Radiotherapy-induced ferroptosis for cancer treatment

Abstract

Ferroptosis is a regulated cell death mechanism controlled by iron, amino acid and reactive oxygen species metabolisms, which is very relevant for cancer therapy. Radiotherapy-induced ferroptosis is critical for tumor suppression and several preclinical studies have demonstrated that the combination of ionizing radiation with small molecules or nano-systems is effective in combating cancer growth and overcoming drug or ionizing radiation resistance. Here, we briefly overview the mechanisms of ferroptosis and the cross-talk existing between the cellular pathways activated by ferroptosis and those induced by radiotherapy. Lastly, we discuss the recently reported combinational studies involving radiotherapy, small molecules as well as nano-systems and report the recent findings achieved in this field for the treatment of tumors.

Full text

Radiotherapy-induced ferroptosis
for cancer treatment
Giovanni L. Beretta and Nadia Zaffaroni*
Molecular Pharmacology Unit, Department of Experimental Oncology, Fondazione IRCCS Istituto
Nazionale dei Tumori di Milano, Milan, Italy
Ferroptosis is a regulated cell death mechanism controlled by iron, amino acid and
reactive oxygen species metabolisms, which is very relevant for cancer therapy.
Radiotherapy-induced ferroptosis is critical for tumor suppression and several
preclinical studies have demonstrated that the combination of ionizing radiation
with small molecules or nano-systems is effective in combating cancer growth
and overcoming drug or ionizing radiation resistance. Here, we briefly overview
the mechanisms of ferroptosis and the cross-talk existing between the cellular
pathways activated by ferroptosis and those induced by radiotherapy. Lastly, we
discuss the recently reported combinational studies involving radiotherapy, small
molecules as well as nano-systems and report the recent findings achieved in this
field for the treatment of tumors.
KEYWORDS
ferroptosis, radiotherapy, reactive oxygen species, nanomedicine, drug combinations,
gene signatures
1 Introduction
The accidental cell death (ACD) and the regulated cell death (RCD) govern the cell fate
(Tang et al., 2019). Necrosis is the best representative of ACD, which is a passive mechanism
allowing plasma membrane rupture, cytoplasm release and inducing inflammation reaction.
Conversely, RCD is an active and regulated cell suicide, which can involve or not
inflammation reaction, playing crucial functions in tissue homeostasis and in the
pathogenesis of several diseases (Galluzzi et al., 2018;Tang et al., 2019). So far, two
RCD categories are reported: apoptotic and non-apoptotic. Necroptosis, pyroptosis,
autophagy and ferroptosis belong to the non-apoptotic RCD and are classified according
to different molecular, morphological, biochemical and functional features (Galluzzi et al.,
2018). RCD pathways are implicated in physiologic processes regulating the development of
multicellular organisms and represent defense mechanisms against cancer transformation/
development as well as against pathogen infections (Galluzzi et al., 2018;Tang et al., 2019).
As suggested by its name, ferroptosis is stimulated by the lipid peroxidation provoked by the
iron accumulated into the cells. Critical for ferroptosis is the content of polyunsaturated-
fatty-acids (PUFA) composing the cellular membrane. PUFA represent a toxic reservoir that
in iron- and reactive oxygen species (ROS)-rich conditions are susceptible to peroxidation,
leading to membrane damage and cell death (Stockwell et al., 2017).
The induction of ferroptosis is a new interesting strategy for fighting cancer (Lei et al.,
2022). This strategy is based on the condition known as iron addiction, which is typical of
cancer cells that need higher levels of iron in comparison with healthy cells (Friedmann
Angeli et al., 2019). Iron addiction renders cancer cells more sensitive to iron and to iron-
induced ROS production (Fenton reaction) than normal cells (Torti and Torti, 2019).
Though ferroptosis induction proved efficacy in overcoming resistance to apoptosis
developed by tumors exposed to anticancer therapy, tumor cells challenged by
OPEN ACCESS
EDITED BY
Guo Chen,
China Pharmaceutical University, China
REVIEWED BY
Ana Cipak Gasparovic,
Rudjer Boskovic Institute, Croatia
Lixia Gao,
Chongqing University of Arts and
Sciences, China
*CORRESPONDENCE
Nadia Zaffaroni,
nadia.zaffaroni@istitutotumori.mi.it
RECEIVED 04 May 2023
ACCEPTED 05 June 2023
PUBLISHED 14 June 2023
CITATION
Beretta GL and Zaffaroni N (2023),
Radiotherapy-induced ferroptosis for
cancer treatment.
Front. Mol. Biosci. 10:1216733.
doi: 10.3389/fmolb.2023.1216733
COPYRIGHT
© 2023 Beretta and Zaffaroni. This is an
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author(s) and the copyright owner(s) are
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which does not comply with these terms.
Frontiers in Molecular Biosciences frontiersin.org01
TYPE Review
PUBLISHED 14 June 2023
DOI 10.3389/fmolb.2023.1216733

ferroptosis inducers can evolve defense mechanisms that counteract
ferroptosis and in turn preserving cell vitality (Stockwell et al., 2017;
Hassannia et al., 2019;Zheng and Conrad, 2020). This implies that
drugs hitting cellular pathways involved in resistance to ferroptosis
potentiate pharmacological interventions (Liang et al., 2019).
Besides chemotherapy, tumors are treated by radiotherapy as
well. The exposure to ionizing radiation (IR) induces DNA damage
leading to cell death (Delaney et al., 2005;Jaffray, 2012). Besides
direct DNA damage, IR hits water molecules contained into the cells
favoring their radiolysis and, together with the activation of specific
enzymes, stimulate ROS production. ROS, including peroxides
(H
2
O
2
, ROOH), free radicals (HO•,HO
2
•,R•,RO•,NO•and
NO
2
•), singlet oxygen (
1
O
2
) and superoxide (O
2
−•), attack DNA,
lipids, and proteins (Azzam et al., 2012;Reisz et al., 2014). DNA
damages include nucleotide base damage, single strand breaks
(SSBs), and double strand breaks (DSBs) (Baidoo et al., 2013).
The cellular response to damaged DNA allows cell-cycle arrest,
cellular senescence, and RCD. Although apoptosis is the most
studied RCD induced by radiotherapy, other types of RCD are
reported in radiotherapy-treated cells, including ferroptosis (Lang
et al., 2019;Adjemian et al., 2020;Lei et al., 2020;Ye et al., 2020).
Since for the treatment of tumors radiotherapy is administered in
combination with chemotherapy, it is conceivable to study the
radiotherapy and ferroptosis pathways as well as their cross-talk
to set up combination strategies maximizing tumor response.
In this review we briefly overview the cellular pathways
implicated in ferroptosis induced by radiotherapy and discuss the
potential combination strategies with small molecules and nano-
systems for enhancing radiotherapy antitumor activity.
2 General overview on ferroptosis
Compared to apoptosis, autophagy and necrosis, ferroptosis
shows peculiar properties. Morphological alterations of
mitochondria, including reduction in volume, increased density
of the mitochondrial membranes as well as reduced
mitochondrial cristae, characterize ferroptotic cells. Cells
undergoing ferroptosis are rounded and floating with intact
nuclei and uncondensed chromatin (Dixon et al., 2012;Stockwell
et al., 2017). Conversely, the typical features of apoptosis (e.g.,
chromatin condensation and the production of apoptotic bodies)
FIGURE 1
Cellular mechanisms of ferroptosis. The cellular pathways involving iron, amino acids and ROS metabolisms are reported. CIP, cellular iron pool; FR,
ferritin; CER, ceruloplasmin; TF, transferrin; TFR, transferrin receptor; FPN, ferroportin; ACSL4, acyl-CoA synthetase long-chain family member4;
LPCAT3, lysophosphatidylcholine acyltransferase 3; ALOXs, arachidonate lipoxygenase; GPX4, glutathione peroxidase 4; ASC, alanine–serine–cysteine
system; SLC7A11, solute carrier family 7 member 11; SLC3A2, solute carrier family 3 member 2; GLS, glutaminases; GSS, glutathione synthetase; GCL,
glutamate-cysteine ligase; PUFAs, polyunsaturated fatty acids; FSP1, ferroptosis suppressor protein 1; TXN, thioredoxin. AA, arachidonoyl; AdA, adrenic
acid; PE, phosphatidylethanolamines; RKIPI, Raf1 kinase inhibitory protein GSH, glutathione; GSSG, glutathione disulfide. The figure is prepared using
tools from Servier Medical Art (http://www.servier.fr/servier-medical-art, accessed on March 2023).
