Jane Esther P, Premkumar Daniel R, Pollack Ian F
Department of Neurosurgery, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute Brain Tumor Center, Pennsylvania, USA.
J Pharmacol Exp Ther. 2006 Dec;319(3):1070-80. doi: 10.1124/jpet.106.108621. Epub 2006 Sep 7.
Mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) are activated in the majority of gliomas and contribute to tumor cell growth and survival. Sorafenib (Bay43-9006; Nexavar) is a dual-action Raf and vascular endothelial growth factor receptor inhibitor that blocks receptor phosphorylation and MAPK-mediated signaling and inhibits growth in a number of tumor types. Because our initial studies of this agent in a series of glioma cell lines showed only partial growth inhibition at clinically achievable concentrations, we questioned whether inhibition of PKC signaling using the PKC-delta inhibitor rottlerin might potentiate therapeutic efficacy. Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with sorafenib and rottlerin as single agents or in combination. Sorafenib and rottlerin reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition. Flow-cytometric measurements of cells stained with Annexin V-propidium iodide and immunocytochemical assessment of cytochrome c and apoptosis-inducing factor release demonstrated that addition of rottlerin resulted in significantly higher levels of apoptosis than sorafenib alone. In addition, the combination of sorafenib and rottlerin reduced or completely inhibited the phosphorylation of extracellular signal-regulated kinase and Akt and down-regulated cell cycle regulatory proteins such as cyclin-D1, cyclin-D3, cyclin-dependent kinase (cdk)4, and cdk6 in a dose- and time-dependent manner. Our results clearly indicate that inhibition of PKC-delta signaling enhances the antiproliferative effect of sorafenib in malignant human glioma cell lines and support the examination of combinations of signaling inhibitors in these tumors.
丝裂原活化蛋白激酶（MAPK）和蛋白激酶C（PKC）在大多数胶质瘤中被激活，促进肿瘤细胞的生长和存活。索拉非尼（Bay43 - 9006；多吉美）是一种双作用的Raf和血管内皮生长因子受体抑制剂，可阻断受体磷酸化和MAPK介导的信号传导，并抑制多种肿瘤类型的生长。由于我们最初在一系列胶质瘤细胞系中对该药物的研究表明，在临床可达到的浓度下仅产生部分生长抑制，我们质疑使用PKC-δ抑制剂rottlerin抑制PKC信号传导是否可能增强治疗效果。对用索拉非尼和rottlerin单独或联合处理的细胞进行增殖测定、凋亡诱导研究和蛋白质免疫印迹分析。索拉非尼和rottlerin单独使用时均可降低所有细胞系的增殖，联合使用则显著增强生长抑制作用。用膜联蛋白V-碘化丙啶染色的细胞进行流式细胞术测量，以及对细胞色素c和凋亡诱导因子释放进行免疫细胞化学评估，结果表明添加rottlerin导致的凋亡水平明显高于单独使用索拉非尼。此外，索拉非尼和rottlerin的联合使用以剂量和时间依赖性方式降低或完全抑制细胞外信号调节激酶和Akt的磷酸化，并下调细胞周期调节蛋白，如细胞周期蛋白D1、细胞周期蛋白D3、细胞周期蛋白依赖性激酶（cdk）4和cdk6。我们的结果清楚地表明，抑制PKC-δ信号传导可增强索拉非尼对恶性人脑胶质瘤细胞系的抗增殖作用，并支持在这些肿瘤中研究信号抑制剂的联合使用。
