The Study Of The Mechanism On GRP78 Mediated Multiple Signal Pathways Mediated By YQCTF To Inhibit Tumor Angiogenesis

ObjectiveBased on the clinical and experimental study of YQCTF,the molecular mechanism of YQCTF on tumor microvascular angiogenesis and the regulation of GRP78 in vascular neonatal signaling Pathway were further investigated by in vivo and in vivo two-level experiments.Clinical application of tumor angiogenesis to provide theoretical and experimental basis.MethodsIn vitro cell experimental part:Application of Guangzhou University of Traditional Chinese Medicine Cancer Center for the treatment of lung cancer commonly used side YQCTF role in human umbilical vein endothelial cells(HUVEC).(2)To determine the effect of different concentrations of YQCTF on the activity of HUVEC in hypoxic microenvironment.(2)To determine the effect of YQCTF on the expression of HUVEC in hypoxic microenvironment,and to investigate the effect of different concentrations of YQCTF on hypoxia microenvironment.The effect of YQCTF on apoptosis and cell cycle progression of HUVEC cells was observed by flow cytometry(FCM).The effect of YQCTF on apoptosis and cell cycle proliferation of HUVEC cells was observed by flow cytometry(FCM).(3)The effects of YQCTF on the angiogenesis of hyooxia HUVEC cells.(4)The effect of YQCTF on the repair ability and cell migration ability of hypoxia HUVEC cells was observed by Transwell assay.(5)The expression of HIF-1 a,GRP78,VEGF/VEGFR,PI3K/AKT,ERK,FAK and phosphorylation in the angiogenesis of hypoxic HUVEC were detected by QPCR and Western Blot methods.(6)Silencing GRP78 by siRNA interference technique to explore the mechanism of angiogenesis and the mechanism of GRP78 involved in regulation;(7)To observe the translocation of GRP78 in HUVEC under hypoxia,YQCTF and silencing GRP78 by immunofluorescence,and to explore the molecular mechanism of YQCTF involved in the regulation of HUVEC angiogenesis.In vitro animal model:SPF grade BALB/c male nude mice were used to construct lung nude mice model of lung adenocarcinoma A549 cells.After the model was successfully divided into Control grouP and YQCTF grouP,YQCTF grouP was given daily 18.45g/kg.d-1 intragastric administration,Control grouP given equal volume of saline saline.The rats were sacrificed at 3 days after treatment.The tumor volume was observed and the tumor volume was observed..The expression of HIF-1 a,GRP78,VEGF/VEGFR,PI3K/AKT,ERK and FAK were detected by immunohistochemistry(IHC)in the microvessel density(MVD)and angiogenesis of lung cancer.And phosphorylation level.To evaluate the effect of YQCTF on neovascularization of tumor.ResultsIn vitro cell experimental part:(1)the results of CCK8 assay showed that hypoxia induced by chemical agent CoCl2 with time and concentration increased,the inhibition of cell viability,cytotoxicity,selection of cell concentration significantly inhibited the viability and proliferation of subsequent experiments(100uM 48h).When 100 ?M of CoCl2 was used to interfere with 48h,the formation,migration and invasion of the cells were enhanced.(2)CCK8 assay showed that YQCTF could significantly inhibit the activity of hypoxia HUVEC cells,and was dependent on the drug concentration,and HUVEC activity decreased with the increase of YQCTF concentration(P<0.05,P<0.01).(3)The morphological changes of HUVEC cells observed by inverted microscope showed that YQCTF could inhibit the proliferation of HUVEC..(4)The results of flow cytometry(FCM)showed that YQCTF induced cell apoptosis in a dose-dependent manner(P<0· 05,P<0.01);The results of Cell DNA Content showed that YQCTF could significantly increase G0/G1-phase cells,and decrease S-phase and G2/M-phase on cell cycle,and inhibit the proliferation of cells(P<0.05,P<0.01).