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Original Articles
Heme oxygenase-1 mediates BAY 11e7085 induced ferroptosis
Ling-Chu Chang
a
,
b
,
*
, Shih-Kai Chiang
c
, Shuen-Ei Chen
c
, Yung-Luen Yu
d
,
e
,
f
,
Ruey-Hwang Chou
d
,
e
,
f
, Wei-Chao Chang
e
a
Chinese Medicinal Research and Development Center, China Medical University Hospital, Taichung 40447, Taiwan
b
Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan
c
Department of Animal Science, National Chung Hsing University, Taichung 40227, Taiwan
d
Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40447, Taiwan
e
Center for Molecular Medicine, China Medical University Hospital, Taichung 40447, Taiwan
f
Department of Biotechnology, Asia University, Taichung 41354, Taiwan
article info
Article history:
Received 1 July 2017
Received in revised form
12 December 2017
Accepted 17 December 2017
Keywords:
BAY 11e7085
Ferroptosis
Reactive oxygen species
Glutathione
Heme oxygenase-1
abstract
Ferroptosis is a form of oxidative cell death and has become a chemotherapeutic target for cancer
treatment. BAY 11e7085 (BAY), which is a well-known I
k
B
a
inhibitor, suppressed viability in cancer cells
via induction of ferroptotic death in an NF-
k
B-independent manner. Reactive oxygen species scavenging,
relief of lipid peroxidation, replenishment of glutathione and thiol-containing agents, as well as iron
chelation, rescued BAY-induced cell death. BAY upregulated a variety of Nrf2 target genes related to redox
regulation, particularly heme oxygenase-1 (HO-1). Studies with specific inhibitors and shRNA in-
terventions suggested that the hierarchy of induction is Nrf2!SLC7A11!HO-1. SLC7A11 inhibition by
erastin, sulfasalazine, or shRNA interference sensitizes BAY-induced cell death. Overexperession of
SLC7A11 attenuated BAY-inhibited cell viability. The ferroptotic process induced by hHO-1 over-
expression further indicated that HO-1 is a key mediator of BAY-induced ferroptosis that operates
through cellular redox regulation and iron accumulation. BAY causes compartmentalization of HO-1 into
the nucleus and mitochondrion, and followed mitochondrial dysfunctions, leading to lysosome targeting
for mitophagy. In this study, we first discovered that BAY induced ferroptosis via Nrf2!SLC7A11!HO-1
pathway and HO-1 is a key mediator by responding to the cellular redox status.
©2017 Elsevier B.V. All rights reserved.
1. Introduction
Ferroptosis is a new form of regulated cell death characterized
by reactive oxygen species (ROS) generation, lipid peroxidation,
and iron accumulation. It is mechanistically different from necrosis/
necroptosis and apoptosis [1e3]. Morphological alterations of fer-
roptosis are manifested through cell volume shrinkage, increased
mitochondrial membrane density, and reduced cristae [3].
Recently, regulating ferroptotic process has become a strategy for
chemotherapy in cancer treatment and several agents have been
shown to trigger cell ferroptosis by acting on system X
c
!
[4],
glutathione peroxidase 4 (GPx4) [3,4], endoplasmic reticulum (ER)
homeostasis, and ferritin degradation through an autophagic pro-
cess [4,5].
Nuclear factor-E2-related factor 2 (Nrf2) is a master regulator in
the cellular defensive response against oxidative or electrophilic
stress. Identified target genes by Nrf2 include NAD(P)H quinone
oxidoreductase 1, heme oxygenase-1 (HO-1), solute carrier family 7
membrane 11 (SLC7A11, xCT), NAD(P)H quinone oxidoreductase 1,
thioredoxin 1, phase II detoxifying enzymes (e.g., glutathione S-
transferase, UDP-glucuronosyltransferase, GPx4, glutathione
reductase, and glutamate cysteine ligase subunits; GCLc and
GCLm), and several multidrug resistance-associated transporters
Abbreviations: 7-AAD, 7-aminoactinomycin D; BAY, BAY 11e7085; BiP, binding
immunoglobulin protein; CHOP, CCAAT/enhancer-binding protein (C/EBP) homol-
ogous protein; DFO, deferoxamine; DTT, dithiothreitol; ER, endoplasmic reticulum;
FBS, fetal bovine serum; GBM, glioblastoma multiforme; GPx4, glutathione perox-
idase 4; GSH, glutathione; GSSG, oxidized
L
-glutathione; HO, heme oxygenase;
KEAP1, Kelch-like ECH-associated protein 1; LIP, labile iron pool;
b
Me,
b
-mercap-
toethanol; NAC, N-acetyl-
L
-cysteine; Nrf2, nuclear factor-E2-related factor 2; PBS,
phosphate-buffered saline; PERK, ER stress-related protein double-stranded RNA-
dependent protein kinase-like ER kinase; ROS, reactive oxygen species; SAS, sul-
fasalazine; SLC7A11, solute carrier family 7 membrane 11; ZnPP, zinc protopor-
phyrin-9.
*Corresponding author. Chinese Medicinal Research and Development Center,
China Medical University Hospital, No. 2, Yude Road, Taichung, 40447, Taiwan
E-mail address: t27602@mail.cmuh.org.tw (L.-C. Chang).
Contents lists available at ScienceDirect
Cancer Letters
journal homepage: www.elsevier.com/locate/canlet
https://doi.org/10.1016/j.canlet.2017.12.025
0304-3835/©2017 Elsevier B.V. All rights reserved.
Cancer Letters 416 (2018) 124e137
[6,7]. Nrf2 is anchored to Kelch-like ECH-associated protein 1
(KEAP1)-Cul3 complex and thus rapidly targeted for degradation. In
response to stimuli such as pro-oxidants or electrophiles, KEAP1 is
modified through oxidation or adduction on cysteine residues,
leading to degradation, and thereby dissociates Nrf2 for trans-
location into the nucleus, where it is activated by dimerization with
a small protein MAF and binds to the antioxidant-responsive ele-
ments (ARE) for target antioxidant genes expression [6,7].
SLC7A11 and chaperone CD98 form the system X
c
!
, a cystine/
glutamine antiporter, which regulates glutathione (GSH) synthesis/
homeostasis and is thus tightly associated with the defensive
response against oxidative insults [8]. Pharmacological inhibition of
system X
c
!
has been shown to trigger cell ferroptosis [1,4]. Heme
oxygenases are the rate-limiting enzymes in heme degradation and
the catabolic products are free iron, carbon monoxide, and bili-
verdin/bilirubin [9]. Since the inducible form, HO-1, rapidly re-
sponds to a variety of stimuli, such as oxidative, hypoxia, and
inflammation [10], it has been shown to function as an antioxidant
and anti-apoptotic molecule [7]. An increase in the cellular HO-1
level is considered to be an antioxidative mechanism to protect
cells from ROS assault. However, overexpression of HO-1 has been
shown to have pro-oxidant effects [11e14]. Therefore, activation of
the Nrf2 pathway, particularly with HO-1 and SLC7A11 induction
through oxidative agents may participate in the iron-dependent
and oxidative cell death.
