International Journal of

Molecular Sciences

Article
Heme Oxygenase Modulation Drives Ferroptosis in TNBC Cells
Valeria Consoli 1 , Valeria Sorrenti 1,2, * , Valeria Pittalà 1,2 , Khaled Greish 3 , Agata Grazia D’Amico 1 ,
Giuseppe Romeo 1,2 , Sebastiano Intagliata 1,2 , Loredana Salerno 1,2 and Luca Vanella 1,2

1 Department of Drug and Health Science, University of Catania, 95125 Catania, Italy;
valeria.consoli@phd.unict.it (V.C.); valeria.pittala@unict.it (V.P.); agata.damico@unict.it (A.G.D.);
giuseppe.romeo@unict.it (G.R.); s.intagliata@unict.it (S.I.); lsalerno@unict.it (L.S.); lvanella@unict.it (L.V.)
2 CERNUT-Research Centre on Nutraceuticals and Health Products, University of Catania, 95125 Catania, Italy
3 Princess Al-Jawhara Centre for Molecular Medicine, Department of Molecular Medicine, School of Medicine
and Medical Sciences, Arabian Gulf University, Manama 329, Bahrain; khaledfg@agu.edu.bh
* Correspondence: sorrenti@unict.it

Abstract: The term ferroptosis refers to a peculiar type of programmed cell death (PCD) mainly char-
acterized by extensive iron-dependent lipid peroxidation. Recently, ferroptosis has been suggested as
a potential new strategy for the treatment of several cancers, including breast cancer (BC). In particu-
lar, among the BC subtypes, triple negative breast cancer (TNBC) is considered the most aggressive,
and conventional drugs fail to provide long-term efficacy. In this context, our study’s purpose was
to investigate the mechanism of ferroptosis in breast cancer cell lines and reveal the significance of
heme oxygenase (HO) modulation in the process, providing new biochemical approaches. HO’s
effect on BC was evaluated by MTT tests, gene silencing, Western blot analysis, and measurement of
reactive oxygen species (ROS), glutathione (GSH) and lipid hydroperoxide (LOOH) levels. In order
to assess HO’s implication, different approaches were exploited, using two distinct HO-1 inducers
(hemin and curcumin), a well-known HO inhibitor (SnMP) and a selective HO-2 inhibitor. The data
obtained showed HO’s contribution to the onset of ferroptosis; in particular, HO-1 induction seemed
Citation: Consoli, V.; Sorrenti, V.;
to accelerate the process. Moreover, our results suggest a potential role of HO-2 in erastin-induced
Pittalà, V.; Greish, K.; D’Amico, A.G.;
ferroptosis. In view of the above, HO modulation in ferroptosis can offer a novel approach for breast
Romeo, G.; Intagliata, S.; Salerno, L.;
Vanella, L. Heme Oxygenase
cancer treatment.
Modulation Drives Ferroptosis in
TNBC Cells. Int. J. Mol. Sci. 2022, 23, Keywords: HO-1; HO-2; cancer; erastin; heme; curcumin; inducers; inhibitors
5709. https://doi.org/10.3390/
ijms23105709

Academic Editor: Ahmad R. Safa

1. Introduction
Received: 26 April 2022 Ferroptosis is a recently identified type of programmed cell death (PCD), and it was
Accepted: 19 May 2022 first described by Dixon et al. in 2012 using RAS-selective lethal (RSL) small molecules,
Published: 20 May 2022 such as erastin and RSL3, as triggers of the process [1]. This form of PCD has characteristic
Publisher’s Note: MDPI stays neutral features that differentiate it from other historically well-known types of cellular death
with regard to jurisdictional claims in such as apoptosis, autophagy, necrosis and necroptosis [2]. Ferroptosis is mainly charac-
published maps and institutional affil- terized by iron accumulation, which leads to greatly increased ROS production and lipid
iations. peroxidation. This particular process involves peculiar features including mitochondrial
shrinkage and dysfunction, together with the rounded morphology of the cell undergoing
ferroptotic death upon erastin exposure [3]. The targets of ferroptosis inducers, for instance
erastin and RSL3, are specific inhibitors of the cystine/glutamate antiporter system Xc−
Copyright: © 2022 by the authors. and glutathione peroxidase 4 (GPX4), respectively. ACSL4 (acyl-CoA synthetase long-
Licensee MDPI, Basel, Switzerland.
chain family member 4) dictates sensitivity to ferroptosis and, usually, its expression is
This article is an open access article
upregulated in some types of cancer rather than in healthy cells, determining a potential
distributed under the terms and
selectivity for cancer cells [4]. Multiple lines of evidence have suggested ferroptosis’ pivotal
conditions of the Creative Commons
role in tumor suppressor pathways, including p53 activation, highlighting its relevance in
Attribution (CC BY) license (https://
the field of antineoplastic therapies [5,6]. Indeed, several studies have demonstrated the
creativecommons.org/licenses/by/
high potential of ferroptosis as a novel therapeutic strategy for the treatment of different
4.0/).

Int. J. Mol. Sci. 2022, 23, 5709. https://doi.org/10.3390/ijms23105709 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 5709 2 of 13

cancers, including hepatic, lung and breast cancer [7–12]. Recent studies have confirmed
that female breast cancer (BC) has overtaken lung cancer as the most commonly diagnosed
cancer, with an estimated 2.3 million new cases (11.7%), closely followed by lung (11.4%),
colorectal (10.0%), prostate (7.3%) and stomach (5.6%) cancers. In women, BC represents
the most common type of cancer diagnosed yearly, and it is the leading cause of cancer
deaths [13]. BC is particularly heterogeneous, showing characteristic morphology, histology
and biochemical features among the different subtypes, which, in turn, are translated into
different prognosis and therapy outcomes. Triple negative breast cancer (TNBC) represents
about 17% of all breast carcinomas and it is characterized by the absence of the estrogen
receptor (ER), the progesterone receptor (PR) and human epidermal growth factor receptor
2 (HER2) [14]. Among other subtypes of BC, TNBC is considered the most aggressive and
it has been associated with worse prognosis and outcome. Studies have shown a high
recurrence potential, with the majority of deaths occurring between the third and the fifth
year after initial treatment [15–18]. Due to the absence of specific molecular targets, TNBC
treatment is essentially based on surgery followed by radio- and chemotherapy. However,
the occurrence of relapses and metastasis spread proves the low long-term efficacy of
conventional chemotherapy and facilitates the insurgence of chemo-resistance, highlighting
the urgency to find alternative therapeutical strategies. Thus, ferroptosis has turned into a
very captivating area of research. Among the regulators of the ferroptotic process, nuclear
factor erythroid 2-related factor (Nrf2) has been gaining a great deal of attention as a multi-
functional regulator of cellular redox balance and protective antioxidant response systems,
including heme oxygenase (HO) enzymes. Nrf2 plays a dual role in tumor progression and
its action is essentially context-dependent. Indeed, sustained Nrf2 pathway activation may
provide an advantageous environment for cancer cells’ growth and survival by buffering
the ROS levels that are often found to be elevated due to their abnormally rapid prolifera-
tion and fast metabolic rate [19]. Nevertheless, cancer cells show a tendency to develop a
redox adaptation by promoting the activation of antioxidant and anti-apoptotic systems, en-
hancing cell survival and chemoresistance [20,21]. Likewise, HO, as the main target protein
of Nrf2, can exert either a cytoprotective action or a detrimental one in cancer, depending
on the specific cellular conditions [22–24]. In particular, HO-1 overexpression is generally
observed in several malignant human neoplastic diseases, serving as a cytoprotective factor
in the early stages of tumorigenesis, but eventually promoting malignant cells’ growth,
proliferation and invasion [25–28]. On the basis of these observations, HO-1 inhibition has
been widely exploited as a valid therapeutic strategy for cancer treatment [29–35]. However,
recent findings seem to propose a different approach for pharmacological modulation of
HO-1 in cancer therapy, displaying a reduction in cancerous cell proliferation following
enzyme induction [36,37]. Hence, the emerging correlation between modulation of the HO
system and ferroptosis can be considered as a potential new path to pursue for treating
specific cancers. Taking that into consideration, in this work, we aimed to understand the
impact of modulation of the HO system in ferroptosis through different approaches in
order to pave the way for future studies on the mechanisms involved in the ferroptotic
process and the eventual development of novel HO modulators as ferroptosis inducers and
anticancer agents.

