Department of Medical Laboratory, Changsha Medical University, Changsha, Hunan 410219, P.R. China.
Department of Clinical Laboratory, The First Affiliated Hospital of Hunan Normal University, Changsha, Hunan 410005, P.R. China.
Int J Mol Med. 2017 Jul;40(1):235-242. doi: 10.3892/ijmm.2017.2989. Epub 2017 May 15.
Purvalanol A is a highly selective inhibitor of Cdc2 [also known as cyclin-dependent kinase 1 (CDK1)]. Taxol is an anti-tumor chemotherapeutic drug which is widely used clinically. In this study, the CDK1 inhibitor, purvalanol A was applied to explore the relevance of Cdc2 signaling and taxol sensitivity through analyses, such as cellular proliferation and apoptosis assays, ELISA, western blot analysis and immunoprecipitation. We demonstrated that purvalanol A effectively enhanced the taxol-induced apoptosis of NCI-H1299 cells, as well as its inhibitory effects on cellular proliferation and colony formation. In combination, purvalanol A and taxol mainly decreased the expression of oncoprotein 18 (Op18)/stathmin and phosphorylation at Ser16 and Ser38, while purvalanol A alone inhibited the phosphorylation of Op18/stathmin at all 4 serine sites. Co-treatment with purvalanol A and taxol weakened the expression of Bcl-2 and activated the extrinsic cell death pathway through the activation of caspase-3 and caspase-8. Further experiments indicated that Cdc2 kinase activities, including the expression of Cdc2 and the level of phospho-Cdc2 (Thr161) were significantly higher in taxol-resistant NCI-H1299 cells compared with the relatively sensitive CNE1 cells before and following treatment with taxol. These findings suggest that Cdc2 is positively associatd with the development of taxol resistance. The Cdc2 inhibitor, purvalanol A, enhanced the cytotoxic effects of taxol through Op18/stathmin. Our findings may prove to be useful in clinical practice, as they may provide a treatment strategy with which to to reduce the doses of taxol applied clinically, thus alleviating the side-effects.
普伐诺醇 A 是一种高度选择性的 Cdc2 [也称为周期蛋白依赖性激酶 1 (CDK1)] 抑制剂。紫杉醇是一种广泛应用于临床的抗肿瘤化疗药物。在这项研究中，通过细胞增殖和凋亡检测、ELISA、western blot 分析和免疫沉淀等分析，应用 CDK1 抑制剂普伐诺醇 A 来探讨 Cdc2 信号与紫杉醇敏感性的相关性。我们证明普伐诺醇 A 能有效增强 NCI-H1299 细胞中紫杉醇诱导的细胞凋亡，以及对细胞增殖和集落形成的抑制作用。联合应用普伐诺醇 A 和紫杉醇主要降低了癌蛋白 18(Op18)/微管蛋白和丝氨酸 16 和丝氨酸 38 磷酸化的表达，而普伐诺醇 A 单独作用时则抑制 Op18/微管蛋白所有 4 个丝氨酸位点的磷酸化。普伐诺醇 A 和紫杉醇联合处理可减弱 Bcl-2 的表达，并通过激活 caspase-3 和 caspase-8 激活外源性细胞死亡途径。进一步的实验表明，Cdc2 激酶活性，包括 Cdc2 的表达和磷酸化 Cdc2(Thr161)的水平，在紫杉醇耐药的 NCI-H1299 细胞中明显高于相对敏感的 CNE1 细胞，无论是在紫杉醇处理之前还是之后。这些发现表明 Cdc2 与紫杉醇耐药的发生呈正相关。Cdc2 抑制剂普伐诺醇 A 通过 Op18/微管蛋白增强了紫杉醇的细胞毒性作用。我们的发现可能在临床上有用，因为它们可能提供一种治疗策略，以减少临床上应用的紫杉醇剂量，从而减轻副作用。
