- Open Access
Valproic acid and butyrate induce apoptosis in human cancer cells through inhibition of gene expression of Akt/protein kinase B
© Chen et al; licensee BioMed Central Ltd. 2006
- Received: 15 August 2006
- Accepted: 11 December 2006
- Published: 11 December 2006
In eukaryotic cells, the genomic DNA is packed with histones to form the nucleosome and chromatin structure. Reversible acetylation of the histone tails plays an important role in the control of specific gene expression. Mounting evidence has established that histone deacetylase inhibitors selectively induce cellular differentiation, growth arrest and apoptosis in variety of cancer cells, making them a promising class of anticancer drugs. However, the molecular mechanisms of the anti-cancer effects of these inhibitors have yet to be understood.
Here, we report that a key determinant for the susceptibility of cancer cells to histone deacetylase inhibitors is their ability to maintain cellular Akt activity in response to the treatment. Also known as protein kinase B, Akt is an essential pro-survival factor in cell proliferation and is often deregulated during tumorigenesis. We show that histone deacetylase inhibitors, such as valproic acid and butyrate, impede Akt1 and Akt2 expression, which leads to Akt deactivation and apoptotic cell death. In addition, valproic acid and butyrate induce apoptosis through the caspase-dependent pathway. The activity of caspase-9 is robustly activated upon valproic acid or butyrate treatment. Constitutively active Akt is able to block the caspase activation and rescues cells from butyrate-induced apoptotic cell death.
Our study demonstrates that although the primary target of histone deacetylase inhibitors is transcription, it is the capacity of cells to maintain cellular survival networks that determines their fate of survival.
- HeLa Cell
- Valproic Acid
- Apoptotic Cell Death
- HDAC Inhibitor
In eukaryotic cells, histones play an important role in transcription control by packing the genomic DNA into an array of nucleosomes and higher order chromatin structure [1–3]. The amino-terminal tails of histones are rich in positively charged lysine residues which form tight contacts with the negatively charged DNA backbones, restricting the accessibility of DNA for the binding of transcription regulators [4, 5]. This restrictive milieu needs to be relieved to allow the regulation of chromatin structure and function, and ultimately for gene activation to occur, which often is achieved through acetylation of the histone tails by histone acetyltransferases [6–9]. On the other hand, deacetylation of the histone tails by histone deacetylases (HDAC) restores the histone restriction, resulting in gene repression.
Malignant cells gain various phenotypic characteristics during the development of cancer, which permit them to proliferate abnormally and eventually invade surrounding tissues. Numerous studies have demonstrated the importance of epigenetic alteration in cancer onset. This has raised the possibility of controlling transcription as a potential approach in cancer therapeutics . Valproic acid, a short-chain fatty acid, is a well established drug in the treatment of epilepsy, migraine, cluster headaches and for the control of a variety of seizures . Butyrate, also a short chain fatty acid, naturally produced by bacterial fermentation in the colon, has been designated as the most potent fatty acid in arresting cell proliferation . Both compounds are classified as HDAC inhibitors [13, 14]. Many studies have shown that HDAC inhibitors selectively induce cellular differentiation, growth arrest and apoptosis in cancer cells, making these inhibitors a promising new class of anticancer drugs [15–17]. However, the molecular mechanisms of the anticancer effects of the HDAC inhibitors have yet to be understood.
Akt, also known as protein kinase B, is a serine/threonine protein kinase and a key player in the regulation of apoptosis, proliferation and tumorigenesis [18–20]. Currently, three mammalian isoforms have been identified, namely Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ . Akt1 is the predominant isoform in most tissues while Akt2 is highly expressed in skeletal muscle, heart, liver and kidney [22, 23]. Akt3 exhibits a more restricted pattern of distribution, mostly found in testis and brain . The phosphorylation of a conserved threonine residue (Thr308 in Akt1, Thr309 in Akt2 and Thr305 in Akt3), upon growth factor stimulation, is required for Akt activation, while the phosphorylation of a serine residue (Ser473 in Akt1, Ser474 in Akt2 and Ser474 in Akt3) is only required for maximal Akt activity [21, 24–26]. Constitutively active Akt can block apoptosis induced by several diverse treatments, such as growth factor withdrawal, UV irradiation, matrix detachment and DNA damage [27–29].
Akt promotes cellular survival by directly phosphorylating transcription factors that control the expression of pro-survival or anti-apoptotic genes. For instance, the forkhead family of transcription factors resides in the nucleus where they induce the expression of pro-apoptotic genes . However, in the presence of active Akt, these transcription factors are exported out of the nucleus and sequestered in the cytoplasm by 14-3-3 proteins . Cytoplasmic retention of these transcription factors acts as a negative regulation of apoptotic machinery. In contrast, NF-kB, a transcription factor involved in promoting cell survival, is positively influenced by Akt that phosphorylates and activates IKKα, leading to the destruction of I-kB and the entry of NF-kB into the nucleus [31–33].