2. Results
2.1. Cellular Screening, Ferroptotic Model Validation and Implications of HO
Based on the preliminary results (Figure 1A), the two BC cell lines MCF-7 and TNBC
MDA-MB 231 showed opposites trends in terms of GPX4 and ACSL4, and low levels of
HO-1. Both cell lines did not show any significant difference in their HO-2 expression levels.
As shown in Figure 1B, the response to erastin (a well-known ferroptosis inducer) was
significantly different in the chosen cell lines; notably, cell viability was greatly decreased
after 48 h of treatment in MDA-MB 231 compared with MCF-7. In order to validate
the ferroptotic model, we treated cells with known ferroptosis inhibitors: deferoxamine
(DFO), ferrostatin-1 (Fer-1) and Trolox. As shown in Figure 1C, all the inhibitors were able
 levels. As shown in Figure 1B, the response to erastin (a well-known ferroptosis inducer)
was significantly different in the chosen cell lines; notably, cell viability was greatly
decreased after 48 h of treatment in MDA-MB 231 compared with MCF-7. In order to
Int. J. Mol. Sci. 2022, 23, 5709 3 of 13
validate the ferroptotic model, we treated cells with known ferroptosis inhibitors:
deferoxamine (DFO), ferrostatin-1 (Fer-1) and Trolox. As shown in Figure 1C, all the
inhibitors were able to reverse erastin’s effect on cell viability. We further examined HO-
to reverse erastin’s effect on cell viability. We by
further examined 0 s involvement in
HO-1protein
1’s involvement in erastin-induced ferroptosis measuring the HO-1 levels and
erastin-induced
enzymatic ferroptosis
activity by measuring
after treatment (Figure the
1D).HO-1
Analysisprotein levels
of the dataand enzymatic
revealed activity
a significant
after treatment (Figure 1D). Analysis of the data revealed a significant
increase in HO-1 levels after 36 h of treatment, as well as an increase in bilirubin formationincrease in HO-
1after
levels after 36 h of treatment, as well as an increase in bilirubin
48 h (data not shown). In agreement with Kwon et al. [38], erastin-mediated formation after 48 h
(data not shown). In agreement with Kwon et al. [38], erastin-mediated
ferroptosis was found to increase HO-1 at both the enzymatic and transcriptional levels. ferroptosis was
found to increase HO-1 at both the enzymatic and transcriptional levels.
Thus, subsequent treatment with an HO inhibitor could have been useful for assessing Thus, subsequent
treatment
HO’s with an
enzymatic HO inhibitor
activity couldCells
contribution. have been
were usefulwith
treated for non-toxic
assessing concentrations
HO’s enzymatic of
activity contribution. Cells were treated with non-toxic concentrations
a well-known HO inhibitor (SnMP) and its enzymatic substrate (hemin). Co-treatment of a well-known
HO inhibitor
with SnMP and (SnMP) and
erastin its enzymatic
surprisingly substrate
reversed (hemin).
erastin’s Co-treatment
cytotoxic with SnMP
effect (Figure and
1E), while
erastin surprisingly reversed erastin’s cytotoxic effect (Figure 1E), while co-treatment
co-treatment with hemin markedly potentiated it (Figure 1F). On the basis of these results, with
heminsiRNA
HO-1 markedly
waspotentiated
used. As shownit (Figure 1F). On
in Figure 1G,the basis oferastin’s
although these results, HO-1
cytotoxic siRNA
effect was
on HO-
used. As shown in Figure 1G, although erastin’s cytotoxic effect on HO-1-silenced cells was
1-silenced cells was slightly but significantly reduced, co-treatment with hemin was
slightly but significantly reduced, co-treatment with hemin was considerably less effective
considerably less effective compared with HO-1-expressing cells.
compared with HO-1-expressing cells.

Figure 1. (A) Basal protein expression screening of HO-1, HO-2, GPX4 and ACSL4 in A549, NCI-H292,
Figure
MCF-7, 1. (A) Basal231
MDA-MB protein expression
and DU145 screening
cell lines. of HO-1,
(B) Evaluation ofHO-2, GPX4
erastin’s and ACSL4
cytotoxicity in A549,
in breast NCI-
cancer cell
H292, MCF-7, MDA-MB
### 231 and DU145 cell lines. (B) Evaluation of erastin’s
lines at 48 h ( p < 0.0005 vs. CTRL MDA-MB 231; *** p < 0.0005 vs. CTRL MCF-7). (C) Ferroptosiscytotoxicity in breast
cancer cell lines aterastin’s
model validation: 48 h (###effect
p < 0.0005 vs. CTRL
was reversed byMDA-MB
DFO (100 231;
µM),*** p < (1
Fer-1 0.0005 vs. CTRL
µM) and TroloxMCF-7).
(100 µM)(C) at
Ferroptosis model validation: erastin’s ### effect was reversed by DFO (100 µM), Fer-1 (1 µM) and
48 h (* p < 0.05, *** p < 0.0005 vs. CTRL; p < 0.0005 vs. ###
erastin). (D) Measurement of HO-1 levels
Trolox (100 µM) at 48 h (* p < 0.05, *** p < 0.0005 vs. CTRL; p < 0.0005 vs. erastin). (D) 0Measurement
after treatment with erastin (5 µM) (* p < 0.05 vs. CTRL). (E,F) Assessment of HO-1 s modulation
of HO-1 levels after treatment with erastin (5 µM) (* p < 0.05 vs. CTRL). (E,F) Assessment of HO-1’s
### < 0.0005 vs.
effect on erastin-induced
modulation effect on erastin-induced p <death
cell death (***cell 0.0005(***
vs.pCTRL;
< 0.0005§ pvs.
< 0.0005
CTRL;vs. < 0.0005 vs.perastin;
§ p erastin; ### p

<erastin). (G)erastin).
0.0005 vs. Effect of (G)
HO-1 silencing
Effect on cell
of HO-1 viability
silencing onafter treatmentafter
cell viability withtreatment
erastin and a erastin/hemin
with erastin and a
48 h (*** p < 0.0005
combination atcombination § p < 0.0005 ### p < 0.0005 vs.###
erastin/hemin at 48 hvs. CTRL;
(*** p < 0.0005 vs. erastin;
vs. CTRL; § p < 0.0005 vs. erastin; erastin/hemin).
p < 0.0005 vs.
erastin/hemin).
The The results
results are expressed asare ± SEM.
expressed
means as means ± SEM.

2.2. Effects
2.2. Effects of
of Hemin
Hemin and
and Erastin
Erastin on
on Ferroptosis-Associated
Ferroptosis-Associated Oxidative
Oxidative Stress
Stress
Measurement of ROS at two different time points displayed
Measurement of ROS at two different time points displayed an an increase
increase after
after 24
24 h
h of
of
erastin treatment, while co-treatment with hemin showed a peak after only 8 h
erastin treatment, while co-treatment with hemin showed a peak after only 8 h (Figure(Figure 2A).
The increase in ROS was associated with a significant decrease in GSH cellular content, as
2A). The increase in ROS was associated with a significant decrease in GSH cellular
shown in Figure 2B, especially for the combination of hemin and erastin. Concurrently, Fe2+
content, as shown in Figure 2B, especially for the combination of hemin and erastin.
and lipid hydroperoxide (LOOH) levels were elevated after 24 h of treatment; in particular,
Concurrently, Fe2+ and lipid hydroperoxide (LOOH) levels were elevated after 24 h of
Fe2+ levels were similar for the erastin and co-treatment groups, while LOOH levels were
significantly increased only after the combination treatment (Figure 2C,D). Hence, inducing
HO-1 may alter redox homeostasis, leading to lipid peroxidation.
 treatment; in particular, Fe2+ levels were similar for the erastin and co-treatment groups,
while LOOH levels were significantly increased only after the combination treatment
Int. J. Mol. Sci. 2022, 23, 5709 4 of 13
(Figure 2C,D). Hence, inducing HO-1 may alter redox homeostasis, leading to lipid
peroxidation.

