In addition, the level of caspase 9 mRNA was also increased in the SiHa cells and the total caspase 9 mRNA of the SiHa cells was about 9 fold higher than that of the HeLa cells. Therefore, we assessed the enzymatic CC 5013 activity of caspase 9 by using a fluorescence based assay. As shown in Fig. 6E, valproic acid or butyrate treatment failed to induce the activity of caspase 9 in the SiHa cells. However, the treatments resulted in a slight increase of the caspase 8 activity but no caspase 3 activa tion. Taken together, these data indicate that the ability of cells to survive valproic acid or butyrate treatment depends on their ability to Constitutively active Akt counteracts butyrate induced apop phosphorylation status of Akt in the SiHa cells was not reduced by the treatments.

Next, we performed quantitative real time RT PCR analysis to assess the mRNA levels of Akt isoforms. As shown in Fig. 5C, both valproic acid and butyrate decreased the levels of Akt1 and Akt2 mRNA to a certain degree, but not significantly. In contrast, the treatments increased the levels of Akt3 mRNA by over 50 fold in comparison to untreated control, which is consistent with the augmented levels maintain the cellular activity of Akt. Discussion HDAC inhibitors, inducers of differentiation or apoptosis of some cervical and ovarian cancer cells, have become a new class of drugs for treatment of a variety of cancers. However, many questions concerning their mecha nisms of action and their therapeutic potentials for differ ent cancers are largely unanswered.

These are intimate related issues due to the heterogeneity of the genetic lesion and epigenetic alteration of the cancer. The molec ular mechanisms of HDAC inhibitors as cancer therapeu tics may be highly dependent on the type or cause of the cancer. Our study demonstrated that HDAC inhibitors, such as valproic acid and butyrate, induce apoptosis in HeLa cervical cancer cells by inhibition of gene expression of Akt1 and Akt2. In addition, the apoptotic cell death induced by valproic acid or butyrate is mediated through the caspase dependent pathways. Inhibition of histone deacetylation and alteration of chro matin structure often lead to transcriptional activation. Numerous studies have shown that, through its chroma tin remodeling activities, HDAC inhibitors are capable of modulating gene transcription involved in various cellu lar processes such as cell cycle progression, differentiation and apoptosis.

Gene silencing or abnormal expres sion is a hallmark of many forms of malignancy. The effi cacy of HDAC inhibitors in cancer therapeutics may well come from restoring silenced gene expression since tran scription is the primary target of HDAC inhibitors. Increasing the expression of some pro apoptotic proteins, such as TRAIL and p21, AV-951 has been shown to be one of the molecular mechanisms by which the HDAC inhibitors induce cancer cell death.