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Contents
DUSP3/VHR: A Druggable Dual Phosphatase for Human Diseases . . . 1

Lucas Falc~ao Monteiro, Pault Yeison Minaya Ferruzo, Lilian Cristina Russo,
Jessica Oliveira Farias, and Fábio Luı́s Forti
Oncotic Cell Death in Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Kep Yong Loh, Ziting Wang, and Ping Liao
Magnesium Extravaganza: A Critical Compendium of Current
Research into Cellular Mg2+ Transporters Other than TRPM6/7 . . . . 65
Martin Kolisek, Gerhard Sponder, Ivana Pilchova, Michal Cibulka,
Zuzana Tatarkova, Tanja Werner, and Peter Racay
Curcumin in Advancing Treatment for Gynecological Cancers
with Developed Drug- and Radiotherapy-Associated Resistance . . . . . 107
Amir Abbas Momtazi-Borojeni, Jafar Mosafer, Banafsheh Nikfar,
Mahnaz Ekhlasi-Hundrieser, Shahla Chaichian, Abolfazl Mehdizadehkashi,
and Atefeh Vaezi
Correction to: Curcumin in Advancing Treatment for Gynecological
Cancers with Developed Drug- and Radiotherapy-Associated
Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
and Atefeh Vaezi
v
Rev Physiol Biochem Pharmacol (2019) 176: 1–36
DOI: 10.1007/112_2018_12
Published online: 2 August 2018
DUSP3/VHR: A Druggable Dual

Phosphatase for Human Diseases
Lucas Falcão Monteiro, Pault Yeison Minaya Ferruzo,

Lilian Cristina Russo, Jessica Oliveira Farias, and Fábio Luís Forti
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Protein Tyrosine Phosphatase (PTP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Dual-Specificity Phosphatase (DUSP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 ADUSP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 DUSP3/VHR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3 Molecular and Biological Functions of DUSP3/VHR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1 DUSP3 in Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2 DUSP3 in Genomic Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3 DUSP3 in Blood-Associated Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4 Overview of Current Knowledge on DUSP3/VHR Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Abstract Protein tyrosine kinases (PTK), discovered in the 1970s, have been
considered master regulators of biological processes with high clinical significance
as targets for human diseases. Their actions are countered by protein tyrosine
phosphatases (PTP), enzymes yet underrepresented as drug targets because of the
high homology of their catalytic domains and high charge of their catalytic pocket.
This scenario is still worse for some PTP subclasses, for example, for the atypical
dual-specificity phosphatases (ADUSPs), whose biological functions are not even
completely known. In this sense, the present work focuses on the dual-specificity
phosphatase 3 (DUSP3), also known as VH1-related phosphatase (VHR), an uncom-
mon regulator of mitogen-activated protein kinase (MAPK) phosphorylation.
DUSP3 expression and activities are suggestive of a tumor suppressor or tumor-
promoting enzyme in different types of human cancers. Furthermore, DUSP3 has
other biological functions involving immune response mediation, thrombosis,
hemostasis, angiogenesis, and genomic stability that occur through either MAPK-
L. F. Monteiro, P. Y. M. Ferruzo, L. C. Russo, J. O. Farias, and F. L. Forti (*)

Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP,
Brazil
e-mail: flforti@iq.usp.br
2 L. F. Monteiro et al.
dependent or MAPK-independent mechanisms. This broad spectrum of actions is

likely due to the large substrate diversity and molecular mechanisms that are still
under scrutiny. The growing advances in characterizing new DUSP3 substrates will
allow the development of pharmacological inhibitors relevant for possible future
clinical trials. This review covers all aspects of DUSP3, since its gene cloning and
crystallographic structure resolution, in addition to its classical and novel substrates
and the biological processes involved, followed by an update of what is currently
known about the DUSP3/VHR-inhibiting compounds that might be considered
potential drugs to treat human diseases.
Keywords Dual-specificity phosphatase 3 (DUSP3) · Mitogen-activated protein

kinases (MAPK) · Pharmacological DUSP3 inhibitors · Phosphatases on human
diseases · Protein tyrosine phosphatases (PTP) · Vaccinia H1-related phosphatase
(VHR)
1 Introduction
In recent years there has been substantial progress in the development of protein
tyrosine phosphatase (PTP) inhibitors suggesting that these enzymes, for long time
considered undruggable, can provide unique solutions for the treatment of human
diseases (Hendriks et al. 2013; Tonks 2013). Many recent strategies have been used
in the development of drugs that target PTPs as ways of expanding the possibilities
of intervention in the function of these enzymes and in biological processes depen-
dent on them. These strategies include (a) orthosteric inhibitors (reversible compet-
itive, bidentate or uncompetitive, and irreversible), (b) allosteric inhibitors,
(c) oligomerization inhibitors, (d) radioimmunotherapy, and (e) PTP receptor
biological decoy (He et al. 2013; Stanford and Bottini 2017).
The continuous generation of more selective probes of high quality for the
activity of individual PTPs is essential for the successful development of inhibitory
drugs of these enzymes. New chemical inhibitors are enhancing performance in
PTPs already considered clinical targets such as PTP1B and SHP-2 and are bringing
into focus new targets such as STEP, PTPN22, VE-PTP, CD45, CDC25A/B/C, and
LMPTP. Advances of functional studies of RPTPs are revealing new opportunities
for inhibiting PTP domains within the receptor structure using small biological
molecules that act to stabilize the oxidation of catalytic intermediates or even the
formation of receptor complexes. But, there are no magic bullets to attack the PTPs,
and a successful strategy for an enzyme from one of the classes may not be as
effective for a member of another class or even for a different member of the same
class (He et al. 2013; Stanford and Bottini 2017).
This is the case, for example, of the dual-specificity phosphatases (DUSPs),
which have a diversity of structural possibility of substrates, many of which have
not yet been identified both structurally and functionally (Tonks 2013). Among the
members of this subclass I of PTPs (Fig. 1), DUSP6 and PRL-1/2/3 are being
considered druggable targets for some deficiencies of the immune system, including
DUSP3/VHR Is a Potential Drug Target 3
Fig. 1 A brief classification of the protein tyrosine phosphatase (PTP) superfamily within the
human genome showing the numerical distribution in the four classes and highlighting the DUSP3
belonging to the atypical dual phosphatases
cancer, and also for melanomas (Stanford and Bottini 2017). This review has as
differential a specific focus on DUSP3, aiming to enlarge the list of druggable targets
of the atypical dual phosphatases (He et al. 2013; Hendriks et al. 2013). We first
describe all its molecular targets and biological functions described in the literature;
and, secondly, we reexamine all inhibitors already developed and discuss their
mechanism of action, specificity, permeability, bioavailability, and potential as a
drug or as a starting point for drug development.
2 Protein Tyrosine Phosphatase (PTP)
PTPs are very specific, non-redundant, and catalytically active enzymes. While
protein tyrosine kinases (PTKs) constitute a superfamily of enzymes with the same
evolutionary origin, PTPs have distinct evolutionary origins (Alonso et al. 2004c;
Manning et al. 2002; Vang et al. 2008). PTPs are classified into four subfamilies
according to amino acid sequences that share their catalytic domain and according to
the presence of cysteine (Cys or C) or aspartate (Asp or D) in the catalytic cleft that
acts as a catalytic amino acid. PTPs expression patterns vary; there are enzymes with
wide distribution and some are specific to certain tissues. Most human cells express
30–60% of all the PTPs genes; neural and hematopoietic cells commonly express
more PTPs than other tissues (Vang et al. 2008).
Three subfamilies (classes I–III) present cysteine-based catalysis, and together,
they make up almost all of the PTPs, comprising 99 proteins (Fig. 1). Class I is
subdivided into both classical tyrosine phosphatases and dual phosphatases, which
are the more diversified phosphatases that dephosphorylate not only the tyrosine
(Tyr) residue (Alonso et al. 2004c). Class II is also Tyr-specific, and its only
representative is the low molecular weight protein Tyr phosphatase (LMWPTP), a
highly conserved evolutionary enzyme that may have broad implications for human
health (Patterson et al. 2009). Class III differs from the others by the presence of a
rhodanese structure. It is composed of cell cycle regulatory proteins, cell division
cycle 25 (Cdc25) phosphatases, which activate cyclin-dependent kinases (CDKs)
when they remove phosphate pools from some Tyr and Thr residues present in their
regulatory sites (Mustelin 2007). The fourth and last subfamily (Class IV) is formed
by phosphatases that have aspartate as a critical residue in the catalytic cleft: (1) EyA
(eyes absent) proteins play an important role in the organogenesis of vertebrates, and
(2) haloacid dehalogenase (HAD) proteins are able to dephosphorylate both Tyr and
Ser/Thr residues from various substrates, including proteins, sugars, nucleotides, and
phospholipids (Fig. 1) (Bayón and Alonso 2010; Mustelin 2007; Patterson et al.
2009).
2.1 Dual-Specificity Phosphatase (DUSP)
The dual-specificity phosphatase (DUSP) group is the largest and most diversified
among the nonclassical PTPs and is composed of 61 proteins (Fig. 1). DUSPs are
able to dephosphorylate both Tyr and Ser/Thr residues due to their catalytic site’s
structure, which is not as deep as and more open than that of classical phosphatases.
The consensus sequence, HC(X)5R, present in the catalytic domain of classical
phosphatases and DUSPs, is highly conserved. At the base of the catalytic cleft is
the Cys residue, which characterizes classes I–III (Alonso et al. 2004c; Farooq and
Zhou 2004; Mandl et al. 2005). DUSP’s catalytic mechanism and that of classical
phosphatases are similar and involve substrate hydrolysis and formation of a stable
phosphoryl intermediate, with an arginine residue near the catalytic slit contributing
directly to the reaction catalysis; a slightly distant aspartate acid protonates the
phosphate group (Fig. 2) (Bayón and Alonso 2010; Denu and Dixon 1995, 1998).
The DUSP subfamily has diverse biological roles as evidenced by its subdivision
into 16 groups. It has been well established that it is involved in mitogen-activated
protein kinase (MAPK) pathway regulation, acting mainly on extracellular-regulated
Fig. 2 Details of the DUSP3/VHR crystal structure presenting the four amino acid residues more
relevant to its enzymatic activity. (a) The catalytic cysteine (Cys) 124 (yellow) is shown in close
proximity to histidine (His) 123 and arginine (Arg) 130 residues (red) comprising the catalytic triad.
(b) The regulatory tyrosine (Tyr) 138 (green) is sitting at a central alpha helix, relatively distant
from the catalytic cysteine (yellow) (modified from 1vhr.pdb) (Yuvaniyama et al. 1996)
kinase (ERK)1/2, jun kinase (JNK), and p38 (Bayón and Alonso 2010; Bermudez
et al. 2010; Nunes-Xavier et al. 2011; Patterson et al. 2009; Pulido and Hooft van
Huijsduijnen 2008). In this context, DUSP subfamily members play important roles
in several cell events: (1) in cell cycle regulation, a function usually performed by the
MAPK phosphatases (MKPs, including PAC1, MKP1–5, MKP7, hVH3, hVH5,
PYST2, and MK-STYX) and some other atypical DUSPs, including DUSP3, the
protein of interest in this work (Nunes-Xavier et al. 2011; Patterson et al. 2009;
Pulido and Hooft van Huijsduijnen 2008), (2) in several types of cancer (MKPs1–3,
MKP8, PAC1, DUSP3 and 5, PTEN, and PRLs) (Arnoldussen and Saatcioglu 2009;
Bermudez et al. 2010; Nunes-Xavier et al. 2011; Pulido and Hooft van Huijsduijnen
2008), (3) in immune responses and inflammation (MKP1, 5, and 6, PAC1, and
DUSP3) (Jeffrey et al. 2007; Lang et al. 2006; Salojin and Oravecz 2007), and
(4) hereditary diseases (MTM and Laforin) (Bayón and Alonso 2010; Patterson et al.
2009; Pulido and Hooft van Huijsduijnen 2008).
DUSP subgroups are organized according to shared sequence similarity and by
the presence of specific structures; for example, the MKPs have the CDC25 homol-
ogy 2 (CH2) at their N-terminal, while the myotubularins (MTM) present a
pleckstrin homology (PH) domain at its N-terminus, which explains the activity of
MTM on lipids (Bermudez et al. 2010; Nunes-Xavier et al. 2011; Patterson et al.
2009). The less characterized and even more diversified subgroup are the small
(generally <250 aa) atypical dual phosphatases or ADUSPs (Fig. 1).
2.2 ADUSP
There are 19 identified proteins in the atypical dual-specificity phosphatase

(ADUSP) group, which has only been discovered within the last 10 years (Fig. 1).
Most of the proteins in this group are small (<27 kDa; <250 aa), and in addition to
dephosphorylating Tyr and Ser/Thr residues, they may have mRNA, lipids, and
glycogen as substrates (Bayón and Alonso 2010; Patterson et al. 2009). ADUSPs
exhibit the consensual catalytic structure of DUSP, which is responsible for phos-
phatase activity, and may also present regulatory and recognition/interaction
sequences (such as myristoylation) in their C- and N-terminal portions (Alonso
et al. 2004a, b; Jeong et al. 2006; Schwertassek et al. 2010). Another feature that
defines ADUSP is lack of the CH2 sequence in the MKPs (Bayón and Alonso 2010;
Patterson et al. 2009), which is the structure that contains the kinase-interacting
motifs responsible for the specificity of MKPs to MAPKs (Bermudez et al. 2010;
Nunes-Xavier et al. 2011; Patterson et al. 2009). The absence of this structure,
together with the negative results for certain ADUSPs as regulators of MAPK
pathways, suggests that ERK1/2, JNK, and p38 are not the characteristic substrates
of this group (Schwertassek et al. 2010; Zhou et al. 2002). It is true and undeniable
that several members within the MKP group share some redundancy in their sub-
strates but are enzymes with differential expression between cells and with varying
regulatory mechanisms (Alonso et al. 2004c; Patterson et al. 2009; Bayón and
Alonso 2010; Farooq and Zhou 2004). In addition, the same can be stated for a
few ADUSPs, such as DUSP3 and DUSP12, that even without such a structure (the
CH2 domain), behave as true MKPs and act to dephosphorylate ERK1/2, JNK, and
even p38 (Bayón and Alonso 2010; Cho et al. 2017).
2.3 DUSP3/VHR
DUSP3 (also known as vaccinia H1-related phosphatase or VHR) was the first dual
phosphatase identified in mammals in 1991 (Ishibashi et al. 1992) and also the first
ADUSP crystallized in 1996 (Fig. 2) (Yuvaniyama et al. 1996). It is constitutively
active, widely expressed in several tissues, and can be found in the nucleus or cytosol
as these locations are important for its functions (Alonso et al. 2001; Rahmouni et al.
2006). Among the roles attributed to DUSP3 (discussed in more detail in the
following sections) are cell cycle control, proliferation, and senescence of some
types of cancer and also in the immune responses in which the majority of cases are
mediated by the MAPK substrates (Fig. 3) (Alonso et al. 2003; Bayón and Alonso
2010; Hoyt et al. 2007; Jeffrey et al. 2007; Kondoh and Nishida 2007; Nunes-Xavier
et al. 2011; Patterson et al. 2009; Rahmouni et al. 2006; Salojin and Oravecz 2007).
DUSP3 is an ~21 kDa enzyme with 185 residues and no known signal sequence
in addition to its basic core in which the HC(X)5R consensus sequence common to
the Class I PTPs is present. DUSP3’s catalytic triad consisting of His-123 (red),
Cell Cycle Regulaon, Proliferaon,
Cyc D1
PKC
Cell Adhesion and Migraon
NF-κB
Phosphatase Acvators
PLCγ
MAPK
STAT5 EGFR
VRK3
Paxillin FAK DUSP3/VHR PTKs
NBS1
NPM1
HRNPC NUCL
ATM cMyc
CHK1/2 ARF
p53 p21
Chromosome Segregaon,
Fig. 3 DUSP3 substrates, validated both in vivo and in vitro (first layer of neighbors), identified
in vitro, and predicted in silico (first and second layer of neighbors), all work in conjunction to
mediate this phosphatase’s diverse biological functions shown in the scheme (magenta periphery).
Under specific conditions, some protein kinases (Tyr or Ser/Thr) can phosphorylate DUSP3 by
increasing its phosphatase activity (in blue on the right) on its phospho-substrates
Cys-124, (yellow), and Arg-130 (red) are highlighted in the Fig. 2a. Its three-
dimensional (3D) structure has a catalytic slit of 6 Å depth, which is shallower
than the classical PTPs (9 Å) but is more open, which explains substrate differences
between these groups and also supports the possibility of other substrates mediating
DUSPs’ activities. This is the case for the ADUSP group, which is generally more
diverse in substrates than PTP classical group. Furthermore, it is noteworthy to
mention that if other ADUSPs share structural similarities and biological function-
alities with DUSP3, the task of dephosphorylating some substrates of DUSP3 could
also be played by these enzymes (Barford et al. 1994; Bayón and Alonso 2010;
Ishibashi et al. 1992).
Unlike most MKPs, DUSP3 is not regulated in response to MAPK activation
(Kang and Kim 2006), and although it does not have the CH2 structure at its
N-terminus, DUSP3 behaves as a true MKP capable of dephosphorylating
ERK1/2, JNK, and, to a lesser extent, p38 (Cerignoli et al. 2006; Hoyt et al. 2007;
Kondoh and Nishida 2007; Todd et al. 1999), which suggest the existence of an
CH2-independent catalytic mechanism. Also, while MKPs have their catalytic
activity increased by binding of their substrates, DUSP3 has an increase in activity
caused by VRK3, Zap70, and Tyk2. These enzymes phosphorylate DUSP3 at its
Tyr-138 residue (shown in green on the Fig. 2) in cells that express them, and it has
been well established that mutations in this residue changes normal DUSP3 function
(Alonso et al. 2003; Hoyt et al. 2007; Kang and Kim 2006). The existence of this
regulatory site in DUSP3 is favorable with respect to the investigation of its potential
as an on-off therapeutic intervention (Bayón and Alonso 2010; Nunes-Xavier et al.
2011).
In addition to the MAPK substrates, the receptor tyrosine kinase ErbB, of
epidermal growth factor (EGF)-2, has been shown to have specific Tyr dephosphor-
ylation by DUSP3 in non-small cell lung cancers (Mustelin 2007). In immune cells
under stimulation by cytokines and growth factors, DUSP3 dephosphorylates the
signal transducer and activator of transcription 5 (STAT5), whose activity and
nuclear translocation are regulated by phosphorylation/dephosphorylation events
on two specific Tyr residues (Bayón and Alonso 2010).
The three classical MAPKs have been shown to be involved in the DNA damage
response and repair pathways activated by different types of genotoxic stress that
culminate in the regulation of ATM (a serine/threonine kinase) or phosphorylation of
the histone, H2AX isoform (Arnoldussen and Saatcioglu 2009; Jeffrey et al. 2007;
Lang et al. 2006; Salojin and Oravecz 2007). Therefore, novel roles for DUSP3 in
the maintenance of genomic stability have emerged, uncovering new biological
functions for this dual phosphatase. More recently, Forti (Forti 2015) and collabo-
rators (Panico and Forti 2013) have suggested several nuclear proteins, involved in
different aspects of the DNA damage response and repair, as putative protein
interactors that could be dephosphorylated by DUSP3, including nibrin (NBS1),
nucleophosmin (NPM), nucleolin (NUCL), heterogeneous nuclear ribonucleopro-
tein (hnRNP) C1/C2, and ATM/ATR (Alonso et al. 2004a, b; Jeong et al. 2006).
New relationships between protein targets and functions will be discussed in the next
sections proposing DUSP3 phosphatase as a good candidate for inhibition in clinical
settings (Fig. 3).
3 Molecular and Biological Functions of DUSP3/VHR
3.1 DUSP3 in Tumorigenesis
Many types of cancers originate from several activating mutations or genomic

aberrations of kinase signaling pathway members, for example, KRAS and EGFR,
which activate downstream MAPK signaling (Herbst et al. 2008), hence the rele-
vance of studies in the regulation of these pathways. DUSP3 is one of the phospha-
tases that has the capacity of regulating MAPKs (ERK1/2, JNK, and p38) and some
growth factor receptors, such as epidermal growth factor receptor (EGFR) and
human epidermal growth factor receptor (HER2) (Rahmouni et al. 2006; Wang
et al. 2011), which have altered activities in specific types of cancer. DUSP3
regulates cell cycle progression and has its levels modulated during the cycle.
Several studies have demonstrated the importance of DUSP3 in proliferation and
invasion of cancer cells (Henkens et al. 2008; Rahmouni et al. 2006). For instance,
Hela cells lacking DUSP3 arrest at the G1/S and G2/M transitions of cell cycle and
initiate senescence (Fig. 3) (Rahmouni et al. 2006; Stein et al. 1991).
MAPK activation has a growth-promoting role during the early G1 phase of the
cell cycle (Rahmouni et al. 2006). On the other hand, constitutively elevated ERK1/2
and JNK activity arrests cells in G2/M phase and G1/S-phase of the cell cycle,
respectively (Chau and Shibuya 1999; Rahmouni et al. 2006). Rahmouni et al. show
that the biphasic DUSP3 expression during cell cycle allows the regulation of the
MAPK activation (Rahmouni et al. 2006). They also demonstrated that low expres-
sion of DUSP3 in early G1 phase permits greater ERK1/2 and JNK activities, and the
continuously rising levels of DUSP3 during the S, G2, and M phase then prevent
MAPKs’ growth-arresting effects during these phases. This result shows the impor-
tance of DUSP3 in the G1 phase, in which there is well-known overexpression of
cyclin D1 (Baldin et al. 1993). This cyclin plays an important role in cell cycle
progression and is involved in many types of cancers (Sicinski et al. 1995; Tetsu and
McCormick 1999) such as breast cancer, in which cyclin D1 expression is
deregulated by several mechanisms (Ahnstrom et al. 2005; Sicinski and Weinberg
1997). One of these DUSP3-associated mechanisms occurs via overexpression of
breast cancer gene BRCA1-IRIS (a splice variant locus of the gene brca1) (ElShamy
and Livingston 2004), which induces cyclin D1 overexpression and increases cell
proliferation. BRCA1-IRIS can transcriptionally induce cyclin D1 expression
through binding and activation of the c-jun/AP1 transcription factor, which induces
its transcription in a DUSP3-independent manner (Nakuci et al. 2006). Moreover,
BRCA1-IRIS can also activate cyclin D1 expression in a breast cancer cell line in a
non-transcriptional fashion, in a DUSP3-dependent manner (Fig. 3) (Hao and
ElShamy 2007).
In the latter scenario, overexpression of the components of the axis BRCA1-
IRIS/EGFR/ErbB2 decreases the expression of DUSP3 in normal (human
mammary epithelial) and breast cancer cell lines (MCF-7 and SKBR3). This
DUSP3 downregulation generates JNK activation and subsequently activation of
c-Jun/AP1 and cyclin D1 overexpression that can transform normal mammary
epithelial cells, whereas overexpressing DUSP3 in breast cancer cells can block
the cyclin D1 overexpression. The downregulation of BRCA1-IRIS reduces the
expression of cyclin D1 by elevating the expression levels of DUSP3 mRNA and
protein, reducing the protein expression of EGFR and ErbB2 (Hao and ElShamy
2007). These results suggest that BRCA1-IRIS overexpression, and consequently
DUSP3 downregulation, play important roles in the development of more aggressive
endocrine-resistant breast cancer phenotypes that depend on the expression and
activation of growth factor or cell membrane receptors. In this regard, DUSP3
restoration might be sufficient to overcome either the cellular over-proliferation or
transformation dependent on Cyclin D1 overexpression (Hao and ElShamy 2007).
Following the same rationale, DUSP3 expression is lower in non-small cell lung
cancer (NSCLC) tissues in comparison with normal lung tissues. Contrary to other
studies, DUSP3 activity in NSCLC cells/tissues had very limited activity against
MAPKs but regulated ErBb and EGFR signaling that is involved in epithelial cell
growth regulation (Wang et al. 2011). These tyrosine kinase receptors are related to
different diseases, including many types of cancer (Hynes and MacDonald 2009;
Hynes and Stern 1994; Nicholson et al. 2001). Wang et al. (2011) demonstrated the
capability of DUSP3 to dephosphorylate EGFR and ErB2 in cell lines. EGF stim-
ulation of DUSP3-expressing NSCLC cells led to a reduced Tyr-992 phosphoryla-
tion on EGFR mainly at the early times of treatment. DUSP3 downregulation
strongly increases phosphorylation at the EGFR Tyr-992 residue, with or without
EGFR stimulation. Therefore, DUSP3 overexpression reduces phosphorylation of
EGFR Tyr-992 and makes the cell less responsive to EGF. DUSP3 also inhibited the
downstream signaling events such as Tyr-783 phosphorylation in phospholipase C
(PLC)γ and protein kinase C (PKC) activation. On the other hand, Tyr-416 phos-
phorylation of Src activation is not affected when H1299 cells are stimulated with
EGF, therefore showing specificity of DUSP3 actions (Fig. 3).
In vitro DUSP3 overexpression could suppress cell proliferation in 2D and 3D
H1290 cell cultures. Xenograft DUSP3 overexpression also has been shown to
reduce tumor size. In patients with non-small cell lung cancer (NSCLC), DUSP3
mRNA and transcribed protein expression is significantly lower in tumor tissues than
in adjacent normal lung tissues. These data suggest that DUSP3 expression is also
particularly downregulated in these cancer cells and its overexpression can suppress
cancer cell growth. A decrease in DUSP3 expression might form part of the
pathogenic-initiating mechanisms of NSCLC. Wagner et al. (2013) tried to explain
this possible mechanism involved in DUSP3 downregulation and the onset of lung
tumorigenesis in NSCLC. They found that KDM2A, an H3 lysine 36 (H3K36)
demethylase (Shi et al. 2007), promotes lung tumorigenesis by epigenetically
enhancing ERK1/2 signaling through DUSP3 downregulation; this finding is in
contrast to those from Wang et al. (2011), who related the low DUSP3 expression
to EGFR pathway overactivation. These differences may probably be due to the
heterogeneous NSCLC molecular etiology (Herbst et al. 2008) and can also be
reinforced by the Lewis lung carcinoma experimental metastasis model, where
DUSP3/ mice developed a metastatic growth dependent on DUSP3 absence
through a mechanism of intense recruitment of macrophages to the lung metastasis
(Vandereyken et al. 2017a).
As many other genes are aberrantly expressed in tumors, dusp3 gene has been
shown overexpressed in some types of cancers. Several studies relate that
overexpression of DUSP3 can be associated with the onset or development of a
carcinogenic phenotype. However this cannot be considered a cause of such pheno-
type especially because other expression levels of other ADUSPs have not been
assessed. Even so, it is important to mention that in cervical cancer cell lines such as
HeLa, SiHa, CaSki, C33, and HT3 and other epithelial cells from the high-grade
squamous intraepithelial lesions (SIL), invasive squamous cell carcinomas, primary
cervical adenocarcinomas, and adenocarcinoma in situ of the uterine cervix, DUSP3
is upregulated when compared with primary keratinocytes and normal tissues. In
these cell lines, DUSP3 is located in the cytoplasm and nucleus, while in normal
keratinocytes, it is mainly cytoplasmic; conversely, in cervix cancer tissues, DUSP3
has a mainly nuclear localization (Henkens et al. 2008). In HeLa cells the
distribution of DUSP3 is cell cycle phase dependent, mostly located in the nucleus of
interphasic cells; during the metaphase DUSP3 is concentrated around the chroma-
tin, and in telophase it is between the daughter chromatids (Rahmouni et al. 2006).
The increased amount of DUSP3 in cell lines and tissues of cervix cancer is not due
to increased expression of DUSP3 mRNA or stabilization of its mRNA, since its
levels in both cancer cell lines and normal keratinocytes are the same, but due to an
increase in DUSP3 protein half-life (posttranslational stabilization). Interestingly,
DUSP3’s half-life also varies in different phases of the cell cycle being lower in the
G1 phase (Henkens et al. 2008; Rahmouni et al. 2006). This dynamic expression and
differential distribution of DUSP3 in cervix cancers allow this phosphatase to
regulate ERK1/2 and JNK activity during the S and G2/M phases of the cell cycle;
excessive activity of these kinases can trigger cell cycle arrest in G1/S or
G2/M. Consequently, cervix cancer cells overexpressing DUSP3 can drive G1
progression despite an inhibitory signal or unfavorable environmental conditions
(Henkens et al. 2008).
As in epithelial cervix cancer cell lines, DUSP3 is overexpressed in prostate
cancer compared with normal prostate (Arnoldussen et al. 2008). In normal prostate
epithelial cells, androgen withdrawal leads to decreased cell proliferation, increased
apoptosis, and atrophy (Mercader et al. 2007). Conversely, tumor prostate
epithelial cells can overcome this deficiency and move to an androgen-independent
state (Feldman and Feldman 2001). When androgen-responsive prostate cancer
cell lines are treated with R1881 (a synthetic androgen), various MKPs are
overexpressed. Furthermore, apoptosis-inducing treatments with TPA (12-O-
tetradecanoylphorbol-13-acetate) or thapsigargin (TG) plus R1881 increase the
expression of these phosphatases. Among these overexpressed phosphatases,
DUSP3 mRNA and protein are significantly augmented. Furthermore, deregulation
of the MAPK pathway is also involved in prostate carcinogenesis (Bakin et al.
2003). For instance, in LNCaP cells (androgen-responsive prostate cancer cell line),
treatment with TPA/TG plus R1881 decreases JNK phosphorylation significantly
(Engedal et al. 2002) but did not affect phosphorylated ERK1/2 levels (Lorenzo and
Saatcioglu 2008), which seem to be caused by cellular stress, while their activation
has also been implicated in apoptosis (Wada and Penninger 2004).
These data suggest that overexpression of DUSP3 specifically inactivates JNK,
but not ERK1/2, what further suggests that DUSP3 can reverse apoptosis induced by
JNK in LNCaP cells when these cells are stimulated with androgens. In vivo,
androgen-sensitive grafts expressing DUSP3 have increased resistance to
castration-induced apoptosis, and tumor regression is inversely correlated to
DUSP3 expression (Shi 2007). This possible mechanism suggests a role for
DUSP3 in prostate cancer progression and also that DUSP3 knockdown in prostate
cancer may activate JNK, leading to apoptosis (Fig. 3). However and again, these
mechanisms were not explored considering the knockdown of other MKPs or
ADUSPs that could overlap their actions on the dephosphorylation of MAPKs.
DUSP3 is also overexpressed in dysplastic nevi (DNs, benign melanocytic
tumors, or lesions with a more hyper-proliferative phenotype); it is expressed in
the epidermis and DN nevus cells, but it is higher in the DN epidermis compared
with the common melanocytic nevus (CMN, lesion considered to be in cellular