Figure 2. (A) Evaluation of the effects of hemin, erastin and an erastin/hemin combination treatments
Figure 2. (A) Evaluation of the effects of hemin, ### erastin and an erastin/hemin combination
on ROS levels (** p < 0.005, *** p < 0.0005 vs. CTRL; p < 0.0005 vs. erastin). (B) Measurement of
treatments on ROS levels (** p < 0.005, *** p < 0.0005 vs. CTRL; ### p < 0.0005 vs. erastin).###(B)
non-protein thiol group (RSH) concentrations after 24 h of treatment (** p < 0.005 vs. CTRL; p<
Measurement of non-protein thiol group (RSH) concentrations after 24 h of treatment (** p < 0.005,
2+ and LOOH levels after 24 h of treatment (** p < 0.005 vs.
0.0005 vs. erastin). (C,D) Evaluation of Fe
*** p < 0.0005 vs. CTRL; ### p < 0.0005 vs. erastin). (C,D) Evaluation of Fe2+ and LOOH levels after 24
CTRL; ### p < 0.0005 vs. erastin). The results are expressed as means ± SEM.
h of treatment (** p < 0.005, *** p < 0.0005 vs. CTRL; ### p < 0.0005 vs. erastin). The results are expressed
as2.3.
means ± SEM.
Effects of Curcumin-Mediated HO-1 Induction
As shown in Figure 3A,B, curcumin (5–50 µM) reduced cell viability in a dose-
2.3. Effects of Curcumin-Mediated HO-1 Induction
dependent manner and, as assessed by an ELISA assay, concurrently increased HO-1
As shown
protein in Figure
expression levels. 3A,B,
Basedcurcumin (5–50 µM)
on these results, reduced
a curcumin cell viabilityofin
concentration 30aµM
dose-
was
Int. J. Mol. Sci. 2022, 23, x dependent mannerthe
used to perform and, as assessed
following by an ELISA
experiments. assay,
As well as concurrently increased
erastin, curcumin HO-1
was able to
5 of 14
protein expression levels. Based on these results, a curcumin concentration
2+
induce intracellular ROS, decrease GSH content and increase both Fe and LOOH levelsof 30 µM was
used to perform
(Figure 3C–F). the following experiments. As well as erastin, curcumin was able to
induce intracellular ROS, decrease GSH content and increase both Fe2+ and LOOH levels
(Figure 3C–F).

Figure 3. (A,B) Evaluation of curcumin (5, 10, 20, 30, 50 µM) treatment on MDA-MB 231 cells’ viability
Figure 3. (A,B) Evaluation of curcumin (5, 10, 20, 30, 50 µM) treatment on MDA-MB 231 cells’
and HO-1 expression levels (*** p < 0.0005 vs. CTRL). (C–F) Effect of curcumin (30 µM) on RSH, ROS,
viability and HO-1 expression levels (*** p < 0.0005 vs. CTRL). (C–F) Effect of curcumin (30 µM) on
Fe2+ and LOOH levels (* p < 0.05, *** p < 0.0005 vs. CTRL). The results are expressed as means ± SEM.
RSH, ROS, Fe2+ and LOOH levels (* p < 0.05, *** p < 0.0005 vs. CTRL). The results are expressed as
means ± SEM.

2.4. Effect of Ferroptosis Inducers on Mitochondrial Dysfunction and Lipid ROS Accumulation
In order to confirm the activation of ferroptosis by the tested compounds, we used
erastin, hemin and curcumin to perform JC-1 assay and measure lipid ROS accumulation
with BODIPY 665/676. As shown in Figure 4A,B, both the erastin/hemin combination and
curcumin markedly decreased mitochondrial membrane potential, as shown by the
 Figure 3. (A,B) Evaluation of curcumin (5, 10, 20, 30, 50 µM) treatment on MDA-MB 231 c
Int. J. Mol. Sci. 2022, 23, 5709
viability and HO-1 expression levels (*** p < 0.0005 vs. CTRL). (C–F) Effect of curcumin
5 of 13
(30 µM
RSH, ROS, Fe2+ and LOOH levels (* p < 0.05, *** p < 0.0005 vs. CTRL). The results are expresse
means ± SEM.

2.4. Effect of Ferroptosis Inducers

2.4. Effect of on Mitochondrial
Ferroptosis Dysfunction and
Inducers on Mitochondrial Lipid ROS
Dysfunction Accumulation
and Lipid ROS Accumulati
In order to confirm
In orderthe
to activation
confirm the ofactivation
ferroptosisofby the testedby
ferroptosis compounds, we used
the tested compounds, we u
erastin, hemin and curcumin
erastin, hemin and to curcumin
perform JC-1 assay and
to perform measure
JC-1 lipid
assay and ROS accumulation
measure lipid ROS accumula
with BODIPY 665/676.
with BODIPY As665/676.
shown As in Figure
shown 4A,B, both4A,B,
in Figure the erastin/hemin combination
both the erastin/hemin combination
and curcumin curcumin markedly decreased mitochondrial membrane potential, by
markedly decreased mitochondrial membrane potential, as shown as the
shown by
reduction in reduction
JC-1 dimerinformation
JC-1 dimer (red fluorescence)
formation and an increase
(red fluorescence) andinangreen monomer
increase in green mono
fluorescence.fluorescence.
Conversely, BODIPY fluorescence
Conversely, BODIPYdisplayed a significant
fluorescence displayedincrease, mainlyincrease,
a significant for ma
the erastin/hemin combination and the curcumin treatment (Figure 4C).
for the erastin/hemin combination and the curcumin treatment (Figure 4C).

Figure 4. (A)
Figure 4. (A) Fluorescence Fluorescence
images imagesafter
of JC-1 staining of JC-1
8 h ofstaining after
treatment. 8 Evaluation
(B,C) h of treatment. (B,C) Evaluatio
of treatment
treatment effects
effects on mitochondrial on mitochondrial
membrane potential and membrane potential and(*lipid
lipid ROS accumulation ROS **
p < 0.05, accumulation
p < 0.005, vs.(* p < 0.05
< 0.005,
CTRL; ## p < 0.005, ###vs.
p <CTRL;
## p < 0.005, ### p < 0.0005 vs. erastin). The results are expressed as means ± SE
0.0005 vs. erastin). The results are expressed as means ± SEM.

2.5. Effects of Ferroptosis Inducers on GPX4, FHC and HO-2 Protein Expression
Western blot analysis was performed for GPX4, FHC (ferritin heavy chain) and HO-2
proteins (Figure 5A). As previously demonstrated [39–41], erastin significantly reduced
GPX4 expression after 24 h of treatment, as well as the erastin/hemin combination, which
did not further enhance the effects of erastin. Hemin treatment caused a significant increase
in GPX4 expression levels, as also reported by Jin et al. [42].
Regarding the curcumin treatment, the 30 µM concentration was not able to reduce
GPX4 levels, while 50 µM was as effective as erastin (Figure 5B).
In our experimental conditions, erastin treatment for 24 h was not able to reduce FHC
levels; instead, the erastin/hemin combination and curcumin (50 µM) treatment produced
a significant decrease (Figure 5C).
Surprisingly, we observed a significant reduction in HO-2 protein levels in the erastin
and erastin/hemin combination groups of 23% and 30%, respectively (Figure 5D), suggest-
ing the possible implication of HO-2 in erastin-induced ferroptosis.

2.6. Investigating HO-20 s Involvement in Erastin-Induced Ferroptosis

Subsequently, we tested an HO-2-selective inhibitor, LS 2/26 [43], to investigate HO-20 s
role in the ferroptotic process. Prior to assessing the effect of the HO-2 inhibitor in erastin-
induced ferroptosis, we determined its cytotoxicity at 5 and 10 µM, then co-treatment with
erastin was examined. After 48 h, an MTT assay was performed to assess cell viability,
and LS 2/26 showed a synergistic effect when combined with erastin, potentiating its
cytotoxicity, as observed in Figure 5E.
 GPX4 levels, while 50 µM was as effective as erastin (Figure 5B).
In our experimental conditions, erastin treatment for 24 h was not able to reduce FHC
levels; instead, the erastin/hemin combination and curcumin (50 µM) treatment produced
a significant decrease (Figure 5C).
Surprisingly, we observed a significant reduction in HO-2 protein levels in the erastin
Int. J. Mol. Sci. 2022, 23, 5709 and erastin/hemin combination groups of 23% and 30%, respectively (Figure6 of 5D),
13
suggesting the possible implication of HO-2 in erastin-induced ferroptosis.

Figure5.5.(A–D)
Figure (A–D) Effects
Effects of hemin
of hemin (5 erastin
(5 µM), µM), erastin
(5 µM),(5theµM), the erastin/hemin
erastin/hemin combinationcombination
and curcuminand
curcumin (30, 50 µM) on GPX4, FHC and HO-2 protein expression levels (* p < 0.05, ** p
(30, 50 µM) on GPX4, FHC and HO-2 protein expression levels (* p < 0.05, ** p < 0.005, *** p < 0.0005< 0.005, ***
p < 0.0005 vs. CTRL). (E) Assessment of the effects of the HO-2 selective inhibitor LS 2/26 on cell
vs. CTRL). (E) Assessment of the effects of the HO-2 selective inhibitor LS 2/26 on cell viability
viability (*** p < 0.0005 vs. CTRL; ## p < 0.005, ### p < 0.0005 vs. erastin). The results are expressed as
(*** p < 0.0005 vs. CTRL; ## p < 0.005, ### p < 0.0005 vs. erastin). The results are expressed as
means ± SEM.
means ± SEM.

