Effect Of PKG Ⅱ On EGF/EGFR-Induced Signal Transduction And Related Biological Activities In Gastric Cancer Cells

Our previous study showed that expression of type Ⅱ cGMP-dependent protein kinase (PKG Ⅱ) was decreased in gastric cancer cells. Increasing the expression and the activity of PKG Ⅱ could inhibit EGF-induced signal transduction of MAPK/ERK-mediated pathway and proliferation of gastric cancer cells. Furthermore, our research revealed that PKG Ⅱ inhibited EGF-induced activation of EGF receptor (EGFR). On these grounds, we speculate that PKG II may inhibit EGF-induced signal transductions through blocking the activation of EGFR and thereby exhibit a fargoing inhibition on related biological activities.Objective:To elucidate the inhibition of PKG II on EGF/EGFR initiated signal transduction of PLC-IP3/DAG-mediated, PI3K-Akt-mediated, and JAKs-STAT-mediated pathways, and the influence of PKG II on EGF/EGFR induced invasion, migration, apoptosis and cell cycle change of gastric cancer cells.Methods:In GES-1cells,8-pCPT-cGMP was used to activate endogous PKG II. Western blotting and Immunoprecipitation (IP) methods were applied to detect the activity of the main signal transduction components of PLC-IP3/DAG-mediated, PI3K-Akt-mediated, JAKs-STAT-mediated and MAPK-mediated pathways. CVTK kit and Transwell migration assay were used to detect the proliferation and migration of the cells.In gastric cancer cell lines AGS and HGC-27, adenoviral construct encoding PKG II cDNA (Ad-PKG II) was used to infect the cells to icrease the expression of PKG II and8-pCPT-cGMP was used to activate the PKG II. Western blotting, IP, ELISA, enzyme-labeled fluorescent and membrane and cytosol protein extraction method were applied to detect the activity of the main signal transduction components of PLC-IP3/DAG-mediated, PI3K-Akt-mediated, and JAKs-STAT-mediated pathways. Transwell migration assay and transwell invasion assay were applied to measure the migration activity and invasion activity of the cells. TUNEL method was used to detect the apoptosis of the cells. Cell cycle change was observed by flow cytometry. Expression or activity of invasion, migration, apoptosis and cell cycle related proteins was detected by Western blotting or activity assay kit. Serine and threonine phosphorylation of EGFR caused by PKG II was detected by IP and Western blotting. The binding between PKG II and EGFR was detected by Co-IP.Results:In GES-1cells, activated endogenous PKG II inhibits EGF-induced Tyr992, Tyr1173and Tyr1068phosphorylation of EGFR, EGF-induced phosphorylation of PLCy1, PI3K, JAK1and ERK1/2, and EGF-induced proliferation and migration.In AGS and HGC-27cells, PKG II inhibits all of EGF/EGFR initiated signal transduction pathways. For PLCγ1-mediated transduction pathway, PKG II inhibits EGF-induced pathway-related Tyr992phosphorylation of EGFR, and PLCy1phosphorylation, DAG formation, Ca2+releasing, PKCa activation and CaMKIIa phosphorylation; For PI3K/Akt-mediated transduction pathway, PKG II inhibits EGF-induced pathway-related Tyr1173phosphorylation of EGFR, PI3K phosphorylation, Akt phosphorylation, mTOR phosphorylation and NF-kB activation; For JAK/STAT-mediated pathway, PKG II inhibits EGF-induced pathway-related Tyr1068phosphorylation of EGFR, JAK1phosphorylation and STAT1phosphorylation. PKG II also inibits EGF induced invasion and migration of the cells, reverses the anti-apoptic effect of EGF and prevents the cells from entering proliferation cycle induced by EGF. Finally, our results show that PKG II binds with EGFR directlly and causes serine and threonine phosphorylation of EGFR, suggesting that PKG II blocks activation of EGFR through binding with and cuasing phosphorylation of the receptor.Conclusions:In GES-1cells, activated endogenous PKG II can inhibit EGF/EGFR initiated signal transduction of PLC-IP3/DAG-mediated, PI3K-Akt-mediated, JAKs-STAT-mediated and MAPK-mediated pathways, and inhibits EGF-induced proliferation and migration.In gastric cancer cell AGS and HGC-27, activated exogenous PKG II can block the activation of EGFR through phosphorylating EGFR, then inhibits EGFR initiated signal transduction of PLC-IP3/DAG-mediated, PI3K-Akt-mediated and JAKs-STAT-mediated pathways, and ultimately inhibits EGF-induced invasion and migration, reverses anti-apoptotic effect of EGF, and prevents cells from entering proliferation cycle induced by EGF. The research results indicate that PKG Ⅱ has cancer inhibition effect and suggest that PKG Ⅱ is an anti-cancer factor.