Cancer Letters 483 (2020) 127–136

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Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

Mini-review

Ferroptosis, a novel pharmacological mechanism of anti-cancer drugs T

a,b,c a,b,c a,b,c a,b,c a,b,c
Yanwei Su , Bin Zhao , Liangfu Zhou , Zheyuan Zhang , Ying Shen ,
Huanhuan Lva,b,c, Luban Hamdy Hameed AlQudsyc, Peng Shangb,c,∗
a
School of Life Science, Northwestern Polytechnical University, Xi'an, Shaanxi, 710072, China
b
Research & Development Institute of Northwestern Polytechnical University in Shenzhen Research & Development Institute of Northwestern Polytechnical University in
Shenzhen, Shenzhen, 518057, China
c
Key Laboratory for Space Bioscience and Biotechnology, Institute of Special Environmental Biophysics, School of Life Science, Northwestern Polytechnical University,
Xi'an, Shaanxi, 710072, China

ARTICLE INFO ABSTRACT

Keywords: Ferroptosis, a form of regulated cell death, is initiated by oxidative perturbations of the intracellular micro-
System xc- environment, which is under the constitutive control of glutathione peroxidase 4 (GPX4). Ferrous iron (Fe2+)
Glutathione peroxidase 4 accumulation and lipid peroxidation are critical events in the induction of ferroptosis, which is inhibited by iron
Iron chelators and lipophilic antioxidants. Ferroptosis terminates in mitochondrial dysfunction and toxic lipid per-
Ferritinophagy
oxidation. It plays a vital role in inhibiting cancer growth and proliferation. It can be induced in cancer cells, and
Reactive oxygen species
certain normal cells, by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3) or clinical
drugs. The purpose of this review is to summarize the various drugs (e.g., sulfasalazine, lanperisone, sorafenib,
fenugreek (trigonelline), acetaminophen, cisplatin, artesunate, combination of siramesine and lapatinib, fer-
umoxytol, and salinomycin (ironomycin)) that could induce ferroptosis in cancer cells and provide an overview
of current knowledge regarding the mechanisms underlying ferroptosis. In future, we anticipate the development
of more ferroptosis-inducing drugs, and the availability of such drugs for the clinical treatment of cancer.

1. Introduction RSL3 treatments do not trigger morphological changes or biochemical

Ferroptosis is a form of cell death which is morphologically, bio-
chemically and genetically distinct from apoptosis, necrosis and au-
tophagy. Ferroptosis was discovered and identified by Dixon in 2012
[1]. It is characterized by an overwhelming, iron-dependent accumu-
lation of lethal lipids and reactive oxygen species (ROS) [2]. In fer-
roptotic cells, the mitochondria appear smaller and have increased
mitochondrial membrane density, and reduced or vanished of mi-
tochondrial cristae. The term ‘ferroptosis’ originated from the fact that
iron chelation, which lowers intracellular iron levels, prevents the
formation of reactive radical species like radical hydroxyl, and protects
cells from death [1].
Essentially, ferroptosis inducers were identified before the concept
of ferroptosis. The first identified ferroptosis inducer was erastin, in
2003, which is a synthetic lethal molecule expressing the engineered
mutant RAS oncogene in human foreskin fibroblasts (BJeLR) [3]. Era-
stin irreversibly binds to SLC7A11, and thereby inactivates it. Ras-se-
lective lethal small molecule (RSL)-3 and RSL5, which selectively killed
BJeLR cells in a non-apoptotic manner, were later identified in 2008 in
a high-throughput small molecule-screening study [4]. Erastin and
processes consistent with apoptosis, like chromatin margination or poly
ADP-ribose polymerase (PARP) cleavage. Moreover, erastin- and RSL3-
induced cell deaths are not attenuated by apoptosis-, necrosis-, ne-
croptosis-, or autophagy-inhibitors. However, antioxidants (e.g., vi-
tamin E) and iron chelators (e.g., deferoxamine mesylate) block RSLs-
induced cell death. Consequently, ferroptosis generally refers to an
iron-dependent, non-apoptotic form of regulated cell death (RCD)
[5,6].
Ferroptosis plays an important role in cancer therapy. As a recently
identified condition that regulates cell death, ferroptosis is a novel
method for the destruction of cancer cells. In this review, we have
summarized the signaling pathways involved in ferroptosis, while fo-
cusing on the mechanisms of ferroptosis-associated drugs against cancer
cells to lay a foundation for cancer treatment.
2. Mechanisms of ferroptosis
Ferroptosis is a novel RCD with its unique morphological, bio-
chemical and genetic hallmarks. Glutathione peroxidase 4 (GPX4) and
system xc- are considered to be the primary signaling pathways

∗
Corresponding author. Research & Development Institute of Northwestern Polytechnical University in Shenzhen, Shenzhen, 518057, China.
E-mail address: shangpeng@nwpu.edu.cn (P. Shang).

