Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide

Article abstract of DOI:10.1016/j.bbrc.2019.08.073

Singlet oxygen causes a cytotoxic process in tumor cells in photodynamic therapy (PDT) and skin photoaging. The mechanism responsible for this cytotoxicity is, however, not fully understood. 1-Methylnaphthalene-4-propionate endoperoxide (MNPE) is a cell-permeable endoperoxide that generates pure singlet oxygen. We previously reported that cell death induced by MNPE did not show the typical profile of apoptosis, and the cause of this cell death remains elusive. We report herein on an investigation of the mechanism for MNPE-induced cell death from the view point of ferroptosis. The findings indicate that the MNPE treatment decreased the viabilities of mouse hepatoma Hepa 1-6 cells in vitro, and that this decrease was accompanied by increases in the concentrations of both intracellular ferrous iron and the level of lipid peroxidation, but that the caspase-mediated apoptotic pathway was not activated. The intracellular levels of cysteine and glutathione were not affected by the MNPE treatment. Importantly, an assay of lactate dehydrogenase activity revealed that the cell death caused by MNPE was suppressed by ferrostatin-1, a ferroptosis-specific inhibitor. Collectively, these results strongly indicate that ferroptosis is the main cell death pathway induced by singlet oxygen.

Full text of DOI:10.1016/j.bbrc.2019.08.073

Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Induction of ferroptosis by singlet oxygen generated from
naphthalene endoperoxide
Takujiro Homma*, Sho Kobayashi, Junichi Fujii
Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata, 990-9585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Singlet oxygen causes a cytotoxic process in tumor cells in photodynamic therapy (PDT) and skin pho-
toaging. The mechanism responsible for this cytotoxicity is, however, not fully understood. 1-
Methylnaphthalene-4-propionate endoperoxide (MNPE) is a cell-permeable endoperoxide that gener-
ates pure singlet oxygen. We previously reported that cell death induced by MNPE did not show the
typical profile of apoptosis, and the cause of this cell death remains elusive. We report herein on an
investigation of the mechanism for MNPE-induced cell death from the view point of ferroptosis. The
findings indicate that the MNPE treatment decreased the viabilities of mouse hepatoma Hepa 1-6 cells
in vitro, and that this decrease was accompanied by increases in the concentrations of both intracellular
ferrous iron and the level of lipid peroxidation, but that the caspase-mediated apoptotic pathway was not
activated. The intracellular levels of cysteine and glutathione were not affected by the MNPE treatment.
Importantly, an assay of lactate dehydrogenase activity revealed that the cell death caused by MNPE was
suppressed by ferrostatin-1, a ferroptosis-specific inhibitor. Collectively, these results strongly indicate
that ferroptosis is the main cell death pathway induced by singlet oxygen.
Received 10 August 2019
Accepted 13 August 2019
Available online xxx
Keywords:
Singlet oxygen
ferroptosis
Lipid peroxidation
ROS
Glutathione
© 2019 Elsevier Inc. All rights reserved.
1. Introduction
available.
1-Methylnaphthalene-4-propionate
endoperoxide
(MNPE) is a cell-permeable endoperoxide that generates pure
singlet oxygen. Using MNPE, we previously reported that singlet
oxygen is highly toxic and causes a non-apoptotic type of cell death
[8e10]. Although this process is accompanied by the release of
cytochrome c from mitochondria, cell death induced by MNPE does
not show the typical profile of apoptosis, but instead appears to
have necrosis-like characteristics [8]. This abortive apoptotic
pathway is mainly due to the direct inhibition of caspase activity
through protein modification by singlet oxygen [10]. Because pro-
teases that contain catalytic cysteine (Cys) residues are highly
sensitive to singlet oxygen-mediated inactivation [9,11], a reactive
Cys at the catalytic center in caspases would also be susceptible to
oxidative modification by singlet oxygen.
