was performed using SAS V9.2 and all reported p values are two sided using an alpha level of 0.05. Results Vorinostat Barasertib Aurora Kinase inhibitor and Aurora Kinase inhibitors curb lymphoma growth singly and together We tested single and combined titrations of MK 0457 or MK 5108 and vorinostat in both cell growth and apoptosis assays with Hodgkin lymphoma cell lines L540 and KM H2 and with non Hodgkin lymphoma cell lines including Daudi, DHL 4 and DHL 6. Figure 1A shows L540 growth inhibition by each drug, as determined by MTS assays. Inhibition was dose dependent and combinations of both drugs inhibited cell growth more than any drug alone at the lower doses. We obtained similar results with the other cell lines tested . Order of addition experiments showed no greater effect than with simultaneous addition of drugs .
These data allowed us to calculate IC50 and Combination Index values. Table 1 shows that for most lymphoma cell lines the IC50s of these drugs were in the sub micromolar range . The few exceptions were in relative sensitivities mk-2866 841205-47-8 to one or the other AKi. For five of six lines tested excepting the DHL 6 cells the IC50,s of MK 0457 were lower than those of MK 5108. We also determined Combination Index values , showing that combining AKi,s MK 0457 or MK 5108 with vorinostat had an additive or frequently synergistic effect . There were no consistent differences in CI values between Aki,s when combined with vorinostat. Apoptosis data suggested the growth inhibition seen in MTS assays was not primarily due to cell cycle arrest or longer cycling times, but to time and dose dependent increases in apoptosis, as assayed by Annexin V cell labeling.
The combination of Kretzner et al. Page 4 Cancer Res. Author manuscript, available in PMC 2012 June 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript vorinostat and an AKi was consistently more effective in promoting cell death than any drug alone in L540 cells, with similar data obtained in Daudi , KMH2 and DHL 4 cells . The extent of apoptosis with vorinostat plus either AKi was from 2 to 7 fold greater than with either AKi alone, presumably because AK inhibition leads primarily to cell cycle arrest rather than cell death. To discriminate between cell cycle arrest and death, we performed cell cycle analysis, with representative results for L540 cells shown in Figure 2. Incubation in 1.
5 μM vorinostat enlarges a modest subpopulation of cells in the sub G1 region, often indicative of dead cells, while treatment with 100 nM MK 0457 produces a large increase in cells arrested in the G2/M phase, as well as a small increase in the sub G1 region. Significantly, the two drugs combined shift a substantial proportion of the L540 cells into the sub G1 population . Percentages of cell populations in each cell cycle phase for various treatments are listed in Supplementary Table 1. We obtained similar results with the HL cell line KM H2 and the NHL cell line Daudi, a Burkitt,s lymphoma . The additivity, or in some cases, synergy of these two drugs is reflected in the enrichment of sub G1 phase cells when both drugs are present.
Cell size determination showed most cells treated with MK 0457 were enlarged, whereas those treated additionally with vorinostat were smaller than control cells , consistent with sub G1 phase dead and/or dying cells. Along with enlargement, there was evidence of endoreduplication in some assays, with small cell populations beyond the G2/M peak . The percentage of apoptosis in each condition exceeds that of cells in sub G1, as Annexin V labels intact cells early in apoptosis as well as further degraded ones. Vorinostat brings about changes in lymphoma cell gene expression We performed real time PCR analysis of drug treated L540 cells to determine reasons for the drugs, effects on the cell cycle and apoptosis. AKi treatment had little effect on expression of the genes we analyzed, in contrast to strong effects seen with HDAC inhibition. Vorinostat led to do