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Int. J. Mol. Sci. 2014, 15, 2660-2671; doi:10.3390/ijms15022660
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Involvement of Hydrogen Peroxide in Safingol-Induced
Endonuclease G-Mediated Apoptosis of Squamous Cell
Carcinoma Cells
Masakazu Hamada *, Ken Wakabayashi, Atsushi Masui, Soichi Iwai, Tomoaki Imai and
Yoshiaki Yura
Department of Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry,
1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; E-Mails: dental_ken@yahoo.co.jp (K.W.);
a-masui@dent.osaka-u.ac.jp (A.M.); s-iwai@dent.osaka-u.ac.jp (S.I.); hsc12@hotmail.com (T.I.);
yura@dent.osaka-u.ac.jp (Y.Y.)
* Author to whom correspondence should be addressed; E-Mail: hmdmskz@dent.osaka-u.ac.jp;
Tel.: +81-6-6879-2941; Fax: +81-6-6879-2170.
Received: 8 November 2013; in revised form: 3 January 2014 / Accepted: 13 February 2014 /
Published: 17 February 2014
Abstract: Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a
mitochondrial apoptogenic mediator—endonuclease G (endo G)—and apoptosis of human
oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown.
The levels of hydrogen peroxide (H
2
O
2
) in cultured oral SCC cells were measured.
Treatment with safingol increased intracellular H
2
O
2
levels but not extracellular H
2
O
2
levels,
indicating the production of H
2
O
2
. The cell killing effect of safingol and H
2
O
2
was
diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine
(NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that
apoptotic cell death occurred by treatment with H
2
O
2
and safingol. The number of apoptotic
cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the
cytoplasm and an association of endo G with mitochondria was observed. After treatment
with H
2
O
2
and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the
nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic
cells and the translocation of endo G. Knock down of endo G diminished the cell killing
effect of H
2
O
2
and safingol. These results suggest that H
2
O
2
is involved in the endo
G-mediated apoptosis of oral SCC cells by safingol.
OPEN ACCESS
Int. J. Mol. Sci. 2014, 15 2661
Keywords: safingol; hydrogen peroxide; apoptosis; endonuclease G
1. Introduction
Apoptosis, the best-described type of programmed cell death, is characterized by cell membrane
blebbing, a reduction in cellular volume, the activation of caspases, chromatin condensation and nuclear
fragmentation [1,2]. Internucleosomal DNA fragmentation is a hallmark of the apoptotic process and at
least two endonucleases, caspase-activated DNase (CAD) and endonuclease G (endo G), are thought to be
important for mammalian DNA fragmentation during apoptosis [3,4]. The best-characterized major
enzyme for DNA fragmentation is the CAD that forms an inactive heterodimer with inhibitor of CAD
(ICAD). Following apoptotic signaling, ICAD is proteolyzed by caspase-3 causing the dissociation of
the CAD/ICAD heterodimer and releasing CAD, which then moves from the cytosol to the nucleus.
Endo G is an endonuclease that is released from the mitochondrial intermembrane space and translocates
to the cell nucleus to induce DNA fragmentation in a caspase-independent manner [4–6].
Safingol, a L-threo-dihydrosphingosine, is a synthetic lipid and functions by targeting the
lipid-binding regulatory domain of protein kinase C (PKC) [7,8]. In previous studies, safingol was used
as a PKCα-selective inhibitor and antitumor activity was demonstrated [8–12]. The cytotoxic effect of
safingol was also attributed to the inhibition of sphingosine kinase 1, thus preventing the formation of
sphingosine-1-phosphate, which is involved in cell proliferation, invasion and angiogenesis [13–15].
Safingol is currently under a phase I clinical trial in combination with cisplatin for the treatment of
advanced solid tumors [16]. Our previous studies indicated that safingol induced apoptosis of oral
squamous cell carcinoma (SCC) cells, accompanied by the nuclear translocation of endo G from
mitochondria in a caspase 3-independet manner, using DNA fragmentation assay, flow cytometric
analysis and immunostaining [17], but upstream mediators remain largely unknown.
Oxidative stress has been implicated in a number of physiological and pathological processes,
including cancer, ischemic injury, neurodegenerative diseases, chronic inflammation, type II diabetes
and arteriosclerosis [18]. Reactive oxygen species (ROS) are recognized as chemical mediators in
deciding the fate of cells, depending on the extent of oxidative damage. In the present study, we
investigated the possible involvement of H
2
O
2
as a ROS in endo G-mediated apoptosis of oral SCC cells
treated with safingol.
