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目的:探讨cGMP依赖性蛋白激酶Ⅱ(cGMP-dependent protein kinaseⅡ,PKGⅡ)对人胃癌SGC-7901细胞增殖相关的丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)下游核糖体S6激酶1(ribosomal S6 kinase1,RSK1)和原癌基因c-Jun、c-Fos的作用。方法:用腺病毒Ad-LacZ和Ad-PKGⅡ感染SGC-7901细胞,使细胞高表达PKGⅡ;用特异性PKGⅡ激活剂8-pCPT-cGMP[8-(p-chlorophenylthio)-cyclic GMP]作用于感染了腺病毒的细胞,再用表皮生长因子(epidermal growth factor,EGF)进行刺激。MTT法检测PKGⅡ对EGF刺激引起的SGC-7901细胞增殖的影响,RT-PCR法和蛋白质印迹法分别检测PKGⅡ对EGF引起的SGC-7901细胞中c-Jun、c-FosmRNA和p-RSK1、c-Jun、c-Fos蛋白表达的影响,免疫荧光显微镜下观察p-RSK1在SGC-7901细胞核内的分布情况。结果:8-pCPT-cGMP作用于感染了Ad-PKGⅡ的SGC-7901细胞后,EGF对SGC-7901细胞的增殖促进作用和EGF诱导的c-Jun、c-FosmRNA和蛋白的表达受到抑制,RSK1(Ser380)磷酸化水平明显降低;EGF刺激后,SGC-7901细胞核内大量累积p-RSK1,8-pCPT-cGMP作用后细胞核内p-RSK1减少。结论:激活的PKGⅡ可有效抑制EGF诱导的胃癌细胞的增殖、RSK1Ser380位点的磷酸化、p-RSK1在细胞核内的累积以及原癌基因c-Jun和c-Fos的表达。
Objective: To investigate the effect of cGMP-dependent protein kinase Ⅱ (PKGⅡ) on mitogen-activated protein kinase (MAPK) / extracellular signal-regulated kinase extracellular signal-regulated kinase (ERK) downstream ribosomal S6 kinase1 (RSK1) and proto-oncogene c-Jun, c-Fos. Methods: SGC-7901 cells were infected with adenovirus Ad-LacZ and Ad-PKG Ⅱ, and PKGⅡwas highly expressed in cells. 8-pCPT-cGMP [8- (p- chlorophenylthio) -cyclic GMP] The adenovirus cells were stimulated with epidermal growth factor (EGF). The effect of PKGⅡ on EGF-induced proliferation of SGC-7901 cells was detected by MTT assay. The expression of c-Jun, c-Fos mRNA and p-RSK1, c -Jun, c-Fos protein expression, the distribution of p-RSK1 in SGC-7901 cells was observed by immunofluorescence microscopy. Results: The effect of EGF on the proliferation of SGC-7901 cells and the expression of c-Jun, c-Fos mRNA and protein induced by EGF were inhibited after 8-pCPT-cGMP treatment on SGC-7901 cells infected with Ad-PKGⅡ. (Ser380) phosphorylation was significantly decreased. After EGF stimulation, p-RSK1 decreased in the nucleus of SGC-7901 cells after massive accumulation of p-RSK1,8-pCPT-cGMP. CONCLUSION: The activated PKGⅡ can effectively inhibit the proliferation of EGF-induced gastric cancer cells, the phosphorylation of RSK1Ser380, the accumulation of p-RSK1 in the nucleus and the expression of proto-oncogene c-Jun and c-Fos.