Study On Anti-tumor Angiogenesis Effect And Mechanism Of Tilianin Based On VEGF-A

Malignancy is one of the major causes of human death.The origin of malignant tumors is not angiogenesis,but when tumor volume reaches 2-3mm~3,tumor can't continue to grow and metastasize without enough nutrition provided by new angiogenesis.However,side effects of most anti-angiogenic agents that are already used in clinical and the high price limit their long-term use.Hence,new antiangiogenic drug with relatively affordable prices urgently needed to find.Flavonoids generally have anti-inflammatory,anti-virus,anti-tumor effect.Uygur ethnic medicine Dracocephalum moldavica contains dozens of flavonoids ingredients,which mainly used for the treatment of respiratory diseases,neurasthenia,anxiety and so on.Tilianin(TIL)is the main active ingredient extracted from Dracocephalum moldavica.Researches have founded that tilianin could inhibit the proliferation and migration of rat vascular smooth muscle cells,inhibit TGF-?/Smad signaling pathway,down-regulate EGFR-MEK-Erk and Akt signaling pathway,inhibit cytokine-induced VCAM-1expression.The purpose of this study is to investigate whether tilianin has tumor suppressor and angiogenesis inhibitory effects,and to reveal its characteristics and clarify related mechanism.In the first part of this study,the construction of non-small cell lung cancer tumor-bearing mouse model was carried out on the basis of the pre-test study to investigate the tumor inhibitory effect of different doses of tilianin.The results showed that low-dose tilianin did not significantly inhibit the tumor growth,but high-dose tilianin could significantly inhibit tumor growth in tumor-bearing mice.In the second part of this project,we used CCK-8 kit to carry out in vitro cytotoxicity test of tilianin,A549 cells and HUVEC cells were the target cells.The results showed that10-160?M tilianin inhibited proliferation of HUVECs by 4.07%-55.86%and 8.76%-65.34%at 24h and 48h,respectively,the IC50 values were 140?M and 102.3?M,respectively.HUVECs proliferation was significantly induced by VEGF-A(P<0.01),the inhibitory rates of 10-160?M tilianin on VEGF-A-induced proliferation of HUVECs were4.26%-77.33%at 24h,5.95%-81.91%at 48h,the IC50 values were 105.2?M(24h)and85.5??(48h).However,tilianin could significantly inhibited A549 proliferation at higher concentrations with IC50 of 557?M(24h)and 310.6?M(48h).These results indicated that HUVECs are more sensitive to tilianin,and that the inhibitory effect of tilianin on tumor growth may be related to the inhibition of the proliferation of vascular endothelial cells and the inhibition of angiogenesis.In the third part of this study,we investigated the effect of tilianin on the major process of angiogenesis in vitro through VEGF-A-induced migration model and tubule formation model of vascular endothelial cells;we investigated the effect of tilianin on angiogenesis in vivo through in vivo angiogenesis model induced by VEGF-A.The results showed that 10-40?M tilianin inhibited VEGF-A induced migration of HUVECs by47.21%-87.15%.Tilianin blocked VEGF-A-induced tube formation in vitro in a concentration-dependent manner,10?M tilianin inhibited more than 40%tubule formation,40?M tilianin almost completely inhibited lumen formation,tilianin not only could inhibit the formation of new tubules,but also could destroy the tubules that had been formed.Compared with the negative control group,VEGF-A could induce in vivo angiogenesis,while the tilianin intervention group could significantly reverse this effect:the microvessel density of the matrigel in VEGF-A-induced group is about nine times of the negative control group;the microvessel density in tilianin intervention group decreased about 8times compared with VEGF-A-induced group.Next tumor tissues obtained from the first part were used for immunohistochemistry experiments,the analysis results of immunohistochemical staining showed that CD31 expression was significantly decreased in high-dose tilianin group compared with control group,and the microvessel density in tumor tissue was lower than that in control group(P<0.01).Tunel fluorescence results of three experimental groups showed that the number of apoptotic cells in the high-dose tilianin experimental group was significantly higher than the other two groups.In the fourth part of the subject,we studied the antiangiogenesis mechanism of tilianin by western blotting.The results showed that 40?M tilianin significantly inhibited the VEGF-A-induced phosphorylation of VEGFR2,Akt and Erk1/2 in HUVECs.40?M tilianin could significantly supressed the expression of VEGF-A,p-Akt,HIF-1?in A549cells;40?M tilianin significantly reduced the accumulation of HIF-1?protein in A549cells under hypoxia.The results of this part showed that the antitumor effect of tilianin was related to regulating protein expression of VEGF-A,p-Akt,HIF-1?in A549 and the inhibitory effect on angiogenesis of tilianin was related to the regulation of p-VEGFR2,p-Akt and p-Erk1/2 in HUVECs.