# Arsenic-Based Antineoplastic Drugs and Their Mechanisms of Action

Authors: Stephen John Ralph
Journal: Metal-Based Drugs
 (2008)

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## Abstract

Arsenic-based compounds have become accepted agents for cancer therapy providing high rates of remission of some
cancers such as acute promyelocytic leukemia (APL). The mechanisms by which arsenic-containing compounds kill cells and
reasons for selective killing of only certain types of cancer cells such as APLs have recently been delineated. This knowledge
was gained in parallel with increasing understanding and awareness of the importance of intracellular redox systems and
regulation of the production of reactive oxygen species (ROS) by controlling mitochondrial function. Many of the targets for
the arsenic-containing compounds are mitochondrial proteins involved in regulating the production of ROS. Inhibition of these
proteins by disulfide linkage of vicinal thiol groups often leads to increased production of ROS and induction of apoptotic
signalling pathways. Sensitivity or resistance to the actions of arsenic-containing compounds on cancer cells and normal
cells depends on the levels of transport systems for their uptake or efflux from the cells as well as their redox defence
mechanisms. The exact mechanisms of arsenic toxicity as well as its anticancer properties are likely to be related and these
aspects of arsenic metabolism are covered in this review. Greater understanding of the mechanisms of action of arsenic will
help determine the risks of human exposure to this chemical. Novel organic arsenic-containing compounds and the lessons
learned from studying their selective sensitivity in targeting dividing endothelial cells to inhibit angiogenesis raise the future
possibility for designing better targeted antineoplastic arsenic-containing compounds with less toxicity to normal cells.