3.2.6. Investigating HO-2’s Involvement in Erastin-Induced Ferroptosis

Discussion
Subsequently,
HO-1 we tested
overexpression hasan HO-2-selective
been demonstrated inhibitor, LS 2/26 [43],
as a mechanism to investigate
of resistance and HO-
ma-
2’s role in the ferroptotic process. Prior to assessing the effect of the
lignancy progression in cancer cells; however, it has been demonstrated that low levels of HO-2 inhibitor in
erastin-induced
HO-1 are associatedferroptosis, we determined
with an increased its cytotoxicity
risk of metastasis in oral at
and5 tongue
and 10squamous
µM, thencell co-
treatment with
carcinoma, erastin
together withwas examined.presence
a significant After 48ofh,undifferentiated
an MTT assay was cellsperformed to assess
[44]. Additionally,
cell analysis
data viability,byand
HanLS et 2/26 showed
al. showed a low
that synergistic
expressioneffect when
levels combined
of HO-1 in renalwith erastin,
carcinoma
potentiating
predicted pooritsprognosis,
cytotoxicity, as observed
which might be in Figure
improved 5E. by activating ferroptosis through
the induction of HO-1 [45]. Furthermore, overexpression of HO-1 in prostate cancer cells
3. Discussion
resulted in markedly reduced cell proliferation and migration [46]. Thus, the influence of
HO-1HO-1 on cancer proliferationhas
overexpression andbeen
metastasis is not univocal
demonstrated and may depend
as a mechanism on the type
of resistance and
of cancer.
malignancy progression in cancer cells; however, it has been demonstrated that low levels
of HO-1The principal aim of
are associated thisan
with work was torisk
increased investigate the implications
of metastasis in oral and of the HO
tongue system
squamous
in
cell carcinoma, together with a significant presence of undifferentiated cellscould
the onset of ferroptosis. In particular, we firstly hypothesized that HO induction [44].
accelerate
Additionally,ferroptosis in cancer
data analysis bycells
Hanexpressing
et al. showedlow levels
that lowof the induciblelevels
expression isoform (HO-1).
of HO-1 in
Next, in order to find the most sensitive cancer cells to ferroptosis, we screened five cell
lines with different tissue origins (A549 and NCI-H292 are lung cancer cell lines, MCF-7
and MDA-MB 231 are breast cancer cell lines, and DU145 is a prostate cancer cell line),
measuring the protein expression levels of the most relevant ferroptotic biomarkers such as
GPX4 and ACSL4, together with HO-1 and HO-2 (Figure 1A). On the basis of the results
obtained from the cell viability assay, we chose to perform further experiments only on the
MDA-MB 231 cell line, which turned out to be the most sensitive. We further examined
HO-10 s involvement in erastin-induced ferroptosis by measuring HO-1 protein levels and
enzymatic activity after treatment. In order to investigate the role of enzymatic modulation
of HO in the ferroptotic process, we treated cells with non-toxic concentrations of a well-
known HO inhibitor (SnMP) and its enzymatic substrate (hemin). SnMP inhibited heme
degradation, leading to a reduction in HO-derived labile iron, which was initially increased
by erastin. Furthermore, hemin, as an HO substrate, is known to induce both HO-1
expression and enhance its enzymatic activity [47,48], resulting in an increase of iron, which
under normal conditions, is not able to induce any toxicity; nevertheless, hemin accelerates
ferroptotic cell death in the presence of erastin. Hemin and ferric ammonium citrate were
Int. J. Mol. Sci. 2022, 23, 5709 7 of 13

found to promote erastin-induced ferroptosis; however, this effect was not observed after
biliverdin or bilirubin treatment [49]. As reported by Naveen Kumar [50], hemin represents
a pro-oxidant molecule that can induce ferroptosis both via proteosome and inflammasome
activation and Fe2+ accumulation via HO-1 induction. Additionally, low levels of hemin
can induce BACH-1 degradation and Nrf2 transcriptional activity in response to augmented
oxidative stress levels. Thus, it seems evident that hemin’s contribution to iron release,
mostly due to HO-1-mediated heme degradation, can synergistically enhance and accelerate
erastin’s cytotoxic effect, leading to a further increase in ROS and a subsequently altered
cellular redox balance, which determines the pro-oxidant cascade initiation that triggers
ferroptotic cell death.
To better understand and elucidate HO-10 s involvement in this process, we decided
to use HO-1 siRNA. The obtained results indicated a potential additional mechanism of
action for the effect of the erastin/hemin combination. Indeed, under our experimental
conditions, the hemin treatment did not affect cell viability; nevertheless, it is known that
hemin is able to induce ROS even at low and/or non-toxic concentrations [51]. Seiwert and
colleagues have shown that HO-1 silencing increased ROS production and enhanced hemin
cytotoxicity [52]. These data are in agreement with our results showing that combination
treatment of erastin and hemin maintains a cytotoxic effect in HO-1-silenced cells besides
HO-1 induction. Taken together, these results highlight the role of HO as one of the many
factors that are able to sustain ferroptosis. Thus, it is worth remarking on the difference
between the two pharmacological strategies used by us and other research groups [29,37,53],
exploiting either the induction or inhibition of the HO system in order to understand their
effectiveness in different cellular and animal models. Low basal HO-1 levels appear to
be critical to predict cells’ sensitivity to ferroptosis, suggesting its potential targeting as a
novel therapeutic approach for BC.
The antineoplastic effect of HO-1 induction is reflected by an increase om oxidative
stress, which drives the progression of ferroptosis. As shown in Figure 2, the erastin
and hemin co-treatment led to markedly increased ROS production, GSH depletion and lipid
peroxidation concurrently with Fe2+ accumulation, reflecting the onset of the ferroptotic process.
Based on data from the literature [54–57], we decided to use curcumin as a naturally
derived HO-1 inducer to explore the possible mechanism of HO-1 from a different per-
spective, which excluded the involvement of the exogenous substrate. Thus, curcumin can
be considered as an inducer of ferroptosis in MDA-MB 231, in agreement with the results
obtained by Cao et al. [58]. The effects of the two ferroptosis inducers on mitochondrial
dysfunction and lipid ROS accumulation were assessed fluorometrically by measuring
lipid ROS accumulation with BODIPY 665/676 and mitochondrial dysfunction via a JC-1
assay. The results confirmed that both curcumin and the erastin/hemin combination are
able to induce ferroptosis. Even though erastin showed a low efficacy in these experiments,
this can be explained by the time point chosen (8 h) for performing the experiments, dis-
playing the higher potency of the erastin/hemin combination and curcumin alone for lipid
ROS accumulation.
Western blot analysis was performed to assess the ferroptosis inducers’ effects on
MDA-MB 231 protein expression levels; in particular, GPX4, FHC and HO-2 were analyzed.
GPX4 is considered one of the principal ferroptotic markers and plays a pivotal role in
the onset of ferroptosis, as its inhibition triggers lipid peroxidation, leading to cellular
death. The results obtained might be a consequence of the cellular compensatory effect
resulting from hemin-induced Nrf2 activation, as demonstrated by several studies [59–61].
Although Nrf2 stabilization occurs following hemin treatment, HO-1 over-activation be-
comes cytotoxic due to an excessive increase in iron beyond the ferritin buffering capacity.
Labile iron is largely considered to be one of the main ferroptotic features, acting as an
intracellular pro-oxidant that is able to trigger the Fenton reaction and, consequently, lipid
peroxidation [6,62,63]. Among the several factors that influence iron metabolism, ferritin
has been studied to understand the release/accumulation mechanism of labile iron. Ferritin
is a cytosolic iron storage protein consisting of two subunits, namely ferritin heavy chain
Int. J. Mol. Sci. 2022, 23, 5709 8 of 13