Direct phosphorylation of proteins involved in apoptosis is another mechanism by which Akt promotes cellular survival. BAD, a member of the Bcl-2 family is sequestered by 14-3-3 proteins in the cytosol upon phosphorylation by Akt . This event prevents BAD from interacting with pro-survival members of the Bcl-2 family at the mitochondrial membrane [24, 25]. Akt prevents cytochrome release by maintaining structural integrity of the mitochondria and is also involved in post-mitochondrial events through phosphorylation of caspase-9 [24, 35–37]. Recent studies demonstrated that Akt positively regulates the protein stability and transcriptional activity of coactivator p300 [38, 39]. Since p300 contains an intrinsic histone acetyltransferase activity, it is possible that Akt also promotes cellular survival through the regulation of chromatin dynamics.
HDAC inhibitors have a range of anti-cancer activities including the induction of apoptosis in transformed culture cells and in cancers. Some studies have observed a decreased Akt activity following treatment with HDAC inhibitors. Here we report that the induction of cancer cell apoptosis by HDAC inhibitors, such as valproic acid and butyrate, is achieved through inhibition of gene expression of Akt1 and Akt2, pro-survival factors involved in many cell signalling pathways. In addition, valproic acid and butyrate induce apoptotic cell death through the caspase-dependent pathway.
Valproic acid and butyrate repress Akt1 and Akt2 expression and induce apoptosis in HeLa cells
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 (data not shown). However, Akt mRNA was reduced in the HeLa cells following butyrate treatment (data not shown). 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 primers 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 compared to untreated control cells. Valproic acid treatment also reduced the levels of the Akt1 and Akt2 mRNA, but to a lesser degree (Fig. 1B). Intriguingly, the level of Akt3 mRNA was basically unaffected by valproic acid or butyrate treatment (Fig. 1B). 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 (Fig. 1C), which is not a normal Akt isoform expression pattern for cervical epithelial cells.
Valproic acid and butyrate induce caspase-3 activation
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 (data not shown). Quantitative real-time RT-PCR analysis confirmed that the capase-9 mRNA of the HeLa cells was indeed increased to about 1.5 fold upon valproic acid or butyrate treatment (Fig. 3B). Next, we assessed the enzymatic 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 [42–44]. To determine the role of the death receptor pathway in HDAC inhibitor induced HeLa cell death, we evaluated the activity of caspase-8 in the HeLa cells by using a fluorescence based assay. Following treatment with valproic acid or butyrate, the activity of caspase-8 was increased to about 2 and 5 fold respectively, but to a lesser degree in comparison to the caspase-9 activity (Fig. 3D). Taken together, our data suggest that the HDAC inhibitor-induced apoptotic cell death is attained by activating both the caspase-8 and caspase-9 activities.
Introduction of constitutively active Akt prevents butyrate-induced apoptosis
Valproic acid and butyrate do not affect the Akt activity and cellular survival of SiHa cells
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 mechanisms of action and their therapeutic potentials for different cancers are largely unanswered. These are intimate related issues due to the heterogeneity of the genetic lesion and epigenetic alteration of the cancer. The molecular mechanisms of HDAC inhibitors as cancer therapeutics 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 chromatin structure often lead to transcriptional activation. Numerous studies have shown that, through its chromatin remodeling activities, HDAC inhibitors are capable of modulating gene transcription involved in various cellular processes such as cell cycle progression, differentiation and apoptosis . Gene silencing or abnormal expression is a hallmark of many forms of malignancy. The efficacy of HDAC inhibitors in cancer therapeutics may well come from restoring silenced gene expression since transcription is the primary target of HDAC inhibitors. Increasing the expression of some pro-apoptotic proteins, such as TRAIL and p21, has been shown to be one of the molecular mechanisms by which the HDAC inhibitors induce cancer cell death [46, 47]. However, HDAC inhibitor treatment can also result in gene repression . Our study demonstrates that indeed this may be an alternative mechanism by which HDAC inhibitors induce cell apoptotic death. Treatment of HeLa cells with HDAC inhibitors such as valproic acid and butyrate leads to inhibition of Akt1 and Akt2 expression, consequently to deactivation of cellular Akt (Fig. 1 and 2).
The link between the action of HDAC inhibitors and the down-regulation of Akt phosphorylation has been reported previously [18, 49, 50]. In both 87MG and PC3 cells, treatment with HDAC inhibitors decreases the levels of Akt phosphorylation . We, for the first time, show that the down-regulation of Akt activity is a consequence of inhibition of Akt1 and Akt2 expression by valproic acid and butyrate. It appears that these inhibitors repress Akt1 and Akt2 transcription thereby depleting the cellular Akt protein. The negative effect of valproic acid or butyrate on Akt expression could be mediated through a direct or indirect mechanism, i.e. through inhibiting deacetylation of nonhistone proteins, such as transcription regulators, or gene activation of some negative regulators of Akt gene transcription. Understanding the molecular basis by which Akt gene expression is regulated will ultimately help us to design better strategies to treat cancer.
