Download Sodium valproate induces mitochondria

Sodium valproate induces mitochondria-dependent apoptosis in
human hepatoblastoma cells
Wei WANG, Xiao-li LIAO, Jing-hong CHEN, Dan-dan LI,Chun-lan LIN, Yu-xia YAN,
Yu-hui TANG and Jian-wei JIANG
Department of Biochemistry, Medical College of Jinan University, Guangzhou
510632, Guangdong Province, China (Wei WANG, Xiao-li LIAO, Jing-hong CHEN,
Chun-lan LIN, Yu-xia YAN, Jian-wei JIANG)
State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-sen
University, 651 Dong feng Road East, Guangzhou 510060,Guangdong Province,
China.( Dan-dan LI)
Department of Biochemistry, Guangzhou University of Chinese Medicine,Guangzhou
510006 Guangdong Province, China (Yu-hui TANG)
Correspondence to: Pro. Jian-wei JIANG, Department of Biochemistry, Medical
College of Jinan University, Guangzhou 510632, Guangdong Province, China (TEL:
+020-85220256,Fax: +020-85221314, E-mail: [email protected])
This study was supported by grants from Sci-Tech Project Foundation of Guangdong
Province, China (2009B080701051) and the National Natural Science Foundation of
China, No. 30973811.
Keywords: hepatoblastoma; sodium valproate; JNK; mitochondria; apoptosis
ABSTRACT
Background Sodium valproate inhibits proliferation in neuroblastoma and glioma cells, and
inhibits proliferation and induces apoptosis in hepatoblastoma cells. Information describing the
molecular pathways of the antitumor effects of sodium valproate is limited; therefore, we explored
the mechanisms of action of sodium valproate in the human hepatoblastoma cell line, HepG2.
Methods The effects of sodium valproate on the proliferation of HepG2 cells were evaluated by
the Walsh–schema transform (WST) and colony formation assays. Sodium valproate-induced
apoptosis in HepG2 cells was investigated with fluorescence microscopy to detect morphological
changes; by flow cytometry to calculate DNA ploidy and apoptotic cell percentages; with western
blot analyses to determine JNK, p-JNK, Bcl-2, Bax, and caspase-3 and -9 protein expression
levels; and using JC-1 fluorescence microscopy to detect the membrane potential of mitochondria.
Statistical analyses were performed using analysis of variance by SPSS 13.0 software.
Results Our results indicated that sodium valproate treatment inhibited the proliferation of HepG2
cells in a dose-dependent manner. Sodium valproate induced apoptosis in HepG2 cells as it:
caused morphologic changes associated with apoptosis, including condensed and fragmented
chromatin; increased the percentage of hypodiploid cells in a dose-dependent manner; increased
the percentage of annexin V-positive/propidium iodide-negative cells from 9.52% to 74.87%;
decreased JNK and increased phosphate-JNK protein expression levels; reduced the membrane
potential of mitochondria; decreased the ratio of Bcl-2/Bax; and activated caspase-3 and -9.
Conclusion Sodium valproate inhibited the proliferation of HepG2 cells, triggered
mitochondria-dependent HepG2 cell apoptosis and activated JNK .
INTRODUCTION
Liver cancer is the third most common cancer in the world and a leading cause of cancer
morbidity and mortality. Hepatocellular carcinoma (HCC) is a primary malignant tumor of the
liver that is associated with poor prognosis. Traditional treatment regimes for HCC include
surgical techniques and various chemotherapy drugs1; however, significant survival benefit from
these protocols is unlikely. In particular, chemotherapy has variable effects as HCC is extremely
chemoresistant; therefore, the search for new reliable chemotherapy regimens must be actively
pursued. Recently, sodium valproate was shown to inhibit the proliferation of cancer cells in
neuroblastoma and glioma 2-5, and effect the proliferation and apoptosis of human hepatoblastoma
HepG2 cells6. Sodium valproate is a histone deacetylase inhibitor that has been used to treat
epilepsy7. Valproic acid possesses anti-manic properties and is an important mood stabilizer in the
treatment of bipolar disorder8. It inactivates the inhibitory neurotransmitter GABA through the
inhibition of GABA transaminase and has acute inhibitory effects on neuronal excitation in the
central nervous system. Presently, information describing the molecular mechanisms of the
antitumor effects of sodium valproate is limited.
Mitogen-activated protein kinases (MAPKs) are a family of proline-directed Ser/Thr kinases
activated by dual phosphorylation. Three MAPK groups have been the focus of intense study: the
extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38
MAPKs9, 10. The ERK cascade is activated by growth factors and is critical for proliferation and
survival11. The p38 and JNK pathways are simultaneously activated in response to a variety of
cellular and environmental stresses such as pro-inflammatory cytokines, genotoxic agents, and
apoptosis12-15.