senescence) (Mitsui et al. 2016). It was found that DUSP3 interacted with the focal
adhesion kinase (FAK) dephosphorylating it at the Tyr-379 residue in H1299 cell
line and in MEFs and lung epithelial cells of DUSP3-null mice. This interaction
involves new functions: (1) DUSP3 knockdown resulting in higher cell motility by
enhancing FAK phosphorylation at focal cell adhesions and (2) increase in migra-
tion. On the other hand, DUSP3 overexpression caused a decrease in phospho-FAK
and inhibited cell migration (Chen et al. 2017). The enhanced motility causes an
increase in tumor cell malignancy, further supporting the fact that DUSP3 plays
important roles in the cancer development (Fig. 3).
DUSP3 affects other important aspect of the tumorigenesis process: the angio-
genesis, which is a crucial for growth and tumor maintenance. Other DUSPs have
already been described to mediate the endothelial cell migration and angiogenesis
(DUSPs 1, 2, 10, and 16). It was recently found that endothelial cells have high
DUSP3 expression, which could be related to tubulogenesis (network length and
number of tube intersections) in vivo via PKC, without being connected to ERK1/2
and JNK phosphorylation. In the same endothelial cells, the DUSP3 knockdown
under β-FGF stimulus strongly blocked the angiogenic sprouting (Amand et al.
2014). In addition, by applying different in vivo and ex vivo models, it was observed
that the DUSP3/ mice (which had normal development and no apparent or
spontaneous pathological phenotypes) had a clear decrease in neovascularization.
Therefore, altogether these data suggest that DUSP3 plays a role in neoangiogenesis
in vivo (Amand et al. 2014) and highlight its relevant involvements in different steps
of the tumorigenesis processes (Fig. 3).
3.2 DUSP3 in Genomic Stability
The roles of DUSP3 in cell cycle checkpoints and its relation to ERK1/2 have been
extensively studied. Although DUSP3 is not upregulated in response to MAPK
activation, it controls the cell cycle transition at G1/S and G2/M in an ERK1/2-
and JNK-dependent manner. DUSP3 knockdown in HeLa cells provokes cell cycle
arrest and senescence in human cancer cell (Rahmouni et al. 2006), associated with
an accelerated death of mitotically arrested cells (Tambe et al. 2016). However, an
unexpected action of the pair DUSP3-ERK1/2 that has been studied very recently is
the effect on genomic stability, which can be investigated by several aspects.
At the outset, inhibition of ERK1/2 has not resulted in defects in chromosomal
events, spindle assembly checkpoint signaling (Foley and Kapoor 2013), or mitotic
exit (Roberts et al. 2002; Shinohara et al. 2006), but instead, its overactivation
induced multipolar spindles and aneuploidy in cells (Eves et al. 2006). Because of
the strong relationship between ERK1/2 and DUSP3, the role of this phosphatase on
the formation of multipolar spindles in cancer cells was also investigated (Tambe
et al. 2016). In early mitotic mammalian cells, both DUSP3 and ERK1/2
(non-phosphorylated) are in the spindle apparatus (Rahmouni et al. 2006; Willard
and Crouch 2001), and the transient inhibition of DUSP3 leads to the formation of
multipolar spindles in human mitotic cancer cells, which is reversed by the silencing
or chemical inhibition of ERK1/2 (Tambe et al. 2016). Additionally, the authors
showed that the ectopic overexpression of DUSP3 reduced ERK1/2 activity and
reversed the DUSP3 siRNA-induced multipolar spindles in HeLa cells (Tambe et al.
2016). Together, these data suggest that the relationship between ERK1/2 and
DUSP3 may have other roles in addition to cell cycle control and proliferation, as
demonstrated with respect to genomic stability (Fig. 3) (Tambe et al. 2016). But
these authors did not explore other relationships between MKPs or ADUSPs and
ERK1/2 regulating the spindle apparatus, suggesting a possible dependence on
substrates phosphorylated by MAPK and not directly to DUSP3 itself. In addition,
it has been shown that other DUSPs are also related to correct cell division. For
example, DUSP5 and DUSP7 are related with genomic stability because they are
connected to other proteins related to spindle formation and nuclear envelop
breakdown, respectively (Matta et al. 2007; Pfender et al. 2015).
Besides that, DUSP3 is extensively present in the nucleus of various cell lines
(Forti 2015; Henkens et al. 2008), especially after a genotoxic stress (Forti 2015).
DUSP3 co-localizes with the three MAPK isoforms (ERK1/2, JNK, p38) in the
nucleus upon gamma radiation-induced damage; it may also indicate that this
phosphatase may have other substrates and roles in genomic stability, indirectly or
completely not related to these kinases. Indeed, a study of our group, through a
computational approach and experimental validation analysis, identified novel
DUSP3 substrates specifically involved in genomic stability or integrity (Forti
2015). After DUSP3 has been found co-localized with phosphorylated H2AX
(S139), a classical marker of DNA strand breaks (that has also been observed for
the DUSP4) (Lawan et al. 2011), a deep bioinformatics analysis of human nuclear
proteins containing the Thr-X-Tyr motif (commonly present in the activation loop of
MAPKs), 121 putative DUSP3 substrates related to DNA damage and response and
repair were identified. Many of these data were experimentally validated in HeLa
cells exposed to ionizing radiation (gamma) sections; DUSP3 was found to
independently co-localize with pATM (S1981), pATR (S428), pBRCA1 (S1423),
BRCA2, centromere protein (CENP)-F, cyclin A, nibrin (NBS1), apurinic/
apyrimidinic endonuclease (APE1), the double-strand break repair protein
(MRE11), DNA repair protein (RAD50), checkpoint kinase (pCHK) 2 (T68), and
p53 (S15) proteins (Forti 2015). These data are consistent with the hypothesis that
DUSP3 could participate in DNA damage repair by interacting and/or
dephosphorylating these proteins with different timing following stress exposure.
According to these data, DUSP3 downregulation (by siRNA silencing or chemical
inhibition, as discussed later) provokes different effects in tumor cell lines under
genotoxic stress, including senescence, decreased cell proliferation/survival associ-
ated with an increased DNA damage, and an impaired and/or delayed DNA strand
break repair (Torres et al. 2017). However, another caveat of this study is that these
T-X-Y-containing substrates could also be dephosphorylated by other MKPs or
ADUSPs acting on MAPKs, and not only necessarily by DUSP3. With this in
mind, our group invested in another strategy, now using proteomics and
interactomes, to identify new substrates of DUSP3 very likely related to genomic

stability (Panico and Forti 2013). From the 45 proteins found interacting with
DUSP3, 50% are involved in DNA/RNA structure and functions. Based on the
presence of phosphorylatable Tyr and Thr residues, in addition to any relationship to
DUSP3-regulated biological processes, nucleophosmin (NPM), nucleolin (NCL),
and heterogeneous ribonucleoprotein (hnRNP) C1/C2 were the three best validated
proteins as novel and direct substrates interacting with DUSP3 (Panico and Forti
2013).
Among many different NCL functions (rRNA synthesis, ribosomal biogenesis,
cell cycle regulation, senescence, proliferation, and survival) (Olson et al. 2000;
Pinol-Roma 1999; Srivastava and Pollard 1999), it was also demonstrated that it
facilitates nucleosomal unfolding through Rad50, removing the H2A/H2B histones
around the DNA double-strand break, which is an important step for efficient DNA
repair by NHEJ (Goldstein et al. 2013). NCL can also directly affect p53 expression
levels and facilitate DNA repair by NER (Takagi et al. 2005). As demonstrated, after
radiation exposure, NCL associates with HAUSP and stabilizes p53 by protecting it
from degradation of MDM2 (Lim et al. 2015); at the same time it translocates from
the nucleolus to the nucleosome through the p53 complex (Daniely et al. 2002). The
hnRNPC complex participates in NHEJ-associated DNA double-strand break repair,
and it is also involved in p53 expression that occurs after DNA damage, therefore
being implicated in the NER pathway (Yano et al. 2008). NPM is related to mRNA
processing, ribosomal biogenesis, cell proliferation, centrosome duplication, and
migration (Grisendi et al. 2006), but it is immediately expressed and related to
DNA repair and survival after UV exposure (Wu et al. 2002a, b). NPM also interacts
with MDM2 (Kurki et al. 2004) and ARF (Lee et al. 2005a), thus protecting p53
from degradation. After UV or gamma irradiation-induced DNA damage, NPM
links to chromatin and regulates p53 expression, mediating DNA repair mechanisms
through the NER pathway (Lee et al. 2005b).
These three proteins show high tyrosine phosphorylation probabilities, and they
are hyperphosphorylated in vivo, so they could be potential targets of dephosphor-
ylation by DUSP3. There is a lack of knowledge about tyrosine phosphorylation of
NCL, hnRNPC, and NPM; however it is important that the level and specificity of
these phosphorylations will orientate the proteins for the different functions. In the
case of threonine residues, the regulation of NCL phosphorylation drives its func-
tions during cell cycle (Srivastava and Pollard 1999) and is closely related to cell
proliferation capacity (Miranda et al. 1995). It has been demonstrated that the
amount of hnRNP C1/C2 phosphorylation regulates p53 gene expression after
DNA damage (Christian et al. 2008), and it is phosphorylated by p38 after UV
radiation-induced DNA damage (Yang et al. 2002). NPM is involved in centrosome
duplication, and its phosphorylation at Thr 234 and 237 residues occurs during
mitosis (Cha et al. 2004), while phosphorylation at the Thr199 residue directs NPM
to DNA strand break repair (Koike et al. 2010); dephosphorylation of these threo-
nines by PP1β after UV radiation promotes DNA repair (Lin et al. 2010).
Therefore, DUSP3’s activity could contribute to NUCL, hnRNP C1/C2, and
NPM tyrosine dephosphorylation under genotoxic stress and promote DNA repair.
Several experimental strategies have been employed by our group to access these
questions, and preliminary results point to a differential phosphorylation of specific
(not all) tyrosines on these proteins, which are dependent on DUSP3 expression
and/or activity (Fig. 3). Also, we are confirming that DUSP3-deficient cells showed
deficient repair of specific DNA lesions promoted by UV radiation, with direct
implications of the NER pathway (Forti et al. unpublished results). Once DUSP3
physically interacts with these three proteins (Panico and Forti 2013) and dephos-
phorylates them, thus increasing/decreasing their activity/function toward different
biological processes, we have now strong evidences that DUSP3 is a player in
genomic stability maintenance, through still unknown mechanisms but very likely
through these new protein partners or substrates (NUCL, hnRNP C1/C2, and NPM),
paving new ways for the drug development and clinical trials targeting DUSP3
independently of its actions on MAPK.
3.3 DUSP3 in Blood-Associated Diseases
The first report about DUSP3’s role and expression in circulatory system cells were
in T lymphocytes (Ishibashi et al. 1992). In thymocytes, DUSP3 mRNA levels are
very low compared to brain and heart tissues, in which DUSP3 expression levels are
already low, and especially when compared to expression of other phosphatases such
as DUSP1, which is considered the most abundant phosphatase for many tissues
(Tanzola and Kersh 2006). However, T cells at resting state constitutively express
DUSP3 (Alonso et al. 2001; Ishibashi et al. 1992), and the activation of T cells does
not induce the expression of this enzyme that primarily reduces ERK1/2 phosphor-
ylation, but this inhibitory effect is more marked to JNK phosphorylation (Alonso
et al. 2001). On the other hand, p38 kinase is also activated in T cells in response to
receptor activation and UV radiation, but the co-expressions of active and inactive
DUSP3 had no significant impact on the regulation of the p38 kinase pathway
(Alonso et al. 2001).
Resting T cells express the hematopoietic protein tyrosine phosphatase (HePTP)
and DUSP3; after TCR stimulation additional MAPK phosphatases are synthesized.
However, compared to VHX and MKP6, two other small dual-specific phosphatase
expressed in T cells, DUSP3 was the most efficient to reduce the TCR-induced
activation of an NFAT-AP-1-driven reporter when the three proteins were expressed
at equal amounts; therefore the phosphatase is more potent to inhibit the TCR
signaling to IL-2 (Alonso et al. 2001). When ectopically overexpressed into Jurkat
cells by transfection, DUSP3 inhibits ERK1/2 activation in response to T cell antigen
receptor (TCR) activation by IL-2 (Alonso et al. 2001), and this effect is dependent
on direct DUSP3 phosphorylation by zeta-chain-associated protein (ZAP)-70 on the
Tyr138 residue (Alonso et al. 2003). The ZAP-70 tyrosine kinase is a key component
of the TCR signaling pathway in which the DUSP3-Y138F mutant was shown to
increase TCR-induced ERK1/2 activation and the induction of the IL-2 gene expres-
sion. Basal activity of ZAP-70 has been shown to be sufficient to stimulate T cell
functions, but when there is TCR stimulation by an antigen, this becomes even more
evident and causes DUSP3 translocation from the cytosol to the T cell pole, thereby
facilitating the phosphorylation of DUSP3 on the Tyr138 residue (Alonso et al.
2003).
DUSP3 also selectively dephosphorylates IFN-β-induced signal transducers and
activators of transcription 5 (STAT5), which leads to subsequent inhibition of
transcription factor activities. In this context, STAT5’s Src homology 2 domain
(SH2) was required for effective dephosphorylation by DUSP3 since the recruitment
of the phosphatase activity on STAT5 requires DUSP3 phosphorylation on the
Tyr138 residue. Tyrosine kinase 2 (TYK2), which mediates STAT5 phosphoryla-
tion, has also been shown to be responsible for DUSP3 phosphorylation on the
Tyr138 residue very likely due to the high homology between this kinase and SYK
and also ZAP-70 (Fig. 2) (Hoyt et al. 2007).
In 2010, a data collection extracted from the RefDIC (Reference Genomics
Database of Immune Cell) database about the expression pattern of genes encoding
PTPs in mice was published. This work showed that DUSP3 expression was
detected mainly in macrophages but also in immature dendritic cells, mast cells,
and neutrophils although these data were all related to the DUSP3 basal expression
in cells that did not receive any type of physical or chemical treatment or differen-
tiation stimulus (Arimura and Yagi 2010). At the level of proteins, monocytes and
macrophages demonstrated higher DUSP3 level expression when compared to
neutrophils and B and T cells (Singh et al. 2015), in addition to platelets, which
also presented DUSP3 levels significantly higher than B and T lymphocytes
(Musumeci et al. 2014). These data bring to light the importance of research about
the role of DUSP3 in circulatory system cells since these cells have different
expression patterns from each other in addition to different physiological functions
(Fig. 3).
In another study, DUSP3 activity was identified as being relevant in the inflam-
matory response to S. aureus, both in humans and mice, through the NF-kB
signaling as a negative feedback component. The cytokine inflammatory response
was improved after DUSP3 knockdown in macrophages as observed by an increase
in pro-inflammatory cytokine production via NF-kB. This cytokine increase may
lead to a hyper-responsiveness of the host’s immune system, leading to the so-called
immune paralysis (Yan et al. 2014). In contrast to its pro-inflammatory properties, it
has been recently shown that DUSP3 deficiency in mice is capable of promoting
tolerance to LPS-induced endotoxin shock and polymicrobial septic shock, which is
mainly dependent on macrophages. This protection is associated with an expressive
increase of anti-inflammatory M2-like macrophages, decreased TNF production, and
impaired ERK1/2 activation. In in vivo experiments, it was observed that after
LPS-induced endotoxic shock, the DUSP3/ mice recovered their normal
temperature, while the DUSP3+/+ mice remained hypothermic. Finally, it was also
demonstrated that resistance to septic shock was transferable through monocytes to
wild-type (WT) mice and was associated with M2-like macrophage dominance
(Singh et al. 2015). Resistance to sepsis in females, but not in males or ovariecto-
mized females (OVX), was associated with decreased ERK1/2, phosphoinositide
kinase (PI3K), and protein kinase B (AKT) activation (Vandereyken et al. 2017b).
DUSP3 expression in peritoneal macrophages was abolished in the mice that
received DUSP3/ bone marrow cells, and after LPS challenge 70% of the female
mice that received DUSP3/ bone marrow cells survived until the end of the
experiment, compared to 9% of the female mice that received DUSP3+/+ bone
marrow cells and male mice that received DUSP3/ and DUSP3+/+ cells that
died within 4 days. These data demonstrate that in the absence of DUSP3, female
sex hormones are involved in the observed resistance of DUSP3/ mice to
LPS-induced lethality, thus suggesting that DUSP3 inhibition combined with estro-
gen administration may lead to protection against septic shock (Vandereyken et al.
2017b).
Another recent study has also elucidated the importance of DUSP3 in platelet
aggregation mechanisms, despite previous researches implicating other non-DUSP
phosphatases in platelet signaling such as CD148, PTP1B, SHP1, and SHP2
(Musumeci et al. 2014; Tautz et al. 2015). This phosphatase seems to be implicated
in platelet signaling through collagen receptor glycoprotein VI (GPVI) and C-type
lectin-like type II (CLECII) receptors to promote reduction of SYK and phospholi-
pase C γ2 (PLCγ2) tyrosine phosphorylation, without affecting the overall tyrosine
phosphorylation (such as ERK1/2 and JNK). In DUSP3 absence, thrombus forma-
tion was significantly impaired without affecting bleeding, suggesting that this
enzyme plays a key role in arterial thrombosis but is not necessary for primary
hemostasis. Ex vivo experiments further demonstrated that DUSP3 deficiency
resulted in defective platelet aggregation, granule secretion, and αIIbβ3 integrin
activation in response to GPVI and CLEC-2 receptor stimulation without affecting
GPCR-mediated platelet activation (such as the purinergic [P2Y12], thromboxane
[TXA2], and thrombin receptors) (Musumeci et al. 2014; Tautz et al. 2015). The
pharmacotherapy currently employed for thrombosis has been mainly based on
G-protein-coupled receptor (GPCR) inhibition and the formation of molecules that
can stimulate these receptors. Although currently available and commonly used
platelet precursors increase patient survival, leading to decreased mortality and
morbidity, these also produce other adverse effects such as increased risk of gastro-
intestinal toxicity, neutropenia, thrombocytopenia, and bleeding in combination with
an increased incidence of arterial thrombosis cases. Because of these and other
reasons, DUSP3 constitutes a potential target for the development of new, safer
therapies in platelet aggregation (Tautz et al. 2015) and, here, through MAPK-
independent mechanisms.
4 Overview of Current Knowledge on DUSP3/VHR

Inhibitors
Pan-inhibitors of protein tyrosine phosphatases have long been established in the

literature. Sodium orthovanadate has been known to inhibit PTPs since the 1980s
(Tamura et al. 1984), and it has become a staple in the field of phosphatase studies.
Other widespread PTP inhibitors include α-Bromo-4-(carboxymethoxy)

acetophenone (also known as PTP inhibitor III) (Arabaci et al. 2002), phenylarsine
oxide (Levenson and Blackshear 1989), and 4-hydroxy-3,3-dimethyl-2H-benz[g]
indole-2,5(3H)-dione (also known as BVT 948) (Liljebris et al. 2004). Although
their breadth of action is wide, they cannot inhibit every PTP. Conversely, the fact
that they inhibit tyrosine phosphatases with little selectivity makes them poor
candidates for clinical trials and probable therapies.
In the face of the roles that have been attributed to DUSP3 in many clinically
relevant cellular processes, as mentioned in previous sections, there have been
considerable efforts to find potent and specific inhibitors of this phosphatase,
which will allow for discovery and better comprehension of its biological functions
in vitro and in vivo (cells and animal models). These compounds may also be
employed as drugs for certain diseases through a mechanism hampering the prolif-
eration of tumors that are sensitive to DUSP3 inactivation: for example, tumors that
undergo cell cycle arrest mediated by MAP kinase overactivation. However, before
starting the timeline description of DUSP3-inhibiting drugs, it is worthy to mention
that most of them have limited potency, because their IC50 values usually do not
overcome micromolar ranges, whereas to be valuable in human clinical trials, they
need to work with IC50 of nanomolar or less. Additionally, they have typically low
selectivity since, in general, they were not tested for all closely related enzyme
(ADUSPs and even MKPs) and furthermore to the aforementioned new protein
targets identified. Anyhow, we intend to inform the reader about structure and
functions of these DUSP3 inhibitors and, at the same time, to warn the readers that
the compounds discussed here are just starting points mainly because they have
numerous side effects from off-targets. This could be better justified and solved if
further studies were carried out to distinguish whether the new substrates of DUSP3
could also be dephosphorylated by some other ADUSPs, especially from in vivo
experiments, thus reducing or eliminating the pharmacological effects of DUSP3
inhibitors. The real specificities of the published DUSP3 compounds toward other
ADUSPs are relatively unknown because most of the works have been done by
using just a few MKPs and/or classical PTPs as controls for in vitro experiments (see
Table 1, column “Activity toward other phosphatases”). Nevertheless, these drugs
(mostly structurally designed from DUSP3 crystal structure) continue to be potential
precursors to more specific molecules.
As most PTPs, it is long time known that DUSP3 is sensitive to oxidative stress,
which causes the thiol moiety in its catalytic cysteine residue (Cys-124) to be
oxidized to sulfenic acid and even further into sulfinic and sulfonic acid, the last
two being irreversible modifications under cellular conditions (Chung et al. 2013).
Kim et al. (2000) have evaluated the inhibitory potential of metallic ions on DUSP3,
finding that Cd2+, Fe3+, and Zn2+ are able to perform reversible inhibition, most
likely due to binding. Cu2+-induced DUSP3 inactivation by oxidation, which could
not be reversed by EDTA chelation similar to other ions, showed that its activity
could be rescued upon dithiothreitol (DTT) addition, indicating that the cysteine
residue was not fully oxidized since DTT can only reduce sulfenic acid forms. The
relevance of a Cu-associated DUSP3 regulatory mechanism in vivo still remains
Table 1 List of DUSP3/VHR inhibitors described up to date in the current literature
Ref# Compound name Structure IC50 (DUSP3) Activity toward other phosphatasesa
1 RK-682 2.0 μM (Hamaguchi et al. Cdc25B, Laforin, PTP1B, CD45
1995) (Ueda et al. 2002)
11.6 μM (Ueda et al. 2002)
2 RK-682 enamine 2c 2.0 μM (Hirai et al. 2011) –

DUSP3/VHR Is a Potential Drug Target
(continued)
19
Table 1 (continued)
20
3 Stevastelin A 2.7 μM (Hamaguchi et al. –
1997)
4 Stevastelin B 10.8 μM (Hamaguchi et al. –

1997)
L. F. Monteiro et al.
5 4-Isoavenaciolide 1.2 μM (Ueda et al. 2002) Cdc25, Laforin, PTP1B (Bermudez
et al. 2010)
6 7-Hydroxy-5,6-dimethoxy-1,4- 3.0 μM (Bae et al. 2004) –

phenanthrenequinone
7 Cinn-GEE 290 μM (Park et al. 2004) N.A.

(equilibrium constant)
8 NSC 357756 N.A. N.A.
9 NU-126 38.1 μM (Lazo et al. 2006) MKP1, PTP1B (Arnoldussen and

Saatcioglu 2009)
10 NU-176 47.4 μM (Lazo et al. 2006) MKP1, MKP3, Cdc25B, PTP1B

(Arnoldussen and Saatcioglu 2009)
21
(continued)
Table 1 (continued)
22
11 GATPT 2.92 μM (Shi et al. 2007) N.A.
12 Benzoic acid 1 3.7 μM (Park et al. 2008) N.A.
13 Benzoic acid 2 4.7 μM (Park et al. 2008) N.A.
14 NSC-87877 7.83 μM (Park et al. 2009) N.A.

15 SA1 18 nM (Wu et al. 2009) –
16 SA2 47 nM (Wu et al. 2009) MKP1, CD45, PTP1B (Jeong et al.

2006)
17 SA3 74 nM (Wu et al. 2009) MKP1, CD45, PTP1B (Denu and

Dixon 1998)
23
(continued)
Table 1 (continued)
24
18 SA4 78 nM (Wu et al. 2009) MKP1, CD45
19 SA5 270 nM (Wu et al. 2009) HEPTP, CD45, PTP1B
a
Within one order of magnitude of DUSP3 IC50 reported value
open to studies, and the use of metal ions in a clinical setting is most likely not
feasible due to the nonspecific nature of the redox reactions.
Other groups have searched for natural or synthetic organic compounds with
DUSP3 inhibitory activity. In 1995, Hamaguchi et al. (Hamaguchi et al. 1995)
isolated an organic acid, RK-682, from a Streptomyces strain via monitoring
DUSP3 inhibitory activity (Table 1, compound 1). The authors identified cellular
phosphotyrosine level enhancement, which was different than the pattern caused by
orthovanadate. The group also evaluated its effect on the human B cell leukemia
(Ball-1) cell cycle, observing an arrest at G1/S in accordance with DUSP3’s loss of
function. They calculated the IC50 to be 2.0 μM, and RK-682 has been since then
considered a classic DUSPase inhibitor with no inhibitory potential against PPases
but also displaying promiscuous binding to other PTPases (Carneiro et al. 2015).
The lactone moiety of this compound is thought to interact with the active site of the
phosphatase, thereby inhibiting its activity (Hirai et al. 2011).
Maintaining the long hydrophobic side chain of RK-682, which was found to be
critical for DUSP3 binding, the group later developed enamine derivatives to
improve permeability and enable in vivo action. One of the derivatives containing
m-methylbenzylamine substitution on the enamine group showed improved perme-
ability and high selectivity toward DUSP3, displaying neglectable IC50 values
toward other selected phosphatases. This compound, called RK-682 enamine 2c
(Table 1, compound 2), at a concentration of 30 μM, caused an increase of pERK
and pJNK and led to an arrest at the G1/S transition in mouse fibroblast cells
(NIH3T3 cells). According to 3D models, the enhanced selectivity of this drug is
due to the interaction of the m-methylbenzylamine group with the margins of the
active pocket (Hirai et al. 2011).
The same group identified and synthesized metabolites from a Penicillium strain.
This class of compounds has been called stevastelins and is composed of valine,
threonine, serine, and a 3,5-dihydroxy-2,4-dimethylstearic acid moiety (Table 1,
compounds 3 and 4). In 1996, they observed that some of these compounds’
derivatives had inhibitory potential against DUSP3 and that the long-chain aliphatic
hydrocarbon group was necessary for inhibition (Hamaguchi et al. 1997). While
stevastelin B had a strong immunosuppressive effect in situ for Jurkat cells, it did not
display in vitro inhibition of DUSP3. Conversely, stevastelin A, a sulfonylated
derivative of stevastelin B, had a strong inhibitory effect on DUSP3 but not CD45
(a PTPase) or PPases. The authors hypothesized that stevastelin B might be
sulfonylated or phosphorylated after incorporation into the cell, turning it into a
biologically active form; in contrast, stevastelin A is not diffusible through the
membrane and is therefore not active in vivo. The effect of stevastelins A and B
on the cell cycle of tsFTZ10 cell lines (mammary carcinoma cdc2 mutant) was also
evaluated, and upon release of a G2 phase arrest, the cells treated with stevastelin B
had an inhibited cell cycle transition (much like those treated with vanadate), while
stevastelin A had no visible effects. However, it would be interesting to investigate
whether DUSP3’s loss of function causes an inhibition of the G2/M transition or
whether stevastelin B causes an arrest at G1/S, which would indicate selective
DUSP3 inhibition (Hamaguchi et al. 1997).
In 2002 a compound from a fungal strain which was identified to be a

stereoisomer of avenaciolide, 4-isoavenaciolide (4-iA) (Table 1, compound 5).
This furofurandione has a highly reactive exo-methylene group that is able to bind
to DUSP3 on two cysteine residues, namely, Cys124 and Cys171, via a Michaelis
addition reaction, therefore inactivating the enzyme. 4-iA showed inhibition of
DUSP3 (IC50 ¼ 1.2 μM), other DSPs, and also PTP1B (a classic PTP). However,
it displayed weak CD45 inhibition and no inhibition of PPases (Ueda et al. 2002).
In 2004, Bae et al. (Bae et al. 2004) reported the isolation of a
phenanthrenequinone from the Dendrobium moniliforme orchid species (Table 1,
compound 6). This compound displayed strong inhibition of DUSP3 and PTP1B
but could not inhibit PP1. In the same year, Park et al. (2004) identified a peptidyl
aldehyde that is a slow-binding inhibitor of DUSP3, and following Bae’s work
had shown that this molecule targeted other PTP and SH2 domains. This was a
cinnamaldehyde and tripeptide Gly-Glu-Glu-NH2 adduct (Cinn-GEE), and it
inhibits DUSP3 with low potency with an overall equilibrium constant of
290 110 μM (Table 1, compound 7). Following mechanistic studies with other
phosphatases, the group hypothesizes that Cinn-GEE covalently modifies a
conserved arginine near the pY-binding pocket, forming a reversible imine/enamine
adduct with the PTP domain of DUSP3 (Park et al. 2004).
In 2006, Lazo et al. (Lazo et al. 2006) worked with analogs of NSC 357756
(Table 1, compound 8), a small molecular MAK kinase (MKP)-3 inhibitor, and
found out that two of them (namely, NU-126 and 176) were able to selectively
inhibit MKP1 and DUSP3 (Table 1, compound 9 and 10). For NU-126, they
assayed the dephosphorylating activity of DUSP3 in vitro against an ERK1/2
phosphopeptide, and they observed a decrease in phosphate release accompanying
higher inhibitor concentrations. However, in vivo assays showed no cellular
changes in response to NU-126 and other analogs, and the authors hypothesize
that the compounds might not be permeable. However, they suggest these inhibitors
should provide a starting point for the development of other, more efficient
pharmacophores.
Shi et al. (2007) later identified a potent, selective, and competitive DUSP3
inhibitor via a docking-based high-throughput screening followed by nuclear mag-
netic resonance (NMR) binding studies, (glucosamine-aminoethoxy)triphenyltin
(GATPT) (Table 1, compound 11). The IC50 value was calculated to be 2.92 μM
in in vitro assays using p-nitrophenyl phosphate. The mechanism of action does not
involve oxidation of the catalytic cysteine but instead binds precisely in the active
pocket and establishes tight hydrogen bonds and hydrophobic interactions with key
catalytic residues, namely, Asp29, Cys124, Glu126, Ser129, and Arg130. In vivo
studies with HeLa cells exposed to GATPT caused pERK and pJNK accumulation,
fairly similar to effects from DUSP3 siRNA knockdown. This result suggests
selective DUSP3 inhibition, showing strong results at a 5.0 μM concentration. Cell
cycle progression was also evaluated, and GATPT was shown to arrest cells at the
G1/S transition, according to what has been observed for DUSP3 loss of function.
Moreover, GATPT did not cause any changes in cell cycle progression in DUSP3
knockdown cells, once again indicating the selective action of this inhibitor (Shi
et al. 2007).
Our group has also assayed the in vivo effects of low doses of GATPT (250 nM)
in HeLa (cervix carcinoma) and MeWo (metastatic melanoma) cells and observed
higher accumulation of DNA damage and higher pERK1/2 and pH2AX levels
following gamma radiation exposure. Also, a downregulation of homologous
recombination (HR) and nonhomologous end joining (NHEJ) pathways of DNA
repair, following pretreatment with GATPT and subsequent gamma radiation of
HeLa cells, was assessed by the use of EJ5-GFP and DR-GFP constructs (Bennardo
et al. 2008). The accumulation of DNA damage, as assessed by the alkaline comet
assay, was also seen in the MRC-5 fibroblast cell line and in the ATM-deficient cell
lines (AT5-BIVA) (Torres et al. 2017), showing the impact of DUSP3 inactivation
on genomic instability.
Park et al. (2008) assayed 194 compounds that scored high on docking simula-
tions against DUSP3 and found 17 molecules that displayed at least 50% inhibition
at 50 μM concentration. The two most potent inhibitors shared a common
[2-(2,5-dioxoimidazolidin-4-ylidenemethyl)-pyrrol-1-yl]-benzoic acid scaffold and
showed IC50 values around 4 μM (Table 1, compound 12 and 13). The authors
explored the features of the lowest-energy conformation of these two compounds,
which are similar to the cases in which the benzoate group points toward the catalytic
residue and is stabilized by four hydrogen bonds involving Arg125 and Arg130.
However, they differ in that the imidazolidine-2,4-dione group is accommodated in a
small binding pocket for the first molecule but is exposed to bulk solvent for the
second one, most likely due to bulky chlorophenyl group substitution.
In 2009, Park et al. (Park et al. 2009) assessed NSC-87877 inhibitory activity
(Table 1, compound 14), a known SHP-2 inhibitor, against DUSP3. They derived
an IC50 value of 7.83 0.98 μM and deduced that this quinolinesulfonic acid acts as
a competitive inhibitor because of binding in the catalytic site. They also performed
in vitro experiments with active, phosphorylated ERK in the presence of DUSP3 and
increasing concentrations of NSC-87877, and they observed dose-dependent inhi-
bition of DUSP3 activity toward ERK. In vivo experiments with EGF-treated HEK
293 cells and transfected with a FLAG-DUSP3 expression plasmid also demon-
strated a rise in phospho-ERK levels as a result of increasing concentrations of the
inhibitor. This compound, however, shows inhibitory potential for several other
PTPs, therefore being considered very nonspecific.
In the same year, a group of researchers at the Burnham Institute (La Jolla, USA)
and Liège University (Liège, Belgium) developed multidentate small molecule
inhibitors with potent and selective activity over DUSP3 (Wu et al. 2009). Starting
from screening of thousands of drug-like molecules, they picked the most active hit,
2-((Z)-4-oxo-5-((E)-3-phenylallylidene)-2-thioxothiazolidin-3-yl)ethanesulfonic
acid. They subsequently searched for analogs of this compound, keeping the oxo-
thioxothiazolidinyl-ethanesulfonic acid moiety as the hydrophilic target for the
catalytic cleft and searching for additional hydrophobic regions to stabilize the
docking into the active site. Five of these structures displayed inhibitory activity
on the nanomolar scale and were at least ten times more potent for DUSP3 than other
PTPs (Table 1, compound 15–19). They also assayed the in vivo performance of
these compounds, finding SA3 to be the most active, as a result of displaying strong
antiproliferative effect over CaSki and HeLa cells and reduction in cell growth and
thymidine incorporation without inducing apoptosis. However, the compounds did
not significantly hamper normal keratinocyte cell growth, indicating they would be
good candidates for cancer treatment. SA3 was also co-crystallized with DUSP3,
showing clear electron density in the active site for the oxo-thioxothiazolidinyl-
ethanesulfonic acid moiety, displaying multiple hydrogen bond interactions within
the P-loop and a salt bridge with the guanidinium group of Arg130 (Wu et al. 2009).
Our group has also assessed in vivo effects of SA3 (Torres et al. 2017); by using
concentrations of 20 μM in HeLa and MeWo cells, we observed an increase in
sensitivity to gamma radiation as assessed by growth curves and senescence-
associated staining. Similar to the results obtained with GATPT pretreatments, we
also saw an increase in DNA damage accumulation after ionizing radiation expo-
sure, as assessed by comet assays, and an increase in pH2AX and pERK1/2 levels.
Also, there was a significant (~50%) reduction in nonhomologous end joining and
homologous recombination pathways of DNA strand break repair in HeLa cells
pretreated with SA3 and following exposure to gamma radiation regimes (Torres
et al. 2017).
The ability that the aforementioned DUSP3/VHR inhibitors present in disrupting
DUSP3-mediated biological effects emphasizes the interest in finding better, safer,
and specific inhibitors for this somewhat elusive enzyme that belongs to the DUSP
subfamily. Obviously, all the published work is just the beginning, and much
remains to be evaluated, namely, the selectivity of these drugs against other
ADUSPs, potential side effects on other key metabolic enzymes, and the availability
of more potent compounds. However, much of these issues would be addressed in
clinical trials, and the properties of these molecules offer a great starting point for the
development of better drugs.
5 Conclusions
DUSP3/VHR-knockout mice have been generated for functional studies of this dual
phosphatase in vivo, and no characteristic disease-associated phenotypes were
observed in these models. However, when these animals or cells derived from
them were challenged with different stimuli or stress, this enzyme deficiency
revealed very promising phenotypes (Amand et al. 2014; Singh et al. 2015;
Vandereyken et al. 2017b). For example, in terms of cancer biology, the majority
of the published reports discussed in this review shows opposite functions for
DUSP3. Whereas DUSP3 loss of function inhibits proliferation and invasiveness
of cancer cell lines in accordance with the oncogene addiction model (Weinstein and
Joe 2008), thus defining this phosphatase as an oncogene that regulates the growth
and survival of cancer cells, in other cases the gain of function of DUSP3 could
generate higher proliferation, migration, and invasiveness capacity of cells; therefore
DUSP3 could be considered a tumor suppressor protein. In addition, the well-
demonstrated effects of DUSP3 on the control of angiogenesis and neoangiogenesis
in vivo strongly support its potential tumorigenesis-promoting activities. However, it

