Curcumin-Induced Apoptosis in Human Hepatocellular
Carcinoma J5 Cells: Critical Role of Ca
+2
-Dependent
Pathway
Wei-HsunWang,
1, 2I-Tsang Chiang,
1Kuke Ding,
3Jing-Gung Chung,
4Wuu-Jyh Lin,
5Song-Shei Lin,
6and Jeng-Jong Hwang
11Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan 2Department of Orthopedic Surgery, Changhua Christian Hospital, Changhua 500, Taiwan
3National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China 4Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
5Division of Radioisotope, Institute of Nuclear Energy Research, Taoyuan 325, Taiwan
6Department of Radiological Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan
The antitumor efects of curcumin, a natural biologically active compound extracted from rhizomes of Curcuma longa, have been
studied in many cancer cell types including human hepatocellular carcinoma (HCC). Here, we investigated the efects of Ca2+
on
curcumin-induced apoptosis in human HCC J5 cells. The abrogation of mitochondrial membrane potential (ΔΨm), the increase
of reactive oxygen species (ROS) production, and calcium release were demonstrated with flow cytometry as early as 15 minutes
after curcumin treatment. In addition, an increase level of cytochrome c in the cytoplasm which led to DNA fragmentation was
observed. To verify the role of Ca2+ in curcumin-induced apoptosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N_,N_-tetraacetic acid
(BAPTA), an intracellular calcium chelator, was applied. Cell viability was increased, but ΔΨm, ROS production, activation of
caspase 3, and cell death were decreased in J5 cells pretreated with BAPTA for 2 h followed by the treatment of 25 μM curcumin.
These results suggest that the curcumin-induced apoptosis in human HCC J5 cells is via mitochondria-dependent pathway and is
closely related to the level of intracellular accumulation of calcium.
1. Introduction
Human HCC treated with chemotherapy often turned out
with poor prognosis [1, 2]. Curcumin, one of phytochemical
compounds, has been shown with chemopreventive and
chemotherapeutic properties against tumors in animal models
and clinical trials [3–5]. Curcumin induces the apoptosis
of tumor cells through mitochondria-dependant pathways,
including the release of cytochrome c, changes in
electron
transport, and loss of mitochondrial transmembrane potential
[6]. Curcumin can stimulate intracellular Ca2+ uptake into the mitochondria [7], resulting in the stimulation of
oxidative phosphorylation, transmission, and amplification
of the apoptotic signal via the suppression of mitochondria
membrane potential and the release of cytochrome c [8].
Apoptosis induced by curcumin in human HepG2 cells has
been shown through mitochondrial hyperpolarization and
DNA damage [9]. Mitochondria are the moderator of intracellular
Ca2+ dynamics and transport through a complex system
consisting of two modes of influx and efux [7]. Oxidative
phosphorylation can be stimulated by the accumulation
of Ca2+ in the mitochondrial matrix, then transmit and amplify the apoptotic signal [8]. Apoptosis induced by
curcumin also has been reported through the prevention of
intracellular Ca2+ depletion and the release of cytochrome c
in mouse melanoma cells [10]. We hypothesize that
curcumin-induced Ca2+ release will result in mitochondrial Ca2+ overuptake to afect mitochondria membrane
potential stability.
To prove this, we choose BAPTA, an intracellular Ca2+ chelator, as the inhibitor formitochondrial Ca2+ uptake [11].
However, previous study indicates that curcumin-induced
apoptosis is through ER stress dependent pathway, that is,
GADD153 transcription activation [12]. In this study, we demonstrated that curcumin-induced apoptosis in human
HCC J5 cells is via Ca2+-regulated mitochondria-dependent
pathway.
2.Materials andMethods
2.1. Cell Culture. The HCC J5 cell line was obtained
from
the Cell Culture Center of the National Taiwan University
(Taipei, Taiwan). Cells were cultured with DMEM supplemented
with 2mM L-glutamine, 1.5 g/L sodium bicarbonate, 10% fetal bovine serum, and 2%
penicillin-streptomycin
(10,000 U/mL penicillin and 10 mg/mL streptomycin in a 5%
CO2 humidified incubator).
2.2. Morphological Study and Cell Viability. The J5
cells were
cultured in 12-well plates at a density of 2 × 105 cells/ well for 24 h, then treated with various
concentrations of curcumin
(0, 10, 15, 20, 25, and 50 μM in 0.1% DMSO) for diferent
time periods. Trypan blue exclusion was used to the cell viability as previously described [13]. In short, approximately
10 μL of cell suspensions in PBS were mixed with 40 μL of trypan blue. The numbers of stained (dead cells)
and unstained cells (live cells) were counted under a light microscope.