BAY 11e7085 (BAY), (E)-3-(4-t-Butylphenylsulfonyl)-2-
propenenitrile, is a well-known irreversible inhibitor of I
k
B
a
phosphorylation, which leads to the blockade of NF-
k
B signaling
[15]. Past studies have shown an anti-proliferation and pro-
apoptotic effect by BAY on colonic epithelial cells [16], keratino-
cytes [17], chondrocytes [18 ], endothelial cells [19], and synovial
fibroblasts [20]. In addition, BAY has been implicated as a potential
agent against colon cancer [12], renal carcinoma [19], and lym-
phoma [21]. Proferroptotic effects by BAY and details of related
mechanisms, however, have never been reported.
The present study aimed to characterize the underlying mech-
anism by BAY and demonstrate potential chemotherapeutic targets
for cancer treatment. For the first time, we showed that BAY
significantly suppressed cell viability of cancer cells, which rely on
ferroptotic cell death induction independently of I
k
B/NF-
k
B
pathway. Pharmacological approaches and genetic gain/loss of
function studies further identified Nrf2!SLC7A11!HO-1 pathway
induction as a key signaling in BAY-induced ferroptosis. Our find-
ings provide new insights to understand the mechanism of HO-1 in
ferroptotic cell death and highlight a chemotherapeutic target for
cancer treatment.
2. Materials and methods
2.1. Reagents and antibodies
DMEM/F12 medium, RPMI-1640 medium, McCoy's 5a Medium, fetal bovine
serum (FBS), and penicillin/streptomycin were purchased from Thermo Fisher Sci-
entific Inc. (Pittsburgh, PA, USA). Beta-mercaptoethanol (
b
Me) and dithiothreitol
(DTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). BAY 11e7085 (BAY), z-
VAD-fmk, N-acetyl-
L
-cysteine (NAC), reduced
L
-glutathione (GSH), oxidized
L
-gluta-
thione (GSSG), and 2
0
,7
0
-dichlorodihydrofluorescein diacetate were obtained from
Enzo Life Sciences (Farmingdale, NY, USA). Monobromobimane, deferiprone,
diphenyl-1-pyrenylphosphine, necrostatin-1, ferrostatin-1, liproxstatin-1, zinc
protoporphyrin-9,
L
-NAME, zileuton, rotenone, erastin, sulfasalazine, and sorafenib
were obtained from Cayman Chemicals (Ann Arbor, MI, USA).Antibodies against NF-
k
B p50 (#12540), NF-
k
B p65 (#8242), phospho-NF-
k
B p65 (Ser536, #3033), I
k
B
a
(#4814), phospho-I
k
B
a
(Ser32, #2859), IKK
a
(2682), IKK
b
(#2370), phospho-IKK
a
/
b
(Ser176/180, #2697), Nrf2 (#12721), KEAP1 (#4678), MEK1 (#8727), H2A.X (#2595),
CHOP (#2895), BiP (#3177), PERK (#5683), PDI (#3501), Myc-Tag (#2278), Flag-Tag
(#8146), SLC7A11 (#12691), Thioredoxin 1 (#2429), and TOM20 (#42406) and
MitoTracker
®
Red CMXRos were purchased from Cell Signaling Technology (Beverly,
MA, USA). The antibody against
b
-actin (MAB1501) was from the EMD Millipore
Corporation. Antibodies against GPx4 (sc-50497), HO-1 (sc-390991), and LAMP-1
(sc-17768) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Other reagents were purchased from Sigma-Aldrich. These compounds were dis-
solved in DMSO (final concentration was less than 0.1% (v/v)).
2.2. Cell culture
All cell lines used in this study were obtained from American Type Culture
Collection (Manassas, VA, USA). Human breast cancer cell lines (MDA-MB-231, MDA-
MB-468, MCF-7, and SKBR3) and a human lung cancercell line (A549) were cultured
in DMEM/F12 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100
m
g/
ml streptomycin. Human hepatocellular carcinoma (HuH-7) and glioblastoma
(DBTRG-05MG) were cultured in RPMI-1640 medium supplemented with 10% FBS,
100 U/ml penicillin, and 100
m
g/ml streptomycin. A human ovarian carcinoma cell
line (SKOV3) was cultured in McCoy's 5a medium supplemented with 10% FBS, 100
U/ml penicillin, and 100
m
g/ml streptomycin. The cells were maintained in a hu-
midified incubator at 37
"
C and 5% CO
2
.
2.3. Cell viability assay
Cell viability was evaluated using the sulforhodamine B colorimetric assay. Cells
were cultured in 96-well plates and treated with the specified compounds for the
indicated times. After treatment, cells were fixed with cold 10% trichloroacetic acid
and kept at 4
"
C for 1 h. After washing the cells, sulforhodamine B solution 0.4% (w/
v) in 1% acetic acid was added to each well. Then, the plates were incubated for
10 min at room temperature. Unbound dye was removed by washing the cells with
1% acetic acid, and the plates were air dried. Bound dyes were dissolved in DMSO
and detected spectrophotometrically at an absorbance of 510 nm.
2.4. Cell morphology observation
Cells were visualized and photographed using a phase-contrast microscope
equipped with a digital camera (Leica Microsystems, Wetzlar, Germany).
2.5. Staining of fluorescent dyes
Fluorescent dyes, 2
0
,7
0
-dichlorodihydrofluorescein diacetate (DCF, for ROS),
diphenyl-1-pyrenylphosphine (for lipid peroxidation), monobromobimane (mBBr,
for glutathione), rhodamine 123 (for mitochondrial membrane potential), and ac-
ridine orange 10-nonylbromide staining (NAO, for mitochondrial mass) were either
measured through an ELISA plate reader or flow cytometry. With an ELISA plate
reader, the cells were seeded in a 96-well black plate (Nunc, Thermo Fisher Scien-
tific, Inc.) 24 h prior to treatment. After treatment, the cells were washed with
phosphate-buffered saline (PBS) three times and fluorescent dyes (final concen-
tration: 10
m
M) were added to the complete medium and incubated for 30 min at
37
"
C. The cells were washed with PBS three times, and the intracellular fluorescence
was measured using a microplate reader (BioTek, Gen5, Winooski, VT, USA).
Detection wavelengths were Ex 485/Em 528 (for DCF) and Ex 380/Em 450 (for
mBBr). The fluorescent levels were expressed as arbitrary units or normalized by the
control group and calculated as a percentage. Using the flow cytometry method,
compound-treated cells were trypsinized and then incubated with 10
m
Mfluores-
cent dyes in PBS at 37
"
C for 30 min. After incubation, the cells were washed with
PBS and quantified by flow cytometry (BD FACSCalibur, Becton Dickinson Inc. San
Jose, CA, USA). The data were analyzed using Cell Quest software (DB Biosciences,
Franklin Lakes, NJ, USA). The fluorescent levels were expressed as the mean fluo-
rescence intensity or calculated by either fold change or percentage.
2.6. Staining of annexin V and 7-aminoactinomycin D (7-AAD)
Ferroptosis was determined followed the method described by Chen et al. [25].
After treatments, cells were collected and stained with Annexin V-FITC reagent
(BioVision) and 7-AAD (Cayman) and subjected to flow cytometry. The percentages
of dead cells were quantified with CellQuest software.