2. Results
2.1. Production of ROS in SCC Cells by Treatment with Safingol
SAS cells were incubated with hydrogen peroxide (H
2
O
2
) or safingol, and extracellular and
intracellular levels of H
2
O
2
were measured using an assay kit for measuring H
2
O
2
concentration [19,20]
12 h later. After treatment with 100 µM H
2
O
2
, the intracellular H
2
O
2
concentration increased, but the
extracellular H
2
O
2
concentration did not (Figure 1A). When SAS cells were treated with safingol at
15 or 25 µM, the H
2
O
2
levels in the cells also increased. The difference between the treated cells and
untreated control was significant. The level of H
2
O
2
in the medium of the cells treated with H
2
O
2
or
Int. J. Mol. Sci. 2014, 15 2662
safingol was not altered. When SAS cells were treated with 15 µM safingol for 6, 12, and 24 h, the
intracellular H
2
O
2
concentration increased and reached a max level of 12 h.
Figure 1. Production of ROS in SCC cells treated with safingol. SAS cells were treated with
H
2
O
2
or safingol, and extracellular and intracellular ROS levels were determined 12 h later
(A); SAS cells were treated with 15 µM safingol, and intracellular ROS levels were
determined 0, 6, 12, 24 h later (B). The data represent the mean ± SD of three
determinations. ** p < 0.01 vs. control.
2.2. Induction of Cell Death by H
2
O
2
and Safingol
Cell death was examined using the trypan blue dye exclusion test. When SAS cells were treated with
100 µM H
2
O
2
for 12 h, the proportion of dead cells increased to 36%, though this increase was
diminished in the presence of a ROS scavenger, N-acetyl-L-cysteine (NAC) [21,22], with 22% of cells
nonviable (Figure 2A). When cells were treated with 15 µM safingol for 12 h, 45% were found to be
nonviable. This value was reduced to 21% by NAC. When another oral SCC cell line HSC-3 was used,
H
2
O
2
and safingol decreased the proportion of viable cells in a similar manner as observed in SAS cells.
The suppressive effect was blunted by NAC (Figure 2B). When 500U PEG catalase (PEG-cat) was used
to delete H
2
O
2
production by safingol, the cell killing effect of safingol was decreased (Figure 2C).
SAS cells were treated with H
2
O
2
or safingol and dual staining with annexin V and propidium iodide
(PI) was performed. Cells stained with annexin V alone were considered to be apoptotic cells.
The percentage of apoptotic cells was also increased by treatment with H
2
O
2
and safingol, up to 34% and
23%, respectively (Figure 3). These values decreased to 14% and 15% in the presence of NAC.
Int. J. Mol. Sci. 2014, 15 2663
Figure 2. Induction of cell death by H
2
O
2
and safingol. SAS (A) and HSC-3 (B) cells were
treated with 100 µM H
2
O
2
or 15 µM safingol alone. Alternatively, they were treated with
H
2
O
2
or safingol in the presence of the ROS scavenger NAC for 12 h. SAS cells were
treated with 100 µM H
2
O
2
or 15 µM safingol alone. Alternatively, they were pretreated
with 500U PEG catalase (PEG-cat) for 2 h and then they were treated with H
2
O
2
or
safingol for 12 h (C). Thereafter, the cells were stained with trypan blue. The percentages
of dead cells were calculated. The data represent the mean ± SD of three determinations.
* p < 0.05, ** p < 0.01 vs. treated group with NAC or PEG-cat.
Figure 3. Induction of apoptotic cells death by H
2
O
2
and safingol. SAS cells were treated
with 100 µM H
2
O
2
or 15 µM safingol alone (A). Alternatively, they were also treated with
H
2
O
2
or safingol in the presence of the ROS scavenger NAC for 12 h. Thereafter, the cells
were stained with annexin V and PI. The percentages of apoptotic cells stained with annexin
V alone were calculated (B). The data represent the mean ± SD of three determinations.
** p < 0.01 vs. treated group with NAC.
2.3. Effect of Endo G Small Interfering RNA (siRNA) on the Cell Death Caused by H
2
O
2
and Safingol
Previously, we reported that safingol induced the translocation of endo G from mitochondria to the
nucleus and induced apoptosis [17]. In the present study, the effect of siRNA on cell viability was
examined. SAS cells were transfected with endo G siRNA and subjected to immunoblotting. The
expression of endo G was downregulated by this treatment, whereas it was maintained after the
transfection of nonsense siRNA (Figure 4A).
