Results Valproic acid and butyrate repress Akt1 selleck screening library and Akt2 expression and induce apoptosis in HeLa cells HDAC inhibitor induced growth arrest and apoptotic cell death have been observed in a variety of solid and hema tological cancers, but the mechanisms of their action remain obscure. To understand the mechanism of apoptotic cell death induced by HDAC inhibitors such as valproic acid and butyrate, we employed HeLa cells derived from a human cervical cancer. As shown in Fig. 1A, butyrate induced significant HeLa cell death after 16 hours of treatment and more significantly after 24 hours as determined by flow cytometry analysis of the subG1 pop ulations. Valproic acid was also able to induce significant HeLa cell death after 24 hours of treatment, but in a lesser degree.

Affymetrix microarray analysis of gene expression profiles revealed that butyrate treatment down regulated pro apoptotic BAD, BAX and BAK, genes associated with the mitochondrial pathway, while genes associated with the death receptor pathway, such as Fas, FasL, Trail and DR5, were basically not affected by the treatment. However, Akt mRNA was reduced in the HeLa cells following butyrate treatment. Since Akt is a key regulator of cellular survival, we studied the effects of HDAC inhibitors on Akt expression by using quantitative real time RT PCR analysis with specific prim ers and corresponding TaqMan probes for different Akt isoforms. As shown in Fig. 1B, following 16 hours of butyrate treatment, the levels of Akt1 and Akt2 mRNA were reduced by about 70% and 50% respectively as com pared to untreated control cells.

Valproic acid treatment also reduced the levels of the Akt1 and Akt2 mRNA, but to a lesser degree. Intriguingly, the level of Akt3 mRNA was basically unaffected by valproic acid or butyrate treatment. Quantification of the relative mRNA levels of different Akt isoforms showed a distinct expression profile in the HeLa cervical cancer cells. Akt3 is the most abundant of Akt isoforms, about 2 and 4 fold more than Akt1 and Akt2 respectively, which is not a normal Akt isoform expression pattern for cervical epithelial cells. Next, we examined whether the abundance of total Akt protein is affected by valproic acid or butyrate treatment. As shown in Fig. 2A, a decline in the abundance of Akt protein was apparent following 16 hours of valproic acid or butyrate treatment, which became more evident after 24 hours of treatment.

On the Batimastat other hand, the levels of SRC protein were not affected by the treatments as previously reported. Quantitative Western blot analysis revealed that cells treated with butyrate exhibit a more significant decrease in Akt protein than the valproic acid treated cells. nearly 40% of Akt reduction was observed after 24 hours of butyrate treatment compared to 30% reduction after valproic acid treatment.