PKG II inhibits EGF/EGFR-induced migration of gastric cancer cells

Abstract

Background: Our previous research results showed that Type II cGMP dependent protein kinase (PKG II) could block the activation of epidermal growth factor receptor (EGFR) and consequently inhibit the proliferation and the related MAPK/ERK-mediated signal transduction of gastric cancer cell line BGC-823, suggesting that PKG II might inhibit other EGFR-triggered signal transduction pathways and related biological activities of gastric cancer cells. This paper was designed to investigate the potential inhibition of PKG II on EGF/EGFR-induced migration activity and the related signal transduction pathways.

Methodology/principal findings: In gastric cancer cell line AGS, expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA (Ad-PKG II) and treating the cells with cGMP analogue 8-pCPT-cGMP. Phosphorylation of proteins was detected by Western Blotting and active small G protein Ras and Rac1 was measured by "Pull-down" method. Cell migration activity was detected with trans-well equipment. Binding between PKG II and EGFR was detected with Co-IP. The results showed EGF stimulated migration of AGS cell and the effect was related to PLCγ1 and ERK-mediated signal transduction pathways. PKG II inhibited EGF-induced migration activity and blocked EGF-initiated signal transduction of PLCγ1 and MAPK/ERK-mediated pathways through preventing EGF-induced Tyr 992 and Tyr 1068 phosphorylation of EGFR. PKG II bound with EGFR and caused threonine phosphorylation of it.

Conclusion/significance: Our results systemically confirms the inhibition of PKG II on EGF-induced migration and related signal transduction of PLCγ1 and MAPK/ERK-mediated pathways, indicating that PKG II has a fargoing inhibition on EGF/EGFR related signal transduction and biological activities of gastric cancer cells through phosphorylating EGFR and blocking the activation of it.

Figure 1. U73122/U1026/PKG II inhibits EGF-induced Cell migration.

Migration activity of AGS cells was analyzed with transwell system. The cells were infected with Ad-LacZ or Ad-PKG II for 48 h and serum starved o/n. In Ad-LacZ+EGF and Ad-PKGII+EGF groups, EGF (100 ng/ml) was added to the culture medium; In Ad-LacZ+cGMP+EGF and Ad-PKGII+cGMP+EGF groups, cells were treated with 8-pCPT-cGMP for 1 h and then EGF (100 ng/ml) was added to the culture medium. The migration time was 12 h. A: Representative figures of migrated-cells stained by Giemsa (×200); B: The number of migrated cells in each group. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate (*P<0.01, compared to LacZ group; ^&P<0.01, compared to LacZ+ EGF group).

Figure 2. PKG II prevents EGF-induced Tyr 992 phosphorylation of EGFR.

AGS cells were infected with Ad-LacZ or Ad-PKG II for 48 h and serum starved o/n. In Ad-LacZ+EGF and Ad-PKGII+EGF groups, cells were incubated with EGF (100 ng/ml) for 5 min. In Ad-LacZ+cGMP+EGF and Ad-PKGII+cGMP+EGF groups, cells were treated with 8-pCPT-cGMP for 1 h and then with EGF (100 ng/ml) for 5 min. Cells were harvested and lysed as described in material and methods. The cell lysate was subjected to Western blotting with antibody against Tyr 992 phospho-EGFR and EGFR. Total EGFR protein levels were used as loading control. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group and PKG II group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Figure 3. PKG II prevents EGF-induced Tyr 1068 phosphorylation of EGFR.

AGS cells were treated same as described in Figure 2. Western blotting was applied to detect Tyr 1068 phosphorylation of EGFR and densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group and PKG II group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Figure 4. PKG II blocks the phosphorylation of PLCγ1.

