purvalanol-a and Lung-Neoplasms

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.

Topics: Carcinoma, Non-Small-Cell Lung; CDC2 Protein Kinase; Cell Line, Tumor; Humans; Lung Neoplasms; Paclitaxel; Purines; Stathmin

Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials.. Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G(2) cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACi-induced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells.. These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Checkpoint Kinase 1; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Lung Neoplasms; Panobinostat; Phosphorylation; Protein Kinases; Purines

Survivin is expressed in most tumor cells and has been associated with both anti-apoptosis and mitotic progression. However, the mechanism of regulation of the survivin expression remains unclear. In this study we investigated the expression and regulation of survivin in the nitric oxide (NO)-exposed human lung carcinoma cells. The lung carcinoma cell lines CL3, H1299, and A549 but not normal lung fibroblast expressed high levels of survivin proteins. NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) decreased the survivin expression. SNAP (0.4 mm, 24h)and SNP (1 mm, 24 h) significantly induced cytotoxicity and apoptosis in lung carcinoma cells. Furthermore, SNAP inhibited the cell growth and increased the fractions of G(2)/M phase. The levels of cyclin B1 and phospho-cdc2-(Thr-161) proteins were inhibited in the NO-exposed cells. The cdc25 phosphatase inhibitors (Cpd 5 and NSC 663284) and the cdc2 kinase inhibitors (alsterpaullone and purvalanol A) enhanced SNP-induced cytotoxicity and the decrease in survivin expression. However, overexpression of survivin by a pOTB7-survivin vector reduced SNP-induced cell growth inhibition and cytotoxicity. In addition, SNP activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The specific p38 MAP kinase inhibitor, SB202190, significantly decreased the cytotoxicity and increased the survivin levels in NO donor-treated and inducible NOS-transfected cells. Conversely, anticancer agents including quercetin, arsenite, and cisplatin but not genistein increased the levels of survivin protein. Our results indicated for the first time that NO inhibited the expression of survivin, which was down-regulated by the p38 MAP kinase pathway.

Topics: Antineoplastic Agents; Apoptosis; Arsenites; Benzazepines; Blotting, Western; Carcinoma; CDC2 Protein Kinase; Cell Cycle; Cell Division; Cell Line, Tumor; Cisplatin; Dose-Response Relationship, Drug; Down-Regulation; Fluorescent Antibody Technique, Indirect; G2 Phase; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Indoles; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; MAP Kinase Signaling System; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Mitosis; Neoplasm Proteins; Nitric Oxide; Nitroprusside; p38 Mitogen-Activated Protein Kinases; Penicillamine; Purines; Pyridines; Quercetin; Quinolones; Quinones; Reverse Transcriptase Polymerase Chain Reaction; Survivin; Time Factors; Transfection