In the present study, we demonstrated that sodium valproate inhibited the proliferation of
human hepatoblastoma HepG2 cells and induced apoptosis by reducing the membrane potential of
mitochondria and activating caspases-3/9. Furthermore, we found that JNK was involved in the
antitumor activity of sodium valproate.
METHODS
Cells and reagents
HepG2 cells (human hepatoblastoma cell line) were maintained in RPMI 1640 (Gibco Corporation,
Carlsbad, California, USA) supplemented with 10 % newborn bovine serum (Gibco Corporation)
at 37ºC and 5% CO2. Sodium valproate was dissolved in PBS to make a stock solution of 30
mmol/L. The primary antibodies used for immunoblot analyses were anti-caspase-3 (#9662; Cell
Signaling, Danvers, Massachusetts, USA), anti-caspase-9 (#9502; Cell Signaling), anti-JNK
(#9525; Cell Signaling), anti-P-JNK (#9251; Cell Signaling), anti-Bcl-2 (#2876; Cell Signaling),
anti-Bax (#2772; Cell Signaling) and anti-GAPDH (#3683 Cell Signaling). Cell lysis buffer was
purchased from Cell Signaling.
WST-8 cell proliferation assay
HepG2 cells were plated at a density of 5 × 104 cells/ml in 96-well plates (Corning, USA). After
24 h, sodium valproate was added at final concentrations of 0, 2, 4, 6, and 8 mmol/L. After 48-h
incubation, 10 μl of cck-8 reagent was added to each well. Following another 1.5 h incubation,
absorbance was determined at 450 nm with a reference wavelength of 570 nm, using a micro titer
plate reader (Bio-Rad, USA). Cell proliferation inhibitory rates were calculated according to the
formula: proliferation inhibitory rates = (A control group-A test group)/A control group × 100%.
Plate colony formation assay
Colony formation was detected using a plate colony formation assay. Logarithmically growing
HepG2 cells were seeded in duplicate at a concentration of 400 cells/well in 12-well flat-bottom
microplates with 2 ml RPMI 1640 containing 15% newborn bovine serum. Cells were incubated
with different concentrations of sodium valproate for 7 days. Cell colonies were fixed in
methanol/acetone (3:1, v/v), stained with crystal violet for 20 min, and photographed.
Flow cytometry
Flow cytometry (Beckman Coulter, Fullerton, California, USA) was used to identify apoptotic
cells by DNA fragmentation analysis, and to determine the apoptotic rate. For DNA fragmentation
analysis, HepG2 cells were incubated with 0, 2, 4, 6, and 8 mmol/L sodium valproate for 48 h,
harvested, washed with PBS, and fixed in 70 % ice-cold ethanol overnight. Fixed cells were
incubated with 20 units/ml RNase I and 50 μg/ml propidium iodide (PI) for 30 min. Cells in the
sub-G1 phase of the cell-cycle were identified as apoptotic. To determine the apoptotic rate,
HepG2 cells were seeded in 6-well plates at a density of 2 × 104 cells/ml, incubated with sodium
valproate for 48 h, and collected. Annexin V- FITC/ PI (Becton Dickinson, USA) staining was
performed following the manufacturer’s protocol. Phosphatidyl serine translocation to the cell
surface is an indicator of early apoptotic cells; therefore, annexin V-positive, PI-negative cells
were identified as apoptotic. The apoptotic rate was determined using CellQuest software (FCM,
Becton Dickinson, USA).
Fluorescence microscopy
Sodium valproate-induced apoptosis in HepG2 cells was visualized using Hoechst 33258 staining.
Cells were treated with 5 mmol/L sodium valproate for 48 h, harvested, and smeared on slides.
The slides were air dried, fixed in methanol/acetone (3:1, v/v), and stained with Hoechst 33258 (5
µg/ml) for 20 min. Nuclear morphological changes were examined using fluorescence microscopy
(DFC480; Leica Microsystems, Wetzlar, Germany).
Immunoblot assay
HepG2 cells were treated with 0, 2, 4, 6, and 8 mmol/L sodium valproate for 48 h. Whole cell
lysates were prepared by adding 2 × SDS sample buffer. Equal amounts of protein were
electrophoresed on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were
blocked for 60 min at room temperature with 5% nonfat dry milk/TBS-Tween 20 (TBST) and
reacted with appropriate antibodies for JNK, phosphate-JNK, Bcl-2, Bax, caspase-9, caspase-3, or
PARP (1:1000 dilution in blocking buffer) overnight at 4ºC. Subsequently, membranes were
washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for
1 h at room temperature. Proteins were visualized by incubation with SuperSignal West Pico
reagent (NCI5079 Thermo) and exposure to radiograph film (Kodak, Rochester, New York,
USA).
