On the other hand, minor is recognized regarding the results of curcumin on other kinds of AML. From the current research, we investigated the effect and mode of action of curcumin on monocytic leukemia THP 1 cells. We initial examined the effect of different concentrations of curcumin on THP one cell apoptosis. Upcoming, interference of your inhibitor of ERK and JNK and PMA handled THP one cells have been used to study the most likely mechanism of curcumin mediated apoptosis. Methods Cell and reagents The THP one cell line, derived from human acute mono cytic leukemia, was obtained from American Form Culture Collection. Cells were cultured in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 1% L glutamine, 1% non crucial amino acids. Curcu min, dimethyl sulfoxide, SP600125, U0126 and phorbol 12 myristate 13 acetate were bought from Sigma.
Antibo dies towards caspase three, cleaved caspase 8, Caspase 9, FoxO4, phospho FoxO4, FoxO3a, FoxO1, phos pho FoxO1, phospho FoxO3a, p85, phospho p85, p110a, PDK1, Phospho PDK1, purchase CGK 733 JunB, c Jun, phospho c Jun Ser63, AKT1, AKT2, AKT3, phospho AKT, phospho AKT, ATF2, phospho ATF2 Thr71, phospho JNK, phospho ERK, ERK, JNK, p38, phos pho p38, caspase 8 and histone H3 have been purchased from Cell signaling laboratory and anti bodies towards PARP one, caspase 3 and GAPDH have been from Epitomics Inc. b actin antibody and phospho JunB were obtained from Sigma and Santa Cruz Biotechnology, respectively. Movement cytometry THP 1 cells, which had been treated with curcumin, were harvested and fixed with 70% ethanol at 4 C overnight. Right after PBS washing, the cells had been incubated with RNase A for five min.
Following incubation with propidium iodide, the cells underwent movement cytometry. For double staining, THP one cells had been initially treated with PhipPhiLux G1D2 cas pase 3 substrate at 3-Deazaneplanocin A ic50 37 C for 45 min. Immediately after washing, the cells had been stained with propidium iodide and analyzed working with flow cytometry. Protein extraction and immunoblotting THP 1 cells had been lyzed with RIPA lysis buffer. Complete cell lysates were extracted as described previously. The lysates had been separated working with polyacrylamide gel electrophoresis. Just after transfer, the membrane was blotted with antibody and designed with an enhanced chemiluminescent kit. Caspase activity assay THP one cells had been handled with DMSO and curcumin while in the presence of U0126 and SP600125 for 10 hours. The cells were subsequently incu bated with Caspase Glo three 7 reagent kit and caspases 3 7 action was detected and analyzed employing a GloMax Multi Detection Program in accordance to the manufacturers guidelines. WST 1 assays THP 1 cells and PMA taken care of tHP 1 cells had been seeded on the density of 50 000 cells cm2 in 96 properly plates. The cells were incubated with DMSO and 50 uM curcumin for 18 hr.