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as well as that of autophagy (e.g., the formation of autophagosomes)
are not reported in ferroptotic cells. Agents that inhibit apoptosis,
autophagy and necroptosis are ineffective on ferroptosis induction.
Therefore, the sensitivity to drugs that induce ferroptosis is
maintained by cells deficient in apoptotic-related factors (e.g.,
BAX, BAK, MLKL, and RIPK1/3). On the contrary, antioxidants
and iron chelators inhibit ferroptosis (Vanden Berghe et al., 2010;
Lei et al., 2019).
Ferroptosis is governed by three main cellular mechanisms,
including i) iron metabolism, ii) amino acid metabolism and iii)
ROS metabolism (Figure 1)(Liang et al., 2019).
2.1 Iron metabolism
Iron transport systems regulate iron accumulation and in turn
ferroptosis induction. These transporters, including ceruloplasmin
(CER), transferrin (TF), transferrin receptor (TFR), ferritin (FR) and
ferroportin (FPN), impact on intracellular levels of iron. Adsorbed
Fe
2+
is oxidized to Fe
3+
by CER and in this form is captured by TF.
The interaction of TF with TFR favors the cellular uptake of iron.
Upon reduction to Fe
2+
by the sixtransmembrane epithelial antigen
of the prostate 3 (STEAP3), iron is bound to FR or stored into the
cellular iron pool (CIP). When cells are saturated by iron, exceeding
amounts of Fe
2+
are oxidized to Fe
3+
and pumped out of the cells by
FPN (Trujillo-Alonso et al., 2019). CIP is controlled by other two
factors: the nuclear receptor coactivator 4 (NCOA4), which is a
specific receptor favoring FR accumulation into the autophagosome,
and the iron-responsive element binding protein 2 (IREB2), which is
a transcription factor controlling the iron metabolism by regulating
the level of FR (Dixon et al., 2012;Mancias et al., 2014;Tang et al.,
2018). Interferences with the iron balance regulated by these
mechanisms (e.g., increased uptake or reduced export) stimulate
iron-mediated lipid peroxidation and ferroptosis (Yang and
Stockwell, 2008;Stockwell et al., 2017).
2.2 Amino acid metabolism
The exchange of cystine/cystathionine across the plasma
membranes depends on the red-ox state of the extracellular
compartment. Under reducing conditions, the intracellular
accumulation of cysteine is controlled by the
alanine–serine–cysteine (ASC) system. Conversely, oxidative
extracellular conditions stimulate the exchange of cystine/
cystathionine with glutamate mediated by Xc–transporter system
(Doll and Conrad, 2017). Two subunits linked via a disulfide bridge
compose the Xc–, including the catalytic subunit solute carrier
family 7 member 11 (SLC7A11) and the regulatory subunit
solute carrier family 3 member 2 (SLC3A2). Intracellular
glutamate levels, which are under the control of the enzymatic
activity degrading glutamine (glutaminolysis) mediated by
glutaminases (GLS) 1 and 2, impact on Xc–activity. Diminished
cellular content of cysteine, which is stimulated by enhanced GLS
activity or reduced SLC7A11 levels as well as reduced activation of
spermidine/spermine N1 acetyltransferase 1 (SAT1), favors lipid
peroxidation and ferroptosis induction (Jiang et al., 2015;Jennis
et al., 2016;Ou et al., 2016;Zhang et al., 2018). Alterations in GLS,
SLC7A11 or SAT1 activities, which result in reduced intracellular
availability of cysteine, negatively impact on glutathione (GSH)
levels triggering ferroptosis (Shah et al., 2017). The synthesis of
GSH needs glutamate, cysteine, and glycine and is catalyzed by
glutamate-cysteine ligase (GCL) and glutathione synthase (GSS).
The erastin-mediated inhibition of Xc–, which reduces cystine
uptake, or the inhibition of GSH biosynthesis via buthionine
sulfoximine, deplete intracellular GSH levels leading to ferroptosis.
2.3 ROS metabolism
Besides DNA damage, ROS stimulate ferroptosis by
provoking alterations in lipid metabolism (D’Herde and
Krysko et al., 2017;Lin et al., 2018). The lipid peroxidation
occurring in PUFA is among the most important type of
cellular damage for ferroptosis induction, and cells with
high levels of PUFA are very sensitive to ferroptosis (Yang
et al., 2016;Yuan et al., 2016). The catalytic activity of two
enzymes, acyl-CoA synthetase long-chain family member 4
(ACSL4) and lysophosphatidylcholine acyltransferase 3
(LPCAT3), which allows the esterification and incorporation
of PUFA into membrane phospholipids, is crucial for
sensitizing cells to ferroptosis. The accumulation of lipid
peroxides enhances the formation of additional ROS that
increases biomacromolecule damage leading to cell
membrane destabilization and micelle formation, in turn
enhancing ferroptosis induction (Gaschler and Stockwell,
2017;Feng and Stockwell, 2018). Two proteins are critical
for ferroptosis induction as well, the small scaffolding
protein Raf1 kinase inhibitory protein (RKIP1) and the
seleno enzyme glutathione peroxidase 4 (GPX4) (Wenzel
et al., 2017;Tang et al., 2018;Liang et al., 2019). By
interacting with the iron-containing enzyme arachidonate
lipoxygenase 15 (ALOX15), RKIP1interferes with the
production of phospholipid alcohols regulating ferroptosis.
Similarly, GPX4 is a detoxifying enzyme catalyzing the
transformation of PUFA into non-toxic phospholipid
alcohols. The red-ox reaction involving the oxidation of
GSH into GSSG is required by GPX4 to accomplish its
catalytic activity, and cells showing high levels of GPX4 are
less susceptible to ferroptosis (Seibtetal.,2019). On the
contrary, compounds impairing the activity of GPX4 by
reducing its expression/activity are typical ferroptosis
inducers (Tang et al., 2018;Liang et al., 2019). Since the
activity of GPX4 needs selenocysteine tRNA, whose cellular
amount is controlled by the mevalonate (MVA) pathway, the
inhibition of MVA pathway favors ferroptosis by reducing the
availability of selenocysteine tRNA (Kryukov et al., 2003).
Another protein controlling GPX4 activity is the ferroptosis
suppressor protein 1 (FSP1). FSP1 inhibits GPX4 and its
expression is high in cells resistant to ferroptosis. Elevated
levels of FSP1 protect cells against compounds that induce
ferroptosis by targeting GPX4 (Bersuker et al., 2019;Doll et al.,
2019). Upon myristoylation, the cytoplasmic FSP1 moves to
the plasma membranes and in this peculiar cellular localization
catalyzes the reduction of coenzyme Q10. This behavior, in
presence of GSH as well as active GPX4, attenuates the
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propagation of lipid peroxide production and reduces
phospholipid peroxidation attenuating ferroptosis (Doll
et al., 2019).
3 Cross-talk between radiotherapy and
ferroptosis in cancer
Besides direct damage of biomacromolecules (e.g., DNA,
proteins and lipids), radiotherapy generates ROS, which are the
most important molecules responsible for lipid peroxide
accumulation and ferroptosis. The relationship between
radiotherapy and ferroptosis is corroborated by several
evidence, including the specific staining (e.g., C11-BODIPY) as
well as the increased expression of specific markers (e.g., MDA, 4-
HNE and prostaglandin-endoperoxide synthase 2, PTGS2)
reflecting lipid peroxidation observed in IR-exposed cancer cell
lines and tumor samples. Moreover, irradiated cells show
morphological alterations of mitochondria typical of ferroptosis
(Lang et al., 2019). Of note, these features depend on IR doses
administered. In support of the cross-talk between radiotherapy
and ferroptosis is the observation that treatment of cells with iron
chelators (e.g., deferoxamine) or with ferroptosis inhibitors (e.g.,
ferrostatin-1 and liproxstatin-1) before exposure to IR partially
rescue their survival, and that this finding is more evident in
comparison to what observed combining IR with compounds
that inhibits other RCD (Lei et al., 2021).