Although singlet oxygen is known to react with multiple cellular
components, its preference for reacting with conjugated double
bonds is very high, and hence it preferentially attacks poly-
unsaturated fatty acids in cells [12]. In fact lipid peroxides are
involved in the cytotoxic action of singlet oxygen released from
MNPE [8,9]. Treatment with vitamin E, a lipophilic antioxidant or
the overexpression of Gpx4 protects cells against MNPE-induced
cell death, while a hydrophilic antioxidant vitamin C fails to sup-
press the cell death.
Ferroptosis is a newly characterized type of non-apoptotic
regulated cell death in which free iron and lipid peroxidation
products are characteristically involved [1]. Ferroptosis appears to
depend on ferrous iron, which can donate an electron to hydrogen
peroxide, resulting in the formation of hydroxyl radicals by the
Fenton reaction, which, in turn, react with polyunsaturated fatty
acids to produce lipid peroxides. Because phospholipid hydroper-
oxide glutathione peroxidase (Gpx4) effectively suppresses fer-
roptosis, it is generally assumed that lipid peroxides function as the
key driver of ferroptosis [2,3].
Singlet oxygen is not a radical but a highly reactive oxygen
species (ROS) and is responsible for skin damage induced by UVA
irradiation [4e6] and for the cytotoxic anti-cancer effect in
photodynamic therapy (PDT) [7]. Despite its significant role in
sunburn and PDT, the biological effects of singlet oxygen are not
fully understood, and one of the reasons for this is that an appro-
priate system for producing pure singlet oxygen is essentially not
* Corresponding author.
E-mail address: tkhomma@med.id.yamagata-u.ac.jp (T. Homma).
https://doi.org/10.1016/j.bbrc.2019.08.073
0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073

2
T. Homma et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
EDTA), supplemented with a protease inhibitor cocktail (Sigma-
Aldrich, St. Louis, MO, USA; P8340). The lysate was centrifuged at
15,000ꢁg for 10 min in a microcentrifuge. Protein concentrations
were determined using a Pierce™ BCA Protein Assay Kit (Thermo
Abbreviations
Cys
cysteine
Fisher
Scientific).
The
proteins
were
separated
on
GSH
Gpx4
glutathione
phospholipid hydroperoxide glutathione
peroxidase
1-methylnaphthalene-4-propionate
endoperoxide
photodynamic therapy
SDSepolyacrylamide gels and blotted onto polyvinylidene difluor-
ide (PVDF) membranes (GE Healthcare, Chicago, IL, USA). The blots
were then blocked with 5% skim milk in Tris-buffered saline con-
taining 0.1% Tween-20 (TBST), and incubated overnight with the
primary antibodies diluted in TBST. The primary antibodies used in
the study were: Caspase-3 (R&D Systems, Minneapolis, MN, USA;
AF-605-NA), cleaved caspase-3 (Cell Signaling Technology, Danvers,
MA, USA; #9664), and GAPDH (Santa Cruz Biotechnology, Dallas,
TX, USA; sc-25778). After three washings with TBST, the blots were
incubated with horseradish peroxidase (HRP)-conjugated anti-goat
(Santa Cruz Biotechnology; sc-2020) or anti-rabbit (Santa Cruz
Biotechnology; sc-2004) secondary antibodies. After further
washing, the bands were detected using the Immobilon western
chemiluminescent HRP substrate (Merck Millipore, Burlington, MA,
USA) on an image analyzer (ImageQuant LAS500, GE Healthcare).
MNPE
PDT
ROS
LC-MS
reactive oxygen species
liquid chromatography-mass spectrometry
Based on these observations, we hypothesized that singlet ox-
ygen causes ferroptosis by means of its ability to trigger lipid per-
oxidation. In the present study, we examined the issue of how
singlet oxygen causes cell death by employing MNPE and the
findings indicate that ferroptosis is actually the main cell death
pathway that is induced by singlet oxygen.
