by annexin V/PI analysis revealed analogous results and V+/PI+ cells of 39–59% for combination treatment versus 5–21% for 100–500 nmol/l belinostat alone respectively, Fig 4B). Similar phenomena were observed in Opioid Receptor three primary AML blast samples, and representative quantitative cell death data are shown in Fig 4C. Moreover, these findings were accompanied by concordant increases in PARP cleavage in the two primary AML blast samples assayed . In addition, the pronounced increase in lethality of the combine regimen toward primary AML cells was also confirmed by evaluating Wright–Giemsa stained cytospin slides under light microscopy, which revealed a marked increase in cells displaying apoptotic morphology following belinostat/bortezomib exposure .
Second, parallel studies were performed in primary blasts from patients with B cell or T cell ALL. As Lacosamide shown in Fig 5A, B, exposure of primary B ALL and T ALL cells to the combination of belinostat and bortezomib resulted in a sharp increase in DiOC6 )/7AAD+ cells and/ or in annexin V+/PI) and /PI+ cells respectively. Quantitation of cell death in primary blasts of B cell and T cell ALL demonstrated a striking increase in lethality with combined belinostat/bortezomib treatment, compared to the effects of each agent administered alone . Notably, Wright–Giemsa staining revealed classical features of apoptosis in both B ALL and T ALL blasts co exposed to belinostat/bortezomib , consistent with the marked increase in apoptosis detected by flow cytometry. Moreover, these results were further confirmed by a clear increase in PARP cleavage in primary B ALL and T ALL blasts .
Finally, to assess the selectivity of the bortezomib/belinostat combination regimen, toxicity toward normal CB CD34+ cells was examined following exposure to 500 nmol/l belinostat, the highest concentration employed in the present studies. Interestingly, in contrast to the pronounced potentiation in cell killing in primary AML and ALL blasts farriers by the combined treatment, co administration of bortezomib failed to increase the lethality of belinostat in normal CD34+ cells, reflected by a minimal increase in annexin V+/PI) and /PI+ cells . Quantitation of cell death demonstrated little or no toxicity of identical treatments toward four in these primary leukaemia samples. Similar changes were observed in another AML specimen .
Analogous to results obtained in leukaemia cell lines, co exposure of primary AML and ALL cells to belinostat and bortezomib resulted in a clear increase in expression of Bim, particularly the BimEL isoform changes in expression of survivin were not apparent. Together, these findings indicate that, as in the case of continuously cultured leukaemia cell lines, co administration of belinostat and bortezomib in primary AML and ALL cells results in a shift from pro survival to pro death signalling pathways. Discussion The results of the present study indicate that low, sub micromolar concentrations of the pan HDAC inhibitor belinostat interact in a highly synergistic manner with nanomolar concentrations of bortezomib to induce apoptosis in human acute myeloid and lymphoid leukaemia cells. Several groups, including our own, have described synergistic interactions between proteasome and HDAC inhibitors.