is not possible to choose which DUSP3 function a drug will inhibit in a human
patient; all of them could actually be affected. Hence, if a DUSP3 inhibitor can have
protumorigenic effects, it will be contraindicated in all patients since we cannot
ensure that any patient does not bear precancerous lesions of any relevant type.
Therefore, it would be relevant to establish in animal models if the existing DUSP3
inhibitors are pro- or antitumorigenic and only if the protumor effects could be
excluded for sure that such inhibitors could be released for clinical usages.
Moreover, several other older – and more recent – studies shed light on the
importance of DUSP3 regulating the physiological and biological roles of the
circulatory system such as immune responses to septic shock and thrombosis
inhibition and provide subsidies for future investigations on the therapeutic potential
of DUSP3 in diseases related to the circulatory system, where regulating its expres-
sion and/or activity by pharmacological drugs or genetic therapies could prove to be
beneficial. According to all of the revisited DUSP3 molecular and biological func-
tions, its potential use as a drug target is the primary focus of many groups working
with medicinal chemistry. But obviously, these DUSP3 promising properties require
proof-of-concept clinical trials to be conducted and probably further investments on
synthesis to develop structural analogs with improved potency, specificity, perme-
ability, and bioavailability. In conclusion, this deep review will contribute to facil-
itate the development of therapies involving DUSP3/VHR inhibition in cases where
the loss of their function impairs the growth of cancer cells, improves the immune
responses against pathogens, or even ameliorates cellular responses against some
existing therapies (chemo- or radiotherapy), especially when the inhibitors do not
affect normal cellular processes or healthy tissues.
Acknowledgments This project was supported by FAPESP (Grants # 2008/58264-5 and # 2015/
03983-0) and CNPq (Grant # 402230/2016-7) to FLF, head of the Laboratory of Signaling in
Biomolecular Systems (LSBS). LCR is a senior postdoctoral fellow from the CAPES-PNPD
program at the Institute of Chemistry, University of Sao Paulo. JOF is a PhD student fellow of
Fapesp (# 2017/16491-4), and PYFM is a master’s fellow of CNPq, both enrolled at the
postgraduation program in Biochemistry and Molecular Biology, Institute of Chemistry, University
of Sao Paulo. LFM is a master’s fellow of CAPES at the Biotechnology program, also in University
of Sao Paulo. All authors thank BO and JRD for technical assistance in the LSBS laboratory.
Financial Support Fapesp, Capes, and CNPq.
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DOI: 10.1007/112_2018_13
Published online: 5 December 2018
Oncotic Cell Death in Stroke
Kep Yong Loh, Ziting Wang, and Ping Liao
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2 Pathways of Sodium Influx in Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.1 Na+/K+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.2 Glutamate Receptor Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3 Acid-Sensing Ion Channels (ASICs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.4 Na+/H+ Exchanger (NHE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.5 Electrogenic Na+/HCO3 Co-transporter 1 (NBCe1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.6 Transient Receptor Potential (TRP) Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3 Pathways of Chloride Influx in Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.1 Na+-K+-2Cl Co-transporter 1 (NKCC1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4 Pathways of Potassium Efflux in Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1 KCNQ (Kv7) K+ Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.2 ATP-Dependent K+ Channels (KATP Channels) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5 Aquaporins (AQPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
6 Mitochondrial Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
7 Key Challenges and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
K. Y. Loh
Duke-NUS Medical School, Singapore, Singapore
Calcium Signalling Laboratory, National Neuroscience Institute, Singapore, Singapore
e-mail: e0011140@u.duke.nus.edu
Z. Wang
Department of Urology, National University Hospital, Singapore, Singapore
e-mail: ziting_wang@nuhs.edu.sg
P. Liao (*)
Duke-NUS Medical School, Singapore, Singapore
Calcium Signalling Laboratory, National Neuroscience Institute, Singapore, Singapore
Singapore Institute of Technology, Singapore, Singapore
e-mail: ping_liao@nni.com.sg
38 K. Y. Loh et al.
Abstract Oncotic cell death or oncosis represents a major mechanism of cell death
in ischaemic stroke, occurring in many central nervous system (CNS) cell types
including neurons, glia and vascular endothelial cells. In stroke, energy depletion
causes ionic pump failure and disrupts ionic homeostasis. Imbalance between the
influx of Na+ and Cl ions and the efflux of K+ ions through various channel proteins
and transporters creates a transmembrane osmotic gradient, with ensuing movement
of water into the cells, resulting in cell swelling and oncosis. Oncosis is a key
mediator of cerebral oedema in ischaemic stroke, contributing directly through
cytotoxic oedema, and indirectly through vasogenic oedema by causing vascular
endothelial cell death and disruption of the blood-brain barrier (BBB). Hence,
inhibition of uncontrolled ionic flux represents a novel and powerful strategy in
achieving neuroprotection in stroke. In this review, we provide an overview of
oncotic cell death in the pathology of stroke. Importantly, we summarised the
therapeutically significant pathways of water, Na+, Cl and K+ movement across
cell membranes in the CNS and their respective roles in the pathobiology of stroke.
Keywords Cerebral oedema · Ion channels · Oncosis · Stroke
Abbreviations
AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

AQP Aquaporin
ASIC Acid-sensing ion channels
ATP Adenosine triphosphate
BBB Blood-brain barrier
CCC Chloride co-transporter
CNS Central nervous system
CSF Cerebrospinal fluid
GABAA γ-Aminobutyric acid type A
iNOS Inducible nitric oxide synthase
MCAO Middle cerebral artery occlusion
NBCe1 Na+/HCO3 co-transporter 1
NHE Na+/H+ exchanger
NKCC1 Na+-K+-2Cl co-transporter 1
NMDA N-methyl-D-aspartate
PI Propidium iodide
ROS Reactive oxygen species
SUR1 Sulfonylurea receptor isoform 1
TNF-α Tumour necrosis factor alpha
TRP Transient receptor potential
UCP Uncoupling protein
Oncotic Cell Death in Stroke 39
1 Introduction
In stroke, cell death exhibits two types of morphological changes: cell swelling, also
known as oncosis or oncotic cell death (Weerasinghe and Buja 2012), and cell
shrinkage, a hallmark of programmed cell death (Majno and Joris 1995). There are
two widely studied programmed cell death pathways: apoptosis and autophagy. In
oncosis, the root cause is increased membrane permeability, leading to cellular
swelling, organelle swelling and blebbing. In apoptosis, cell shrinkage and fragmen-
tation into membrane-bound apoptotic bodies are typical features. Following oncosis
or apoptosis, cells undergo irreversible necrosis and phagocytosis, which are uni-
versal processes in all cell death pathways (Fig. 1) (Lipton 1999). In stroke, there
have a heterogeneous distribution of oncotic and apoptotic processes across affected
brain regions. Cell apoptosis predominates in regions of reduced severity of ische-
mic damage (penumbra) as compared to the infarct core and following a shorter
period of ischaemia as compared to prolonged ischaemia (Charriaut-Marlangue et al.
1996). Pertinent factors influencing the outcome of cell death include cell type, age
and state of the cell at the point of insult (Martin et al. 1998). This review will discuss
oncotic cell death in stroke. Other types of cell death can be found in previous
excellent reviews (Fricker et al. 2018; Lipton 1999).
All types of cells in the brain including neuron, glia and vascular cells can
undergo oncosis following stroke. Buja et al. summarized the changes of cellular
oncosis into three stages (Buja 2005): In stage 1, ionic pumps fail to maintain ionic
balance across cell membrane due to a depletion of adenosine triphosphate (ATP),
and cell swelling ensues as water fluxes into the cell accompanying ions. It has been
predicted that when energy depletion lowers pump strength to less than 65% of
baseline, membrane potential is no longer preserved, and ionic homeostasis is
disrupted, leading to rapid influx of sodium and chloride ions (Dijkstra et al.
2016). During stage 2, membrane damage is irreversible, and more severe, allowing
large molecules such as propidium iodide (PI) and trypan blue to enter the cells.
Stage 3 represents the eventual physical disruption of the cell membrane, suggesting
that the cells are in the necrotic phase. Stage 3 is not unique to oncosis, but a
common pathway for all types of cell death.
The specific ionic composition of the cytosol is significantly different from
extracellular fluid (Fig. 2a). Cytosolic fluid contains a higher concentration of K+
and negatively charged protein, whereas extracellular fluid contains a higher con-
centration of Na+, Ca2+ and Cl . This ionic gradient is critical for cellular functions
including the formation of membrane potential. Various pumps consume ATP to
maintain this ionic gradient. After stroke, ATP depletion causes the functional failure
of the pumps, leading to an ionic flux down their respective gradients across the
cytosolic membrane (Fig. 2b). Na+, Ca2+ and Cl flow into the cell, whereas K+ ions
flow outside the cell. During the early stage of ATP depletion, cytosolic membrane is
relatively intact and impermeable to the intracellular negatively charged protein.
Therefore, no transmembrane movement of protein occurs. If ionic influx is greater
than efflux, a higher intracellular osmotic pressure is created. Water thus moves into
40 K. Y. Loh et al.
Fig. 1 Two major types of morphological cell changes in cell death: oncosis and apoptosis, as
characterised by cell swelling and shrinkage, respectively, with a common downstream pathway of
necrosis and phagocytosis (Reproduced with permission from Majno and Joris 1995)
the cell to balance the osmotic pressure, leading to cell swelling and oncosis. Given
that the extracellular Ca2+ concentration (1–2 mM) is much lower than that of Na+
(140–145 mM) and Cl (110 mM) concentrations, the osmotic pressure change
caused by Ca2+ influx is expected to be less prominent than Na+ and Cl influx.
Therefore, inhibition of sodium chloride influx following stroke could play a critical
role in preventing oncosis.
a Intracellular Extracellular
Na+ 5-15 mM Na+ 140-145 mM
K+ 140-150 mM K+ 4-5 mM
Ca2+ 0.1-1.5 μM Ca2+ 1.8 mM
Cl- 4-30 mM
Negavely charged proteins Cl- 110 mM
b Intracellular Space Extracellular Space
Na+
K+
Cl-
Ca2+
Fig. 2 (a) Ionic composition of intracellular and extracellular environment with the higher ionic
concentrations in bold. (b) Net ionic movement across cell membranes in ischaemia
Cerebral oedema is a well-documented complication of stroke (Stokum et al.

2016). There are two types of cerebral oedema in stroke: cytotoxic and vasogenic
oedema. Oncosis is the direct cause of cytotoxic oedema and indirectly involved in
vasogenic oedema. In cytotoxic oedema, the pumps in the cell membrane of
astrocytes and neurons cease to function, causing electrolyte accumulation in the
cell and subsequent cell swelling or oncosis. As cytotoxic oedema represents the
redistribution of water from extracellular to intracellular space, the total water
content does not change if the blood-brain barrier (BBB) is intact. Cytotoxic oedema
affects both grey matter and white matter as astrocytes are involved (Chu et al.
2007). During stroke, oxygen and glucose depletion also leads to cell death in the
vasculature, causing BBB disruption. Blood content including water is thus leaked
into the brain parenchyma, resulting in vasogenic oedema. Migration of water
outside vasculature can result in a rapid increase in intracranial pressure and may
cause severe complications such as brain herniation. Oncosis participates in
vasogenic oedema indirectly by causing endothelial cell death (Khanna et al.
2014). In cerebral ischemia, cessation of energy supply leads to oncosis in endothe-
lium, in a similar manner to astrocytes and neuronal oncosis. Endothelial cell death
increases BBB permeability, resulting in vasogenic oedema. It should be noted that
vasogenic oedema requires some blood flow. If blood flow ceases completely, no
water is able to migrate outside of the vasculature, even under the circumstances of
BBB disruption. This explains why cerebral oedema is less pronounced in the infarct
core as compared to the penumbral regions due to paucity of blood flow and why
oedema can be exacerbated by reperfusion therapies.
In the following sections, we summarise the pathways of water and major
electrolyte movements across the cell membrane during stroke, namely, Na+, Cl
influx and K+ efflux.
42 K. Y. Loh et al.
2 Pathways of Sodium Influx in Stroke
In stroke, the primary initiating event is the arrest of oxidative phosphorylation and
reduction in ATP production. The resultant deactivation of ATP-dependent Na+
pumps, activation of glutamate-dependent channels, pH-sensitive proton-dependent
channels and other Na+- conducting ion channels culminate in uncontrolled accu-
mulation of intracellular Na+, causing anoxic depolarization. Pharmacological inhi-
bition of Na+ fluxes has exhibited neuroprotective effects early in ischaemic insult up
to 2 h post-ischaemia, demonstrating that the deleterious role of Na+ flux extends
well into the post-ischaemic period (Crumrine et al. 1997). Possible mechanisms of
Na+-induced damage in ischaemia include stimulation of glutamate release, increas-
ing intracellular Ca2+ and depletion of ATP via activation of the Na+/K+ pump
(Lipton 1999). Importantly, the influx of NaCl can generate a transmembrane
osmotic gradient, leading to water movement into the intracellular environment,
causing cell swelling and oncotic cell death (Trump et al. 1997; Weerasinghe and
Buja 2012). Here, we examine the major pathways of sodium influx in stroke which
is of therapeutic significance.
2.1 Na+/K+-ATPase
Na+/K+-ATPase, a member of the P-type ATPase protein family, is found univer-

sally in all mammalian cell membranes, including neurons and glial cells. It consists
of heterodimeric α, β and γ subunits (Moseley et al. 2003). Functionally, it maintains
the resting membrane potential by actively exchanging 3 Na+ from the intracellular
environment for 2 K+ from the extracellular environment. It utilises ATP as a major
source of energy and is exquisitely sensitive to ATP depletion (Glushchenko and
Izvarina 1997; Pietrini et al. 1992). Diminished oxygen and ATP in ischaemic stroke
causes pump failure, culminating in a rapid accumulation of cytosolic Na+ and Ca2+
and depletion of cytosolic K+. Expression of Na+/K+-ATPase was found to be
reduced in the ischaemic penumbra following focal cerebral ischaemia in rats
(Jung et al. 2007). This process represents one of the earliest cellular events in
ischaemic stroke. As such, Na+/K+-ATPase pump failure inescapably plays a
significant role in cellular oncosis, apoptosis and necrosis in ischaemic stroke
(Hu and Song 2017; Fann et al. 2013; Small et al. 1999; Jung et al. 2007).
Given its critical role in the pathogenesis of ischaemic stroke, Na+/K+-ATPase
represents a potential therapeutic target. Theoretically, pharmacological Na+/K+-
ATPase inhibition is assumed to mimic ischaemic pump failure, resulting in
uncontrolled ionic influx and oncosis, resulting in exacerbation of ischaemic cell
damage. However, mounting evidence has suggested that Na+/K+-ATPase inhibi-
tion, in fact, decreases cell death mainly through (1) ATP conservation (Liu and
Yenari 2007; De Angelis and Haupert 1998) and (2) inhibition of autophagy (Hu and
Song 2017; Liu et al. 2013). This was supported by studies demonstrating the
beneficial effects of cardiac glycosides, a Na+/K+-ATPase inhibitor, in neuro-

protection, by inhibition of autophagic cell death in a cerebral hypoxia-ischaemic
model (Liu et al. 2013).
In addition, the postulation that Na+/K+-ATPase pump function is disrupted in
stroke has been challenged by previous studies demonstrating increased or
unchanged pump activity in ischaemic stroke models (Goldberg et al. 1984;
MacMillan 1982). This contradicts the understanding of the pathological role of
Na+/K+-ATPase pump dysfunction and the resultant dysregulation of ionic flux,
oncosis and apoptosis in stroke. Further evidence is necessary to further define the
pathological role of Na+/K+-ATPase in stroke. This will provide greater clarity to
the neuroprotective effects of Na+/K+-ATPase pump inhibition.
2.2 Glutamate Receptor Channels
Glutamate is the main excitatory neurotransmitter in the central nervous system. It is

the key mediator of essential brain functions, synaptic transmission and plasticity
and neurodevelopment (Frenguelli 2013). Important ionotropic glutamate receptors
which have been extensively studied in stroke pathology include N-methyl-D-
aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) receptors. These receptors are ubiquitous in neurons and glial cells (astro-
cytes and oligodendrocytes) (Dzamba et al. 2013), playing particularly essential
roles in ischaemic axonal injury and hypoxic-ischaemic oligodendrocyte damage
(Domercq et al. 2005).
Glutamate-dependent excitotoxicity has been regarded as the chief cytotoxic
mechanism in stroke (Chamorro et al. 2016). In the ischaemic core, anoxic depolar-
ization ensues, causing enhanced glutamate release and decreased reuptake. Gluta-
mate accumulation in the synaptic and extracellular spaces results in excessive
stimulation of NMDA and AMPA receptors (Choi and Rothman 1990). Upon
activation, NMDA receptors permit the flow of K+, Na+ and Ca2+ ions. AMPA
receptors conduct K+ and Na+ ions, and additionally, Ca2+ in the absence of
the GluR2 subunit (Traynelis et al. 2010). Intracellular accumulation of Ca2+
ions activates calcium-dependent enzymes which digest essential structural
proteins, leading to loss of cellular integrity and neuronal death (Lai et al. 2014).
Acute excitotoxic neurodegeneration is also contributed by the drastic influx of Na+
and Cl ions, likely attributed to oncosis (Walz 1992; Rothman 1985). Inhibition of
NaCl influx was found to eliminate NMDA-mediated neurodegeneration (Hasbani
et al. 1998).
Unsurprisingly, given its key role in the pathophysiology of stroke, antagonism of
glutamate receptors has been the major subject of translational stroke research for the
past decades (Hu and Song 2017). Despite triumphs in the use of memantine in
dementia, and plausible benefits in other central nervous system (CNS) pathologies
including Parkinson’s disease and epilepsy, success has been elusive in the realm of
stroke research (Chen et al. 1992; Geter-Douglass and Witkin 1999; Merello et al.
44 K. Y. Loh et al.
1999; Liang et al. 2007). Limitations in the glutamate-based approach could be

explained by the transient and self-limiting nature of glutamate-dependent
excitotoxicity upon synaptic vesicle depletion, leaving a narrow window of thera-
peutic opportunity (Leiva-Salcedo et al. 2017). This is supported by the fact that
positive results from preclinical trials involved drug administration at an early stage
of stroke onset (Chamorro et al. 2016; Lai et al. 2014), and late administration may
be injurious to the post-stroke neuro-recovery process (Song et al. 2017). In addition,
non-selective blockade of glutamate receptors outside the ischaemic area could
generate severe side effects (Yu et al. 2013). Other important mechanisms may
also contribute to the pathological pathway in stroke. Targeting the glutamate-
independent process of excitotoxicity which is regulated by the sulfonylurea receptor
isoform-1 (SUR1)-regulated NCCa-ATP (Simard et al. 2006) and transient receptor
potential (TRP) channels represent an interesting alternative therapeutic approach
(Leiva-Salcedo et al. 2017) and will be reviewed in the following sections.
2.3 Acid-Sensing Ion Channels (ASICs)
ASICs are proton-gated cation channels belonging to the degenerin/epithelial Na+

channel superfamily. Six protein subunits have been identified which are encoded
by four genes: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4 which
co-assemble to form heteromeric and homomeric channels. These channels are
highly expressed in peripheral sensory neurons and CNS neurons. Peripherally,
these channels are distributed in the dorsal root and trigeminal ganglion. Centrally,
they are located in the cerebral cortex, cerebellum, hippocampus, amygdala and
olfactory bulb (Wemmie et al. 2003; Xiong et al. 2008; Waldmann et al. 1997).
ASIC1a has been found to be the major subunit in CNS neurons. ASIC channels in
CNS neurons are predominantly ASIC1a homomeric channels and ASIC1a/2a
heteromeric channels, with ASIC1a chiefly responsible for generating current
amplitude (Baron et al. 2002; Askwith et al. 2004).
In stroke, the anoxic environment leads to a predominance of anaerobic respira-
tion, resulting in the accumulation of lactic acid and protons, fall in pH and
subsequent activation of ASICs. All ASICs conduct an inward sodium current in
response to extracellular protons, which result in neuronal membrane depolarization
and excitation. ASIC1a homomeric channels are additionally permeable to Ca2+.
ASIC1a is also functionally coupled with NMDA receptors by the activation of the
Ca2+/calmodulin-dependent protein kinase II cascade. These potentiate the role of
ASIC1a in intracellular Ca2+ and Na+ accumulation and acidotoxicity in ischaemic
conditions (Radu et al. 2016; Xiong et al. 2008).
The role of ASICs in stroke pathology has been further supported by studies
which showed that ASIC1a gene knockout has a neuroprotective effect in ischaemic
neurons. Pharmacological inhibition of ASIC1a using amiloride also successfully
reduced infarct volume in transient and permanent rodent stroke models (Miao et al.
2010; Pignataro et al. 2007; Xiong et al. 2004). Flurbiprofen, a non-steroidal
anti-inflammatory drug which inhibits ASIC1a, has been shown to be beneficial in

both acute and late phases of stroke (Mishra et al. 2010). Recognising the coupling
of ASIC1a and NMDA in causing intracellular Ca2+ accumulation, co-inhibition of
the two channels has been examined with encouraging results. Co-inhibition of the
two channels provides superior neuroprotective effect compared to singular inhibi-
tion, and ASIC1a blockage prolongs the therapeutic time window of NMDA block-
age (Pignataro et al. 2007). In addition to stroke, ASICs have also been targeted in
other CNS pathologies, including epilepsy, migraine and neurodegenerative diseases
(multiple sclerosis, Parkinson’s disease and Alzheimer’s disease). Peripherally,
ASIC inhibition has been investigated in the treatment of peripheral pain and
diabetic neuropathy (Radu et al. 2016).
2.4 Na+/H+ Exchanger (NHE)
Mammalian NHEs are integral membrane ion channels that catalyse the exchange of
intracellular H+ for Na+. They perform an essential role in the regulation of intra-
cellular pH, Na+ content, volume and cellular proliferation in epithelial and
non-epithelial cells. Nine NHEs have been identified, and they can be classified as
plasma membrane NHEs (NHE1–5) and organellar NHEs (NHE6–9) (Ohgaki et al.
2011).
NHE1 is expressed ubiquitously in the CNS and functions to regulate intracellular
pH and volume in neurons and glial cells (Ma and Haddad 1997). Increased NHE1
expression was detected in the ischaemic penumbra in a rat model of focal cerebral
ischaemia (Jung et al. 2007). NHE1 activity is enhanced following intracellular
acidosis in stroke as a major pathway of proton extrusion, functioning as a protective
mechanism. The increase in NHE1 expression, on the other hand, contributes to
intracellular Na+ accumulation and resultant cell death in ischaemia. NHE1 inhibitor
cariporide has been successfully shown to reduce neuronal cell death and decrease
intracellular Na+ and Ca2+ concentrations in cultured mouse cortical neurons (Luo
et al. 2005). Furthermore, a reduced infarct volume was observed in NHE1 knockout
mice as compared to wild type following transient ischaemia and reperfusion (Luo
et al. 2005). These evidences support the role of NHE1in ischaemic neuronal injury.
NHE1activity is also enhanced in other ischaemic pathologies, notably, in cardiac
myocytes during ischaemic-reperfusion injury to maintain intracellular pH (Avkiran
2001; Avkiran et al. 2001).
Of note, NHE also functions physiologically to regulate vectorial ion transport
across epithelia, being expressed in both apical and basolateral membranes. In
particular, NHE1 and NHE2 isoforms have been found in the luminal membrane
of endothelial cells constituting the BBB (Wang et al. 2003; Noel et al. 1996;
Mokgokong et al. 2014). BBB Na+ transporters have been implicated in early
ischaemia-induced cerebral and astrocytic oedema in stroke prior to breakdown of
the BBB (Schielke et al. 1991). Studies have shown that NHE1 and NHE2 are
upregulated in cerebral microvascular endothelial cells in response to hypoxia and
46 K. Y. Loh et al.
aglycaemia, implying a contributory role of NHE in early cerebral oedema in stroke

(Lam et al. 2009; Mokgokong et al. 2014). This is further supported by successful
reduction of cerebral oedema in middle cerebral artery occlusion (MCAO) rat
models with the use of NHE-blocking SM20220 (Suzuki et al. 2002; Kitayama
et al. 2001).
2.5 Electrogenic Na+/HCO3 Co-transporter 1 (NBCe1)
NBCe1, a bicarbonate-dependent acid-based membrane transporter, is another

important pH regulator, expressed in epithelial cells and the CNS (Schrodl-Haussel
et al. 2015). It is found predominantly in astrocytes (Theparambil et al. 2014) but
also expressed in neurons in the CNS (Svichar et al. 2011). The voltage-gated
NBCe1 functions to conduct Na+ and HCO3 with a stoichiometry of 1:2, respec-
tively, in both directions across the glial cell membranes, hence moderating both
intracellular and extracellular pH (Brookes and Turner 1994; Brune et al. 1994).
Changes in expression of NBCe have been observed in CNS pathologies including
stroke (Jung et al. 2007) and seizures (Kang et al. 2002). NBCe also mediates
important renal and extra-renal functions, and dysfunction is associated with pathol-
ogies including proximal renal tubular acidosis, short stature, migraine and ocular
and enamel abnormalities (Seki et al. 2013).
In stroke, membrane depolarization induced by extracellular K+ accumulation is
the key activator of glial NBCe1, augmenting the inward Na+ and HCO3 current
(Bevensee et al. 1997; Schrodl-Haussel et al. 2015). In the early stage of ischaemia,
this alkalinising mechanism performs a key role in the maintenance of K+ homeo-
stasis by astrocytes. The alkalinising mechanism supports the activity of Na+/K+-
ATPase under ischaemic conditions, therefore sustaining active transport of K+ from
the external environment into glial cells (Leis et al. 2005). Coupled with its intra-
cellular pH-moderating effects, this suggests a protective function of NBCe1
alkalinising current in ischaemia (Kumar et al. 2007). NBCe1 expression has been
found to be upregulated in the ischaemic penumbra after focal cerebral ischaemia in
rats (Jung et al. 2007). Inhibition of NBCe1 was shown to exacerbate ischaemia-
induced astrocyte death in vitro, although the exact mechanism has not been clarified
and may not be related to dysregulation of intracellular pH (Yao et al. 2016).
However, conflicting findings in literature have challenged the alleged protective
role of NBCe1 in ischaemia. Other work has shown that ischaemic cardiac injury
was reduced by inhibition of NBCe (Fantinelli et al. 2014; Khandoudi et al. 2001).
With prolonged ischaemia, the alkalinising mechanism of NBCe1 may contribute to
accumulation of intracellular Na+, leading to the deleterious cascade of
excitotoxicity and oncotic cell death (Jung et al. 2007; Leis et al. 2005). This is
apparent in the observation of decreased buffering capacity of reactive astrocytes in
the proximity of the ischaemic core as compared to regions further away (Leis et al.
2005).
As such, this evidence suggests that early inhibition of NBCe1 may be

deleterious, whereas late inhibition post-stroke may be beneficial in achieving
neuroprotection. Further studies are required to better characterise the conflicting
role of NBCe in stroke, focusing on the temporal effects of NBCe1 inhibition.
2.6 Transient Receptor Potential (TRP) Channels
The TRP channel superfamily consists of about 30 members grouped into

6 subfamilies based on amino acid sequence homology: TRPA (ankyrin), TRPC
(canonical), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin) and
TRPV (vanilloid) (Wu et al. 2010). They perform a key role in regulation of
intracellular Ca2+ concentrations, and hence, its role in stroke pathology has been
actively investigated. These channels are widely expressed in the CNS, including
neurons, astrocytes, oligodendrocytes, microglia, ependymal cells, cerebral vascular
endothelium and smooth muscles. The subtypes TRPC3/4/6, TRPM2/4/7 and
TRPV1/3/4 have been implicated in ischaemic cell death (Zhang and Liao 2015).
Of note, the TRPM4 channel represents a pathway of unchecked Na+ entry and
contributes to oncotic cell death in stroke. TRPM4 is a non-selective, monovalent
cation-conducting channel that is activated by increased intracellular Ca2+ concen-
trations, inhibited by adenosine triphosphate (ATP) and altered by reactive oxygen
species (Vennekens and Nilius 2007). It has been established that TRPM4
co-assembles with sulfonylurea receptor isoform 1 (SUR1) to form a unique
SUR1-TRPM4 channel analogous to the SUR1-NCCa-ATP channel. SUR1-NCCa-ATP
channel activity is enhanced in numerous CNS pathologies including stroke,
traumatic brain injury, subarachnoid haemorrhage and spinal cord injury. SUR1-
TRPM4 was reported to be upregulated in neurons, astrocytes and cerebral vascular
endothelium (Simard et al. 2012; Woo et al. 2013). Under ischaemic conditions,
activation of SUR1-TRPM4 channel is purported to cause a sustained, uncontrolled
Na+ influx, causing excitotoxicity via a glutamate-independent mechanism and
leading to oncotic cell death (Leiva-Salcedo et al. 2017).
SUR-1 inhibition by antidiabetic glibenclamide has shown promise in achieving
neuroprotection in stroke (Simard et al. 2009, 2014). TRPM4 was revealed to be
upregulated in the cerebral vascular endothelium of the penumbra in a permanent rat
stroke model, peaking at 1 day post-occlusion. siRNA-mediated inhibition of
TRPM4 facilitated angiogenesis and improved vascular integrity. Furthermore,
there was an observed reduction in infarct volume and improvement in motor
function in siRNA-treated rats. By protecting the BBB, TRPM4 inhibition may be
clinically useful in reducing risks of cerebral reperfusion therapies including cerebral
oedema and haemorrhage, hence potentially increasing the time window for reper-
fusion therapy (Loh et al. 2014). The therapeutic effect of TRPM4 blockade in
reduction of reperfusion injury was further elucidated in a transient MCAO rat
model. Following reperfusion, siRNA treatment reduced cerebral oedema shown
48 K. Y. Loh et al.
via multimodal imaging, reduced infarct volume, improved motor function and
demonstrated vascular protective effects (Chen et al. 2018).
Although TRPM4 inhibition has been shown to protect vascular integrity and
reduce brain injury following acute ischaemic stroke, the underlying mechanisms
other than oncosis have not been fully elucidated. Numerous studies have shown that
inflammation plays a crucial role in ischaemic-reperfusion injury in stroke (Anrather
and Iadecola 2016; Pan et al. 2007; Leiva-Salcedo et al. 2017). Under hypoxic
conditions in stroke, necrotic cells and BBB leakage release damage signals which
initiate a cascade of inflammatory response via recruitment of cellular and humoral
components of the immune system, which is facilitated by reperfusion. Conse-
quently, this creates an outburst of pro-inflammatory mediators and reactive
oxidative species surrounding the ischaemic region, resulting in amplification of
ischaemic damage (Anrather and Iadecola 2016; Kim et al. 2014; Lakhan et al. 2009;
Tuttolomondo et al. 2009; Vidale et al. 2017; Wang et al. 2007). TRPM4 inhibition
has been shown to reduce neuroinflammation in animal models of subarachnoid
haemorrhage (Tosun et al. 2013) and experimental autoimmune encephalomyelitis
(Makar et al. 2015). The role of TRPM4 inhibition in neuroinflammation in stroke
needs further investigation.
3 Pathways of Chloride Influx in Stroke
3.1 Na+-K+-2Cl Co-transporter 1 (NKCC1)
The chloride co-transporters (CCCs) catalyse the coupled transport of Na+, K+ and
Cl across cell membranes and modulate the chloride electrochemical gradient. The
chloride gradient across neuronal membranes then determines the direction of
chloride current flow through the γ-aminobutyric acid type A (GABAA) receptors,
either leading to cellular hyperpolarization through chloride influx or cellular
depolarization through chloride efflux (Kaila et al. 2014).
The CCCs consist of three members based on their homeostatic functions: Na+-
K -2Cl co-transporters (NKCC) (two isoforms NKCC1 and NKCC2), Na+-Cl
+
co-transporter and K+-Cl co-transporters (four isoforms KCC1 to KCC4). Other

than the Na+-Cl co-transporter and NKCC2 which are mainly renally expressed, the
rest of the CCCs are expressed in the mammalian nervous system (Martin-Aragon
Baudel et al. 2017).
The NKCC1 isoform plays a key role in regulation of intracellular chloride
concentration and cell volume in the CNS. It is expressed in glia, neurons, cerebral
vascular endothelial cells and choroid plexus epithelial cells (Gerelsaikhan and
Turner 2000). In physiological cellular ionic homeostasis in the CNS, Cl influx is
mediated by NKCC1 via electroneutral co-transportation of K+ and Na+ ions, which
is driven by the Na+ electrochemical gradient established by the Na+/K+-ATPase
pump. Consequently, the Cl influx promotes GABAA-mediated Cl extrusion,
resulting in cell depolarization (Martin-Aragon Baudel et al. 2017). This homeostatic