https://doi.org/10.1016/j.canlet.2020.02.015
Received 13 December 2019; Received in revised form 12 February 2020; Accepted 12 February 2020
0304-3835/ © 2020 Elsevier B.V. All rights reserved.
Y. Su, et al.
associated with ferroptosis [1]. System xc- belongs to the family of
heterodimeric amino acid transporters. SLC7A11, an amino acid
transporter, functions to exchange L-cystine and L-glutamate [7]. Ad-
ditionally, iron metabolism and lipid peroxidation are two essential
events in ferroptosis. Erastin, a small molecule acting as a ferroptosis
inducer, inhibits the function of system xc- and depletes glutathione
(GSH), finally inactivating GPX4 [1]. Inactivation of GPX4 leads to the
accumulation of lipid peroxides, which further leads to an increase in
ROS [8]. Circulating iron, ferric ion (Fe3+), is imported into cells by the
transferrin receptor (TFR), and subsequently converted to Fe2+ in the
endosome [9]. Excessive amounts of Fe2+ leads to the accumulation of
lipid ROS through the Fenton reaction, which in turn causes ferroptosis
(Fig. 1) [1].
2.1. Inhibition of system xc- - glutathione/GPX4 axis triggers ferroptosis
2.1.1. Inhibition of system xc- triggers ferroptosis
The first mechanism under consideration for the induction of fer-
roptosis is the inhibition of system xc- (e.g., mediated by erastin), which
indirectly inhibits GPX4 [1,10]. Erastin, a classic ferroptosis agonist,
inhibits the function of system xc-, thereby causing GSH depletion and
GPX4 inactivation, and subsequently triggering ferroptosis (Fig. 1)
[1,6,8].
System xc-, a cystine/glutamate antiporter on the plasma mem-
brane, imports extracellular cystine in exchange for intracellular glu-
tamate. It plays a key role in maintaining the intracellular GSH levels
and extracellular cystine/cysteine redox balance [7]. Furthermore, the
system xc- is a hetero-dimeric cell surface amino acid antiporter, which
consists of the twelve-pass transmembrane transporter protein
SLC7A11, coupled to the single-pass transmembrane regulatory protein
SLC3A2, by a disulfide bond [11]. Cysteine is a precursor of GSH
synthesis, and cystine can be reduced by it. It is produced during me-
thionine metabolism in tissues (such as liver), by trans-sulfuration
pathways including the cleavage of cystathionine by gamma-cy-
stathionase to alpha-ketobutyrate and cysteine [12]. In contrast, certain
types of cancer cells, including leukemia and lymphomas, are unable to
generate cysteine. Likewise, to maintain growth, amino acids need to be
taken from their micro-environment [13]. When the cysteine con-
centrations are relatively low in in vivo circulation, intracellular
128
Cancer Letters 483 (2020) 127–136
Fig. 1. Mechanisms of ferroptosis. The inhibition of
system xc- leads to GSH depletion and subsequent
inactivation of GPX4, finally leading to the accu-
mulation of lethal lipid peroxides and initiation of
ferroptosis in the presence of iron. Excessive
amounts of Fe2+ leads to the accumulation of lipid
ROS through the Fenton reaction, which in turn
causes ferroptosis. (GSH, glutathione; GS-SG, oxi-
dized glutathione; GPX4, glutathione peroxidase 4;
ROS, reactive oxygen species; PUFA, poly-
unsaturated fatty acids; NCOA4, nuclear receptor
co-activator 4; IREB2/IRP2, iron responsive element
binding protein 2; TFR, transferrin receptor; FPN,
ferroportin; DMT1, divalent metal transporter 1;
STEAP3, six-transmembrane epithelial antigen of
prostate 3)
cysteine is supplemented by system xc--mediated cysteine secreted from
the nearby somatic cells (e.g., fibroblasts, activated macrophages, or
dendritic cells) in their micro-environment [14]. Cancer cells expres-
sing the system xc- transporter directly take up cystine from their micro-
environment. Therefore, system xc- transporter is a potential target for
the treatment of cancers in which the growth and survival of cancer
cells are heavily dependent on the uptake of amino acids.
Additionally, GPX4 uses GSH as a basic co-factor to catalyze the
reduction of hydrogen peroxide and organic peroxides, especially lipid
peroxides, to water and the corresponding alcohols. Therefore, the in-
hibition of system xc- leads to the depletion of GSH, and subsequent
inactivation of GPX4, finally leading to the accumulation of lethal lipid
peroxides and initiation of ferroptosis, in the presence of iron.
Inhibition of system xc--mediated GSH synthesis is a major mechanism
which induces the accumulation of lipid peroxides, and the occurrence
of ferroptosis.
2.1.2. Roles of GPX4 in ferroptosis
Another molecular mechanism of ferroptosis is the direct inhibition
of GPX4 through the loss of its activity, or by promoting its degradation.
GPX4 is a unique member of the selenium-dependent glutathione per-
oxidase family in mammals, with a pivotal role in the inhibition of lipid
peroxides during ferroptotic cell death [8]. GPX4 functions to remove
lipid peroxides from phospholipid membranes [15]. It reduces toxic
lipid peroxides to non-toxic lipid alcohols in the presence of GSH [8].
The classical ferroptosis inducer RSL3 covalently targets the seleno-
cysteine in its active site, in an irreversible manner, thereby inhibiting
GPX4 enzyme activity [8,16]. Inactivation or loss of GPX4 leads to the
accumulation of lipid peroxides, which is considered as a lethal signal
for ferroptosis (Fig. 1) [16,17]. GPX4 has been identified as a negative
regulator of ferroptosis since it limits the production of lipid ROS [8].
However, the GPX4-mediated regulatory mechanism of ferroptosis is
still largely unknown.
2.1.3. Roles of lipid peroxidation in ferroptosis
Lipid peroxidation is a key event in ferroptosis. The production of
ROS caused by excessive iron in turn causes an increase in lipid per-
oxides. The production of lipid peroxides in cell membranes is also a
source of lipid ROS, which in turn leads to ferroptosis [18]. The
Y. Su, et al.
overwhelming accumulation of lipid ROS leads to the execution of
ferroptosis, which can be prevented by lipophilic antioxidants. Nicoti-
namide adenine dinucleotide phosphate (NAPDH) oxidases are re-
sponsible for the accumulation of ROS in erastin-induced ferroptosis
[1]. Under normal conditions, fatty acid hydroperoxides are converted
to fatty acid alcohols by GPX4. However, the occurrence of ferroptosis
inhibits the activity of GPX4. Accumulated fatty acid peroxides are
further catalyzed into lipid peroxide free radicals in the lipid-mediated
Fenton reaction. The most susceptible lipids are the polyunsaturated
fatty acid-containing phospholipids (PUFA-PLs) which result in sub-
sequent cell death [19]. These lipid free radicals extract protons from
adjacent PUFAs, triggering a new round of lipid oxidation, further
spreading oxidative damage from one lipid to another, and accelerating
the production of lipid ROS. PUFA oxidation and free radical-mediated
damage ultimately results in the fragmentation of PUFA into a variety
of products [20]. PUFA peroxides, as well as their final product re-
actants, such as malondialdehydes and 4-hydroxynonenal, are dama-
