The Cdc 2 / Cdk 1 inhibitor , purvalanol A , enhances the cytotoxic effects of taxol through Op 18 / stathmin in non-small cell lung cancer cells in vitro

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 taxolresistant 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. Introduction Cdc2, also known as cyclin dependent kinase 1 (CDK1), controls the cell cycle entry from the G2 to the M phase and promotes the commencement of mitotic phase events (1), the abnormal activation of which directly causes aberrant cell proliferation, and malignant transformation and tumorigenesis in prostate cancer cells (2-4). Cdc2 activation also depends on the phosphorylation of Thr161, and CDC25-mediated dephosphorylation at Thr14 and Tyr-15, which exhibits enzymatic activity when only phospho-Thr161 remains (5). Purvalanol A is a selective inhibitor of Cdc2, which strongly inhibits Cdc2 kinase activity at a low concentration of 2 μM (6,7). Investigators have identified that purvalanol A effectively suppresses Cdc2 activity and the progression from the G2 phase to mitosis, which leads to the loss of clonogenicity and cellular apoptosis in both MKN45 and MKN28 X-irradiated gastric cancer cells (8). Oncoprotein 18 (Op18)/stathmin is a small molecule weight phosphoprotein which is highly expressed in cancer cells. Its main functions are to regulate the equilibrium of microtubule (MT) dynamics and control cell cycle progression, which is closely associated with the maintenance of tumor malignant phenotypes (9-12). Op18/stathmin has 4 phosphoserine sites (p-Ser16, p-Ser25, p-Ser38 and p-Ser63), which integrates and relays various signals from intraor extacellular stimuli through phosphorylated inactivation and dephosphorylated activation (13-15). In a previous study, we found that Epstein-Barr virusspecific protein-latent membrane protein 1 (LMP1) regulates the Op18/stathmin signaling pathway by mediating Cdc2, which accelerates cell cycle progression and promotes cell proliferation (16). In another recent study of ours, we confirmed that human NCI-H1299 non-small cell lung cancer cells highly expressing Op18/stathmin were the most highly resistant to taxol among 5 different cancer cells originating from epithelia, including CNE1, Hep3B-2, MGC, MCF-7 and NCI-H1299 (17). In this study, NCI-H1299 cells were employed to clarify the association between Cdc2 signaling and taxol resistance, and to elucidate the related molecular mechanisms. The Cdc2/Cdk1 inhibitor, purvalanol A, enhances the cytotoxic effects of taxol through Op18/stathmin in non-small cell lung cancer cells in vitro XIAN CHEN1, YING LIAO1, DAN LONG1, TING YU1, FANG SHEN2 and XUECHI LIN1,3 1Department of Medical Laboratory, Changsha Medical University, Changsha, Hunan 410219; 2Department of Clinical Laboratory, The First Affiliated Hospital of Hunan Normal University, Changsha, Hunan 410005; 3Department of Anatomy, Histology and Embryology, Institute of Neuroscience, Changsha Medical University, Changsha, Hunan 410219, P.R. China Received August 1, 2016; Accepted May 3, 2017 DOI: 10.3892/ijmm.2017.2989 Correspondence to: Professor Xuechi Lin, Department of Medical Laboratory, Changsha Medical University, Leifeng Road, Wangcheng, Changsha, Hunan 410219, P.R. China E-mail: xuechilin71@126.com