TS at the mRNA level measured Apixaban by quantitative PCR . Synergistic anti proliferative eVects of PXD101 and 5 FU in vitro In order to determine if there was any beneWt to combining PXD101 with 5 FU on inhibition of proliferation, a constant ratio combination design based on each compound’s EC50 value was used. The maximum concentrations of PXD101 and 5 FU were determined by multiplying their EC50 values by 5.0625, followed by constructing a titration curve using 1.5 fold dilutions. This ensured that all concentrations used were on the linear range of their individual titration curves. Co incubation of PXD101 and 5 FU for 48 h produced synergy over a wide range of concentrations with only one combination point being slightly antagonistic .
Furthermore, the synergistic eVects were improved by pre incubation with PXD101 for 24 h followed by 5 FU for 48 h alone . Strong synergy could be produced with this schedule, using adjusted PXD101 concentrations to take into account the shorter exposure time. Again, only one combination point showed some mild antagonism. Synergistic proteasom inhibitor list anti clonogenic eVects of PXD101 and 5 FU in vitro The eVects of PXD101 and 5 FU alone and in combination on the clonogenicity potential of HCT116 cells were tested in vitro. Both PXD101 and 5 FU proved to be eVective inhibitors of colony formation with EC50 values of 13 and 45 M after 24 h incubation, respectively .Co incubation of PXD101 and 5 FU for 24 h produced synergy to strong synergy over a wide range of concentrations .
Additionally, the synergistic eVects were slightly improved by pre incubation with PXD101 for 24 h followed by 5 FU for 24 h alone , with one combination point showing very strong synergism. Incubation with PXD101 and 5 FU produces enhanced apoptosis over single agent incubations in vitro HCT116 Decitabine price cells were incubated with PXD101 and 5 FU either alone or in combination for 48 h and whole cell lysates prepared. Western blots were then produced using these lysates and probed with antibodies that detect PARP cleavage. Cleavage of PARP by Caspase 3 occurs early in the apoptotic response . Incubation of HCT116 cells with either PXD101 or 5 FU alone for 48 h produced low levels of PARP cleavage while co incubation enhanced this eVect substantially . In contrast, incubating in PXD101 for 24 h followed by a further 48 h in 5 FU alone did not lead to enhanced cleavage over either compound alone .
The eVects of either compound alone on the TUNEL assay in HCT116 cells were enhanced when PXD101 and 5 FU were combined. TUNEL detects apoptosis induced DNA nicks, which is also one of the hallmarks of apoptosis . Cells were treated with 0.9 M PXD101 for 24 Decitabine ic50 h followed by a further 48 h in 11 M 5 FU alone, and the TUNEL assay was performed. PXD101 and 5 FU produced 1.4 and 1.8 fold shifts in the TUNEL positive population, respectively, whereas a 3.4 fold shift was produced when they were combined . In contrast to the PARP cleavage experiments a co incubation schedule only produced a minor shift in TUNEL positive population of around 1.6 fold compared to 1.3 fold using single compound . This in line with the data demonstrating that incubation with PXD101, followed by 5 FU, achieved a greater degree of synergy in their signs anti proliferative eVects.