mechanism is essential in early neurodevelopment (Pfeffer et al. 2009).
Under hypoxic conditions in stroke, NKCC1 activity is enhanced by phosphor-
ylation, induced by multiple factors including depleted intracellular Cl concentra-
tions, accumulation of intracellular Ca2+ and increased β-adrenergic activity (Liang
et al. 2007). This increased NaCl inflow causes cytotoxic oedema and oncotic cell
death. Numerous studies have provided evidence in support of NKCC1’s role in
oedema and cell death in stroke. NKCC1 expression was enhanced in cortical
neurons and rat brain lysates from cerebral cortex and striatum in a transient stroke
model (Yan et al. 2003; Wang et al. 2014). Luminal NKCC1 in the BBB may also
mediate vasogenic oedema due to increased NaCl secretion into the brain during
ischaemia (O’Donnell et al. 2004). Notably, the use of NKCC1 antagonist,
bumetanide, significantly decreased sodium overload and cell death and attenuated
infarct volume and cerebral oedema in animal stroke models (Chen et al. 2005; Chen
and Sun 2005; Yan et al. 2003; Wang et al. 2014). In addition, delayed and
prolonged administration of bumetanide post-stroke was shown to promote neuro-
recovery and behavioural improvement in rats (Xu et al. 2017). The role of NKCC1
in neurogenesis is further supported by a study demonstrating positive effects in the
setting of traumatic brain injury through the HIF-1α pathway (Lu et al. 2015). These
highlight the potential therapeutic role of NKCC1 blockade in stroke.
4 Pathways of Potassium Efflux in Stroke
In the ischaemic brain, disruption of the Na+/K+-ATPase and upregulation of

selective K+ channel activity lead to excessive K+ efflux and increased extracellular
K+ concentrations. Marked accumulation of extracellular K+ concentrations and
decrease in intracellular K+ concentrations are recognised as the key steps leading
to cell volume shrinkage and apoptotic cell death (Remillard and Yuan 2004). This
mechanism constitutes the process of ‘spreading depression’ in the brain post-
ischaemia and is injurious to brain tissues (Somjen 2001). It also represents the
early event in programmed cell death before other death activators including caspase
activation (Gomez-Angelats et al. 2000). This pathological mechanism has been
identified in neurons and non-neuronal cells in ischaemia, as well as under other
pathological circumstances (Song and Yu 2014). At the same time, K+ efflux in acute
phase of excitotoxicity may be protective due to hyperpolarization which reduces
cell excitation (Weston and Edwards 1992). We review the significant K+ channels
involved in stroke.
4.1 KCNQ (Kv7) K+ Channels
KCNQ (Kv7) channels consist of four subunits anchored to a central K+-conducting

pore, with each subunit consisting of six transmembrane segments. KCNQ2–5
50 K. Y. Loh et al.
channels mediate M-type currents in the CNS (Wang and Li 2016). M-type currents
are time- and voltage dependent and perform a key role in regulation of neuronal
excitability by maintenance of resting membrane potential, modulating excitability
thresholds and spike generations. Activation of M-type currents results in a
hyperpolarising effect, while inhibition produces neuronal excitation (Bierbower
et al. 2015). KCNQ activity is controlled by numerous signalling pathways (Wang
and Li 2016). Retigabine, an M-channel activator and an anti-epileptic drug, has
been known to yield the therapeutic effect via hyperpolarising neuronal membrane
potential (Miceli et al. 2008).
Early in stroke, M-current is activated by reactive oxidative species and is thought
to be neuroprotective in response to cellular hypoxia (Boscia et al. 2006). M-channel
activators achieved neuroprotection, effecting a reduction of infarct volume and
neurological deficits in mouse photothrombotic and MCAO stroke models. This
has been attributed to the hyperpolarising effect of K+ efflux which reduces neuronal
hyper-excitability in ischaemia. Interestingly, in the same study, it was found that the
neuroprotective effects of M-channel activators were more pronounced when admin-
istered early (0–3 h post-stroke compared to 6 h), indicating a probable therapeutic
time window of efficacy (Boscia et al. 2006). This was further supported by another
in vitro study which demonstrated the oxidative stress enhancement of KCNQ-
mediated current, and inhibition of which during 30 min of oxygen-glucose depri-
vation exacerbated cell death. These imply the early neuroprotective effects of
enhanced K+ efflux in the acute phase of excitotoxicity in stroke.
However, other studies have shown that activation of KCNQ-mediated K+-efflux
may contribute to apoptosis in the CNS. KCNQ2/3 activators resulted in dose-
dependent K+ efflux, intracellular decrease in K+, caspase activation and cell death
in hippocampal neurons, which was attenuated by a KCNQ inhibitor. In Chinese
hamster ovarian cells, KCNQ activators initiate the cascade of events leading to cell
apoptosis, which was, again, prevented by KCNQ inhibitors (Zhou et al. 2011; Song
and Yu 2014). It is possible that KCNQ-mediated K+ efflux is beneficial in the acute
stage of stroke by inhibiting neuronal excitotoxicity. However, with prolonged
ischaemia, disruption of cellular membrane integrity and persistent channel activa-
tion could result in dysregulation of K+ homeostasis and yield a pro-apoptotic effect
which overcomes its neuroprotective effect. Such temporal effect of KCNQ inhibi-
tion/activation in stroke requires further clarification.
4.2 ATP-Dependent K+ Channels (KATP Channels)
KATP channels are expressed widely in cells of the CNS, including neurons, astro-
cytes, microglia, vascular smooth muscles and endothelium (Sun and Hu 2010).
KATP channels generally consist of two types of subunit: (1) a member of the K+-
inward rectifier family (Kir6.x) forming the central pore and (2) the SURx which
forms the regulatory subunit (Ashcroft and Gribble 1998). The four subunits present
in the CNS include Kir6.1, Kir6.2, SUR1 and SUR2, and these are heterogeneously
associated to form KATP channels in different CNS cell types. For example, the
Kir6.2 forms the central pore in most neurons, while Kir6.1 is predominantly
expressed in astrocytes and microglia (Thomzig et al. 2001, 2005). KATP regulates
neuronal, astrocytes, microglial and BBB functions in stroke, thus providing a
valuable therapeutic target (Sun and Hu 2010).
Under ischaemic conditions, with a decreased ATP:ADP ratio, activation of KATP
promotes a hyperpolarising K+ efflux current, reduces intracellular Ca2+ accumula-
tion, decreases excitatory glutamate release and the arrest of the ischaemic cascade,
providing a protective effect against neuronal excitotoxicity (Seino 2003). Iptakalim,
a KATP opener, reduced glutaminergic activity, promoted neuro-recovery and
decreased neuronal necrosis and apoptosis in in vivo and in vitro experiments
(Wang et al. 2004). The presence of the Kir6.2 subunit in mice showed a reduction
in period of neuronal depolarization post-ischaemia and neuroprotective effect,
while those without the subunit displayed more severe ischaemic neuronal damage
(Sun et al. 2006) and exquisite sensitivity to hypoxia-induced seizures (Yamada and
Inagaki 2005).
Besides reducing neuronal excitation, KATP may also reduce brain damage by
modulating other key players in the ischaemic cascade. Astrocytic gap junctions are
important in the transfer of toxic and beneficial molecules between cells in the
ischaemic core and penumbra. KATP opening was found to support the function of
gap junctions in astrocytes and may be beneficial in ischaemia (Sun and Hu 2010).
Activated microglia, the resident CNS macrophage, constitutes the early immune
response in stroke and exerts a deleterious effect by producing inflammatory and
cytotoxic mediators (Smith et al. 1998). Diazoxide, a KATP activator, was effective
in reducing microglial activation post-carotid artery occlusion in rat brain (Farkas
et al. 2005). In a Parkinsonian rat model, KATP activation suppressed microglial
inflammatory activity and inhibited the production of pro-inflammatory mediators
including inducible nitric oxide synthase (iNOS), tumour necrosis factor alpha
(TNF-α) and prostaglandin E2 (Zhou et al. 2008). Importantly, KATP may play a
critical role in regulating BBB integrity, as shown by the use of diazoxide post-
ischaemia-reperfusion to ameliorate cerebral oedema (Lenzser et al. 2005).
5 Aquaporins (AQPs)
AQPs are integral membrane proteins. Structurally, they consist of monomeric

subunits which assume a tetrameric arrangement to form channel pores in the cell
membrane. These pores constitute selective channels which allow transportation of
water molecules and small neutral solutes across membranes (Verkman 2013). The
AQP family consists of 13 subtypes (AQP0–12), which can be further divided into
3 subclasses based on the type of solute transported: (a) AQPs involved in water
transport only (AQP0,1,2,4,5), (b) aquaglyceroporins involved in water and small
solute transport (AQP3,7,9,10) and (c) unorthodox AQPs with unknown function
52 K. Y. Loh et al.
(AQP6,8,11,12) (Meli et al. 2018). AQPs are widely distributed in cells and tissues
and play essential roles in maintaining water homeostasis.
AQP4 is the major subtype found in the central nervous system. They are found
exclusively in astrocyte membranes, specifically the perivascular end-feet and glial-
limiting membranes at the boundary between the brain parenchyma and the
subarachnoid cerebrospinal fluid (CSF) compartment and below the ependyma at
the boundary of the brain parenchyma and the ventricular CSF compartment. AQP4
have also been found in microglial membranes following lipopolysaccharide
induction (Papadopoulos and Verkman 2007).
AQP4 performs a bimodal role in the pathology of cerebral oedema in ischaemic
stroke. In the early post-ischaemic period, AQP4 contributes to oncotic cell death
and cytotoxic oedema by facilitating water influx into astrocytes following
dysregulation of ionic flux as mentioned previously. This causes extensive astrocyte
swelling which exacerbates brain injury through vascular congestion. Additionally,
astrocyte swelling can increase cellular permeability to excitatory mediators such as
glutamate through opening of volume-regulated ion channels, enhancing excitotoxic
cell damage. Following the initial post-ischaemic period, with increased tissue death
and breakdown of the BBB, leakage of serum proteins set off the process of
vasogenic oedema. This can potentially cause significant brain swelling, leading to
catastrophic brain herniation and death. AQP4 performs a protective role in resolu-
tion of vasogenic oedema by mediating transcellular movement of water from the
astrocyte cell membranes of the glial limitans into the subarachnoid CSF, and from
the ependyma and sub-ependymal astrocyte membranes into ventricular CSF, and
lastly through astrocyte pericapillary foot processes into serum. Hence, inhibition of
AQP4 early post-ischaemia may attenuate the deleterious effects of cytotoxic
oedema and astrocyte swelling, while promotion of AQP4 activity in late ischaemic
stroke or haemorrhagic conversion may enhance oedema clearance and decrease
neuroinflammation by protecting BBB integrity with decreased activation of inflam-
matory cells. AQP4 has also been implicated in other neuropathologies including
Parkinson’s disease and neuromyelitis optica (Vella et al. 2015).
The potential therapeutic effect of targeting AQP4 in ischaemic stroke has been
shown in various studies. AQP4 knockout mice has been associated with decreased
mortality and improved motor recovery 3–14 days post-MCAO with decreased
infarct volume, neuronal cell death and neuroinflammation compared to wild type.
However, oedema was not shown to be reduced in AQP4 knockout mice (Hirt et al.
2017). Another study employed the use of AQP4 inhibitor TGN020 15 min post-
MCAO in a rat model. TGN020 successfully reduced oedema, gliosis and apoptosis
3 and 7 days post-occlusion (Pirici et al. 2017). The discrepancy in effects on
cerebral oedema in the above-mentioned studies may be explained by the bimodal
effects of AQP4 in different phases post-stroke. Persistent cerebral oedema in AQP4
knockout mice may highlight the importance of AQP4 in oedema resolution in the
delayed post-ischaemic phase.
Although AQP4 is a promising target in the treatment of ischaemic stroke, it is
pertinent to further clarify the time point at which AQP4 inhibition or activation
provides the optimal therapeutic outcome. Furthermore, heterogeneity in AQP4
Table 1 Summary of ion channels and transporters involved in oncotic cell death in ischaemic
stroke
Ion
channels/
transporters Conduction Cell type Pharmacological agent
Sodium-conducting channels
Na+/K+- Na+, K+ Neurons, glial Cardiac glycosides (Liu et al. 2013)
ATPase
AMPA Na+, K+, Ca2+ Neurons, glial Memantine (Chen et al. 1992)
NMDA
ASIC1a Na+, Ca2+ Neurons Flurbiprofen (Mishra et al. 2010)
Amiloride (Miao et al. 2010)
NHE1 Na+, H+ Neurons, glial, vascular Cariporide (Luo et al. 2005)
endothelial SM20220 (Suzuki et al. 2002)
NBCe1 Na+, HCO3 Neurons, astrocytes S0859 (Yao et al. 2016)
TRPM4 Na+, K+, Cs+ Neurons, astrocytes, Glibenclamide (Simard et al. 2009)
vascular endothelial TRPM4-siRNA (Loh et al. 2014;
Chen et al. 2018)
Potassium-conducting channels
KCNQ K+ Neurons Retigabine (Miceli et al. 2008)
(Kv7)
KATP K+ Neurons, glial, vascular Iptakalim (Wang et al. 2004)
endothelial, vascular
smooth muscles
Chloride-conducting channels
NKCC1 Cl , Na+, K+ Neurons, glial, vascular Bumetanide (Chen et al. 2005;
endothelial, choroid plexus Yan et al. 2003; Wang et al. 2014;
epithelial Xu et al. 2017)
Aquaporins
AQP4 Water Astrocytes, microglial TGN020 (Pirici et al. 2017)
expression and dynamic changes in pathological states pose a difficult challenge in

development of AQP4 as a therapeutic target in stroke. Distribution of AQP4 is both
spatially and temporally dependent on the types of stroke models employed and
regions of brain injury, adding to the complexity of defining the target through
animal studies (Badaut et al. 2011) (Table 1).
6 Mitochondrial Dysfunction
Mitochondria are the main source of cellular energy production in the form of ATP
by oxidative phosphorylation, alongside other important roles in cell metabolism,
calcium regulation, apoptosis and free radical production. Mitochondria in the
central nervous system are exquisitely susceptible to ischaemic damage. In early
54 K. Y. Loh et al.
stages of reduced cerebral perfusion, even in the absence of energy deficits, mito-
chondrial respiratory activities can exhibit severe alterations (Fiskum et al. 1999).
In ischaemic cell death, mitochondrial dysfunction disrupts the process of oxida-
tive phosphorylation and ATP production, increases generation of reactive oxygen
species (ROS) and impairs calcium buffering. In addition, the opening of mitochon-
drial permeability transition pores facilitates the release of pro-apoptotic mitochon-
drial proteins into the cytoplasm which activates both caspase-dependent
(cytochrome c) and caspase-independent (calpain) apoptotic pathways (Lipton
1999; Watts et al. 2013). In the ischaemic brain, mitochondrial dysfunction repre-
sents the key step to oncotic cell death. Accounting for more than 90% of cellular
ATP production, mitochondrial dysfunction results in the significant reduction of
ATP availability. This causes the disruption of normal ionic and water flux across
membranes, causing cell swelling and the ensuing process of oncosis in ischaemic
stroke (Mills et al. 2002).
Given the vital role of the electrochemical gradient across the mitochondrial
membranes in oxidative phosphorylation, it has been proposed that a loss in the
electrochemical gradient heralds the cellular changes in oncosis (Mills et al. 2002).
The uncoupling protein (UCP) family performs a central role in regulation of the
mitochondrial membrane potential. These proteins are expressed in the inner mito-
chondrial membranes. They regulate the membrane potential by facilitating entry of
protons which are driven out from the matrix during oxidative respiration. Hence,
they reduce the proton gradient required for generation of ATP in the oxidative
phosphorylation process (Klingenberg et al. 2001). The UCP-1 is highly expressed
in the inner mitochondrial membrane of brown adipocytes in animals. UCP-1
reduces ATP production and functions to divert energy use for cold-induced ther-
mogenesis (Ricquier and Bouillaud 2000b). Another member of the UCP family,
UCP-2, which is expressed widely in other tissues, has been examined for its role in
regulation of the mitochondrial membrane potential (Ricquier and Bouillaud 2000a).
In vitro studies have revealed that an increased expression of UCP-2 significantly
reduces mitochondrial membrane potential and ATP production and results in a
mechanism of cell death which is morphologically similar to oncosis (Mills et al.
2002). Inhibition of these uncoupling proteins has the potential to prevent oncotic
cell death by maintaining mitochondrial membrane potential and ATP production in
hypoxic conditions.
However, the role of UCP-2 in ischaemic stroke remains controversial. In vivo
studies with UCP-2 knockout mice paradoxically resulted in increased infarct
volume associated with suppression of anti-oxidant, cell-cycle and DNA-repair
genes and increased expression of inflammatory mediators (Haines et al. 2010).
The purported neuroprotective effect of UCP-2 has been attributed to its anti-oxidant
effect which reduces oxidative stress during cerebral ischaemia-reperfusion. UCP-2
overexpressing mice demonstrated decreased neuronal damage as compared to wild
type in cerebral ischaemia-reperfusion and had lower ROS levels (Haines and Li
2012). The role of UCP-2 on oncosis remains poorly understood.
The diverse functions of mitochondrial and pathological roles in cerebral ischae-
mia provide a potential novel target in stroke treatment. In addition to UCP-2, other
anti-apoptotic proteins such as inhibitor-of-apoptosis proteins, peroxisome

proliferator-activated receptors and anti-oxidant proteins superoxide dismutase
2 have also been studied (Yang et al. 2018). Further elucidation of mitochondrial
protective mechanisms is imperative to the development of new therapeutic options
in ischaemic stroke.
7 Key Challenges and Future Directions
The myriad of possible therapeutic targets in oncotic cell death represent viable
alternatives in treatment of ischaemic stroke. However, despite promising results in
many preclinical studies, none of the experimented agents have successfully
progressed down the drug developmental pathways. To facilitate the development
of new therapies in stroke, several key issues need to be addressed.
Firstly, the expression and functions of the implicated ion channels exhibit
dynamic temporal changes following ischaemic stroke. The activation or inhibition
of the channels may or may not produce the desired neuroprotective effect
depending on the phases of ischaemic stroke when pharmacological intervention is
initiated. The temporal expression and role of the target channels at different phases
of stroke needs to be better characterised. This will aid in defining the optimal time
period post-stroke in targeting these ion channels.
Secondly, these ion channels are not unique to the central nervous system and are
expressed in other tissues. Ensuring selectivity in targeting the CNS represents
another key challenge. Many of the pharmacological agents that have been used in
preclinical studies represent currently available drugs used for other medical indi-
cations. Examples include glibenclamide, a diabetic medication, in targeting
TRPM4, and the use of bumetanide, a loop diuretic, in targeting NKCC1. Potential
adverse effects on other organ systems need to be carefully evaluated before the use
of these drugs in ischaemic stroke. While a more expensive option compared to
using existing medication, future directions in development of stroke therapies could
involve designing targeted therapies in the form of small molecules or other novel
blockers. Additionally, it is important to achieve good CNS penetration and stability
in the pharmacological design of the drugs.
The last issue involves the translation of preclinical stroke research to clinical
trials. Despite promising results in preclinical trials, many of the novel
neuroprotective agents failed at clinical trials. These have been attributed to poor
trial designs and questionable preclinical data. More stringent regulation of preclin-
ical animal studies is key to providing accurate and reproducible data to facilitate
progress to clinical trials. The Stroke Therapy and Academic Industry Roundtable
has implemented guidelines for preclinical stroke studies to ensure study quality.
Even so, compliance of studies to guidelines remains a problem. In addition,
inappropriate animal stroke models used may influence success at human trials
despite full compliance to the guidelines. Future efforts should also focus on
evaluating the suitability of various animal stroke models in mimicking the human
diseased state (Herson and Traystman 2014).
56 K. Y. Loh et al.
8 Conclusion
Oncosis represents a major type of cell death in stroke, occurring directly after
energy depletion. Being closely linked to transmembrane electrolyte movements,
blocking or delaying such movements will ameliorate oncotic cell death. As the
process involves multiple ions and their respective transmembrane transporters, a
better strategy is to target multiple pathways simultaneously, yielding a synergistic
effect in preventing oncotic cell death. In comparison to apoptosis and autophagy,
where key players of the death pathway are located intracellularly, the mediators of
oncosis largely consist of transmembrane channels and transporters, suggesting that
they are a superior and more accessible class of target for intervention. Furthermore,
as oncosis is involved in cell death in major CNS cell types, namely, astrocytes,
neurons and vascular cells, inhibition of oncosis will ameliorate both cytotoxic
and vasogenic oedema. With a stabilised BBB, reduction of subsequent neuro-
inflammation is expected. It should be noted that most ion channels and transporters
are constitutively expressed in the healthy brain and other non-CNS tissues and
organs. The development of drugs that selectively act on hypoxic CNS cells without
affecting healthy tissues remains a challenge. With further advancements in drug
discovery, targeting of oncosis may form a powerful strategy in achieving
neuroprotection in a post-stroke setting.
Acknowledgements This work was supported by grant NMRC/CIRG/1425/2015 and NMRC/

CIRG/1469/2017 from the Singapore Ministry of Health’s National Medical Research Council to P.L.
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DOI: 10.1007/112_2018_15
Published online: 8 November 2018
Magnesium Extravaganza: A Critical

Compendium of Current Research
into Cellular Mg2+ Transporters Other
than TRPM6/7
Martin Kolisek, Gerhard Sponder, Ivana Pilchova, Michal Cibulka,

Zuzana Tatarkova, Tanja Werner, and Peter Racay
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
1.1 Toward the Identification of Mammalian/Human Mg2+ Transporters/Mg2+
Homeostatic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
2 Novel Mammalian Magnesiotropic Genes Encode for Electrogenic Mg2+ Transporters
(Channels) When Expressed in Xenopus laevis Oocytes but Not in Homologous Expression
Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3 Expression Profiles of Novel Magnesiotropic Genes and Their Protein Products . . . . . . . . . . 73
4 Cellular Compartmentalization and Functional Characterization of Proteins Encoded by
the Putative and Confirmed Mg2+ Transporters/Mg2+ Homeostatic Factors . . . . . . . . . . . . . . . . 73
4.1 Mg2+ Transporters and/or Mg2+ Homeostatic Factors Localized to Mitochondria . . . 75
4.2 Mg2+ Transporters and/or Mg2+ Homeostatic Factors Localized to Endoplasmic
Reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3 Mg2+ Transporters and/or Mg2+ Homeostatic Factors Localized to the Golgi
Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.4 Mg2+ Transporters and/or Mg2+ Homeostatic Factors Localized to Lysosomes . . . . . . 84
4.5 Mg2+ Transport Across Nuclear Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.6 Putative and Confirmed Mg2+ Transporters/Mg2+ Homeostatic Factors with Unclear
Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
M. Kolisek (*), I. Pilchova, and P. Racay

Biomedical Center Martin, Division of Neurosciences, Jessenius Faculty of Medicine in Martin,
Comenius University in Bratislava, Martin, Slovakia
e-mail: kolisek@jfmed.uniba.sk
G. Sponder
Institute of Veterinary Physiology, Veterinary Medicine, Free University of Berlin, Berlin,
Germany
M. Cibulka and Z. Tatarkova
Institute of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius
University in Bratislava, Martin, Slovakia
T. Werner
NuOmix Research k.s., Martin, Slovakia
66 M. Kolisek et al.
4.7 Putative and Confirmed Mg2+ Transporters/Mg2+ Homeostatic Factors with Plasma
Membrane Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4.8 MagT1 (Magnesium Transporter Subtype 1) and TUSC3/N33 (Tumor Suppressor
Candidate 3): Mg2+ Transporter Candidates Which Turned to Have Other but
Not Mg2+ Transport Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Abstract Magnesium research has boomed within the last 20 years. The real
breakthrough came at the start of the new millennium with the discovery of
a plethora of possible Mg homeostatic factors that, in particular, included putative
Mg2+ transporters. Until that point, Mg research was limited to biochemical and
physiological work, as no target molecular entities were known that could be used to
explore the molecular biology of Mg homeostasis at the level of the cell, tissue,
organ, or organism and to translate such knowledge into the field of clinical medicine
and pharmacology. Because of the aforementioned, Mg2+ and Mg homeostasis, both
of which had been heavily marginalized within the biomedical field in the twentieth
century, have become overnight a focal point of many studies ranging from primary
biomedical research to translational medicine.
The amount of literature concerning cellular Mg2+ transport and cellular Mg
homeostasis is increasing, together with a certain amount of confusion, especially
about the function(s) of the newly discovered and, in the majority of instances, still
only putative Mg2+ transporters/Mg2+ homeostatic factors. Newcomers to the field
of Mg research will thus find it particularly difficult to orient themselves.
Here, we briefly but critically summarize the status quo of the current under-
standing of the molecular entities behind cellular Mg2+ homeostasis in mammalian/
human cells other than TRPM6/7 chanzymes, which have been universally accepted
as being unspecific cation channel kinases allowing the flux of Mg2+ while consti-
tuting the major gateway for Mg2+ to enter the cell.
Keywords Carrier · Channel · Ion transporter · Magnesium (Mg) · Mg homeostasis
1 Introduction
A chronic magnesium (Mg or when ionized Mg2+) deficiency is associated with

the progressive deterioration of basal molecular processes and their physiological
functions in the cell.
Indeed, Mg is an essential factor having its role in fundamental processes
including the synthesis of bio-macromolecules, energy metabolism, the building
and remodeling of the cytoskeleton and cell motility, the regulation of cell growth
and proliferation, the modulation of cellular signaling, the regulation of apoptosis
and cell death, the stabilization of biomembranes and regulation of their fluidity, and
the regulation of transport of other solutes into and within the cell (Nishizawa et al.
Magnesium Extravaganza: A Critical Compendium of Current Research into. . . 67
2007; Romani 2007, 2011). Many of the functions of Mg2+ result from its being a
natural antagonist of Ca2+ in a plethora of cellular processes (Nishizawa et al. 2007).
The basal concentration of bioavailable intracellular Mg2+ ([Mg2+]i) ranges from
0.2 to 1 mM in various mammalian cell types, a concentration that is enormous when
compared with resting [Ca2+]i, which ranges from only 10s to ~100 nM (Romani
2007, 2011; Romani and Scarpa 2000; Delva et al. 1996, 2006; Zhou and Clapham
2009; Dragileva et al. 1999).
Basal [Mg2+]i is highly stable. Even when exposed to unphysiologically high
(e.g., 10–30 mM) or low (0–0.3 mM) extracellular [Mg2+] ([Mg2+]e), the cell
maintains its [Mg2+]i close to its basal value. Furthermore, the electrochemical
equilibrium potential (VeqMg2þ ) would be reached at the [Mg2+]i of ~50 mM in most
eukaryotes under resting conditions, but in reality, cells keep [Mg2+]i 50–100 times
lower than that. This suggests the existence of effective cellular regulatory (Mg2+
transport) mechanisms that maintain intracellular Mg2+ homeostasis (Nishizawa
et al. 2007; Romani 2007, 2011; Schweigel-Röntgen and Kolisek 2014; Flatman
1984).
The [Mg2+]i (cytosol) of most mammalian cells types is almost equimolar to
the concentration of [Mg2+]e (extracellular fluid; [Mg2+]e ¼ approximately
0.8–1 mM vs. [Mg2+]i ¼ approximately 0.2–1 mM) (Nishizawa et al. 2007; Romani
2007, 2011; Romani and Scarpa 2000; Delva et al. 1996; Schweigel-Röntgen and
Kolisek 2014). Similarly, a Mg2+ concentration gradient is almost nonexistent
between [Mg2+]i (cytosol) and [Mg2+]io (intracellular organelles) (Nishizawa et al.
2007; Romani 2011; Rutter et al. 1990). This emphasizes the importance of the
electrical component of the electrochemical gradient for Mg2+ transport and further
suggests that a primary active (ATP-dependent) or secondary active (dependent on
Na+- or H+-motive forces) Mg2+ transport mechanism would be of great importance
for the maintenance of cellular Mg2+ homeostasis and transcellular Mg2+ transport
(Nishizawa et al. 2007; Romani 2011).
Several Mg2+ transport mechanisms have been proposed to exist based on
biochemical and physiological evidence. Among these are the following: Mg2+ ion
channel(s), Na+/Mg2 and H+/Mg2+ exchangers, Mn2+/Mg2+ and Ca2+/Mg2+
exchangers, choline/Mg2+ exchanger, and Mg2+ HCO3 a Mg2+ Cl symporter
(Romani 2011; Schweigel-Röntgen and Kolisek 2014; Günther et al. 1990; Ebel
et al. 2002; McGuigan et al. 2002; Günther 1993).
1.1 Toward the Identification of Mammalian/Human Mg2+

Transporters/Mg2+ Homeostatic Factors
Pioneering work in the world of Mg2+ transporters in prokaryotes was carried out by
Michael E. Maguire and his colleagues; they described three prominent classes of
bacterial Mg2+ transporters (CorA, MgtA, and MgtE) long before the discovery of
eukaryotic Mg2+ transporters (Hmiel et al. 1986, 1989; Smith et al. 1995). The major
bacterial Mg2+ transporters CorA and MgtE have a wide phylogenetic distribution,
whereas MgtA occurs only in some bacteria (Maguire 2006; Groisman et al. 2013).
MgtA and MgtB are P-type ATPases that mediate Mg2+ influx. Both are exclu-
sive to bacteria (Groisman et al. 2013; Maguire 1992). On the other hand, eukaryotic
homologs of CorA and MgtE have been identified (Groisman et al. 2013).
The first Mg2+ transporters ever described in eukaryotic cells (yeast:
Saccharomyces cerevisiae) were the CorA homologs Mrs2 and Alr1 (Bui et al.
1999; Gregan et al. 2001a; Graschopf et al. 2001). Whereas the Mrs2 gene is nuclear,
the Mrs2 protein operates in mitochondria, namely, in the inner mitochondrial
membrane (IMM; Fig. 1) (Gregan et al. 2001a; Wiesenberger et al. 1992; Kolisek
et al. 2003; Schindl et al. 2007). Mrs2 is now widely accepted as being an essential
component of the mitochondrial Mg2+ homeostatic network from yeast to man
(Kolisek et al. 2003; Schindl et al. 2007; Zsurka et al. 2001; Piskacek et al. 2009;
Sponder et al. 2013a; Yamanaka et al. 2016; Merolle et al. 2018). Alr1 is a Mg2+
transporter integral to the cytoplasmic membrane of S. cerevisiae (Graschopf et al.
2001).
Fig. 1 Cellular Mg2+ transport mechanisms and/or Mg2+ homeostatic factors. The confirmed Mg2+
transporters/Mg2+ homeostatic factors are shown in color. The putative Mg2+ transporters/Mg2+
homeostatic factors are shown in white. MagT1 and TUSC3/N33 are crossed (red cross) because
they were already disproved to function as Mg2+ transporters. SLC41A2, CNNM1, CNNM3, and
CNNM4 are not depicted due to ongoing controversy either on their localization or function(s) or
both. CNNM2 is depicted as a Mg2+ homeostatic factor
Almost in parallel with Mrs2, chanzymes TRPM6/7 were discovered and iden-
tified as unique natural fusion proteins comprising a functional channel domain and a
functional kinase domain (Montell 2003; Nadler et al. 2001; Schlingmann et al.
2002). The abilities of TRPM6/7 to conduct Mg2+ transport have been robustly
researched over the last 15 years, and many reviews are available online. Hence, in
the following text, we will only mention TRPM6/7 when relevant.
The real momentum to the otherwise rather small field of Mg2+ transport research
was provided by the work of Angela Goytain and Gary A. Quamme. At the turn of
the millennium, they designed an elegant set of Mg2+ starvation experiments
utilizing mice and MDCT (mouse distal convoluted tubule) cells. By using oligo-
nucleotide microarray technology, quantitative real-time RT-PCR, and in silico
similarity searches, they identified magnesiotropic genes (MgG(s), namely, genes
reactive to changing [Mg2+]i at the level of gene expression) encoding for 15 novel
putative Mg2+ transporters (npMgT; Fig. 1) in mouse (SLC41A1, SLC41A2,
SLC41A3, NIPA1, NIPA2, NIPA3 (human NIPAL1), NIPA4 (human NIPAL4),
MagT1, TUSC3/N33, MMgT1, MMgT2, CNNM2, HIP14 and HIP14L, and
MagC1) (Quamme 2010). All of these mouse MgGs, except for MMgT2, also
have human homologs (Table 1; https://www.genecards.org; https://www.uniprot.
org). Most of the proteins encoded by mouse MgGs have been characterized as
encoding for “Mg2+ transporters with channel-like properties” (Quamme 2010;
Goytain and Quamme 2005a, b, c, d, 2008; Goytain et al. 2007, 2008a, b). Thus,
the group led by Goytain and Quamme revolutionized the whole field of mamma-
lian/human magnesium research by suggesting molecular identities of the entities
constituting the cellular Mg homeostatic machinery.
2 Novel Mammalian Magnesiotropic Genes Encode

for Electrogenic Mg2+ Transporters (Channels) When
Expressed in Xenopus laevis Oocytes but Not
in Homologous Expression Systems
The patch clamp is an electrophysiological technique standardly used to character-

ize/confirm the electrogenic transport of any ion across a specific ion channel.
By using a single channel patch clamp, Schindl and coworkers showed bona fide
that the mitochondrial Mrs2 forms a Mg2+-selective channel of high conductance
(155 pS) in giant liposomes constituted from submitochondrial particles
(S. cerevisiae overexpressing Mrs2) fused with asolectin vesicles (Schindl et al.
2007). Groups that characterized TRPM6/7 transporters as being plasma membrane-
localized unspecific divalent-cation-conducting chanzymes also used the patch
clamp method in its various configurations (Fig. 1) (Nadler et al. 2001; Penner and
Fleig 2007; Monteilh-Zoller et al. 2003; Voets et al. 2004). We should also mention
that the Mrs2 channel and the TRPM6/7 chanzymes have been primarily electro-
physiologically characterized in homologous expression systems (yeast Mrs2 in
Table 1 Mammalian (human) Mg2+ transport systems/Mg homeostatic factors