2.7. Immunofluorescence staining
Cells were grown on sterile coverslips embedded in a 6-well plate. After treat-
ments, cells were fixed with 3.7% (w/v) paraformaldehyde and permeabilized with
0.2% (v/v) Triton X-100. After blocking with 2% (w/v) bovine serum albumin in PBS,
the protein was detected using anti-PDI and anti-HO-1 antibodies followed by re-
action with Alexa Fluor
®
488- conjugated and Alexa Fluor
®
594- conjugated sec-
ondary antibodies (Invitrogen). For the determination of mitochondrial morphology,
treated cells were incubated with MitoTracker
®
Red CMXRos for 30 min prior to the
end of the experiment and then fixed and permeabilized. After blocking, the cells
were stained using LAMP-1 antibody followed by a reactionwith Alexa Fluor
®
488-
conjugated secondary antibody. Coverslips were mounted and fluorescence images
were taken by a Leica Microsystems TCS SP8 Confocal Spectral microscope (Leica
Microsystems, Wetzlar, Germany).
2.8. Determination of the labile iron pool
The total cellular labile iron pool was detected based on the calcein-
acetoxymethyl ester method [22]. After trypsinization, the cells were washed
twice with PBS followed by incubation of 0.2
m
M calcein-acetoxymethyl ester (Enzo)
for 15 min at 37
"
C. Then, the cells were washed with PBS and incubated with or
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137 125
without deferiprone (100
m
M) for 1 h at 37
"
C. The cells were analyzed by flow
cytometry. The levels of the liable iron pool were calculated by the difference in
cellular mean fluorescence with and without deferiprone incubation.
2.9. Quantitative real-time reverse transcription PCR (qRT-PCR) and XBP1 mRNA
splicing
Total RNA was extracted using TRIzol
®
Reagent (ThermoFisher). Five
m
g of total
RNA was used as a template for cDNA strain synthesis using M-MLV reverse tran-
scriptase (ThermoFisher) according to the manufacturer's instructions. The qRT-PCR
analysis was performed by a LightCycler
®
480 II Real-Time PCR system (Roche
Applied Sciences, Manheim, Germany) using KAPA SYBR FAST qPCR Master Mix
(Kapa Biosystems). PCR primers were shown in Supplementary methods Table S1.
The expression levels of mRNA were normalized against the level of
b
-actin mRNA in
the same sample.
The XBP1 mRNA splicing assay was detected by Reverse Transcription-PCR using
XBP1 specific primers and
b
-actin-specific primers as a control. The PCR conditions
were as follows: 95
"
C for 3 min, followed by 35 cycles of 95
"
C for 30 s, 55
"
C for 30 s,
72
"
C for 30 s. The PCR products were separated on a 3% agarose gel and visualized
using a GelDoc Imaging System (Bio-Rad).
2.10. Gene knockdown by shRNA
The pCMV-
D
R8.91, pMD.G, and specific short hairpin PLKO.1 plasmids were
purchased from the National RNAi Core Facility Academia Sinica (Taiwan). The
shRNA clones used in this study are described in Supplementary methods Table S2.
The 293T cells were transiently transfected with specific shRNA and packaging
vectors (pCMV-
D
R8.91 and pMD.G) using Lipofectamine 2000 transfection reagent
(Invitrogen) in 293T cells. Forty-eight h after transduction, lentivirus particles in the
medium were filtered with a 0.22
m
mfilter and used for infection. Cancer cells were
infected with specific shRNA viral-contained supernatant in the presence of poly-
brene (8
m
g/ml). After 24 h of incubation at 37
"
C, the medium was replaced with
complete medium containing puromycin (2
m
g/ml). The cells were prepared for tests
and harvested based on the experiments that were required.
2.11. Overexpression of HO-1 and SLC7A11
Cancer cells were cultured in a culture dish or a culture plate and allowed to
attach overnight. The empty vector, pCMV-XL5 (OriGene) or pcDNA3.1
þ
/C-(K)DYK
(GeneScript), or the vector containing human HMOX1 (OriGene) or human SLC7A11
(GeneScript) were transfected into cells for 24 h.
2.12. Isolation of cytosolic and nuclear protein
After treatment, cells were washed with PBS and trypsinized with trypsin. Cells
were then gently suspended in a hypotonic buffer (10mM HEPES, pH 7.9, 1.5 mM
MgCl
2
, proteinase inhibitors, and phosphatase inhibitors) and kept on ice for 15 min.
After the addition of 0.5% NP-40, the cell suspension was vigorously vortexed for 10 s
to lyse the cells and release the cytoplasm. The cellular mixtures were then
centrifuged at 10,000 $gfor 2 min at 4
"
C to separate the cytoplasmic components
(supernatant) from the nuclei fraction (pellet). For the purification of nuclear pro-
teins, the nuclear pellets were washed with hypotonic buffer three times and sus-
pended in PBS containing proteinase inhibitors and phosphatase inhibitors. The
suspension was further sonicated and centrifuged at 12,000 $gat 4
"
C for 15 min.
The isolated fractions were stored at !80
"
C for further analysis.
2.13. Isolation of mitochondrial and cytosolic protein fractions
Mitochondria were isolated based on the method by Frezza et al., 20 07 [23].
Briefly, cells were suspended in Isolation Buffer (10 mM Tris/MOPs, 1 mM EGTA/Tris,
200 mM sucrose, 1 mM Na
3
VO
4
, 5 mM NaF, 1 mM PMSF,10
m
g/ml each of leupeptin,
aprotinin, and pepstatin A) and homogenized using a Teflon pestle. The homoge-
nates were centrifuged at 600 $gfor 10 min at 4
"
C. The supernatant was further
centrifuged at 7000 $gfor 10 min at 4
"
C. The pellets containing the mitochondria
were stored for the mitochondria assays.
2.14. Western blot analysis
Cells were suspended in PBS containing proteinase inhibitors (1 mM PMSF,
10
m
g/ml each of leupeptin, aprotinin, and pepstatin A) and phosphatase inhibitors
(1 mM Na
3
VO
4
, 1 mM NaF) and then sonicated. Protein concentrations were
measured using the protein assay from Bio-Rad (Hercules, CA, USA). Lysate proteins
were separated by SDS-PAGE gels and transferred onto FluoroTrans
®
PVDF Transfer
Membranes (Pall Corporation). The membranes were then blocked with Tris-
buffered saline with 0.1% Tween 20 and 5% skim milk before they were incubated
with the appropriate primary antibodies. The membranes reacted with horseradish
peroxidase-conjugated secondary antibodies (EMD Millipore). Signaling was visu-
alized using the Clarity™Western ECL Substrate (Bio-Rad) and measured using the
luminescence image analyzer ImageQuant LAS 4000 (GE Healthcare Life Sciences).
2.15. Statistical analysis
All experiments were conducted at least three times, and the data are presented
as the mean ±SEM. Statistical significance among different treatments was deter-
mined by a t-test. A Pvalue <.05 was considered to be statistically significant.