2.4. Detection of fragmented DNA by agarose gel electrophoresis
2. Materials and methods
DNA was isolated using a conventional procedure of phenol-
chloroform extraction and isopropanol precipitation. The DNA
samples were electrophoresed on a 1% agarose gel in Tris-acetate-
EDTA buffer, then visualized on ImageQuant LAS500 after staining
with 0.5 mg/ml ethidium bromide.
2.1. Cell culture and treatment
Hepa 1-6 cells, a mouse hepatoma-derived cell line, were ob-
tained from the RIKEN Bioresource Center (Tsukuba, Japan) and
were utilized in this study as described in a previous report [13].
Briefly, the cells were maintained in Dulbecco's Modified Eagle's
Medium (DMEM; FUJIFILM Wako Pure Chemical, Osaka, Japan;
044e29765) supplemented with 10% fetal bovine serum (FBS;
Biowest, Riverside, MO, USA) and a penicillin-streptomycin solu-
tion (FUJIFILM Wako Pure Chemical) at 37 ꢀC in a 5% CO2 incubator.
Where indicated, the cells were treated with dimethyl sulfoxide
2.5. Liquid chromatography-mass spectrometry (LC-MS) analyses
LC-MS analyses of the intracellular contents of Cys and GSH
were performed as described previously [13]. Briefly, the collected
cells were homogenized in 200
bonate, pH 8.0, containing 20 mM N-ethylmaleimide (NEM; FUJI-
FILM Wako Pure Chemical). A 50 l aliquot of the sample was mixed
with 100 l of methanol containing 5 M N-methylmaleimide
(NMM)-derivatized GSH as an internal standard and an additional
ml of 50 mM ammonium bicar-
(DMSO; FUJIFILM Wako Pure Chemical) as
a vehicle, 1-
m
methylnaphthalene-4-propionate endoperoxide (MNPE; Waken B
Tech., ltd, Kyoto, Japan), ferrostatin-1 (Cayman Chemical, Ann Ar-
bor, MI, USA), or staurosporine (FUJIFILM Wako Pure Chemical). For
the MNPE treatment, Hepa 1-6 cells were initially treated with
MNPE for 2 h, after which the media was exchanged for fresh me-
dia, and the preparation then incubated further until cell harvest, as
previously described [8].
m
m
100 ml of chloroform, the mixture was thoroughly stirred and
centrifuged at 12,000ꢁg for 15 min at 4 ꢀC. The upper aqueous layer
was filtered through 0.45 mm filters (Millex®-LH, Merck Millipore).
A 90
ml aliquot of the filtrate was lyophilized, the residue dissolved
in 30
ml of Milli-Q water, and then subjected to LC-MS analyses. A Q
Exactive Hybrid Quadruple-Orbitrap mass spectrometer (Thermo
Fisher Scientific) was operated in the positive ionization mode. The
Ultimate 3000 liquid chromatography system consisted of a WPS-
3000 TRS autosampler, a TCC-3000 RS column oven, and an HPG-
3400RS quaternary pump (Dionex, Sunnyvale, CA). A SeQuant®
2.2. Evaluation of the viability and cytotoxicity of cells
Cells were seeded at an initial density of (1 ꢁ 105/ml). Cell
viability was determined using a CellTiter-Blue® Cell Viability
Assay (Promega, Madison, WI, USA) according to the manufac-
turer's instructions. Fluorescence intensity was measured using a
microplate reader Valioskan Flash (Thermo Fisher Scientific, Wal-
tham, MA, USA) with an excitation wavelength of 560 nm and an
emission wavelength of 590 nm. Cytotoxicity was determined by
means of a lactate dehydrogenase (LDH) assay as described previ-
ously with minor modifications [8]. The reaction mixture contained
ZIC®-pHILIC column (2.1 ꢁ 150 mm, 5
mm particle size; Merck
KGaA, Germany) was maintained at 30 ꢀC. Mobile phase A was
20 mM ammonium bicarbonate, pH 9.8, and mobile phase B was
100% acetonitrile. System control, data acquisition, and quantitative
analysis were performed with the Xcalibur 2.2 software. Standard
curves for GSH-NEM, and Cys-NEM showed linearity in the con-
centration ranges examined.
