Studies On Anti-angiogenesis Activity And Its Mechanism Of MT189,a New Microtubulin Inhibitor

Angiogenesis is essential to tumor progression.Accordingly,angiogenesis inhibition has become an attractive target for suppressing tumor growth,and angiogenesis inhibitors have been utilized clinically in cancer therapy.Tubulin inhibitors are widely used in the clinic for cancer therapy,and have potential angiogenesis inhibition.Our laboratory has elucidated that MT series compound MT189 is a new microtubulin inhibitor targeting colchicine sites.Previous studies have shown that MT189 has a broad spectrum of potent inhibition on tumor cell proliferation and has equivalent potency on multidrug resistant cells.MT189 activates the JNK pathway,down-regulates MCL-1 protein levels,induces apoptosis that resulting in anti-tumor effect.Based on these research results,we further studied the role of MT189 in inhibiting angiogenesis and the mechanism behind this effect.In this study,we used in vitro,ex vivo and in vivo models to systematically investigate the effect of MT189,confirming the inhibitory effect of MT189 on neovascularization.In vitro proliferation inhibition experiments showed that MT189 can effectively inhibit the proliferation of vascular endothelial cells in a time-and concentration-dependent manner.Under the condition of no obvious cytotoxicity,transwell and tube formation data indicated MT189 exhibited inhibitory effect on the migration and tube formation of vascular endothelial cells,and the effects were not attributed to the inhibitory effect on proliferation.We utilized both rat aortic ring sprouting and CAM neovascularization models to further test anti-angiogenic effect of MT189 in the whole process of angiogenesis.MT189 inhibited microvessel sprouting of the rat aortic ring in a concentration-dependent manner.Similarly,MT189 reduced CAM(chorioallantoic membrane)neovascularization in a dose-dependent manner.Further studies have showed that the anti-angiogenic effect of MT189 is associated with VEGF and its receptor VEGFR2.The stimulation of VEGF can promote endothelial cell proliferation,migration,and tube formation.However,MT189 reversed these effects induced by VEGF in a concentration-dependent manner.In tumor cells,MT189 prevented hypoxia-induced increases in VEGF transcription and secretion in a concentration-dependent manner.Additionally,MT189 lowered levels of the HIF-1? protein,a master transcription factor that promotes the transcription of Vegf.At normoxic conditions,MT189 also significantly reduced transcription and secretion of the VEGF protein in tumor cells.In addition,endothelial cells can also produce VEGF by autocrine,and MT189 also inhibited the transcription and secretion of VEGF in the endothelial cells.VEGF binds to and activates VEGFR2 by phosphorylation,subsequently activating its downstream Src kinase.In the current study,MT189 decreased the phosphorylation levels of VEGFR2,and Src kinase,also in a concentration-dependent manner.A variety of Src-targeted molecules are involved in the regulation of adhesion junctions.Therefore,our data suggest that MT189 might affect cell adhesion.MT189 can change the distribution of proteins involved in cell adhesion and affect cell adhesion.For the cell-cell adhesion,we found that MT189 only increased the amount of ?-catenin protein in a concentration-and time-dependent manner,while the protein levels of VE-cadherin,Vinculin,and the F-Actin remained unchanged.Further investigation showed that treatment with MT189 led to apparent rearrangement of F-Actin,stress fibers increased with increased concentrations of MT189.Vinculin was shown to disperse throughout the whole cell,and treatment with MT189 changed its distribution by moving it to the membrane of cells in a concentration-dependent fashion.In addition,MT189 increased ?-Catenin expression was primarily located at the cell membranes,where the co-localization of ?-Catenin and F-Actin was also enhanced in a concentration-dependent manner.Moreover,MT189 significantly enhanced the co-localization of VE-Cadherin and Vinculin,VE-Cadherin and ?-Catenin in a concentration-dependent manner.For the cell-matrix adhesion,due to the reduction of Src phosphorylation induced by MT189,we further found that MT189 decreased the phosphorylation levels of both FAK and Paxillin.Further,MT189 changed the distribution of Paxillin by driving the protein to the cell membrane and reduced its co-localization with FAK.In contrast,MT189 increased the co-localization of Paxillin and Vinculin.These results suggest that MT189 may enhance cell-cell junctions by promoting linkage of cell adhesion proteins and F-actin,inhibit the turnover of focal adhesion by reducing the phosphorylation of both FAK and Paxillin,and promote the binding of Paxillin to Vinculin,consequently suppressing ECs migration.Subsequent studies indicate that MT189-induced activation of JNK pathway is associated with angiogenesis inhibitory effects.Here,we further found that the JNK inhibitor SP600125 partially reversed MT189-mediated inhibition of endothelial cell migration,and MT189-mediated decreases in VEGF secretion.Moreover,the JNK inhibitor decreased MT189-induced phosphorylation of JNK and c-Jun,and partially reversed the downregulation of Src and FAK phosphorylation.Under hypoxic conditions,MT189 also increased the phosphorylation of JNK and c-Jun,decreased the phosphorylation of Src and FAK.From the data,MT189 can also activate P38,and SP600125 has also been reported to inhibit CDK1.Therefore,we added another JNK inhibitor(JNK-IN-8),the P38 inhibitor SB203580,and the CDK1 inhibitor RO3306 in subsequent experiments.The results showed that only two JNK inhibitors reversed the inhibitory effects of MT189 on tube formation in endothelial cells,VEGF expression in two cancer cells,and MT189-triggered increase in the phosphorylation of JNK and c-Jun in these cells.These results show that MT189 inhibits angiogenesis by selectively activating JNK pathway.In summary,this study demonstrates that MT189 suppresses the proliferation,migration,tube formation of ECs,and the whole process of angiogenesis.For the mechanism,MT189 causes JNK activation and subsequent inhibition of the VEGF signaling pathway that results in the inhibition of endothelial cell proliferation,migration,tube formation,and these effects can be translated into the final suppression of angiogenesis.Importantly,the inhibition of VEGF and its downstream Src Kinase leads to F-Actin rearrangement,strengthening cell and cell junctions,and inhibiting focal adhesion turnover,and then,these actions result in MT189-induced suppression of ECs migration contributing to the inhibitory effect of MT189 on angiogenesis.These new findings confirm and elucidate the inhibitory effects and unique mechanisms of the new microtubulin inhibitor MT189,and provide a basis for novel cancer drug development studies utilizing MT189.