Western further blot analysis using an antibody specific for phosphorylated Ser473 residue in Akt demonstrated that the phosphorylation level of Akt protein started to decline after 16 hours of butyrate treatment, and again valproic acid had a weaker effect. Quantification of the Western blots verified that the levels of Akt phos phorylation were reduced by about 60% and 40% after 24 hours of butyrate and valproic acid treatment respectively, as compared to untreated control cells. Taken together, these data suggest that the down regulation of Akt activity by HDAC inhibitors, such as valproic acid and butyrate, is achieved through inhibition of gene expres sion of Akt1 and Akt2. Valproic acid and butyrate induce caspase 3 activation Programmed cell death can be mediated through either caspase dependent or independent mechanisms.

To determine the molecular pathway of cell apoptotic death induced by HDAC inhibitors, such as valproic acid and butyrate, we examined the active status of caspase 3 fol lowing the treatment of HeLa cells. Western blot analysis with an antibody specifically against caspase 3 showed that the HeLa cells contain an abundance of the precursor caspase 3. However, the cleaved or activated form of caspase 3 was only observed following valproic acid or butyrate treatment. Again, butyrate had a stronger effect than valproic acid on inducing caspase 3 activity, correlating directly with its higher efficacy than valproic acid on the deactivation of Akt and on the induc tion of cell apoptotic death.

Taken together, these data suggest that apoptotic cell death induced by HDAC inhibitors, such as valproic acid and butyrate, is mediated through the caspase dependent pathway. Valproic acid and butyrate activate both the caspase 8 and caspase 9 Caspase 3 is an executor caspase that can be cleaved or activated by either caspase 9, the initiator caspase of the mitochondrial pathway, or caspase 8, the initiator caspase of the death receptor pathway. Affymetrix microarray analysis showed that butyrate treatment increased the level of caspase 9 mRNA to a small extent. Quantitative real time RT PCR analysis con firmed that the capase 9 mRNA of the HeLa cells was indeed increased to about 1. 5 fold upon valproic acid or butyrate treatment. Next, we assessed the enzy matic activity of caspase 9 by using a fluorescence based assay in the presence or absence of LEHD CHO, a specific inhibitor for the caspase 9.

As shown in Fig. 3C, valproic acid or butyrate treatment induced the caspase 9 activity and again butyrate induced activity of the caspase 9 more robustly than valproic acid, about 9 fold versus 4 fold. Caspase 8 can be activated by HDAC inhibitors through the death receptors in several cancer cell lines. To determine the role of the death receptor pathway in HDAC Entinostat inhibitor induced HeLa cell death, we evaluated the activity of caspase 8 in the HeLa cells by using a fluo rescence based assay.