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Introduction
Sodium butyrate is a short chain, fatty acid sodium salt
that was produced by the carbohydrate fermentation of
bacteria. The concentration of sodium butyrate can attain 5
mmol/L in the human colon. Evidence has shown that
sodium butyrate was the major fuel for colonic epithelial cells,
and it can influence cell proliferation by the release of growth
factors or gastrointestinal peptides, such as gastrin, or by
the modulation of mucosal blood flow[1]. It was suggested
that sodium butyrate had a weak toxicity to normal cells.
Sodium butyrate causes the histone acetylation by
inhibiting the activation of histone deacetylase. Sodium
butyrate is not only a deacetylase inhibitor, but also has the
function of demethylation[2_4].
Sodium butyrate could induce the transformation of cell
differentiation or apoptosis[2,3]. Heerdt
et al demonstrated that sodium butyrate and its analogue could arrest MCF-7
cell growth. They prompted cell apoptosis by the
mitochondrial pathway[2]. Duan et
al also demonstrated that sodium butyrate and trichostatin A (TSA) induced apoptosis and
downregulated the transcription of
Bcl-2[5].
Recently, Earel et al showed that sodium butyrate
and trichostatin A (TSA) increased TNF-related
apoptosis-inducing ligand receptor 2 gene transcription to accelerate the
death-inducing signaling complex formation, caspase
activation, and loss of mitochondrial membrane potential of
tumor cells[6].
The above results showed that sodium butyrate was a
novel chemotherapeutic drug with weak toxicity; however,
the precise mechanism has not been elucidated.
Raji cells were derived from a Burkitt lymphoma. Normally,
Raji cells are suspended and grow in medium.
Death-associated protein kinase (DAPK), which is a
Ca2+/CaM regulation protein kinase and executes program cell death in various
signal transduce pathways, was silenced in the Raji cells
because of the methylation of the DAPK gene
promoter[7]. In our earlier studies, we reported that Raji cells expressed
DAPK and displayed protrusions to adhere to matrices
induced by sodium butyrate[8].
In the present study, we determined whether DAPK
expression would prompt Raji cell apoptosis and whether
adhesion-growth Raji cells would be more sensitive to anoikis
than suspended-growth Raji cells, thus, we investigated its
possible mechanism.
Materials and methods
Cell culture The Raji cell line, offered by Sun Yat-Sen
University Cancer Center (Guangzhou, Guangdong, China)
was cultured in the RPMI-1640 medium (Gibco BRL, Grand
Island, NY, USA) containing penicillin 100 µg/mL and
streptomycin 100 µg/mL and supplemented with 10% calf blood
serum (Sijiqing Laboratories, Hangzhou, China) at 37 °C in a
humidified atmosphere with 5% CO2.
Effect of sodium butyrate on the morphology of Raji cells
The Raji cells were cultured in 6-well plates
(3.0×106 cells/well) and treated with 3 mmol/L sodium butyrate (Sigma, St
Louis, MO, USA) at 37 °C in a humidified atmosphere with
5% CO2 for 2 d. Cell morphology was observed with a
scanning electron microscope (JSM-T300, Shimadzu,Kyoto,
Japan) at the Guangzhou Medical College Electron
Microscope Center (Guangzhou, China).
Effects on apoptosis by preventing Raji cells adhesion
Each well of the 24-well plates was covered with 1mL
polyHEME (10 mg/mL; Sigma, USA) dissolved in ethyl
alcohol. After the ethyl alcohol had been air-dried, each well
of the 24-well plates was washed with
1×phosphate-buffered saline
(PBS)[9].
The Raji cells were divided into 3 groups. The first group
was cultured in polyHEME-coated, 4-well plates and named
the polyHEME group; the second group was cultured in the
normal 24-well plates and named the non-polyHEME group;
the third group was the control group. The cell number of
every well was 2.0×105 cells. The polyHEME and
non-polyHEME groups were treated with 3 mmol/L sodium
butyrate for 2, 4, 6, 8, and 10 d, separately. The control group
was cultured in the normal medium. The cells in the control
group were supplied with 0.5 mL medium every 4 d.
Simultaneity, the sodium butyrate-treatment group cells were
supplied with 0.5 mL medium containing 3 mmol/L sodium
butyrate.
Some cells samples were taken from each well to
determine cell viability by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA) assay
every 2 d[10]. Briefly, the number of viable cells in each well was
estimated by adding 10 µL 0.5 mg/mL MTT solution. The
cells were dissolved with 100 µL solution that contained
20% SDS and 50% dimethyl formamide after the cells had
been incubated for 4 h at 37 oC. The optical densities were
quantified at 570 nm (Bio-Rad model 540, Hercules, CA, USA).