(5)The results of tube Formation showed that YQCTF remarkablely suppressed the lymphatic vessel formation ability(P<0.05,P<0.01).(6)Wound healing suggested that YQCTF significantly inhibited the migration of the HUVEC cell lines(P<0.05,P<0.01).(7)Transwell assay suggested that YQCTF remarkedly inhibit cell migration and cell invasion of HUVEC in a dose-dependent manner(P<0.05,P<0.01).(8)Western Blot and QPCR showed that YQCTF significantly down-regulated the protein and mRNA expression of HIF-1?,GRP78,VEGF/VEGFR,PI3K/AKT,ERK1/2,FAK in hypoxia HUVEC(P<0.05,P<0.01).These results suggest that YQCTF can improve the microenvironment of hypoxia and inhibit the angiogenesis of hypoxia HUVEC,and the mechanism may be through the regulation of multiple signaling pathways.(9)Immunofluorescence showed that HUVEC expression was elevated in GRP78 under hypoxia.The fluorescence intensity of GRP78 in the cell and the cell membrane decreased after YQCTF intervention.(9)Immunofluorescence showed that GRP expression of HUVEC increased in hyPoxic microenvironment,and GRP78 expression level in cell membrane increased,suggesting that hypoxia could cause cell translocation(P<0.05,P<0.01).(10)siRNA transfected HUVEC silenced GRP78 expression,the results showed that transfection of siRNA GRP78,HUVEC lumen formation,migration and invasive ability was significantly reduced;silent GRP78,angiogenesis related to angiogenesis(P<0.05),after treatment of GRP78,The exPression of HIF-1? and VEGF/VEGFR was down-regulated,and the phosphorylation of AKT,ERK1/2 and FAK decreased,suggesting that GRP78 was involved in the regulation of angiogenesis.GRP78 plays an important role in tumor angiogenesis and growth mechanism.(11)The results of immunofluorescence showed the expression of GRP78 were decreased not only intracellular and membrane when silencing GRP78,suggesting that GRP78 might be an important target signal molecule in angiogenesis.In vivo animal experimental part:(1)The body weight of two groups of lung cancer A549 cells transplanted in nude mice showed no significant inhibition of YQCTF on the growth of nude mice during this experimental Period.YQCTF could inhibit the tumor volume(P<0.05,P<0.01),indicating that YQCTF can inhibit the growth of lung cancer cell transplanted tumor(P<0.01).(2)Immunohistochemical results showed that the microvessel density(MVD)of YQFTF group was significantly decreased(P<0.01),suggesting that YQCTF could inhibit the angiogenesis of tumor cells in vivo.Compared with Contol group,YQCTF group could significantly inhibit the expression of HIF-1 a,GRP78,VEGF/VEGFR,PI3K/AKT,ERK1/2,FAK and AKT,ERK and FAK phosphorylation in transplanted tumor cells(P<0.05,P<0.01),suggesting that YQCTF inhibits the exPression of HIF-1?,GRP78,VEGF/VEGFR,PI3K/AKT,ERK and FAK signaling pathways in vivo.Consistent with the results of in vivo cell experiments.Conclusion1.Successfully constructed hypoxic endothelial cell model and lung cancer cell transplantation tumor animal model.2.In vitro HUVEC can enhance the angiogenesis in hypoxic condition,and YQCTF can decrease HUVEC angiogenesis by inhibiting hyPoxia microenvironment,inhibiting HUVEC Proliferation,lumen formation,cell migration and invasion ability.In vivo YQCTF can significantly inhibit the growth of lung cancer cells,reduce the microvessel density of transplanted tumor,inhibit angiogenesis.3.The mechanism of anti-tumor angiogenesis in YQCTF can be expressed by multi-target and multi-target inhibition of HIF-1 ?,GRP78,VEGF/VEGFR,PI3K/AKT,ERK1/2 and FAK signaling Pathway.4.GRP78 is involved in the regulation of angiogenesis.The expression of GRP78 may be involved in tumor angiogenesis by regulating HIF-1?,VEGF/VEGFR,PI3K/AKT,ERK and FAK signaling pathway.