3. Results
3.1. BAY induces an NF-
k
B-independent cell death
Treatment of BAY drastically inhibited cell viability in MCF-7,
MDA-MB-231, MDA-MB-468, and SKBR3 breast cancer cells
(Fig. 1A) with IC
50
(the concentrations of fifty percent inhibition)
approximately 7.03 ±0.07, 4.31 ±0.6, 5.21 ±0.14, and
4.10 ±0.01
m
M for 24 h of treatment, respectively. Morphological
examinations showed that numerous vacuoles were accumulated
in the perinuclear and cytoplasmic areas (Fig. 1B). Suppression of
cell viability by BAY was also observed in other cancer cells,
including SKOV3 ovary cells, A549 lung cells, HuH-7 hepatoma
cells, and DBTRG-05MG glioblastoma multiforme (GBM) cells with
an IC
50
value approximately 7.31 ±0.09, 4.66 ±0.1, 5.84 ±0.03, and
5.53 ±0.11
m
M for 24 h of treatment, respectively (Fig. 1A). Treat-
ment of BAY resulted in rapid inhibition of I
k
B
a
and the NF-
k
B
subunit p65 phosphorylation without changes in the I
k
B
a
and p65
protein levels, whereas neither phosphorylation nor protein levels
of IKK
a
and IKK
b
were significantly affected by BAY
(Supplementary Fig. S1). However, in cells with a shRNA inter-
vention for I
k
B
a
and p65 knockdown, BAY remained effective to
inhibit cell viability in a concentration-dependent manner
(Fig. 1CeD). The intervention of I
k
B
a
and p65 knockdown slightly
suppressed cell death (Fig. 1CeD). Therefore, NF-
k
B pathway was
excluded in BAY-mediated cell death.
3.2. BAY suppresses cell viability through ROS generation
Various pharmacological inhibitors, including apoptosis (z-
VAD-fmk), necrosis/necroptosis (necrostatin-1), ferroptosis (fer-
rostatin-1 and liproxstatin-1), antioxidant (N-acetyl-
L
-cysteine,
NAC), and nitric oxide synthase (
L
-NAME) were used to define the
type of cell death by BAY. BAY-induced cell death was significantly
attenuated in the presence of ferrostatin-1, liproxstatin-1, and NAC,
but not by z-VAD-fmk, necrostatin-1, and
L
-NAME (Fig. 2A). Cell
viability was also determined by annexin V/7-AAD and analyzed by
flow cytometry [24]. As shown in Fig. 2B, BAY significantly
increased the 7-AAD-positive cell population, but little in Annexin
V staining. The addition of ferrostatin-1, liporxstatin-1, and NAC
effectively suppressed BAY-induced cell death (Fig. 2B). Zileuton, a
5-lipoxygenase inhibitor, suppresses lipid and protein oxidation
[25], and ferroptosis [8,26] also significantly reversed cell death by
BAY (Supplementary Fig. S2). These results excluded the involve-
ment of apoptosis and necrosis/necroptosis in BAY-induced cell
death and suggested that there was an alternative type of cell death
which is dependent on ROS induction and lipid peroxidation.
Ferroptosis depends on ROS for its cytotoxicity [1]. In the pres-
ence of fluorogenic dye, 2
0
,7
0
-dichlorofluorescein diacetate that can
monitor total cellular ROS generation, BAY rapidly and significantly
triggered ROS production in both MDA-MB-231 and MDA-MB-468
breast cancer cells in a concentration- and time-dependent manner
(Fig. 2C). Treatment of BAY also significantly increased lipid per-
oxidation which slightly less than H
2
O
2
, an oxidant, but stronger
than erastin, a ferroptosis inducer [1,4]. Rotenone, a mitochondrial
complex I inhibitor which enhances ROS generation [27], has no
effect on lipid peroxidation (Fig. 2D). Furthermore, under I
k
B
a
and
p65 knockdown, BAY maintained effective induction on ROS gen-
eration (Fig. 2E, Supplementary Fig. S3). Furthermore, NAC treat-
ment completely prevented phosphorylation of I
k
B
a
and p65
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137126
suppression by BAY in as little as 0.25 h (Fig. 2F), suggesting that
BAY exerts its cytotoxicity by operating through ROS generation.
3.3. BAY upregulates the redox-responsive proteins
Treatment of BAY caused a concentration-dependent suppres-
sion of cellular GSH levels (Fig. 3A). The supplementation of
reduced GSH but not oxidized GSH (GSSG) completely abolished
cell death by BAY (Fig. 3B). Treatment of the thiol-containing
reducing agents,
b
-mercaptoethanol (
b
Me) or dithiothreitol (DTT),
blunted ROS generation and rescued cell viability (Fig. 3CeD). In
addition,
b
Me and DTT attenuated suppression of I
k
B
a
and p65
phosphorylation by BAY (Supplementary Fig. S4), suggesting that
the oxidative stress-related events also mediate the inhibitory ef-
fect of BAY on I
k
B
a
/NF-
k
B phosphorylation, and the induction of
oxidative cell death is irrelevant to I
k
B
a
/NF-
k
B activation. These
results indicated that BAY exerts its cytotoxic effect by acting on
cellular redox regulation, leading to the ferroptotic cell death.
We next examined the expression of genes related to cellular
redox response, as well as autophagy and ER stress that has been
shown responsive to ferroptotic agents [2,5]. Treatment of BAY
upregulated gene expression related to oxidative stress and ER
stress responses including HMOX1,SLC7A11,HSPA5, and DDIT3, as
well as to cellular redox regulation and autophagy pathways
including GSTp,GSR,GCLC,NFE2L2,TXNRD1,TXNIP,NQO1,XPB1,
GADD45A,HSP90B1, and SQSTM1 (Fig. 3E). HO-1 and SLC7A11
abundance (encoded by HMOX1 and SLC7A11, respectively)
increased at 4 h and sustained to 8 h after BAY treatment (Fig. 3F).
Nrf2, a known upstream of HO-1 and SLC7A11 in response to ROS
and electrophilic stress [6,7], exhibited a dramatic increase at 1 h
and sustained to 8 h, whereas Nrf2 repressor KEAP1 decreased after
1h (Fig. 3F). GPx4, a ferroptosis repressor [3] that provides an
antioxidative defense to prevent lipid peroxidation, showed little
change (Fig. 3F).
3.4. BAY induces cell death via upregulation of HO-1
Both pharmacological inhibitor and knockdown approach with
shRNA were used to examine the role of HO-1 in BAY-induced
ferroptosis. Treatment of ZnPP (zinc protoporphyrin-9), a specific
inhibitor of HO-1, effectively attenuated BAY-induced cell death
(Fig. 4A). Defective HO-1 expression significantly rescued cell sur-
vival suppressed by BAY (Fig. 4B). We next examined the upstream
molecules levels of HO-1. Defective Nrf2 expression blunted
SLC7A11 and HO-1 levels (Fig. 4C), but the deficiency of SLC7A11
only abolished the increase of the HO-1 level by BAY without effects
on Nrf2 (Fig. 4D). The defective HO-1 expression had no marked
Fig. 1. BAY induces an NF-
k
B-independent cell death. (A) BAY effectively inhibits cell viability. Cancer cells were treated with the indicated concentrations of BAY for 24 h. Cell
viability was assessed by a sulforhodamine B colorimetric reaction. (B) Cancer cells were treated with vehicle or BAY (5
m
M or 10
m
M) for 24 h and the morphology was photo-
graphed using a phase-contrast microscope (magnification, 100 $). Representative images from three independent experiments are shown. (C, D) Cancer cells were transfected
with shRNA of I
k
B
a
(encoded by NFKBIA) or NF-
k
B p65 (encoded by RELA), then followed by 8 h treatment with 10
m
M BAY and used for cell viability analysis. Cells were harvested
for protein determination using Western blot analysis. All values are the mean ±SEM from three independent experiments. NS, no significant difference compared to the control
(CTL).