20
ml of the culture medium, 0.3 mM NADH,1 mM sodium pyruvate,
and 200 mM sodium phosphate buffer, pH 7.4 in total of 100
ml.
Initial activities were calculated from the rate of disappearance of
NADH during the starting linear phase of the reaction by moni-
toring the absorbance at 340 nm.
2.6. Flow cytometry
Cells were incubated with 10 mM C11-BODIPY 581/591 (Thermo
Fisher Scientific) in the culture medium for 1 h and then washed
with PBS. After trypsinization, the cells were collected and sub-
jected to flow cytometry (FACSCanto™ II, BD Biosciences, Tokyo,
Japan) at an excitation wavelength of 488 nm and an emission
wavelength of 517e527 nm.
2.3. Western blotting
Cells were lysed in cold lysis buffer (50 mM Tris-HCl pH 7.5,
150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 2 mM
Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073

T. Homma et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
2.7. Detection of intracellular ferrous iron under fluorescent
microscopy
software (San Diego, CA, USA). A P-value of less than 0.05 was
considered to be significant.
Cells were incubated with 5
mM FeRhoNox™-1 (Goryo Chemical,
3. Results
Sapporo, Japan) according to the manufacturer's instructions. The
cells were then washed with PBS and images were obtained using a
BZ-X700 microscope (KEYENCE, Osaka, Japan).
3.1. MNPE induces ferroptosis
Our objective was to investigate the cytocidal effect of singlet
oxygen released from MNPE in Hepa 1-6 cells in which ferroptosis
had been previously implicated [13]. We examined MNPE-induced
changes in the viabilities of Hepa 1-6 cells by using phase-contrast
microscopy and CellTiter-Blue® Cell Viability Assays. Compared
with DMSO-treated cells, the viabilities of the Hepa 1-6 cells were
drastically decreased by MNPE in a dose-dependent manner
(Fig. 1A and B). An assay for LDH release revealed that MNPE
induced cell death during a 24 h period of incubation while MNP, a
precursor of MNPE, had no effect on LDH release under these
conditions, suggesting that the decreased viability of Hepa 1-6 cells
was caused by cell death triggered by singlet oxygen generated
from MNPE (Fig. 1C). Importantly, the lethal effects of MNPE were
suppressed by ferrostatin-1, a ferroptosis-specific inhibitor that
functions by preventing the accumulation of lipid peroxides [1],
suggesting that MNPE-induced cell death is associated with
ferroptosis.
2.8. Immunostaining
Cells were washed with PBS and fixed in 4% formaldehyde for
15 min at room temperature. After washing twice with PBS, the
cells were permeabilized for 5 min with 0.5% Triton X-100 in PBS,
then blocked for 30 min by treatment with 1% BSA in PBS at room
temperature, and incubated overnight with anti-HNE (JaICA,
Fukuroi, Japan, dilution 1:500) antibodies at 4 ꢀC. After three
washes in PBS, the cells were further incubated with a goat anti-
mouse IgG (H þ L), Alexa Fluor® 488 conjugate antibody (Thermo
Fisher Scientific, dilution 1:500) for 60 min at room temperature.
All images were obtained using a BZ-X700 microscope (KEYENCE).
2.9. Statistical analysis
Statistical analyses were performed using the GraphPad Prism 6
A
DMSO
Fer-1
MNPE (2 mM)
MNPE (4 mM)
MNPE (2 mM) + Fer-1
MNPE (4 mM) + Fer-1
B
C
(−) Fer-1
150
100
50
n.s.
150
100
50
(+) Fer-1
n.s.
*** ***
***
***
***
n.s.
