Exploring The Anti-angiogenesis Effect Of Nitidine Chloride Based On MTOR Signal Pathway

Nitidine chloride(NC)is a kind of benzophenanthridine alkaloid derived from the parts such as the root of Zanthoxylum nitidum(Roxb.)DC.,having excellent anti-tumor effect.Previous studies have indicated that NC can inhibit tumor angiogenesis as well as proliferation and migration of human umbilical vein endothelial cells(HUVECs),down-regulating the expression of vascular endothelial growth factor(VEGF)as well as signal transducer and activator of transcription 3(STAT3).However,the related molecular mechanism has not been clarified yet.Mammalian target of rapamycin(m TOR)signal cascade regulates the stimulation and secretion of VEGF and matrix metalloproteinase(MMP)that induce tumor angiogenesis.The result of our previous enzyme assay has showed that NC can inhibit the phosphorylation of m TOR kinase which would be the potential target of anti-angiogenesis of NC.Therefore,in this thesis we have preliminarily demonstrated that the mechanism of anti-angiogenesis of NC based on m TOR signal pathway,providing experimental evidence for future development of NC as a multitarget antitumor drug.Objective:1.To evaluate the effect of nitidine chloride(NC)on neovascularization in zebrafish larvaes and angiogenesis in human hepatocellular carcinoma SMMC7721 xenografts.2.To study the effect of NC on the angiogenesis induced by human hepatocellular carcinoma cells.3.To explore the regulatory effect of m TOR signaling on the anti-angiogenesis of NC.Methods:1.Transgenic AB zebrafishes(Danio rerio)(Genotype: mp805a)whose vasculature has been labeled by green fluorescent protein(GFP)were used as testing animals to evaluate the inhibitory effect of NC on the development of the subintestinal veins of zebrafish larvaes.2.The xenograft model of human hepatocellular carcinoma SMMC7721 cells was established in nude mices and four groups including Control group,NC group,rapamycin(Rapa)group and inhibitor of STAT3(S3I-201)group were set and the treatment period was 15 days.When the period was ended,the nude mices were sacrificed after anesthetization and tumor tissues were collected.And hematoxylin & eosin(HE)staining was used to assess the the pathological changes of tumor tissue,and transmission electron microscopy(TEM)was used to observe the ultrastructural changes of tumor tissue.Western blotting was employed to determine the effect of NC on the protein levels of VEGF,MMP2,m TOR,p-m TOR,HIF-1?,STAT3,the phosphorylation of STAT3 on its Tyr705 residue(p-STAT3-Tyr705)in tumor tissues.3.Transwells were used to simulate the proliferation and migration of HUVECs induced by human hepatic carcinoma SMMC7721 cells and study theinhibitory effect of NC on tumor angiogenesis stimulated by hepatic carcinoma cells.And cellular immunofluorescence technique was employed to detect the effect of NC on expression of VEGF in SMMC7721 cells.4.Western blotting technique was used to determine the effect of NC on the protein levels of VEGF,MMP2,m TOR,p-m TOR,HIF-1?,STAT3,p-STAT3-Tyr705 and the phosphorylation of STAT3 on its Ser727 residue(p-STAT3-Ser727)in SMMC7721 cells.5.Enzyme assay was carried out to detect the inhibitory effect of NC on the phosphorylation of m TOR kinase.And the Surflex-Dock module of the computer aided drug design(CADD)software SYBYL2.0 was used to study the interaction between ligand NC and receptor m TOR kinase.Results:1.Compared with Control group,the development of the subintestinal veins of zebrafish larvaes in NC group was obviously inhibited,and the difference between the two groups was statistically significant(P <0.001).2.Compared with Control group,the average weight and volume of the solid tumors were significantly decreased(P <0.001).The result of HE staining indicated that the neovascularization and the tabular vascular endothelial cells could be found in the tumor tissue of both Control group and S3I-201 group,without any necrosis.Compared with Control group,a large area of necrosis tissue can be seen in the tumor tissues of NC group and Rapa group.TEM observation showed normal morphology of cells,integrated mitochondrias and homogeneous chromatin in both Control group and S3I-201 group,but mitochondrial injury and nuclear chromatin pyknosis in NC group and Rapa group.3.The results of western blotting indicated that the expression of fourproteins including VEGF,MMP2,p-m TOR and HIF-1? in NC group and Rapa group decreased significantly compared with control group.However,the levels of the above 4 protein in S3I-201 group was significantly increased.Furthermore,expression of p-STAT3-Tyr705 in NC group,Rapa group,S3I-201 group significantly decreased.All these differences above were statistically significant(P <0.001).4.The results of Transwell assay showed that both NC and Rapamycin can inhibit the migration of HUVECs triggered by SMMC7721 cells,compared with Control group.And immunofluorescent assays showed an inhibited expression of VEGF in NC group SMMC7721 cells.5.The results of western blotting of cell assays indicated that the expression of four proteins including VEGF,m TOR,p-m TOR and HIF-1? in NC group and Rapa group decreased significantly compared with Control group(P <0.05).And compared with Control group,expression of p-STAT3-Tyr705 in NC group,Rapa group and S3I-201 group decreased significantly(P <0.05).6.The results of enzyme assay indicated that NC could inhibit the phosphorylation of m TOR.And the result of the molecular docking showed that NC interacted with m TOR by hydrogen bonds and van der Vaals forces.Conclusions:1.NC have inhibitory effect on the angiogenesis in zebrafish larvaes.2.NC can inhibit the growth of human hepatic carcinomas xenograft in nude mices and the angiogenesis.3.NC inhibits the migration of HUVECs induced by SMMC7721 cells by down-regulate the VEGF level of human hepatic SMMC7721 cells.4.The m TOR kinase is the target of the anti-angiogenesis effect of NC,and NC inhibits the angiogenesis through m TOR/ HIF-1? signal pathway.