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## Body

## 1. Arsenic and Arsenic-Containing Compounds in the Natural Environment or as a Result of Human Application

Arsenic  is a toxic metalloid [1, 2]
that exists throughout nature
in organic and inorganic forms.  It is
commonly present in soils on average at several mg/kg, as well as in marine
sediments, and is enriched in mineral deposits as oxides and sulfides. The
basic element, arsenic, exists in either of three allotropic forms: yellow,
black, or grey with the stable, semimetallic form having a silver/steely-grey
colour as a brittle, crystalline solid. The semimetallic form oxidises rapidly
in air, and at high temperatures produces a white cloud of arsenic trioxide (As2O3). Arsenic, with its variety of
chemical forms and oxidation states, is listed in Table 1. The current IUPAC
nomenclature is listed
in [3] as well as the more common names of the arsenic-based compounds which
are mainly used throughout this review.Table 1
Common names and fully systematic
(additive) names for Arsenic oxoacid and related structures.
Some organic derivative names still contain the
word “acid,” as in the following derivatives of arsonic acid = H2AsHO3 =
[AsHO(OH)2], for example,  PhAsO(OH)2 phenylarsonic acid.
Example of alternative names for Arsenic species usedCommon nameAbbreviationIUPAC Fully systematic additive nameChemical formulaArsenite, Arsenous acid, Arsorous acidAs(III), AsIIITrihydroxidoarsenicH3AsO3  =    [As(OH)3]Arsinious acidAs(III), AsIIIDihydrohydroxidoarsenicHAsH2O
=  [AsH2(OH)]Arsonous acidAs(III), AsIIIHydridodihydroxidoarsenicH2AsHO2 =  [AsH(OH)2]Arsenate, Arsenic acid, Arsoric acidAs(V), AsVTrihydroxidooxidoarsenicH3AsO4 =   AsO(OH)3Arsinic acidAs(V), AsVDihydridohydroxidooxidoarsenicHAsH2O2 = [AsH2O(OH)]Arsonic acidAs(V), AsVHydridodihydroxidooxidoarsenicH2AsHO3 =  [AsHO(OH)2]Monomethylarsonic acidMMA(V), MMAVMethanedihydroxidooxidoarsenicCH3AsO(OH)2Monomethylarsonous acidMMA(III), MMAIIIMethanedihydroxidoarsenicCH3As(OH)2Dimethylarsinic acidDMA(V), DMAVDimethanehydroxidooxidoarsenic(CH3)2AsO(OH)Dimethylarsinous acidDMA(III), DMAIIIDimethanehydroxidoarsenic(CH3)2AsOHWhen absorbed at toxic
levels, arsenic causes severe health problems, including cancer. Acceptable
levels were lowered from 50μg/L to 10 μg/L in drinking water by the WHO [4]. Arsenic-containing compounds are applied in
plant pesticides and insecticides and arsenic environmental contamination represents
a global health problem, particularly from leaching into ground water. When the
soluble levels exceed 50 μg/L
in drinking water as in many regions of Bangladesh, arsenic becomes a
particular health concern as it has recently been associated with increased
cancer rates appearing after consumption with a lag time up to 10–20 years [5].Whilst the carcinogenic
aspects of arsenic compounds are not the focus of this review, nevertheless this
undesirable aspect needs to be raised. Thus, arsenic and its methylated species
are known carcinogens but this description is probably inaccurate as they act
more as cocarcinogens by facilitating/promoting the induction of tumors of the
skin, urinary bladder, and lung rather than directly inducing cell transformation
and oncogenesis [2, 6]. As cocarcinogens, mechanisms include indirect effects
such as DNA damaging genotoxicity by altered DNA methylation as well as
inducing high levels of oxidative stress leading to altered cell proliferation and
tumor promotion (detailed in [7]).In terms of chronic toxicity, the 50% lethal dose of arsenic as arsenic trioxide
in mice received by the oral route varies from 15–48 mg/kg, whereas the acute
lethal dose in humans varies from 1–3 mg/kg body weight [4].  Despite its toxicity, arsenic has been applied
in Chinese medicines for thousands of years as refined preparations of realgar
(As4S4) or orpiment (As2S3) [8]
which are two rare mineralised forms. Realgar, easily discerned by its bright
red and orpiment with a browny yellow appearance are ores often occurring close
to each other in hydrothermal veins of precious metal sulfide ores or hot
spring deposits as volcanic sublimate products (crystallized from gases). Hence,
arsenic is a common waste product from the mining of metal ores and the name
realgar is possibly derived from Arabic words for “powder of the
mine” (rahj al ghar).The long history of medical applications has included treating many
diseases such as cancers administering micromolar levels in patients and, in
particular, arsenic compounds have proven to be very effective against certain
hematological malignancies (reviewed in [9, 10]). Thus, arsenic oxides and
derivatives have been established as effective treatments for acute
promyelocytic leukemia (APL), and they are being tested as therapies for a
range of other hematological cancers including myelodysplastic syndromes,
multiple myeloma, and chronic myelogenous leukemia (CML). The results of
clinical trails using arsenic-based drugs in cancer therapy have been
extensively reviewed [8–12]. Hence, this review mainly concerns those factors
that explain the antineoplastic mechanism of action of arsenic-based drugs,
distinguishing their selectivity for killing certain types of cancer cells from
their potential for toxicity to normal cells in the body. The reason for their efficacy
and selectivity in treating certain hematological malignancies is covered in
the last section of this review. However, the mechanisms of action of arsenic-basedcompounds
on cells must first be described in detail to provide sufficient understanding
to enable their selective targeting of specific types of cancer cells to be
duly evaluated.Water distributes the majority of
inorganic arsenic in the biosphere either as   pentavalent
arsenate (As5+, As(V)) or trivalent arsenic (As3+,
As(III) [2]. In solution, the pH and redox conditions affect
the chemical state of
arsenic that predominates. Thus, in highly oxidised environments, the arsenate (As5+)
form predominates as one of four major species of arsenic acid; H3AsO4,
H2AsO4−, HAsO42−, and
AsO43−  [13].  However,
mildly reducing conditions such as those generally present inside all mammalian
cells [14] will favour the reduction of the pentavalent arsenate (As5+)
to the trivalent arsenic (As3+). Arsenous acid with the formula As(OH)3 results
after the slow hydrolysis of arsenic trioxide in water. As the pH increases,
arsenous acid is converted to the arsenic oxide ions [AsO(OH)2]-,
[AsO2(OH)]-2, and [AsO3]-3 [13].