At least, 5000 cells were counted. The cell viability is calculated using the following formula:
2.3. Comet Assay. 2 × 105 J5 cells/well were grown in 12-well
plates and treated with curcumin at 0, 25, and 50
μMfor 24h,
then examined for DNA damage using Comet assay. Cells
were harvested and mixed with low melting point agarose.
The mixture was then placed in the solid normal melting
point agarose on the slide covered with coverslip. The coverslip
was removed after the agarose was gelled at 4◦C. The
slide
was transferred to the lysis bufer at 4◦C for 1 h
before putting
in alkaline bufer for electrophoresis (25V, 300 mA). The
slide was washed with neutralized bufer and stained with
PI after electrophoresis [13].
grown in
6-well plates and treated with 25 μMcurcumin for 12, 24, 36,
and 48 h. The fragmented DNA was extracted using a cell
genomic DNA purification kit (Genemark). The DNA extracted
procedures followed the protocols provided by the manufacture.
The DNA fragmentation was assayed with 1.5% agarose gel electrophoresis.
2.5. Caspase-3 Activity Assay. 2 × 105 J5 cells/well were cultured
in 12-well plates and treated with 25 μM curcumin for various time periods. Cells were harvested in a 15-mL
centrifuge tube by centrifugation. 50 μL of 10μM PhiPhilux
solution, a substrate for caspase-3, was added to each well
and incubated at 37◦C for 1 h. Cells were then
washed once
with 1mL of ice-cold PBS and resuspended in fresh 1mL
PBS. Caspase-3 activity was analyzed by flow cytometry
(Becton-Dickinson, CA, USA) equipped with an argon ion laser at 488nm wavelength [13]. In addition, J5 cells were
pretreated with 10 μM 1,2-bis(o-
aminophenoxy)ethane-N,N,N_,N_-tetraacetic acid (BAPTA), a calcium chelator,
for
2 h, then were assayed for caspase-3 activity as described in
the above.
2.6. Detection of Reactive Oxygen Species (ROS). 2 ×
105 J5
cells/well in 12-well plates were incubated with 25 μM curcumin
for diferent time periods to detect the changes of ROS. Cells were harvested and washed twice, resuspended
in 500 μL of 10μM 2,7-dichlorodihydrofluorescein diacetate
(DCFH-DA), and incubated at 37◦C for 30 min, then
analyzed
by flow cytometry [13].
2.7. Detection of Mitochondrial Membrane Potential (ΔΨm).
2 × 105 J5 cells/well in 12-well plates were incubated with
25 μM curcumin for diferent time course to determine the
changes in ΔΨm. Cells were harvested and washed twice, resuspended
in 500 μL of 4 μMDiOC6, and incubated at 37◦C
for 30min, then analyzed by flow cytometry [13].
2.8. Cell Viability, ROS Production, ΔΨm Levels in J5
cells
Pre-Treated with BAPTA. 2 × 105 J5 cells/well in 12-well plates
were pre-treated with 100 μM BAPTA for 2 h, then treated
with 25 μM curcumin for 24 h. Cells were harvested and
washed twice, half of cells were analyzed for cell viability with
PI staining, the rest was resuspended in 4 μM DiOC6 and
10 μM DCFH-DA before incubated at 37◦C for 30 min,
then
analyzed by flow cytometry.
2.9. Determination of Ca2+ Concentration. 2 × 105 J5 cells/well in 12-well plates were incubated with 25 μM curcumin
for various time intervals to determine the Ca2+ levels.
Cells were harvested and washed twice, resuspended in
3 μg/mL Indo 1/AM, incubated at 37◦C for 30 min, and
analyzed by flow cytometry.