Analysis of mitochondrial membrane potential
HepG2 cells were treated with 0, 2, and 4 mmol/L sodium valproate for 48 h, harvested, washed
with PBS, fixed in JC-1 solution for 30 min at 37ºC, and smeared on slides. The membrane
potential of mitochondria was examined under a fluorescence microscope.
Statistical analysis
All assays were performed in triplicate. Data are expressed as the mean ± SD. Statistical analyses
were performed using analysis of variance by SPSS 13.0 software. A value of P < 0.05 was
considered statistically significant.
RESULTS
Sodium valproate inhibits HepG2 cell proliferation
The proliferation of HepG2 cells was significantly inhibited in a dose-dependent manner by
exposure to sodium valproate for 48 h (P < 0.05 vs. the control group; Fig 1.A). The IC50 value of
sodium valproate for HepG2 cells was 5.38 mmol/L. In addition, colony formation was
significantly decreased when HepG2 cells were exposed to various concentrations of sodium
valproate for 7 days (Fig 1.B).
Sodium valproate induces apoptosis in HepG2 cells
Inhibition of cell proliferation could result from the induction of apoptosis, cell growth arrest, and/
or the inhibition of growth. We investigated whether sodium valproate induced apoptosis in
HepG2 cells. The percentage of cells in the hypodiploid DNA peak (sub-G1 population) and
annexin V-FITC/PI double staining were used as indicators of apoptosis. Sodium valproate
treatment increased the sub-G1 cell population and the percentage of annexin V-positive/
PI-negative cells (from 9.52% to 74.87%) (Fig. 2.B).
In addition, cells were stained with Hoechst 33258 to detect the morphologic changes of
apoptotic nuclei using fluorescence microscopy. Following exposure to 5 mmol/L sodium
valproate (approx. IC50) for 48 h, HepG2 cells exhibited typical apoptotic characteristics
including cell shrinkage, apoptosis-chromatin condensation, and apoptotic body formation (Fig.
2.C).
Effect of sodium valproate on intracellular signaling protein expression levels
To explore the molecular mechanisms of sodium valproate-induced apoptosis, HepG2 cells were
treated with different concentrations of sodium valproate for 48 h, and JNK and p-JNK protein
expression levels were detected. Immunoblot assays indicated that sodium valproate increased the
phosphorylation of JNK and decreased JNK expression in a dose-dependent manner (Fig.3.A). As
the intrinsic pathway of apoptosis is regulated by members of the Bcl-2 protein family, Bcl-2 and
Bax protein expression levels were also analyzed. Immunoblot assays revealed that sodium
valproate caused a significant decrease in Bcl-2 expression, and that Bax was unaffected. The ratio
of Bcl-2/Bax was reduced (Fig. 3.B).
Sodium valproate activates caspases-3 and -9 and changes the membrane
potential of mitochondria in HepG2 cells
To investigate the role of caspases in sodium valproate-induced apoptosis, we investigated the
activation of caspase-3 and -9 in HepG2 cells treated with different concentrations of sodium
valproate for 48 h. The results showed that sodium valproate activated caspase-3 and -9 in a
dose-dependent manner (Fig. 4.A).
In addition, cytofluorometry was used to analyze the membrane potential of the mitochondria
in HepG2 cells after sodium valproate treatment using the lipophilic cationic probe,
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1)16. This
membrane-permeable fluorochrome can penetrate cells and its fluorescence is a reflection of the
mitochondrial membrane potential. When live cells are incubated with JC-1, mitochondrial
membrane polarization allows JC-1 to selectively enter mitochondria which causes the reversible
formation of JC-1 aggregates. JC-1 aggregates show a spectral shift in emitted light from 530 nm
(emission of JC-1 monomeric form; green) to 590 nm (emission of J-aggregate; green-orange)
upon excitation at 490 nm. Our data showed reduced levels of orange fluorescence in HepG2 cells
treated with 2 and 4 mmol/L sodium valproate for 48 h compared with the control (Fig.4.B).
DISCUSSION
In this study, we investigated the effect of sodium valproate on the human hepatoblastoma HepG2
cell line. We found that sodium valproate effectively inhibited the proliferation of HepG2 cells
and induced apoptosis in a dose-dependent manner. Our results suggest that sodium valproate has
potential as an antitumor drug.
Previous studies indicate that sodium valproate induces cell death17. In accordance with these
reports, our results showed that sodium valproate induced apoptosis and caused typical apoptotic
morphology including cell shrinkage, nuclear condensation, and nuclear fragmentation in HepG2
cells. Taken together, these data indicate that the antitumor effect of sodium valproate on HepG2
cells is due to the induction of cell apoptosis.
