Effect And Molecular Mechanism Of PKGâ…¡ On VEGFR2-mediated Signal Transduction In Gastric Cancer Cells

Objective:To study the effect of Type II cGMP-dependent protein kinase(PKG II) on vascular endothelial growth factor receptor 2(VEGFR2) mediated signal transduction pathways and biological activities in human gastric cancer cells, and to explore its molecular mechanism. Methods:The gastric cancer cell lines HGC-27 and AGS were infected with adenoviral constructs encoding the cDNA of PKG II(Ad-PKG II) to increase the expression of PKG II, treated with 8-pCPT-cGMP to activate PKG II and treated with vascular endothelial growth factor A( VEGF-A) to activate VEGFR2. The following methods were applied in the study: Western Blot assay was used to analyze the expression and phosphorylation of the related proteins; MTT assay, Transwell assay, Annexin V-FITC/PI method and Matrigel tube formation assay were used to measure cells proliferation, migration, apoptosis and angiogenesis respectively; The interaction between VEGFR2 and PKG II was detected by Co-immunoprecipitation; The phosphorylation of serine/threonine(Ser/Thr) on VEGFR2 caused by PKG II was detected by Immunoprecipitation and Western Blotting; The mutation of the threonine 393(Thr393), threonine 439(Thr439) and serine1231(Ser1231) of VEGFR2 amino acid chain into alanine acid were used to elucidate the specific phosphorylating site of PKG II on VEGFR2.Results:1. PKG II inhibited VEGF-A-triggered tyrosine phosphorylation/activation of VEGFR2, and inhibited VEGF-A/VEGFR2 mediated phosphorylation/activation of ERK1/2, MEK, Akt and mTOR in the gastric cancer cells.2. PKG II inhibited VEGF-A induced cell proliferation, migration, angiogenesis; It also reversed anti-apoptosis effect of VEGF-A.3. PKG II bound with VEGFR2 and caused its serine/threonine phosphorylation.4. PKG II blocked VEGF-A-triggered VEGFR2 activation through phosphorylation Thr439 and Ser1231 of VEGFR2. Conclusions:PKG II bound with VEGFR2 and caused its phosphorylation at Thr439 and Ser1231, then blocked VEGF-A induced activation of VEGFR2, inhibited VEGF-A/VEGFR2 initiated singal transductions, and finally inhibited VEGF-A/VEGFR2 induced proliferation and migration of gastric cancer cells. PKG II also inhibited VEGF-A/VEGFR2 induced angiogenesis and reversed anti-apoptosis effect of VEGF-A/VEGFR2. These results elucidate PKG II as a valid anti-cancer factor.