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137 127
effects on Nrf2 and SLC7A11 levels (Fig. 4E). These results suggested
a Nrf2!SLC7A11!HO-1 hierarchy as a response to BAY induction.
Furthermore, BAY treatment increased cytosolic Nrf2 and HO-1
translocation into the nuclei, whereas NF-
k
B subunit p50 and p65
levels remained still in the cytosol (Fig. 4F). BAY also significantly
increased HO-1 compartmentalization within mitochondrial frac-
tions and suppressed the abundance of cytosolic thioredoxin 1 and
mitochondrial outer membrane TOM20 [28](Fig. 4G). NAC,
b
Me,
and DTT treatments blunted the BAY-induced increase of Nrf2, HO-
1, and SLC7A11 abundance (Supplementary Fig. S5A!B) as well as
nuclear translocation of Nrf2 and HO-1 (Supplementary
Fig. S5C!D). However, BAY remained effective in promoting Nrf2
and suppressing the KEAP1 in I
k
B
a
- and p65-knockdown cells
(Supplementary Fig. S6).
3.5. SLC7A11 inhibition sensitizes cancer cells to ferroptotic death
induction by BAY
SLC7A11 play an essential role in the process of ferroptosis [1,4].
We further examined the impacts of SLC7A11 status on BAY-
induced cell death. Erastin, sulfasalazine (SAS), and sorafenib act
as SLC7A11 inhibitors and cause the compensated upregulation of
SLC7A11, could lead to ferroptosis by blockade of cysteine uptake
and GSH synthesis [4]. Compared to erastin and SAS, BAY induced a
more substantial increase of SLC7A11, whereas sorafenib is variable
in MDA-MB-231 and DBTRG-05MG (Fig. 5A). However, all three
ferroptosis-inducing agents failed to elevate HO-1 expression
(Fig. 5A). BAY-induced HO-1 remained unchanged while combi-
nation with erastin and SAS, but suppressed by the presence of
sorafenib (Fig. 5A). Moreover, the combination with erastin and SAS
increased BAY-induced cell death (Fig. 5B). Deficient SLC7A11
Fig. 2. BAY suppresses cell viability through ROS generation. (A, B) Cancer cells were pre-incubated with various inhibitors, z-VAD-fmk (VAD, 50
m
M), necrostatin-1 (Nec, 50
m
M),
ferrostatin-1 (Ferro, 1
m
M), liproxstatin-1 (Lipro, 1
m
M), N-acetyl-
L
-cysteine (NAC, 5 mM), or
L
-NAME (NAME, 1 mM) for 30 min followed by BAY (10
m
M) treatment for 8 h. Cell
viability was assessed by a sulforhodamine B colorimetric reaction (A) or Annexin V/7-AAD staining (B). For Annexin V/7-AAD staining, cells were collected and the proportion of 7-
AAD-positive cells (dead) was determined by flow cytometry. All values are the mean ±SEM from three independent experiments. **P<.01, compared to the BAY alone. (C) Cancer
cells were loaded with 20,70-dichlorofluorescein diacetate (DCF) and exposed to BAYat the indicated concentrations and time intervals for ROS generation analysis. The fluorescent
signal was detected by a fluorescent plate reader. (D) Cancer cells were treated with vehicle (DMSO, CTL), BAY (10
m
M), hydrogen peroxide (H
2
O
2
, 0.6 mM), erastin (10
m
M), or
rotenone (1
m
M), then used for lipid peroxidation analysis with the fluorescent dye (diphenyl-1-pyrenylphosphine) staining and flow cytometry. *P<.05; **P<.01, compared to the
control (CTL). (E) I
k
B
a
knockdown cancer cells were treated with 10
m
M BAY for 8 h and used for ROS generation analysis by the DCF staining and flow cytometry. NS, no significant
difference compared to the control (shLUC). (F) MDA-MB-231 breast cancer cells were pretreated with 5 mM NAC followed by 10
m
M BAY for 6 h and used for protein determination
in a Western blot analysis. The representative figure is one of three independent experiments.
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137128
expression through shRNA interference significantly exacerbated
cell death by BAY in both MDA-MB-231 and DBTRG-05MG cells
(Fig. 5C).
Efficacy of erastin and sorafenib is dependent on SLC7A11 in
gliomas [29]. The increase of SLC7A11 levels ameliorates sorafenib
and erastin-induced ferroptosis [30]. We next tested whether the
BAY-inhibited cell viability is reversed by the overexpression of
SLC7A11. Overexpression of human SLC7A11-Flag (hSLC7A11)
significantly blunted BAY-increased HO-1 and BAY-induced cell
death (Fig. 5DeE). Thereby, the abundance of SLC7A11 affects the
sensitivity of cell viability inhibition by BAY.
3.6. HO-1 upregulation elicits cellular ferrous accumulation to
mediate ferroptosis by BAY
Overexpression of HO-1 has been shown to exert pro-oxidant
effects [11e14] and ferroptosis by erastin was associated with
upregulated HO-1 expression [31]. Since the knockdown of HO-1
significantly blocked BAY-induced ferroptosis, the role of HO-1 in
BAY-mediated ROS generation and lipid peroxidation was further
investigated. Compared to the control vector treatment, enforced
expression of human HO-1-myc (hHO-1) caused a concentration-
dependent decrease in cell viability (Fig. 6A!B). The HO-1
expression has been shown to regulate cellular iron homeostasis
[14,32], and both free and chelatable iron, which is the so-called
labile iron pool (LIP), contributes to the ferrous accumulation that
leads to cell cytotoxicity via ROS generation [33]. LIP levels were
elevated by BAYand hHO-1-overexpression (Fig. 6CeD). Treatment
of ZnPP significantly prevented the increase of LIP level by BAY and
hHO-1 overexpression (Fig. 6CeD). Knockdown of HO-1 blunted
BAY-elicited LIP levels (Fig. 6E). Treatment of deferoxamine (DFO),
an iron chelator, significantly ameliorated BAY-induced cell death
(Fig. 6F). These results indicated that HO-1 mediates BAY-induced
ferroptosis by promoting cellular ferrous accumulation.
Intracellular iron levels are maintained by the transferrin re-
ceptor and ferroportin for iron import and export, and particularly
Fig. 3. BAY upregulates the redox-responsive protein expression. (A) Cancer cells were treated with indicated concentrations (CTL, 2.5, 5, 7.5, or 10
m
M) of BAY for 4 h. The levels of
glutathione were determined using monobromobimane staining. All values are the mean ±SEM from three independent experiments. *P<.05; **P<.01, compared to the control
(CTL). (B) Cancer cells were pre-incubated with 2 mM GSH or 2 mM GSSG for 30 min, then induced by BAY (10
m
M) for 8 h and used for cell viability analysis. **P<.01. (C, D) Cancer
cells were pretreated with
b
Me (100
m
M) or DTT (100
m
M) for 30 min and followed by BAY (10
m
M) treatments. ROS generation (C) was detected at 4 h and cell viability (D) was
detected 8 h after BAY stimulation. All values are the mean ±SEM from three independent experiments. **P<.01. (E) Total RNA extracted from BAY (10
m
M for 4 h)-treated cancer
cells were subjected to qRT-PCR analysis for gene expression analysis. The expression levels of mRNA were normalized to
b
-actin. (F) Cancer cells were treated with 10
m
M BAY for
the indicated time intervals. Cells were harvested for protein determination using Western blot analysis. The representative figure is one of three independent experiments.