(FHC) and ferritin light chain (FTL). FHC has ferroxidase activity and is able to convert
Fe2+ to Fe3+ in order to ensure iron’s entrance to its core [64]. Recently, new evidence has
shown the significance of ferritin degradation in erastin-induced ferroptosis, highlighting
the involvement of a novel identified autophagic process known as ferritinophagy, which
was found to promote ferroptosis through extensive ferritin iron release [65,66]. Ultimately,
we wanted to investigate the potential role of HO-2, the constitutive enzymatic isoform,
whose involvement in cancer and ferroptosis is still poorly understood. The data obtained
suggest a plausible implication of HO-2 in erastin’s mechanisms of action; once again, the
effects of the hemin/erastin combination were more accentuated. Although few studies
have focused their attention on HO-20 s role in cancer, our results seem to be in accordance
with previous in vitro and in vivo evidence on HO-2 deletion, which is associated with an
increase in superoxide and elevated oxidative stress levels [67–69].
In conclusion, ferroptosis has emerged as promising alternative to conventional cancer
treatments and for its potential use in overcoming multidrug resistance, which often occurs
following apoptosis-based gold standard protocols. To date, however, a full understanding
of ferroptosis mechanisms is still lacking, and much more is needed to actually be able to
modulate and exploit it as alternative strategy for cancer treatment. Our study focused on
the modulation of one of the possible factors implicated in ferroptosis, the heme oxygenase
system. Recent studies have pointed out HO’s possible involvement in this process, as
it is the principal enzyme involved in heme catabolism and its activity is strictly related
to intracellular iron release. Our results confirmed the initial hypothesis that cells with
low HO expression can be more sensitive to ferroptosis, and that enzyme induction in
this context can be useful to accelerate it. Indeed, HO can be considered as a modulator
factor in ferroptosis, also representing a marker of cells’ responsiveness to this type of
PCD. Several natural compounds have found to possess strong potential for clinical use as
promoters or inhibitors of ferroptosis [70], but few studies have focused their attention on
the involvement of the HO system. Although a substantial amount of data on ferroptosis
inhibitors already exist [71], for inducers such as curcumin, more extensive studies need
to be performed. Furthermore, it was interesting to notice that HO-2, the constitutive
isoform of the enzyme whose role in cancer is still unclear, seems to be involved in the
erastin-triggered process. Of course, these findings may lay the foundations for future
studies to deepen our understanding of the role of both HO-1 and HO-2 in ferroptosis,
making HO modulation a possible novel therapeutic strategy.

4. Materials and Methods

4.1. Cell Culture and Viability Assay
Experiments were conducted on the human breast adenocarcinoma cell lines MCF-
7 and MDA-MB 231 (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM), high glucose (HG) supplemented with 10% FBS and
1% penicillin–streptomycin, and maintained at 37 ◦ C and 5% CO2 . Both cell lines were
treated with erastin (Sigma, St. Louis, MO, USA) at 1, 5, 10, and 50 µM for 48 h hours, then
only MDA-MB 231 cells were treated with erastin (1, 5, 10, 50 µM) with deferoxamine (DFO
100 µM), ferrostatin-1 (1 µM) and Trolox (100 µM); hemin (5 µM), tin mesoporphyrin (SnMP,
10 µM) or curcumin (5, 10, 20, 30, 50 µM) for 48 h. Cells were also treated with a selective
HO-2 inhibitor ((2-[[4-(1 H-imidazol-1-yl)butyl]thio]-5-chlorobenzothiazole)) (5, 10 µM), as
previously reported by Salerno et al. [43]. In order to evaluate cell viability, cells were seeded
into 96-well plates at a density of 7.0 × 103 cells/well in 100 µL of the culture medium.
After 24 h, treatments were administered using DMEM supplemented with 1% FBS and
after 48 h, 100 µL of 0.25 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (ACROS Organics, Antwerp, Belgium) solution was added to each well,
and the cells were incubated for 2 h at 37 ◦ C and 5% CO2 . After incubation, the supernatant
was removed, and 100 µL of DMSO was added to each well to dissolve the formazan salts
produced by mitochondria. The amount of formazan was proportional to the number
of viable cells in the sample. Ultimately, absorbance (OD) was measured in a microplate
Int. J. Mol. Sci. 2022, 23, 5709 9 of 13

reader (Biotek Synergy-HT, Winooski, VT, USA) at λ = 570 nm. Eight replicate wells were
used for each group, and at least two separate experiments were performed.

4.2. Cell Transfection

MDA-MB 231 cells were seeded 24 h prior to transfection using an Opti-MEM medium
supplemented with 10% FBS and without antibiotics. Upon reaching 70–80% confluence,
the cells were transfected with Lipofectamine 2000 following the manufacturer’s instruc-
tions. siRNA against HMOX-1 (50 -CCUCAAAUGCAGUAUUUUUtt-30 , Ambion by Life
Technologies, Carlsbad, CA, USA) was resuspended in RNAse-DNAse free H2 O and di-
luted in Opti-MEM medium without antibiotics, then the siRNA–Lipofectamine complex
(ratio 1:1) was obtained after mixing and incubation for 5 min. After incubation, the culture
medium was replaced and the siRNA–Lipofectamine complex was maintained for 6 h,
then the cells were treated for 48 h and an MTT assay was performed. Validation of gene
silencing was obtained by performing qRT-PCR.

4.3. Western Blot Analysis

A preliminary Western blot analysis was conducted to investigate the basal levels of
HO-1, HO-2 and the ferroptosis markers GPX4 and ACSL4 in the A549 (ATCC, CCL-185),
NCI-H292 (ATCC, CRL-1848), MCF-7, MDA-MB 231 and DU145 (ATCC, HTB-81) cell lines
in order to choose the most sensitive to ferroptosis. Subsequently HO-2, GPX4 and FHC
levels were evaluated after treatment with erastin, hemin, an erastin/hemin combination
and curcumin. Samples were processed as previously described by Sorrenti et al., [37].
Membranes were incubated overnight with HO-1 (GTX101147, diluted 1:1000, GeneTex,
Irvine, CA, USA), HO-2 (SPA897, diluted 1:2000, Enzo Life Sciences, Farmingdale, NY,
USA), GPX4 (ab125066, diluted 1:1000, Abcam, Cambridge, UK), ACSL4 (PA5–27137,
diluted 1:1000, Thermo Fisher Scientific, Rodano, MI, Italy), FHC (ab81444, diluted 1:1000)
and β-actin (GTX109639, diluted 1:7000, GeneTex) primary antibodies. Goat anti-rabbit
secondary antibody was used to detect blots (diluted 1:7000). Blots were scanned, and
densitometric analysis was performed with the Odyssey Infrared Imaging System (LI-COR,
Milan, Italy). Values were normalized to β-actin.

4.4. Measurement of HO Enzymatic Activity

HO’s enzymatic activity was measured in cell lysates as the difference in absorbance
between 464 and 530 nm of the bilirubin produced. The reaction mixtures consisted of
20 mM Tris-HCl at pH 7.4 (2 mg/mL), the cell lysate, 0.5–2 mg/mL biliverdin reductase, 1
mM NADPH, 2 mM glucose 6-phosphate (G6P), 1 U G6P dehydrogenase and 25 µM hemin.
Incubation was carried out in a circulating water bath in the dark for 1 h at 37 ◦ C. The
reaction was stopped by adding chloroform. After recovering the chloroform phase, the
amount of bilirubin that was formed was measured with a double-beam spectrophotometer
at OD 464–530 nm (extinction coefficient: 40 mM/cm−1 for bilirubin). One unit of the
enzyme was defined as the amount of enzyme catalyzing the formation of 1 nmol of
bilirubin/mg protein/h.

4.5. Determination of HO-1 Levels (ELISA)

HO-1 levels were assessed using Simple Step ELISA (ab207621, Abcam, Cambridge,
UK) according to the manufacturer’s instructions. A microplate reader was used to measure
the absorbance (OD) at λ = 450 nm. All samples were measured in triplicate and the results
are expressed as a percentage of the control.

4.6. Measurement of Intracellular Fe2+ Content

The intracellular content of ferrous iron was determined in cells treated for 18 h using
the colorimetric iron assay kit from Abcam (ab83366, Abcam, Cambridge, UK) following
the manufacturer’s instructions. The assay was performed in triplicate for every sample.
The results are expressed in picomoles/µg of proteins.
Int. J. Mol. Sci. 2022, 23, 5709 10 of 13

4.7. Measurement of Lipid Peroxidation

Levels of LOOH were evaluated through the oxidation of Fe2+ to Fe3+ in the presence
of xylenol orange (12709580). The assay mixture contained 200 µg of the sample (total
cell lysate), 100 µM xylenol orange, 250 µM ammonium ferrous sulphate, 90% ethanol,
4 mM butylated hydroxytoluene and 25 mM H2 SO4 . Samples were incubated at room
temperature for 30 min, and the absorbance was finally measured at λ = 560 nm using a
microplate reader (Biotek Synergy-HT, Winooski, VT, USA). Calibration was performed
using hydrogen peroxide (0.2–20 µM). The results from at least two experiments were
expressed as a percentage of the control. In order to assess the lipid peroxidation levels,
cells were maintained for 30 min with fluorescent staining with BODIPY 665/676 (5 µM),
then washed with PBS twice. Fluorescence was measured with a VariosKan plate reader
(Thermo Fisher Scientific, Waltham, MA, USA), and the results were expressed as the
fluorescence intensity (AU).