ging to cells. Lipid peroxidation is the oxidative degradation of lipids,
and it plays an important role in inducing ferroptosis by increasing li-
potoxicity [21].
2.2. Activation of iron axis triggers ferroptosis
2.2.1. The roles of iron in ferroptosis
Iron is an essential nutrient implicated in the synthesis of iron-sulfur
cluster (Fe–S), heme, and other cofactors, in all eukaryotes and most
prokaryotes. Ferroptosis is an iron-dependent, non-apoptotic form of
regulated cell death, which requires abundant available-cellular iron
[9,22]. It has been verified that iron overload contributes to ferroptosis
in cancer cells. Iron metabolism-related proteins like transferrin (TF),
transferrin receptor 1 (TFR1), ferroportin (FPN), divalent metal trans-
porter 1 (DMT1), ferritin heavy chain 1 (FTH1), and ferritin light chain
(FTL), are vital mediators of ferroptosis [9]. TFR1 mediates a great
majority of the cellular iron uptake, by binding to iron-transferrin at the
cell surface, which is internalized by endocytosis through receptor-
binding. Iron, stored in the form of ferritin, is used up during low iron
levels. Degradation of ferritin in the lysosomes causes an increase in
iron levels, which further leads to the accumulation of intracellular
ROS, and ultimately cell death [23]. Ferritin is the major intracellular
iron storage protein complex, and includes FTL1 and FTH1. Further-
more, FPN is the only iron efflux transporter in the plasma membrane,
which releases iron from cells. Summarizing, iron levels are actively
regulated in cells by TFR, which transports iron into cells, and FPN,
which exports iron from the cell to the outside (Fig. 1).
Ferroptosis, induced by erastin or cysteine deletion, can be pre-
vented by TFRC gene silencing, and by the iron chelator, deferoxamine
(DFO) [4,24,25]. Conversely, supplementation with iron-bound trans-
ferrin or a bioavailable form of iron (e.g., ferric ammonium citrate),
rather than other divalent metals, accelerates erastin-induced ferrop-
tosis [1,24]. Excess iron catalyzes the Fenton reaction, producing highly
reactive hydroxyl radicals, which enhance lipid ROS production, and
lead to ferroptosis [26].
Furthermore, iron regulatory proteins (IRPs), including IRP1 and
IRP2, bind to the mRNA of iron response elements (IRE) and regulate
the expression of DMT1, TFR1, ferritin and FPN1 [27]. IRP1 is regu-
lated by an abnormal iron-sulfur cluster switch [28]. Iron response
element binding protein 2 (IRE-BP2) regulates iron metabolism. It is
considered as an important gene in the induction of ferroptosis [29].
IRP2, an RNA-binding protein which controls the translation of a set of
mRNAs involved in iron homeostasis, reduces iron uptake by reducing
the expression of TFRs through the ubiquitin-proteasomal system in
iron-replete cells. It sequesters free Fe2+ by inducing ferritin expression
[30]. In iron-depleted cells, IRP binds to IREs, which inhibit the
translation of RNA elements in the mRNA of ferritin, TFR, and many
other transcripts, or prolong the half-life of mRNA. Consequently, iron-
starved cells increase the transferrin-bound iron absorption ability of
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Cancer Letters 483 (2020) 127–136
TFR, and reduce the chelation of ferritin during storage [31].
2.2.2. Ferritinophagy
Essentially, ferroptosis is an autophagic cell death process [32]. It
was previously suggested to be an autophagy-independent process [1].
However, by using various concentrations of erastin and measuring
ferroptosis at different time periods in two different cell types (MEF and
HT1080 cells), Gao et al. [32] that the effect of autophagy-inhibition on
ferroptosis was more obvious in the early stages of ferroptosis when the
induction was weak, and tended to be stable when induction was
strong. Consequently, ferroptosis was previously considered to be in-
dependent of autophagy.
Intriguingly, autophagy plays an important role in ferroptosis,
through the regulation of cellular iron homeostasis and cellular ROS
[32]. Increased autophagy degrades ferritin, which leads to increased
iron levels, resulting in oxidative injury by Fenton reaction, an essential
factor for ferroptosis [33,34]. This process named ferritinophagy has
also been demonstrated to serve as a bridge between ferroptosis and
autophagy [33]. Autophagy exerts positive effects on the regulation of
ferroptosis. Remarkably, nuclear receptor coactivator 4 (NCOA4) is a
selective cargo receptor for the selective autophagic turnover of ferritin.
Using NCOA4 as the cargo receptor, ferritinophagy recognizes ferritin
and delivers it for lysosomal degradation, resulting in the release of free
iron, thereby increasing the iron levels (Fig. 1) [35].
3. Ferroptosis and cancer
Cell death is essential for normal development, homeostasis, and
prevention of hyper-proliferative diseases like cancer. Despite success
in clinical cancer treatments, resistance to existing chemotherapeutic
drugs owing to genetic changes remains a problem [36]. Ferroptosis is
associated with a variety of physiological and pathological processes
and diseases, especially the treatment of multiple types of cancers.
Numerous studies have confirmed that ferroptosis plays a key role in
killing tumor cells and inhibiting tumor growth. Ferroptosis was iden-
tified as the cause for the death of several types of tumorigenic cells like
non-small cell lung cancer [37], breast cancer [38], leukemia [39],
pancreatic cancer [40], and hepatocellular carcinoma Table 1 [41].
Consequently, ferroptosis induction may be a novel therapeutic strategy
for cancer treatment.
Fortunately, some clinical drugs approved by the U.S. Food and
Drug Administration (FDA) (e.g., sorafenib, sulfasalazine and artesu-
nate) can induce ferroptosis in several cancer types, thereby making the
application of ferroptosis in pre-clinical and clinical settings feasible
Table 1. Moreover, ferroptosis inducers (like erastin, piperazine erastin,
and RSL3) were found to prevent tumor growth in the xenograft model
of HT-1080 cells in vivo [8]. Collectively, in the clinical setting, there is
an urgent need for the discovery of ferroptosis-inducing drugs, for use
in the treatment of tumors, especially drug-resistant tumors.
4. Ferroptosis-associated anti-cancer drugs
4.1. Sulfasalazine
Sulfasalazine, an azo bridge-linked anti-inflammatory agent, was
originally synthesized from an antibiotic, sulfapyridine, in 1940. It is
commonly used to treat chronic inflammatory diseases like in-
flammatory bowel (Crohn's) disease and rheumatoid arthritis [42,43].
In Crohn's disease, 5-aminosalicylic acid and sulfasalazine appear to
have equivalent activity, each superior to sulfapyridine, while sulfa-
pyridine is the active component in rheumatoid arthritis treatment
[43].
In 2001, a study showed that sulfasalazine was a potent inhibitor of
system xc- [13]. Subsequently, Sulfasalazine was found to inhibit cy-
stine absorption, leading to the attenuation of GSH, and ultimately
resulting in the death of certain types of cancer cells, both in vitro and in
Y. Su, et al. Cancer Letters 483 (2020) 127–136