## 2. Methylated Forms of Arsenic Show Increased Cytotoxicity inside Cells

Arsenic combines readily
with many elements; and bacteria have evolved systems to detoxify inorganic
arsenic to form organic arsenic-containing compounds such as methylated forms. Plant
and insect pesticides have been made using organic arsenic derivatives; and monomethylarsonic acid
(MMA(V)) and dimethylarsinic acid (DMA(V)) are used in products for weed
control. Also, MMA(V) and DMA(V) are metabolites of inorganic arsenic, formed
intracellularly in mammals, primarily in the liver. The metabolic process of
inorganic arsenic conversion is known as biotransformation and appears to
enhance the excretion of arsenic from the body, involving formation of
methylated compounds of trivalent arsenic as intermediates. The metabolism
involves reduction to a trivalent state and oxidative methylation to a
pentavalent state. In addition,
reductases present in cells and other reactions facilitate the reduction of
arsenic acid [As(V)] to the arsenous [As(III)] form including methylation of the
arsenous form, mainly via the liver, to produce mono-, di-, and trimethylated
species [2]. The main enzyme involved in methylation of As(III) is arsenic
methyltransferase (As3MT) that requires glutathione (GSH) to promote the
reaction and a cycling redox system such as thioredoxin (reviewed in [15]) to
detoxify arsenic-containing compounds. Many animal species convert inorganic arsenic-containing
compounds into mono-, di- and tri-methylated arsenic species which are mostly
then secreted in the urine [16]. This is because the methylated forms are not
as well absorbed by cells compared to the inorganic forms, although they have
much greater cytotoxicity if they do enter cells [15, 17].The reductive metabolism of arsenic has an
important role in its toxicity. The trivalent arsenic-containing compounds, including
the methylated organic forms have much greater potency than the pentavalent arsenic-containing
compounds as cytotoxics and carcinogens [18–23]. For example, in cytotoxicity
assays, the IC50 values for cultures of primary hepatocytes, keratinocytes, and
epithelial cells ranged from 3 μM to well over 20 μM for trihydroxidoarsenic salts whereas for monomethylarsonous
acid [MMA(III)], the values were consistently much lower at only several μM for the equivalent normal cell types [24–26]. Organic
arsenic-based ingredients are
commonly used as feed additives in poultry farming to
increase weight gain by preventing bacterial and parasitic infections, thereby
increasing feed efficiency and improving pigmentation.  The three major arsenic-containing compounds
used in this manner are arsenilic acid (p-aminophenyl arsonic acid), roxarsone
(4-hydroxy-3-nitrophenylarsonic acid), and nitarsone (4-nitro-phenylarsonic
acid) [22, 23].  The metabolism of these arsenic-containing
compounds and waste products produced by birds and mammals consuming them is
still uncertain at present and what the global environmental impact might be.