Int. J. Mol. Sci. 2014, 15 2664
Treatment with 300 µM H
2
O
2
and 15 µM safingol increased the percentage of dead cells to 33% and
27%, respectively, in the cultures transfected with nonsense siRNA. In endo G siRNA-transfected cells,
these values decreased to 14% and 17%, respectively, indicating the involvement of endo G in the H
2
O
2
-
and safingol-induced cell death (Figure 4B).
Figure 4. Effect of endo G siRNA transfection on cell viability. (A) SAS cells were
transfected with endo G siRNA or nonsense siRNA and cultured for 24 h. They were
subjected to an immunoblot analysis. At least three determinations were performed.
A representative result is shown; (B) Endo G siRNA- or nonsense siRNA-transfected SAS
cells were treated with 300 µM H
2
O
2
or 15 µM safingol for 12 h and subjected to a trypan
blue dye exclusion test. The data represent the mean ± SD of three determinations.
** p < 0.01 vs. treated group with endo G siRNA transfection.
2.4. Translocation of Endo G by H
2
O
2
and Safingol
The effect of H
2
O
2
and safingol on the localization of mitochondria and the expression of endo G
were examined using immunofluoresent antibody staining. In untreated SAS cells, the mitochondria
were filamentous with a tubular appearance and often interconnected forming a network. Most
cytoplasmic staining of endo G was co-localized with mitochondria, and specific nuclear staining was
not observed (Figure 5). After treatment with 100 µM H
2
O
2
, the localization of mitochondria
was unchanged, but endo G showed diffuse distribution. Inconsistent with the results for
4',6-diamidino-2-phenylindole (DAPI) staining, nuclear staining of endo G was observed. When cells
were treated with H
2
O
2
in the presence of NAC, the endo G was confined to the cytoplasm and nuclear
localization was not observed. Safingol also induced nuclear staining of endo G, which was completely
blocked by NAC.
Int. J. Mol. Sci. 2014, 15 2665
Figure 5. Translocation of endo G by H
2
O
2
and safingol. SAS cells were treated with
100 µM H
2
O
2
or 15 µM safingol in the presence or absence of NAC for 12 h. They were
subjected to immunofluorescent staining using DAPI, antibody against endo G and
Mitotracker Red CMXRos DAPI. Untreated cells were also stained. The imaging of each
staining was merged. At least three measurements were performed. A representative result
is shown.
3. Discussion
We had shown that safingol could induce cell death with characteristics of apoptosis at a
concentration of 25 µM in a caspase three-independent manner [23]. At 10 µM, however, though a
proportion of cells detached, they reattached on the plate after prolonged incubation. The cell killing
effect of safingol was marginal at this concentration [17,23]. On the other hand, safingol was reported to
exert an inhibitory effect on sphingosine kinase 1 at concentrations below 10 µM [14,16]. Since our
previous study was undertaken at higher concentrations, safingol would affect sphingosine kinase 1 as
well as PKC, to induce apoptosis of oral SCC cells.
Activation of apoptosis is associated with the generation of ROS [24]. Indeed, some anticancer drugs
induce production of ROS during apoptosis [25–27]. Mizutani et al. [27] reported that the critical
apoptotic trigger of doxorubicin, a topoisomerase II inhibitor, was oxidative DNA damage from
doxorubicin-induced H
2
O
2
production and that the oxidative damage caused the indirect generation of
H
2
O
2
through poly (ADP-ribose) polymerase (PARP) and nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase activation, leading to an increase in mitochondrial membrane permeability.
To determine the possible involvement of ROS in safingol-induced cell death in oral SCC cells, we
Int. J. Mol. Sci. 2014, 15 2666
examined the levels of intracellular and extracellular ROS and found that safingol as well as H
2
O
2
increased intracellular H
2
O
2
level. When cell death was estimated using the trypan blue dye exclusion
test, H
2
O
2
and safingol induced cell death and the killing effect was efficiently blocked by a ROS
scavenger, NAC, in SAS and HSC-3 cells. Apoptotic cells stained with annexin V alone were also
reduced by NAC in SAS cells. The difference observed in trypan blue and annexin V staining especially
in safingol-treated cells may represent apoptotic cells with necrotic degradation, because apoptotic cells
looks like necrotic cells at an advanced stage. Thus, it can be stated that safingol produces ROS
including H
2
O
2
, which is responsible for the induction of apoptotic cell death in oral SCC cells.