vivo [43]. Sulfasalazine induces ferroptosis by inhibiting the function of

Reference
system xc- (Fig. 2) [1]. Sulfasalazine has been reported to suppress drug

[77,81]
[10,41]
[1,13]

[37]

[38]

[39]
[96]
[49]

[41]
[52]
resistance in certain lung adenocarcinoma and breast cancer cells in
vitro [44,45]. Sulfasalazine is considered to be non-toxic, and therefore
it can be combined with traditional drugs to reduce drug resistance and

Mutationally-active KRas pancreatic ductal adenocarcinoma (PDAC) cell

side effects [46]. Okazaki and his colleagues demonstrated that the
combination of dyclonine and sulfasalazine co-operatively suppressed
the growth of head and neck squamous cell carcinoma or gastric tumors
highly expressing ALDH3A1, which were resistant to sulfasalazine
Hepatocellular Carcinoma Cells; head and neck cancer cells

monotherapy [47]. Moreover, Yamaguchi et al. [40] found that a triple

GSH, glutathione; ROS, reactive oxygen species; NRF2, The nuclear factor erythroid 2-related factor 2; IREB2/IRP2, Iron response element binding protein 2; TFRC, transferrin receptor.
combination treatment with piperlongumine, cotylenin A, and sulfa-
Lymphoma; small-cell lung cancer; Prostate Cancer

salazine was highly effective against pancreatic cancer.

Non-small cell lung cancer (NSCLC) cell lines

4.2. Lanperisone

The FDA-approved drug lanperisone (LP), a modified form of tol-

Hepatocellular Carcinoma Cells

perisone, which was originally developed as a muscle relaxant, has

been clinically tested and found to safely and effectively target Ras-
Primary hepatocytes cells
Kras-mutant tumor cells

mutant cancers [48]. The mechanism of cancer cell destruction by LP is

similar to the iron- and ROS-dependent mechanisms in K-ras mutant
Cancer/cancer cells

Breast cancer cells

Cancer stem cells

cells, which leads to oxidative stress and ultimately cell death [30,49].
Leukemia cells

Furthermore, it is possible that LP inhibits the function of system xc- or

other targets in the ferroptotic pathway (Fig. 2) [1]. In terms of the
underlying mechanism of action, LP appears to be similar to erastin. By
lines

binding to the mitochondrial voltage-dependent anion channels

(VDACs), erastin alters VDAC gating, leading to mitochondrial dys-
Inhibits the absorption of cystine by system xc-, causes GSH depletion; activates NRF2 against ferroptosis

function, production of ROS, and ultimately triggering ferroptosis [49].