The results were estimated by absorbency. In each well, the
remains of the cells were centrifuged and washed twice in 1×
PBS and incubated in 1× PBS containing 100 µg/mL propidium
iodide (PI) (Sigma, USA) and 200 µg/mL RNase after the cells
were fixed with ice-cool 70% ethanol overnight. The cells
were then analyzed at an excitation wavelength of 488 nm by
flow cytometry (EPICS XL, Coulter, Fullerton, CA, USA).
The percentage of degraded DNA was determined by the
number of cells with subdiploid DNA divided by the total
number of cells examined under each experimental
condition[10].
A morphological study was performed as described
previously[11]. After being treated with sodium butyrate, the
cells were collected, washed with 1× PBS, and incubated in
1×PBS containing 10 µg/mL Hoechst33258 (Sigma, USA) for
10 min. Nuclear morphology was examined using
fluorescence microscopy (Olympus DX71, Olympus Corporation,
Tokyo, Japan).
DAPK and FAK expression analysis in Raji cells
The cells were loaded with cell decomposition buffer (pH 8.0)
that contained 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5
mmol/L EDTA, 1% NP40, 0.05% phenylmethanesulfonyl
fluoride(PMSF), 2 µg/mL aprotinin (Sigma, USA), and 2 µg/mL
leupeptin (Sigma, USA) after centrifugation and washed twice
in 1×PBS. The cells were then treated 3 times for 10 s with
ultrasonic processing on ice. The proteins were determined
as described previously by Western
blotting[12] using the DAPK monoclonal antibody (clone-55, Sigma, USA), rabbit
antihuman FAK (C-20, Santa Cruz Biotechology, Santa Cruz,
California USA), anti-actin (pan Ab-5, clone actn05, Lab
Vision, Fremont, CA, USA), and Western blotting luminol
reagent (Amersham Biosciences, Uppsala, Sweden).
Construction of expression plasmid for C-terminal
peptides of DAPK The C-terminal coding peptides of DAPK
were amplified from
pcDNA3.1(+)-DAPK[13] with the PCR method and subcloned into the expression vector
pcDNA3.1(+), forming
pcDNA3.1(+)-DCTP[13].
The Raji cells were transfected with a pcDNA3.1(+)-DCTP
using Lipofectamine 2000 transfection reagent (Invitrogen,
Carlsbad, California, USA) according to the manufacturer's
protocol. Stable cell lines were cultured in medium
containing 1000 µg/mL G418 (Invitrogen, USA). Three weeks later,
the cells were cultivated in the medium that contained
500µg/mL G418. The Raji cells transfected with pcDNA3.1(+)-DCTP
were induced by sodium butyrate. The morphology of the
cells was observed under a microscope (Olympus DX71) and
cell apoptosis was detected by flow cytometry.
Data analysis Data were expressed as mean±SD.
Statistical significance was evaluated using the Student's
t-test. P<0.05 was considered to be statistically significant.
Results
Effect of sodium butyrate on Raji cells After being treated
with sodium butyrate, the suspended-growth Raji cells
adhered to the bottom of the culture flask (Figure 1). The
newborn fibrous pseudopods structures occurred on the cell.
Effect on cell growth by preventing Raji cell adhesion
After being treated with sodium butyrate, the survival of the
Raji cells was reduced in a time-dependent manner if cell
adhesion was suppressed (Figure 2). In contrast, the cells
treated with sodium butyrate continued to grow slowly if the
cell adhesion was permitted. The control group cells
proliferated so rapidly that they attained to plainness phase in 4 d.
Suppressing adherence increased the apoptotic Raji cell
ratio The apoptotic cells induced by sodium butyrate
increased in a time-dependent manner. Significant DNA
condenses were observed (Figure 3). The cells that were
suppressed from adhesion were more sensitive to apoptosis than
free cells (Figure 4). DNA degradation examined by flow
cytometry showed more apoptotic cells when the Raji cells
were suppressed from adhesion.
