Antitumor Activity And Mechanism Of Kinase Inhibitors C0129 And C0198

Cancer is one of the major causes of human death and affects of human health seriously.According to the International Agency for Research on Cancer（IARC）,by2030 cancer patients will total to 22.2 million worldwide.Currently,treatments for cancer include surgery,radiotherapy and chemotherapy,among which chemotherapy has been deemed effective but accompanied with defects including low efficacy,poor prognosis,serious side effects,and drug resistance.The prominent feature of cancer is the out-of-control growth of cancer cells,which destroys normal tissues and organs,making them structurally and functionally damaged,and ultimately affects normal body mechanisms.Recently years,pharmaceutical scientists have been concentrating on the development of novel anti-tumor drugs targeting kinases,receptors,or cytokines which are expressed in the specific phases of cancer progression,in order to improve drug selectivity,safety and tolerability,reduce drug toxicity and ensure drug clinical efficacy.Aurora kinases and vascular endothelial growth factor receptor 2（VEGFR2）are two important anti-tumoral targets.Aurora kinases belong to serine/threonine kinases,which play important roles in centrosome replication,bipolar spindle formation,chromosomal rearrangement and mitotic checkpoints surveillance during mitosis.VEGFR2,an important receptor tyrosine kinase,induces angiogenesis,promotes proliferation and metastasis,and inhibits apoptosis of tumor cells.Yet no Aurora kinase inhibitor has been approved in the clinic,and the narrow anti-tumoral spectrum,drug-resistance and severe side-effects of VEGFR2 receptor kinase inhibitors such as sorafenib,gefitinib,etc.also limit their further application despite their already proved clinical efficiency.In our previous work,we have found that compounds C0129 and C0198 had potent Aurora kinase-inhibitory and tumor-suppressive activities,in this thesis we will further identify their anti-tumoral effects and study mechanism in vitro and in vivo.Compound C0129 is a stable nitroxyl radical-labeled thymidine derivative,and showed potent inhibitory activities toward not only Aurora A,but also VEGFR2kinase,with the IC₅₀ values for Aurora A kinase and VEGFR2 being 0.061 nM and1.469 nM respectively.Compound C0129 effectively inhibited the proliferation of HT-29,HepG2,A549,HeLa and PC-3 tumor cells,with the IC₅₀ values determined were 2.9?0.5μM,3.2?0.7μM,4.5?0.6μM,0.9?0.3μM and 2.8?0.5μM in CCK-8 assay,respectively.Simultaneously,C0129 exhibited less toxicity to normal HRMC cells than that of VX680.Furthermore,Edu assay showed that C0129inhibited the DNA replication of HeLa cells.On one hand,flow cytometry and western blot assays demonstrated that C0129 hindered spindle formation,affected chromosomal stability and regulated the cdc2/cyclinB1 complex activities in HeLa cells,and thus arrested HeLa cells at G2/M phase.On the other hand,C0129 induced HeLa cell apoptosis,and regulated the expression of apoptosis-related factors BAD,Bax,Bcl-2,caspase-3 and caspase-9.Moreover,C0129 regulated PI3K/Akt/mTOR signaling pathway in HeLa cells.In addition,C0129 inhibited the migration and invasion of HeLa cells by regulating the expression of FAK,VE-cadherin,YB-1,p38MAPK,etc.Also C0129 was involved in the regulation of Ras/Raf/MEK/ERK signaling pathway.Pharmacokinetic experiments of C0129 showed that the t_1/2 of C0129 was 4.12 h after oral administration,and the T_max was 4 h.The acute toxicity assay results showed that the LD₅₀ of C0129 was higher than 1000 mg/kg.C0129exhibited significant anti-tumoral activities in HeLa（70%TGI）xenograft models compared with the vehicle-treated control group at the concentration of 50 mg/kg,which was higher than that of VX680（50%TGI）.IHC results demonstrated that C0129 had low toxicities for heart,liver,lung,spleen and kidney.In conclusion,C0129 was a dual Aurora A/VEGFR2 kinase inhibitor,which displayed significant anti-mitotic and anti-angiogenetic effects and satisfying anti-tumoral activities both in vitro and in vivo,and has the potential for further development.C0198 is a phthalazinone Aurora kinase inhibitor with the IC₅₀ values for Aurora A and Aurora B being 118 nM and 80 nM,and effectively inhibited the proliferation of HCT-116,PC-3,HeLa,A549 and LoVo tumor cells,with the IC₅₀ values being0.83?0.2μM,1.75?0.6μM,3.2?0.5μM,2.8?0.5μM and 2.2?0.7μM in CCK-8 assay,respectively.C0198 showed potent anti-proliferative effects in HCT-116 and PC-3 tumor cells,which were achieved by its suppression of cell mitotic G2/M transition,as well as low toxicity to normal HRMC cells.The anti-mitotic effects of C0198 are closely associated with its regulation of Aurora A and Aurora B.On one hand,C0198 decreased the phosphorylation of Aurora A at Thr288 and CDC25B at Ser353,and thus reduced the activities of cdc2/CyclinB1complex,leading to the malfunction of centrosomes and the malformation of spindles.On the other hand,C0198 reduced the phosphorylation of Aurora B at Thr232,which resulted in the damaged centromeric functions,the losses and damages of chromosomes,and the disruption of chromosomal replication and segregation.The combined effects on Aurora A and Aurora B of C0198 arrested HCT-116 and PC-3 cells at G2/M phase,and suppressed tumor cell growth.Pharmacokinetic studies of C0198 showed that the t_1/2 of C0198 was 21 h after oral administration,and the T_max was 1.6 h.The acute toxicity assay results showed that the LD₅₀ of C0198 was higher than 1000 mg/kg.C0198 exhibited significant antit-tumoral activities in HCT116（75%TGI）and PC-3（66%TGI）xenograft models compared with the vehicle-treated control group at the concentration 90mg/kg,which were higher than those of VX680 in HCT116（60%TGI）and PC-3（55%TGI）.IHC results showed C0198 had low toxicities for heart,liver,lung,spleen and kidney.Altogether,C0198 inhibits the activities of Aurora kinases in HCT116 and PC-3 cells,and as a anti-mitotic drug,C0198 suppresses tumor growth both in vivo and in vitro.In summary,this thesis studied the anti-tumoral activities and the related mechanisms of two kinase inhibitors C0129 and C0198,these results suggested that two compounds are potential anticancer agents for further development.