Increases levels of IREB2/IRP2 and TFRC along with a rapid degradation of the iron storage protein
Decreases the expression of ferroportin and ferritin and increasing the expression of transferrin to

Whether LP interacts with VDAC in K-ras mutant cells, which leads to

mitochondrial dysfunction, remains undetermined. In in vivo studies, LP
was showed the ability to inhibit tumor growth in a KRAS-mutant
mouse model [49]. There are few studies on the ferroptosis of cancer
cells caused by LP. However, the specific mechanism needs more re-
Conjugates with GSH to form the Pt-GS complex causing extensive GSH depletion

search.
Promotes ferritin degradation and releases lysosomal iron, reacts with ROS

4.3. Acetaminophen
Inhibits the absorption of cystine by system xc-, causes GSH depletion
Inhibits the absorption of cystine by system xc-, causes GSH depletion

Acetaminophen (APAP) is widely used in the treatment of pain and

Two forms excess of iron produce harmful ROS and cell death

fever in humans. The highly reactive metabolite of acetaminophen is

Reacts rapidly with GSH causing extensive GSH depletion

the N-acetyl-p-benzoquinone imine (NAPQI). An early study had shown

that the early hypothermia caused by acetaminophen in mice was due
to the parent drug, and not its toxic reactive metabolite [50]. Owing to
the complexity of human thermoregulatory systems, the exact me-
chanism of hypothermia caused by acetaminophen is unclear [51].
Lorincz et al. [52] showed that, in addition to necroptosis and
apoptosis, ferroptosis was also involved in acetaminophen-induced cell
Possible mechanisms of ferroptosis

death in primary hepatocytes. However, this phenomenon was not

found in the hepatoma cell line, HepG2. It is well known that HepG2
cells practically do not express phase I enzymes, and therefore cannot
form the reactive metabolite NAPQI [53]. The highly reactive meta-
bolite NAPQI reacts rapidly with GSH, causing extensive GSH deple-
increases iron

tion, and increasing liver damage (Fig. 2) [54]. Thus, the cell death
Blocks NRF2

induced by APAP can be characterized by GSH depletion and GPX in-

ferritin

hibition. The process is independent of caspase, but involves ferroptosis

[55]. Excessive use of APAP can cause liver failure in patients, which is
aggravated by liver ferroptosis [56]. However, this hypothesis is yet to
System xc-,
System xc-
System xc-

be confirmed by in vivo and in vitro studies.

Targets
Drugs associated with ferroptosis.

NRF2
NRF2
GSH
GSH
Iron

Iron

Iron
Iron

4.4. Cisplatin
Salinomycin (ironomycin)
Siramesine and lapatinib
Fenugreek (trigonelline)

Cisplatin is an extremely effective and widely used anti-cancer drug

for the treatment of solid tumors [57]. Previously, the anti-cancer
mechanism of cisplatin was known to be primarily mediated by the
Acetaminophen
Sulfasalazine