Three major pathways regulate IR-mediated ferroptosis
induction (Lang et al., 2019;Lei et al., 2020;Ye et al., 2020)
(Figure 2), including 1) ROS and ACSL4. Increased ROS
produced by IR are responsible for the formation of PUFA
radicals that, following the interaction with oxygen (Fenton
reaction), generate lipid hydroperoxides (PUFA-OOH) (Lei et al.,
2020). This pathway is powered by the IR-induced
ACSL4 expression (Figure 1) that, together with LPCAT3, favors
the synthesis of phospholipid containing PUFA, which are also liable
of peroxidation; 2) GSH and GPX4. IR exposure depletes GSH
leading to reduced activity/expression of GPX4 and in such a way
attenuating the GPX4-mediated detoxification functions leading to
increased toxic effects of lipid peroxides (Ye et al., 2020) and 3)
SLC7A11. Reduced levels of SLC7A11 favor ferroptosis by
downregulating cystine uptake and in turn GSH synthesis and
GPX4 functions (Figure 1). Through the activation of ataxia
telangiectasia mutated serine/threonine kinase (ATM), IR
represses SLC7A11 levels stimulating ferroptosis. Other studies
have underlined that the expression of SLC7A11 is increased
upon IR exposure leading to the interpretation that an adaptive
cellular response to IR rescues the SLC7A11 expression or that the
level of SLC7A11 upon IR exposure depends on a peculiar cellular
context (Xie et al., 2011;Lei et al., 2020).
The activation of the cellular pathways induced by the DNA
damage together with the stimulation of signaling pathways
associated with alterations of lipids contained into the cellular
membranes occurring upon radiotherapy exposure synergize each
FIGURE 2
Cellular pathways implicated in radiotherapy-induced ferroptosis. The figure reports the cross-talk occurring between ionizing radiation and
ferroptosis pathways, including ROS/ACSL4, GSH/GPX4, SLC7A11, ATM/ATR, and AMPK pathways. The figure is prepared using tools from Servier Medical
Art (http://www.servier.fr/servier-medical-art, accessed on March 2023).
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other leading to enhanced tumor growth inhibition (Lei et al., 2020).
IR-induced DNA damage increases the expression of sensor
proteins, including ATM and ataxia telangiectasia and
Rad3 related serine/threonine kinase (ATR) that recognize the
altered DNA and stimulate DNA damage response signaling
cascades (DDR). DDR activate checkpoint kinases 1/2 (chk1/2)
and in turn stimulate the phosphorylation of p53 inducing cell-
cycle arrest. The block of the cell-cycle is required by the cells to test
the severity of the damage and “decide”their fate, 1) survive, upon
the activation of the DNA repair machine in case the damage can be
corrected, or 2) comit suicide, in case the damage is irreparable or
not correctly repaired, triggering RCD, including ferroptosis (Maier
et al., 2016;Huang and Zhou, 2020). Upon p53-mediated cell-cycle
arrest, irradiated cells mostly activate senescence (Bieging et al.,
2014;Maier et al., 2016). P53 mutation, which is a very common
condition in tumors, engage an alternative senescence checkpoint
protein, p16-retinoblastoma (RB) (Sabin and Anderson, 2011).
Senescence can coexists with apoptosis and in case of prolonged
p53 activation, IR preferentially stimulates both intrinsic (e.g.,
PUMA, BAX, NOXA, cytochrome C and caspase-9/3/7) and
extrinsic apoptosis (e.g., FAS/CD95, DR5, FAS ligands and
caspase-8) rather than senescence (Sheikh and Fornace, 2000;
Aubrey et al., 2018;Mijit et al., 2020). Besides the above
mentioned RCD, radiotherapy can induce autophagy and
necroptosis. Since autophagy has both pro-survival and pro-cell
death properties, controversial and not completely elucidated is its
role in IR response (Hu et al., 2016). Similarly, also the role of
necroptosis in radiation response is ambiguous (Adjemian et al.,
2020).
ATM activated by DNA damage increases p53 expression that
reduces the levels of SLC7A11 via a repressing interaction with
SLC7A11 promoter or by stimulating USP7-mediated proteasome
degradation of SLC7A11, in turn leading to ferroptosis (Kang et al.,
2019;Wang et al., 2019). Similarly, p53 induces ferroptosis by
stimulating the expression of SAT1, GLS2 or ferredoxin reductase
(FDXR) (Hu et al., 2010;Ou et al., 2016;Zhang et al., 2017). In
addition, p53 controls the expression of MDM2 that, via regulating
lipid metabolism and FSP1 expression, favors ferroptosis (Venkatesh
et al., 2020). Conversely, ferroptosis is attenuated upon p53-mediated
upregulation of p21. Metabolic stress induced by cystine deprivation
stimulates the p53-p21 axis preserving GSH levels and attenuating
ferroptosis (Tarangelo et al., 2018). Another protein involved in IR-
induced ferroptosis is the AMP-activated protein Kinase (AMPK) (Lei
et al., 2020;Ye et al., 2020). This protein can either stimulate or inhibit
ferroptosis. By stimulating the phosphorylation of beclin 1, which in
turn favors the downregulation of Xc–, activated AMPK induces
ferroptosis (Song et al., 2018). In the other hand, AMPK activates
the biosynthesis of PUFA containing phospholipids inhibiting
ferroptosis (Lee et al., 2020). IR stimulates the expression of heme
oxygenase 1 (HMOX1) and FR, which trigger the release of iron and in
such a way the induction of ferroptosis (Chiang et al., 2018).
Conversely, the exposure to IR upregulates FR heavy chain (FTH1)
and reduces oxidative stress in turn attenuating ferroptosis and
promoting radiation resistance (Choudhary et al., 2020). These
findings indicates an intersection between cellular pathways
implicated in DNA damage response and RCD, which
includes, besides ferroptosis, the immunogenic cell death (ICD)
mechanisms embracing apoptosis, necroptosis and autophagy.
Upon radiotherapy-mediated ICD stimulation, T-cells recruited into
the tumor microenvironment (TME) promote ferroptosis (Lang et al.,
2019).
3.1 Combination strategies for enhancing
ionizing radiation antitumor activity and for
overcoming radiation resistance
Radiation resistance of tumors is an urgent clinical problem
responsible for treatment failure. The understanding of the
molecular mechanisms subtending radiation resistance is critical
for setting up medical strategies, including drug combinations,
aimed at improving rediotherapy response (Table 1).
Radiotherapy is used for the clinical management of patients with
different tumor types including nasopharyngeal carcinoma (NPC) and
the development of radiation resistance is the main cause of treatment
failure in NPC-suffering patients. Huangetal.reportthatthesepatients
show increased expression of m6A mRNA demethylase fat mass and
obesity-associated protein (FTO) and that this feature correlates with
radiation resistance and poor prognosis (Huang et al., 2023). In support
of this observation is the finding that increased levels of FTO
characterize NPC cell lines resistant to IR (C666-1R, HONE1R)
compared to the corresponding sensitive counterparts (C666-1,
HONE1). NPC cells exposed to IR show morphological changes
typical of ferroptosis, increased MDA levels and reduced GSH
cellular content. The treatment with FB23-2 (a FTO inhibitor)
counteractsthisbehavior,which is reversed following FTO
overexpression or upon exposure to ferrostatin-1. Moreover, the IR
response of resistant cells is significantly improved by FB23-2 treatment.
The exposure to IR and FB23-2 increases DNA damage in vitro and this
observation is corroborated in vivo in xenograft HNE1R-bearing mice.
TheauthorsspeculatethatalinkexistsbetweenFTOandOTU
deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1). Molecularly,
via its demethylase activity, FTO produces m6A modification of the
OTUB1 transcript stimulating the expression of OTUB1 protein and its
interaction with SLC7A11, in turn activating SLC7A11 and leading to
ferroptosis inhibition and radiation resistance. These interpretations are
supported in vivo in xenograft HONE1R-bearing mice and PDX-mouse
models exposed to the combination erastin/IR. Compared to IR alone,
the combination with the ferroptosis inducer erastin enhances the
radiosensitivity of HONE1R tumors.
