Reports revealed synergistic effects of celecoxib and imatin

studies unmasked synergistic effects of celecoxib and imatinib in inducing apoptosis in IR K562 cells, with a mechanism relating to the down regulation of MDR 1 and inhibition of COX 2 expression. Phosphate buffered saline, RPMI medium, fetal bovine serum were purchased from Gibco BRL. MTT 2,5 diphenyl tetrazolium bromide, propidium iodide, TMB/H2O2 were from Sigma Aldrich. Nitrocellulose purchase Avagacestat membrane was from Millipore. Mouse monoclonal antibody against cytochrome was from Chemicon. Monoclonal anti-bodies of anti and PARP Tyr were from Upstate. The rest of the chemicals and reagents were obtained from local organizations and are of molecular biology grade. Imatinib was a gift from Natco Pharma Ltd., India. Cells were grown in RPMI 1640 supplemented with ten percent heat inactivated fetal bovine serum, 100 IU/ml penicillin, 100 mg/ml streptomycin and 2mMl glutamine. K562 cells were grown in RPMI medium and IR K562 cells grown in medium containing 1 M imatinib. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 C. The cultured cells were subcultured Eumycetoma twice each week, seeding at a density around 2 103 cells/ml. Cell viability was determined by the trypan blue dye exclusion method. A stock solution of 10 2M celecoxib and imatinib was prepared in DMSO freshly for every test. Cells were grown for 1 week at each concentration of imatinib. Resilient cells were separated by centrifugation through Ficoll Hypaque gradient, washed with RPMI medium and were maintained in RPMI1640 medium supplemented with 10 % FBS and 1 M imatinib. The IC50 of the immune cells towards imatinib was calculated by MTT assay and the flip weight was calculated. IR MAPK inhibitors review K562 cells were confronted with celecoxib, imatinib and combination of imatinib and celecoxib for 2-4 h. Cells after treatment were observed for morphological changes under inverted microscope. DNA laddering was detected by separating fragmented DNA using the SDS/proteinase K/RNase An extraction process, allowing the isolation of only fragmented DNA without contaminating genomic DNA. IR K562 cells were incubated with imatinib, celecoxib and imatinib and celecoxib simultaneously for 24 h. After treatment cells were washed in cold PBS and lysed in a buffer containing 50mM Tris HCl, 1-mm EDTA, 0. 14 days Triton X 100 for 2-0 min at 4 C. After centrifugation at 14,000for 1-5 min, the supernatant was treated with proteinase K and 1000 SDS for 1 h at 5-0 C. DNA was extracted twice with phenol and precipitated with 2 vol and 140mM NaCl. of ethanol at?20 C over-night. DNA precipitates were washed twice in 700-watt ethanol, dissolved in TE buffer, and treated for 1 h at 37 C with RNase A. Eventually, DNA products were electrophoresed in one of the agarose gels, stained with ethidium bromide and visualized under UV light.

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