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137 129
through sequestration into ferritin as a storage form as well as
ferritinophagy for iron release from ferritin [34e36]. Treatment of
BAY upregulated TF (encodes transferrin) and FTH1 (encodes
ferritin heavy chain), downregulated SLC40A1 (encodes ferro-
portin), but had no effects on TFRC (encodes transferrin receptor)
and NCOA4 (encodes NCOA4, nuclear receptor coactivator 4)
expression (Fig. 6G), which is a cargo protein to mediate ferritin
autophagic degradation due to cysteine deprivation or low iron
levels [32]. The hHO-1 overexpression increased TF,TFRC,FTH1, and
NCOA4 expression and decreased SLC40A1 expression (Fig. 6G).
These results suggested that in addition to heme degradation, irons
from exogenous origins and endogenous pools may also contribute
to the cellular ferrous accumulation by BAY/hHO-1 overexpression,
leading to the ferroptotic cell death.
Fig. 4. BAY induces cell death via upregulation of HO-1. (A) Cancer cells were preincubated with zinc protoporphyrin-9 (ZnPP, 3 or 10
m
M) for 30 min followed by 10
m
M BAY
treatment. Cell viability was detected 8 h after BAY treatment. All values are the mean ±SEM from three independent experiments. *P<.05; **P<.01, compared to the BAY alone. (B)
HO-1 (shHMOX1) knockdown cancer cells were treated with 5
m
M or 10
m
M for 8 h followed by cell viability assay. *P<.05; **P<.01. (C!E) Nrf2 (shNFE2L2), SLC7A11 (shSLC7A11),
and HO-1 (shHMOX1) knockdown cancer cells were treated with 10
m
M BAY for 6 and used for protein determination with Western blot analysis. (F) Cancer cells were treated with
10
m
M BAY for the indicated time intervals. Cells were harvested for fractionation. Isolated cytoplasmic and nuclear fractions were used for protein determination through Western
blot analysis. H2A.X and MEK1 served as nuclear and cytoplasmic markers, respectively. (G) Cancer cells were treated with 10
m
M BAY for 6 h. Cytoplasmic (Cyto), mitochondrial
(Mito), and nuclear (Nu) fractions were isolated and used for protein determination by Western blot analysis. Thioredoxin 1 (Trx1), TOM20, and H2A.X served as cytoplasmic,
mitochondrial, and nuclear markers, respectively. The representative figure is one of three independent experiments.
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137130
3.7. BAY induces ER stress involving HO-1 activation
HO-1 is an ER-anchored heme protein [37]. Since GSH depletion
by the system X
c
!
blockade has been shown to enhance ER stress
[4], BAY might be proposed to act on system X
c
!
to elicit ER stress in
favor of the ferroptotic process. Treatment of BAY significantly
promoted ER stress gene expression, including XPB1,GADD45A,
ATF4,ATF6,HSPB1, and HSP90B1, particularly HSPA5 (encodes BiP,
binding immunoglobulin protein) and DDIT3 (encodes CHOP,
CCAAT/enhancer-binding protein homologous protein)(Fig. 3E).
Similar to the results of ER stress induced by other agents [38], a
robust increase in cellular BiP and CHOP level at 4 h was observed
(Fig. 7A). Immunostaining of HO-1 showed that BAY increased HO-
1 which was distributed in the perinuclear region and nuclei
(Fig. 7B). Increased HO-1 co-localized with protein disulfide isom-
erase (PDI), an ER luminal protein [39](Fig. 7B), indicating HO-1
within ER also elevated by BAY. During ER stress, PERK is dimer-
ized and activated by BiP. ATF4 and CHOP thereby are upregulated
by elF2
a
, a downstream effector of PERK [38]. XBP1 (X-box binding
protein 1) is an important transcription factor that regulates genes
responsible for ER-associated degradation [38]. Under ER stress,
XBP mRNA was spliced by IRE1
a
endoribonuclease, resulting in a
translational frame-shift that unspliced XBP1 (uXBP1, inactive form)
converts into spliced XBP1 (sXBP1, activate form) [40]. Treatment of
ZnPP attenuated HO-1, CHOP abundance, and XBP1 splicing
induced by BAY, but had no effects on BiP expression and PERK
phosphorylation (Fig. 7C and D). Overexpression of hHO-1
increased CHOP levels (Fig. 7E) and enhanced XPB1 splicing
(Fig. 7F), which suggested that HO-1 also mediates ER stress along
with the BAY-induced ferroptotic process.
Fig. 5. SLC7A11 inhibition sensitizes cancer cells to ferroptotic death induction by BAY. (A, B) Cancer cells were preincubated with erastin (Era, 10
m
M), sulfasalazine (SAS, 500
m
M),
or sorafenib (Sor, 10
m
M) for 30 min followed by 10
m
M BAY treatment. (A) After 6 h of BAY treatment, cells were harvested for protein determination through Western blot analysis.
Band intensities were quantified and presented below blots. All values are the mean from three independent experiments. Representative images from three independent ex-
periments are shown. (B) Cell viability was detected 8 h after BAY treatment. All values are the mean ±SEM from three independent experiments. *P<.05, compared to the BAY
alone. NS, no significant difference compared to the BAY alone. (C) SLC7A11 (shSLC7A11) knockdown cancer cells were treated with 5
m
M or 10
m
M BAY for 8 h followed by cell
viability assay. *P<.05; **P<.01. (D, E) Cancer cells were transfected with the human SLC7A11-expressed plasmid (hSLC7A11) or control vector (Vec) for 24 h followed by 6 h (D) or
8 h (E) treatment of BAY (10
m
M). Cells were collected for protein determination (D) and cell death (E). Cell death was assessed by a sulforhodamine B colorimetric reaction. *P<.05;