Sodium butyrate induced DAPK expression and
decreased the expression level of total FAK in Raji cells
The results showed that the expression of DAPK increased, while
that of total FAK decreased in the presence or absence of
poly-HEME (Figure 5). It was suggested that sodium
butyrate induced the expression of DAPK and then caused the
protein levels of total FAK to decrease, prompting cell
apoptosis. In addition, the results showed that the change
of DAPK and FAK expression induced by sodium butyrate
was identical in the presence or absence of poly-HEME.
pcDNA3.1(+)-DCTP suppresses apoptosis induced by
sodium butyrate It was found that pcDNA3.1(+)-DCTP
reduced the pseudopod structure of Raji cells induced by 3
mmol/L sodium butyrate treatment for 4 d (Figure 6). In
addition, we discovered that the Raji cells transfected with
pcDNA3.1(+)-DCTP did not affect the protein levels of total
DAPK and FAK in normal medium. However, it could
suppress the decrease of the total FAK protein levels induced
by sodium butyrate (Figure 7).
After being transfected with pcDNA3.1(+)-DCTP, cell
apoptosis decreased (P<0.05). The apoptosis ratio of the
pcDNA3.1(+)-DCTP transfected group was much lower than
the pcDNA3.1(+) group (P<0.05) if cell adhesion was
prevented. The apoptosis ratio of the Raji cells transfected
with pcDNA3.1(+)-DCTP showed no significant differences
between the polyHEME-treated and untreated groups. It
was indicated that the pcDNA3.1(+)-DCTP group could
resist the anoikis induced by sodium butyrate. On the
contrary, the Raji cells transfected with pcDNA3.1(+) were
susceptive to anoikis (P<0.05; Figure 8).
Discussion
Benjamin had indicated that sodium butyrate caused
histone acetylation by suppressing the activity of histone
deacetylase. Simultaneity, sodium butyrate induced
promoter demethylation and reversed the methylation-mediated
suppression of gene expression[3]. Similarly, we previously
reported that sodium butyrate could induce demethylation
of the DAPK gene promoter to inhibit cell growth. Our
experiments proved that Raji cell growth was inhibited
significantly when treated with sodium butyrate at the
concentration of 0.75_6 mmol/L. Sodium butyrate trapped the cell
cycle at the G0/G1
phase[8]. Recently, Zhang et al reported
that histone deacetylase inhibitor TSA improved the
chemotherapeutic efficacy of gastric carcinoma when treated in
combination with anticancer drugs (5-FU, PTX, and SN38)
by the upregulation of DAPK[15]. They indicated that the
histone deacetylase inhibitor could induce cells to express
DAPK and prompt cell apoptosis. The histone deacetylase
inhibitor may be a potential chemotherapeutic drug.
The DAPK expression default causes tumor cells to lose
sensibility to anoikis, so as to enable the
anchor-independent survival of tumor
cells[16]. Previous reports showed that the methylation of the DAPK gene promoter in Raji cells
could silence DAPK expression. It would promote the
apoptosis of Raji cells when the expression of DAPK was
resumed in Raji cells[7,8]. Our results showed that sodium
butyrate induced DAPK expression to promote Raji cell
apoptosis.
DAPK is a multi-domain kinase located in myosin-II
regulatory light chains (MLC). Through its
cystoskeleton-binding region, DAPK phosphorylates MLC and changed the
cell morphology[17].
It had been observed that the cells could have obvious
morphological changes in DAPK-promoted HeLa cells and
293 cells. HeLa and 293 cells were induced to grow many
fibrous pseudopod structures. There were many plate-type
foot structures on the cell surface[18], which was in
accordance with the results drawn by Kuo et
al[19]. Our study showed that the rounded Raji cells grew into many fibrous
pseudopod structures. The fibrous pseudopods converted
the suspended growth to the adhesive growth of the Raji
cells. Meanwhile, some cells underwent apoptosis when the
cells adhered to the bottom of the culture flask. However, if
cell adhesion was suppressed by polyHEME, the
percentage of the apoptotic cells would increase. The results showed
that the Raji cells induced by sodium butyrate became more
susceptive to anoikis grown in an anchor-dependent manner.
Previous reports have shown that DAPK is an important
apoptosis inducer[7,12_17]. Recently, Kuo
et al reported that DAPK blocked migration and invasion independently of
DAPK-induced apoptosis and DAPK regulated cell polarity
to induce cell morphological change during migration, which
may act together with its apoptotic function to suppress
tumor progression[19]. It seemed that sodium butyrate
induced DAPK expression in the Raji cells so as to cause
change of cell morphology. In addition, Raji cells can resist
DAPK-induced anoikis by adhesion on the culture flask. If
the cells lost their ability to adhere onto the culture flask,
they would undergo apoptosis.