Another key protein implicated in radiation resistance by
inhibiting ferroptosis is angiopoietin-like 4 (ANGPTL4) (Zhang
et al., 2022). High levels of ANGPTL4 associate with poor
prognosis of patients suffering from lung adenocarcinoma and
adrenocortical carcinoma. A549 and H1299 lung cancer cell lines
cultured under hypoxic condition show an increased expression of
ANGPTL4 and radiation resistance. Moreover, compared to cells
cultured under physiologic conditions, hypoxic cells show higher
levels of ANGPTL4 accumulated into released exososmes. Upon
silencing of ANGPTL4, A549 cells restore the sensitivity to IR, while
reacquire IR resistance after ANGPTL4 ectopic expression. The
relationship between ANGPTL4 and ferroptosis is demonstrated in
ANGPTL4-overexpressing cells cultured under normoxia, which
show increased expression of GPX4, SLC7A11, FTH1, and FTL (e.g.,
reduced ferroptosis and enhanced radiation resistance). Conversely,
the above ferroptosis-associated proteins are decreased in
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ANGPTL4-silenced cells cultured under hypoxic conditions (e.g.,
increased ferroptosis and radiosensitivity). In vivo experiments in
xenograft A549-bearing mice treated with ANGPTL4 enriched
exosomes and exposed to IR support in vitro data confirming the
relationship between radiation resistance and ferroptosis.
Radiation resistance accounts for the failure of radiotherapy in
hepatocellular carcinoma (HCC) as well and Chen and colleagues
have studied the role played by ferroptosis in radiosensitizing HCC
using a panel of radiosensitive (SK-Hep and HepG2) and
radioresistant (SK-Hep-1R and HepG2-1R) HCC cell lines (Chen
et al., 2023). By comparing the gene expression profiles of SK-Hep
and SK-Hep-1R cells, SOCS2, and SMOX genes were identified as
differentially expressed. The expression of SOCS2 gene was reduced
in SK-Hep-1R, while SMOX was increased. Gene expression data
from GEPIA and HCCDB databases and Kaplan-Meier (K-M)
analysis showed that low expression of SOCS2 and high
expression of SMOX correlate with poor prognosis of HCC
patients. The radiation resistance observed in resistant cells is
overcome upon SOCS2 ectopic expression. Conversely, radiation
resistance is enhanced in SK-Hep and HepG2 cells following
SOCS2 silencing. In vivo experiments confirm the in vitro results.
Moreover, immunohistochemical staining of irradiated explanted
tumors shows an increased expression of SOCS2 paralleled by a
modulation of ferroptosis markers, including a downregulation of
GPX4 and SLC7A11, and an increased expression of 4-HNE. This
observation is confirmed in a set of clinical tissues in which the levels
of ferroptosis markers correlate with radiotherapy response. Co-
immunoprecipitation assays demonstrate a protein interaction
between SOCS2 and SLC7A11. Following the protein interaction,
the E3-ubiquitin ligase activity of SCOS2 allows ubiquitination and
proteasomal degradation of SLC7A11 leading to ferroptosis.
Iron homeostasis is controlled by copper (Cu) and elevated
levels of this micronutrient impact on tumorigenesis, resistance to
treatments and prognosis of HCC suffering patients (Yang et al.,
2022). HepG2 and MHCC-97H cells exposed to IR downregulates
the copper metabolism MURR1 domain 10 (COMMD10)
increasing the cellular accumulation of Cu and leading to
radiation resistance. These results are corroborated by gain and
TABLE 1 Combination strategies for enhancing ferroptosis induced by ionizi ng radiation.
Tumor type Cell lines Pathway involved In vivo evaluation Combination strategy References
Nasopharyngeal carcinoma C666-1 FTO/OTUB1/SLC7A11 HONE1R IR/FB23-2 Huang et al. (2023)
HONE1
C666-1R
HONE1R
Lung cancer A549 ANGPL4/GPX4/SLC7A11 A549 IR/ANGPTL4 enriched exosomes Zhang et al. (2022)
H1299
Hepatocellular carcinoma SK-Hep SOCS2/SLC7A11 SK-Hep-1 SK-Hep-1R IR/SOCS2-overexpressing plasmid Chen et al. (2023)
HepG2
SK-Hep-1R
HepG2-1R
Hepatocellular carcinoma HepG2 COMMD10/HIF1α/
SLC7A11
HepG2 IR/Cu chelator Yang et al. (2022)
MHCC-97H
Oral squamous cell carcinoma SCC15-S GPX4 SCC15-R IR/Hyperbaric oxygen (HBO) Liu et al. (2022)
SCC15-R
Colorectal cancer MC38 ATF3-SLC7A11-GPX4 MC38 IR/niraparib Shen et al. (2022)
CT26
HT29
Esophageal squamous cell carcinoma KYSE30 SCD1 N.D. IR/MF-438 Luo et al. (2022)
KYSE70
KYSE140
KYSE150
KYSE410
KYSE450
KYSE510
N.D., not defined.
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loss of function experiments performed in a panel of HCC cell lines
showing that increased COMMD10 levels parallel with reduced
cellular accumulation of Cu and radisensitivity. Conversely, reduced
expression of COMMD10 increases Cu accumulation leading to
radiation resistance. The role played by Cu in tumor growth is
confirmed in vivo in HepG2-bearing mice fed with Cu-rich food
upon irradiation. Compared to mice fed under normal conditions,
animals fed with Cu-rich food show increased tumor volume, which
is reduced following the administration of a Cu chelator. Moreover,
immunohistochemistry analysis of HCC clinical samples shows that
the levels of COMMD10 in patients sensitive to radiotherapy are
higher than that measured in samples from radioresistant subjects.
Proteomic analysis performed in HepG2 and COMMD10-depleted
HepG2 cells reveals a differential expression of genes implicated in
cell death. These findings, together with the observation that the
treatment with ferrostatin-1 and liproxstatin-1 increases the growth
of COMMD10 overexperring cells, indicate that ferroptosis is
critical for radiosensitivity. Based on the observation that the
sequence of the promoter of SLC7A11 contains binding sites for
HIF1-α, the authors propose an interplay between COMMD10,
SLC7A11, and HIF1-αas a molecular mechanism supporting the
cellular response. By interacting with HIF1-α, COMMD10 impedes
HIF1-αnuclear translocation and reduces SLC7A11 expression. The
IR-mediated reduced expression of COMMD10 favors the cellular
accumulation of Cu that stabilizes HIF1-αand inhibits its ubiquitin
degradation. Following nuclear translocation, HIF1-αstimulates
CER and SLC7A11 transcription inhibiting ferroptosis.
The effect of the co-exposure of IR and hyperbaric oxygen
(HBO) on oral squamous cell carcinoma (OSCC) cells is studied
by Liu and co-workers (Liu et al., 2022). Compared to the exposure
to IR, the combination IR/HBO increases the cytotoxicity in SCC15-
S cells, which is only in minimal part mediated by apoptosis.
Conversely, the combination enhances the levels of ferroptosis
markers (iron, ROS and MDA). Upon IR exposure, cells increase
the levels of ACSL4 and SLC7A11 (two ferroptosis promoters) as
well as that of GPX4 (a ferroptosis blocker), the latter being the
major factor accounting for radiation resistance. The treatment with
IR/HBO is ineffective on ACSL4 and SLC7A11 levels, while reduces
the expression of GPX4 and in such a way shifts the equilibrium
towards the induction of ferroptosis. These findings are
corroborated by the observation that the transfection of SCC15-S
cells with GPX4-overexpressing plasmid or the treatment with
ferrostatin-1 reverse the effects of the combination. The authors
also show that IR/HBO exposure sensitizes the radio-resistant
SCC15-R cells to IR. Although less sensitive to ferroptosis,
SCC15-R cells exposed to the combination significantly reduce
the expression of GPX4 in turn favoring ferroptosis, and these
results are confirmed in vivo in xenograft SCC15-R-bearing mice.
Compared to mice treated with IR, animals exposed to IR/HBO
show a significant reduction in tumor growth. In support of the
ferroptosis induction as a mechanism of tumor growth delay is the
observation that the treatment with ferrostatin-1 counteracts the
antitumor activity of IR/HBO exposure. A deeper investigation on
clinical samples (tumor and normal tissues as well as serum) from
38 OSCC patients shows that, compared to adjacent normal oral
tissues, the expression of GPX4 is increased in tumors. In addition,
the serum levels of GPX4 in cancer patients are increased in
comparison to healthy donors. Of note, high levels of GPX4 (e.g.,
reduced ferroptosis) are accompanied with poor chemo-
radiotherapy outcome.
