Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells

BACKGROUND: The human organic cation transporter-1 (OCT-1) is the primary active protein for imatinib uptake into target BCR-ABL-positive cells. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used by chronic myeloid leukaemia (CML) patients on imatinib to manage musculoskeletal complaints. METHODS: Here we investigated the impact of NSAIDs on functional activity of the OCT-1 (OCT-1 activity; OA) in CML cells. RESULTS: Although ten of twelve NSAIDs tested had no significant impact on OA (P>0.05), we observed increased OA (27% increase in K562; 22% increase in KU812 cells, P<0.05) and reduced IC50(imatinib) when treated with diclofenac. Co-incubation with imatinib and diclofenac resulted in a significantly lower viable cell number compared with imatinib alone. In contrast, ibuprofen led to a significant decrease in OA, an increase in IC50(imatinib) and thus reduced the cytotoxicity of imatinib. In primary CML samples, diclofenac significantly increased OA, particularly in patients with low OA (<4 ng per 200 000 cells), and significantly decreased IC50(imatinib). Ibuprofen induced significant decreases in OA in CML samples and healthy donors. CONCLUSION: On the basis of the expected impact of these two drugs on OA, ibuprofen should be avoided in combination with imatinib. Further studies are warranted regarding the potential benefit of diclofenac to improve OA in a clinical setting.J. Wang, T.P. Hughes, C.H. Kok, V.A. Saunders, A. Frede, K. Groot-Obbink, M. Osborn, A.A. Somogyi, R.J. D’Andrea and D.L. Whit

Celecoxib sensitizes Staphylococcus aureus to antibiotics in macrophages by modulating SIRT1.

We have previously shown that celecoxib in combination with an antibiotic, increase the bacterial sensitivity to antibiotics. However, the underlying molecular mechanism remained elusive. Efficacy of the combinatorial treatment of celecoxib and ampicillin in vitro was evaluated on macrophage-phagocytosed S. aureus. To elucidate the mechanism, signaling pathway of infection and inflammation involving TLR2, JNK, SIRT1 and NF-κB was studied by FACS, Western blot, ELISA and activity assays. Combinatorial treatment of ampicillin and celecoxib reduced the bacterial load in the macrophages. Further studies clearly suggested the activation of the master regulator of oxidative stress and inflammation SIRT1, by celecoxib when used alone and/or in combination with ampicillin. Also, the results indicated that celecoxib inhibited JNK phosphorylation thereby stabilizing and activating SIRT1 protein that inhibited the COX-2 gene transcription with a significant decrease in the levels of protein inflammatory cytokines like IL-6, MIP-1α and IL-1β via inhibition of NF-κB. SIRT1 activation by celecoxib also resulted in increase of catalase and peroxidase activity with a decrease in Nitric oxide levels. In conclusion, we demonstrate a novel role of celecoxib in controlling inflammation as an enhancer of antibiotic activity against bacteria by modulating SIRT1

Inhibition of Bacterial Multidrug Resistance by Celecoxib, a Cyclooxygenase-2 Inhibitor▿

Multidrug resistance (MDR) is a major problem in the treatment of infectious diseases and cancer. Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)-specific inhibitor celecoxib would not only inhibit COX-2 but also help in the reversal of drug resistance in cancers by inhibiting the MDR1 efflux pump. Here, we demonstrate that celecoxib increases the sensitivity of bacteria to the antibiotics ampicillin, kanamycin, chloramphenicol, and ciprofloxacin by accumulating the drugs inside the cell, thus reversing MDR in bacteria

Celecoxib-induced SIRT1 activity inhibited TLR2-JNK-NF-κB signaling.

<p>A. Flowcytometer analysis of TLR2 expression in RAW-264.7 cells phagocytosed with <i>S aureus</i> (1) and treated with ampicillin (2) or celecoxib (3) or both in combination (4). B. Western blot of levels of p-JNK and JNK in <i>S aureus</i> phagocytosed cells (Lane 1) and treated with ampicillin 100 µg/ml (Lane 2), celecoxib 10 µM (Lane 3), and combination of celecoxib and ampicillin (Lane 4), Resveratrol 50 µM (Lane 5) Suramin 25 µM (Lane 6) and RAW-264.7 cells (Lane 7). C. Western blot of levels of Ac- NF-κB and NF-κB in cell lysates of cells without (Lane 1) and with phagocytosed bacteria (Lane 2) treated with ampicillin (Lane 3) or celecoxib (Lane 4) or both (Lane 5) or Resveratrol (Lane 6) or suramin (Lane 7). D. NF-κB p65 levels in cytoplasmic and nuclear isolates of cells treated with/without, and ampicillin and celecoxib. infected without or with <i>S aureus</i> and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7.</p

Combinatorial treatment with celecoxib activated antioxidant enzymes <i>via</i> SIRT1.