Confirmed with known mechanism of operation
Confirmed with partly understood mechanism of operation
Confirmed with unknown mechanism of operation
Unconfirmed Mg2+ transport function
Gene Function Cellular Cytogenetic location Citation

localization
TRPM6 UCChz CM 9q21.13 Montell (2003), Schlingmann et al.
(2002), and Voets et al. (2004)
TRPM7 UCChz CM 15q21.2 Montell (2003), Nadler et al. (2001),
Penner and Fleig (2007), and Monteilh-
Zoller et al. (2003)
hsaMRS2 MgCh IMM 6p22.3 Kolisek et al. (2003), Schindl et al.
(functional ortholog of yeast (2007), Zsurka et al. (2001), and
MRS2) Piskacek et al. (2009)
SLC41A1a,b NME CM 1q32.1 Quamme (2010), Goytain and Quamme
(2005a), Kolisek et al. (2008, 2012,
2013a), and Wabakken et al. (2003)
SLC41A3a,b NME IMM 3q21.2–q21.3 Quamme (2010) and Mastrototaro et al.
(2016)
SLC41A2 a,b
MgT 12q23.3 Quamme (2010), Goytain and Quamme
(2005b), and Sahni et al. (2007)
MagT1a c
MgCh CM Xq21.1 Zhou and Clapham (2009), Quamme
oxidoreductase in (2010), Goytain and Quamme
OST complex (2005c), Shrimal et al. (2015), and
Cherepanova and Gilmore (2016)
c
MgCh CM 8p22 Zhou and Clapham (2009), Quamme
TUSC3/N33 a,b oxidoreductase in (2010), Goytain and Quamme (2005c),
OST complex Shrimal et al. (2015), and Cherepanova
and Gilmore (2016)
NIPA1/SLC57A1a,b PMgT CM 15q11.2 Quamme (2010) and Goytain et al.
(2007)
NIPA2/SLC57A2a PMgT CM 15q11.2 Quamme (2010) and Goytain et al.
(2008a)
NIPAL1a,b (NIPA3) PMgT CM 4p12 Quamme (2010)
NIPAL4a,b (NIPA4) PMgT CM 5q33.3 Quamme (2010)
MMgT1a PMgT GA Xq26.3 Quamme (2010) and Goytain and
Quamme (2008)
MMgT2a, d PMgT GA Not in human Quamme (2010) and Goytain and
Quamme (2008)
CNNM2/ACDP2 a
PMgT/MgHF CM,N,IMS 10q24.32 Quamme (2010), Goytain and Quamme
(2005d), Sponder et al. (2010b, 2016),
Stuiver et al. (2011), Wang et al. (2003,
2004), Yamazaki et al. (2013), Arjona et
al. (2014), and Hirata et al. (2014)
CNNM1b PMgT CM,N 10q24.2 Wang et al. (2003, 2004) and Hirata et
al. (2014)
CNNM3b PMgT CM,N 2q11.2 Quamme (2010), Wang et al. (2003,
2004), and Hirata et al. (2014)
CNNM4 b
PMgT CM,N 2q11.2 Wang et al. (2003, 2004), Yamazaki et
al. (2013), and Hirata et al. (2014)
ZDHHC17/HIP14a MgChz GA/PGV 12q21.2 Quamme (2010), Goytain et al. (2008b),
PA Ducker et al. (2004), Yanai et al. (2006),
and Butland et al. (2014)
ZDHHC17/HIP14 La,b MgChz GA/PGV 11p15.1 Quamme (2010) and Goytain et al.
(2008b)
MagC1a PMgT Data not available 16q3.3 Quamme (2010)
SLC25A24 APC, SCaMC IMM 1p13.3 Traba et al. (2009) and Tewari et al.
(2012)
SLC25A25 APC, SCaMC IMM 9q34.11 Traba et al. (2009) and Tewari et al.
(2012)
(2012)
(2012)
ATP13A4 PMgP ER 3q29 Schultheis et al. (2004)
ATP13A2 (PARK9, functional PMgP Lys 1p36.13 Lambie et al. (2013)
ortholog of C-ATP6 in
C. elegans)
XK (Kell/XK complex) PMgT CM Xp.21.1 Rivera et al. (2013) and Lee et al. (2000)
APC ATP-Mg/Pi carrier, CM cytoplasmic membrane, ER endoplasmic reticulum, GA/PGV Golgi

apparatus/post-Golgi vesicles, IMM inner mitochondrial membrane, IMS intracellular membranous
structures, Lys lysosome, MgCh Mg2+ channel, MgChz Mg2+ chanzyme, MgHF Mg homeostatic
factor, MgT Mg2+ transporter, N nucleus, NME Na+/Mg2+ exchanger, OST oligosaccharyl-
transferase, PA palmitoyl acyltransferase, PMgP putative Mg2+ pump, PMgT putative Mg2+
transporter, SCaMC short Ca2+-binding mitochondrial carrier, UCChZ unspecific cation chanzyme
a
Magnesiotropic genes identified by the group around Quamme
b
Predicted in silico based on similarity search
c
Other function attributed, but not Mg2+ transport
d
Not present in human (identified in mouse)
yeast mitochondria-derived giant liposomes; human TRPM6/7 in human-derived

cell lines (e.g., HEK293)) (Schindl et al. 2007; Nadler et al. 2001; Penner and Fleig
2007; Monteilh-Zoller et al. 2003; Voets et al. 2004).
To demonstrate the ability of novel putative Mg2+ transporters to transport Mg2+,
the group of Goytain and Quamme prepared X. laevis oocytes expressing the
respective novel putative Mg2+ transporters (npMgT(s); mouse origin) and further
examined them with two-electrode voltage clamp (TEV) (Quamme 2010; Goytain
and Quamme 2005a, b, c, d, 2008; Goytain et al. 2007, 2008a, b). Surprisingly, all of
the npMgTs conducted the electrogenic transport of Mg2+ and, as is shown in
Table 2, also that of other cations (except for MagT1) (Quamme 2010; Goytain
and Quamme 2005a, b, c, d, 2008; Goytain et al. 2007, 2008a, b). Our group
attempted to reproduce the electrophysiology data of Goytain and Quamme’s
group by using whole-cell mode patch clamping in HEK293 cells with the inducible
overexpression of human SLC41A1 or human CNNM2 (Kolisek et al. 2008;
Sponder et al. 2016). In both cases, we were unable to detect any SLC41A1- or
CNNM2-related Mg2+ conductance in the homologous expression system. More-
over, Müller’s group was unable to detect any human CNNM2-related Mg2+
currents in HEK293 cells overexpressing CNNM2 (Stuiver et al. 2011). Results
for SLC41A2 resembled those for SLC41A1 and CNNM2. In contrast to the
previous report of Goytain and Quamme’s group showing the ion channel activity
associated with SLC41A2 expression in oocytes, investigations of whole cell cur-
rents in SLC41A2-expressing DT40 cells (Gallus gallus) as performed by Sahni and
colleagues revealed no novel currents of any type associated with SLC41A2 expres-
sion (Sahni et al. 2007). Although not tested electrophysiologically, human
SLC41A3 was identified as being an integral protein to IMM to operate as a
mitochondrial Na+-dependent Mg2+ efflux system in a homologous expression
system (Fig. 1) (Mastrototaro et al. 2016).
The discrepancy between the electrophysiology data obtained by Goytain and
Quamme’s group and the data originating from other research groups might result
from the following: (1) the heterologous expression of mammalian npMgTs in
X. laevis oocytes, (2) the use of an unsuitable electrophysiological technique to
establish permeation profiles, or (3) a combination of both.
Despite several advantages, the overexpression of mammalian ion transporters in
X. laevis oocytes may also have significant disadvantages (Goldin 2006). Perhaps
the most serious pitfall is that oocytes (amphibian) might lack innate regulatory
systems important for the activation and/or proper function of the alien ion
Table 2 Permeation profiles established for Mg2+ transporters by two-electrode voltage clamp
(green) or by patch clamp technique (violet)
Protein Permeation profile Citation
TRPM6 Px/PCa (Hs): Ba2+ ≥ Ni2+ > Mg2+ > Ca2+ Voets et al. (2004)
TRPM7 Px/PCa (Hs): Zn2+ ≈ Ni2+ Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥≥ Sr2+ ≥ Cd2+ ≥ Ca2+ Penner and Fleig (2007) and
Monteilh-Zoller et al. (2003)
Mrs2 Px/PMg (Sc): Mg2+ > Ni2+ Schindl et al. (2007)
NT (not transported): Ca2+, Mn2+, Co2+
SLC41A1 Px/PMg (Mm): Mg2+ > Sr2+ ≈ Fe2+ > Ba2+ > Cu2+ > Zn2+ ≈ Co2+ > Cd2+ > Mn2+ Quamme (2010) and Goytain
NT: Ni2+, Ca2+, Gd3+ and Quamme (2005a)
SLC41A3a Px/PMg (Mm): Ba2+ > Mg2+ > Ni2+ ≈ Zn2+ > Sr2+ ≈ Fe2+ > Mn2+ > Cu2+ ≈ Co2+ Quamme (2010)
NT: Ca2+, Cd2+
SLC41A2 Px/PMg (Mm): Mg2+ > Ba2+ > Ni2+ > Co2+ > Sr2+ ≈ Fe2+ > Mn2+ Quamme (2010) and Goytain
NT: Ca2+, Cu2+, Zn2+, Cd2+ and Quamme (2005b)
MagT1 (Mm) Highly selective for Mg2+ Quamme (2010) and Goytain
and Quamme (2005c)
TUSC3/N33 a Px/PMg (Mm): Mg2+ > Mn2+ > Cu2+ > Fe2+ > Ba2+ ≈ Co2+ > Sr2+ ≈ Zn2+ ≥ Ni2+ > Ca2+ Quamme (2010)
NIPA1 Px/PMg (Mm): Mg2+ > Sr2+ > Co2+ > Zn2+ ≈ Fe2+ > Ni2+ ≈ Ca2+ ≈ Ba2+ ≈ Cu2+ ≈ Mn2+ Quamme (2010) and Goytain
NT: Cd2+ et al. (2007, 2008a)
NIPA2 Px/PMg (Mm): Mg2+ Sr2+ ≈ Co2+ ≈ Zn2+ ≈ Fe2+ ≈ Cd2+ ≈ Ni2+ ≈ Ca2+ ≈ Ba2+ ≈ Cu2+ ≈ Mn2+ Quamme (2010) and Goytain
et al. (2008a)
2+
NPAL3 Px/PMg (Mm): Mg > Sr2+ > Ba2+ > Fe2+ ≈ Mn2+ > Cu2+ ≈ Co2+ > Zn2+ ≈ Cd2+ > Ni2+ ≈ Ca2+ Quamme (2010) and Goytain
et al. (2008a)
NPAL4/Ichthyin Px/PMg (Mm): Mg2+ > Ba2+ > Sr2+ > Fe2+ > Cu2+ ≈ Ca2+ ≈ Zn2+ ≥ Co2+ ≈ Mn2+ ≈ Ni2+ Quamme (2010) and Goytain
NT: Cd2+ et al. (2008a)
MMgT1 Px/PMg (Mm): Mg2+ ≥ Sr2+ ≥ Fe2+ > Co2+ > Cu2+ > Ba2+ > Ca2+ > Zn2+ > Mn2+ > Ni2+ Quamme (2010) and Goytain
and Quamme (2008)
MMgT2 Px/PMg (Mm): Mg2+ ≥ Sr2+ > Ba2+ > Cu2+ > Mn2+ > Co2+ > Ni2+ > Fe2+ > Zn2+ ≥ Ca2+ Quamme (2010) and Goytain
and Quamme (2008)
CNNM2 Px/PMg (Mm): Mg2+ ≥ Co2+ > Ba2+ ≥ Mn2+ ≈ Gd3+ ≥ Sr2+ > Cu2+ ≥ Fe2+ Quamme (2010) and Goytain
NT: Zn2+, Cd2+, Ni2+, Ca2+ and Quamme (2005d)
The permeation profile in original paper (Goytain and Quamme 2005d) does not match
the permeation profile published in review (Quamme 2010)
CNNM1 Px/PMg: not available
CNNM3a Px/PMg (Mm): Mg2+ > Fe2+ > Cu2+ > Co2+> Ni2+ > Ca2+ Quamme (2010)
NT: Sr2+, Ba2+, Mn2+, Zn2+, Cd2+
CNNM4 Px/PMg: not available
HIP14 Px/PMg (Mm): Mg2+ ≈ Sr2+ > Ni2+ > Ba2+ > Zn2+ ≥ Mn2+ > Fe2+ Quamme (2010) and Goytain
NT: Co2+, Cu2+, Ca2+ et al. (2008b)
HIP14L Px/PMg (Mm): Mg2+ ≈ Sr2+ > Ni2+ ≥ Mn2+ > Cu2+ ≈ Ba2+ ≈ Zn2+ Quamme (2010) and
NT: Fe2+, Co2+, Ca2+ Goytain et al. (2008b)
a
Indicates that the data were introduced in the review paper and not in original research paper.
Please note that the npMgT permeation profiles were reconstructed mostly from the graphical
content published in indicated papers of the group around Quamme (green) (Quamme 2010;
Goytain and Quamme 2005a, b, c, d, 2008; Goytain et al. 2007, 2008a, b). Due to frequent lack
of statistics in the original works, it is not being reflected in our interpretation of these permeation
profiles
transporter (mammalian/murine in the case of npMgTs). If the transporter builds

functional complexes with another proteins and/or factors that play essential roles in
the maintenance/regulation of its normal physiological function, and if these
proteins/factors are not present in X. laevis oocytes, then the transporter might act
in a mode different (deviated) from its normal function or it might be completely
dysfunctional (Goldin 2006).
Of note, the principles of TEV do not allow the establishment of a true permeation
profile of any transporter/channel because of the lack of any control for the intra-
cellular ion milieu (Kolisek et al. 2008; Goldin 2006; Fleig et al. 2013).
Considering latter, the functional data concerning npMgTs as acquired with TEV
in X. laevis oocytes must thus be interpreted cautiously. Further verification/exam-
ination of these data in homologous expression systems with appropriate electro-
physiological techniques should therefore be carried out.
3 Expression Profiles of Novel Magnesiotropic Genes

and Their Protein Products
The expression profiles of the novel magnesiotropic genes in various human organs
or tissues are summarized in Table 3. At the level of mRNA, the majority of the
novel magnesiotropic genes are expressed in most of the tested organs/tissues
(Table 3). A ubiquitous and/or almost ubiquitous expression of MgGs might indicate
their importance for overall cellular physiology. However, at the protein level,
TRPM6, CNNM1, CNNM4, NIPAL4, HIP14, and HIP14L are only detected in
specific organs/tissues that have absorptive functions, re-absorptive functions, bar-
rier functions, or secretory functions, or in those that are metabolically highly active
(https://www.proteinatlas.org) (Romani 2011; Quamme 2010).
4 Cellular Compartmentalization and Functional

Characterization of Proteins Encoded by the Putative
and Confirmed Mg2+ Transporters/Mg2+ Homeostatic
Factors
All proteins encoded by the novel MgGs possess membrane-spanning α-helices in

their structures, and thus, all are expected to reside in the cytoplasmic membrane or
in the membranes of intracellular compartments.
In general every cell type is assumed to possess plasma membrane transporters
allowing the influx of Mg2+ and transporters that mediate its efflux (Nishizawa et al.
2007; Romani 2011). In particular, the simultaneous presence of both Mg2+ influx-
and Mg2+ efflux-mediating transport systems is of eminent importance in Mg2+
absorptive and re-absorptive epithelia (Nishizawa et al. 2007; Romani 2011; de
Baaij et al. 2015; Schweigel et al. 1999, 2000, 2006, 2009; Schweigel and Martens
2003). In cells that transport Mg2+ via the transcellular pathway, a clear polarized
distribution has been demonstrated for influx- and efflux-mediating Mg2+ transport
mechanisms. For example, the apical membrane of ruminal epithelial cells (REC)
contains primarily influx-mediating Mg2+ transport mechanisms, whereas the
basolateral membrane contains primarily Mg2+ efflux-mediating transport mecha-
nisms (Schweigel et al. 1999, 2000; Schweigel and Martens 2000). In non-polarized
cells, both influx and efflux mechanisms are assumed to be present in the cytoplas-
mic membrane but with no clearly traceable pattern of their distribution (Nishizawa
et al. 2007; Romani 2011).
Furthermore, the cells conducting the transcellular transport of Mg2+ must con-
tain mechanism(s) that sort(s) the pool of the total cellular Mg2+ into two smaller
pools; the first is intended for transcellular transport, and the second is intended for
the use of the cells themselves. This mechanism(s) must be regulated and at the same
time, very flexible. The existence of such mechanism(s) is only possible in the
Table 3 Organ/tissue-specific expression of confirmed and putative human magnesiotropic genes

according to www.proteinatlas.org
RNA detected in (HPA, GTEx,
Gene FANTOM5) Protein detected in
TRPM6 Ubiquitous with the highest expression Colon, rectum, small intestine,
level in colon, kidney duodenum, parathyroid gland, kidney,
testis
TRPM7 Ubiquitous Ubiquitous
Mrs2 Ubiquitous Ubiquitous
SLC41A1 Ubiquitous Ubiquitous
SLC41A3 Ubiquitous Ubiquitous
SLC41A2 Ubiquitous Almost ubiquitous
MagT1 Ubiquitous Almost ubiquitous
NIPA1/ Ubiquitous with the highest expression Almost ubiquitous
SLC57A1 level in brain
NIPA2/ Ubiquitous Almost ubiquitous
SLC57A2
NIPAL3 Ubiquitous Almost ubiquitous
NIPAL4 Ubiquitous Parathyroid gland, adrenal gland,
liver, gastrointestinal tract, testis,
placenta, skin
TUSC3/ Almost ubiquitous with the highest Ubiquitous
N33 expression level in brain and urogenital
tract
MMgT1 Ubiquitous Ubiquitous
MMgT2 Ubiquitous Data not available
CNNM2/ Ubiquitous with the highest level of Ubiquitous
ACDP2 expression in brain, kidney, and testis
CNNM1 Brain, testis Brain, kidney, testis, skin
CNNM3 Ubiquitous Ubiquitous
CNNM4 Ubiquitous with the highest level of Not ubiquitous, the highest level in
expression in brain, colon, rectum, small brain, stomach, small intestine, colon,
intestine rectum
ZDHHC17/ Almost ubiquitous with the highest Not ubiquitous, the highest level in
HIP14 expression level in brain brain and urogenital tract
ZDHHC17/ Almost ubiquitous with the highest Not ubiquitous, the highest level in
HIP14L expression level in brain stomach and testis
MagC1 Data not available Data not available
SLC25A41 Cerebral cortex, cerebellum, testis Inconclusive data set
ATP13A4 Brain, lungs, thyroid glands, esophagus, Inconclusive data set
skin, epididymis, breast
ATP13A2 Ubiquitous Ubiquitous
(PARK9)
XK Ubiquitous (low levels); enhanced Almost ubiquitous (low levels)
expression in small intestine
presence of (a) intracellular compartments dedicated to the storage of Mg2+ (intra-

cellular Mg2+ stores); (b) a functional network of Mg2+ transporters allowing for
(1) the uptake of Mg2+ from the extracellular fluid, (2) its deposition and reposition
within the intracellular compartments (which is essential for maintaining all the
functions of Mg2+ in the cell), and (3) its extrusion from the cell into the extracellular
fluid; and (c) a battery of regulatory components able to signal and to orchestrate the
function of the network (see b) according to the demands of the cell.
Given the importance of Mg2+ for the plethora of basal physiological processes in
cells, probably all mammalian/human somatic cells and not only those intended for
the absorption and transcellular transport of Mg2+ possess intracellular Mg2+ trans-
port mechanisms allowing the deposition and reposition of Mg2+ within the cell and
its organelles according to the actual needs of the cell (Nishizawa et al. 2007;
Romani 2011).
Among the npMgTs/Mg2+ homeostatic factors, not only the plasma membrane
but also the mitochondrial (SLC41A3) and Golgi-localized (HIP14/HIP14L,
MMgtT1, and MMgt2) npMgTs have been identified (Quamme 2010; Goytain and
Quamme 2008; Goytain et al. 2008b). This is not surprising, as the mitochondria,
endoplasmic reticulum (ER), and Golgi apparatus (GA) were long suspected of
having functions including that of accumulating and storing Mg2+. The function of
mitochondria in the storage of Mg2+ was experimentally established by Kubota and
colleagues in PC12 cells, by Mastrototaro and colleagues in HEK-293 cells, and by
Kolisek and colleagues in the mitochondria of yeasts (S. cerevisiae) (Kolisek et al.
2003; Kubota et al. 2005; Mastrototaro et al. 2015).
4.1 Mg2+ Transporters and/or Mg2+ Homeostatic Factors

Localized to Mitochondria
In mammalian mitochondria, the concentrations of the matrix Mg2+ ([Mg2+]m) have

been estimated to range between 0.2 and 1.5 mM (Kolisek et al. 2003; Jung et al.
1990; Rodríguez-Zavala and Moreno-Sánchez 1998). The [Mg2+]m measured in
cardiac and liver mitochondria lie in range from 0.8 to 1.2 mM. Thus, [Mg2+]m
has a same range to that of [Mg2+]i in the cytoplasm (Romani 2011; Rutter et al.
1990; Jung et al. 1990). Rudolf J. Schweyen’s group has demonstrated that the
mitochondria of wild-type yeast (S. cerevisiae) are able to accumulate Mg2+ up to a
final concentration of approximately 5 mM (Kolisek et al. 2003).
At a transmembrane voltage (IMM) of 130 to 160 mV (Fig. 2) and an [Mg2+]e
of 10 mM, the driving force for Mg2+ influx (ΔμMg) is extremely strong (140 to
169 mV) and theoretically would result in an [Mg2+]m of 1,450 mM instead of the
5 mM observed in the yeast mitochondria under assay conditions as used by Kolisek
and colleagues in their study (Kolisek et al. 2003; Rodríguez-Zavala and Moreno-
Sánchez 1998; Iwatsuki et al. 2000). Therefore, Mg2+ self-evidently does not
equilibrate freely across the IMM, suggesting the existence of a regulated Mg2+
Fig. 2 The distribution of membrane potential across major intracellular compartments. ER

endoplasmic reticulum, GA Golgi apparatus, IMM inner mitochondrial membrane, LYS lysosome
influx mechanism. Essentially similar conclusions have been drawn from the studies
of Jung et al. (1990, 1997). As an alternative to a regulated Mg2+ influx mechanism,
Kolisek and colleagues have proposed the existence of a Mg2+ efflux mechanism
capable of balancing for Mg2+ influx (Kolisek et al. 2003).
Kolisek and colleagues have identified Mrs2 (mitochondrial RNA splicing 2) to
be a channel responsible for Mg2+ flux mediation into mitochondria (Figs. 1, 3, and
4) (Kolisek et al. 2003). Furthermore, they have demonstrated that the Mrs2-
mediated Mg2+ influx is entirely driven by the mitochondrial membrane potential
(ΔψIMM) and inhibited by cobalt(III)hexaammine, an inhibitor of CorA (distant
prokaryotic homolog of Mrs2) (Kolisek et al. 2003). These findings have been
verified by the study of Schindl and coworkers who have shown by means of
electrophysiology that Mrs2 fulfills all the prerequisites for a superconductive
mitochondrial Mg2+ channel (Schindl et al. 2007). The depolarization of IMM
(and thus a decrease of ΔψIMM) leads to the inevitable suspension of Mrs2 function
(Kolisek et al. 2003). This is coherent with Mg2+ release in response to the depo-
larization of IMM (Kubota et al. 2005). Trapani and Wolf have quoted, in their
review, the work of Kubota and colleagues stating that Mrs2-mediated Mg2+ efflux
might take place in mitochondria upon the depolarization of IMM (Kubota et al.
2005; Trapani and Wolf 2015). Considering the enormous Δψ on IMM, a very
Fig. 3 Recycling of Mg2+, Na+, and Ca2+ on inner mitochondrial membrane. MCU mitochondrial
Ca2+ uniporter, NCE Na+/Ca2+ exchanger, NHE Na+/H+ exchanger, NME Na+/Mg2+ exchanger,
Mrs2 mitochondrial Mg2+ channel
strong ΔμMg and an almost negligible Δ[Mg2+] between the intermembrane space
[Mg2+]is and the matrix [Mg2+]m, scenario in which Mrs2 act as a Mg2+-efflux
mechanism is almost impossible, even during an event of drastic depolarization
(Kolisek et al. 2003; Vergun et al. 2003; Corkey et al. 1986). Furthermore, the
overall structure of the Mrs2 channel, the organization of the selective filter, and the
complex gating mechanism are not in support of the hypothesis that the channel may
conduct Mg2+ efflux under certain conditions (Sponder et al. 2013a; Khan et al.
2013).
The functionally inactive, mutant Mrs2 causes in dmy/dmy rats a mitochondrial
disease hallmarked by the demyelination of the neurons (Kuramoto et al. 2011;
Kuwamura et al. 2011).
The proton (H+) gradient across the IMM generates the major motive force
powering the transport of a plethora of solutes across this membrane. Thus, the
mitochondrial Mg2+ efflux system was assumed to be directly or indirectly coupled
to H+ influx. The experimental work of Rutter and colleagues using rat heart
mitochondria supports the existence of a mitochondrial H+/Mg2+ exchanger
(Fig. 4) (Rutter et al. 1990). Furthermore, long-chain fatty acids induce the
rapid release of Mg2+ from rat liver mitochondria in alkaline media, presumably
via a Mg2+/Me+ or an H+/Mg2+ exchanger (Schönfeld et al. 2002). However, until
Fig. 4 The mitochondrial (IMM) Mg2+ transport circuit (Mrs2, SLC41A3, APC, and HME). APC
ATP-Mg/Pi carrier, HME H+/Mg2+ exchanger (?, mechanism encoded by yet unknown molecular
entity), IMM inner mitochondrial membrane, Mrs2 Mg2+ channel, NME Na+/Mg2+ exchanger
now the molecular identity of the mitochondrial transporter capable of Mg2+ efflux
via a mechanism of H+/Mg2+ exchange remains elusive.
Recently, the group of Sponder has provided experimental evidence that
npMgT SLC41A3 is a Na+-coupled Mg2+ efflux system (very likely a Na+/Mg2+
exchanger) that resides in the IMM (Figs. 1, 3, and 4) (Mastrototaro et al. 2016). This
finding is also supported by the observation that the 25Mg2+ uptake in wild type
(SLC41A3+/+) and SLC41A3/ mice is alike (de Baaij et al. 2016). The exact nature
of the Na+-Mg2+ transport coupling via SLC41A3 and the stoichiometry of this
process remains to be addressed. Perhaps, the further characterization of SLC4A3
will shed more light on the observation of Zhang and Melwin that the elevation of
[Na+]i induces an efflux of Mg2+ from intracellular pool(s) (mitochondria) in rat
sublingual mucous acini (Zhang and Melvin 1996).
Moreover, an increase of the classic cytosolic secondary messenger cAMP has
been reported to activate the release of Mg2+ from mitochondria (Romani et al.
1991). cAMP is an important cofactor/activator of PKA. Whether PKA plays a role
in the activation of SLC41A3 remains to be examined. However, the cAMP-
dependent activation of PKA followed by the phosphorylation of the plasma mem-
brane Na+/Mg2+ exchanger SLC41A1 has clearly been demonstrated as being an
essential step toward the release of Mg2+ from the cell (Mastrototaro et al. 2015;
Kolisek et al. 2013a; Sponder et al. 2017). Therefore, keeping in mind the shared
ancestry of SLC41A1 and SLC41A3, it would be unsurprising if SLC41A3 also
undergoes PKA-mediated activation (Fig. 5). Recently, this hypothesis has become
even more feasible because of the discovery of A-kinase-anchoring proteins
(AKAP), namely, AKAP121 and SPHKAP/SKIP (sphingosine kinase type-1
anchor/interacting protein), which are known to tether PKA to the outer mitochon-
drial membrane (OMM) and intermembrane space within the proximity of its local
targets (Fig. 5) (Feliciello et al. 2005; Kovanich et al. 2010; Means et al. 2011;
Lefkimmiatis et al. 2013).
The existence of an independent intramitochondrial cAMP signaling circuit
consisting of the bicarbonate-activated soluble adenylyl cyclase (sAC), PKA holo-
enzyme, and phosphodiesterase 2a (PDE2A) has also been reported (Lefkimmiatis
et al. 2013; Wuttke et al. 2001; Zippin et al. 2003; Sardanelli et al. 2006; Acin-Perez
et al. 2009, 2011a, b). Hence, strictly hypothetically, even a matrix-based cAMP-
PKA regulation of SLC41A3-mediated Mg2+ would be possible (Fig. 5).
Fig. 5 Strictly hypothetical model of SLC41A3 regulation via cAMP-PKA and Akt/PKB signaling
pathways in mitochondria. AC adenylyl cyclase, Akt/PKB protein kinase B, AKAP1 A-kinase-
anchoring protein 1, APC ATP-Mg/Pi carrier, CM cytoplasmic membrane, G glucagon, GP G
protein, HME H+/Mg2+ exchanger (?, mechanism encoded by yet unknown molecular entity), IMM
inner mitochondrial membrane, INS insulin, Mrs2 mitochondrial Mg2+ channel, NME Na+/Mg2+
exchanger, OMM outer mitochondrial membrane, PDE2a phosphodiesterase 2a, PKA protein
kinase A, RTK receptor tyrosine kinase, sAC soluble adenylyl cyclase, SKIP sphingosine kinase
type 1 interacting protein
Insulin (INS) decreases the concentration of the cytosolic cAMP via the classic
INS-signaling cascade IR-PI3K-Akt/PKB by the activation of phosphodiesterase 3b
(PDE3b) and consequently limits the activation of the cytosolic PKA (Mastrototaro
et al. 2015).
INS promotes the efflux of Mg2+ from mitochondria (and perhaps also other
intracellular Mg2+ stores) via a cAMP-PKA-independent mechanism concomitant
with the translocation of PKB into mitochondria (Fig. 5) (Mastrototaro et al. 2015;
Bijur and Jope 2003). The exact nature of the INS action on the process of
mitochondrial Mg2+ release and its correlation with the translocation/accumulation
of Akt/PKB into mitochondria remain elusive. Without doubt, PKA and Akt/PKB
play fundamental roles in the regulation of cytosolic and mitochondrial [Mg2+] and
the regulation of mitochondrial homeostasis per se. Furthermore, both of these
kinases significantly influence cellular energetics and metabolic activity. Thus,
further research aimed at explaining the role of PKA and Akt/PKB in the regulation
of the mitochondrial accumulation of Mg2+ and its release from mitochondria is
urgently needed (Zhang et al. 2017a; Manning and Toker 2017).
Recently, van Ooijen and colleagues have explained a possible molecular back-
ground behind the involvement of [Mg2+]i and its oscillations in the regulation of the
circadian clock in eukaryotes (Feeney et al. 2016). Interestingly, PKA and Akt/PKB
have both been substantially implicated in the regulation of the circadian clock
(Huang et al. 2007; Noguchi et al. 2018; Luciano et al. 2018). Thus, we can assume
that except for SLC41A1, also the components of mitochondrial Mg2+ homeostasis
(Mrs2, SLC41A3 and APC) and their regulatory network (very likely PKA and
Akt/PKB) form an intricate biological mechanism making mitochondria not only
“a battery” but also “a tuning mechanism” for the circadian clock. Further research
into the intersection between mitochondrial homeostasis and cellular/mitochondrial
Mg2+ homeostasis is undoubtedly of great importance, especially for the field
of chronomedicine and for research into degenerative diseases, in which
CHRONO-component plays an important role.
ATP-Mg/Pi carrier (APC; or short Ca2+-binding mitochondrial carrier, SCaMC;
or solute carrier family 25 member A24, A25, A23, A41 (SLC25A24, SLC25A25,
SLC25A23, SLC25A41)), which is localized in the IMM, facilitates an
electroneutral reversible exchange between MgATP2 and HPO42 (Figs. 1 and
4) (Run et al. 2015; Traba et al. 2009; Joyal and Aprille 1992). APC remains inactive
unless stimulated by a cytosolic Ca2+ signal (Nosek et al. 1990). While under
physiological conditions, the preferred substrates for the APC are MgATP2 and
Pi (HPO42), but in the absence of Mg2+, ADP (HADP2) can also be transported
via the APC (Joyal and Aprille 1992; Tewari et al. 2012). Nevertheless, this
transporter does not transport ionized Mg2+ alone; complexed with ATP, its impact
on the modulation of mitochondrial or cytosolic [Mg2+] might be significant (Tewari
et al. 2012; Kun 1976). However, no information has been acquired about any
possible crosstalk between APC and other mitochondrial Mg2+ transporters.
The ADP/ATP carrier (AAC, or solute carrier family 25 member A4, A5, A6
(SLC25A4–6)) is the major carrier that exports ATP out of the matrix for energy
consumption while importing ADP for the production of new ATP by the ATP
synthase, and its functional defects can be detrimental for the cell (Run et al. 2015;
Fiore et al. 1998; Klingenberg 2008). Whereas AAC accounts for the bulk of
ADP/ATP recycling in the matrix, APC is important for mitochondrial activities in
the matrix that depend on adenine nucleotides, such as gluconeogenesis and mito-
chondrial biogenesis (Run et al. 2015). Quoting Romani, Trapani, and Wolf have
stated that, although the AAC substrate of choice is ATP, it might change to MgATP
in some cases, e.g., following cAMP or thyroid hormone stimulation (Romani 2011;
Trapani and Wolf 2015). However, this statement is contradicted by several works
concluding that MgATP or MgADP is not transported through AAC (Run et al.
2015; Pfaff et al. 1969; Nury et al. 2006). Therefore, currently, questions remain as
to whether AAC, alone or in the mitochondrial permeability transition pore complex,
contributes to Mg2+ transport across IMM (Karch and Molkentin 2014).
Pharmacological experiments with SNAP, 8-Br-cGMP, diazoxide, and several
inhibitors, performed by Yamanaka and colleagues, have revealed that the NO –
cGMP – protein kinase G (PKG) signaling pathway triggers an increase in [Mg2+]i
and that Mg2+ mobilization is attributable to the Mg2+ release from mitochondria
induced by mitoKATP channel opening (Yamanaka et al. 2013). Furthermore, Mg2+
release is potentiated by the positive feedback loop including mitoKATP channel
opening, mitochondrial depolarization, and PKC activation (Yamanaka et al. 2013).
Despite the ongoing debate about the physical existence of the mitoKATP channel,
the recreation of the experiments of Yamanaka and colleagues in rodent or human-
derived cells/cell lines with silenced expression or overexpression of mitochondrial
Mg2+ extruder SLC41A3 would be of interest (Yamanaka et al. 2013; Garlid and
Halestrap 2012).
Yeast (S. cerevisiae) Lpe10 (or MFM1; Mrs2 function modulating factor 1)
belongs to the CorA superfamily of Mg2+ transporters. Moreover, it is homologous
to yeast Mrs2 (approximately 32% sequence homology and presence of the G-M-N
Mg2+ binding motive) (Gregan et al. 2001b). Sponder and colleagues have utilized
an intriguing set of complementation experiments in order to demonstrate that both
Lpe10 and Mrs2 are functionally related and that they form complexes together but
cannot substitute for each other (Sponder et al. 2010a). Deletion of Lpe10 leads to a
rapid loss of the membrane potential on IMM, a phenomenon otherwise not seen
when only Mrs2 is deleted (Sponder et al. 2010a). Lpe10 alone is not able to mediate
the high-capacity Mg2+ influx otherwise seen with Mrs2. When coexpressed with
Mrs2, they yield a unique reduced Mg2+ conductance in comparison with that of
Mrs2 channels (Sponder et al. 2010a). A homolog of Lpe10 is not found in
mammalian/human mitochondria. However, we cannot exclude that, during the
evolution, the Mrs2-modulating role of Lpe10 was overtaken by an as yet unknown
mitochondrial protein innate to mammalian/human mitochondria. Only further
targeted research may address this issue.
Recently, a mitochondrial Mg2+ efflux system constituted by Mme1 (mitochon-
drial Mg2+ exporter 1) protein has been described in S. cerevisiae, followed by the
discovery of its ortholog, dMme1 protein, in Drosophila melanogaster (Cui et al.
2015, 2016). No human ortholog of Mme1 or dMme1, if any, has yet been identified.
An alteration in the expression of dMme1, although only resulting in a change of
about 10% in mitochondrial Mg2+ levels in either direction, leads to a significant

survival reduction in Drosophila (Cui et al. 2016). Despite the excitement brought
about by the discovery of Mme1 and dMme1, further experiments confirming its role
in the mitochondrial Mg2+ homeostasis of single-cellular and lower multicellular
eukaryotes are necessary. In particular, the mode of operation of both orthologs
should be explained. Testing its ability to functionally complement for the deletion
of SLC41A3 would also be of interest.