Another interesting combination for sensitizing colorectal
cancer (CC) to IR is proposed by Shen and colleagues (Shen
et al., 2022). To protect themselves against DNA damages, cancer
cells stimulate poly (ADP-ribose) polymerase (PARP)-1 and PARP-
2 activities favoring the activation of DNA repair pathways. This
scenario supports the rational of combining radiotherapy with
PARP inhibitors (PARPi) to potentiate IR-mediated DNA
damage and in turn increasing cell death. Compared to CC cell
lines (murine MC38 and CT26 cells as well as human HT29 cells)
exposed to IR, cells exposed to the combination of the PARPi
niraparib with IR show increased levels of DNA DSBs. The
combination significantly increases the cell death in vitro and
enhances antitumor effects in vivo. The exposure to PARPi
induces the cyclic GMP-AMP synthase (cGAS) and stimulator of
interferon genes (STING), allowing the activation of cGAS-STING-
TBK1-IRF3 signaling that stimulates IFNB1 transcription and the
release of IFNβ, CXCL10, CCL5, and MX1. These findings are also
observed in vivo in M38 tumor-bearing mice. The role played by
cGAS is corroborated by the observation that cGAS-silenced
M38 cells are less sensitive to the combination treatment. The
levels of ferroptosis markers (increased MDA and PTGS2 levels,
reduced SLC7A11 and GPX4 expression) reflect the induction of
ferroptosis as a mechanism of cell death. The analysis of the gene
expression of cGAS-silenced M38 cells in comparison to cGAS
normal expressing cells supports a critical role for the activating
transcription factor 3 (ATF3) and underlines the existence of a
ATF3-SLC7A11-GPX4 axis controlling ferroptosis induction upon
exposure to IR and niraparib. In addition, cGAS depletion in
M38 tumor-bearing mice abolishes the IR-induced infiltration of
CD8+T, CD8+GZMB + T-cells leading to reduced antitumor
efficacy, thus corroborating the role of cGAS for the combination
efficacy. The analysis of tumor samples before and after
radiotherapy from 32 patients affected by CC reveals that
increased expression of cGAS, ATF3, and PTGS2 as well as an
high density of CD8+T-cells associate with a high disease-free
survival rate.
The enzyme stearoyl-CoA desaturase (SCD1) catalyzes the
formation of oleic acid and palmitoleic acid and plays a critical
role in IR response. Increased levels of SCD1 are observed in a panel
of esophageal squamous cell carcinoma (ESCC) cell lines and the
targeting of SCD1 by MF-438 is pursued by Luo and colleagues for
increasing IR potency (Luo et al., 2022). Cells treated with MF-438
reduce cell growth and the combined exposure to IR and subtoxic
concentrations of MF-438 results in synergistic antiproliferative
activity. The synergism is attenuated upon silencing of SCD1 as
well as following exposure to the ferroptosis inhibitor RLS3. The
MF-438-mediated inhibition of SCD1 increases lipid peroxidation,
ATP and HMGB1 release into the extracellular compartment, and
this behavior is enhanced upon exposure to the combination IR/MF-
438. Since similar results are observed in cells exposed to the
combination IR/RLS3, it is likely that the induction of ferroptosis
is the key mechanism of antitumor activity. These observations are
corroborated by the finding that cells exposed to exogenous oleic
acid or palmitoleic acid undergo ferroptosis. In vitro data are
confirmed in vivo in ESCC-bearing mice exposed to IR/MF-438.
The authors analyze the expression of SCD1 in ESCC patients from
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GEPIA database and stratify them in high and low expression
groups. Compared to normal epithelium, SCD1 is significantly
increased in tumor tissues and high SCD1 expressing patients
experience a shorter disease-free survival.
3.2 Nano-systems for enhancing ionizing
radiation antitumor activity
Another strategy for potentiating IR-mediated ferroptosis
induction implies the use of metal-based nanoparticles (NPs)
(Table 2). These NPs exploit the enhanced permeability retention
effect to selectively induce ferroptosis in tumors. The tumor
selectivity is also enhanced by the in local IR exposure, and these
features allow reduced treatment toxicity.
3.2.1 Iron-based nanosystems
Lin and coworkers have assembled hemin, PX-12 (a TRX-1
inhibitor) and human serum albumin to built HPNPs (Lin et al.,
2023).TheseNPsarestableinphysiologicsolutionandinbloodand
rapidly release PX-12 under acidic conditions (e.g., pH 5). Acidic
conditions, recapitulating acidic TME, favor the production of OH•
by HPNPs and stimulate Fenton reaction. In vitro experiments
performed in mouse melanoma B16F10 cells and normal mouse
L929 fibroblasts show that HPNPs are better internalized in tumor
compared to normal cells. The combination HPNPs/radiotherapy
improves ROS production leading to increased cytotoxicity with
respect to HPNPs administered alone. Besides the increased ROS
production (mediated by both hemin and radiotherapy), the
combination HPNPs/radiotherapy implements MDA, reduces the
levels of antioxidants (GSH and TRX-1) and attenuates the
GPX4 activity, in turn stimulating ferroptosis. These findings are
corroborated by the observation that the treatment with ferrostatin-1
counteracts this behavior. In vivo experiments in B16F10 tumor-suffering
mice demonstrate that HPNPs are biocompatible. Moreover, compared
to animals treated with HPNPs, the tumor growth of mice exposed to the
combination HPNPs/radiotherapy is significantly reduced. Ex vivo
analysis evidences reduced GSH and GPX4 levels as well as increased
MDA content in tumors exposed to the combination with respect to
those treated with HPNPs alone.
To potentiate radiotherapy efficacy in breast cancer, Hou et al.
propose multifunctional NPs composed by a shell of platinum
decorated with hyaluronic acid (HA) encapsulating a core of
Fe(III)-polydopamine (FPH) (Hou et al. 2023). FPH are stable at
pH 7.4 and show photothermal properties upon 808 nm irradiation.
Conversely, in acidic conditions NPs dissociate and release Fe
3+
.The
red-ox reaction Fe
3+
/Fe
2+
converts GSH into GSSG allowing GSH
depletion and H
2
O
2
hydrolysis producing O
2
and OH•(Fenton
reaction). Since the depletion of GSH is increased at 50°C, it is
conceivable that the photothermal-mediated hyperthermia
improves antitumor potency of FPH. An additional property of
FPH is their ability to produce O
2
by catalyzing the hydrolysis of
TABLE 2 Nano-systems for enhancing the antitumor activity of ionizing radiation.
Nano-system Type of metal Cell lines In vivo evaluation References
HPNPs Iron Mouse melanoma B16F10 cells Mouse melanoma B16F10 tumors Lin et al. (2023)
Mouse normal L929 fibroblasts
FPH Iron Mouse mammary carcinoma 4T1 cells Mouse mammary 4T1 tumors Hou et al. (2023)
Mouse RAW264.7 macrophages
Fe
2
O
3
@TA-Pt Iron Mouse mammary carcinoma 4T1 cells Mouse mammary 4T1 tumors Jiang et al. (2022)
Normal human HUVEC cells
GOD@FeN
4
-SAzyme Iron Mouse mammary carcinoma 4T1 cells Mouse mammary 4T1 tumors Zhu et al. (2022)
SPIONC Iron Human lung cancer NCI-H460 cells Human lung cancer NCI-H460 tumors Li et al. (2022)
iCoDMSN Cobalt Mouse mammary carcinoma 4T1 cells Mouse mammary 4T1 tumors Zhao et al. (2023)
Human breast cancer MCF-7 cells
Human lung cancer A549 cells
Human colorectal cancer Caco-2 cells
Human gastric carcinoma SGC-7901 cells
AGulX Gadolinium Human breast cancer MDA-MB-231 cells Human breast cancer MDA-MB-231 tumors Sun et al. (2022)
Human breast cancer MDA-MB-468 cells
3a Gold Human cervical carcinoma HeLa cells Human cervical carcinoma HeLa cells transplanted in
zebrafish
Yang et al. (2022)
Human cervical carcinoma SiHa cells
PBmB-DOX Bismuth Mouse mammary carcinoma 4T1 cells Mouse mammary 4T1 tumors Hou et al. (2022)
Frontiers in Molecular Biosciences frontiersin.org08
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H
2
O
2
by Pt nanoenzyme. In vitro experiments carried out in mouse
4T1 breast cancer cells and in RAW264.7 macrophage cells show a
preferential accumulation of FPH in cancer cells, likely dependent on
the interaction of HA with CD44. In addition, compared to the
treatment with FPH, near infrared radiation (NIR, 808 nm) or IR
alone, the exposure to FPH/NIR and FPH/IR significantly potentiates
cytotoxicity in 4T1 cells. The improved cytotoxicity observed in cells
treated with the combinations correlates with increased depletion of
GSH levels and increased ROS and DNA damages. This behavior is
counteracted by the treatment with ferrostatin-1. In vivo experiments
performed in 4T1 tumor-bearing mice corroborate in vitro results and
demonstrate the biocompatibility of FPH. NIR absorbance, Pt-
mediated X-ray attenuation and enhanced permeability retention-
mediated tumor selectivity render FPH a very useful tool for imaging
as well.