Raveh et al reported that the expression of the C-terminal
of DAPK suppressed the apoptosis that was promoted by
DAPK. It was speculated that the C-terminal fragments
suppressed the activity of DAPK by interacting with DAPK
molecules. However, the precise mechanism has not been
elucidated[20].
Kozak et al suggested that the translational initiation
region should be in accordance with "A/GNNATGG"
sequences in mRNA[21]. We screened the DAPK gene and
identified 4231_4631 bp regions of coding sequence that
contained 17 amino acid residues of C-terminal coding
sequence tail of DAPK. There were "GGCATGG" sequences,
which were a potential translational initiation region,
according to Kozak et al's theory, in 4231_4631 bp regions of
coding sequence. Our previous study showed that
pcDNA3.1(+)-DCTP did not affect the proliferation of human
embryonic lung fibroblasts. However, pcDNA3.1(+)-DCTP could
inhibit the toxicity of TNF-α[14].
Our results showed that the DAPK and FAK expression
levels in the Raji cells transfected with pcDNA3.1(+)-DCTP
was the same as normal Raji cells. It was suggested that
pcDNA3.1(+)-DCTP did not affect the expression of DAPK
and the protein levels of total FAK without sodium butyrate.
In addition, pcDNA3.1(+)-DCTP did not affect sodium
butyrate-induced DAPK expression. However, it could
suppress the decrease of total FAK protein levels induced by
sodium butyrate.
Our experiments also indicated that the Raji cells
transfected with the pcDNA3.1(+)-DCTP were able to resist the
apoptosis induced by sodium butyrate. In addition, the Raji
cells, transfected with the pcDNA3.1(+)-DCTP, lost
sensitivity to anoikis. The results demonstrated that DAPK
induced anoikis together with a reduction in cell pseudopod
quantity, indirectly implying that DAPK participated in
cytomorphology change. However, Lebakken et
al found that Raji cells transfected with the syndecan-1 gene could
quickly stick to the matrix[22]. Obviously, there are other
factors affecting the Raji cell cytomorphological change in
addition to DAPK expression.
FAK participates in the signal conduction pathway of
cell adhesion between cells and the extracellular matrix, and
tightly combines some cell skeleton structure proteins,
including fibronectin, laminin, actin, and fodrin. FAK is a
survival protein that suppresses apoptosis and maintains
cell suspended growth without
anchor[23]. Wang et al found that DAPK could affect cell adhesion by changing the
conformation of integrin. DAPK restrained the activity of
integrin and blocked the cell survival signal induced by
integrin. Simultaneously, it activated the apoptosis signal
mediated by p53, disturbed the function of FAK, and
downregulated the survival signal that FAK participated
in[24]. Our experiment also demonstrated that sodium
butyrate induced the Raji cells to restore DAPK expression,
while the expression of FAK was downregulated in the Raji
cells. DAPK expression degraded the survival signal of the
cells and prompted the cells to die.
Wen et al showed that FAK was cleaved by caspases
early in the apoptotic process. They demonstrated that
Apo-2L and Fas induced the sequential proteolysis of FAK in
Jurkat T cells. It was shown that the disruption of FAK
contributed to the morphological changes observed in
apoptotic suspension and adherent
cells[25]. Kurenova et al also demonstrated that caspase-8 and the Fas-associated
death domain(FADD)-dependent pathway mediated apoptosis by the attenuation of FAK expression in tumor
cells[26]. It was well known that DAPK could activate the
caspase-dependent pathway to promote cell
apoptosis[16,17,24], so we thought that DAPK may mediate the decrease of the
total protein of FAK by the caspase-dependent pathway in
sodium butyrate-induced Raji cell apoptosis.
The Raji cells transfected with pcDNA3.1(+)-DCTP
resisted the downregulation of the FAK protein and enabled
the cells' survival. We thought that pcDNA3.1(+)-DCTP
expressed peptides to specifically suppress the DAPK
activity, so the DAPK-mediated caspase-dependent apoptosis signal conduction pathway was reduced. The
cells were able to maintain the total FAK protein level. Based
on our experimental results, DAPK expression and the
decrease of total FAK protein played a vital role in Raji cells
susceptibility to anoikis.
In this paper we have shown that: (i) sodium butyrate
induced DAPK expression and caused Raji cells to display
many protrusions all around the cells; (ii) DAPK expression
was an important reason to induce the suspended-growth
Raji cells to adhere onto the culture flask; and (iii) DAPK
expression prompted apoptosis by decreasing the total
protein of FAK.
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