Ferumoxytol

production of nuclear DNA adducts, leading to apoptosis [58]. Un-

Lanperisone

Artesunate
Sorafenib

Cisplatin

fortunately, many cancers often develop resistance in response to cis-

Table 1

Drugs

platin treatment [59]. Recent research has indicated that cisplatin is an

inducer of both ferroptosis and apoptosis in the non-small cell lung

130
Y. Su, et al.
cancer (NSCLC) cell lines A549 and HCT116 cells [37].
Platinum compounds, including cisplatin, have a higher affinity for
thiol-rich biomolecules. GSH is one of the most abundant non-protein
thiols in cells. In the cytoplasm, the major part of intracellular cisplatin
is found conjugated to GSH, to form the Pt-GS complex (Fig. 2) [60].
Similar to erastin, GSH depletion along with the inactivation of GPXs
was the underlying mechanism of action of cisplatin. However, the
inhibitory effect of cisplatin on GPXs was weaker than that of erastin on
GPXs. Additionally, Guo et al. [37] showed that the antitumor activity
of cisplatin combined with erastin was superior to that of cisplatin
alone. Considering the different mechanisms of these two inducers,
combining erastin with cisplatin may be a better strategy to improve
the efficacy of cisplatin treatment [37,61]. Drug resistance was found to
affect cisplatin-induced apoptosis, but not cisplatin-induced ferroptosis.
Therefore, ferroptosis provides a novel approach for the clinical ap-
plication of cisplatin, and a bright future for tumor resistance to cis-
platin.
4.5. Sorafenib
Sorafenib, an inhibitor of oncogenic kinases, is an FDA-approved
drug for the treatment of patients with advanced renal cell carcinoma
(RCC), advanced hepatocellular carcinoma (HCC), and other solid tu-
mors [62]. The antitumor efficiency of sorafenib correlates with the
inhibition of molecular components in the Raf-MEK-ERK signaling
pathway, and several receptor tyrosine kinases including the vascular
endothelial growth factor receptor (VEGFR), thereby inhibiting
neoangiogenesis [63].
Recently, Louandre et al. [64] found that sorafenib had the ability
induce ferroptosis. However, the induction of ferroptosis was unrelated
to the RAF kinase inhibitory effect of sorafenib [65]. There are two
main mechanisms by which sorafenib effects ferroptosis (Fig. 3). Like
erastin, sorafenib inhibits system xc--mediated cystine import, leading
to endoplasmic reticulum stress (ER stress), GSH depletion and the iron-
dependent accumulation of lipid ROS [10]. Another mechanism is as-
sociated with the p62-Keap1-NRF2 pathway (Fig. 3) [41]. The nuclear
factor erythroid 2-related factor 2 (NRF2) is a key factor in determining
the therapeutic response of ferroptosis-targeted therapies in HCC cells.
Under the treatment of sorafenib, the expression of the substrate
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Cancer Letters 483 (2020) 127–136
Fig. 2. Inhibition of system xc--glutathione/GPX4
axis triggers ferroptosis. Sulfasalazine inhibits the
absorption of cystine by system xc-. Lanperisone
induces a non-apoptotic, iron-dependent death in
mouse KRAS-mutant tumor cells that may inhibit
the function of system xc- or other targets in the
ferroptotic pathway. The highly reactive metabolite
NAPQI of acetaminophen rapidly reacts with GSH
causing extensive GSH depletion. Cisplatin is con-
jugated with GSH to form the Pt-GS complex.
(NAPQI, N-acetyl-p-benzoquinone imine; GSH, glu-
tathione; GS-SG, oxidized glutathione; GPX4, glu-
tathione peroxidase 4; ROS, reactive oxygen species;
PUFA, polyunsaturated fatty acids)
adaptor p62 protein prevents NRF2 degradation, and enhances sub-
sequent NRF2 nuclear accumulation by the inactivation of Kelch-like
ECH-associated protein 1 (Keap1) [41]. Additionally, NRF2 protein
activates MafG, and induces multiple genes including quinone oxidor-
eductase 1 (NQO1), heme oxygenase-1 (HO1), and ferritin heavy chain
1 (FTH1) [66]. Metallothionein-1G (MT-1G) is a new regulator of fer-
roptosis in HCC cells [67]. Metallothioneins (MT) are a family of high-
level small stress response proteins, which are induced by oxidative
stress. They can effectively scavenge ROS [68]. On treatment with
sorafenib, under the high expression of NRF2, the expression of MT-1G
(but not other MTs) in HCC cells was significantly up-regulated. MT-1G
inhibits ferroptosis by blocking GSH depletion-mediated lipid perox-
idation, a process that promotes sorafenib resistance in HCC cells [67].
In summary, NRF2 is a master transcription factor which prevents
sorafenib-induced ferroptosis by regulating in redox and iron metabo-
lism [67].
Ferroptosis is a potential target in the treatment of HCC, thereby
opening new avenues for the optimum usage of sorafenib in tumors
[64]. Sorafenib is the first clinically approved anticancer drug, which
induces ferroptosis [65]. The combination of erastin and sorafenib may
prove to be a promising therapeutic strategy for cancer treatment,
especially in cases of sorafenib resistance [36]. Moreover, NRF2 and
MT-1G inhibit sorafenib-induced ferroptosis, and the inhibition of these
regulators can increase the resistance to sorafenib, thereby providing a
promising strategy for HCC treatment. Conversely, haloperidol pro-
motes sorafenib-induced ferroptosis, thereby presenting a novel
strategy for the treatment of HCC with sorafenib [69].
4.6. Fenugreek (trigonelline)
Trigonelline, the main pharmacological ingredient of a traditional
Chinese herbal medicine Trigonella foenum-graecum L. (fenugreek), is an
alkaloid present in considerable amounts in coffee and fenugreek seed.
Fenugreek plays an important role in the treatment of diabetes, com-
plications of diabetes, and central nervous system disorders.
Furthermore, trigonelline has been shown to reduce diabetic auditory
neuropathy and platelet aggregation, by affecting β cell regeneration,
insulin secretion, activities of enzymes associated with glucose meta-
bolism, ROS, axonal extension, and neuron excitability [70]. Recently,
Y. Su, et al.
trigonelline has been reported to act against NRF2 (Fig. 3) [71,72].
Inhibition of NRF2 by the alkaloid trigonelline was found to sig-
nificantly enhance the anticancer activity of erastin and sorafenib in
HCC cells and tumor xenograft models [41]. On inhibition of NRF2 by
trigonelline, the expression of MT-1G was blocked, which reduced the
content of GSH and further caused ferroptosis [41].
4.7. Artesunate
