Cyclic GMP/protein kinase G type‐Iα (PKG‐Iα) signaling pathway promotes CREB phosphorylation and maintains higher c‐IAP1, livin, survivin, and Mcl‐1 expression and the inhibition of PKG‐Iα kinase activity synergizes with cisplatin in non‐small cell lung cancer cells

Abstract

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-Iα (PKG-Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG-Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti-apoptotic Bcl-2-related proteins in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocks endogenous NO-induced activation of cGMP/PKG-Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c-IAP1, livin, and survivin. DT-2 (inhibitor of PKG-Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c-IAP1, livin, and survivin expression. Gene knockdown of PKG-Iα expression using small-interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c-IAP1, livin, survivin, and Mcl-1 expression. Inhibition of PKG-Iα kinase activity with DT-2 dramatically enhanced pro-apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT-2 and cisplatin increased apoptosis compared with cisplatin or DT-2 alone, showing a synergistic effect. The data suggest that the PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c-IAP1, livin, survivin, and Mcl-1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin.