Study On The Directed Screening Of Vascular Targeted Active Ingredients Of T.hemsleyanum And Its Influences On Tumor Angiogenesis

Malignant tumors are often growth in an invasively style and were always spread through the blood vessels in long distance.This makes cancer one of the most difficultdiseases to betherapy.At present,anti-angiogenesis was always used as animportant strategyof tumor control and was applid in tumor therapy.Vascular endothelial growth factor(VEGF)is a target factor involved in angiogenesis which paly roles in tumor proliferation and metastasis.Tetrastigma hemsleyanum Diels et Gilg is a effectively anti-cancer medicinein folk,and also has been used in clinical,a large number of studies shows that T.hemsleyanum could control thegrowth of tumor cells,but there were no reports aboutthe interaction betwee T.hemsleyanumand VEGF which was related tothe formation of blood vessels.In this study,we used the affinity column to screen the active factors that could specifically bind to VEGF,and then the specific factors that directly bind to VEGF in T.hemsleyanum were further analyzed by high performance liquid chromatography(HPLC)and mass spectrometry(MS).The cell proliferation was analyzed by MTT assay and crystallographic violet assay in human umbilical vein endothelial cells(HUVEC)after T.hemsleyanum and Procyanidin B1 and catechin treatment;the ability of migration and invasion was analyzed by wound-healing assay and Transwell assay in HUVEC cells after T.hemsleyanum and Procyanidin B1 and catechin treatment;the change of tube formation ability of HUVEC cells was analyzed by tube formation assay after T.hemsleyanum and procyanidin B1 and catechins treatment;the expression of VEGF,(P)AKT,(P)ERK1/2 and Ras protein in HUVEC cells after T.hemsleyanum and procyanidin B1 and catechin treatment were detected by Western blotting,and the effects on MAPK / ERK and PI3 K / AKT pathways were examined;the expression of CD34,CD31 and VEGF in tumor tissue after T.hemsleyanum treatment was detected by immunohistochemistry.The main results were as follows:1.Procyanidin B1 and catechin can be specifically combined with VEGF affinity column,and Procyanidin B1 can be eluted,catechins are tightly bound almost without elution;2.The survival and proliferation of HUVEC cells were not obvious inhibited by T.hemsleyanum and procyanidin B1,while the proliferation of HUVEC cells were significantly inhibited by catechins,and this inhibitory effect was in a dose-dependent manner;3.The capacity of migration and invasion of HUVEC cells were significant inhibited by T.hemsleyanum,procyanidins B1 and catechins;4.HUVEC cells tuber formation treated with 30 ?g/mL T.hemsleyanum and 30 ?mol/L procyanidins B1 and catechins were significantly decreated;5.The ability of inhibiting the formation of tumor blood vessels was effected by inhibiting the MAPK/ERK,PI3K/AKT pathway,which effected by inhibiting the expression of VEGF,(P)AKT,(P)ERK1/2and Phosphorylation processafter T.hemsleyanumand Procyanidin B1 and catechin treatment;6.T.hemsleyanum has a significant inhibitory effect on the angiogenesis of tumor tissue in a dose-dependent manner,the expression of CD34,CD31 and VEGF in the tumor tissue was significantly inhibited.Experiments show that VEGF affinity column can screen the anti-tumor angiogenesis active ingredients of T.hemsleyanum: proanthocyanidins B1 and catechins,these activity factors can inhibit the activity of VEGF to inhibit cells migration,invasion and tubular formation ability,inhibit the expression of MAPK/ERK,PI3K/AKT pathway,thereby inhibiting tumor angiogenesis.The study provides a simple and convenient method for screening natural anti-tumor drugs with anti-angiogenic function.