<p>A. RT-PCR and Western blot analysis of SIRT1 in RAW 264.7 cells (Lane 1) and <i>S aureus</i> phagocytosed cells (Lane 2) treated with ampicillin 100 µg/ml (Lane 3), celecoxib 10 µM (Lane 4), and combination of celecoxib and ampicillin (Lane 5), Resveratrol 50 µM (Lane 6) and Suramin 25 µM (Lane 7). GAPDH and β-actin served as controls for RT-PCR and Western blot respectively. B. Activity assay of SIRT1 immunoprecipitated from whole cell lysates of cells infected without or with <i>S aureus</i> and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. C. Nitric Oxide levels in the culture supernatants of cells infected without or with <i>S aureus</i> and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. D. Anti-oxidant enzymes, catalase and peroxidase, activity in the whole cell lysates of cells infected without or with <i>S aureus</i> and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7.</p

Celecoxib inhibits intracellular bacteria.

<p>A. Effect of NSAIDs such as Indomethacin (Ind), Ibuprofen (Ib), Flurbiprofen (Fb), gallic acid (GA) and celecoxib (Ce) and antibiotic ampicillin (Amp) on the growth of <i>Staphylococcus aureus</i>. DMSO and MeOH (methanol) are used as solvent controls. * p-value<0.01 compared to Control. B. Effect of celecoxib, ampicillin and their combination on the survival of intracellular bacteria as measured by the CFU. * p-value<0.01 compared to corresponding Control.</p

Celecoxib activates SIRT1 in a biochemical, <i>in silico</i> and <i>in vitro</i> cell-based assay.

<p>A. A representative graph of three independent experiments each with duplicates showing the enzyme activity of SIRT1 in presence and absence of different concentrations of celecoxib. Suramin, a known inhibitor of SIRT1 is taken as an assay control. * p-value<0.01 compared to Control. B. Left - Model of Celecoxib bound to SIRT1 protein (1–217 residues, proposed N-terminal activation domain of SIRT1). Right - Model showing the interactions of Celecoxib with SIRT1 protein. C. A representative Western blot showing a dose-dependent decrease of Ac-H3K9 levels in presence of celecoxib. * p-value<0.01 compared to Control. D. A representative Western blot showing the signal compensation of Ac-H3K9 levels on co-treatment of cells with celecoxib and suramin at 25 µM concentration each. * p-value<0.01 compared to Control Lane 1. 5 mM sodium butyrate treated cells. Lane 2. Suramin 25 µM. Lane 3. Celecoxib 25 µM. Lane 4. Celecoxib and Suramin 25 µM. Lane 5. Resveratrol 50 µM.</p

Celecoxib-induced SIRT1 activity inhibited proinflammatory gene expression.

<p>A. COX-2 RNA and protein expression in cell lysates of cells without (Lane 1) and with phagocytosed bacteria (Lane 2) treated with celecoxib (Lane 3) or ampicillin (Lane 4) or both (Lane 5) or Resveratrol (Lane 6) or suramin (Lane 7) B. PGE₂ levels in the culture supernatants of cells infected without or with <i>S aureus</i> and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol andSuramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. C. Levels of IL-6, IL-1β, MIP-1α and IL-2 in the culture supernatants of cells infected without or with <i>S aureus</i> and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol andSuramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. D. Total p53 and Ac-p53 levels in cells treated without (Lane 1) and with phagocytosed bacteria (Lane 2) treated with ampicillin (Lane 3) or celecoxib (Lane 4) or both (Lane 5) or Resveratrol (Lane 6) or suramin (Lane 7).</p

Schematic representation of mechanism of action of combinatorial treatment in host cells.

<p>Schematic representation of mechanism of action of combinatorial treatment in host cells.</p

Overexpressed HDAC8 in cervical cancer cells shows functional redundancy of tubulin deacetylation with HDAC6

Abstract Background Histone deacetylases (HDACs) are involved in epigenetic gene regulation via deacetylation of acetylated lysine residues of both histone and non-histone proteins. Among the 18 HDACs identified in humans, HDAC8, a class I HDAC, is best understood structurally and enzymatically. However, its precise subcellular location, function in normal cellular physiology, its protein partners and substrates still remain elusive. Methods The subcellular localization of HDAC8 was studied using immunofluorescence and confocal imaging. The binding parterns were identified employing immunoprecipitation (IP) followed by MALDI-TOF analysis and confirmed using in-silico protein-protein interaction studies, HPLC-based in vitro deacetylation assay, intrinsic fluorescence spectrophotometric analysis, Circular dichroism (CD) and Surface Plasmon Resonance (SPR). Functional characterization of the binding was carried out using immunoblot and knockdown by siRNA. Using one way ANOVA statistical significance (n = 3) was determined. Results Here, we show that HDAC8 and its phosphorylated form (pHDAC8) localized predominantly in the cytoplasm in cancerous, HeLa, and non-cancerous (normal), HEK293T, cells, although nucleolar localization was observed in HeLa cells. The study identified Alpha tubulin as a novel interacting partner of HDAC8. Further, the results indicated binding and deacetylation of tubulin at ac-lys40 by HDAC8. Knockdown of HDAC8 by siRNA, inhibition of HDAC8 and/or HDAC6 by PCI-34051 and tubastatin respectively, cell-migration, cell morphology and cell cycle analysis clearly explained HDAC8 as tubulin deacetylase in HeLa cells and HDAC6 in HEK 293 T cells. Conclusions HDAC8 shows functional redundancy with HDAC6 when overexpressed in cervical cancer cells, HeLa, and deacetylaes ac-lys40 of alpha tubulin leading to cervical cancer proliferation and progression