We investigated the mechanism of action of sodium valproate on HepG2 cells. Sodium
valproate phosphorylated and activated JNK, inhibited Bcl-2 protein expression, and had no effect
on Bax. Bcl-2 and Bax are members of the Bcl-2 family of proteins which are central regulators of
the mitochondrial pathway of apoptosis. The Bcl-2 family is composed of pro- and antiapoptotic
proteins. Antiapoptotic members such as Bcl-2 promote cell survival by inhibiting the functions of
the proapoptotic proteins. Under normal conditions, Bax is localized to the cytosol; however, in
response to death stimuli, Bax undergoes a conformational change that triggers its translocation to
and insertion into the outer mitochondrial membrane. This leads to the permeabilization of the
outer mitochondrial membrane and the release of proapoptotic proteins, including cytochrome c.
Released cytochrome c interacts with apoptotic protease activating factor-1 and pro-caspase-9 to
form the apoptosome. This generates mature caspase-9 and begins a proteolytic cascade,
ultimately resulting in cell death18-20. We observed a sodium valproate-induced decrease in the
ratio of Bcl-2/Bax in HepG2 cells and propose that this resulted in the initiation of the apoptotic
pathway.
Mitochondria play a crucial role in the apoptotic signal transduction pathway21. The change
in membrane potential of mitochondria activates both caspase-3 and -9 and is an important
upstream event of mitochondrial-dependent apoptosis22. In our study, sodium valproate activated
caspase-3 and-9 in HepG2 cells in a dose-dependent manner. This suggests that sodium
valproate-induced apoptosis in hepatoblastoma cells results from caspase activation and the
mitochondrial apoptotic pathway.
In summary, we found that sodium valproate inhibits the proliferation and induces apoptosis
of HepG2 cells through an intracellular signaling pathway involving the activation of JNK, the
Bcl-2 proteins, and caspases-3 and-9. Further studies are needed to fully understand the
mechanism of action of sodium valproate and should investigate the expression of other Bcl-2
anti-apoptosis family members such as Bcl-xl, Mcl-1, and the BH-3 domain-only proteins; the
translocation of Bax from the cytosol to mitochondria; and the relationship between JNK
activation, Bcl-2 proteins, and the mitochondria-dependent cell apoptosis signaling pathway. Our
data identify sodium valproate as a potential novel therapeutic agent for use against human
hepatoblastoma cells. Furthermore, as sodium valproate is a drug traditionally used to treat
epilepsy, our study may provide useful information describing the relationship between nervous
disorders and cancer.
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Figure 1. A: Sodium valproate-induced inhibition of HepG2 cell proliferation. HepG2 cells were incubated with
sodium valproate for 48 h. Cell survival rates were determined and an IC50 value was calculated. Values represent
the means ± SD of three independent experiments; P < 0.05 vs. the control group. B: Sodium valproate-induced
inhibition of HepG2 cell colony formation. Data represent one of three experiments yielding similar results.
Figure 2. Induction of apoptosis in HepG2 cells by sodium valproate. A: Sodium valproate-induced apoptosis was
determined by annexin V-FITC and PI staining. HepG2 cells were treated with 0, 2, 4, 6, and 8 mmol/L sodium
valproate for 48 h. Data shown are representative of 3 different experiments. B: HepG2 cells were treated with 0, 2,
4, 6, and 8 mmol/L sodium valproate for 48 h. Data represent one of three experiments yielding similar results. C:
Hochest 33258 staining detected morphological changes in the nuclei of control HepG2 cells and those treated
with 5 mm/L sodium valproate for 48 h. The images were captured by fluorescence microscopy. (Crystal violet
staining; original magnification X 100)
Figure 3. Effects of sodium valproate on intracellular signaling protein expression levels in HepG2 cells. A:
Sodium valproate activates the JNK pathway in HepG2 cells. HepG2 cells were treated with 0, 2, 4, 6, and 8
mmol/L sodium valproate for 48 h. The effects of sodium valproate on JNK protein expression levels were
evaluated by immunoblotting. B: Sodium valproate caused a significant decrease in Bcl-2 but not Bax protein
levels. HepG2 cells were treated with 0, 2, 4, 6, and 8 mmol/L sodium valproate for 48 h. Bcl-2 and Bax protein
expression levels were detected by western blot. Data represent one of three experiments yielding similar results.
Figure 4. Sodium valproate activates caspases-3 and -9 and changes the membrane potential of mitochondria in
HepG2 cells. A: Sodium valproate activates caspase-3 and 9. HepG2 cells were treated with 0, 2, 4, 6, and 8
mmol/L sodium valproate for 48 h. The effects of sodium valproate on caspase-3 and -9 protein expression levels
were evaluated by immunoblotting. B: Sodium valproate changes mitochondrial membrane potential. HepG2 cells
were treated with 0, 2, and 4 mmol/L sodium valproate for 48 h. The change in mitochondrial membrane potential
was evaluated by fluorescence microscopy. Data represent one of three experiments yielding similar results.
(Crystal violet staining; original magnification X100)
.