## 3. Cellular Actions of Arsenic-Containing Compounds

### 3.1. Inhibitors of Energy Metabolism: Effects on Glycolysis and Oxidative
Phosphorylation

At the
biochemical level, inorganic arsenic in the pentavalent state (As(V), arsenate, AsO4) resembles a
phosphate (PO4) group in structure and it can replace phosphate in many
reactions. The mitochondria is a
major intracellular site where arsenate is metabolised, taken up As(V), rapidly
reducing it, and exporting the As(III) product back into the cytosol [27].  The specific location of the site(s) for
arsenic reduction in the mitochondria has not yet been defined.  However, arsenate can affect oxidative phosphorylation
by binding to the Fo/F1 ATP synthase [28]. Arsenate can be used by the ATP
synthase more efficiently than phosphate depending on the Ca2+ levels [29],
producing ADP-arsenate which unlike ATP becomes rapidly hydrolysed and unable
to form stable high-energy compounds [30]. It is suggested that the structure
and charge similarities of PO43−, AsO43−,
and SO42− result in
indiscriminate binding to at least two sites located in the mitochondrial
matrix [31]. At one site, occupation by any of these three anions results in
protection against uncoupling of the mitochondrial proton gradient by K+;
at the second site, in the Fo/F1 ATP synthase,  AsO43− and SO42− compete for binding against PO43−, leading to the inhibition of ATP production [31].Intriguingly, As2O3,
whilst shown to have no effect on oxidative phosphorylation levels in HeLa [10 μM] and AS-30D [100 μM] hepatoma cancer cells, significantly inhibited
glycolysis, particularly during the exponential growth phase of these cells
when they were actively respiring and producing 70% of their ATP from
mitochondria [32]. Several other studies have also shown that arsenic compounds
do not affect oxidative phosphorylation. Thus, with submitochondrial particles
in the presence of an ATP regenerating system, 20 mM arsenate had no effect on
NADH formation, ATP hydrolysis, and Pi  ↔  H20 exchange [33]. In a related study with
isolated liver mitochondria, As2O3 was again shown to
have no effect on oxygen consumption, or the respiratory control ratio at a concentration
(10 μM) found to be maximally effective in promoting
apoptosis in whole cells [34]. However, at higher concentrations (>50 μM), pyruvate/malate-supported
respiration (via complex I) became blocked, but there was no effect on either
complex II or IV.  The inhibitory effect on complex
I was reversed by the addition of the reducing agent, dithiothreitol,
indicating that direct oxidative damage was involved.  In addition, it was shown that even with the
block in complex I, the cells continued to maintain cellular ATP levels through
glycolysis, and hence, depletion of cellular ATP was not the cause for the
cytotoxicity of As2O3 [34]. Probably the most definitive evidence
for the importance of mitochondria in mediating As2O3 killing
of cancer cells comes from studying cells lacking mitochondrial function [35].
A subclone of mitochondrial respiration deficient cells was derived from the
HL-60 human leukaemia
cell line by growth in the presence of ethidium bromide to mutate the
mitochondrial genome, and these cells are known as HL-60 “ρ0” cells [35]. Due to the lack of
mitochondrial respiration, ρ0cells depend on glycolysis for
their energy source and, as would be expected, produced substantially less
superoxide radicals (∼20% of the control cells). When these ρ0 cells were incubated with ∼10 μM As2O3, they were resistant
to the drug, revealing that mitochondrial respiratory function is required for
the cytotoxic actions of As2O3 [35].The
inhibition of glycolysis by arsenic-based drugs appears unlikely to be a significant
factor involved in the drug-induced killing of cancer cells. Thus, incubating
cells in glucose-deficient medium to block glycolysis had no significant effect
on theAs2O3− [30 uM] mediated levels of cell death in
the Jurkat cell line [36]. However, when glycolysis was blocked and
mitochondrial respiration inhibited using oligomycin A, the cells became very
sensitive to As2O3− mediated cell death.  In this regard, it is also worth noting that studies
of individual glycolytic enzymes analysed in purified form in vitro have shown arsenite
and arsenate to be relatively weak inhibitors. Hence, for hexokinase (IC50:
15 mM for arsenate [37, 38]), phosphofructokinase (IC50 > 5 mM for arsenite; [39])
and  pyruvate dehydrogenase (PDH IC50:
80–120 μM arsenite; [40]), relatively large concentrations
were required to inhibit these enzymes. In fact, arsenate has been shown to stimulate
the activities of the two important glycolytic enzymes, hexokinase [37] and
GAPDH [41], by overcoming product inhibition in these reactions. In the case of
GAPDH, arsenate acts catalytically to promote the oxidation of phosphoglycerate
[42] and the reaction involved the formation of an arsenate analogue of the
phosphate ester as an intermediate which rapidly hydrolysed, helping to drive
the reaction forward [33, 43]. This process has been commonly described in
relation to the effects of arsenate on numerous enzymatic reactions involving
phosphate and has been termed “arsenolysis” [33].Given the relative
insensitivity to the direct effects of arsenic compounds shown by the
glycolytic pathway, it follows that it is unlikely that glycolytic inhibition
results from  direct binding and
modification of the enzymes in this pathway by arsenic-based drugs. More likely,
the inhibition of glycolysis results from an indirect effect, caused by the
actions of arsenic compounds in modifying mitochondrial respiration leading to
production of ROS which then acts to inhibit the glycolytic enzymes. Additional
support for the mitochondrial-mediated ROS involvement in the action of arsenic
to inhibit glycolysis comes from studies where As2O3 was
found to be ∼38 times more potent in cells than in the pure preparation at
inhibiting pyruvate dehydrogenase [40]. Also, inhibiting mitochondrial
respiration suppressed the resulting inhibition of pyruvate dehydrogenase activity
and H2O2 production by this
drug. Furthermore, the inhibition of pyruvate dehydrogenase by As2O3 was shown to require the Fenton reaction occurring via hydroxyl radical intermediates
[40]. The mitochondrial effects of arsenic compounds are detailed later in this
review and to reiterate at this point, the evidence indicates that the actions
of arsenic compounds on glycolysis are not the main cause for the cytotoxic
effects of these drugs at clinically relevant concentrations (1–6 μM) required in plasma for the killing of cancer
cells in APL patients [8, 44].


