Effect And Mechanism Of COH-2-3 In Human Promyelocytic Leukemia HL60 Cells

ObjectivesThe purpose of this study was to identify the effect and mechanism of COH-2-3 in human promyelocytic leukemia HL60 cells.Methods1,MTTThe cytotoxic reaction was assessed using the MTT (3-(4, 5-dimethylthythiazol-2-yl) 2,5-diphenyl-tetrazoliumbromide) assay method. MTT (5mg/mL) was dissolved in phosphate buffered saline (PBS) and filtered. Cells at a density of 1,000 cells per 200μL medium per well were seeded in a 96-well plate. Cells were then treated with 0,0.01,0.03,0.1,0.3,1,3μg/mL COH-2-3 for 44h at 37℃in 5% CO2. After incubation,20μL MTT solutions (final concentration 0.5mg/mL) was added to each well and incubated at 37℃for a further 4h. The 96-well plate containing the cells was centrifuged at 1000 rpm for 5 min at 4℃. The MTT solution was removed from the wells and the formazan crystals were dissolved in 150μL DMSO. After 10min, the plates were read on a microplate reader at 570nm wavelength.2,Flow cytometryThe effect of COH-2-3 on growth inhibition was assessed as the percentage of inhibition in cell growth. The experiments were repeated thrice. Analysis of DNA content by Flow cytometry, cells treated with 0,0.1,0.3, 1μg/mL COH-2-3 for 24h or 1μg/mL COH-2-3 for 0,6,12,24h were collected by centrifugation, washed twice with PBS, and then fixed with 70% ethanol at 4℃for over night. The fixed cells were collected by centrifugation at 1000 rpm for 5 min and washed once with PBS. After treatment with RNase (20μg/mL) for 30min at 37℃, cells were stained with propidium iodide (50μg/mL) for 30min at 4℃in the dark and analyzed with a flow cytometer. The experiments were repeated thrice.3,AO/EB flurescence stainingAO/EB flurescence staining to detect apoptosis, cells were treated with 1μg/mL COH-2-3 for 0,6,12,24h. The morphological changes were analyzed by fluorescent microscopy using acridine orange (AO) and ethidium bromide (EB) staining. Briefly, 1μL of a stock solution containing 100μg/mL each of AO and EB was added to 25μL of cell suspension. Viable cells were colored green with intact nuclei. Apoptotic cell was demonstrated by the appearance of cell shrinkage with condensation and fragmentation of nuclei and the formation of apoptotic body.4,ImmunofluorescenceCells treated with 1μg/mL COH-2-3 for 24h were harvested and then fixed in 4% paraformaldehyde for 10 min at room temperature. After washing in PBS for 5 min, cells were permeabilized in 0.5% Triton X-100 in PBS for 30 min. After blocking with 5% BSA for 2h at 37℃, cells were incubated with 1:50 mouse anti-p-tubulin monoclonal antibody for overnight at 4℃. After primary antibody incubation, cells were rinsed in PBS three times and then incubated with 1:50 FITC-conjugated secondary anti-mouse 1gG antibody for 2h at 37℃in the dark. DNA was counterstained with 1:100 Hoechst 33342 for 5 min at room temperature in the dark. Stained cells were visualized under a fluorescence microscope.5,Western blotCells treated with various concentrations of COH-2-3 were harvested. Protein extracts (80μg) prepared with RIPA lysis buffer were separated on an 8-12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membrane. After blocking with 5% nonfat dry milk for 2h, the membranes were probed with primary antibodies at 4℃overnight and then HRP-labeled secondary antibodies at room temperature for 2.5h. Proteins were visualized using enhanced chemiluminescence (ECL). The experiments were repeated thrice.ResultsCOH-2-3 and Cisplatin were found to inhibit the growth of HL60 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M-phase arrest and apoptosis in HL60 cells after exposure to COH-2-3. Morphological changes such as the presence of chromatin condensation and nuclear fragmentation after AO/EB staining were observed. We observed that the normal arrangement and organization of microtubule networks were destroyed. Analysis of the cell cycle-related proteins demonstrated that COH-2-3 increased the protein level of cyclin B. COH-2-3 also caused a conspicuous increase in apoptosis, which was associated with activation of caspase-3 and down-regulation the protein levels of Bcl-2.ConclusionOur data demonstrated that COH-2-3 can induce G2/M-phase arrest in HL60 cells through up-regulating G2/M-phase regulatory proteins and inhibiting tubulin polymerization. Apoptosis induced by COH-2-3 may be through the mitochondrial pathways and caspase-dependent manner.