Choi Yoon Jung, Kim So Young, Oh Jung Min, Juhnn Yong Sung
Department of Biochemistry and Molecular Biology, Laboratory of Cellular Signaling, Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Korea.
Exp Mol Med. 2009 Aug 31;41(8):592-600. doi: 10.3858/emm.2009.41.8.065.
Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.
刺激性异三聚体GTP结合蛋白（Gs蛋白）响应各种信号刺激cAMP生成，并调节多种细胞现象，如增殖和凋亡。本研究旨在探讨Gs蛋白对γ射线诱导的肺癌细胞凋亡的影响及其分子机制，试图开发一种新策略来提高γ射线放疗的疗效。通过克隆形成试验、PI染色细胞的FACS分析以及对H1299人肺癌细胞中细胞色素C的细胞质转位、半胱天冬酶-3和聚（ADP-核糖）聚合酶（PARP）的裂解进行蛋白质印迹分析评估，Gsα亚基组成型活性突变体（GalphasQL）的表达通过线粒体依赖性途径增强了γ射线诱导的凋亡。GalphasQL在蛋白质和mRNA水平上调了Bak的表达。用PKA抑制剂（H89）、SP600125（JNK抑制剂）和CRE诱饵处理可阻断GalphasQL刺激的Bak报告基因荧光素酶活性。GalphasQL的表达增加了cAMP反应元件结合蛋白（CREB）和AP-1的基础活性以及γ射线诱导的荧光素酶活性，以及CREB和AP-1与Bak启动子的结合。此外，发现Gs激活信号前列腺素E2增强了γ射线诱导的凋亡，而用前列腺素受体拮抗剂处理可消除这种增强作用。这些结果表明，在H1299肺癌细胞中，Gs通过CREB和AP-1上调Bak表达来增强γ射线诱导的凋亡，提示通过调节Gs信号通路可能提高肺癌放疗的疗效。