4.8. Measurement of Mitochondrial Membrane Potential

Following the treatments, cells were incubated with a 3 µM JC-1 staining solution
(T3168, Invitrogen, Waltham, MA, USA) at 37 ◦ C for 20 min and then washed with PBS
in order to visualize the fluorescence with a fluorescence microscope (EVOS Fl AMG).
The JC-1 probe aggregates to form a polymer in the mitochondrial matrix of healthy
cells, producing a strong red fluorescence (Ex = 585 nm, Em = 590 nm). Otherwise,
dysfunctional mitochondria present JC-1 monomers, resulting in the emission of a green
fluorescent signal (Ex = 514 nm, Em = 529 nm). The results are expressed as the ratio of
red/green fluorescence.

4.9. Determination of Thiol Groups

The concentration of non-protein thiol groups (RSH), reflecting about 90% of the GSH
cellular content, was measured in total cell lysates. After 18 h of treatment, RSH levels
were evaluated by a spectrophotometric assay based on the reaction of thiol groups with
2,2-dithio-bis-nitrobenzoic acid (DTNB). A DTNB solution and the samples were mixed
and incubated at room temperature for 20 min in the dark until the noticeable appearance
of a yellow color. After incubation, samples were centrifuged at 3000 rpm for 10 min.
The supernatant was collected and set in a black 96-well plate for measurement of the
absorbance in a microplate reader (Biotek Synergy-HT, Winooski, VT, USA) at λ = 412 nm.
Experiments were conducted in quadruplicate. The results are expressed in pmoles/µL.

4.10. Measurement of ROS Levels

Levels of reactive oxygen species (ROS) were determined using the fluorescent probe
20 ,70 -dichlorofluorescein diacetate (DCFH-DA). Cells were washed with a 0.1% Triton solu-
tion to enhance cellular probe permeation, then 100 µL of a DCFH-DA working solution
(200 µM) was added to each well and incubated at 37 ◦ C for 30 min. After incubation, fluo-
rescence was measured in a microplate reader (excitation, λ = 488 nm; emission, λ = 525 nm).
Eight replicate wells were used for each group. The results are expressed as fluorescence
intensity (AU)/proteins (mg/mL).

4.11. Statistical Analysis

At least three independent experiments were performed for each analysis. The sta-
tistical significance (p < 0.05) of the differences between the experimental groups was
determined by Fisher’s method for analyses of multiple comparisons. For comparison
between treatment groups, the null hypothesis was tested by either a single-factor analysis
of variance (ANOVA) for multiple groups or an unpaired t-test for two groups, and the
data are presented as means ± SEM.

Author Contributions: Conceptualization, L.V., V.S. and V.C.; methodology, V.C. and L.V.; formal
analysis, V.C., G.R. and A.G.D.; resources, K.G., L.S., L.V., V.P. and S.I.; data curation, V.C., L.V. and
Int. J. Mol. Sci. 2022, 23, 5709 11 of 13

V.S.; writing—original draft preparation, V.C. and L.V.; writing—review and editing, V.S., G.R., L.S.,
S.I., A.G.D., V.P. and K.G.; supervision, L.V.; project administration, V.P.; funding acquisition, V.P. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the University of Catania, “Programma Ricerca di Ateneo
UNICT 2020-22 linea 2”, “META” Project.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this paper.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Dixon, S.J.; Lemberg, K.M.; Lamprecht, M.R.; Skouta, R.; Zaitsev, E.M.; Gleason, C.E.; Patel, D.N.; Bauer, A.J.; Cantley, A.M.; Yang,
W.S.; et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell 2012, 149, 1060–1072. [CrossRef] [PubMed]
2. Mou, Y.; Wang, J.; Wu, J.; He, D.; Zhang, C.; Duan, C.; Li, B. Ferroptosis, a new form of cell death: Opportunities and challenges in
cancer. J. Hematol. Oncol. 2019, 12, 34. [CrossRef] [PubMed]
3. Xie, Y.; Hou, W.; Song, X.; Yu, Y.; Huang, J.; Sun, X.; Kang, R.; Tang, D. Ferroptosis: Process and function. Cell Death Differ. 2016,
23, 369–379. [CrossRef] [PubMed]
4. Zhao, Y.; Li, Y.; Zhang, R.; Wang, F.; Wang, T.; Jiao, Y. The Role of Erastin in Ferroptosis and Its Prospects in Cancer Therapy.
OncoTargets Ther. 2020, 13, 5429–5441. [CrossRef] [PubMed]
5. Jiang, L.; Kon, N.; Li, T.; Wang, S.J.; Su, T.; Hibshoosh, H.; Baer, R.; Gu, W. Ferroptosis as a p53-mediated activity during tumour
suppression. Nature 2015, 520, 57–62. [CrossRef]
6. Kim, M.J.; Yun, G.J.; Kim, S.E. Metabolic Regulation of Ferroptosis in Cancer. Biology 2021, 10, 83. [CrossRef]
7. Liang, C.; Zhang, X.; Yang, M.; Dong, X. Recent Progress in Ferroptosis Inducers for Cancer Therapy. Adv. Mater. 2019,
31, e1904197. [CrossRef]
8. Nie, J.; Lin, B.; Zhou, M.; Wu, L.; Zheng, T. Role of ferroptosis in hepatocellular carcinoma. J. Cancer Res. Clin. Onco.l. 2018,
144, 2329–2337. [CrossRef]
9. Zou, J.; Wang, L.; Tang, H.; Liu, X.; Peng, F.; Peng, C. Ferroptosis in Non-Small Cell Lung Cancer: Progression and Therapeutic
Potential on It. Int. J. Mol. Sci. 2021, 22, 13335. [CrossRef]
10. Li, K.; Lin, C.; Li, M.; Xu, K.; He, Y.; Mao, Y.; Lu, L.; Geng, W.; Li, X.; Luo, Z.; et al. Multienzyme-like Reactivity Cooperatively
Impairs Glutathione Peroxidase 4 and Ferroptosis Suppressor Protein 1 Pathways in Triple-Negative Breast Cancer for Sensitized
Ferroptosis Therapy. ACS Nano 2022, 16, 2381–2398. [CrossRef]
11. Jin, L.Y.; Gu, Y.L.; Zhu, Q.; Li, X.H.; Jiang, G.Q. The role of ferroptosis-related genes for overall survival prediction in breast cancer.
J. Clin. Lab. Anal. 2021, 35, e24094. [CrossRef] [PubMed]
12. Yao, X.; Xie, R.; Cao, Y.; Tang, J.; Men, Y.; Peng, H.; Yang, W. Simvastatin induced ferroptosis for triple-negative breast cancer
therapy. J. Nanobiotechnology 2021, 19, 311. [CrossRef] [PubMed]
13. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [CrossRef]
[PubMed]
14. Foulkes, W.D.; Smith, I.E.; Reis-Filho, J.S. Triple-negative breast cancer. N. Engl. J. Med. 2010, 363, 1938–1948. [CrossRef]
15. Ovcaricek, T.; Frkovic, S.G.; Matos, E.; Mozina, B.; Borstnar, S. Triple negative breast cancer—prognostic factors and survival.
Radiol. Oncol. 2011, 45, 46–52. [CrossRef]
16. Dent, R.; Trudeau, M.; Pritchard, K.I.; Hanna, W.M.; Kahn, H.K.; Sawka, C.A.; Lickley, L.A.; Rawlinson, E.; Sun, P.; Narod, S.A.
Triple-negative breast cancer: Clinical features and patterns of recurrence. Clin. Cancer Res. 2007, 13, 4429–4434. [CrossRef]
17. Li, Y.; Kong, X.; Wang, Z.; Xuan, L. Recent advances of transcriptomics and proteomics in triple-negative breast cancer prognosis
assessment. J. Cell Mol. Med. 2022, 26, 1351–1362. [CrossRef]
18. Gierach, G.L.; Burke, A.; Anderson, W.F. Epidemiology of triple negative breast cancers. Breast Dis. 2010, 32, 5–24. [CrossRef]
19. Xiang, M.; Namani, A.; Wu, S.; Wang, X. Nrf2: Bane or blessing in cancer? J. Cancer Res. Clin. Oncol. 2014, 140, 1251–1259.
[CrossRef]
20. Consoli, V.; Sorrenti, V.; Grosso, S.; Vanella, L. Heme Oxygenase-1 Signaling and Redox Homeostasis in Physiopathological
Conditions. Biomolecules 2021, 11, 589. [CrossRef]
21. Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?
Nat. Rev. Drug Discov. 2009, 8, 579–591. [CrossRef] [PubMed]
22. Na, H.K.; Surh, Y.J. Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1. Free Radic. Biol. Med. 2014,
67, 353–365. [CrossRef] [PubMed]
23. Loboda, A.; Damulewicz, M.; Pyza, E.; Jozkowicz, A.; Dulak, J. Role of Nrf2/HO-1 system in development, oxidative stress
response and diseases: An evolutionarily conserved mechanism. Cell. Mol. Life Sci. 2016, 73, 3221–3247. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2022, 23, 5709 12 of 13

