Right after serum deprivation for 48 hrs, MV4-11 parental cells and MV4-11-R cells were transferred into complete medium for an extra 24 hours. Flow cytometric examination exposed that MV4-11 parental cells had a substantially decreased S-phase population , but a considerably enhanced G2/M phase population as compared with MV4-11-R cells. In addition, there were 4.five instances more dead cells in MV4-11 cells than in MV4-11-R cells as established through the trypan blue dye exclusion system with the end of serum Nutlin-3 548472-68-0 selleckchem depletion for 48 hrs. Taken with each other, these benefits recommend that overexpression of survivin in MV4-11-R cells prospects to accelerated S-phase shift and resistance to apoptosis. FLT3 ligand?mediated STAT actions and survivin expression To mimic the overexpression of FLT3LG during the resistant cells, we cultured the parental MV4-11 cells with raising concentration of FLT3 ligand from the cell culture for 48 hrs. Supplemental FLT3 ligand stimulation fairly elevated the expression level of p-STAT1, p-STAT3, and p-STAT5. The expression of survivin was also enhanced in the concentration-dependent method in response to FLT3 ligand stimulation.
To test no matter whether leukemia cells might be protected by FLT3 ligand, we taken care of MV4-11 cells together with the very same panel of FLT3 inhibitors from the presence of 50 ng/mL FLT3 ligand in culture medium for 48 hours. Adding FLT3 ligand rendered MV4-11 cells resistance to all the FLT3 inhibitors examined, despite the fact that the degree of IC50 increment varied. The FLT3 ligand exists in membrane-bound and soluble kinds, that are each biologically energetic.
To test irrespective of whether the secreted soluble kind of FLT3 ligand by tumor kinase inhibitor MV4-11-R cells contributes to resistance, we 1st harvested conditioned medium from MV4-11-R cells incubated in full medium for twelve hrs. Then, MV4-11 cells had been washed twice with 1_ phosphate-buffered saline and cultured in conditioned medium for 2, four, and 6 hours, followed by Western blot analysis. As proven in Figure 3B, incubation in conditioned medium resulted in elevated expression of p-FLT3, p-STATs, and survivin.We noticed the expression of p-STAT5 somewhat dropped at six hrs, despite the fact that survivin continued to boost. To investigate the impact of down-regulation of FLT3 ligand, MV4-11-R cells had been handled which has a FLT3 ligand neutralizing antibody for 48 hours, and cell viability was analyzed. Figure 3C showed the viable cell quantity was appreciably decreased and apoptotic cell number was appreciably elevated in FLT3 ligand neutralizing antibodytreated samples as in contrast with untreated or isotype management?handled samples. As anticipated, in neutralizing antibody-treated samples, the expression of p-FLT3, p-STATs, and survivin was decreased. These information propose that FLT3 ligand plays an essential purpose in mediating the resistance to FLT3 inhibitors.