Artesunate (ART), a water soluble derivative of artemisinin, is a
first-line drug in the treatment of malaria [73]. ARTs are sesquiterpene
lactones with a 1, 2, 4-trioxane core incorporating an endoperoxide
linkage [74]. The mechanism of action of ART against malaria is still
controversial. A widespread assumption is that ART derivatives are pro-
drugs which are activated by the cleavage of the endoperoxide bridge,
in the presence of iron or heme, through Fenton reaction. This reaction
produces ROS and carbon-centered free radicals, which impose direct
toxicity on the intra-erythrocytic parasites [74,75].
Recent studies have revealed that ART selectively induces ferrop-
tosis in mutationally-active KRAS pancreatic ductal adenocarcinoma
(PDAC) cell lines [76]. This process is blocked by the iron chelator DFO,
and enhanced by exogenous lysosomal iron. In addition to the iron
chelator DFO, ROS inhibitors (e.g., trolox and ferrostatin-1) except
necrostatin-1, were found to significantly inhibit ART-induced ferrop-
tosis in PDAC cells. Moreover, ART is preferably accumulated in the
lysosomes, and activates lysosomal function via the promotion of ly-
sosomal V-ATPase assembly. Furthermore, lysosomes function up-
stream of the mitochondria in the production of ROS. ART treatment
activates lysosomal function, and promotes ferritin degradation [77].
Lysosomal iron has been shown to react with ROS, leading to the for-
mation of free radicals via Fenton reaction, which results in lysosomal
bursting and cell death [78]. It has been reported that artesunate se-
lectively killed head and neck cancer (HNC) cells by inducing the iron-
dependent, ROS-accumulated ferroptosis. Artesunate induces ferrop-
tosis via the accumulation of iron, leading to elevated lipid peroxides
and reduced antioxidant capacity [79,80]. Surprisingly, artesunate also
triggers ferritinophagy accompanied by the up-regulation of NCOA4
(Fig. 4) [80].
Additionally, a study showed that 10 artemisinin derivatives,
132
Cancer Letters 483 (2020) 127–136
Fig. 3. Inhibition of NRF2 pathway triggers ferrop-
tosis. Treatment with sorafenib effects ferroptosis by
two main mechanisms. First, sorafenib inhibits the
function of system xc- to induce ferroptosis. Second,
the p62-Keap1-NRF2 pathway inhibits sorafenib-
induced ferroptosis in HCC. Fenugreek (trigonel-
line) induces ferroptosis by inhibiting NRF2. (NRF2,
nuclear factor erythroid 2-related factor; Keap1,
Kelch-like ECH-associated protein 1; MT-1G,
Metallothionein-1G; NQO1, quinone oxidoreductase
1; HO1, heme oxygenase-1; FTH1, ferritin heavy
chain 1; GSH, glutathione; GPX4, glutathione per-
oxidase 4; ROS, reactive oxygen species) (The solid
line represents what has been reported, while the
dotted line is a prediction.)
including ART, altered the mRNA levels of numerous iron-related
genes, which contribute to cell death in cancer cells [81]. This process
may also be involved in ferroptosis, and requires more research.
4.8. Siramesine and lapatinib
Siramesine, a sigma-2 receptor ligand, is a lysosomotropic agent
that was originally used to treat depression [82]. Siramesine-treated
tumor cells displayed lysosomal membrane permeabilization, which
lead to the release of cathepsin B and an increase in ROS, resulting in
cell death [83]. Siramesine has earlier been shown to be well tolerated
by humans. Lapatinib is a potent double tyrosine kinase inhibitor of the
epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2 [84]. Both
in vitro and in vivo studies demonstrated the ability of lapatinib to in-
hibit the proliferation of ErbB2 and EGFR-overexpressing cancer cells
[85]. Although lapatinib presents a new treatment choice for ErbB2-
positive cancer, lapatinib monotherapy frequently demonstrated only
modest activity in intermediate ErbB2-positive breast cancer cells [86].
A recent study showed that the combination of siramesine and la-
patinib acted synergistically and induced ferroptosis-mediated cell
death in breast cancer cells [26,38]. Ma et al. [38] found that the
combination of siramesine and lapatinib induced ferroptosis in MDA
MB 231 and SKBR3 cells by lowering the expression of ferroportin and
ferritin, and increasing the expression of transferrin, thereby increasing
the free iron content. Subsequently, the excess iron catalyzes the Fenton
reaction, which produces extremely active hydroxyl radicals, and raises
ROS production, leading to ferroptosis (Fig. 4). However, this process is
associated with autophagy, and further research is required to de-
termine if ferritinophagy is involved [1,38]. The ferroptosis triggered
by the combination of lapatinib and siramesine acts independent of the
downstream targets (EGFR family members) and cathepsin B [38]. This
indicates the involvement of other targets of siramesine and lapatinib in
ferroptosis. Therefore, more in-depth research is required to explore the
targets of siramesine and lapatinib associated with ferroptosis. These
results provide us with a novel way to induce ferroptosis - by altering
the transport of iron-utilizing clinical drugs – aiding in the development
of new therapeutic strategies to overcome the apoptosis resistance of
breast cancer.
Y. Su, et al.
4.9. Ferumoxytol
Ferumoxytol (Feraheme), an FDA-approved super-paramagnetic
iron oxide nanoparticle with a carbohydrate shell composed of poly-
glucose sorbitol carboxymethyl ether (PSC), is clinically used in the
treatment of iron deficiency associated anemia in patients in the USA
[87]. Ferumoxytol is produced by AMAG pharmaceuticals, Inc. (Cam-
bridge, MA), with a colloidal particle size of 30 nm and a molecular
weight of 750 kDa [88]. Ferumoxytol reduces potential toxicity by se-
parating bioactive iron cores from plasma components, until the iron-
PSC complex enters the reticuloendothelial system macrophages [89].
Ferumoxytol is a sterile liquid with a neutral pH which is isotonic with
mannitol. Preliminary data indicates that it contains less free iron
compared to other intravenous iron preparations [88]. Therefore, fer-
umoxytol can be administered quickly in relatively high doses [90].
Ferumoxytol has been widely considered as safe when given as a single
infusion.
Recently, Trujillo-Alonso et al. [91] found that ferumoxytol showed
anti-leukemic effect in vitro and in vivo. Low expression of ferroportin
results in the susceptibility of leukemia cells to ferumoxytol through an