**P<.01.
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137 131
3.8. Upregulation of HO-1 is associated with BAY-mediated
alteration of mitochondrial homeostasis
Iron overloading can lead to mitochondrial damage due to lipid
peroxidation in the membranes [41], and mitochondrial
morphological changes by ferroptosis are characterized by
condensation of membrane density, rupture of outer membrane,
and reduction/loss of the cristae [1,6]. Treatment of BAY also caused
a loss of mitochondrial membrane potential (Fig. 8A), which was
one characteristic identified in ferroptosis [1]. In NAO staining,
Fig. 6. HO-1 upregulation elicits cellular ferrous accumulation to mediate ferroptosis by BAY. (A, B) Cell viability and morphological observations were assessed 24 h after
transfection with the human HO-1-expressed plasmid (hHO-1) or control vector (Vec). Representative images from three independent experiments are shown. (C, D) Cancer cells
were pretreated with 10
m
M zinc protoporphyrin-9 (ZnPP) for 30 min followed by 10
m
M BAY (C) or transfection of the hHO-1-expressed plasmid (D). After 6 (C) or 24 h (D)
treatment, intracellular iron content was determined with calcein-acetoxymethyl ester labeling methods. The results are expressed as the mean ±SEM from three independent
experiments. *P<.05; **P<.01. (E) HO-1 knockdown cells were treated with 10
m
M BAY for 6 h, followed by determination of intracellular iron content. **P<.01. (F) Cancer cells
were pretreated with 20
m
M deferoxamine (DFO) for 30 min followed by BAY treatment for 6 h **P<.01. (G) Cancer cells were treated with 10
m
M BAY for 4 h or transfected with
2
m
g of control vector (Vec) or human HO-1 plasmid (hHO-1) for 24 h. Total RNA was extracted and subjected to qRT-PCR analysis. The expression levels of mRNA were normalized to
b
-actin.*P<.05; **P<.01, compared to vehicle (DMSO, CTL) or the control vector (Vec) group. The representative figure is one of three independent experiments.
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137132
which is a metachromatic dye that does not alter mitochondrial
membrane potential [42], a decrease of mitochondrial mass by BAY
was observed (Fig. 8B). Through MitoTracker staining, mitochon-
dria initially formed a filamentous network throughout the cytosol,
but were scattered inwards with a discontinuous distribution that
was retracted to the perinuclear region after BAY treatment
(Fig. 8C). BAY induced mitochondrial colocalization with the lyso-
somal protein, LAMP-1, as shown in the overlapping areas within
the perinuclear region (Fig. 8C), which suggested there was an
autophagic process with damaged mitochondria, specifically
mitophagy. Knockdown of HO-1 significantly attenuated BAY's
effects on mitochondrial biology, including, membrane potential,
mass, distribution, and mitophagy (Fig. 8AeC), whereas over-
expression of hHO-1 mimicked the effects of BAY on mitochondrial
aberrances (Fig. 8DeF). Collectively, these results demonstrated
that HO-1 mediates mitochondrial damage and follows mitophagy,
leading to ferroptosis.
4. Discussion
In this study, we first identified BAY as a new ferroptosis inducer
through a novel mechanism. We confirmed the results and
Fig. 7. BAY induces ER stress involving HO-1 activation. (A) Cancer cells were treated with 10
m
M BAY for the indicated time intervals. Cells were collected for protein determination
by Western blot analysis. (B) Cancer cells were treated with 10
m
M BAY for 6 h. HO-1 expressed in ER was determined by immunostaining with an anti-PDI and anti-HO-1 antibodies
and imaged by a confocal microscopy at 400x magnification. (C, D) Cancer cells were pretreated with 10
m
M ZnPP for 30 min and then stimulated with 10
m
M BAY for 6 h or 8 h for
protein determination (C) and an XBP1 mRNA splicing assay (D), respectively. XBP1 mRNA splicing assay was determined using reverse transcription-PCR. Unspliced (uXBP1) and
spliced (sXBP1) forms of XBP1 are shown. (E, F) Twenty-four hours after transfection with the human HO-1 plasmid (hHO-1) or control vector (Vec), the cells were collected and
used for protein determination (D) and an XBP1 mRNA splicing assay (F). The representative figure is one of three independent experiments.
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137 133
Fig. 8. Upregulation of HO-1 is associated with BAY-mediated alteration of mitochondrial homeostasis. (A!C) HO-1-knockdown cancer cells were treated with 10
m
M BAY for 6 h
and used for mitochondrial membrane potential (A), mass (B), and mitophagy (C) analysis. Mitochondrial membrane potential and mass were assessed by rhodamine 123 and
acridine orange 10-nonyl bromide staining (NAO) staining and quantified according to intensity using flow cytometry. The results are expressed as the mean ±SEM from three
independent experiments. *P<.05; **P<.01,compared to the BAY control (shLUC). Mitophagy was determined with MitoTracker staining and immunostaining with an anti-LAMP-1
antibody and imaged by a confocal microscopy at 400 x magnification. Scale bar ¼10
m
m. (D!F) Twenty-four hours after transfection with the human HO-1 plasmid (hHO-1) or
control vector (Vec), cancer cells were collected and used for mitochondrial membrane potential (D), mass (E), and mitophagy (F) analysis. *P<.05; **P<.01, compared to the
control (Vec). Representative images from three independent experiments are shown.
specifically demonstrated that the HO-1 mediates BAY-induced
ferroptosis by dysregulating cellular redox regulation. Higher HO-
1 level was prone to oxidative insults [11e14], whereby the defect
of HO-1 modulates BAY's effect on cell death. The knockdown of
Nrf2 and SLC7A11 prevented HO-1 induction by BAY, but HO-1
knockdown had no change in Nrf2 and SLC7A11 abundance.
These results suggested that the regulation hierarchy is
Nrf2!SLC7A11!HO-1.
The recognized ferroptotic compounds include system X
c
!
in-
hibitors (e.g., erastin, sulfasalazine, and buthionine sulfoximine)
known as Class I inducers, which cause cellular GSH depletion and
redox status imbalance [1], and Class II inducers, specifically GPx4
inhibitors (e.g., RSL3 and DPI derivatives), act on lipid peroxidation
[26]. Some small molecules, however, such as sorafenib and arte-
misinin, also ignite the ferroptotic process in a system X
c
!
- and
GPx4-independent manner but their operative mechanisms remain
elusive [1e3]. Pharmacological inhibition of system X
c
!
in cancer
cells has been shown a compensatory increase in SLC7A11
expression [4]. Based on the effects of ROS generation, glutathione
depletion, iron accumulation, and a compensatory upregulation of
SLC7A11, BAY should be categorized into Class I ferroptosis
inducers.
Additive death effect was found in the combination of BAY with
ferroptotic inducers, erastin and SAS, and SLC7A11 shRNA inter-
ference. Moreover, the increase of SLC7A11 significantly delayed
BAY-induced ferroptosis. The SLC7A11 might affect the sensitivity
of BAY's toxicity, however, its mechanism remains further investi-
gation. Noteworthy, erastin has been demonstrated that targeting
inhibition on system L related transporters [4]. SAS has more effect
on NF-
k
B activity inhibition [43].
Both Nrf2 and NF-
k
B pathways are critical for regulating cellular
redox status, and a variety of molecular interactions depending on
cell types occur and allow for cross-talk between the two pathways
[44]. The inhibitory effect of BAY on p65 and I
k
B
a
phosphorylation
was prevented by NAC and
b
Me within 0.25 h, which suggests that
this cell death process occurred in a redox-dependent manner.
Furthermore, I
k
B
a
and p65 knockdown by shRNA intervention only
caused very slight suppression on cell survival. BAY treatment
greatly enhanced cell death but failed to affect p65 and p50
translocation, and this suppression was mimicked by enforced
overexpression of hHO-1. Therefore, NF-
k
B signaling was excluded
from BAY-induced ferroptosis despite iron accumulation and the
resulting oxidative insults occurred due to activation of HO-1 and
decreased KEAP1 levels, which can regulate I
k
B
a
-NF-
k
B activation
[44].