Fe
2
O
3
@TA-Pt are NPs containing a core of Fe
2
O
3
covered by
platinum and tannic acid (TA-Pt) (Jiang et al., 2022). Fe
2
O
3
@TA-Pt
arestableinmouseserum,whileacidicpH(5.5),mimickingTME,favors
the disassembling of the TA-Pt envelop releasing Fe
2
O
3
. In presence of
H
2
O
2
, which is abundant in TME, Fe
3+
is converted in Fe
2+
generating O
2
and OH•(Fenton reaction). Fe
2
O
3
@TA-Pt better accumulate into
4T1 cells with respect to normal HUVEC cells, and this observation
correlates with the increased cytotoxicity in tumor cells. The analysis of
the DNA damage upon NPs exposure reveals that, besides the ROS-
mediated DNA damage, Pt-DNA adducts, which reflect the release of the
Pt by NPs, are observed. Additionally, Fe
2
O
3
@TA-Pt treatment enhances
the radiotherapy sensitivity of 4T1 cells and potentiates ferroptosis
induction, as demonstrated by the reduced levels of GSH and
GPX4 observed upon combination exposure. In vivo experiments in
4T1 tumor-bearing mice show the preferential accumulation of NPs in
liver and tumor. Moreover, the tumor volume of mice exposed to
radiotherapy upon treatment with Fe
2
O
3
@TA-Pt is significantly
reduced with respect to that of animals singly treated with NPs or
radiotherapy. The combination Fe
2
O
3
@TA-Pt/IR is well tolerated with
nosignsoftoxicity,reducesthetumorrecurrenceaswellasthe
pulmonary metastasis and enhances the survival of mice.
Single-atom nanozymes (SAzymes) are enzyme-based drugs
containing a single metal atom in their active sites that are
interesting for anticancer therapy. Critical for the antitumor
properties of these nano-systems is the presence into the TME of
a specific enzymatic activity as well as H
2
O
2
.Zhu et al. (2022) have
engineered a SAzyme based on FeN
4
and glucose oxidase (GOD)
(GOD@FeN
4
-SAzyme) for radio-enzymatic therapy. The elevated
glucose level in tumor over the normal cells allows the production of
H
2
O
2
via GOD of the GOD@FeN
4
-SAzyme and this results in
sustained production of OH•and O
2
as well as in GSH
depletion. The enzymatic cascade triggered by GOD is enhanced
by IR, which favor the conversion Fe
3+
/Fe
2+
implementing the
generation of OH•and potentiating apoptosis and ferroptosis.
Cytotoxic experiments performed in 4T1 cells show that the
killing activity of GOD@FeN
4
-SAzyme is enhanced upon
exposure to IR. Compared to the treatment with GOD@FeN
4
-
SAzyme or IR separately, the combination GOD@FeN
4
-SAzyme/
IR significantly increases the DNA damages (increased γ-H2AX
signals) as well as ferroptosis (increased lipid peroxidation and OH•,
reduced GSH and GPX4 levels accompanied by mitochondria
membrane alterations). Besides ferroptosis, treated 4T1 cells show
apoptosis induction (e.g., PARP and caspase 3 activation).
Intravenous and intratumoral injection are used for in vivo
administration of GOD@FeN
4
-SAzyme in 4T1-tumor bearing
mice. Magnetic resonance imaging (MRI) reveals that GOD@
FeN
4
-SAzyme accumulate into the tumor. Compared to the
exposure to GOD@FeN
4
-SAzyme or IR, a significant reduction of
tumor volume is observed in animals treated with the combination
GOD@FeN
4
-SAzyme/IR. Ex vivo investigation recapitulates in vitro
findings (e.g., increased γ-H2Ax and reduced GSH). GOD@FeN
4
-
SAzyme are biocompatible and the combination is well tolerated
as well.
The pH-sensitive supramagnetic iron oxide nano-clusters (SPIONC)
are proposed by Li et al. (2022) for enhancing radiation sensitivity in lung
cancer. Under acidic conditions, which recapitulate TME and
intracellular compartment conditions, SPIONC decompose and
generate OH•via Fenton reaction. SPIONC efficiently accumulate in
NCI-H460 cells. Compared to SPIONC or IR individual exposure, the
combination SPIONC/IR is more effective in inhibiting cell proliferation.
Cells exposed to the combination increase iron, lipid peroxides, ROS and
γ-H2AX levels. These features reflect apoptosis (reduced expression of
Bcl2 and increased caspase 3 activation) and ferroptosis induction
(reduced expression of SLC7A11 and GPX4). The involvement of
ferroptosis in SPIONC/IR response of tumor cells is corroborated by
the observation that the pre-treatment with ferrostatin-1 attenuates
cytotoxicity. In vivo experiments in orthotopic mice model of NCI-
H460 cells in which SPIONC are injected by both intravenous and intra-
tracheal delivery show that intra-tracheal delivery is to prefer for MRI
analysis.Moreover,theexposuretothecombinationSPIONC/IRresults
in increased tumor volume inhibition and survival rate with respect to the
administration of SPIONC or IR separately. No important toxic side
effects are reported upon treatment. The analysis of explanted tumors
from euthanized mice confirms the molecular alterations observed in
vitro.
3.2.2 Nano-systems based on cobalt, gadolinium,
gold, and bismuth
Another ferroptosis-stimulating nano-system has been recently
proposed by Zhao and colleagues (Zhao et al., 2023). Starting from
the observation that high level of cobalt (Co) in tumors associates
with a good prognosis, the authors have engineered Co oxide
nanodots by assembling bovine serum albumin and CoCl
2
. These
nanodots are conjugated with iRGD peptides and encapsulated into
dentritic mesoporus silica nanoparticles (iCoDMSN). Acidic
conditions stimulate the release of Co
2+
by iCoDMSN and the
presence of iRGD favors their tumor penetration. iCoDMSN are
cytotoxic on a panel of different tumor cell lines, including murine
4T1 cells and human MCF-7 breast cancer cells, human A549 lung
cancer cells, human Caco-2 colorectal cancer cells and human SGC-
7901gastric carcinoma cells. Therefore, these nanostructures show
interesting photoacoustic imaging ability under 808 nm irradiation
and in vivo experiments in 4T1-tumor bearing mice show that
iCoDMSN are well tolerated (e.g., no important signs of liver and
kidney toxicity), preferentially co-localize with lysosomes and that
iRGD favors tumor accumulation. Proteomic studies performed on
4T1 cells exposed to iCoDMSN underline that ferroptosis pathways
play a critical role for cell response. These findings are supported by
the increased lipid peroxidation, MDA and iron levels observed in
iCoDMSN-treated 4T1 cells, and by the observation that this
behavior is counteracted upon exposure to ferrostatin-1.
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Proteomic analysis also underlines an increased level of HMOX1,
which controls Fe
2+
accumulation and ferroptosis induction by
increasing the expression of TFR as well as by reducing solute
carrier family 40 member 1 (SLC40A1). Upon treatment,
accumulated iCoDMSN perturb the KEAP1/NRF2/HMOX1 axis.
This axis is governed by the level of nuclear factor erythroid 2-
related factor 2 (NRF2, Bellezza et al., 2018), which is a transcription
factor for HMOX1. These results are confirmed in vivo in 4T1-
tumor bearing mice exposed to the combination iCoDMSN/
radiotherapy. Compared to mice treated with iCoDMSN, a
increased tumor volume inhibition and survival rate are observed
in mice treated with iCoDMSN/radiotherapy. Ex vivo analysis
confirms the results obtained in vitro (reduced KEAP1, increased
NRF2, HMOX1, iron, and MDA).
Aimed at overcoming radiation resistance and reducing
radiotherapy damages to normal tissues, Sun and colleagues have
proposed gadolinium (Gd)-based NPs (AGulX) (Sun et al., 2022).