0
0
MNPE (mM)
0
1
2
4
Fig. 1. MNPE induces ferroptotic cell death. (A) Representative phase-contrast images of cells after treatment with MNPE. Hepa 1-6 cells were treated with 2 or 4 mM MNPE for 2 h
and then incubated in fresh medium for an additional 22 h in the presence or absence of 10 M ferrostatin-1 (Fer-1). (B) Viability of cells was assessed by CellTiter-Blue® Cell
Viability Assay. Hepa 1-6 cells were treated with indicated doses of MNPE for 2 h and then incubated in fresh medium for an additional 22 h in the presence or absence of 10
m
mM
ferrostatin-1 (Fer-1). Data represent the mean SEM (n ¼ 3). ***P < 0.001 (Tukey's test). n. s., not significant. (C) Cytotoxicity of cells was assessed by measuring released LDH
activity. Hepa 1-6 cells were treated with 4 mM MNPE or MNP (a precursor of MNPE) for 2 h and then incubated in fresh medium for an additional 22 h in the presence or absence of
10
m
M ferrostatin-1 (Fer-1). MNP was prepared by incubating MNPE at 37 ꢀC for 3 h. Data represent the mean SEM (n ¼ 3). ***P < 0.001 (Tukey's test). n. s., not significant. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073

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T. Homma et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
We then further characterized the cellular damage induced
We also evaluated the intracellular levels of ferrous iron,
another hallmark of ferroptosis, using the specific fluorescent probe
FeRhoNox™-1 [14] under a fluorescent microscopy. The results
showed that the MNPE treatment resulted in an increase in the
fluorescent intensity of the cells, confirming an increase in the
concentration of intracellular ferrous iron (Fig. 3D).
by MNPE treatment. DNA fragmentation, one of the represen-
tative criteria of apoptosis, was examined by examining DNA
extracted from the cells that had been treated with MNPE or
staurosporine, a well-known apoptotic inducer, on agarose gels.
DNA fragmentation was observed only in the cells that had been
treated with staurosporine, and not in the cells that had been
treated with MNPE (Fig. 2A). Since DNA fragmentation and the
subsequent apoptotic process are triggered by the activation of
caspases, we analyzed the proteolytic activation of caspase 3, an
executioner caspase of apoptosis. As we expected, the cleavage
of procaspase 3 was observed only after treatment with staur-
osporine, but not in response to MNPE (Fig. 2B). These results
indicate that MNPE is capable of inducing Hepa 1-6 cell death
without activating caspases, consistent with previously reported
findings [8].
3.3. MNPE does not alter Cys-GSH status
Glutathione (GSH), a Cys-centered tripeptide, has pleiotropic
functions in redox homeostasis, and the availability of Cys limits the
extent of GSH synthesis under conditions of cell culture. Because
cells with a GSH insufficiency are susceptible to ferroptotic cell
death, we measured the intracellular levels of Cys and GSH in the
cells that had been treated with MNPE. As a result, we found that
they were not significantly affected by MNPE (Fig. 4A and B). It
therefore appears that singlet oxygen released from MNPE induces
ferroptosis without affecting Cys-GSH status.
3.2. MNPE induces lipid peroxidation
We next investigated the issue of whether MNPE causes lipid
peroxidation, a hallmark of ferroptosis. After treating Hepa 1-6 cells
with MNPE, the increase in fluorescence intensity of the lipid per-
oxidation probe C11-BODIPY was monitored by flow cytometry
(Fig. 3A and B) and fluorescent microscopy (Fig. 3C). The MNPE-
treated cells showed an increase in the fluorescence at 24 h after
the treatment, and this increase was suppressed by a co-treatment
with ferrostatin-1. We also examined the oxidation of intrinsic
lipids in the MNPE-treated cells by determining the accumulation
of 4-hydroxynonenal (4-HNE), a predominant lipid peroxidation
product, using a specific antibody, by fluorescent microscopy
(Fig. 3C). MNPE-treated cells showed a greater increase in 4-HNE
adducts compared to the control cells. Co-treatment with
ferrostatin-1 again significantly suppressed lipid peroxidation in
the cells.