Pharmacological approaches and enforced overexpression
further demonstrated that HO-1 mediates BAY-induced ferroptosis
in cancer cells by regulating cellular iron and redox homeostasis.
Overload of oxidative stress has been shown to massively upregu-
late HOs, which thereafter act as a pro-oxidant to exert detrimental
effects on cell functionality [11e14 ]. The underlying cytotoxic
mechanisms operated by HO-1 are thus attributed to iron accu-
mulation through HO-1 catalysis, which further induces ROS pro-
duction and leads to oxidative stress, lipid peroxidation, protein
and genomic damages [45,46]. Carbon monoxide, another catalytic
product of HO-1, may also contribute to BAY-induced ferroptosis
that is similar to fibrosarcoma cells through erastin induction [30].
Compartmentalization of HO-1 is also involved in cell ferroptosis
since mitochondria-targeted HO-1 has been shown to cause a loss
in cytochrome coxidase activity and induce higher ROS generation
to lead to mitochondrial dysfunction and a higher recruitment of
LC3B which is an autophagy marker [11 ]. In astrocytes, over-
expression of HO-1 has been shown to induce cytoplasmic vacuo-
lation and mitochondrial macroautophagy [11]. HO-1-mediated
heme degradation and iron release are required for SQSTM1 cargo
vesicle formation containing aggregated proteins [47]. SQSTM1
gene expression increased after BAY treatment, suggesting that
autophagic regulation could be a potential target in BAY-induced
ferroptosis. Additionally, nuclear compartmentalization of HO-1
per se instead of its enzymatic activity mediates cytotoxicity
through certain oxidative compounds that lead to ER stress,
genomic instability, and cell death in myeloma cells [48]. Whether
compartmentalization of HO-1 by BAY contributes partially to its
ferroptotic induction remains further investigation.
Iron participates in various biological functions, such as oxygen
transportation, DNA synthesis, and metabolic processes, as a co-
factor and/or component of proteins to regulate mitochondrial
tricarboxylic acid cycle and the electron transport chain [49].
However, excessive iron, particularly divalent irons, may provoke
uncontrolled generation of ROS via the Fenton reaction, leading to
lipid peroxidation, proteotoxicity, genotoxicity, and even cell death
[45,46]. In astrocytes, overexpression of hHO-1 promoted cellular
oxidative stress, and the opening of mitochondrial permeability
transition pore, leading to iron influx into the matrix and ultimately
mitochondrial cristae disruption, degeneration, and macro-
autophagy [14]. Local HO-1 compartmentalized in the mitochon-
drion leads to iron liberation, mitochondrial oxidative damages,
and mitochondrial macroautophagy [11,14,50].
Normally, mitochondria are associated with cytoskeletons to
form a network complex, and the association is critical for cellular
calcium homeostasis, ATP and ROS production, mitochondrial
shape biogenesis, and degradation. Thus, it governs death signaling
and cell fate [1,51e53]. HO-1 has been shown to modulate the
integrity of the cellular cytoskeleton and filopodia formation as
well as ameliorate aggressive migration by regulating key marker
gene expressions related to cell adhesion and cell-cell communi-
cation in human PCa prostate cancer cells [54]. A protective role of
HO-1 to maintain mitochondrion homeostasis by modulating the
fission/fusion process and related mediator expressions were
observed in endotoxin-injured rat lungs, normal and RAW 264.7
macrophages as well as cardiomyocytes [11,55,56]. The cytoskele-
tons associated with mitochondrion homeostasis downstream of
HO-1 mediation in BAY-induced perturbation of mitochondrial
homeostasis require further investigation. In astrocytes, over-
expression of hHO-1, however, promoted cellular oxidative stress,
and the opening of mitochondrial permeability transition pore,
leading to iron influx into the matrix and ultimately mitochondrial
cristae disruption, degeneration, and macroautophagy [14]. The
present results demonstrated that HO-1 mediates aberrant mito-
chondrial biology. Consistent with previous reports [11,14,49],
mitochondrial macroautophagy by HO-1 was further shown to
depend on iron liberation and mitochondrial oxidative damages,
and it was likely associated with local HO-1 compartmentalized in
the mitochondrion.
Mitochondria play a central role in ferroptosis conduction.
Transcription of several mitochondrial genes, including RPL8,IREB2,
CS,ATP5G3,TTC35, and ACSF2 are involved in ferroptosis [1]. Erastin,
a ferroptosis activator, can bind to VDACs (voltage-dependent anion
channels) and alter the selectivity of VADCs to increase iron as well
as other anion movements into the mitochondria that lead to
impaired mitochondrial membrane permeability and attenuation
of NADH oxidation as well as promoting ROS production and fer-
roptosis [51,57]. In human hepatocellular carcinoma cells, inhibi-
tion of retinoblastoma protein (Rb) expression was shown to
mediate sorafenib-increased local ROS production within the
mitochondria and induce ferroptosis [58]. CDGSH iron sulfur
domain 1 (CISD1) can limit mitochondrial lipid peroxidation by
repressing iron uptake in the mitochondria [58]. Acyl-CoA syn-
thetase long-chain family 4 (ACSL4) also contributes to ferroptosis
by activating 5-lipooxygenase and promoting 5-
L.-C. Chang et al. / Cancer Letters 416 (2018) 124e137 135
hydroxyeicosatetraenoic acid (5-HETE) production [59]. In the
study, zileuton, a 5-lipooxygenase inhibitor, significantly attenu-
ated BAY-induced ferroptosis, which suggested there was an
involvement of ACSL4 [60]. Therefore, BAY may also disturb mito-
chondrial homeostasis via the mitochondrial compartmentation of
HO-1.
Ferroptosis induction has been regarded as a therapeutic strat-
egy in cancer treatments. Our present results indicated that HO-1
mediates redox regulation in cancer cell ferroptosis involving ER
stress and mitochondrial homeostasis (Fig. 9), and that over-
expression of hHO-1 induces ferroptosis in cancer cells is a novel
strategy for cancer treatment. The present results reveal a new
mechanism in ferroptotic induction of cancer cells as well as a new
therapeutic target for cancer treatment of triple-negative breast
cancer and GBM.
Author contributions
LCC and SKC designed the study, carried out the experiments,
and conducted data collection. LCC, SKC, SEC, YLY, RHC, and WCC
analyzed the data and discussion. LCC, SKC, and SEC wrote the
manuscript. All authors read and approved the final manuscript.
Statement of conflicts of interest
The authors declare no conflicts of interests.
Acknowledgements
This work was supported by the Ministry of Science and Tech-
nology of the Republic of China, Taiwan (MOST 104-2314-B-039-
034 and MOST 105-2628-B-039-004-MY3 to L.-C. Chang, NSC 100-
2321-B-005-008-MY3 to S.-E. Chen). We thank the Graduate
Institute of Cancer Biology and Center for Molecular Medicine,
China Medical University, Taichung, Taiwan, for providing reagents,
materials, and analysis tools. We appreciate Mr. Ru-Chun Tai, the
Medical Research Core Facilities Center, Office of Research and
Development at China Medical University, Taichung, Taiwan, for
helping and assisting with the Confocal Spectral microscope.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.canlet.2017.12.025.
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