2.10. Western Blotting. 2 × 105 J5 cells/well in 12-well plates were treated with 25 μM curcumin for 0, 6, 12, 24,
and 48 h. The level of cytochrome c in the cytosol was
isolated according to the manufacturer’s protocol (A cytosol/
International,
Temecula, CA, USA). The total proteins of cells were extracted with cell lysis bufer (50mMTris-HCL pH8.0,
120mM NaCl, 0.5% NP-40, 1mM PMSF), and 40 μg of protein
extract was separated by 10% SDS-PAGE, then transferred
to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), blocked with 5%nonfatmilk in TBSTween bufer
(0.12M Tris-base, 1.5M NaCl, 0.1% Tween20) for 1 hour at
room temperature, and incubated with the appropriate antibody
overnight at 4◦C, then incubated with horseradish
peroxidase
conjugated secondary antibody for 30min at room temperature. The bound antibody was detected with
peroxidase-conjugated anti-rabbit antibody (1 : 10000) or antimouse
antibody (1 : 10000) followed by chemiluminescence (ECL System) and exposed by autoradiography. The following
primary antibodies except cytochrome c (1 : 500) (Oncogene
Research Products): β-actin (1 : 10000), Bcl-2 (1 : 1000),
Bcl-xl (1 : 1000), Fas (1 : 1000), caspase-8 (1 : 1000),
caspase-12 (1 : 1000), and catalase (1 : 1000) were purchased from
Upstate, Millipore.
2.11. Statistics. Student’s t-test was used to evaluate
the
significance or P values between groups (∗P < 0.05, ∗∗P <
0.01). Standard errors of mean values were depicted as error
bars in all figures.
3. Results
3.1.Morphological Study and Cell Viability. The
morphology
of J5 cells induced by curcumin was remained
unchanged,
but the apoptotic bodies could be observed (Figure 1(a)),
and increased with times. Figure 1(b) shows the viability of
J5 cells are decreased with the increase of curcumin concentration
(10–50 μM).
3.2. Ca2+ Production, Mitochondria Membrane
Potential
(ΔΨm), and Production of Reactive Oxygen Species
(ROS)
Afected by Curcumin in J5 Cells. Figure 2(a) showed
that
Ca2+ production was significantly enhanced from 15 min up
to 720 min by 25 μM curcumin treatment, while the mitochondria
membrane potential (ΔΨm) was significantly decreased
(Figure 2(b)) as compared with that of the control. Reactive oxygen species (ROS) was also significantly increased
and reached the highest levels at 15–60 min after 25 μM curcumin treatment (Figure 2(c)).
3.3. The Release of Cytochrome c and Apoptotic-Associated
Proteins Afected by Curcumin in J5 Cells. To
characterize the
molecular mechanisms of curcumin-induced apoptosis in J5
cells, the expressions of apoptotic-associated proteins were
examined with Western blotting. Figure 3(a) showed that
cytochrome c was released from the mitochondria to the
cytosol in J5 cells treated with 25 μM curcumin for diferent
time periods (6–48 h). On the other hand, the protein levels
of Bcl-2, Bcl-xL, and Fas were decreased. Both caspase-12 and
catalase were increased after curcumin treatment for 6 and
12 h, but decreased for 24 and 36 h, then increased again for
48 h. Procaspase-8 were not afected by curcumin treatment.
3.4. DNA Damage and Fragmentation Caused by Curcumin in
J5 Cells. DAPI staining was used to detect the DNA
damage
in J5 cells treated with curcumin. Figure 4(a) showed that the
nuclei of control cells were round and smaller as compared
with the condensed and larger nuclei of cells exposed to 25
and 50 μM curcumin for 24 h. The DNA damage induced by
curcumin was in a dose-dependent manner. The Comet assay
also showed the similar results. The 50 μM curcumin treatment
showed a longer DNA migration smear (Figure 4(b)), indicating that more DNA was damaged in the cells. DNA
fragmentations were found in J5 cells after12, 24, 36, and
48 h of continuous exposure to 25 μM curcumin as shown
in Figure 4(c). The induction of DNA fragmentation by curcumin
was in a time-dependent manner.
3.5. Efects of Calcium Chelator BAPTA on Cell Viability, ΔΨm,
ROS Production, and Caspase-3Activity Induced by Curcumin
in J5 Cells. J5 cells were pretreated with 100 μM
BAPTA
for 2 h, followed by incubation with 25 μM curcumin for
diferent time periods. Cell viability, ΔΨm, ROS, and
caspase-3 activity were analyzed by flow cytometry. Figure 5(a)
showed that BAPTA could rescue the cell death from curcumin
treatment. The recovery of mitochondria membrane
potential ΔΨm and the inhibition of ROS by BAPTA were
shown in Figures 5(b) and 5(c), respectively. In addition,
caspase-3 activity increased by 25 μMcurcumin was inhibited by
BAPTA.