AGS cells were grown in 100-mm plates and infected with either Ad-LacZ or Ad-PKG II. Then, the cells were serum-starved o/n and treated differently: in Ad-LacZ group, no drug treatment; in Ad-LacZ+EGF group, the cells were incubated with EGF(100 ng/ml) for 5 min; in Ad-PKG II+ cGMP+EGF group, cells were incubated with 250 µM 8-pCPT-cGMP for 1 h and followed by incubating with EGF (100 ng/ml) for 5 min. Immunoprecipitation with antibody against PLCγ1 was performed to precipitate PLCγ1 and the phosphorylation of precipitated PLCγ1 was analyze by Western blotting with antibody against phospho- PLCγ1(Tyr783). The results shown are representative of three independent experiments.

Figure 5. PKG II suppresses the formation of DAG.

AGS cells were treated same as described in Figure 4. The concentration of DAG in the cell extracts was measured by ELISA. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate [*P<0.05, compared to LacZ group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group].

Figure 6. PKG II inhibits the release of Ca²⁺ from the endoplasmic reticulum (ER).

Either Ad-PKG II-infected or Ad-LacZ-infected cells growing in a 96-well plate were serum-starved for 12 h, loaded with 5 µM of membrane permeable calcium indicator fluo-3/AM for 30 min at 37°C in DMEM. After loading with the fluo-3/AM, cells were washed with PBS solution and suspended in DMEM, and then incubated with 8-pCPT-cGMP (100 µΜ and 250 µΜ) for 30 min, and then stimulated with EGF (100 ng/ml) for 5 min. Fluorescence measurements were performed using an Olympus Fluoview-500 confocal system. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate [*P<0.05, compared to LacZ group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group].

Figure 7. PKG II prevents the activation of PKCα.

AGS cells were treated same as described in Figure 4. Subcellular fractionation into cytosol and membrane fractions was performed by using Membrane and Cytosol Protein Extraction Kit. Western blotting was used to detect PKCα either on the membrane or in the cytosol. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group; ^&P<0.05, compared to LacZ+EGF group).

Figure 8. PKG II blocks the phosphorylation of CAMK Iiα.

AGS cells were treated same as described in Figure 2. Western blotting was applied to detect the phosphorylation of CAMK IIα. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Figure 9. PKG II blocks the phosphorylation of ERK.

AGS cells were treated same as described in Figure 2. Western blotting was applied to detect the phosphorylation of ERK. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Figure 10. PKG II inhibits EGF-induced Ras activation.

The “pull-down” method was used to detect the activated Ras. Cell lysate was prepared and equal amounts of protein were incubated with GST-RBD beads as described in materials and methods. Binding complexes were collected by centrifugation, resolved by SDS-PAGE, transferred onto PVDF membrane and probed with anti-pan Ras antibody. The results shown are representative of three independent experiments.

Figure 11. U73122/U1026/PKG II inhibits EGF-induced activation of RAC1.

The “pull-down” method was used to detect the activated RAC1. Cell lysate was prepared and equal amounts of protein were incubated with GST-PBD beads as described in materials and methods. Binding complexes were collected by centrifugation, resolved by SDS-PAGE, transferred onto PVDF membrane and probed with anti-pan RAC1 antibody. The results shown are representative of three independent experiments.

Figure 12. PKG II binds with EGFR and causes Threonine phosphorylation of it.

A: Results of Co-immunoprecipitation. AGS cells were grown in 100-mm plates and infected with Ad-PKG II. After being serum-starved o/n, treated with 250 µM 8-pCPT-cGMP for 1 h, and incubated with EGF (100 ng/ml) for 5 min, the cells were lysed and the lysate was immunoprecipitated with anti-PKG II antibody or isotype-matched IgG. The precipitates were probed with anti-EGFR antibody. Five percentage of cell lysate was used as a protein input control. The contrary experiment, i.e. immunoprecipitated with anti-EGFR antibody and probed with anti-PKG II antibody, was also performed. B: Results of immunoprecipitation and Western blotting. AGS cells were treated same as A, and the lysate was immune-precipitated with antibody against EGFR to enrich the protein. The precipitates were subjected to Western blotting with pan anti-Threonine phosphorylation antibody. The results shown are representative of three independent experiments.