24. Ryter, S.W.; Tyrrell, R.M. The heme synthesis and degradation pathways: Role in oxidant sensitivity. Heme oxygenase has both
pro- and antioxidant properties. Free Radic. Biol. Med. 2000, 28, 289–309. [CrossRef]
25. Noh, S.J.; Bae, J.S.; Jamiyandorj, U.; Park, H.S.; Kwon, K.S.; Jung, S.H.; Youn, H.J.; Lee, H.; Park, B.H.; Chung, M.J.; et al.
Expression of nerve growth factor and heme oxygenase-1 predict poor survival of breast carcinoma patients. BMC Cancer 2013,
13, 516. [CrossRef]
26. Maines, M.D.; Abrahamsson, P.A. Expression of heme oxygenase-1 (HSP32) in human prostate: Normal, hyperplastic, and tumor
tissue distribution. Urology 1996, 47, 727–733. [CrossRef]
27. Yin, H.; Fang, J.; Liao, L.; Maeda, H.; Su, Q. Upregulation of heme oxygenase-1 in colorectal cancer patients with increased
circulation carbon monoxide levels, potentially affects chemotherapeutic sensitivity. BMC Cancer 2014, 14, 436. [CrossRef]
28. Tsuji, M.H.; Yanagawa, T.; Iwasa, S.; Tabuchi, K.; Onizawa, K.; Bannai, S.; Toyooka, H.; Yoshida, H. Heme oxygenase-1 expression
in oral squamous cell carcinoma as involved in lymph node metastasis. Cancer Lett. 1999, 138, 53–59. [CrossRef]
29. Fallica, A.N.; Sorrenti, V.; D’Amico, A.G.; Salerno, L.; Romeo, G.; Intagliata, S.; Consoli, V.; Floresta, G.; Rescifina, A.; D’Agata,
V.; et al. Discovery of Novel Acetamide-Based Heme Oxygenase-1 Inhibitors with Potent. J. Med. Chem. 2021, 64, 13373–13393.
[CrossRef]
30. Salerno, L.; Vanella, L.; Sorrenti, V.; Consoli, V.; Ciaffaglione, V.; Fallica, A.N.; Canale, V.; Zajdel, P.; Pignatello, R.; Intagliata, S.
Novel mutual prodrug of 5-fluorouracil and heme oxygenase-1 inhibitor (5-FU/HO-1 hybrid): Design and preliminary. J. Enzym.
Inhib. Med. Chem. 2021, 36, 1378–1386. [CrossRef]
31. Ciaffaglione, V.; Intagliata, S.; Pittalà, V.; Marrazzo, A.; Sorrenti, V.; Vanella, L.; Rescifina, A.; Floresta, G.; Sultan, A.; Greish, K.;
et al. New Arylethanolimidazole Derivatives as HO-1 Inhibitors with Cytotoxicity against MCF-7 Breast Cancer Cells. Int. J. Mol.
Sci. 2020, 21, 1923. [CrossRef] [PubMed]
32. Mucha, O.; Podkalicka, P.; Mikulski, M.; Barwacz, S.; Andrysiak, K.; Biela, A.; Mieczkowski, M.; Kachamakova-Trojanowska,
N.; Ryszawy, D.; Białas, A.; et al. Development and characterization of a new inhibitor of heme oxygenase activity for cancer
treatment. Arch. Biochem. Biophys. 2019, 671, 130–142. [CrossRef] [PubMed]
33. Okunlola, F.O.; Soremekun, O.S.; Olotu, F.A.; Soliman, M.E.S. East to West not North-West: Structure-Based Mechanistic
Resolution of 8-Hydroxyl Replacement and Resulting Effects on the Activities of Imidazole-Based Heme Oxygenase-1 Inhibitors.
Protein. J. 2021, 40, 166–174. [CrossRef] [PubMed]
34. Rahman, M.N.; Vukomanovic, D.; Vlahakis, J.Z.; Szarek, W.A.; Nakatsu, K.; Jia, Z. Structural insights into human heme oxygenase-
1 inhibition by potent and selective azole-based compounds. J. R. Soc. Interface 2013, 10, 20120697. [CrossRef] [PubMed]
35. Salerno, L.; Floresta, G.; Ciaffaglione, V.; Gentile, D.; Margani, F.; Turnaturi, R.; Rescifina, A.; Pittalà, V. Progress in the development
of selective heme oxygenase-1 inhibitors and their potential therapeutic application. Eur. J. Med. Chem. 2019, 167, 439–453.
[CrossRef]
36. Tertil, M.; Golda, S.; Skrzypek, K.; Florczyk, U.; Weglarczyk, K.; Kotlinowski, J.; Maleszewska, M.; Czauderna, S.; Pichon, C.;
Kieda, C.; et al. Nrf2-heme oxygenase-1 axis in mucoepidermoid carcinoma of the lung: Antitumoral effects associated with
down-regulation of matrix metalloproteinases. Free Radic. Biol. Med. 2015, 89, 147–157. [CrossRef] [PubMed]
37. Sorrenti, V.; D’Amico, A.G.; Barbagallo, I.; Consoli, V.; Grosso, S.; Vanella, L. Tin Mesoporphyrin Selectively Reduces Non-Small-
Cell Lung Cancer Cell Line A549 Proliferation by Interfering with Heme Oxygenase and Glutathione Systems. Biomolecules 2021,
11, 917. [CrossRef]
38. Kwon, M.Y.; Park, E.; Lee, S.J.; Chung, S.W. Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death. Oncotarget 2015,
6, 24393–24403. [CrossRef]
39. Lee, N.; Carlisle, A.E.; Peppers, A.; Park, S.J.; Doshi, M.B.; Spears, M.E.; Kim, D. xCT-Driven Expression of GPX4 Determines
Sensitivity of Breast Cancer Cells to Ferroptosis Inducers. Antioxidants 2021, 10, 317. [CrossRef]
40. Shibata, Y.; Yasui, H.; Higashikawa, K.; Miyamoto, N.; Kuge, Y. Erastin, a ferroptosis-inducing agent, sensitized cancer cells to
X-ray irradiation via glutathione starvation in vitro and in vivo. PLoS ONE 2019, 14, e0225931. [CrossRef]
41. Pan, X.; Lin, Z.; Jiang, D.; Yu, Y.; Yang, D.; Zhou, H.; Zhan, D.; Liu, S.; Peng, G.; Chen, Z.; et al. Erastin decreases radioresistance of
NSCLC cells partially by inducing GPX4-mediated ferroptosis. Oncol. Lett. 2019, 17, 3001–3008. [CrossRef] [PubMed]
42. Jin, Z.L.; Gao, W.Y.; Liao, S.J.; Yu, T.; Shi, Q.; Yu, S.Z.; Cai, Y.F. Paeonol inhibits the progression of intracerebral haemorrhage by
mediating the HOTAIR/UPF1/ACSL4 axis. ASN Neuro 2021, 13, 17590914211010647. [CrossRef] [PubMed]
43. Salerno, L.; Pittalà, V.; Romeo, G.; Modica, M.N.; Marrazzo, A.; Siracusa, M.A.; Sorrenti, V.; Di Giacomo, C.; Vanella, L.; Parayath,
N.N.; et al. Novel imidazole derivatives as heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) inhibitors and their cytotoxic
activity in human-derived cancer cell lines. Eur. J. Med. Chem. 2015, 96, 162–172. [CrossRef] [PubMed]
44. Yanagawa, T.; Omura, K.; Harada, H.; Nakaso, K.; Iwasa, S.; Koyama, Y.; Onizawa, K.; Yusa, H.; Yoshida, H. Heme oxygenase-1
expression predicts cervical lymph node metastasis of tongue squamous cell carcinomas. Oral. Oncol. 2004, 40, 21–27. [CrossRef]
45. Han, S.; Lin, F.; Qi, Y.; Liu, C.; Zhou, L.; Xia, Y.; Chen, K.; Xing, J.; Liu, Z.; Yu, W.; et al. HO-1 Contributes to Luteolin-Triggered
Ferroptosis in Clear Cell Renal Cell Carcinoma via Increasing the Labile Iron Pool and Promoting Lipid Peroxidation. Oxidative
Med. Cell. Longev. 2022, 2022, 3846217. [CrossRef]
46. Gueron, G.; De Siervi, A.; Ferrando, M.; Salierno, M.; De Luca, P.; Elguero, B.; Meiss, R.; Navone, N.; Vazquez, E.S. Critical role of
endogenous heme oxygenase 1 as a tuner of the invasive potential of prostate cancer cells. Mol. Cancer Res. 2009, 7, 1745–1755.
[CrossRef]
Int. J. Mol. Sci. 2022, 23, 5709 13 of 13