AGulX are based on polysiloxane covering Gd entrapped by the
chelator dodecane tetraacetic acid (DOTA) moieties that functionalize
the polysiloxane. Upon IR exposure, AGulX produce secondary and
Auger electrons as well as free radicals. These NPs are under clinical
investigation in brain, lung and pancreatic cancers. In the study by Sun
et al., AGulX are evaluated in triple negative breast cancer cells (MDA-
MB-231 and MDA-MB-468 cell lines) in vitro and in vivo.Comparedto
the treatment with AGulX or IR, the combination AGulX/IR reduces cell
growth as well as cell migration and invasion capability. Additionally,
cells exposed to the combination enhance ROS production, DNA
damage (increased number of γ-H2AX foci) as well as G2/M cell-
cycle arrest. Molecularly, the combination stimulates the
phosphorylation of ATR and Chk1 (e.g., G2/M block) and reduces
the phosphorylation of ATM and Chk2 (e.g., reduced homologous
recombination repair ability). Moreover, by diminishing the activation
of the MRN-ATM-Chk2 axis, the combination also impairs the non
homologous end-joining, which is reflected by the reduced
phosphorylation of p53 and BRCA1. Besides apoptosis induction
(e.g., PARP and caspase 3 activation), cells exposed to AGulX/IR
reduce the expression of NRF2 favoring ferroptosis by attenuating
SLC7A11 activity, in turn reducing GSH synthesis and GPX4 activity.
The increased levels of lipid perixidation and MDA support the
induction of ferroptosis as a mechanism of cell death. These findings
are confirmed by the observation that ferroptosis is attenuated by the
siRNA-mediated silencing of NRF2, SLC7A11 and GPX4 as well as by
the treatment with ferrostatin-1. The in vitro results parallel the in vivo
observations in MDA-MB-231 tumor-bearing mice. No important signs
of toxicity are reported in treated animals.
A series of metal-biotin-conjugated nano-structures based
on different metals endowed with radiosensitizer properties is
proposed by Yang and colleagues (Yang et al., 2022). Among the
different nano-systems, the gold derivative 3a is selected for
further investigations. 3a contains a biotin moiety for favoring
tumor selectivity and uptake, a triply bonded dicarbon alkynyl
amide linker joining biotin to Au, which is hidden by a lipophilic
phosphine residue to increase membrane solubility. Compared
to the auranofin (the reference), 3a shows similar
antiproliferative potency on human cervical carcinoma HeLa
and SiHa cells. Moreover, the tumor selectivity of 3a, which is
dependent on biotin, is supported by the observation that 3a
uptake is higher in Hela cells (expressing high levels of biotin
receptor) with respect to normal human cervical epithelial
H8 cells (expressing low levels of the receptor). Au-
containing compounds, including auranofin, inhibit
thioredoxin reductases (TRXR) and 3a-treated cells show
reduced TRXR enzymatic activity. This finding is
corroborated by docking studies showing that Au binds the
selenium of the TRXR. The inhibition of the detoxification
properties of TRXR stimulates ROS production and favors
G2/M cell-cycle arrest as well as apoptosis (e.g., reduced
Bcl2, increased Bax and alteration of the mitochondria
membrane potential). Gene expression analysis performed on
HeLa cells exposed to 3a shows a differential expression of genes
involved in ferroptosis, includingTXNRD1,HMOX1,SLC7A11,
GCLM, FTH1, FTL, GPX1, GPX1P1, and GPX4. The exposure of
HeLa cells to IR following 3a treatment stimulates DNA damage
(increased γ-H2AX levels) and downregulates GPX4 expression,
leading to reduced cell survival. This behavior is counteracted by
the exposure to N-acetyl L-cysteine or ferrostatin-1. The
radiosensitizing properties of 3a are confirmed in vivo in
zebrafish transplanted with Hela cells exposed to the
combination 3a/IR.
Along this way, Hou et al. have reported the synthesis and the
antitumor properties of PBmB-DOX NPs (Hou et al., 2022). PBmB-DOX
include a core of Bi
2
S
3
covered by PEGylated doxorubicin (DOX).
Moreover, PBmB-DOX contain-Mn-O- bonds that are sensitive to
high GSH level, which is typical of the TME, allowing tumor
selectivity and release of DOX under acidic conditions. The PBmB-
DOX disassembling mediated by high levels of GSH favors the release of
Mn
2+
that, besides stimulating Fenton reaction and potentiating DOX-
mediated antitumor activity, allows magnetic resonance contrast
enhancement. The downregulation of GSH reduces 4T1 cell
proliferation, while no important changes are evidenced in the growth
of normal PBmB-DOX-treated HUVECs cells. The depletion of GSH
upon treatment reduces the expression of GPX4 and increases lipid
peroxidation. PBmB-DOX are more potent than free DOX on 4T1 cells
and, compared to the exposure to IR or PBmB-DOX as single treatments,
the combination PBmB-DOX/IR increases the amount of γ-H2AX foci.
In vivo studies in 4T1 tumor-bearing mice demonstrate that PBmB-DOX
are biocompatible with no important toxic effects reported for major
organs, and that they preferentially accumulate into the tumor. Longer
circulation time in plasma is reported for PBmB-DOX with respect to free
DOX, and PBmB-DOX more efficiently suppress tumor growth with
respect to free DOX. The exposure to IR upon PBmB-DOX treatment
significantly improves antitumor activity with no important signs of
toxicity. Histological analysis of tumors explanted from mice shows
increased expression of γ-H2AX and reduced GPX4 levels following
the exposure to the combination. Lastly, a remarkable signal
enhancement in tumor is evidenced in MRI after 6 h post-injection of
PBmB-DOX, thus confirming the theranostic properties of the nano-
system.
4 Ionizing radiation-associated gene
signatures
Recently, results from investigations focused on the studies of
ferroptosis-associated gene signatures for predicting radiotherapy
patient outcomes have been reported (Table 3).
Frontiers in Molecular Biosciences frontiersin.org10
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The combination of temozolomide and radiotherapy is used
for the treatment of malignant glioblastoma (GBM). Though
effective, the combination is not curative and the identification
of radiosensitive-associated biomarkers is an urgent need for
predicting prognosis and therapy outcome. In the study by Xie
et al. (2022), expression profiles of genes involved in radiation
response and ferroptosis-associated pathways of GBM patients
and healthy subjects from The Cancer Genome Atlas (TCGA)
database are analyzed. Among the differentially expressed genes
(DEGs) intersecting the two pathways, seven genes (MAPK1,
ZEB1, MAP1LC3A, HSPB1, CA9, STAT3, and TNFAIP3)
overlap and the analysis of the protein-protein interaction
network indicates STAT3 as the hub gene. The application of
the Least Absolute Shrinkage and Selection Operator (LASSO)
and Cox regression analysis defines a risk score that stratifies
patients in low- and high-risk groups. Patients in the high-risk
group show low overall survival (OS) and high mortality.
Receiver Operating Characteristic (ROC) curve and K-M
analysis confirm the power of the signature in predicting
patients’survival. The prognostic model is validated by data
from Chinese Glioma Genome Atlas (CGGA) database used as
an external independent validation cohort. Functional
enrichment analyses defined by Gene Ontology (GO) and
Kyoto Encyclopedia of Genes and Genomes (KEGG) as well
as immune cell infiltration patterns analysis from single-sample
Gene Set Enrichment Analysis (ssGSEA) allow the identification
of the most represented pathways in high-risk group, including
IL-17, cytokine-cytokine receptor interaction, TNF signaling
pathways, DCs, macrophages, Tumor-infiltrating lymphocytes
(TIL) and Treg cells. In vitro experiments performed in
glioblastoma U87 and U251 cells treated with the
combination erastin/IR support the relationship between
radiosensitivity and ferroptosis.
Gene expression profiles of breast cancer and normal tissues
as well as the survival and clinical information from TCGA
database are analyzed by Liu and colleagues (Liu et al., 2022).
Among the DEGs associated with ferroptosis, SLC7A11 is the
most upregulated in tumors compared to normal tissues.