4. Discussion
The findings reported herein indicate that an MNPE treatment
induces cell death (Fig. 1), which had the following characteristics:
a necrotic pattern with caspase-independency (Fig. 2) and increases
in the levels of lipid peroxidation and ferrous iron (Fig. 3). This cell
death and lipid peroxidation were rescued by ferrostatin-1. Based
on these observations, it is conceivable that singlet oxygen released
from MNPE caused ferroptosis in Hepa 1-6 cells. It is also note-
worthy that the MNPE treatment did not affect the Cys-GSH status
(Fig. 4), which, at first glance, appears to not be consistent with the
original findings of ferroptosis [1] and many other cases of fer-
roptosis [15]. However, considering that singlet oxygen peroxidizes
unsaturated fatty acids [12] and that lipid peroxides are direct
mediators of ferroptosis [16], the ferroptotic pathway could be
A
B
kDa
35
Full-length
Caspase-3
25
bp
25
15
1500
1000
Cleaved
Caspase-3
500
100
GADPH
35
Fig. 2. Effects of MNPE on hallmarks of apoptosis. (A) Effect of MNPE on DNA ladder formation. Hepa 1-6 cells were treated with 4 mM MNPE for 2 h and then incubated in fresh
medium for an additional 10 h. For a positive control, cells were treated with 1 M staurosporine (Stauro) for 12 h. DNA was extracted from the cells, separated on a 1% agarose gel
m
and stained with ethidium bromide (B) Western blot analysis of caspase-3 in cells. Hepa 1-6 cells were treated under the same conditions as (A) and lysates were blotted for full
length caspase-3, cleaved caspase-3, and GAPDH.
Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073

T. Homma et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
n.s.
A
B
150
100
50
**
**
300
250
DMSO
MNPE
200
150
MNPE+Fer-1
100
50
0
0
10
10
C11-BODIPY
C
DMSO
MNPE
MNPE + Fer-1
D
DMSO
MNPE
Fig. 3. Effects of MNPE on the hallmarks of ferroptosis.(A) Lipid ROS production assessed by flow cytometry using a C11-BODIPY 581/591. Hepa 1-6 cells were treated with 2 mM
MNPE for 2 h and then incubated in fresh medium for an additional 22 h in the presence or absence of 10 M ferrostatin-1 (Fer-1). (B) Quantitative analysis of Lipid ROS levels from
(A). Data are presented as the mean SEM (n ¼ 3). **P < 0.01 (Tukey's test). n. s., not significant. (C) Hepa 1-6 cells were treated with 4 mM MNPE for 2 h and then incubated in fresh
medium for an additional 22 h in the presence or absence of 10 M ferrostatin-1 (Fer-1), and then a C11-BODIPY and 4-HNE-adducts were visualized. Bars: 100 m. (D) Hepa 1-
6 cells were treated with 4 mM MNPE for 2 h and then incubated in fresh medium for an additional 22 h, and intracellular ferrous iron was then visualized. Bars: 100 m.
m
m
m
m
activated by singlet oxygen during the process of lipid peroxidation,
downstream of the Cys-GSH axis.
generators of singlet oxygen. Because not only singlet oxygen but
also other ROS (including free radicals) are generated during
photosensitization [7], different types of cell death may occur
simultaneously under these experimental conditions. Compared to
other ROS, the reactions of singlet oxygen with biological molecules
are actually rather specific [12]. Therefore, caspase activation under
these conditions may not represent the result of singlet oxygen, but
the combined effects of ROS. Because MNPE generates ‘‘pure’’
singlet oxygen with no radical formation through thermal decom-
position under physiological conditions [21e23], MNPE would be a
reliable tool for investigating the specific function of singlet oxygen
The MNPE treatment did not activate the caspase-mediated
apoptotic pathway (Fig. 2) but induced ferroptosis (Figs. 1 and 3).