47. Wagener, F.A.; da Silva, J.L.; Farley, T.; de Witte, T.; Kappas, A.; Abraham, N.G. Differential effects of heme oxygenase isoforms on
heme mediation of endothelial intracellular adhesion molecule 1 expression. J. Pharmacol. Exp. Ther. 1999, 291, 416–423.
48. Li, Z.; Wang, Y.; Man, R.Y.; Vanhoutte, P.M. Upregulation of heme oxygenase-1 potentiates EDH-type relaxations in the mesenteric
artery of the spontaneously hypertensive rat. Am. J. Physiol. Heart Circ. Physiol. 2013, 305, H1471–H1483. [CrossRef]
49. Tang, D.; Chen, X.; Kang, R.; Kroemer, G. Ferroptosis: Molecular mechanisms and health implications. Cell Res. 2021, 31, 107–125.
[CrossRef]
50. NaveenKumar, S.K.; Hemshekhar, M.; Kemparaju, K.; Girish, K.S. Hemin-induced platelet activation and ferroptosis is mediated
through ROS-driven proteasomal activity and inflammasome activation: Protection by Melatonin. Biochim. Biophys. Acta Mol.
Basis Dis. 2019, 1865, 2303–2316. [CrossRef]
51. Fiorito, V.; Chiabrando, D.; Petrillo, S.; Bertino, F.; Tolosano, E. The Multifaceted Role of Heme in Cancer. Front. Oncol. 2019,
9, 1540. [CrossRef] [PubMed]
52. Seiwert, N.; Wecklein, S.; Demuth, P.; Hasselwander, S.; Kemper, T.A.; Schwerdtle, T.; Brunner, T.; Fahrer, J. Heme oxygenase 1
protects human colonocytes against ROS formation, oxidative DNA damage and cytotoxicity induced by heme iron, but not
inorganic iron. Cell Death Dis. 2020, 11, 787. [CrossRef] [PubMed]
53. Sorrenti, V.; Raffaele, M.; Vanella, L.; Acquaviva, R.; Salerno, L.; Pittalà, V.; Intagliata, S.; Di Giacomo, C. Protective Effects of
Caffeic Acid Phenethyl Ester (CAPE) and Novel Cape Analogue as Inducers of Heme Oxygenase-1 in Streptozotocin-Induced
Type 1 Diabetic Rats. Int. J. Mol. Sci. 2019, 20, 2441. [CrossRef] [PubMed]
54. Mohammed, F.; Rashid-Doubell, F.; Taha, S.; Cassidy, S.; Fredericks, S. Effects of curcumin complexes on MDA-MB-231 breast
cancer cell proliferation. Int. J. Oncol. 2020, 57, 445–455. [CrossRef]
55. Lima, C.F.; Pereira-Wilson, C.; Rattan, S.I. Curcumin induces heme oxygenase-1 in normal human skin fibroblasts through redox
signaling: Relevance for anti-aging intervention. Mol. Nutr. Food Res. 2011, 55, 430–442. [CrossRef]
56. Balogun, E.; Hoque, M.; Gong, P.; Killeen, E.; Green, C.J.; Foresti, R.; Alam, J.; Motterlini, R. Curcumin activates the haem
oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem. J. 2003, 371, 887–895. [CrossRef]
57. Scapagnini, G.; Foresti, R.; Calabrese, V.; Giuffrida Stella, A.M.; Green, C.J.; Motterlini, R. Caffeic acid phenethyl ester and
curcumin: A novel class of heme oxygenase-1 inducers. Mol. Pharmacol. 2002, 61, 554–561. [CrossRef]
58. Cao, X.; Li, Y.; Wang, Y.; Yu, T.; Zhu, C.; Zhang, X.; Guan, J. Curcumin suppresses tumorigenesis by ferroptosis in breast cancer.
PLoS ONE 2022, 17, e0261370. [CrossRef]
59. Jang, H.Y.; Hong, O.Y.; Chung, E.Y.; Park, K.H.; Kim, J.S. Roles of JNK/Nrf2 Pathway on Hemin-Induced Heme Oxygenase-1
Activation in MCF-7 Human Breast Cancer Cells. Medicina 2020, 56, 268. [CrossRef]
60. Georgiou-Siafis, S.K.; Tsiftsoglou, A.S. Activation of KEAP1/NRF2 stress signaling involved in the molecular basis of hemin-
induced cytotoxicity in human pro-erythroid K562 cells. Biochem. Pharmacol. 2020, 175, 113900. [CrossRef]
61. Song, X.; Long, D. Nrf2 and Ferroptosis: A New Research Direction for Neurodegenerative Diseases. Front. Neurosci. 2020, 14, 267.
[CrossRef] [PubMed]
62. Imoto, S.; Sawamura, T.; Shibuya, Y.; Kono, M.; Ohbuchi, A.; Suzuki, T.; Mizokoshi, Y.; Saigo, K. Labile iron, ROS, and cell death
are prominently induced by haemin, but not by non-transferrin-bound iron. Transfus. Apher. Sci. 2021, 61, 103319. [CrossRef]
63. Liang, D.; Minikes, A.M.; Jiang, X. Ferroptosis at the intersection of lipid metabolism and cellular signaling. Mol. Cell 2022.
[CrossRef] [PubMed]
64. Chen, X.; Yu, C.; Kang, R.; Tang, D. Iron Metabolism in Ferroptosis. Front. Cell Dev. Biol. 2020, 8, 590226. [CrossRef] [PubMed]
65. Gao, M.; Monian, P.; Pan, Q.; Zhang, W.; Xiang, J.; Jiang, X. Ferroptosis is an autophagic cell death process. Cell Res. 2016,
26, 1021–1032. [CrossRef] [PubMed]
66. Gryzik, M.; Asperti, M.; Denardo, A.; Arosio, P.; Poli, M. NCOA4-mediated ferritinophagy promotes ferroptosis induced by
erastin, but not by RSL3 in HeLa cells. Biochim. Biophys. Acta Mol. Cell Res. 2021, 1868, 118913. [CrossRef]
67. Sodhi, K.; Inoue, K.; Gotlinger, K.H.; Canestraro, M.; Vanella, L.; Kim, D.H.; Manthati, V.L.; Koduru, S.R.; Falck, J.R.; Schwartzman,
M.L.; et al. Epoxyeicosatrienoic acid agonist rescues the metabolic syndrome phenotype of HO-2-null mice. J. Pharmacol. Exp.
Ther. 2009, 331, 906–916. [CrossRef]
68. Intagliata, S.; Salerno, L.; Ciaffaglione, V.; Leonardi, C.; Fallica, A.N.; Carota, G.; Amata, E.; Marrazzo, A.; Pittalà, V.; Romeo, G.
Heme Oxygenase-2 (HO-2) as a therapeutic target: Activators and inhibitors. Eur. J. Med. Chem. 2019, 183, 111703. [CrossRef]
69. Vanella, L.; Di Giacomo, C.; Acquaviva, R.; Barbagallo, I.; Li Volti, G.; Cardile, V.; Abraham, N.G.; Sorrenti, V. Effects of ellagic
Acid on angiogenic factors in prostate cancer cells. Cancers 2013, 5, 726–738. [CrossRef]
70. Ge, C.; Zhang, S.; Mu, H.; Zheng, S.; Tan, Z.; Huang, X.; Xu, C.; Zou, J.; Zhu, Y.; Feng, D.; et al. Emerging Mechanisms and Disease
Implications of Ferroptosis: Potential Applications of Natural Products. Front. Cell Dev. Biol. 2021, 9, 774957. [CrossRef]
71. Xie, Y.; Song, X.; Sun, X.; Huang, J.; Zhong, M.; Lotze, M.T.; Zeh, H.J.; Kang, R.; Tang, D. Identification of baicalein as a ferroptosis
inhibitor by natural product library screening. Biochem. Biophys. Res. Commun. 2016, 473, 775–780. [CrossRef] [PubMed]