### 3.2. Interconversion of As(III)↔     As(V) in Cells

Under conditions of
high mitochondrial respiration inside cells, it is possible that trivalent
arsenicals inducing significant production of ROS as superoxide, peroxide, and
hydroxyl radicals can also result in oxidation to produce arsenate (AsO43−)
ionic species [45, 46].  Consequently, the
impact that arsenic compounds
will have on any given cell will most likely depend on the state of
cellular respiration and production of ROS affecting the arsenic speciation and
whether the cell is dependant on glycolysis versus mitochondrial respiration
for its ATP synthesis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the
glycolytic enzyme abundantly found in all cells and especially blood cells and
liver, is a major intracellular arsenate reductase [47] requiring GSH, NAD, and
glycolytic substrate [48, 49]. Given that the levels and specific activity of
GAPDH is much higher in malignant cells than in normal cells [50], this could
contribute to the rapid reduction of As(V) species in their cytosol into more
toxic As(III) forms.In the GAPDH reaction,
As(V) reduction may take place during, or as a consequence of the arsenolytic
cleavage of the thioester bond formed between the enzyme's Cys149 residue and
the 3-phosphoglyceroyl moiety of the substrate. Hydrolysis of
1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of
the normal substrate 1,3-diphosphoglycerate under the same conditions [51].
Hence, GAPDH is proposed as one of the key cellular converting enzymes for
reducing As(V) to As(III). Although purine nucleoside phosphorylase was
proposed to be an arsenate reductase [52], this was later refuted [53].  The other major class of As(V) reductases in
cells are the glutathione S transferases and of these, the omega form or GSTO1
appears to be most important. Thus, GSTO1 can reduce arsenate to arsenite,
MMA(V) to MMA(III), and DMA(V) to DMA(III) and deletion of the GSTO1 gene in
mice reduced the extent of biotransformation by 30–80% in most tissues examined
[54].