Localized to Endoplasmic Reticulum
The high metabolic activity and the large estimated ΔψER of 75 to 95 mV across
the membrane of ER make this cellular compartment, similar to mitochondria, not
only “a Mg2+ consumer” but also a suitable compartment for storing Mg2+ (with a
total Mg concentration estimated as being between 14 and 18 mM) (Fig. 2) (Romani
2011; Qin et al. 2011). Unfortunately, among npMgTs characterized by Goytain and
Quamme’s group, no candidates have been predicted to localize to ER compartment
(Quamme 2010).
In the regnum of plants, Li and colleagues have mapped the Mg2+ transporter
AtMGT4 (Arabidopsis thaliana Mg2+ Transporter 4), a distant homolog of yeast
Mrs2 and a member of the CorA superfamily, to ER (Li et al. 2015). There is no
reason to suggest that the ER of animal cells differ from the ER of plant cells with
respect to the existence of ER-localized Mg2+ transporters/Mg2+ homeostatic factors.
Presently, many indirect indices argue for the existence of an ER-localized Mg2+
transporter in mammalian/human cellular systems; however, only future experimen-
tal attempts will uncover their identities.
ATP13A4, a member of the subfamily of P5-type ATPases, is thought to act as a
cation transporter and can serve as an example of a promising candidate (Schultheis
et al. 2004). Subcellularly, it localizes to the ER when expressed in COS-7 cells
(Fig. 1) (Vallipuram et al. 2010). Will and colleagues have hypothesized that
ATP13A4 transports Mg2+ (Will et al. 2010). However, the same authors stress
that the substrate specificity of ATP13A4 is as yet only poorly understood.

Localized to the Golgi Apparatus
In contrast to ER, a screen performed by Goytain and Quamme’s group has revealed
the identities of four genes encoding for proteins putatively localized to the GA and
post-Golgi vesicles, namely, MMgT1, MMgT2 (not found in the human genome but
present in mouse), and HIP14 and HIP14L (Quamme 2010; Goytain and Quamme
2008; Goytain et al. 2008b).
Murine MMgT1 (membrane Mg2+ transporter 1) and MMgT2 (membrane Mg2+

transporter 2) are proteins putatively localized in the GA and post-Golgi vesicles and
possess two transmembrane helices but show no structural similarities to other
known Mg2+ transporters (Fig. 1) (Quamme 2010; Goytain and Quamme 2008).
Murine MMgT1 and MMgT2 are 97 and 81% similar to human MMgT1 (Quamme
2010; Goytain and Quamme 2008). When expressed in X. laevis oocytes, they
conduct the electrogenic transport of Mg2+ as determined with TEV analysis and
fluorescence measurements (apart from other cations, Table 2) (Quamme 2010;
Goytain and Quamme 2008). The observed Mg2+ transport was saturable with a
Km ¼ 1.47 0.17 mM (MMgT1) or 0.58 0.07 mM (MMgT2) (Quamme 2010;
Goytain and Quamme 2008). As it is generally assumed that proteins with two
membrane-spanning helices are unable to support the transport of any ion/solute,
Goytain and Quamme have concluded that MMgT1 and MMgT2 form
homooligomeric or heterooligomeric complexes to sustain their transport function
(Quamme 2010; Goytain and Quamme 2008). The ability to transport Mg2+ by
MMgT1 and/or MMgT2 has not yet been demonstrated in a homologous (mamma-
lian) expression system. Furthermore, the previously discussed concerns regarding
the use of TEV for the establishment of the ion permeation profile should be
considered before any data is used for the design of future studies. The localization
data of both putative Mg2+ transporters are based only on confocal microscopy
(Goytain and Quamme 2008). To show bona fide the cellular localization of MMgT1
and MMgT2, a complementary experimental approach leading to the purification of
GA (e.g., differential and density gradient centrifugation) and the immunodetection
of both proteins in the purified GA fraction should be utilized.
HIP14 (huntingtin-interacting protein 14) and HIP14L (HIP14-like protein, 64%
homologous to HIP14) have been proposed to reside in the membrane of GA (HIP14
is predicted to contain five or six transmembrane helices and HIP14L seven trans-
membrane helices) enabling the electrogenic transport of Mg2+ (Fig. 1) (Quamme
2010; Goytain et al. 2008b; Ducker et al. 2004). This conclusion has been made
based on the data from TEV experiments performed on X. laevis oocytes expressing
murine HIP14 or HIP14L (Quamme 2010; Goytain et al. 2008b). The ion selectivity
of HIP14 and HIP14L is quite broad (Table 2); however, showing a clear preference
for Mg2+ and Sr2+ (Quamme 2010; Goytain et al. 2008b). The Mg2+ transport
recorded by Goytain and colleagues is saturable with Km ¼ 0.87 0.02 mM
(HIP14) or 0.74 0.07 mM (HIP14L) (Quamme 2010; Goytain et al. 2008b).
HIP14 has been characterized as palmitoyl acyltransferase essential for the traffick-
ing and functioning of huntingtin with substantial involvement in the ethiopathology
of Huntington disease (Ducker et al. 2004; Yanai et al. 2006; Butland et al. 2014).
Considering the latter and their own data, Goytain and colleagues have concluded
that HIP14 and HIP14L fulfill the requirements for being chanzymes (natural fusion
protein consistent of ion channel and enzyme) (Quamme 2010; Goytain et al.
2008b). Unfortunately, although no doubt exists about HIP14/HIP14L functioning
as a palmitoyl acyltransferase, clear evidence is lacking that it operates as a Mg2+
transporter in GA of mammalian/human cells.
Schapiro and Grinstein have assumed that ΔψGA of GA membrane is small

almost close to 0 mV (Fig. 2) (Schapiro and Grinstein 2000). ΔψGA around 0 mV
and the presumable lack of an inwardly oriented Mg2+ concentration gradient
between the cytosol and the lumen of GA would clearly not be in favor of
GA-localized Mg2+ channels/chanzymes as predicted by Goytain and colleagues
(Schapiro and Grinstein 2000; Günther 2006a). Instead, the existence of
GA-localized Mg2+ transport mechanism(s) powered by ATP (Mg2+ pump) or a
secondary motive force (H+, Na+) would be more sensible.

Localized to Lysosomes
The lysosomal membrane potential ΔψLYS was determined to be, on average,

+19 mV (positive on the luminal side, Fig. 2) (Koivusalo et al. 2011). Therefore,
on the presumption of the equimolar distribution of Mg2+ between the cytosolic and
lysosomal compartments, any transport of positively charged Mg2+ into lysosomes
can be supported only via primary or secondary active Mg2+ transport mechanisms.
The Caenorhabditis elegans ortholog of human P-type ATPase (subfamily P5B)
ATP13A2 (PARK9), CATP-6, is speculated to function as a lysosomal Mg2+
transporter (Lambie et al. 2013). Whether human ATP13A2 functions as a Mg2+
transporter/lysosomal Mg2+-mediating pump remains to be further researched and
convincingly demonstrated (Fig. 1).
4.5 Mg2+ Transport Across Nuclear Membrane
In their most recent work, Maeshima and colleagues have concluded that Mg2+
released from the MgATP complex after its hydrolysis contributes to mitotic
chromosome condensation with increased rigidity, suggesting a novel regulatory
mechanism for higher-order chromatin organization by the intracellular MgATP
complex balance (Maeshima et al. 2018). Because of DNA and RNA metabolism,
which is known to be Mg2+-dependent, Mg2+ is of high demand by the nucleus
(Nishizawa et al. 2007). The major nuclear Mg2+ influx/efflux mechanism is
believed to be constituted by the nuclear pore that allows Mg2+ to move freely
across the porous nuclear membranes simply by diffusion (Romani 2011).
4.6 Putative and Confirmed Mg2+ Transporters/Mg2+

Homeostatic Factors with Unclear Localization
4.6.1 SLC41A2 (Solute Carrier Family 41 Member A2)
Member A2 of the solute carriers family 41 (SLC41A2), which like other members
of this family is distantly homologous to the bacterial Mg2+ transporter MgtE, was
predicted by Sahni and colleagues to reside in cytoplasmic membrane, as based on
results from experimental evidence (Sahni et al. 2007). The protein comprises
11 transmembrane helices with an “N-terminus outside and C-terminus inside”
orientation of its termini (Sahni et al. 2007). Since the orientation of SLC41A2
appears to be the opposite of that predicted by the structure of prokaryotic MgtE
proteins or SLC41A1, the same group has subsequently speculated that the observed
plasma membrane localization of SLC41A2 reflects aberrant cell surface targeting
attributable to the overexpression of the protein (Sahni et al. 2007; Sahni and
Scharenberg 2013). Despite the targeting and placement of SLC41A2 to the cyto-
plasmic membrane having not been completely rejected, the hypothesis that
SLC41A2 plays its roles in intracellular membranous compartments and vesicles
is currently better accepted (Sahni et al. 2007; Sahni and Scharenberg 2013).
Similar to other npMgTs functionally characterized by the group of Goytain
and Quamme, SLC41A2, when overexpressed in X. laevis oocytes, also
conducts the electrogenic transport of Mg2+ and of other cations (Table 2) (Quamme
2010; Goytain and Quamme 2005b). Sahni and colleagues have attempted to
identify Mg2+-specific currents in DT40 cells overexpressing human SLC41A2
with patch clamp; however, these attempts remain unsuccessful (Sahni et al. 2007).
Although the data on the mode of Mg2+ transport via SLC41A2 are puzzling, we
can safely say that, with high probability, SLC41A2 is a Mg2+ transporter. This
assumption is underpinned by the ability of SLC41A2 to complement the growth/
proliferation defect of DT40 TRPM7-KO cells, when they are cultured at physio-
logical [Mg2+]e. TRPM7-deficient DT40 cells induced to express SLC41A2 prolif-
erate more slowly than wild-type DT40 cells in culture medium containing
physiological levels of Mg2+, but in contrast to the TRPM7-deficient cells, they
are able to proliferate continuously (Sahni et al. 2007). Furthermore, Sahni
and colleagues have determined the Mg2+ uptake (measured by the ratio of 26Mg2+/
24
Mg2+) to be approximately twofold to threefold higher in SLC41A2-
overexpressing cells as compared with the cells without induced expression of
transgenic SLC41A2 and even greater than that observed in the wild-type DT40
cells (Sahni et al. 2007). The last mentioned results provide direct evidence that
SLC41A2 overexpression correlates with enhanced Mg2+ accumulation.
Whether the gene SLC41A2 is truly magnesiotropic remains controversial.
Goytain and Quamme have used real-time RT-PCR analysis of MDCT cells cultured
in medium with [Mg2+]e of 1 mM or in Mg2+ free medium for 16 h and found no
change in SLC41A2 expression; the same result was found to be the case with kidney
cortical tissue harvested from mice fed with a normal or low Mg2+ diet for 5 days
(Goytain and Quamme 2005b). In a review on “the modern Mg2+ transporters,”

Quamme has however postulated that “SLC41A1–3 transcripts are present in many
tissues of normal mice. Furthermore, the mRNAs in these tissues are increased with
low Mg2+” (Quamme 2010).
Despite rather strong experimental evidence, especially from the laboratory of
Sahni and colleagues, SLC41A2 is an only marginally researched Mg2+ transporter,
which certainly deserves much more attention, as it could be a key player in the
cellular Mg2+ homeostasis, like members A1 and A3 from the same family of solute
transporters.
4.6.2 The CNNM (Cyclin and CBS Domain Divalent Metal Cation
Transport Mediator) Family, “Enfant terrible” Among Mg2+
Transporters/Mg2+ Homeostatic Factors
The CNNM family consists of four members (CNNM1–CNNM4) that were for-
merly known as ancient conserved domain proteins (ACDP). Their original name
was based on the observation that these genes/proteins possessed a highly conserved
domain also found in other species from bacteria to mammals. The most conserved
regions of CNNMs are the two cystathionine beta-synthase (CBS) domains and the
DUF21 domain that are both also found in bacterial CorC (Wang et al. 2003).
The high homology of ACD proteins to the bacterial CorC protein which is
involved in Mg2+ and Co2+ efflux early led to the speculation that these proteins
are involved in ion transport (Gibson et al. 1991; Wang et al. 2004). Although
sequence conservation among the family members is remarkably high (for the
human and mouse family members: 55.3% of amino acid identity and 83.3% of
amino acid homology in the conserved region), their expression pattern, localization
within the cell (Fig. 1), and their function appear to be divergent and are still a matter
of debate (Wang et al. 2004).
CNNM2 (Cyclin and CBS Domain Divalent Metal Cation Transport

Mediator 2)
CNNM2 is the best studied representative of this family and, at the same time, is the
most controversial. Wang and colleagues have demonstrated the expression of
CNNM2 in most mouse tissues; however, the expression levels are generally low,
except in the brain, kidney, and liver (Wang et al. 2003). This finding has subse-
quently been confirmed in mice by Goytain and Quamme who have found the
highest expression in kidney and brain (Goytain and Quamme 2005d). Abundant
amounts have also been detected in the heart and liver, with lower expression in the
small intestine and colon. As mentioned previously, the expression of CNNM2 is
influenced by the food Mg content and displays increased expression in the kidneys
of mice fed with a low Mg diet and in cultured MDCT cells cultured in low Mg
medium (Goytain and Quamme 2005d). Mouse CNNM2, when expressed in
Xenopus oocytes, exhibits currents for Mg2+ and other divalent cations (Table 2) and
therefore behaves as a rather unspecific cation transporter. However, as the Km was
within a physiological range only for Mg2+, Goytain and Quamme concluded that
CNNM2 primarily acts as a Mg2+ transporter (Quamme 2010; Goytain and Quamme
2005d). The assumption that CNNM2 acts as a Mg2+ transporter seems to be
supported by a study of Sponder and colleagues who have demonstrated that the
longest splice variant of human CNNM2 (875 amino acids) partially complements
the Mg-dependent growth defect of Salmonella strains MM281 (deficient for MgtA,
MgtB, and CorA). Only the somewhat shorter splice variant 2 fails to do so (Sponder
et al. 2010b).
Further evidence for the involvement of CNNM2 in Mg2+ transport has
come from a study that has identified a connection between CNNM2 and
dominant hypomagnesemia. This rare disorder is characterized by severely lowered
serum Mg2+ levels most probably caused by defective tubular reabsorption. Such a
notion is also supported by the localization of CNNM2 on the basolateral membrane
of DCT cells and its strong expression in the kidney (Stuiver et al. 2011; de Baaij
et al. 2012).
Interestingly, the electrophysiological characterization of CNNM2 in HEK293
cells yields Mg2+-sensitive Na+ currents and thereby divergent results from those
observed in Xenopus oocytes. The authors have consequently concluded that
CNNM2 is a regulatory factor rather than a direct mediator of Mg2+ transport
(Stuiver et al. 2011). Currents for Na+ have also been reported by another group
when CNNM2 is overexpressed in HEK293 cells (Yamazaki et al. 2013).
Arjona and colleagues have used zebrafish as a model system to study the
function of CNNM2 and have reported disturbed brain development and reduced
body Mg content as consequences of the deletion of the gene. They have furthermore
demonstrated, with the aid of stable isotope 25Mg2+, that the overexpression of
mouse CNNM2 in HEK293 cells increases the cellular Mg content. However,
since this effect is abolished by the TRPM7 inhibitor 2 APB, the authors have
concluded that this effect is indirect and presumably mediated by a regulatory
effect of CNNM2 on the Mg2+ permeable channel TRPM7 (Arjona et al. 2014).
This is in contrast to the data obtained by Hirata and colleagues with the Mg2+
indicator Magnesium Green, which has shown the strong and remarkably fast efflux
of Mg2+ within seconds when CNNM2 is expressed in HEK293 cells. They have
further found the CBS domains that directly bind ATP in a Mg2+-dependent manner
as being indispensable for Mg2+ efflux (Hirata et al. 2014).
The same group of Miki has subsequently used mice to investigate the function of
CNNM2 in an animal model. Homozygous knockout mice have an embryonic lethal
phenotype; heterozygous mice are viable and exhibit impaired Mg reabsorption with
reduced serum Mg levels. The same phenotype is observed in mice with a kidney-
specific deletion of CNNM2 (Funato et al. 2017).
In contrast to these functional data from mice, Sponder and colleagues have
employed mag-fura-2, Mg2+-sensitive fluorescent dye, in an in vitro model to
investigate the Mg2+ transport activity of two of the three known splice variants of
human CNNM2 under conditions favoring both Mg2+ influx and efflux. Although
the same expression system, HEK293 cells as in previous studies, were used neither
electroneutral nor was electrogenic Mg2+ transport detected for the two splice
variants in the overexpressing cells. The authors have concluded that CNNM2 acts
as Mg2+ homeostatic factor without being a Mg2+ transporter itself (Sponder et al.
2016).
Recently, a role of CNNM2 in tumorigenesis has been proposed. The protein
interacts with PRL-1, a member of the so-called phosphatases of regenerating liver;
these phosphatases exhibit high expression in most solid tumors and hematological
cancers and are considered to be highly oncogenic. Binding between the two
proteins is mediated via the interaction of an amino acid in the CBS domain of
CNNM2 and the catalytic domain of the phosphatase. The authors speculate that this
interaction increases the cellular magnesium content, thereby aggravating tumor
progression and metastasis formation (Giménez-Mascarell et al. 2017).
In summary, great efforts have been undertaken to elucidate the molecular
function of CNNM2. In view of the connection between mutations in CNNM2
and patients suffering from hypomagnesemia and knockdown experiments in
mouse and zebrafish, the involvement of CNNMs in cellular Mg2+ homeostasis is
unequivocal. However, the strongly divergent behavior of CNNM2 in various
expression systems and experimental setups complicates rather than clarifies the
situation and has led to much conflicting and contradictory data. A clear conclusion
cannot therefore be drawn about the function of this protein in cellular Mg2+
homeostasis.
CNNM4 (Cyclin and CBS Domain Divalent Metal Cation Transport

Mediator 4)
CNNM4 has a relatively broad expression pattern with its highest expression in
intestinal epithelia (Wang et al. 2003; de Baaij et al. 2012).
The first indication for a function in metal ion homeostasis came from the
observation that the expression of the protein in HEK293 cells resulted in an
increased sensitivity to copper, manganese, and cobalt. The toxicity of these metal
ions was aggravated by the coexpression of CNNM4 together with COX11
suggesting a possible functional dependence of CNNM4 on other proteins (Guo
et al. 2005). Mutations in CNNM4 do not influence blood Mg concentrations and
cause Jalili syndrome in humans, a combination of recessively inherited cone-rod
dystrophy and amelogenesis imperfecta. Given the broad expression pattern, the
findings that only the retina and ameloblasts are affected by the expression of mutant
variants of CNNM4 and that therefore phenotypic consequences are restricted to
retinal function and tooth biomineralization are of interest (Parry et al. 2009).
CNNM4 has also appeared together with CNNM2 and CNNM3 in a genome-
wide association study on common single nucleotide polymorphisms SNPs being
associated with serum magnesium concentrations (Meyer et al. 2010).
Direct evidence for an involvement of CNNM4 in Mg2+ homeostasis has come
from Miki’s group who have characterized CNNM4 in mice. Expression of the
protein has been found to be high in the intestine where it localizes to the basolateral
membrane. No expression in the kidney has been detected. CNNM4 knockout mice
are viable with no obvious phenotype. However, the animals exhibit lower serum
Mg concentration, a result that has been attributed to impaired intestinal Mg2+
absorption. The transport function was directly assessed by the use of the Mg2+
indicator Magnesium Green. When CNNM4 was expressed in HEK293 cells,
the protein mediated a very rapid efflux of Mg2+ that was dependent on extracellular
Na+. Similar to CNNM2, the CBS domains are also essential for the transport
activity of CNNM4 (Hirata et al. 2014). From their data, the authors conclude that
CNNM4 forms a high capacity Mg2+ efflux system that is localized in the basolateral
membrane of the intestine and that acts as a counterpart to the TRPM6/7 channel-
mediated Mg2+ uptake system on the apical side. Of note is the observation that the
knockout of CNNM4 in mice results in amelogenesis imperfecta. However, in
contrast to descriptions of Jalili syndrome in humans, the retina remains unaffected
(Yamazaki et al. 2013).
CNNM1 and CNNM3 (Cyclin and CBS Domain Divalent Metal Cation
Transport Mediator3 and 4)
Knowledge about the two other members of this family, namely, CNNM1 and
CNNM3, is still scarce. Wang et al. have reported the high expression of CNNM1
in the brain, whereas only low levels have been detected in the kidney, testis, and
most other tested tissues. For CNNM3, the highest expression has been observed in
the brain, kidney, liver, and heart and very low levels in skeletal muscles (Wang et al.
2003). Together with CNNM2 and CNNM4, CNNM3 has been also found in the
aforementioned genome-wide association study as being linked to the serum mag-
nesium concentration (Meyer et al. 2010). Studies directly investigating the possible
transport activity of the two proteins are rare. In a study on the pufferfish Takifugu
obscurus, CNNM3 was upregulated in the kidney when the animals were kept in salt
water. Immunohistochemical investigations revealed expression in the proximal
tubule where CNNM3 was localized to the lateral membrane. The expression of
CNNM3 in Xenopus oocytes resulted in a significant decrease of the cellular Mg2+
concentration (Islam et al. 2014).
The only study in a mammalian model system was performed in the laboratory of
Miki. They investigated the importance of the CBS domains for all four members of
the CNNM family.
CNNMs were expressed in HEK293 cells, and Magnesium Green was used for
intracellular Mg2+ detection. In contrast to the aforementioned strong activity of
CNNM2 and CNNM4, only a weak efflux was detected for CNNM1, and no activity
in cells expressing CNNM3 was observed in this experimental setup (Hirata et al.
2014).
Interestingly, for CNNM3 an interaction with two members of the aforemen-
tioned phosphatases of regenerating liver (PRLs), namely, PRL-2 and PRL-3, has
also been reported suggesting a role of CNNM3 in tumor development and progres-
sion (Zhang et al. 2017b).
4.7 Putative and Confirmed Mg2+ Transporters/Mg2+

Homeostatic Factors with Plasma Membrane
Localization
Several Mg2+ influx and/or efflux mechanisms have been foreseen to exist in the
cytoplasmic membrane (Nishizawa et al. 2007; Romani 2011; Schweigel et al.
2000). Presently, only chanzymes TRPM6/7 and Na+/Mg2+ exchanger SLC41A1
are well characterized at the molecular level (Schweigel-Röntgen and Kolisek 2014;
Penner and Fleig 2007; Sponder et al. 2017; Cabezas-Bratesco et al. 2015). It is
universally accepted that both chanzymes represent a channel component responsi-
ble for the transport of the majority of extracellular Mg2+ into the cell.
In vitro, also SLC41A1 has been shown to mediate Mg2+ uptake under conditions
strongly supporting Mg2+ influx. However, it has to be stressed out that these
conditions were far from physiological to most of the tested cell types (Schweigel-
Röntgen and Kolisek 2014; Kolisek et al. 2008, 2012; Fleig et al. 2013). Whether
SLC41A1 mediates Mg2+ influx also in vivo under physiological conditions is
unclear and a subject of ongoing debates.
Unequivocally, confirmed by several independent and unbiased studies, SLC41A1
was shown to be an ubiquitously expressed (Table 3), major cellular Mg2+ efflux
system functionally conserved from fish to Man (Schweigel-Röntgen and Kolisek
2014; Kolisek et al. 2008, 2012; Fleig et al. 2013; Islam et al. 2013; Hurd et al. 2013;
Lin et al. 2014).
Thus, it could be stated with confidence that TRPM6/7 chanzymes together with
Mg2+ efflux-mediating carrier SLC41A1 constitute the Mg2+ transport circuit of
cytoplasmic membrane (Fig. 6).
4.7.1 SLC41A1 (Solute Carrier Family 41 Member A1)
SLC41A1 is a plasma membrane-localized protein (Fig. 1) possessing 10 or

11 transmembrane helices with either both termini oriented intracellularly (10 trans-
membrane helices model) or with the N-terminus oriented toward the inside and an
extracellular C-terminus (11 transmembrane helix model) (Kolisek et al. 2008;
Sponder et al. 2013b; Mandt et al. 2011). The putative role of SLC41A1 in cellular
Mg2+ homeostasis, more precisely in Mg2+ transport, was assumed by Wabakken
and colleagues based on its distant homology to the bacterial MgtE family of Mg2+
transporters (Wabakken et al. 2003). Shortly after, it was recognized by Goytain and
Quamme as being an MgG, and by using TEV, mouse SLC41A1 has been shown to
mediate electrogenic Mg2+ influx (but also influx of other cations; Table 2) when
overexpressed by X. laevis oocytes (Quamme 2010; Goytain and Quamme 2005a).
Kolisek and colleagues have utilized the patch clamp technique attempting to
identify SLC41A1-specific Mg2+ currents in HEK293 cells overexpressing human
SLC41A1. However, these attempts remained unsuccessful. Instead of identifying
a SLC41A1-connected Mg2+ conductance, the currents induced by SLC41A1
Fig. 6 Current model of the Mg2+ transport circuit on cytoplasmic membrane (CM). It consists of a
Mg2+ ion channel allowing for Mg2+ influx (chanzymes TRPM6/7) and a Na+/Mg2+ exchanger
(NME; SLC41A1) mediating Mg2+ efflux
overexpression have been identified as endogenous Cl currents, recruited by

depletion of intracellular Mg2+ and blockable by the broad-spectrum Cl transport
antagonist DIDS (Kolisek et al. 2008). Nevertheless, in a strain of Salmonella
enterica MM281 exhibiting disruption of all three distinct magnesium transport
systems (CorA, MgtA, and MgtB), overexpression of human SLC41A1 functionally
substituted for these transporters and restored the growth of the mutant bacteria at
[Mg2+] otherwise nonpermissive for growth (Kolisek et al. 2008). Mandt and
colleagues have also performed functional complementation experiments showing
the ability of SLC41A1 to functionally substitute for TRPM7 in TRPM7-KO DT40
cells with heterologous expression of wild-type SLC41A1 (Mandt et al. 2011).
Furthermore, Kolisek and colleagues have shown that overexpression of SLC41A1
provided HEK293 cells with an increased Mg2+ efflux capacity. With outwardly
directed Mg2+ gradients, a SLC41A1-dependent reduction of the [Mg2+]i accompa-
nied by a significant net decrease of the total cellular [Mg] ([Mg]t) could be observed
in HEK293 cells overexpressing SLC41A1 (Kolisek et al. 2008). SLC41A1 activity

was temperature-sensitive and insensitive to the Mg2+ channel blocker, cobalt(III)
hexaammine (Kolisek et al. 2008). Thus, it has been concluded that SLC41A1 is a
bona fide Mg2+ carrier mediating Mg2+ efflux (Kolisek et al. 2008).
In follow-up work, Kolisek and colleagues, by using the fluorescent probe
mag-fura-2 to measure [Mg2+]i changes in transgenic HEK293 cells with inducible
overexpression of SLC41A1, demonstrated that SLC41A1-mediated Mg2+ efflux is
strictly dependent on [Na+]e and reduced by 91% after complete replacement of Na+
with N-methyl-D-glucamine (Kolisek et al. 2012). Also imipramine and quinidine,
known unspecific Na+/Mg2+ exchanger inhibitors, led to a strong 88–100%
inhibition of SLC41A1-related Mg2+ extrusion (Kolisek et al. 2012). Furthermore,
these authors have demonstrated the cAMP-mediated regulation of the transport
activity of SLC41A1 via phosphorylation by cAMP-dependent protein kinase
A. Thus, all together, it has been concluded that SLC41A1 met all the characteristics
of the Na+/Mg2+ exchanger, which has been formerly described in many scientific
reports before its molecular identity had been uncovered (Nishizawa et al. 2007;
Romani 2007, 2011; Romani and Scarpa 2000; Delva et al. 2006; Schweigel-
Röntgen and Kolisek 2014; Günther 1993, 2006b, 2007; Fleig et al. 2013; Kolisek
et al. 2012).
The cytosolic N-terminal flanking region of SLC41A1 plays a key role in the
regulation of SLC41A1 function and turnover. Not only it possesses multiple
phosphorylation hotspots for various kinases, but Mandt and colleagues have also
shown that the deletion of the cytosolic N-terminal sequence (92 amino acids long)
of the protein suspends Mg2+-dependent regulation of SLC41A1-mediated Mg2+
transport via the endosomal protein recycling in the cell (Kolisek et al. 2012; Mandt
et al. 2011). They have proposed a model, in which independent of [Mg2+]e,
SLC41A1 is constitutively endocytically recycled from the cell surface. When the
[Mg2+]i is low, recycling of endocytosed SLC41A1 to the cell surface is favored over
degradation at the lysosome. When the [Mg2+]i is high, lysosomal degradation is
favored (Mandt et al. 2011). However, this model is not coherent with the core
function of SLC41A1, to conduct Mg2+ extrusion. If that were the case, it would
mean that the cellular capacity to extrude Mg2+ increases with lowering [Mg2+]i, a
rather strange thought.
The importance of cAMP in regulation of SLC41A1-mediated Mg2+ efflux has
been underlined by the work of Mastrototaro and colleagues (Mastrototaro et al.
2015). They have corroborated an inhibitory effect of insulin ([cAMP]i limiting
hormone) on the SLC41A1-dependent Mg2+ efflux and clarified the role of the
insulin signaling cascade with the end effector PDE3b (phosphodiesterase 3b)
for regulation of the Mg2+extruder. Recently Sponder and colleagues correlated
overexpression of SLC41A1 with the attenuation of pro-survival Akt/PKB–Erk1/2
signaling in HEK293, HeLa, and SH-SY5Y cells and thus, from the larger perspec-
tive, demonstrated that SLC41A1 expression status and [Mg2+]e (and consequently
also [Mg2+]i) modulate the activity of kinases involved in anti-apoptotic and, hence,
pro-survival events in cells (Sponder et al. 2017).
SLC41A1 has been implicated in the pathoetiology of various disease conditions.

