Research On Anti-tumor Effect And Molecular Mechanism Of Novel Vandetanib Derivative In Vitro Under Normoxia And Hypoxia

Objective:The progression of solid tumors is associated with overexpression of epidermal growth factor receptor（EGFR）and vascular endothelial growth factor receptor（VEGFR）.Vandetanib,a dual tyrosine kinase inhibitor of EGFR and VEGFR,has shown significant efficacy in a variety of solid tumors,but hypoxia resistance is an important factor hindering its development.Hypoxia is an important feature of solid tumors.Hypoxia mediated resistance to radiotherapy and chemotherapy by affecting many tumor biological factors,which seriously affects the anti-tumor efficacy.Compound 39 is a new compound synthesized by adding a nitroimidazole group on the basis of vandetanib.In vitro anti-tumor effect and its molecular mechanism were studied under normoxia and hypoxia and its hypoxic targeting was mainly explored,which aim to provide new strategies for clinical solid tumors.Methods:1.In vitro bioactivity evaluation of compound 39（1）The anti-proliferation effect of compound 39 on A549,HT-29,Mc F-7,Hep G2 and He La tumor cells under normoxia and hypoxia was determined by Cell Counting Kit-8（CCK-8）experiment,using vandetanib as positive control.The inhibitory effect of compound 39 on the growth of A549 and HT-29 cells was evaluated by colony formation assay.（2）In vitro kinase activity assay was conducted to explore the in vitro inhibitory activity of compound 39 against EGFR and VEGFR-2.2.Antitumor mechanism of compound 39 under normoxia and hypoxia（1）Annexin VFITC/PI double staining and cell scratches assay were used to evaluate the effects of compound 39 on apoptosis and migration of A549 and HT-29 cells under normoxia and hypoxia.（2）Western Blot was used to determine the effect of compound 39 on the expression of apoptosis-related proteins in A549 and HT-29 cells under normoxia and hypoxia.（3）The effects of compound 39 on intracellular reduced glutathione（GSH）and reactive oxygen species（ROS）levels under normoxia and hypoxia were determined using microplate GSH assay kit and ROS assay kit.（4）The effect of compound 39 on the expression of hypoxia-inducible factor（HIF-1α）was determined by Western Blot.Results:1.In vitro bioactivity evaluation of compound 39（1）Compared with vandetanib,compound 39 significantly inhibited the proliferation of 5 tumor cells and remarkable hypoxia-selectivity was observed in A549 cells(IC₅₀ value was 1.55μM under normoxia and 0.31μM under hypoxia,respectively)and HT-29 cells(IC₅₀ value was12.89μM under normoxia and 3.47μM under hypoxia,respectively).（2）Compound 39 exhibited more potent EGFR inhibitory activity with IC₅₀ value of 9.11n M,which was 2 folds lower than that of vandetanib(IC₅₀=19.76n M)and relatively enhanced inhibitory activity against VEGFR-2 kinase(IC₅₀=16.32n M)than vandetanib(IC₅₀=33.26n M).2.Antitumor mechanism of compound 39 under normoxia and hypoxia（1）Compound 39 induced apoptosis and inhibited cell migration of A549 and HT-29 cells under normoxia and hypoxia,which was stronger than that of vandetanib.（2）Compound 39 down-regulated the expression of Bcl-2 protein,up-regulated the expression of Bax protein,and induced the cleavage of ADP-ribose polymerase PARP.（3）Compound 39 significantly decreased the level of GSH and increased the level of ROS in HT-29 cells in a concentration and time dependent manner under hypoxia condition.（4）Compound 39 significantly inhibited the expression of HIF-1αin A549 and HT-29 cells.Conclusions:Compared with its parent drug vandetanib,compound 39 significantly inhibited the proliferation and migration of A549 and HT-29 cells,and induced apoptosis of A549 and HT-29 cells by changing the expression of Bax/Bcl-2 and PARP.Compound 39 showed significant hypoxia targeting by affecting GSH and ROS levels and inhibiting the expression of HIF-1α.