largazole induced TK expression to a moderately high level . Scriptaid, MK-8669 SAHA, and VA induced TK expression only marginally above untreated cells. LBH589, PXD101, and apicidin were extremely toxic to the cells at the highest concentrations tested, which limited our ability to assess their potency in this assay. Therefore, these results demonstrate that most of the structurally diverse HDAC inhibitors used in this study are inducers of EBV TK gene expression and are therefore inducers of EBV lytic phase gene expression. Cell growth analysis with HDAC inhibitors in combination with GCV Because most of the HDAC inhibitors efficiently induced TK expression in P3HR1 cells, we next analyzed whether the presence of the antiherpes virus drug GCV during this induction of lytic phase gene expression would facilitate killing of the EBVinfected cells.
We exposed the cells to the individual HDAC inhibitor, GCV, or the combination for 72 hours. The HDAC inhibitors were then removed and the cells were maintained in fresh medium containing GCV for a further 72 hours. The short chain Daptomycin molecular weight fatty acid class HDAC inhibitor VA showed significant growth inhibition in combination with GCV, because only 29% of cells survived when treated with VA and GCV together, compared with 84% when treated with GCV alone . However, a relatively high concentration Lapatinib price of VA , was necessary to achieve this effect. VA was therefore similar to butyrate, another short chain fatty acid, in its potency. Higher concentrations of VA were cytostatic for the P3HR1 cells.
The cyclic tetrapeptide apicidin also was efficient in cell killing, but the effective dose range was very limited. At 100nM, 34% of the cells survived with the combination treatment, whereas 59% Rolipram ic50 survived with GCV treatment alone. Higher concentrations of apicidin were toxic to the cells. In contrast, the benzamide HDAC inhibitor MS275 was relatively nontoxic to the cells at the concentration ranges effective to induce sensitivity to GCV. In our assays, MS275 at 500nM was found to have optimal cell killing activity when used together with GCV .We also tested 3 cyclic depsipeptide HDAC inhibitors of the largazole class. Largazole was originally isolated from a marine cyanobacterium and showed strong cytotoxic activity, particularly against tumor cells.34 The 3 compounds largazole, largazole A, and largazole B were prepared by total synthesis, as described previously.
32 Chemical structures of the largazole and its synthetic analogs, as well as all of the other HDAC inhibitors used in the study, are shown in Figure 1. Of these 3 largazole derivatives, the parent natural product showed the most significant cell killing effect at a low concentration . Largazole holistic analog B also showed cytotoxic activity in combination, albeit at a lower level. At least 10 other largazole derivatives were tested for their ability to sensitize EBV infected tumor cells to GCV, but only one other largazole analog had activity comparable to parent largazole . Four additional HDAC inhibitors of the hydroxamic acid class were tested in the same assays. Of these 4 inhibitors, SAHA had little effect on cell killing in the presence of GCV. Scriptaid produced strong cytotoxic activity, requiring concentrations in the range of 1M or higher.
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