Noteworthy is the chromosomal localization of SLC41A1 within the Parkinson
disease (PD) susceptibility locus PARK16. Moreover, several PD-associated muta-
tions have been detected in SLC41A1. Regarding PD, it is assumed that gain of
function mutations (e.g., p.A350V) in SLC41A1 might contribute to an increased
risk of developing PD and vice versa – loss of function mutations in the Mg2+
extruder might decrease the risk of developing PD (Kolisek et al. 2013a; Bai et al.
2017).
Furthermore, SLC41A1 has also been associated with preeclampsia/eclampsia in
pregnant women, kidney complications, diabetes mellitus, osteoporosis, and cardio-
vascular complications (Mastrototaro et al. 2015; Hurd et al. 2013; Kolisek et al.
2013b; Tsao et al. 2017; Yu et al. 2014).
4.7.2 KelI/XK
Although primarily expressed in erythroid tissues, Kell and XK are also present in
many other tissues (Table 3) (Rivera et al. 2013; Lee et al. 2000). XK and Kell are
linked close to the membrane surface by a single disulfide bond between Kell
cysteine 72 and XK cysteine 347 (Fig. 1) (Lee et al. 2000). While Kell is an
endothelin-3-converting enzyme, XK has structural characteristics of prokaryotic
and eukaryotic transport proteins (Ho et al. 1994; Zhu et al. 2009). Rivera and
colleagues studied the activity of Na+/Mg2+ exchanger in erythrocytes of Kell-KO,
XK-KO, and Kell/XK-KO mice (Rivera et al. 2013). They found that Na+/Mg2+
exchange was significantly reduced by the absence of XK and increased in Kell-KO
animals compared to wild-type mice. However, in Kell/XK-KO, the Na+/Mg2+
exchange activity resembled that of Kell-KO Na+/Mg2+ exchange activity
suggesting that Kell may act as a Na+/Mg2+ exchanger regulatory unit (Rivera
et al. 2013).
Deletion or loss of function mutations within XK leads to McLeod syndrome.
It is a rare and progressive disease that shares important similarities with Huntington
disease but has widely varied neurologic, neuromuscular, and cardiologic manifes-
tations. Patients with McLeod syndrome have a distinct hematologic presentation
with specific transfusion requirements (Roulis et al. 2018).
4.7.3 NIPA1–NIPA4 (Non-imprinted in Prader-Willi/Angelman

Syndrome 1–4)
The human NIPA family comprises four members, namely, NIPA1, NIPA2,
NIPAL1 (NIPA3), and NIPAL4 (NIPA4). Members 1–4 (mouse) of the NIPA
family have been characterized as putative Mg2+ transporters with channel-like
properties when expressed in X. laevis oocytes and characterized with TEV and
mag-fura-2-assisted fluorescence spectrometry (Table 1) (Quamme 2010; Goytain
et al. 2007, 2008a). NIPA1–NIPA4 mediate Mg2+ uptake that is electrogenic,
voltage-dependent, and saturable with a Km ¼ 0.31, 0.66, 0.9, and 0.36, respectively
(Quamme 2010; Goytain et al. 2007, 2008a). All four NIPA proteins exhibit quite
broad substrate specificity and thus behave like unspecific cation channels (Table 2)
(Quamme 2010; Goytain et al. 2007, 2008a). NIPA1–NIPA4 seem to be ubiqui-
tously expressed, but NIPA1 has its highest expression level in the brain (Table 3).
The alteration of [Mg2+]i induces the redistribution of NIPA1 and NIPA2 between
the endosomal compartment and the plasma membrane (Fig. 1). High [Mg2+]e results
in diminished cell surface NIPA1 and NIPA2, whereas low [Mg2+]e leads to an
accumulation of NIPA1 and NIPA2 in early endosomes and their recruitment to the
plasma membrane (Quamme 2010; Goytain et al. 2007, 2008a).
Unfortunately, all NIPA-related Mg2+ transport data have been acquired in a
heterologous expression system, and thus we cannot exclude that NIPA1–NIPA4 do
not transport Mg2+ in homologous expression systems.
NIPA1 has been implicated in autosomal-dominant hereditary spastic paraplegia
and NIPA2 in childhood absence epilepsy (Svenstrup et al. 2011; Xie et al. 2014). If
the Mg2+ transport function of NIPA proteins is confirmed in a homologous
expression system, this would represent direct evidence of the involvement of
cellular Mg homeostasis in the pathoetiology of these serious neurological ailments.
4.8 MagT1 (Magnesium Transporter Subtype 1) and TUSC3/

N33 (Tumor Suppressor Candidate 3): Mg2+ Transporter
Candidates Which Turned to Have Other but Not Mg2+
Transport Functions
MagT1 (magnesium transporter subtype 1) was screened among the magnesiotropic

genes described by Goytain, Quamme, and colleagues (Quamme 2010; Goytain and
Quamme 2005c). Its first functional characterization was produced by the same
group. MagT1 expressed in X. laevis oocytes conducts the electrogenic transport of
Mg2+ as revealed by TEV. Permeation of MagT1 is limited strictly to Mg2+
(Quamme 2010; Goytain and Quamme 2005c). Zhou and Clapham have demon-
strated the plasma membrane localization of human MagT1 and its human homolog
TUSC3/N33 (tumor suppressor candidate 3; found by in silico analysis, MagT1 and
TUSC3 share 66% identity in the amino acid sequences) and have examined with
whole-cell mode patch clamp net currents resulting from the overexpression of these
proteins in HEK 293T cells. No MagT1 and/or TUSC3/N33-specific Mg2+ conduc-
tance has been recognized (Zhou and Clapham 2009). However, by using the
new generation Mg2+ probe KMG-104 AM, these authors have demonstrated
that siRNA-mediated MagT1 and/or TUSC3/N33 silencing leads to a reduction of
the Mg2+ influx capacity. Interestingly, the knockdown of MagT1 results in about
the same reduction of Mg2+ influx capacity as has been seen for TUSC3/N33; the
simultaneous knockdown of both Mg2+ transporter candidates, namely, MagT1 and
TUSC/N33, has no additive effect on the reduction of Mg2+ influx capacity of

HEK293 cells (Zhou and Clapham 2009).
Zhou and Clapham have also performed a functional complementation test and
shown that, when expressed in the alr1Δ S. cerevisiae strain JS74B, human MagT1
and TUSC3/N33 sp.v. 2 can complement its growth deficiency upon the presence of
normal Mg2+ in the cultivation medium (otherwise this strain proliferates only when
50–100 mM Mg2+ is provided to the cells) (Zhou and Clapham 2009).
The sequences of MagT1 and TUSC3/N33 have no similarity to any known
bacterial or eukaryotic (mammalian) genes encoding for Mg2+ transporters
(Quamme 2010; Goytain and Quamme 2005c). MagT1 and TUSC3/N33 are
orthologs of the yeast proteins Ost3 and Ost6 and have recently been found to be
the oxidoreductase subunits of the ER-localized STT3B (catalytic subunit of the
oligosaccharyltransferase (OST) complex) complex (Shrimal et al. 2015). The
STT3B complex contains either MagT1 or TUSC3/N33, which have a role in
substrate recruitment (Cherepanova et al. 2016; Mohorko et al. 2014).
Obviously, the Mg2+ transport function and localization proposed for MagT1 and
TUSC3/N33 by Goytain and Quamme or Zhou and Clapham are clearly not in
agreement with their oxidoreductase function or being part of the ER-localized OST
complex (Zhou and Clapham 2009; Quamme 2010). The bifunctionality of MagT1
and TUSC3/N33 as OST subunits and plasma membrane Mg2+ transporters is in
conflict with the observation that MagT1 and TUSC3/N33 proteins are not detect-
able in STT3B(/) HEK293-derived cell line, as these OST subunits are unstable in
the absence of STT3B (Cherepanova and Gilmore 2016). Currently, the most
feasible explanation for the involvement of MagT1 and TUSC3/N33 proteins in
cellular Mg homeostasis is the one offered by Cherepanova and colleagues who
hypothesize that the link between MagT1 or TUSC3 expression and Mg homeostasis
probably occurs by an indirect mechanism involving the STT3B-complex-
dependent glycosylation of a protein that is needed for Mg2+ transport activity
(Cherepanova et al. 2016).
5 Summary
Approximately two decades ago, no eukaryotic Mg2+ transporters/Mg2+ homeostatic

factors had been identified at the molecular level. Today, we recognize about two
dozen of them (either confirmed or putative) with their identities revealed to scien-
tists and biomedical professionals. At first glance, this is a remarkable move forward,
but is this true?
Without a doubt, the identification of Mrs2, TRPM6/7, and the novel MgGs
(mostly discovered and characterized by Goytain and Quamme) have brought “fresh
breeze” and momentum to the field of Mg research that was, at that time, limited
“only” to biochemical and physiological studies because of a lack of molecular
targets and because no genetic manipulation, or in-depth characterization of molec-
ular constituents of cellular Mg homeostasis were possible.
However, with hindsight, the selection of X. laevis oocytes for the characteriza-
tion of mammalian npMgTs (encoded by MgGs) in combination with TEV was not
the best move. Unfortunately, not a single mammalian/human npMgT assumed to be
an ion channel transporting Mg2+ (apart of other cations) based on TEV character-
ization in X. laevis oocytes has proven to be one in homologous expression systems.
Thus, strictly speaking, out of the total of 24 candidate Mg2+ transporters/Mg2+
homeostatic factors, only approximately one third has been experimentally
established as being directly involved in the Mg2+ homeostatic network of the cell,
either as Mg2+ transporters or as Mg2+ homeostatic factors. The other two thirds have
been studied only poorly, sometimes being limited to a single report.
Some of the pnMgTs have over the time proven not to be Mg2+ transporters at all.
For example, MagT1 and TUSC3/N33 are still often quoted as being Mg2+ channels,
despite the fact that they have never been shown to be channels in a homologous
expression system. Moreover, large amounts of solid evidence exist showing that
MagT1 and TUSC3/N33 are oxidoreductases of the ER-localized OST complex.
Nowadays, the proteins constituting the cytoplasmic membrane Mg2+ transport
circuit (TRPM6/7 and SLC41A1; Fig. 6) and the mitochondrial (IMM) Mg2+
transport circuit (Mrs2, SLC41A3 and APC/SCaMC; Figs. 3 and 4) have been
demonstrated by independent studies to be the primary constituents of the cellular
Mg2+ homeostasis. They all act like Mg2+ transporters (channels, carriers/
exchangers). The position of CNNM2 and CNNM4 in cellular Mg2+ homeostasis
is a matter of controversy, and the debate continues as to whether these proteins
are Mg2+ transporters per se or whether they play the role of “true” Mg2+ homeo-
static factors without the ability to transport Mg2+ (meaning sensors of [Mg2+]e
and/or [Mg2+]i and regulators of other components of Mg2+ homeostatic network
that are transporting Mg2+).
Nevertheless, no doubt exists that CNNM2 is crucial for Mg2+ homeostasis at the
level of the cell and also of the organism, as mutations in the gene encoding for
CNNM2 have been identified that lead to severe systemic hypomagnesaemia and
renal Mg2+ wasting.
The other putative Mg2+ transporters/homeostatic factors described in this man-
uscript must be robustly researched before uncertainties can be lifted to enable a
comprehensive elucidation of their functions with respect to cellular Mg2+
homeostasis.
Acknowledgment Our gratitude is due to Dr. Theresa Jones for linguistic corrections. This work
was supported by the grant “Return Home” issued by the Government of Slovak Republic to MK
and also by the project “Creating a New Diagnostic Algorithm for Selected Cancer Diseases”
(ITMS: 26220220022) co-financed from EU sources and the European Regional
Development Fund.
The authors declare no conflict of interests. All authors read and approved the final version of
the manuscript.
Contributions MK and GS wrote the manuscript, and IP, MC, ZT, TW, and PR contributed to the
manuscript writing.
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DOI: 10.1007/112_2018_11
© Springer International Publishing AG, part of Springer Nature 2018
Published online: 5 May 2018
Curcumin in Advancing Treatment

for Gynecological Cancers with Developed
Drug- and Radiotherapy-Associated
Resistance

and Atefeh Vaezi
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2 Curcumin in the Treatment of Gynecological Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
The original version of this chapter was revised. A correction to this chapter is available at
DOI: 10.1007/112_2018_14.
A. A. Momtazi-Borojeni (*)
Nanotechnology Research Center, Bu-Ali Research Institute, Mashhad University of Medical
Sciences, Mashhad, Iran
Department of Medical Biotechnology, Student Research Committee, Faculty of Medicine,
Mashhad University of Medical Sciences, Mashhad, Iran
e-mail: momtaziaa921@mums.ac.ir; abbasmomtazi@yahoo.com
J. Mosafer
Research Center of Advanced Technologies in Medicine, Torbat Heydarieh University of
Medical Sciences, Torbat Heydarieh, Iran
B. Nikfar (*)
Pars Advanced and Minimally Invasive Medical Manners Research Center, Pars Hospital, Iran
University of Medical Sciences, Tehran, Iran
e-mail: banafsheh.nikfar@gmail.com
M. Ekhlasi-Hundrieser
Werlhof-Institut, Hannover, Germany
S. Chaichian
Minimally Invasive Techniques Research Center in Women, Tehran Medical Sciences Branch,
Islamic Azad University, Tehran, Iran
A. Mehdizadehkashi
Endometriosis Research Center, Iran University of Medical Sciences, Tehran, Iran
A. Vaezi
Department of Community Medicine, School of Medicine, Isfahan University of Medical
Sciences, Isfahan, Iran
108 A. A. Momtazi-Borojeni et al.
3 Curcumin in Restoring Platinum Drug-Induced Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

4 Molecular Evidences of Synergistic Anticancer Features of Curcumin and Paclitaxel
In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5 Curcumin and Paclitaxel in the Form of Nanoformulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
6 The Radiosensitizing Function of Curcumin in Gynecological Cancers . . . . . . . . . . . . . . . . . 118
7 Approaches to Enhance Curcumin Anticancer Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
8 Exploitation of Molecular Pathways Modulated by Curcumin in Gynecological
Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
9 Curcumin Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
10 Curcumin Nanoformulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
11 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Abstract The development of resistance toward current cancer therapy modalities is

an ongoing challenge in gynecological cancers, especially ovarian and cervical malig-
nancies that require further investigations in the context of drug- and irradiation-
induced resistance. In this regard, curcumin has demonstrated beneficial and highly
pleiotropic actions and increased the therapeutic efficiency of radiochemotherapy. The
antiproliferative, anti-metastatic, anti-angiogenic, and anti-inflammatory effects of
curcumin have been extensively reported in the literature, and it could also act as a
chemopreventive agent which mitigates the out-of-target harmful impact of chemo-
therapeutics on surrounding normal tissues. The current review discussed the modu-
lating influences of curcumin on some cell and molecular features, including the cell
signaling and molecular pathways altered upon curcumin treatment, the expression of
target genes involved in the progression of gynecological cancers, as well as the
expression of genes accountable for the development of resistance toward common
chemotherapeutics and radiotherapy. The cell molecular targets implicated in
curcumin’s resensitizing effect, when used together with cisplatin, paclitaxel, and
irradiation in gynecological cancers, are also addressed. Finally, rational approaches
for improving the therapeutic benefits of curcumin, including curcumin derivatives
with enhanced therapeutic efficacy, using nanoformulations to advance curcumin
stability in physiological media and improve bioavailability have been elucidated.
Keywords Cervical cancer · Cisplatin · Curcumin · Nanoformulation · Ovarian

cancer · Paclitaxel
Abbreviations
CDK Cyclin-dependent kinase

COX-2 Cyclooxygenase-2
Cur Curcumin
DNMTs DNA methyltransferases
GSTs Glutathione S-transferases
HDAC Histone deacetylases
IAPs Inhibitor of apoptosis family of proteins
ICAM-1 Intercellular adhesion molecule 1
Curcumin Effect on Gynecological Cancers 109
IKK IκB kinase

iNOS Inducible nitric oxide synthase
MT Metallothionein
P-gp P-glycoprotein
PI3K Phosphatidylinositide 3-kinase
VEGF Vascular endothelial growth factor
1 Introduction
Cancer is defined as the uncontrolled growth of particular cells inside an organism,

where the outgrowth of cells eventually causes serious complications (Peng et al.
2003). Considering that cancer cells are derived from a normal cell or tissue
following the accumulation of mutations imparting aggressive features, there is
almost as many kinds of cancer cells as there are normal tissues (Abouzeid et al.
2014; Chen et al. 2015a; Ganta and Amiji 2009; Montopoli et al. 2009; Nessa et al.
2012; Peng et al. 2003). The prominent types of cancers are derived from tissues
with high rate of cell division or great exposure to potential mitogenic compounds.
According to the Global Burden of Disease Study (GBD), gynecological cancers and
breast cancer are ranked among the top kinds of cancers causing death, together,
both accounted for 4.29% mortality among women worldwide in 2017 (https://
vizhub.healthdata.org/gbd). Although the targeted treatment of cancer cells has
remained a difficult task, thanks to the numerous attempts of scientists, the treatment
modalities have greatly improved. In this regard, combined-therapy modalities have
been shown to give superior successful results compared to single-treatment modal-
ities in controlling different types of cancers, including gynecological cancers
(Abouzeid et al. 2014; Aqil et al. 2017; Ganta and Amiji 2009; Huq et al. 2014;
Punfa et al. 2012; Saengkrit et al. 2014; Sarisozen et al. 2014).
The sequential mode of therapy in gynecological cancers begins with the surgical
removal of tumors followed with radiotherapy and chemotherapy, to eliminate
any remaining cancer cells. However, the management of cancer therapy faces
difficulties upon relapse with more refractory tumors (Watson et al. 2010; Zaman
et al. 2016; Zhang et al. 2017). For example, in ovarian cancer, more than 70% of the
first diagnosed patients are found resistant to taxane treatment, and finally all of
them are left resistant upon relapse (Watson et al. 2010). Although combination
chemoradiotherapy improves the survival rate, it also increases the chance of
dose-limiting toxicities (Watson et al. 2010). In this regard, curcumin, a polyphenol
derived from Curcuma longa plant, has shown beneficial anti-inflammatory
(Abdollahi et al. 2018; Aggarwal and Harikumar 2009; Momtazi-Borojeni et al.
2017), antitumor (Kuttan et al. 2007; Momtazi et al. 2016), anti-metastatic, anti-
angiogenic (Kuttan et al. 2007), antioxidant (Ak and Gülçin 2008), and chemopre-
ventive (Duvoix et al. 2005; Kawamori et al. 1999; Rezaee et al. 2016) properties,
when added to the current regimens. The molecular targets of curcumin are report-
edly very diverse, including various kinases, gene modulators, transcription factors,
varying growth factors, and cell membrane receptors (Hajavi et al. 2017; Kasinski
et al. 2008; Pan et al. 2008; Soflaei et al. 2017; Watson et al. 2010; Zhang et al.
2017). Curcumin with very wide pleiotropic functions holds the key to modifying
the trend of cancer therapy and advanced development of current cancer therapy
modalities. Regarding gynecological cancers, curcumin has demonstrated opposing
typical cervical cancer risk factors in advancing molecular alterations toward
cancer incidence or progression, including human papilloma virus infections (typi-
cally HPV16 and 18), estrogen, smoking, and obesity (Maruthur et al. 2009; Zaman
et al. 2016). For instance, it has been shown that curcumin could inhibit the
expression of E6 and E7 oncoprotein, reduce estrogen-induced DNA damage, and
mitigate adipose-related inflammation and estrogen production (Zaman et al. 2016).
The current review presents an assortment of studies on curcumin ameliorating
functions in gynecological cancer progression and metastasis. The sensitizing influ-
ence of curcumin treatment, when combined with routine chemotherapeutic agents
like cisplatin and paclitaxel as well as irradiation, has been addressed, together with
the molecular pathways associated with the drug-induced resistance which curcumin
counteracts. Moreover, the approaches adopted to advance curcumin anticancer
potential, stability, and bioavailability have also been discussed thoroughly in
terms of effectiveness, which includes various curcumin formulations and curcumin
derivatives.
2 Curcumin in the Treatment of Gynecological Cancers
It has been shown that curcumin could act as an anti-metastatic agent and inhibit
endometrial carcinoma (EC) cell migration and invasion in vitro through decreasing
the expression and activity of the matrix metalloproteinases (MMP)-2 and MMP-9.
These enzymes that degrade the extracellular matrix in tumors make the metastasis
of cancer cells possible and are believed to drive deep myometrial cancer invasion
and metastasis in lymph node in type II EC. The reduced expression of these
enzymes by curcumin was also found to occur through suppression of the ERK
signaling pathway (Chen et al. 2015b). Curcumin-induced apoptosis in ovarian
cancer cells was found to be independent of p35, as it displayed the same cytotoxic
activity in cells with reduced or knockdown p53 expression, as shown in the
wild-type p53 cells. Nuclear condensation and fragmentation, DNA fragmentation,
and poly(ADP-ribose) polymerase-1 cleavage were the cell features in HEY cells
treated with curcumin which denoted cell apoptosis. Furthermore, it was found
that both the intrinsic and extrinsic pathways of apoptosis could be activated by
curcumin. The enhanced activity of p38 mitogen-activated protein kinases (MAPK)
reduced the expression of antiapoptotic regulators of survivin and Bcl-2, and the
suppression of prosurvival Akt signaling was also found to be involved in curcumin-
mediated anticancer cell death in various ovarian cancer cells (Watson et al. 2010).
It has been shown that curcumin could partially suppress urokinase-type plas-
minogen activator (uPA) expression in the highly invasive human ovarian cancer
cell line, HRA, which is involved in cancer cell metastasis. The expression of the
serine protease uPA has been determined to be fed through Src-MAPK/ERK-PI3K/

Akt-NF-kB and the Src-MAPK/ERK-AP-1 pathways in response to TGF-B1, where
curcumin is reported to only abrogate the formation of the AP-1 complex (Tanaka
et al. 2004). Given the beneficial influences of curcumin in limiting invasive cancer
cell progression, the use of curcumin in the commonly used therapy regimen for
advanced gynecological cancer types could help to improve the outcome of therapy.
This paper presents studies on gynecological cancerous tumors, in which curcumin
addition has been demonstrated to improve the therapeutic window and oppose
drug-resistant cancer cell types.
3 Curcumin in Restoring Platinum Drug-Induced

Resistance
Platinum drugs such as cisplatin and oxaliplatin are the first-line chemotherape-
utics against ovarian, bladder, and testicular cancers, and their administrations are
frequently faced with the development of resistant tumors (Montopoli et al. 2009).
The loss of platinum uptake by cells through gated channel-facilitated diffusion, p53
gene implication in DNA damage repair, and enhanced intracellular level of gluta-
thione, responsible for platinum inactivation and removal, are among the underlying
mechanisms for the drug resistance. To evade the platinum drug-induced resistance,
extensive studies have been conducted, in which a combination therapy with phyto-
chemicals has been shown to be highly effective. In this regard, curcumin has been
utilized in combination with platinum drugs like cisplatin and oxaliplatin to enhance
their anticancer properties.
It has been shown that curcumin could resensitize cisplatin-resistant ovarian
cancer cells, and it suppresses DNA damage responses against these DNA cross-
linking agents. It has been found that curcumin treatment downregulates the Fanconi
anemia (FA)/BRCA pathway-related DNA damage repair responses, such as
FANCD2 protein mono-ubiquitination, which is the prerequisite step for the DNA
damage repair complex to form and relocate into chromatin of the DNA lesion sites
(Chen et al. 2015a). Therefore, curcumin could reverse the acquired resistance in
cancer cells, which lies in the enhanced activation of the FA/BRCA pathway, in
response to DNA cross-linking agents in long-term administration (Fig. 1).
Moreover, curcumin could suppress cisplatin resistance development through
extracellular vesicle-mediated cell-cell communication. It is believed that the extra-
cellular vesicles, known as exosome, transfer some proteins, mRNAs, and non-
coding RNAs from donor cells to recipient cells, and this communication leads
to the development of a drug-resistant cell population in various cancers (Zhang
et al. 2017). Curcumin could limit such exosome-mediated chemoresistance by
changing their contents. For instance, curcumin treatment has been shown to be
accompanied with the restoration of MEG3 long noncoding (lnc) RNA levels
(Fig. 1), upregulation of miR-29a and miR-185, and downregulation of miR-124
Fig. 1 A conclusive view of main mediators in curcumin-mediated resensitizing platinum-resistant

ovarian cancer cells. Curcumin has been found to restore platinum drug-induced resistance through
suppressing the Fanconi anemia (FA)/BRCA pathway-related DNA damage repair responses, such
as FANCD2 protein. Curcumin can also exert resensitizing effect through upregulation of MEG3
lncRNAs levels that inhibit drug resistance. Furthermore, curcumin enhances the ROS production
via reduction of cellular levels of IL-6, NF-Kb, and GSH
and DNA methyl transferases (DNMTs) in cisplatin-resistant cells and exosomes,

resulting in chemo-sensitization in A2780cp ovarian cancer cells. In this regard,
curcumin-mediated overexpression of miR-29a and miR-185 has been shown to
reduce DNMT1, 3A, and 3B levels and downregulate miR-124 expression, which is
an mRNA regulator acting through the PTEN/Akt pathway and the P53/Nanog axis,
and could reduce the survival of ovarian cancer cells, cisplatin resistance, and stem
cell development (Zhang et al. 2017).
Curcumin has been found to enhance the cell killing potential of chemotherapeu-
tics by increasing the generation of reactive oxygen species (ROS) in cancer cells.
It has been reported that curcumin could reduce the level of intracellular thiol of
GSH, which is known to be involved in cisplatin deactivation (Yunos et al. 2011)
(Fig. 1). It has been shown that pretreatment with cisplatin for 4 h before curcumin
addition results in a synergistic cytotoxicity in some ovarian cancer cells, where
curcumin could potentiate the ROS-mediated cell death triggered by cisplatin. The
curcumin efficiency in suppression of cancer cell growth was found to be positively
correlated with the basal levels of ROS in cancer cells. Interestingly, curcumin
exhibited chemopreventive effect on normal tissues by activating the NF-kB
pathway where the ROS intracellular level is decreased, whereas high curcumin
concentrations (>15 μM) were found to enhance ROS levels in these tissues.
Conversely, in cancer cells with previously high ROS concentrations, curcumin
was shown to enhance ROS levels by inactivating the NF-kB pathway (Sreekanth
et al. 2011; Yunos et al. 2011). Moreover, it appears that the reduced ROS and the
increased GSH basal levels are the main hallmark of the development of resistance to
cisplatin in cisplatin-resistant ovarian cancer cells, which could be linked to the more
active NF-kB pathway in these cells. In summary, it is plausible for curcumin to
contribute much greater to induce ROS generation in cisplatin-treated cancer
cells than in non-treated ones, indicating that curcumin could act as a modifier in
chemotherapy (Yunos et al. 2011) (Fig. 1).
In addition to the NF-kB transcription factor discussed above, it was found
that the expression of many other proteins was altered upon curcumin treatment
(Nessa et al. 2012). A total of 59 proteins were found to be associated with platinum
resistance in ovarian cancer cells, juxtaposing 2D gel electrophoresis from A2780
tumor model with that of resistant tumor cells (Huq et al. 2014). These included
cytoskeletal proteins involved in cell invasion and metastasis, stress-related proteins
and molecular chaperones, proteins involved in detoxification and metabolic pro-
cesses, as well as a set of mRNA processing proteins (for a complete list, refer to
Huq et al. 2014). The inhibition of inflammatory cytokines (e.g., TNF-α, IL-1, IL-6)
and enzymes (e.g., cyclooxygenase or COX-2 and inducible nitric oxide synthase or
iNOS), suppression of angiogenic factors (e.g., vascular endothelial growth factor or
VEGF), and modulation of other signaling proteins [e.g., the upregulation of serine/
threonine-specific protein kinase (AKT)] have also been reported in curcumin-
treated cancer cells (Nessa et al. 2012).
It has also been shown that curcumin-mediated sensitization to cisplatin is
associated with its anti-inflammatory activities in resistant cancer cells. It has
been revealed that IL-6 reduction in curcumin-treated CAOV3 and SKOV3 ovarian
cancer cell lines is accompanied by increased sensitivity to cisplatin, where it is
believed that the overproduction of pro-inflammatory cytokines as such by the tumor
cells drives drug resistance and tumor invasion (Chan et al. 2003). The production
of IL-6 could also induce drug resistance in other cancer cells, including myeloma,
lung, breast, prostate, and colon cancer cells. It has been found that multiple
molecular targets are affected in IL-6-induced platinum resistance in various tumor
cells. Mechanistically, IL-6 could reduce cisplatin accumulation in tumor cells
through induction of multidrug-resistant proteins (MRPs) and P-glycoprotein
(Pgp) in human hepatoma and renal carcinoma cells, induce the expression of
glutathione S-transferase involved in ROS scavenging in breast cancer cells, and
enhance the expression of metal-detoxifying protein of metallothionein in ovarian
cancer cells. It has also been proposed to be involved in enhancing the invasiveness
of ovarian cancer cells, where the induced transcription factor NF-kB results to
the expression of additional inflammatory cytokines (Chan et al. 2003).
The modulation of epigenetic regulators could lead to the emergence of cancer
cells, where curcumin has also been shown to counteract them (Roy and Mukherjee
2014). In cervical cancer cases, the human papilloma virus is putatively known as
the causative agent of cancer emergence and development, in which curcumin has
been effective in suppressing the expression of viral oncoproteins of HPV-E6 and
HPV-E7. Curcumin treatment has also been reported to mitigate cell molecular
modulations derived from the activity of HPV-E6 and HPV-E7 proteins. Curcumin
could inhibit p53 ubiquitin-dependent proteasomal degradation driven by HPV-E6
and act against HPV-E7-reduced pRb functionality. Curcumin could result in cell
cycle arrest at the G1/S phase through the modification of regulatory proteins
involved in cell cycle. It could inhibit the histone deacetylases (HDACs) that are
activated by HPV. The acetylation and upregulation of p53 proteins; increased pRb,
p21, and p27; and the corresponding suppression of cylin D1 and CDK4 have also
been shown in both cisplatin-sensitive and cisplatin-resistant cancer cell line SiHa
upon curcumin treatment, which are known to occur during cancer cell apoptosis
(Roy and Mukherjee 2014).
Figure 2 depicts the summary of the abovementioned multiple pathways and
molecular targets where curcumin exerts opposing influence over cisplatin-induced
resistance. In summary, it appears that curcumin is a modifier of multiple cellular
pathways deregulated during cancer cell progression and, therefore, it could con-
tribute to the advanced antiproliferative responses of other chemoagents applied for
cervical and ovarian cancers. Such an improvement might hinder the development of
resistance toward these agents. Paclitaxel is another chemotherapeutic agent that is
administered for different sorts of gynecological cancers, and it has been shown that
co-treatment with curcumin and paclitaxel could promote antitumor responses in
comparison with paclitaxel alone. In this context, curcumin formulations could alter
the expression of multiple cellular proteins and provide resistance to paclitaxel,
which are discussed in the following two sections.
4 Molecular Evidences of Synergistic Anticancer Features

of Curcumin and Paclitaxel In Vivo
In a study conducted by Sreekanth et al. (Sreekanth et al. 2011), a comprehensive

picture of the molecular evidences pertaining to the chemosensitizing effectiveness
of liposomal curcumin in paclitaxel therapy has been presented, in which a curcumin
liposomal formulation, consisting of phosphatidylcholine and cholesterol, was
injected intraperitoneally every other day at a dose of 25 mg/kg to tumor-burden
mice treated with paclitaxel (i.p. dose of 10 mg/kg, twice weekly). The tumors were
mouse squamous cervical carcinoma model induced by 3-methylcholanthrene
(3-MC), a carcinogen, and a xenograft model of a human cervical cancer (HeLa
cells) in nonobese diabetic mice having severe combined immunodeficiency
(NOD-SCID). It was found that the co-treatment with curcumin and paclitaxel
resulted in a synergistic reduction in tumor emergence and tumor volume as
compared to the single treatments. To determine the underlying cell and molecular
mechanisms, a large collection of molecular targets were examined, including
Cyclin D1, ICAM-1

Cell survival VEGF
DNA repair MMP-2, MMP-9
P53 Cox-2
Bcl-2
Rb Survivin
XIAP
Angiogenesis c-IAP
VEGF
IkBa
ion
MEG3 IKK
lat
DNMT3a
thy
PP
me
mi-R29a
A
DNMT1
po
DN
mRNA
hy
PP p21
mi-R185 Cur ReLA p50
PIP2 027
PI–3K Cyclin D1
ne
mi-R124
PTEN
ge
CDK4
Cell survival
proliferation
et
PIP3
mi-R-21
rg
Ac
Ta
mobility
se
Ac
era
RE
Akt P53
NA
pathway
lym
IL-1 TNF-a Ac Ac mRNA
rD
po
a
Ac
cle
A
RN
Nu
TGF-Beta IL-6 HDACs
iNOS
E6
MT Cox-2
D
Dr rug P-gp E7
ug d E2F
ef eto GST Rb E2F
flu xif
x ica transcription
tio E7
n P53 Degradation factor
Rb
Fig. 2 A summary of the molecular pathways by which curcumin treatment could lead to the
sanitization of cancer cells in the cisplatin-induced resistant cancer cells. Curcumin could modulate
the content of exosomes that contain molecular messengers driving the development of resistance
toward cisplatin in the recipient cells (yellow sect). By inactivating the NF-kB pathway, curcumin
could modify many molecular targets that are involved in cancer progression and metastasis (pink
sect). Curcumin could reduce inflammatory cytokine secretion and the enzymes producing inflam-
matory compounds. Through IL-6 downregulation, moreover, curcumin could reduce the expres-
sion of metallothionein, glutathione S-transferase and P-glycoproteins, involved in the scavenging
of superoxide radicals, drug detoxification, and drug efflux from cells, respectively (Green section).
Finally, by counteracting HPV-E6 and HPV-E7 oncoprotein, curcumin could restore the level of
antiapoptotic protein p53, inactivate histone deacetylase involved in chromatin condensation, and
limit E2F transcription factor translocation to the nucleus, where the expression of target genes
drives cell division and growth
the NF-kB activation status and the expression of NF-kB target genes involved
in inflammation and tumor aggressiveness (such as Cox-2, ICAM-1, cyclin D1,
VEGF, MMP-2, and MMP-9); the expression of antiapoptotic proteins that are
transactivated by NF-kB (Bcl-2, c-IA P1, survivin, and XIAP); the expression and
activation of three vital MAP kinases – i.e., c-Jun-NH2 kinase (JNK), extracellular
signal-regulated protein kinase (ERK), and p38; as well as the cleavage and activity
of procaspases 9, 8, 7, and 3. All these molecular evidences indicated that curcumin
could tackle cancerous tumors by modulating various cell signaling pathways and
kinases (Sreekanth et al. 2011), which could promote the efficiency of treatment
in combination with paclitaxel. Similarly, it has been reported that the combination
of curcumin (5 μM) and paclitaxel (5 nM) could augment anticancer responses more
efficiently than paclitaxel alone in HeLa cells, without any synergistic effect on
normal cervical cells, the 293 cell line (Bava et al. 2005). It has been proposed that
the curcumin-induced sensitization to paclitaxel could be related to the opposite
effect of curcumin on the NF-kB activation status. It was identified that curcumin
could suppress NF-kB and Akt pathways, augment the activation of caspases and
cytochrome c release. Moreover, it was discovered that curcumin opposed the
NF-kB activation induced by paclitaxel and reduced the phosphorylation of Akt,
which is a survival signal regulated by NF-kB (Bava et al. 2005). However, at low
concentrations (5 μM), curcumin could not interfere with the tubulin-polymerization
action of paclitaxel and could not further augment the cell cycle protein Cdc2, which
increased during paclitaxel-induced mitotic arrest. This indicates that paclitaxel-
induced resensitization by curcumin is independent of the classic function of taxols
(or paclitaxel).
To exert the abovementioned influences in cancer cells in vivo, it is required to
improve the efficiency of drug delivery to these cells through the application of
various drug delivery systems. Curcumin and paclitaxel have been shown to have
poor pharmacokinetic profiles, which necessitates the use of appropriate formula-
tions to help in attaining the required dose of the drug at tumor sites.
5 Curcumin and Paclitaxel in the Form

of Nanoformulations
According to the recent version of the National Comprehensive Cancer Network

