The Study Of Inhibition Of Tumor Angiogenesis And Inducing Apoptosis Of Colon Cancer Cells By Cinobufagin

ObjectiveColorectal cancer(Colorectal cancer,CRC)is a common digestive system malignant tumor.Distant metastasis is the leading cause of death in patients with colorectal cancer.Tumor metastasis is closely related to angiogenesis.There are few reports about the mechanism of anti-tumor and anti-angiogenesisObjectiveThis Study is to explore the mechanism of cinobufagin on tumor angiogenesis and inhibiting the proliferation of colon cancer cells,providing theoretical basis for the clinical treatment of cinobufagin in treatment of colorectal cancer.Methods1.To observe the effect of the cinobufagin on the angiogenesis by the chick embryo chorioallantoic membrane and the tubular formation experiment2.MTT assay was used to observe cell viability of HUVEC and colon cancer cells SW480 After the treatment of cinobufagin;transwell carbin test was used to observe the impact of cinobufagin on migration of HUVEC and SW480.Hoechst 33342 observed HUVEC and SW480 cell morphology.Hoechst staining was used to detect the morphology of HUVEC and SW480 by cinobufagin.The apoptosis ratio of HUVEC and SW480 was caculated by Flow cytometry.Reactive oxygen species and mitochondrial membrane potential were observed by Flow cytometry.Western blot was used to analysis the expression of protein related to apoptosis and angiogenesis.3.to observe the effect of tumor growth and angiogenesis by orthotopic transplantation tumor model of colon cancer.Results1.Cinobufagin can inhibits chick chorioallantoic membrane neovascularization.Furthermore,the number of vascular has been decreased while concentration of cinobugin raised.The number of primary vascular,secondary vascular,thirdly vascular is less than those in the control group.The tube-formation of HUVEC was obviously inhibited after the treatment of cinobufagin;the number was reduced when HUVECs were treated with cinobufagin.2.Cinobufagin has the ability of inhibition cell viability of human umbilical vein endothelial cells and colon cancer cell SW480.The migration of endothelial cells and SW480 could be inhibited by cinobufagin.After the treatment of cinobufagin,there were obvious nuclear condensation and apoptotic bodies on endothelial cells and colon cancer cells.The ratio of apoptosis of HUVEC and SW480 was increased with the concentration of cinobufagin raised.Cinobufagin decreased mitochondrial membrane potential and increased producttion of ROS in human umbilical vein endothelial cells and SW480.The results of western blot showed that the proportion of Bax/Bcl-2 increased after the treatment of cinobufagin on human umbilical vein endothelial cells and colon cancer cells SW480.In addition,Caspase-3,caspase-9 and PARP were fragmented.The expression of phosphorylated AKT,phosphorylated mTOR,hypoxia inducible factor Hif-1α,MMP-9 were decreased after the treatment with cinobufagin on HUVEC and SW480.3.The volume and weight of tumor were significantly less than those in control(P<0.05).At the same time,the density of microvessel density was significantly lower than that of control group(P<0.05).There was a phenomenon of apoptosis in the tumor cells of cinobufagin group and cisplatin group.The results of Western blot showed that caspase-9,caspase-3 and PARP were fragmented.The phosphorylation of AKT,phosphorylation of mTOR and the expression of Hif-1 were decreased.Conclusions1.Cinobufagin can inhibit angiogenesis and induce the apoptosis of colon cancer cells SW480.2.cinobufagin inhibites angiogenesis by decreasing Hif-1α and phosphorylation of AKT and mTOR3.cinobufagin can enhance the expression of Bax,inducing apoptosis of colon cancer cells.