Sodium butyrate-induced DAPK-mediated apoptosis in human gastric cancer cells

Epigenetic mechanisms of histone acetylation/deacetylation play an important role in the regulation of gene expression associated with the cell cycle and apoptosis. Recently, sodium butyrate, a histone deacetylase (HDAC) inhibitor, has been shown to exhibit anticancer effects via differentiation and apoptosis of cancer cells. Sodium butyrate may be a potential anticancer chemotherapeutic drug; however, the precise mechanism underlying the anticancer effects of sodium butyrate has not been clearly elucidated. In the present study, we investigated the role of death-associated protein kinase (DAPK) on the apoptosis of human gastric cancer cells induced by sodium butyrate. We observed that sodium butyrate induced apoptosis in human gastric cancer cells. Treatment with the HDAC inhibitor sodium butyrate increased the expression of caspase-3 and DAPK1/2 genes but decreased the expression of Bcl-2 in human gastric cancer cells. The expression of DAPK3, p53 and p21 were not altered by sodium butyrate treatment. Analysis of the general expression patterns revealed that sodium butyrate increased the expression of DAPK1/2 but decreased the expression of FAK and induced changes in the proliferation of apoptosis-related genes in human gastric cancer cells. These data suggest that DAPK expression prompts apoptosis by reducing the FAK protein level in sodium butyrate-induced apoptosis of human gastric cancer cells.


Introduction
Histone deacetylase (HDAC) inhibitors represent a structurally diverse group of compounds that inhibit the deacetylation of histones, permitting the chromatin scaffolding to assume a more relaxed, open conformation, which generally promotes gene transcription. HDAC inhibitors induce apoptosis in cancer cells through multiple mechanisms, and thus are emerging as a promising new therapeutic tool for the treatment of a variety of human cancers (7). Sodium butyrate (NaB), a short chain fatty acid, occurs naturally in the body and is synthesized through the acetyl-CoA-dependent catabolic oxidation of long chain saturated fatty acids (1,2). Sodium butyrate, acting as an HDAC inhibitor, is known to exhibit anticancer effects via differentiation of carcinoma cells (3)(4)(5)(6). NaB is a novel chemotherapeutic agent as it is able to activate a variety of anticancer mechanisms, including cell cycle arrest and cellular differentiation (8)(9)(10). Sodium butyrate induces apoptosis, decreases Bcl-2 transcription (11), increases TNF-related apoptosis-inducing ligand receptor 2 gene transcription to accelerate the deathinducing signaling complex formation, activates caspase, and inhibits the mitochondrial membrane potential of cancer cells (12). These results suggest that NaB may represent a potential new class of anticancer agents with low toxicity; however, the mechanisms of action are not fully understood.
DAPK induces programmed cell death through various signaling pathways. FAK participates in signaling pathways involved in adhesion between cells and the extracellular matrix, including proteins such as fibronectin, laminin, actin, and fodrin. FAK is a survival protein that suppresses apoptosis and maintains cell suspended growth (26). In this study, we attempted to elucidate NaB-induced death-associated protein kinase expression and its association with human gastric cancer cell apoptosis.

Sodium butyrate-induced DAPK-mediated apoptosis in human gastric cancer cells
RNA isolation and reverse transcription polymerase chain reaction. Total RNA was prepared using TRIzol reagent (Invitrogen). The RNA was reverse transcribed using oligo (dT) primers and SuperscriptT™ Ⅱ reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Primers used for PCR amplification of this cDNA were caspase-3

Sodium butyrate inhibited cell proliferation.
We examined the effects of NaB on human gastric cancer cell proliferation.  (Fig. 1).
Sodium butyrate induced apoptosis of human gastric cancer cells. Cell apoptosis was determined by counting sub-G1 phase cells with flow cytometry analysis. After 48 h of NaB treatment, cell apoptosis significantly increased from 0.05 to 7.2% in AGS and from 0.07 to 5.83% in MKN45 (both P<0.05). These findings suggested that NaB induced cell apoptosis (Fig. 2).
Effect of sodium butyrate on apoptosis-regulated protein expression. Sodium butyrate increased the expression of caspase-3, DAPK1, and DAPK2 but decreased the expression of Bcl-2 in both AGS and MKN45 cells. The expression of DAPK3, p53 and p21 genes was not altered by NaB treatment (Fig. 3). These finding suggests that NaB induces DAPK1/2 expression in human gastric cancer cells.

Sodium butyrate increased DAPKs expression and decreased the expression of FAK in human gastric cancer cells.
To confirm DAPKs expression induced by NaB treatment, Western blot analysis was performed 48 h after treatment with NaB. Expression of DAPK1/2 was increased, while that of FAK was decreased in human gastric cancer cells (Fig. 4). These finding suggest that NaB induced the expression of DAPK1/2, leading to decreased protein levels of FAK, prompting cell apoptosis.

Increased DAPKs expression and decreased FAKs expression induced by sodium butyrate treatment demonstrated by immunofluorescence.
To confirm DAPKs expression induced by NaB, an immunofluorescence assay was analyzed 48 h after treatment of cells with NaB. Sodium butyrate increased the expression of the DAPK1/2 protein but decreased the expression of FAK. These findings suggest that NaB induced the
In the current study, we aimed to assess sodium butyrateinduced death-associated protein kinase expression, which promotes human gastric cancer cell apoptosis. We first sought to assess the role of sodium butyrate in human gastric cancer cells using several independent approaches. Our results suggest that sodium butyrate inhibits cell proliferation and induces cell apoptosis in human gastric cancer cells.
The level of caspase-3 mRNA expression increased in human gastric cancer cells following treatment with sodium butyrate. Although no studies support the fact that the transcription of caspase-3 can be upregulated directly by histone acetylation, recent evidence suggests that HDAC inhibitors also enhance the acetylation of non-histone proteins (24,25). Down-regulation of Bcl-2 was present after 48 h of exposure to sodium butyrate. These findings suggest that caspase-3 was increased by sodium butyrate in human gastric cancer cells, but not Bcl-2. The p53 gene is not involved in butyrateinduced growth inhibition of breast cancer cells (26). The current study also demonstrates that sodium butyrate did not induce p53 or p21 expression. Recently, it has been reported that the histone deacetylase inhibitor, TSA, could induce cells to express DAPK and promote cell apoptosis (24). DAPK expression causes tumor cells to lose sensitivity to anoikis, enabling anchor-independent survival of tumor cells (25). Our results found that DAPK1/2, but not DAPK3, expression was increased by sodium butyrate treatment in human gastric cancer cells. DAPK1/2 is responsible for the induction of apoptosis, while DAPK3 usually induces morphological changes in apoptosis.
FAK is involved in adhesion between cells and the extracellular matrix, and joins cytoskeletal proteins. FAK is a survival protein that suppresses apoptosis (23). Our experiments demonstrate that sodium butyrate induced DAPK1/2 expression but down-regulated FAK expression in human gastric cancer cells. These findings suggest that a sodium butyrate-induced DAPK-dependent decrease in FAK via the caspase-dependent pathway leads to apoptosis in human gastric cancer cells. In conclusion, sodium butyrate induced DAPK expression in human gastric cancer cells and this expression prompted apoptosis by decreasing FAK levels.