We have consistently demonstrated that singlet oxygen released
from MNPE actually inhibits the action of caspases and results in
the abortion of the apoptotic process [8,10]. These findings may
give an erroneous impression as compared with the previous re-
ports showing that caspase activation during cell death is induced
by singlet oxygen [17e20]. All of these studies involved the use of
photosensitizers including rose bengal and methylene blue as
Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073

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T. Homma et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
A
B
n.s.
n.s.
1.5
1.0
0.5
0.0
80
n.s.
n.s.
60
40
20
0
12 h
24 h
12 h
24 h
Fig. 4. Effects of MNPE on GSH levels during ferroptosis. Hepa 1-6 cells were treated with DMSO or MNPE at indicated conditions (first 2 h for treating) and then the intracellular
contents of (A) Cys and (B) GSH were measured. Data are presented as the mean SEM (n ¼ 3e4). n. s., not significant (Student's t-test).
in biological systems.
oxygen production. Naphthalene endoperoxides are clearly
helpful for understanding singlet oxygen-mediated ferroptosis,
which may contribute to therapeutics for singlet oxygen-involved
pathogenesis.
Having confirmed the fact that MNPE-derived singlet oxygen
triggers ferroptosis, we directed our attention to the underlying
mechanism for this process. For convenience, ferroptosis-inducing
agents can be divided into two classes [15]; class 1 inducers
decrease intracellular GSH (e.g. buthionine sulphoximine, erastin)
and class 2 inducers directly inhibit Gpx4 through active site in-
hibition (e.g. RSL3). While the MNPE treatment had no effect on the
Cys-GSH status (Fig. 4), it is interesting to note that an endoper-
oxide 1,2-dioxolane FINO2 also has been reported to induce fer-
roptosis without decreasing intracellular GSH [24,25]. Different
from MNPE, however, the endoperoxide moiety of FINO2 does not
generate singlet oxygen. Because the endoperoxide bond is highly
reactive, FINO2 directly binds to iron and disturbs its homeostasis.
Other compounds with endoperoxide moiety, artemisinin and its
derivatives, are Chinese anti-malarial drugs and enhance the
sensitivity of cells to ferroptosis [26]. They cause an increase in the
concentration of intracellular ferrous iron but have no effect on GSH
content. Thus FINO2 and artemisinin may induce ferroptosis by
initiating a multifaceted mechanism. Compared to these com-
pounds, however, singlet oxygen is released from MNPE during
cultivation and appears to induce ferroptosis, suggesting that their
mechanisms of action are substantially different.
Considering the fact that MNPE induces ferroptosis without
mediating the depletion of GSH (Fig. 4), it is possible that singlet
oxygen triggers the release of free iron and/or inactivates Gpx4 in
cells. In support of this possibility, it has been reported that singlet
oxygen is capable of irreversibly inactivating glutathione reductase,
thioredoxin reductase, and Gpx in a cell-free system [27,28],
although photosensitizers were employed in these studies. Sele-
nocysteine, which plays catalytic roles in Gpx and thioredoxin
reductase, is more sensitive to oxidation than Cys. However, a
recent in-vivo study revealed that selenocysteine-containing wild-
type Gpx4 is capable of confering resistance to irreversible over-
oxidation caused by peroxides [29]. Thus, the issues of how singlet
oxygen affects iron status or Gpx4 activities remain unclear at
present and further clarification is awaited.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
This work was partly supported by the YU-COE [C31-3] program
of Yamagata University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.08.073.
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In summary, evidence is provided for the first time, to show that
singlet oxygen, which is generated from naphthalene endoperoxide
in pure form, triggers ferroptosis in cultivated cells. Different from
apoptosis, ferroptosis stimulates the release of intracellular bio-
logical molecules, which may be involved in aggravating inflam-
mation or autoimmune responses under conditions of singlet
Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073

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Please cite this article as: T. Homma et al., Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide, Biochemical
and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.08.073