increase in intracellular iron. Ferumoxytol's iron core consists of 5874
iron atoms, and is a mixture of the ferrous and ferric forms of iron.
During Fenton reaction and in the presence of peroxides, the two forms
of iron of the ferumoxytol overproduce harmful ROS which causes cell
death (Fig. 4) [92]. Ferumoxytol treatment significantly reduces the
disease burden in the mouse leukemia model, as well as patient-derived
xenografts of leukemia cells with low ferritin expression [39]. The
mechanism of action of ferumoxytol in destroying cancer cells may be
associated with ferroptosis.
4.10. Salinomycin (ironomycin)
Salinomycin is a monocarboxylic polyether antibiotic isolated from
the bacterial strain Streptomyces albus. It is a broad-spectrum anti-
bacterial agent against gram-positive bacteria, fungi and parasites
[93,94]. Salinomycin is a selective agent for cancer stem cells (CSCs)
through an elusive mechanism. Hamai and colleagues developed a
synthetic derivative of salinomycin – ironomycin - which is at least 10-
fold more potent than salinomycin against CSCs both in vitro and in vivo
133
Cancer Letters 483 (2020) 127–136
Fig. 4. Activation of iron axis triggers ferroptosis.
Artesunate treatment activates lysosomal function
and promotes ferritin degradation. Artesunate also
triggers ferritinophagy by up-regulating NCOA4.
The combination of siramesine and lapatinib in-
duces ferroptosis by decreasing the expression of
ferroportin and ferritin, and increasing the expres-
sion of transferrin, to increase the free iron level in
MDA MB 231 and SKBR3 cells. Ferumoxytol directly
increases intracellular iron in leukemia cells.
Salinomycin (Ironomycin) affects the rise in IREB2/
IRP2 and TFRC/CD71 levels as well as the rapid
degradation of the iron storage protein, ferritin.
More free iron is released, further leading to in-
tracellular ROS accumulation, which ultimately
leads to ferroptosis.(NCOA4, nuclear receptor co-
activator 4; IREB2/IRP2, iron responsive element
binding protein 2; TFRC/CD71, transferrin receptor;
TFR, transferrin receptor; FPN, ferroportin; DMT1,
divalent metal transporter 1; STEAP3, six-trans-
membrane epithelial antigen of prostate 3; ROS,
reactive oxygen species)
[95]. Salinomycin and ironomycin prevented the movement of iron
from the lumen to the cytosol, thereby triggering an iron depletion
reaction. This response was characterized by increased levels of iron
responsive element binding protein 2 (IREB2/IRP2) and transferrin
receptor (TFRC/CD71), and rapid degradation of the iron storage pro-
tein, ferritin (Fig. 4) [95,96]. Ironomycin was physically accumulated
in lysosomes, which induced iron loading in the lysosomes, and the
production of ROS, by Fenton reaction. These effects of ironomycin -
the induction of excess iron and ROS - imply the activation of ferrop-
tosis [97].
5. Conclusions and perspectives
In summary, the aforementioned drugs induce ferroptosis by in-
hibiting the system xc- -glutathione/GPX4 axis or by regulating iron
homeostasis. However, the exact mechanism of ferroptosis induction by
these drugs needs further clinical validation. This review provides a
comprehensive summary of drugs that can induce ferroptosis, laying
the foundation for future clinical treatment of cancer Table 1.
Ferroptosis was previously thought to be a non-apoptotic, non-au-
tophagy, non-necrotic RCD. However, there is growing evidence that
ferroptosis is associated with autophagy [25]. Beclin 1 (BECN1) is a key
player of macroautophagy/autophagy. Song et al. [98] found that
BECN1 was a novel driver of ferroptosis in 2018. It promoted ferrop-
tosis by directly blocking system xc- activity by binding to SLC7A11
[99]. The specific link between ferroptosis and autophagy requires
more in-depth research. Further studies are required to decipher if
BECN1 could link the two pathways of cell death. Therefore, further
research is required to identify the differences and associations between
ferroptosis and other regulated cell death pathways (Fig. 5).
There are many kinds of ferroptosis-inducers, which act by the in-
hibition of system xc-, or by inhibiting GSH, or by directly inhibiting
GPX4, or by altering intracellular iron levels. Generally, ferroptosis is
achieved in two ways, either by affecting lipid oxidation or affecting
iron metabolism. Depending on the type of cancer, physiological con-
dition, and even individual lifestyle, different cancer cells respond
differently to ferroptosis inducers and have different sensitivities to
ferroptosis. Researchers need to explore more drugs targeting these two
important events in ferroptosis to fight cancer (Fig. 5). Simultaneously,
Y. Su, et al.
Fig. 5. Perspective of ferroptosis. Many unsolved mysteries like the relationship
between ferroptosis and other RCD, and the relationship between cell hetero-
geneity and ferroptosis, are yet to be solved. There is an urgent need to discover
new ferroptosis inducers and new effective combination regimens. Currently,
no accurate method or indicator exists to determine the occurrence of ferrop-
tosis in vivo. (RCD, regulated cell death)
there is an urgent need to discover new ferroptosis inducers or effective
combination regimens, as well as more effective and safe chemotherapy
to better utilize ferroptosis to treat cancer (Fig. 5).
Ferroptosis is also involved in cancer development and therapeutic
response and contributes to tumor suppression activities. Although
many in vitro experiments have shown that ferroptosis can be detected
by several measurements like cell viability, iron levels, and ROS levels,
there are no accurate methods or indicators to identify the presence of
ferroptosis in vivo (Fig. 5). Further research is required to understand
the mechanisms of ferroptosis. A deeper understanding of ferroptosis is
certainly beneficial to the treatment of related diseases.
Funding
This work is supported by grants from the National Natural Science
Foundation of China (51777171, 81803032), the Northwestern
Polytechnical University Foundation for Fundamental Research
(3102018JGC012), Natural Science Foundation of Shaanxi Province
(NO. 2019JQ632), and Science and Technology Planning Project of
Shenzhen of China (JCYJ20170412140904406).
Declaration of competing interest
The authors declare no conflict of interest.
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