Numerous clinic-pathologic properties associate with
SLC7A11 levels, including the expression of estrogen
receptor (ER). ER-positive tissues show lower levels of
SLC7A11 (e.g., increased ferroptosis) with respect to ER-
negative samples. The univariate Cox regression for OS
model demonstrates that high SLC7A11 levels associate with
worse OS. In vitro experiments carried out in a panel of breast
cancer cell lines (ER-positive MCF7 and ZR-75-1 as well as ER-
negative MDA-MB-231) treated with ferrostatin-1 or erastin in
combination with IR support the critical role played by
SLC7A11 in regulating IR-induced ferroptosis in ER-positive
cells. The study also shows a positive correlation between the
expression of estrogen receptor 1 (ESR1) and SLC7A11 and the
analysis by K-M predicts poor prognosis for patients with high
levels of ESR1. Molecularly, IR exposure stimulates the
expression of ESR1 that, in turn, increases SLC7A11 levels
attenuating ferroptosis. This finding is supported by the
observationthatuponESR1/SLC7A11knockdowninER-
TABLE 3 Gene signatures associated to ionizing radiation.
Gene signature Tumor types Cell line validation Pathway involved References
MAPK1 Malignant glioblastoma U87 IL-17 Xie et al. (2022)
ZEB1 U251 Cytokine-cytokine receptor interaction
MAP1LC3A TNF signaling pathways
HSPB1 DCs
CA9 Macrophages
STAT3 TIL
TNFAIP3 Treg cells
SCL7A11 Breast cancer MCF7 SLC7A11/ESR1 Liu et al. (2022)
ZR-75-1 SLC7A11/NEDD4L
MDA-MB-231
ACSL3 Prostate cancer No Epithelial–mesenchymal transition Feng et al. (2022)
EPAS1 Allograft rejection
FASN Fc gamma R-mediated phagocytosis
GSTP1 TGF beta signaling
LDHB ECM receptor interaction
NEDD4L Adipocytokine signaling
Androgen response
Notch signalling
Frontiers in Molecular Biosciences frontiersin.org11
Beretta and Zaffaroni 10.3389/fmolb.2023.1216733

positive cells, IR-induced ferroptosis is enhanced.
Immunoprecipitation assay revels that no direct protein-
protein interaction occurs between ESR1 and SLC7A11.
Conversely, a protein interaction involving SLC7A11 and the
E3 ubiquitin ligase neural precursor cell expressed
developmentally downregulated gene 4-like (NEDD4L) is
critical for stimulating the proteasome-mediated degradation
of SLC7A11. Based on these findings, the authors suggest that
two pathways, including ESR1/SLC7A11 and SLC7A11/
NEDD4L, control SLC7A11 level and regulate ferroptosis
induced by IR exposure.
By analyzing expression data (mRNA and lncRNA) from
Gene Expression Omnibus (GEO) database of normal and
prostate cancer tissues of patients treated with radical
radiotherapy and intersecting them with ferroptosis-related
genes, Feng and co-workers construct a gene signature,
including ACSL3, EPAS1, FASN, GSTP1, LDHB, and
NEDD4L. This signature allows the definition of a
ferroptosis-related gene prognostic index (FGPI) useful for
predicting biochemical recurrence (BR) and radiation
resistance of prostate cancer suffering patients (Feng et al.,
2022). FGPI allows the stratification of the patients in high-
and low-risk groups. Although ROC curve poorly discriminates
BR patients from patients who do not experience BR, it
evidences that FGPI potentially reflects radiation resistance.
Indeed, compared to no BR patients, a significant higher
FGPI is observed for BR patients treated with radical
radiotherapy. The application of the K-M curve shows that
FGPI is an independent risk factor for biochemically relapse
(BCR) and metastasis-free survival (MFS) in patients treated
with radical radiotherapy. Moreover, compared to low-risk,
high-risk group patients treated with radical prostatectomy
are at higher risk of metastasis. The application of the
GeneMANIA database and ceRNA network assigns a critical
role to lnRNAPART1 in controlling ACSL3 and
EPAS1 expression via a intricate crosstalk involving
60 different miRNAs. Gene Set Enrichment Analysis (GSEA)
shows differences in pathway enrichment in high-risk
(epithelial–mesenchymal transition, allograft rejection, Fc
gamma R-mediated phagocytosis, TGF beta signaling
pathway, and extracellular matrix receptor interaction) with
respect to low-risk patients (adipocytokine signaling pathway,
androgen response and notch signalling). Drug and
immunologic analysis as well as TME analysis resulting from
the application of dedicated softwares, which consider the
expression of ACSL3 and EPAS1, underline potential
sensitivity to nine drugs (OSI-027, OSI-930, PAC-1, PHA-
793887, PI-103, PIK-93, SNX-2112, TPCA-1, and UNC0638)
for high-risk patients. Compared to no BR patients, BR patients
group shows lower expression levels of METTL14, which predict
sensitivity to methylating agents, and higher expression of
PDCD1LG2 (PD-L2) and CD96. However, only CD96 is
significantly associated with BCR-free survival. Regarding the
results of TME analysis, cancer-related fibroblasts,
macrophages, stromal score, immune score, estimate score,
and tumor purity are risk factors for BCR closely associated
to BCR-free survival.
5 Conclusion
Although radiotherapy is the firstchoiceforthetreatmentof
different tumors types, the development of radiation resistance
impairs its effectiveness and medical strategies aimed at
overcoming this drawback are urgent. Among these strategies,
the combination of IR with ferrptosis inducers proved to synergize
thus potentiating radiotherapy. Moreover, since the activation of
defense cellular pathways in response to IR exposure attenuates
radiotherapy effectiveness, deeper investigations aimed at studying
the involved pathways as well as at developing ferroptosis inducers
or novel combinatorial strategies are intriguing ways to pave for
combating radiation resistance. Besides the use of small molecules
inducing ferroptosis, the combination of IR with nano-systems
endowed with both diagnostic and therapeutic potential is
promising. In spite of their capability to overcome the
resistance to apoptosis developed by tumors exposed to
conventional chemotherapeutics, ferroptosis inducers show
drawbacks typical of small molecules, including low solubility,
limited tumor targeting, and toxic side effects that have often
impeded their clinical evaluation. These drawbacks have been
tackled by NPs that, functioning like a “Trojan horse”, localize
into the tumors via the enhanced permeability retention effect and
are entered into the therapeutics armamentarium for fighting
tumors. NPs ameliorate the circulation time of encapsulated
drugs and stimulate anticancer immunity at the tumor site.
Specific decoration aimed at targeting peculiar tumor-expressing
molecules as well as exploiting non physiologic conditions typical
of TME potentiate the tumor selectivity of NPs. Tumor targeting is
also in part guaranteed by the local irradiation of the tumor. NPs
are designed to disassemble themselves under peculiar conditions
(e.g., the acidic pH as well as the presence of specificenzymes),
ameliorating the selectivity of the cargo release. Therefore, the
magnetic properties of the metal composing the structure of the
NPs account for their theranostics potential. Among the
combinations including small molecules here reported, only IR/
niraparib is currently under investigation in phase I-II clinical
trials (https://www.clinicaltrials.gov)intriplenegativebreast
cancer (NCT03945721 and NCT04837209), pancreatic tumors
(NCT04409002), prostate cancer (NCT04194554,
NCT04037254), glioblastoma (NCT05666349) and head and
neck squamous cell carcinoma (NCT05784012) patients.
Regarding the clinical studies exploiting NPs, although all the
NPs considered have been tested in vivo showing interesting
antitumor profile, only AGluX is under phase I-II clinical
evaluation in patients with brain tumors and brain metastasis,
gynecologic cancers, non small cell lung cancers, and pancreatic
cancers (NCT04899908, NCT03308604, NCT02820454,
NCT04789486, NCT03818386, and NCT04784221. https://www.
clinicaltrials.gov). Lastly, the continuous implementation of the
gene signatures predicting IR response via ferroptosis-
mediated cell death is expected to positively impact on patient`s
health.
In conclusion, despite the preclinical success achieved with
the combination strategies, additional efforts and clinical
investigations are required in the future to demonstrate their
safety profile as well as their antitumor effectiveness.
Frontiers in Molecular Biosciences frontiersin.org12
Beretta and Zaffaroni 10.3389/fmolb.2023.1216733

Author contributions
GB and NZ wrote the review manuscript. All authors
contributed to the article and approved the submitted version.
Funding
The APC was funded by Ricerca Corrente from Italian Ministry
of Health.
Acknowledgments
This work was supported by Ricerca Corrente funds from Italian
Ministry of Health.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their
affiliated organizations, or those of the publisher, the
editors and the reviewers. Any product that may be
evaluated in this article, or claim that may be made by
its manufacturer, is not guaranteed or endorsed by the
publisher.
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