(NCCN) guidelines for cervical cancer, cisplatin and paclitaxel are recommended as
the first-line combination therapy for cervical cancer metastasis (Li et al. 2017).
However, the drug-related side effects (nephrotoxicity and hepatotoxicity) and
development of resistance to the therapy have raised serious concerns in clinics
(Li et al. 2017). As mentioned for cisplatin, some similar mechanisms are also
accountable for the development of paclitaxel-induced resistance, including the
upregulation of transmembrane-associated multidrug resistance proteins (P-gp,
MRP-1, and ABCG2) and activation of major cell signaling pathways, most impor-
tantly, the NF-kB upregulation at the heart of many cellular responses related to
cancer cell evasion. The upregulation of cytoprotective pathways like Akt and
mitogen-activated protein kinase (MAPK), changes in the frequency of b-tubulin
isotypes (Giannakakou et al. 1997; Yusuf et al. 2003), and changes in topoisomerase
II (Topo IIa) activity and GST activities have also been reported for paclitaxel-
induced resistance. Under these conditions, finding a chemopreventive agent like
curcumin that could concomitantly confer cancer cell’s drug resensitization is the
rational approach in dealing with invasive tumors. It has been shown that not
only can curcumin reverse the multidrug resistance of cancer cells, it also could
reduce the nephrotoxicity of paclitaxel, thereby enhancing the chemotherapeutic

window. Similar findings have also been presumed for the paclitaxel and curcumin
combination therapy, especially in the forms of various nanoformulations to enhance
their stability and tumor delivery of these hydrophobic agents.
Paclitaxelis is commonly prescribed against a wide spectrum of epithelial cancers
(Sreekanth et al. 2011), where the hydrophobic nature of the drug promotes drug
assimilation into tissues. However, when the drug is intended to reach a local tumor
far from the injection site, hydrophobic chemoagents are required to be loaded in a
vehicle with a hydrophilic outer layer to prevent the drug from rapid elimination and
uptake by neighboring cells. The same strategy is also needed for the hydrophobic
curcumin, where it is assumed to impart chemoprevention and chemo-sensitization
in normal tissues and tumors, respectively (Ganta and Amiji 2009).
Apart from cellular mechanisms of drug resistance and in vivo poor drug distri-
bution, the high interstitial fluid pressure prevents the drug from moving toward
cancer cells, where the presentation of the drug in the form of nanoformulation could
overcome it (Abouzeid et al. 2014). Moreover, many researchers have attempted to
apply various formulations, sometimes with excipients that impart some cellular
modulating effect resulting to facilitated cell apoptosis (Ganta and Amiji 2009).
For instance, it was reported that poly (ethylene oxide)-modified poly-(epsilon-
caprolactone) (PEO-PCL) nanoparticles encapsulating paclitaxel (PTX) and C6-
ceramide, a lipid potentiating the apoptotic signal responses, could enhance both
the efficiency of paclitaxel transfer to tumor and anticancer responses in SKOV3
human ovarian cancer cells in xenograft tumors, inoculated to female nu/nu mice.
Through the encapsulation of curcumin along with paclitaxel in flaxseed oil cont-
aining nano-emulsion, it was found that curcumin could block NF-kB pathways and
ABC transporter expression in both the wild-type and paclitaxel-resistant SKOV3
cells and enhance the death of cancer cells (Ganta and Amiji 2009). Furthermore, it
has also been shown that the nano-emulsion, rich in omega-3 and omega-6 unsatu-
rated fatty acids, promotes the cellular delivery of curcumin and paclitaxel. Although
the author did not present any data regarding the residence time of curcumin and
paclitaxel in the serum or blood and only focused on the mitigation of MDR features
and cellular apoptosis, it was found that the nanoformulation was physically stable in
size (~150 nm in diameter), and it could entrap large concentrations of paclitaxel and
curcumin. Since both curcumin and paclitaxel are hydrophobic in nature, it was
proposed that the inner oil phase could solubilize them and the outer hydrophilic
shell of the droplets due to the presence of dense PEG chains, could enhance their
physical stability, and apparently would promote drug delivery to tumor, if they are
injected in vivo through the circulatory system (Ganta and Amiji 2009).
The improved solubility and stability of paclitaxel and curcumin together with
the slow drug release feature have also been reported for other types of nano-
formulations, where the entrapment of drug in nanoparticles bypasses the drug efflux
course regulated by MDR proteins and curcumin downregulates MDR proteins such
as P-gp. For instance, Liu et al. developed a complex system of PLGA-phospholipid-
PEG nanoparticles (PLGA stands for poly-[lactic-co-glycolic]-acid polymer) from
paclitaxel and curcumin, where nanoparticles comprised a PLGA core containing the
drug and curcumin, the thin phospholipid interfacial layer, and the PEG hydro-
philic outer layer. It has been found out that this system was more effective in
controlling drug release compared to simple PLGA systems and could retain pacli-
taxel up to 72 h in PBS. Moreover, it has also been shown that the PLGA nano-
particles containing curcumin and paclitaxel are more efficacious in decreasing the
expression of P-gp compared to free curcumin (Liu et al. 2016). Polyethylene glycol-
phosphatidylethanolamine (PEG-PE) micelles targeted with transferrin (TF) are
another example of nanoparticles that have been used to promote paclitaxel and
curcumin delivery to tumor sites and enhance the efficacy of tumor therapy. These
micelles were evaluated against resistant ovarian cancers in a cancer cell culture
grown in multicellular three-dimensional spheroids and in vivo tumors. When
paclitaxel was co-delivered with curcumin in the form of micelles, an increase was
recorded in the cytotoxicity of paclitaxel. In addition, transferrin modification of the
micelles could assist in significantly deeper micelle penetration into the spheroids
and tumors (Sarisozen et al. 2014).
These studies all stated that curcumin could significantly enhance the antitumor
potential of paclitaxel against resistant cancer cells, when curcumin is added into the
chemotherapy regimen. Radiotherapy is also applied along with platinum-based
agents in the treatment of advanced ovarian and cervical cancers. As previously
discussed, curcumin could overcome these drug-induced resistances, and it has been
shown to overcome radiotherapy-induced resistance as well.
6 The Radiosensitizing Function of Curcumin

in Gynecological Cancers
Radiation therapy is an efficient intervention to control cancer cell growth evasion

and metastasis, especially when they are combined with chemotherapeutic agents
capable of inducing radiosensitization such as carboplatin, cisplatin, 5-fluorouracil,
ifosfamide, etoposide, and most taxanes. For instance, chemotherapy involving
cisplatin and 5-fluorouracil is the present chemotherapy regimen for patients suffer-
ing stage IIA to IVA cervical cancer, which are administered together with radio-
therapy. Although the combined chemoradiotherapy improves survival rate, such
combination also increases the probability of chemotherapy-related toxicities includ-
ing gastrointestinal and hematological toxicities. In this regard, the introduction of a
safe agent like curcumin that is capable of inducing radiosensitization without a
significant toxicity on normal tissues could enhance the therapeutic window and
bring benefits for patients with advanced cervical cancers. Phase I clinical trials
proved that the oral administration of curcumin is totally safe up to 12,000 mg/day
(Javvadi et al. 2008; Lao et al. 2006) and could reduce histological lesions related
to cancer evasion in some patients (Cheng et al. 2001).
It has been shown that pretreatment with curcumin could enhance the cell growth-
inhibiting impact of radiotherapy in cervical carcinoma cells (HeLa and SiHa cells)
and spare the normal fibroblast the trouble of increased radiation toxicity through a
modulating intracellular ROS level (Javvadi et al. 2008). This is of importance as it

has been shown that curcumin could act differently on normal and cancer cells,
encouraging ROS generation in cancer cells while acting as antioxidant in normal
cells. The elevated ROS in cancer cells could lead to the long-term activation
of extracellular signal-regulated kinases (ERK1/ERK2) which encourage radio-
sensitization (Javvadi et al. 2008). Conversely, no evidence of NF-kB and Akt
involvement in improving the irradiation therapy of cervical cancer cells was
found on curcumin treatment, although curcumin has been shown to reduce the
activity of NF-kB and Akt in prostate and colorectal cancers (Javvadi et al. 2008).
Moreover, as explained above, curcumin has been shown to resensitize paclitaxel-
induced resistant cells through the downregulation of NF-kB and Akt signaling
pathways. These studies revealed that apparently, the downregulating effect of
curcumin on these pathways is either dependent on cell type or therapy regime,
although the implication of NF-kB and Akt activation in cancer cell survival has
been firmly established. What is consistent is that the curcumin prooxidant activity
could promote the anticancer effect of irradiation by contributing to induced ROS
generation in cancer cells, regardless of cancer cell type or therapy regimen.
Following their studies on the function of ROS in inducing cancer cell apoptosis
(Javvadi et al. 2010; Javvadi et al. 2008), they pinpointed thioredoxin reductase-1
(TxnRd1), a cytosolic antioxidant enzyme scavenging IR-induced ROS, to mediate
curcumin-sensitizing effect on irradiated cancer cells. They confirmed that the
curcumin-mediated inhibition of TxnRd1 activity could result in increased radio-
sensitization in cancer cells, since TxnRd1 overexpression has been shown to
terminate radiosensitization in cancer cells, in response to curcumin treatment
(Fig. 3).
Taken together, the pleiotropic function of curcumin on cancer cells and its
diverse utility to be introduced in the treating schedule of different types of gyne-
cological tumors, where curcumin by affecting a wide variety of cell signaling
modulators, could counteract the phenomena responsible for the development of
chemotherapy and/or IR-induced resistance in cancer cells, which is very common in
these cancers. However, as earlier mentioned, the cancer-targeted functions of
curcumin occur at high concentrations which are often very difficult to reach in
tumors in vivo; therefore, an alternative set of investigations has focused on enhanc-
ing the efficiency of curcumin in cancer cell treatment.
7 Approaches to Enhance Curcumin Anticancer Efficacy
Although curcumin’s multi-therapeutic function has drawn many researchers to

investigate this polyphenolic compound, the insufficient therapeutic benefits of
curcumin have prompted many researchers to look for various approaches to
promote the anticancer potential of curcumin (Table 1). These include a set of
investigations to enhance the pharmacokinetic profile of curcumin using various
formulations, examination of curcumin analogs with the hope to discover derivatives
Fig. 3 Curcumin-mediated radiosensitization in gynecological cancers. Curcumin can sensitize

gynecological cancers to radiation therapy through inhibiting thioredoxin reductase-1 (TxnRd1)
that is a cytosolic antioxidant enzyme scavenging IR-induced ROS
with more anticancer potency, and basic studies to unravel the molecular pathways
modulated with curcumin treatment in order to find a combination therapy capable
of tackling various malignancies.
8 Exploitation of Molecular Pathways Modulated by

Curcumin in Gynecological Cancers
It has been reported that the elevated levels of intracellular sphingosine/ceramides

could promote curcumin-induced inhibition of cell growth and apoptosis in ovarian
cancer cells (Yang et al. 2012). Sphingosine-1-phosphate, sphingosine, and cera-
mides are the metabolites of sphingolipids acting as messengers in cancer cell
progression. While sphingosine/ceramides encourage cell apoptosis, sphingosine-
1-phosphate potentiates cancer cell survival, and the balance between sphingosine/
ceramides and sphingosine-1-phosphate determines the fate of cells. It has been
shown that the curcumin-mediated cell apoptosis in ovarian cancer cells could
be enhanced by the inhibition of sphingosine kinase-1 (SphK1) by the pharmaco-
logical inhibitor (SB 203580). Moreover, the inhibition of ceramide production
by fumonisin B1 terminated the ShK1-induced cancer cell growth. As a result, the
Table 1 Rational approaches to overcome curcumin insufficient efficacy in cervical and ovarian
cancer cells
Curcumin derivatives Description Ref.
3,5-bis (2-flurobenzylidene) Increased cytotoxicity, inhibition of Kasinski et al.
piperidin-4-one NF-kB nuclear translocation, TNF-a- (2008)
induced IkB phosphorylation and
degradation, and IKK inactivation
1,5-bis(22-hydroxyl)21,4- In silico study proposed improved Singh and Misra
pentadiene interaction with HPV16-E6 protein (2013)
active site and p53 restoration
1,5-bis(2-hydroxyphenyl)2 Increased cytotoxicity, DNA Wang et al. (2011)
1,4-pentadiene-3-one fragmentation, and decreased HPV16-
and HPV18-associated E6 and E6
oncoproteins
Dimethoxycurcumin Increased cytotoxicity and Wang et al. (2011)
downregulation of cyclin D1
Curcumin conjugation
Curcumin-piperic acid In silico studies assumed increased Mishra et al.
toxicity in cervical cancers (2005b)
Dipiperoyl and diglycinoyl Increased cytotoxicity and ROS Mishra et al.
curcumin generation in histiocytoma cells, but it (2005a)
may be efficient against cervical and
ovarian cancer cells?
Curcumin-chlorogenic acid In silico study proposed increased Singh and Misra
cytotoxicity and HPV15-E6 (2013)
downregulation
Curcumin nanoformulations
Liposomal curcumin Including DDAB, cholesterol, and a Saengkrit et al.
nonionic surfactant like Montanov82 (2014)
increased cytotoxicity and cell pene-
tration of curcumin
Niosomal curcumin Including nonionic surfactants of Singh and Misra
Span80, Tween80, and Poloxamer (2013)
188 enhanced cytotoxicity and
controlled curcumin release
Milk-derived exosome Tumor growth inhibition following Aqil et al. (2017)
oral administration
PLGA nanoparticles conjugated Targeted delivery of curcumin to Punfa et al. (2012)
to anti-Pgp proteins cervical cancer cell line of KB-V1,
expressing highly P-gp
Naïve PLGA nanoparticles Enhanced cell apoptosis, reduced Zaman et al. (2016)
tumor burden, and suppressed
HPV-E6 and HPV-E7 oncoprotein
expression
Fe3O4 nanoparticles coated Enhanced curcumin entrapment in the Kumar et al. (2014)
layer by layer with dextran and particles, increased cell penetration, and Mancarella
polylysine films and enhanced cytotoxicity et al. (2015)
balance was shifted toward ceramide accumulation which pushes cancer cells toward
apoptosis and may be useful to cumulatively enhance antiproliferative response in
combination with curcumin.
In addition to ceramide accumulation, curcumin has been shown to result in the
modulations of other cell signaling molecules. It has been found that the activation of
AMP-activated protein kinase (AMPK) could induce cell death and suppress cell
progression in a variety of cancer cells, and CaOV3 ovarian cancer cell pretreatment
with an AMPK inhibitor attenuates curcumin-induced cell death. Moreover, p38
activation and Akt inhibition are other changes which occur in apoptotic cancer cells
treated with curcumin. Considering all the mentioned cell signaling effectors, every
agent that could contribute to these modulations has been proven to enhance the
anticancer potential of curcumin (Pan et al. 2008), and maybe their topical admin-
istration combined with curcumin as an ointment could exhibit therapeutic response
in gynecological cancers that is worth being investigated.
9 Curcumin Derivatives
Molecular docking studies of curcumin analogs with various functional group sub-
stitutions were conducted on prospective targets like EGFR tyrosine kinases, where
the potential analogs were tested on various cancer cells with the hope of unraveling
the relationship between curcumin structure and its activity (Sharma et al. 2015).
Sometimes, these studies culminate in the discovery of more potent analogs as
compared to curcumin.
As discussed previously, the NF-kB signaling pathway plays a central role
in governing cancer cell progression and metastasis, where curcumin has exhibited
cancer-therapeutic values via NF-kB inactivation. In this regard, Kasinski et al.
(2008) presented a synthetic monoketone analog of curcumin-termed 3,5-bis(2-
flurobenzylidene) piperidin-4-one – with enhanced anticancer activity against a
variety of cancer cells, including ovarian and cervical cancer cells. In comparison
with curcumin, this analog exhibited enhanced cancer cell growth inhibition up to
tenfold in comparison with curcumin. Likewise, the analog rapidly inhibited the
nuclear translocation of NF-kB at a dose tenfold lower than that of curcumin. In
mechanism, NF-kB inhibition was found to result from the strong analog-IKK
interaction which resulted in cancer cell apoptosis.
1,5-bis(22-hydroxyl)21,4-pentadiene, as a curcumin derivative lacking a diketone
site and methoxy functional groups, has been found to exert more antiproliferative
effect than curcumin on different cervical cancer cells (Singh and Misra 2013).
The curcumin analog 1,5-bis(2-hydroxyphenyl)2 1,4-pentadiene-3-one could induce
apoptosis more efficiently than curcumin, and it downregulates the expression of
oncogenes E6 and E7 in HPV16- and HPV18-infected cervical cancer cells,
known as risk factors of cervical cancers (Paulraj et al. 2015). It has been shown
that dimethoxycurcumin is a more stable analog of curcumin in physiological media
and could exert improved anticancer effect on multiple cervical cancer cells
(Teymouri et al. 2018). These are shining examples of curcumin derivatives with
enhanced efficacy, which begin with in silico studies on curcumin analogs with
successful in vitro improved potency. However, the translation of such a potency to a
real clinical setting is yet to be fully fulfilled. The low water stability and in vivo
bioavailability of curcumin are the main setbacks of curcumin therapy. It has been
shown that curcumin conjugation to hydrophilic molecules like amino acid, piperic
acid, and chlorogenic acid could increase the stability of curcumin in physiological
media (Singh and Misra 2013). Curcumin conjugation to piperic acid could enhance
the cell penetrability of curcumin, and its administration with chlorogenic acid might
fully restrict cancer cell proliferation in estrogen-responsive cervical cancer cells,
where curcumin has been found to be partially effective in comparison (Mishra et al.
2005b; Singh and Misra 2013).
Apart from the conjugation of curcumin to small molecules, it has been shown
that curcumin entrapment in various nanoparticulate systems could improve its
efficacy and tissue distribution in cervical and ovarian cancer cells, as discussed in
the following section.
10 Curcumin Nanoformulations
So far, a range of beneficial functions of curcumin has been presented, although

much diverse biological actions remained unstated as they are out of scope in this
review (Panahi et al. 2018; Teymouri et al. 2018; Teymouri et al. 2017). However, as
already stated, there are some limitations associated with the therapeutic translation
of curcumin in clinics such as low stability at physiological pH, hydrophobic nature,
rapid elimination from circulation, hepatic metabolism, etc. (Aqil et al. 2017; Garcea
et al. 2004). To overcome these limitations, there are numerous investigations, in
which various lipid-based formulations and polymeric-based nanoparticles are uti-
lized for curcumin delivery (Abouzeid et al. 2014; Aqil et al. 2017; Ganta and Amiji
2009; Kumar et al. 2014; Punfa et al. 2012; Saengkrit et al. 2014; Sarisozen et al.
2014; Xu et al. 2016). The list is very long, but as the scope of the current review is
restricted to “curcumin in treating gynecological cancers,” this paper presented
curcumin-loaded nanoparticles that have been tested against these cancers.
It has been shown that lipid-based nanoparticles, including liposomes and
niosomes, enhance curcumin stability in aqueous medium as they could accommo-
date hydrophobic curcumin in their membrane and prevent curcumin from degrada-
tion and precipitation (Saengkrit et al. 2014). In this regard, it has been shown that
the nonionic surfactant (Montanov82®) could decrease liposomal curcumin agglom-
eration and restrict liposomal enlargement and precipitation in long-term storage.
It could also enhance curcumin entrapment efficiency in liposomes. Likewise,
the addition of cholesterol has also been reported to improve the entrapment
efficiency of curcumin and limit the release of curcumin. Although introducing
didecyldimethylammonium bromide (DDAB) imparts a positive charge to lipo-
somes that increases cell uptake in vitro, positively charged liposomes have been
determined to be rapidly removed by the neighboring blood cells at the injection site
when they are intravenously administered. As a result, special consideration should
be given to the route intended for liposomal curcumin administration. If liposomal
curcumin is administered intravenously, where the liposomes are required to travel a
long distance before they reach tumor and accumulate there, negative-to-neutral-
charged liposomes would be probably more successful in reaching the tumor
(Teymouri et al. 2016; Teymouri et al. 2015). However, when the intention is to
enhance curcumin delivery via topical application, for example, as cream or an
ointment in cervical cancers, positively charged liposomal curcumin would promote
curcumin delivery to tissues as well as the therapeutic outcome due to increasing cell
internalization of liposomal curcumin (Debata et al. 2013; Song and Kim 2006).
Another issue that should be taken carefully into account is that the DDAB-
containing liposomes per se have been proven toxic to both cancerous and normal
cells. DDAB together with DOPE at DDAB/DOPE at 40 μM have been found to be
potentially harmful to CasKi cells. It is necessary to investigate whether these
liposomal components will cause unwanted suffering before deciding about their
application in clinics, so as to avoid the possible undesired side effects (Saengkrit
et al. 2014).
The curcumin-niosome system has also been shown to possess high entrapment
efficiency and lead to superior apoptotic rate in ovarian cancer A2780 cells com-
pared with the free curcumin dispersed in dimethyl sulfoxide (Xu et al. 2016). The
niosome system consisted of a nonionic surfactant of Span 80, Tween 80, and
Poloxamer 80 plus additives of cholesterol was examined in terms of entrapment
efficiency and curcumin delivery. It was found that the system is a highly superior
version of liposomal curcumin in terms of curcumin entrapment efficiency. How-
ever, whether niosomal curcumin would be a safer and more successful delivery
system, impart improved pharmacokinetic profiles, and result in higher tumor
accumulation of curcumin than liposomal curcumin is an interesting contrastive
study to be undertaken. Moreover, the surfactant-related hemolysis should be con-
sidered when optimizing these formulations of curcumin (Xu et al. 2016). Given
such serious complications that liposome or noisome ingredients might carry for
clinical application, searching for drug delivery systems that are perfectly safe is
highly desirable.
Milk-derived exosomes loaded with curcumin have been demonstrated to surpass
the low bioavailability of oral curcumin and resulted in three to five times increased
delivery of curcumin to various organs, as compared to free curcumin (Aqil et al.
2017). The exosomal curcumin exhibited increased antiproliferative and anti-
inflammatory activity against multiple cancer cell lines, including breast, lung, and
cervical cancer cells. The underlying mechanism for the promoted efficacy of
curcumin might lie in the fact that exosomes enter via endocytosis and go through
the endosomal pathway, where curcumin activity would be preserved in the desir-
able acidic media of endosomes. Apart from this, exosomes alone have been shown
to possess a moderate intrinsic antiproliferative and anti-inflammatory activity
leading to tumor growth inhibition, which is hardly believed to be achieved by
immune factors, miRNAs, and proteins derived from exosomes. Furthermore, high
contents of proteins would provide a vast area of hydrophobic domains on the

surfaces of exosomes for the lipophilic curcumin to interact and trap in exosomes.
These exosomes were shown to protect curcumin from degradation throughout the
gastrointestinal tract and enhance curcumin intake, indicating that exosomal
curcumins are highly successful oral formulations (Aqil et al. 2017).
Besides lipid-based nanoparticles, various polymer-based nanoparticles have
been utilized to achieve the prolonged stability and enhanced efficacy of curcumin.
For instance, PLGA-based curcumin nanoparticles have been reported to signifi-
cantly decrease the tumor volume in an orthotopic mouse model of cervical cancer
when it is injected intratumorally. The reduction of oncogenic miR-21, suppression
of beta-catenin, and abrogation of E6/E7 HPV oncoproteins were found in tumor
cells treated with curcumin-containing PLGA nanoparticles (Zaman et al. 2016). The
surface modification of curcumin-containing PLGA nanoparticles like the attach-
ment of anti-P-glycoproteins further enhanced curcumin delivery and cytotoxicity in
the resistant cells overexpressing P-gp (Punfa et al. 2012). Poly(2-hydroxyethyl
methacrylate) [PHEMA] with hydrophilic surface and Fe3O4 superparamagnetic
nanoparticles (SPION) coated layer by layer with positively charged Poly(L-lysine)
and negatively charged dextran is another example of polymer-based nanoparticles
that has been applied for improving curcumin delivery to cervical cancer cells
(Kumar et al. 2014; Mancarella et al. 2015). There are many other nanoparticulate
systems and formulations that have been applied for improving curcumin delivery to
various cancer cells, some tested in vitro and others in vivo (Teymouri et al. 2017).
However, to determine which of these systems are fully applicable in clinic settings
requires more in-depth investigation of curcumin nanoparticulate implications in
cervical cancer tumors.
11 Conclusion
The current review attempted to present an appropriate assortment of reports on

the therapeutic and adjuvant potential of curcumin for resistant cancer malignancies,
by placing emphasis on the studies conducted on gynecological cancers. The cur-
rent chemotherapy and radiotherapy have frequently been reported to face refractory
tumors upon relapse in ovarian and cervical cancers with a minor chance of treat-
ment. In this regard, multiple cell and molecular evidences imply that curcumin
could indeed resensitize the cells to chemotherapeutics and irradiation as evidenced
by the enhanced therapeutic index of these regimens, when they are administered in
combination with curcumin. These include suppression of the pathways involved
in DNA damage repair, the inactivation of major transcription factors capable
of promoting cell survival like NF-kB, AP-1, intracellular ROS elevation, down-
regulation of MDR-related proteins, and inhibition of inflammatory responses. Apart
from these potential benefits of curcumin combination therapy, it was discussed that
curcumin has the potential to act as an antitumor agent alone in these cancer types.
Finally, various strategies of enhancing the therapeutic potency of curcumin ranging
from alterations of curcumin molecular structure and curcumin encapsulation in

varying nanoformulations have been presented in a hope to discover a more target-
directed approach to tumor eradication. Curcumin concentration in tissues and tumor
is a defining factor with respect to the intrinsic ROS scavenging potency of cells.
Therefore, it is highly important to investigate the therapeutic benefits of adminis-
tering a given curcumin formulation combined with a chemodrug formulation
followed by radiotherapy, to harness the cancer-treating potential of curcumin at
most. Studies in this direction are highly necessary to translate the in vitro features of
curcumin successfully into clinics, in managing the treatment of resistant cancer
tumors of gynecological cancers.
Acknowledgment The authors would like to say special thanks to Dr. Amir Saberi-Demneh and
Dr. Leila Ghalichi for their guidance and kindness.
Conflict of Interest The authors declare that they have no conflicts of interest about this report.
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DOI: 10.1007/112_2018_14
© Springer International Publishing AG, part of Springer Nature 2018
Published online: 8 December 2018
Correction to: Curcumin in Advancing

Treatment for Gynecological Cancers
with Developed Drug- and Radiotherapy-
Associated Resistance

and Atefeh Vaezi
Correction to:
Chapter “Curcumin in Advancing Treatment
for Gynecological Cancers with Developed Drug-
and Radiotherapy-Associated Resistance” in:
A. A. Momtazi-Borojeni et al., Rev Physiol Biochem
Pharmacol,
DOI: 10.1007/112_2018_11
The affiliation of the 6th author Dr. Abolfazl Mehdizadehkashi was incorrect. It has
been corrected to Endometriosis Research Center, Iran University of Medical Sci-
ences, Tehran, Iran.
The updated online version of this chapter can be found at

DOI: 10.1007/112_2018_11

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Reinhard Jahn Roland Lill

ISSN 0303-4240 ISSN 1617-5786 (electronic)

© Springer Nature Switzerland AG 2019

DUSP3/VHR: A Druggable Dual Phosphatase for Human Diseases . . . 1

DUSP3/VHR: A Druggable Dual

Lucas Falcão Monteiro, Pault Yeison Minaya Ferruzo,

L. F. Monteiro, P. Y. M. Ferruzo, L. C. Russo, J. O. Farias, and F. L. Forti (*)

dependent or MAPK-independent mechanisms. This broad spectrum of actions is

Keywords Dual-speciﬁcity phosphatase 3 (DUSP3) · Mitogen-activated protein

2 Protein Tyrosine Phosphatase (PTP)

2.1 Dual-Speciﬁcity Phosphatase (DUSP)

There are 19 identiﬁed proteins in the atypical dual-speciﬁcity phosphatase

Cell Cycle Regulaon, Proliferaon,

3 Molecular and Biological Functions of DUSP3/VHR

3.1 DUSP3 in Tumorigenesis

Many types of cancers originate from several activating mutations or genomic

with the common melanocytic nevus (CMN, lesion considered to be in cellular

3.2 DUSP3 in Genomic Stability

interactomes, to identify new substrates of DUSP3 very likely related to genomic

3.3 DUSP3 in Blood-Associated Diseases

4 Overview of Current Knowledge on DUSP3/VHR

Pan-inhibitors of protein tyrosine phosphatases have long been established in the

Other widespread PTP inhibitors include α-Bromo-4-(carboxymethoxy)

2 RK-682 enamine 2c 2.0 μM (Hirai et al. 2011) –

4 Stevastelin B 10.8 μM (Hamaguchi et al. –

6 7-Hydroxy-5,6-dimethoxy-1,4- 3.0 μM (Bae et al. 2004) –

7 Cinn-GEE 290 μM (Park et al. 2004) N.A.

8 NSC 357756 N.A. N.A.

9 NU-126 38.1 μM (Lazo et al. 2006) MKP1, PTP1B (Arnoldussen and

10 NU-176 47.4 μM (Lazo et al. 2006) MKP1, MKP3, Cdc25B, PTP1B

12 Benzoic acid 1 3.7 μM (Park et al. 2008) N.A.

13 Benzoic acid 2 4.7 μM (Park et al. 2008) N.A.

14 NSC-87877 7.83 μM (Park et al. 2009) N.A.

16 SA2 47 nM (Wu et al. 2009) MKP1, CD45, PTP1B (Jeong et al.

17 SA3 74 nM (Wu et al. 2009) MKP1, CD45, PTP1B (Denu and

19 SA5 270 nM (Wu et al. 2009) HEPTP, CD45, PTP1B

In 2002 a compound from a fungal strain which was identiﬁed to be a

in vivo strongly support its potential tumorigenesis-promoting activities. However, it

Financial Support Fapesp, Capes, and CNPq.

Alonso A, Rahmouni S, Williams S, van Stipdonk M, Jaroszewski L, Godzik A, Abraham RT,

Hirai G, Tsuchiya A, Koyama Y, Otani Y, Oonuma K, Dodo K, Simizu S, Osada H, Sodeoka M

Pulido R, Hooft van Huijsduijnen R (2008) Protein tyrosine phosphatases: dual-speciﬁcity

Oncotic Cell Death in Stroke

Kep Yong Loh, Ziting Wang, and Ping Liao

Keywords Cerebral oedema · Ion channels · Oncosis · Stroke

AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

b Intracellular Space Extracellular Space

Cerebral oedema is a well-documented complication of stroke (Stokum et al.

2 Pathways of Sodium Inﬂux in Stroke

Na+/K+-ATPase, a member of the P-type ATPase protein family, is found univer-

beneﬁcial effects of cardiac glycosides, a Na+/K+-ATPase inhibitor, in neuro-

2.2 Glutamate Receptor Channels

Glutamate is the main excitatory neurotransmitter in the central nervous system. It is

1999; Liang et al. 2007). Limitations in the glutamate-based approach could be

2.3 Acid-Sensing Ion Channels (ASICs)

ASICs are proton-gated cation channels belonging to the degenerin/epithelial Na+

anti-inﬂammatory drug which inhibits ASIC1a, has been shown to be beneﬁcial in

2.4 Na+/H+ Exchanger (NHE)

aglycaemia, implying a contributory role of NHE in early cerebral oedema in stroke

2.5 Electrogenic Na+/HCO3 Co-transporter 1 (NBCe1)

NBCe1, a bicarbonate-dependent acid-based membrane transporter, is another

As such, this evidence suggests that early inhibition of NBCe1 may be