Mitochondria itself produces ROS, but H
2
O
2
that was added to the cell culture induced apoptosis in the
present study. Cytoplasmic H
2
O
2
must be the inducer of release of apoptogenic mitochondrial factors.
Ling et al. [28] indicated that safingol caused time-and concentration-dependent production of ROS in
MDA-MB-231 and HT-29 cells, suggesting ROS to be a mediator of safingol-induced cancer cell death.
They cultured the cells for 48 h at 10 µM and found necrosis and autophagy, but they did not examine
DNA fragmentation observed in apoptosis. Since safingol at 10 µM did not induce cell death in most
SAS cells, we have not examined the role of autophagy as observed in MDA-MB-231 and HT-29 cells.
Intrinsic apoptosis is critically dependent on mitochondrial outer membrane permeabilization,
which results in the release of mitochondrial intermembrane space proteins, such as cytochrome c, and
endo G [29,30]. In the present study, we found that the effect of H
2
O
2
and safingol was blocked by the
downregulation of endo G expression, indicating that endo G is required for the cell death by the
treatment. We also found that H
2
O
2
as well as safingol induced translocation of endo G to the nucleus.
The expression of endo G was not necessarily correlated with the intensity of mitochondrial staining, but
was present in the cytoplasm of untreated oral SCC cells, representing the synthesis of the mitochondrial
protein in the cytoplasm. After treatment with H
2
O
2
or safingol, the nuclear accumulation of endo G
occurred, but cytoplasmic staining was preserved. Apoptotic signal would stimulate the release of endo
G from mitochondria to the cytoplasm and then the endo G comcomitantly with the preexisting
cytoplasmic endo G move to the nucleus for DNA fragmentaion. It should be also stated that
NAC clearly blocked the alteration of endo G staining by H
2
O
2
and safingol. In a neuronal system,
Higgins et al. [31] found that oxidative stress triggered neuronal caspase-independent cell death and the
translocation of endo G. Treatment caused the redistribution from mitochondria of both endo G and
cytochrome c. Kim et al. [32] treated head and neck cancer cells with cisplatin and found mitochondrial
outer membrane permeabilization, the nuclear translocation of endo G and apoptosis. Together,
we firstly suggest that the expression and translocation of endo G are required for the inducation of cell
death of oral SCC cells by safingol and that H
2
O
2
is one of the upstream factors in this event.
4. Experimental Section
4.1. Cell Culture
The human oral SCC cell line SAS and HSC-3 were obtained from the Japanese Collection of
Research Bioresources (Tokyo, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 µg/mL penicillin and
100 µg/mL streptomycin and grown in an incubator at 37 °C in a humidified atmosphere with 5% CO
2
.
Int. J. Mol. Sci. 2014, 15 2667
4.2. Reagents
Safingol was obtained from Calbiochem-Novabiochem (San Diego, CA, USA). H
2
O
2
and NAC were
obtained from Wako (Osaka, Japan) and PEG-cat was obtained from Sigma (St. Louis, MO, USA).
4.3. Measurement of H
2
O
2
The concentration of H
2
O
2
was determined using a colorimetric assay. Cells were plated in 48-well
plates at a density of 2 × 10
4
cells/well and treated with H
2
O
2
or safingol for 12 h. The supernatant of SAS
cells was harvested as an extracellular sample. Cells were dissociated with an ethylenediaminetetraacetic
acid (EDTA)-trypsin solution, subjected to three cycles of freezing and thawing and used as an
intracellular sample for the H
2
O
2
assay [19,20]. Ten microliters of sample was mixed with 100 µL of
Bioxytech H
2
O
2
-560 (OXIS International, Portland, OR, USA) and incubated for 30 min at room
temperature. Measurements were made using a Benchmark plus microplate spectrophotometer
(Bio-Ras Laboratories, Hercules, CA, USA) at a wavelength of 560 nm.
4.4. Trypan Blue Staining
Cell viability was determined by the trypan blue dye exclusion test. Cells were plated in 6-well plates
at a density of 1 × 10
6
cells/well, cultured for 24 h and treated with 100 µM H
2
O
2
or 15 µM safingol for
12 h. They were dissociated by the EDTA-trypsin solution, and cells were centrifuged suspended in
phosphate-buffered saline (PBS) without Ca
2+
and Mg
2+
. The pellets were then mixed with an equal
volume of PBS without Ca
2+
and Mg
2+
containing 0.4% trypan blue and observed with a microscope.
We counted the numbers of stained and unstained cells. Results were compared to those for the
untreated controls and a percentage was calculated.
