Nevada Cancer Institute, Las Vegas, Nevada.
Mol Cancer Res. 2010 Apr;8(4):578-91. doi: 10.1158/1541-7786.MCR-09-0178. Epub 2010 Apr 6.
Previously, we showed that basal activity of nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis in human ovarian cancer cells. The present study determines whether basal PKG kinase activity regulates Src family kinase (SFK) activity and proliferation in these cells. PKG-Ialpha was identified as predominant isoform in both OV2008 (cisplatin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. In both cell lines, ODQ (inhibitor of endogenous NO-induced cGMP biosynthesis), DT-2 (highly specific inhibitor of PKG-Ialpha kinase activity), and PKG-Ialpha knockdown (using small interfering RNA) caused concentration-dependent inhibition of DNA synthesis (assessed by bromodeoxyuridine incorporation), indicating an important role of basal cGMP/PKG-Ialpha kinase activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on SFK activity, determined using highly selective SFK inhibitor, 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). Studies using DT-2 and PKG-Ialpha small interfering RNA revealed that SFK activity was dependent on PKG-Ialpha kinase activity. Furthermore, SFK activity contributed to endogenous tyrosine phosphorylation of PKG-Ialpha in OV2008 and A2780cp cells. In vitro coincubation of recombinant human c-Src and PKG-Ialpha resulted in c-Src-mediated tyrosine phosphorylation of PKG-Ialpha and enhanced c-Src autophosphorylation/activation, suggesting that human c-Src directly tyrosine phosphorylates PKG-Ialpha and the c-Src/PKG-Ialpha interaction enhances Src kinase activity. Epidermal growth factor-induced stimulation of SFK activity in OV2008 cells increased PKG-Ialpha kinase activity (indicated by Ser(239) phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein), which was blocked by both SKI-1 and SU6656. The data suggest an important role of Src/PKG-Ialpha interaction in promoting DNA synthesis/cell proliferation in human ovarian cancer cells. The NO/cGMP/PKG-Ialpha signaling pathway may provide a novel therapeutic target for disrupting ovarian cancer cell proliferation.
先前，我们表明一氧化氮（NO）/环鸟苷酸（cGMP）/蛋白激酶 G（PKG）信号通路的基础活性可防止自发性细胞凋亡，并赋予人类卵巢癌细胞对顺铂诱导的细胞凋亡的抗性。本研究旨在确定基础 PKG 激酶活性是否调节这些细胞中的Src 家族激酶（SFK）活性和增殖。在 OV2008（顺铂敏感，野生型 p53）和 A2780cp（顺铂耐药，突变型 p53）卵巢癌细胞中均鉴定出 PKG-Ialpha 作为主要同工型。在这两种细胞系中，ODQ（内源性 NO 诱导的 cGMP 生物合成抑制剂），DT-2（PKG-Ialpha 激酶活性的高特异性抑制剂）和 PKG-Ialpha 敲低（使用小干扰 RNA）引起 DNA 合成的浓度依赖性抑制（通过溴脱氧尿苷掺入评估），表明基础 cGMP / PKG-Ialpha 激酶活性在促进细胞增殖中起着重要作用。OV2008 细胞中的 DNA 合成取决于 SFK 活性，这是通过使用高度选择性的 SFK 抑制剂 4-（4'-苯氧基苯胺基）-6,7-二甲氧基喹唑啉（SKI-1）来确定的。使用 DT-2 和 PKG-Ialpha 小干扰 RNA 的研究表明，SFK 活性依赖于 PKG-Ialpha 激酶活性。此外，SFK 活性有助于 OV2008 和 A2780cp 细胞中内源性酪氨酸磷酸化的 PKG-Ialpha。重组人 c-Src 和 PKG-Ialpha 的体外共孵育导致 c-Src 介导的 PKG-Ialpha 酪氨酸磷酸化和增强的 c-Src 自身磷酸化/激活，表明人 c-Src 直接将 PKG-Ialpha 酪氨酸磷酸化，并且 c-Src / PKG-Ialpha 相互作用增强了Src 激酶活性。表皮生长因子诱导的 OV2008 细胞中 SFK 活性的刺激增加了 PKG-Ialpha 激酶活性（由 PKG 底物血管扩张刺激磷蛋白的 Ser（239）磷酸化表示），这被 SKI-1 和 SU6656 阻断。数据表明 Src / PKG-Ialpha 相互作用在促进人类卵巢癌细胞中的 DNA 合成/细胞增殖中起重要作用。NO / cGMP / PKG-Ialpha 信号通路可能为破坏卵巢癌细胞增殖提供新的治疗靶标。
