Ⅱ型cGMP依赖性蛋白激酶抑制VEGFR2介导的人胃癌HGC-27细胞增殖

Type ⅡcGMP-dependent protein kinase inhibits proliferation of gastric cancer cell line HGC-27 mediated by vascular endothelial growth factor receptor 2

目的:探讨Ⅱ型c GMP依赖性蛋白激酶(typeⅡc GMP-dependent protein kinase,PKGⅡ)对血管内皮生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR2)介导的人胃癌HGC-27细胞增殖的影响及其作用机制。方法:应用编码PKGⅡc DNA的腺病毒(Ad-PKGⅡ)感染HGC-27细胞,使其高表达PKGⅡ,并以特异性激动剂8-p CPTc GMP激活PKGⅡ。应用血管内皮生长因子-A(vascular endothelial growth factor A,VEGF-A)激活VEGFR2。MTT法检测细胞增殖;蛋白质印迹法检测p-VEGFR2、磷酸化蛋白激酶B(phosphorylated-protein kinase B,p-PKB/p-Akt)和磷酸化细胞外信号调节激酶1/2(phosphorylated-extracellular signal-regulated kinase,p-ERK1/2)表达;免疫共沉淀法检测PKGⅡ与VEGFR2的结合;免疫沉淀法联合蛋白质印迹法检测PKGⅡ对VEGFR2丝氨酸/苏氨酸(Serine/Threonine,Ser/Thr)的磷酸化作用。结果:VEGF-A可促进HGC-27细胞增殖,并诱导胞内p-VEGFR2、p-Akt和p-ERK1/2表达水平明显升高;以Ad-PKGⅡ感染HGC-27细胞使其高表达PKGⅡ并激活后,VEGF-A引起的细胞增殖受到明显抑制,pVEGFR2、p-Akt和p-ERK1/2表达水平显著降低;PKGⅡ可与VEGFR2相互作用,使后者丝氨酸/苏氨酸磷酸化。结论:激活的PKGⅡ可通过使VEGFR2发生丝氨酸/苏氨酸磷酸化抑制人胃癌HGC-27细胞的VEGFR2酪氨酸磷酸化,进而抑制其下游的增殖相关信号转导,最终抑制该细胞的增殖。

Objective： To investigate the influence of type Ⅱc CMP-dependent protein kinase（PKG Ⅱ ） on proliferation mediated by vascular endothelial growth factor receptor 2 （VEGFR2） and its related mecha- nism in human gastric cancer HGC-27 cell line. Methods： The HGC-27 cell line was transfected with ade- noviral constructs encoding the cDNA of PKG Ⅱ （Ad-PKG Ⅱ ） to increase the expression of PKG Ⅱ , and treated with 8-pCPT-cGMP to activate the PKG Ⅱ and treated with vascular endothelial growth factor A （VEGF-A） to activate VEGFR2. MTT assay was applied to measure the proliferation activity; Western blotting was used to evaluate the phosphorylation of VEGFR2, Akt and ERK1/2; the interaction between VEGFR2 and PKG Ⅱ was detected by co-immunoprecipitation ;the phosphorylation on serine/threonine（ Set/ Thr） of VEGFR2 caused by PKG Ⅱ was detected by immunoprecipitation and Western blotting. Results： VEGF-A treatment caused obvious increase of proliferation and phosphorylation （activation） of VEGFR2, Akt and ERKI/2 in HGC-27 cells. In cells transfected with Ad-PKG Ⅱ to highly express PKG Ⅱ and treated with 8-pCVF-cGMP, the VEGF-A induced increase of proliferation and level of phosphorylation of VEGFR2,Akt and ERK1/2 was significantly inhibited. PKG Ⅱ could bind with VEGFR2 and cause serine/threonine phosphorylation of the receptor. Conclusion： Activated PKG Ⅱ inhibits the tyrosine phosphorylation （ activa- tion） of VEGFR2 through phosphorylating its serine/threonine, subsequently inhibits the proliferation relat- ed signal transduetion, and finally inhibits the proliferation of human gastric cancer cell line HGC-27.