Antitumor Activity And Mechanism Of TY-011 Against Gastric Cancer By Inhibiting Aurora And VEGFR2 Kinases

Background:In China,the gastric cancer incidence has reached the second place in a variety of malignant tumors.The patients number of gastric cancer has more than one third of the all gastric cancer patients number in the world.The gastric cancer hazards to human life and health seriously.Overexpression of Aurora and VEGFR2 have been reported in the gastric cancer tissue.In clinical,the single target inhibitors of Aurora and VEGFR2 have poor effective.It’s important to find the inhibitor of dual target to Aurora and VEGFR2 kinases.Here we cooperated with medicinal chemistry team,designed and synthesized a series of compound by targeting mitotic kinases（Aurora and VEGFR2）,and identified a novel compound TY-011 targeting Aurora A/B and VEGFR2 with promising antitumor activity in cellular and animal level.Methods:1.HTRF?KinEASE?assay was used to screen the compound that inhibit the activities of Aurora A/B.Then we found the compound named TY-011 has the obvious inhibition for Aurora A and Aurora B.And we detect the effect of TY-011against Aurora A/B and VEGFR2 activities,in a concentration-dependent manner.2.MTT assay was used to test the inhibition for human hepatocellular carcinoma cells,human gastric carcinoma cells and human colon cancer cells growth.3.Gastric cancer cell xenograft mouse models were used to evaluate the anti-tumor activity of TY-011 in vivo study.4.Docking simulation study was performed to predict the binding mode of TY-011with Aurora A and B kinases.5.Immunofluorescence and western blot were used to test protein expression.6.Cell cycle and cell apoptosis was analyzed by flow cytometry.7.TUNEL kit was used to determine the apoptosis of tumor tissues.8.HUVEC（Human Umbilical Vein Endothelial Cell）tube formation assay were performed to determine the anti-angiogenesis ability in cellular.9.Immunohistochemistry analysis was performed to determine the anti-angiogenesis ability in vivo.10.The proteomics was used to detect the expression of protein between control group and TY-011 treatment group in gastric cells.Results:1.TY-011 was identified as a potential Aurora A/B and VEGFR2 inhibitor by HTRF?KinEASE?assay.It effectively inhibited Aurora A/B and VEGFR2activities in vitro,with IC₅₀ values of 102.1±10.1 nM,93.9±33.7 nM and 103.4 nM±6.2nM,respectively.2.MTT assay was proved that TY-011 demonstrated prominent inhibitory effects on proliferation of gastric cancer cells with IC₅₀ values of 1μM level.And it also has good anti-tumor activity in part of human hepatocellular carcinoma cells.3.Meaningfully,orally administration of TY-011 demonstrated superior efficacy against the tumor growth in gastric cancer cell xenograft,with⁹0%inhibition rate and 100%tumor regression at 9 mg/kg dose,and TY-011 did not affect the body weight of mice.4.The molecular docking analysis demonstrated that TY-011 occupied the ATP-binding site of both Aurora A and B kinases.These results indicated that TY-011is a potent and novel pan Aurora A/B kinases inhibitor.5.With the treatment of TY-011,the phosphorylation of Aurora A and Aurora B decreased in a concentration-dependent manner.Moreover,the phosphorylation of Aurora kinases inhibition by TY-011 was visualized by immunofluorescence.6.TY-011 treatment induced an obvious accumulation of cells at G2/M phase and a modest increase of cells with>4 N DNA content,which then underwent apoptosis.Moreover,TY-011-induced polyploidy was further confirmed by microscopic inspection.Compared with control cells,chromatins of cells treated with TY-011 were not segregated properly,therefore leading to formation of polyploidy.7.Tumor tissues were then assayed for apoptosis using a TUNEL kit which labels apoptotic nuclei with a fluorescent maker.The tumor tissues from animal receiving TY-011（9 mg/kg）treatment showed an obvious increase in apoptosis.8.HUVEC tube formation assay were performed to evaluate the effect of TY-011 on tube formation.In DMSO treated group,intact tube meshes was formed.However,these tube meshes were disturbed after exposure to TY-011 for 6 h.TY-011 didn’t affect HUVEC proliferation under this treatment condition indicating that the effect of TY-011 to antagonize the angiogenesis was not resulted from its toxic effect on HUVEC cells.9.We have noticed that tumors in TY-011-treated mice had lower blood vessel density than control group,which was further confirmed by the immunohistochemistry analysis of CD31 marker.10.The proteomics was used to detect the expression of protein in gastric cells.We found that transcriptional intermediary factor（TIF1β）was cuted in TY-011 treament group,which caused by the activity of cleaved caspase-3.Conclusions:In conclusion,the compound TY-011,as dual target kinases inhibitor,has obvious inhibitory activity for Aurora and VEGFR2 kinases.TY-011 has good anti-tumor effective for gastric cancer in vitro and in vivo by inhibiting proliferation of gastric cancer cells and tumor angiogenesis.Above all,TY-011 has good prospect for medicine,and provides strong preclinical support for cancer researches and clinical cancer therapeutic schemes.