4. Discussion
We have demonstrated that DNA damage and endoplasmic
reticulum (ER) stress-mediated curcumin-induced cell cycle
arrest and apoptosis are through the activation of caspases,
and mitochondria-dependent pathways in A549 cells [13]; here we further show the similar finding in human
hepatocellular carcinoma J5 cells. Mitochondrial dysfunction
associated with apoptosis is characterized with the loss of mitochondrial membrane potential (ΔΨm), permeability
transition, and the release of cytochrome c from the mitochondria into the cytosol [14]. We also show that
curcumin induces apoptosis in human HCC J5 cells via mitochondrial-dependent pathway with the
suppression of
both mitochondria membrane potential (ΔΨm) and the induction
of cytochrome c release; nevertheless, the ROS production is induced and the Ca2+ in cytoplasm is accumulated.
Other than mitochondrial dysfunction, the mechanisms
responsible for curcumin-induced apoptosis in diferent
cancer cell types may also involve the activation of caspases,
and the inhibition of antiapoptotic Bcl-2 family proteins
[15–17]. We also found that curcumin decreased the protein levels of Bcl-2 and Bcl-xL in this study. Dr¨oge et al.
and
result in the cell death [18]. Our result indicates that ROS
production in J5 cells with the highest levels at 15–60 min
after 25 μM curcumin treatment. Both superoxide dismutase
(SOD) and catalase of ROS scavenger reduced ROS production
[19]. We also found that curcumin increased protein levels of catalase after curcumin treatment for 6 and 12 h in J5
cells. ΔΨm depletion, cytochrome c release, ROS production,
and DNA damage caused by curcumin all have contribution on the cell death. However, neither of the aforementioned results,
in which multiple related mechanisms of curcumin-induced
apoptosis was revealed, indicate the key molecule with
potential to steer the pharmacologic efect of curcumin.
Intracellular-free calcium ([Ca2+]i) is a universal signaling
molecule regulating many cellular functions including apoptosis. In addition, Ca2+-dependent processes are closely
related with the mainstream apoptosis executioners, that is,
caspases [20]. It is also shown to activate and modulate the
execution of a nonapoptotic cell death in C. elegans [21].
Both the overload and the depletion of endoplasmic reticulum
Ca2+ pool result in the induction of ER stress, and further
initiate the apoptotic pathway via activation of
procaspase-12, which is transferred to the ER membrane during ER stress in response to the mobilization of
intracellular Ca2+
stores [20, 22]. Once activated, caspase-12 acts on the efector
caspases to induce apoptosis [23]. We also found the highest
protein level of caspase-12 at 48 h after curcumin treatment
in this study. Furthermore, the disruption of mitochondrial
membrane potential and the disturbance of intracellular free Control 25 μM 50 μM (a) Control 25 μM 50 μM (b) M 0 12 24 36 48 (h) (c)
Figure 4: DNA damage and DNA fragmentation were induced
by curcumin in J5 cells. Cells were incubated with 0, 25, and 50 μM curcumin for 24 h, and DNA damage was examined by (a)
DAPI staining, (b) comet assay, and photographed by fluorescence
microscope. (c) J5 cells were treated with 25 μM curcumin for 0,
12, 24, 36, and 48 h, and DNA fragmentation was determined with
DNA gel electrophoresis.
Ca2+ concentration were also found to be afected by curcumin
[24].
In order to elucidate the mechanism that how Ca2+ was
involved in the curcumin-induced cell death, human HCC
J5 cells were pretreated with BAPTA, a calcium chelator,
followed by the curcumin treatment. The result showed that
BAPTA could reverse curcumin-induced cell death, despite
the fact that ER stress is able to activate apoptosis [25].
Although the previous study of curcumin-induced apoptosis
GADD153 [12], our finding implicates the major significance
of Ca2+-dependent mechanism in curcumin-induced apoptosis. A similar result has been suggested by Bae et al.
in human leukemia cell line as well [7]. Notably, BAPTA also inhibited the depletion of mitochondria membrane potential,
ROS production, and capase-3 activation in human HCC J5 cells. In conclusion, our results suggest that the
apoptosis induced by curcumin in human HCC J5 cells is
through mitochondria-dependent pathway, in which Ca2+
release plays an important role.
Conflict of Interests
The authors declare no conflict of interests.
Author’s Contribution
W.-H.Wang and I.-T. Chiang contributed equally to this paper.
Acknowledgment
This paper was supported by Grant NSC99-2623-E-010–
001-NU from National Science Council, Taipei, Taiwan.
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