4.5. Annexin V and PI Staining
To identify apoptotic cells, annexin V and PI staining was performed using Vybrant Apoptosis Assay
Kit (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s directions.
After treatment with H
2
O
2
or safingol, floating cells were harvested with medium and attached cells
were dissociated with EDTA-trypsin solution. These cells were collected by centrifugation at 1000 rpm
for 5 min. The cell pellets were suspended in 100 µL binding buffer (10 mM HEPES, 140 mM NaCl,
2.5 mM CaCl
2
, pH 7.4) and incubated with 5 µL FITC Annexin V and 1 µL of a propidium iodide
(100 µg/mL) solution for 15 min at room temperature. Staining for annexin V and propidium iodide was
observed under a fluorescence microscope (Microphoto FXA; Nikon, Tokyo, Japan). The percentages of
apoptotic cells stained with annexin V alone were calculated. At least 3 samples and 1000 cells were
counted for determination of the percentage of apoptotic cells.
4.6. Immunoblot Analysis
Cells were washed in PBS and lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 0.1% SDS,
1% TritonX-100, 1% sodium deoxycholate and protease inhibitor cocktail. After sonication on ice and
subsequent centrifugation at 15,000× g for 10 min at 4 °C, the supernatant was collected and the
Int. J. Mol. Sci. 2014, 15 2668
protein concentration was determined using a Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
Sample protein (15 µg) was electrophoresed through a polyacrylamide gel and transferred to a
polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) by electroblotting. The membrane
was probed with antibodies and antibody-binding was detected using an enhanced chemiluminescence
kit (GE Healthcare, Amersham, Buckinghamshire, UK) according to the manufacturer’s instructions.
The antibodies used were a rabbit polyclonal antibody against endo G (Sigma, St. Louis, MO, USA),
and β-actin (Sigma, St. Louis, MO, USA). The secondary antibodies used were horseradish
peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, Beverly, MA, USA) and
peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, USA).
4.7. siRNA Transfection
Chemically synthetic siRNA against endo G and AllStars negative control siRNA (nonsense siRNA)
were purchased from Qiagen (Valencia, CA, USA). The target sequence of the siRNA for endo G was
5'-AAAUGCCUGGAACAACCUUGA-3'. Cells were plated in 6-well plates at a density of
1 × 10
5
cells/well, cultured for 24 h, and transfected with 40 nM endo G siRNA or nonsense siRNA
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
directions. The medium was replaced with DMEM after 3 h and cells were used for experiments at
24 h after transfection.
4.8. Confocal Laser Microscopic Analysis
Cells were treated with H
2
O
2
or safingol for 12 h. Thereafter, they were fixed with 2%
paraformaldehyde, permealized with 0.1% Triton X-100 in PBS and incubated with 25 nM
Mitotracker Red CMXRos (Molecular Probes, Eugene, OR, USA) at 37 °C for 45 min. They were
fixed in 4% paraformaldehyde phosphate buffer solution (WAKO, Osaka, Japan), permealized with
0.1% Triton X-100 in PBS and incubated with a rabbit polyclonal antibody against endo G (Sigma,
St. Louis, MO, USA) diluted 1:200 in PBS for 1 h at room temperature. After washing, the cells were
incubated with Alexa Fluor 488 goat antirabbit antibody (Life Technologies Corporation, Carlsbad,
CA, USA) diluted 1:500 in PBS for 1 h. After washing, coverslips were mounted onto microslides
using a ProLong Gold Antifade Reagent with DAPI (Life Technologies Corporation, Carlsbad, CA,
USA). The slides were analyzed under a confocal laser-scanning microscope Leica TCS SP8
(Leica Microsystems, Mannheim, Germany).
4.9. Statistical Analysis
The statistical analysis was performed using a Student’s t test with Microsoft Excel (Windows vista,
Microsoft, Redmond, WA, USA). The results were expressed as the mean ± SD. The differences were
considered significant at p < 0.05.
Int. J. Mol. Sci. 2014, 15 2669
5. Conclusions
Safingol mimics H
2
O
2
in the ability to induce the death of oral SCC cells. H
2
O
2
as a ROS is
suggested to act as upstream mediators to induce translocation of endo G which can directly contribute
to the cleavage of nuclear DNA.
Acknowledgments
This work was supported in part by a Grant-in Aid for Scientific Research from the Japan Society
for the Promotion of Science (No.22791972, No.25861929).
Conflicts of Interest
The authors declare no conflict of interests.
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