The molecular basis for the action of HDAC inhibitors in caner therapeutics is one of the important questions remaining to be answered. If transcription is the primary target of HDAC inhibitors, then normal cells should be equally if not more susceptible toward the HDAC inhibitors. Our study demonstrated that the key answer may reside in the competency of signaling pathways to maintain the cellular activity of Akt in response to the treatment. Being a key pro-survival factor, Akt is often deregulated during tumorigenesis . For example, HeLa cells exhibit an abnormal expression pattern of Akt isoforms, in which Akt3 is the most abundant of Akt isoforms (Fig. 1C). This deregulation of Akt may sensitize cancer cells to HDAC inhibitors. Without Akt to mediate its pro-survival activity, the cells may be more prone to apoptosis.
The role of caspase in HDAC inhibitor-induced apoptosis has been controversial or may be dependent on cellular context. Some studies have established clear roles for the death receptors and caspase-8 in this process [42, 43, 47, 51]. Our study demonstrated that valproic acid and butyrate promote apoptosis in HeLa cervical cancer cells through a caspase-dependent mechanism (Fig. 3). Most intriguingly, the caspase-9 activity is more robustly activated than caspase-8 upon the valproic acid or butyrate treatment (Fig. 3). In addition, introduction of constitutively active Akt blocks caspase-3 activation and rescues cells from apoptotic death upon butyrate treatment (Fig. 4). Akt directly regulates caspase-9 activity and promotes cellular survival at post-mitochondrial level [35, 36]. It is likely that deactivation of Akt plays a primary role in the activation of caspase-9 during the HeLa apoptotic death induced by valproic acid or butyrate.
Apparently, apoptosis induced by HDAC inhibitors is associated with various disarrays of different cell signaling pathways. The precise molecular mechanisms involved in the cancer therapeutics of HDAC inhibitors may depend highly on the cellular context or the genetic lesion and epigenetic background of the cancer. For targeted or customized cancer therapy, it is essential to understand the distinct mechanisms of apoptotic cell death induced by HDAC inhibitors. Our study demonstrates that although the primary target of HDAC inhibitors may be transcription, it is the cellular environment, or the ability of cells to maintain their survival protein networks that determines their fate, to die or to survive in response to the treatment.
Cell culture and reagents
The cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (Invitrogen) at 37°C with 5% CO2. Valproic acid and sodium butyrate were purchased from Sigma. Antibodies against Akt, phosphor-Akt and caspase3 were obtained from Cell Signalling.
Flow cytometry analysis
Following exposure to valproic acid (2 mM) or sodium butyrate (5 mM) for 8, 16 or 24 hours, cells were detached from tissue culture dishes by trypsinization, combined with floating cells and fixed in 70% ethanol for 30 minutes at -20°C. After washing with phosphate-buffered saline, the cells were incubated with 50 μg/ml of RNase A and 50 μg/ml of propidium iodide for 30 minutes at 37°C. DNA contents of the cells were then profiled by fluorescence activated cell analyzer (Beckman Coulter) to determine the distribution of cells in different phases of the cell cycle .
Quantitative real-time RT-PCR
Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse transcribed using random primers and Superscript II (Invitrogen). Serial dilution of the cDNA was used to determine the appropriate concentration required for the real-time PCR amplification which was carried out by using TaqMan reporter assay with a 7500 Fast Real-Time PCR System (Applied Biosystems). Gene specific primers and TaqMan probes used for the amplification were ordered from Applied Biosystems. Quantification of mRNA levels was performed by using 18S rRNA as an internal control.
Protein extraction and Western blot analysis
Following various treatments, cells were washed and harvested. The cell pellets were suspended and incubated for 30 minutes at 4°C in whole-cell extraction buffer consisting of 10% glycerol, 50 mM Tris-HCl (pH 7.6), 400 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40. The lysates were then centrifuged at 14,000 g for 10 minutes at 4°C. Protein concentrations were determined by Bradford assay (Bio-Rad) using bovine serum albumin as standard. PerkinElmer Life Sciences ECL system was used for the detection and Scion Image software was used to quantify the Western blots .
Caspase activity assay
Following various treatments, cells were assayed for caspase activities by using fluorescent assay kits (Clontech) and a microplate fluorometer (Molecular Devices). The caspase-8 and caspase-9 activities were determined with the same batch of lysates simultaneously and were measured from zero time to 2 hours as the manufacturer recommended. The specificity of the fluorescent signal was determined by IETD-fmk and LEHD-CHO, the specific inhibitors for the caspase-8 and caspase-9 respectively.
We thank Dr. Morris Kates for critical reading of the manuscript. This work is sponsored by operating grant from Canadian Institutes of Health Research (CIHR). During the course of this study, Q.L. is supported by investigator award from CIHR.
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