r 315 and Ser46, was also inhibited by MK 0457 in the presence of Roscovitine CDK inhibitor nocodazole. Vorinostat and AKi treatments lead to changes in micro RNA levels Micro RNAs are key regulators of cell growth and differentiation by virtue of post transcriptional inhibition of mRNA stability and/or translation . Myc transcriptionally activates the miRNA 17 92 cluster . Since vorinostat repressed cmyc message and protein , we tested expression levels of three members of this miRNA cluster, miR 17.5p, miR 17.3p, and miR 18, in response to vorinostat, the unrelated HDACi trichostatin A , and MK 0457 in L540 and DHL 4 cells . Treatment with HDACi resulted in decreases in all three miRNAs relative to untreated cells HDACi also induced changes in three miRNAs that are not myc regulated, miR 15b, miR 34a, and miR 155, as shown in Figures 5B and Supplemenary Figure 5B for L540 and DHL 4 cells, respectively.
The two cell types have distinct changes in the expression of these miRNAs, NPI-2358 714272-27-2 possibly reflecting biological differences between the different lymphoma types involved . Role of Myc downregulation and Mxd1 upregulation by vorinostat Aki combination Lastly, we sought to determine the importance of HDACi induced c myc downregulation in lymphoma cell responses to combined HDAC/AK inhibition. To model this downregulation in the absence of HDACi, we transfected L540 cells with small interfering RNA directed at c myc mRNA, and/or over expressed the Myc antagonist, Mxd1, followed by titration of either MK 0457 or MK 5108 . Knock down of c myc Kretzner et al. Page 6 Cancer Res. Author manuscript, available in PMC 2012 June 1.
NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript message was difficult in L540 cells, typically reaching a 50% decrease at 48 72 hours . Thus, siRNA myc had only a small negative effect on cell survival in response to MK 0457 and a slightly greater effect with MK 5108 . Mxd1 overexpression led to similar results. However, upon combining myc knock down with Mxd1 expression L540 cells clearly became more sensitive to both AKi,s. The IC50 of MK 0457 was lowered from ~0.5 μM to 0.1 μM by combined treatment, while that of MK 5108 went from ~1.8 μM to ~0.35 μM. Thus, combining myc knock down with Mxd1 over expression recapitulates the synergistic effect of combining vorinostat with the AKi,s, which we postulate is due in part to decreased myc levels after treatment.
Discussion We have studied the effects of MK 0457 and MK 5108, prototype aurora kinase inhibitors, in combination with histone deacetylase inhibitor vorinostat. Both drugs inhibit AK A andMK 0457 also inhibits AK B, alone their Aki activity exerts strong negative cell cycle effects on both HL and NHL cells, but has modest consequences for overall cell growth and survival . We hypothesized that AKi induced arrest of cells in G2/M phase results in activated intracellular stress signaling pathways, but that in cancer cells this cellular response is blunted by epigenetic silencing of tumor suppressor and pro apoptotic genes. Thus, the HDACi vorinostat could potentially exert a synergistic or at least additive effect when combined with AKi,s.
This proves to be the case in lymphoma cells, as also seen in acute and chronic myelogenous leukemia cells when combining vorinostat and MK 0457 . Given the similar responses of cells treated with both MK 0457 and MK 5108, we hypothesize that it is inhibition of aurora kinase A that is central to the activity in lymphoma cell lines. The effects of aurora kinase inhibition on gene expression levels are modest, while those of vorinostat are extensive. Key effects of HDAC inhibition were downregulation of c Myc, hTERT, Bcl XL, Mcl 1 and FoxO3A, and upregulation of cell cycle inhibitors p21 and p27 and the pro apoptotic genes Bad, Bid, and Noxa, seen in both qPCR and immunoblot assays. Immunoblotting also demonstrated post translational effects of vorinostat and MK 0457 on p53, leading to stabilization and increased act