-1 cells.4-OH-Tempo and 4-OH-TempOH had the suppression effects on the migration of HMEC-1 cells;so did 4-O-Tempo and 4-O-TempOH.However,4-OH-TempH and 4-O-TempH hardly did not suppress the migration of HMEC-1.Additionally,4-OH-TempOH and 4-O-TempOH markedly inhibited the MMP-2 expression of HMEC-1 cells while 4-OH-Tempo,4-O-Tempo,4-OH-TempH and 4-O-TempH did not have obvious effects on the MMP-2 expression.4-OH-Tempo and 4-OH-TempOH inhibited the tube formation in a concentration-dependent way,so did 4-O-Tempo and 4-O-TempOH.But 4-OH-TempH and 4-O-TempH did not inhibit the tube formation.Moreover,4-OH-Tempo and 4-OH-TempOH reduced the cellular ROS generation in a concentration-dependent way,so did 4-O-Tempo and 4-O-TempOH,while 4-OH-TempH and 4-O-TempH did not affect the ROS generations.4-OH-Tempo and 4-OH-TempOH concentration-dependently inhibited the activation of VEGF-induced VEGFR2 and could entirely inhibit the activation at 400μg/ml,so did 4-O-Tempo and 4-O-TempOH.However,4-OH-TempH and 4-O-TempH did not inhibit the phosphorylation of VEGFR2.4-OH-Tempo,4-OH-TempOH,4-O-Tempo,4-OH-TempH and 4-O-TempH did not influence on the phosphorylation of ERK1/2 induced by VEGF while 4-O-TempOH inhibited the phosphorylation ERK1/2.Furthermore,4-OH-Tempo,4-OH-TempOH,4-O-Tempo and 4-O-TempOH concentration-dependently induced the apoptosis of HMEC-1 cells.Cell cycle studies indicated biphasic effects of 4-OH-Tempo and 4-O-Tempo:on one hand,the proportion of cells in the G1 phase increased and then did not change or reduced;on the other hand,the proportion of cells in the S phase increased and then reduced.4-OH-TempOH and 4-O-TempOH could reduce the proportion of cells in the G1 phase and increase the proportion of cells in the S phase.4-OH-TempH and 4-O-TempH did not have influence on the cell cycles of HMEC-1.Conclusions:1.Nitroxides and their hydroxylamines could obviously inhibit the growth of A549,SW620 cells and HMEC-1;2.Nitroxides and their hydroxylamines play the similar roles in the inhibition of migraition,tube formation and the phosphorylation of VEGFR2 of HMEC-1 induced by VEGF,but the tetramethylpiperidines did not have these effects.The above dose-effect relationships were similar to that in reducing cellular ROS level induced by VEGF.It is indicated that ROS takes part in the whole processes of cell growth and angiogenesis.