### 3.3. Structure and Reactivity of Arsenic-Containing
Compounds with Reduced Thiols

Arsenic has a high affinity for sulfur
and hence, reactive sulfur-containing molecules such as reduced thiols with an
available sulfur atom have a significant propensity for binding to arsenic [55, 56].
As(III)-containing compounds exist as trigonal pyramidal structures and this is
also the structure formed upon binding of arsenic ions to cellular proteins in
vivo where the sulphur atoms of thiolate groups act as coordinating ligands. The
resulting arsenic-thiol linkages are mainly responsible for the ability of
arsenic to modulate the function of various key molecules, enzymes, and ion
transporters inside cells and this intracellular action of arsenic is discussed
in detail in this section.  Arsenic-containing compounds react with mono- and dithiols,
particularly the latter when two thiols are located in close proximity, acting
to cross-link the thiols together.Some debate exists
about the structures and speciation of arsenic-containing compounds (both
inorganic and organic forms) in solution.
In the absence of sulfide, As(III) hydroxide complexes are the major
arsenic-containing species and these structures probably adopt a trigonal
pyramidal structure with the arsenic atom at the apex [57–59].  This trigonal pyramidal structure provides
the potential for As(III) to coordinate linkages with several proximal thiol
groups.  This is believed to be the case
in bacterial enzyme systems such as the ArsR repressor protein where it is
likely to bind three Cys atoms [60]. Thus, in the more toxic form as the
trivalent state (As(III)) inorganic and organic
(methylated) arsenic reacts with critical thiols in proteins, inhibiting their
function, as is the case with the bacterial ArsR protein. For example, As(III) was shown to target the
reactive sulfhydryl group at the active site of thiolase(s) involved in ketogenesis
from acetyl Coenzyme A [61].The
pentavalent species of inorganic arsenate (AsO43−)
favoured to exist in oxidised environments, as well as organic forms of As(V)
have a different structure with a trigonal-bipyramidal shape where the As(V)
atom is located at the centre, co-ordinating to three equatorial and two polar
atoms [62, 63]. In comparing the two different structural states of As(III) and
As(V), it is not yet clear why the trivalent methylarsenic-containing compounds
show a much higher toxicity than their pentavalent analogues [24, 64]. However,
it could relate to cellular uptake as trivalent organoarsenic compounds are more membrane
permeable than the pentavalent species [65]. Also, trivalent arsenic bonded at
a phenyl ring is able to form much more stable covalent cross-links to cysteine
residues compared to arsenic in small molecules such as arsenious acid or arsenite
[66].  Furthermore, the
organic trivalent arsenic-containing compound, phenylarsine oxide [0.1–0.5 μM], is much more potent than the simple arsenite
[1–10 μM] in its cytotoxic activity in APL cells [67].One major
drawback with phenylarsine oxide as a potential cancer therapy is its high
toxicity in vivo and its nonselectivity for cancer versus normal cells,
resulting in cytotoxicity in normal endothelial cells in the same concentration
range (0.2μM) [68].  Thus, phenylarsine oxide, is precluded from
application in clinical cancer therapy, without being further modified as a
drug. The
greater reactivity of phenylarsene oxide and associated cytotoxicity is in
agreement with the results outlined above [66] where mass spectrometric
analysis of different complexes of peptides and proteins with arsenic-containing
species revealed that inorganic arsenite or arsenates did not interact well
with cysteine or glutathione, whereas the organic phenylarsine (3+) oxide did.
In addition, three different phenyl arsenic acids and dimethylarsinic acid that
all contained As(V) also formed complexes with glutathione [66]. Hence, the bulky hydrophobic groups with
electron withdrawing π  orbitals (in the case of phenyl groups) may
promote more stable bonds between the arsenic atom and sulfur groups inside
cells, modulating a larger range of enzymes and proteins with important
functions for maintaining cell viability.In
cells, the most common reactive species that are available for interaction with
arsenic are the abundant free thiol moieties in the tripeptide glutathione (γGlu-Cys-Gly,
GSH) and the free amino acid, cysteine.  Thus, arsenic-based drugs can react by
coordinating binding to free (reduced) thiol groups such as those on cysteine,
particularly those of thioredoxin and glutathione as the major intracellular thiol
species important in cellular redox regulation. It was observed early on that arsenite
and phenylarsine oxide in particular, but not arsenate, reacted with vicinal
thiol groups on proteins [69, 70].  Since
demonstrating that phenylarsine oxide was particularly effective at cross-linking
vicinal thiols in the active site of tyrosine phosphatases [71], the range of
proteins -containing vicinal cysteine residues with which phenylarsine oxide reacts
is increasing. Recent examples include the small GTP binding Rho
protein family [72] and the mitochondrial
carnitine/acylcarnitine transporter [73]. Phenylarsine
oxide, as a strong inhibitor of tyrosine phosphatases would increase tyrosine
phosphorylation levels of enzymes in cellular growth signalling pathways.
Whether the inhibition of tyrosine phosphatases and other enzymes contributes
to the toxic effects of arsenic-containing compounds in normal cells is not
clear, but is likely an important contributing factor to its general
cytotoxicity making phenylarsine oxide unsuitable for cancer therapy.Analysis
of the interactions of As(III) with glutathione or cysteine in vitro in aqueous
solutions by equilibrium binding and use of biophysical techniques including
NMR, electronic spectroscopy, and potentiometry revealed that As(III) binds either
of glutathione or cysteine with similar equilibrium constants [74].  However, several analytical studies by mass
spectroscopy have revealed that arsenate and arsenite do not complex readily
with glutathione or cysteine, but prefer to react with the thiols on reduced
thioredoxin molecules [reviewed in [66]]. This was confirmed by analysis of more
biologically relevant samples from the intracellular environment of HeLa cells
where cytosolic thioredoxin 1 (TRX1) and, in particular thioredoxin 2 (TRX2) in
the mitochondria, was shown to be highly reactive with arsenite (10 μM)
whereas little reactivity was detected with cellular GSH/GSSG [75].Studies
with thioredoxin reductase (TxR) purified from mouse liver showed that arsenic-containing
compounds with As(III) and arsinothiols (complexes of As(III) with GSH or L-cysteine)
were extremely potent inhibitors of this enzyme [24]. Methylarsenic(III)oxide
was most potent with a  Ki∼100 nM, as an irreversible competitive inhibitor. The
effects on purified glutathione reductase (GR) showed that the levels of
inhibition were not as marked with inorganic As(III) and As(V) oxides showing
IC50s in the 10–50 mM range, whereas for methylarsinic(III)oxide it was ∼9 μM. Studies
on this enzyme in whole cells as hepatocytes showed the IC50 to be reduced to
∼3 μM
for methylarsinic(III) oxide and for As2O3>100 μM
[24]. Hence, these observations strongly support a role for the components
thioredoxins and the thioredoxin reductase system as providing cellular targets
that are very sensitive to inhibition by arsenic-based drugs in the low
micromolar range. It also explains the importance of studying the chemistry of arsenic-containing
compounds in the context of both purified enzymes as well as whole cells as biological
systems in order to obtain meaningful results. Thus, although the
GSH/glutathione transferase system undoubtedly plays an important role in
arsenic sensitivity of cells
(see below in Section 4 for details), it   would appear that the thioredoxin system might
represent the most immediate point of sensitive reactivity in relation to
cytotoxicity.


### 3.4. The Major Mechanism of Action of Arsenic-Containing Compounds: Modifying
Mitochondrial Function and Redox Regulation of the Production of Reactive Oxygen Species

Mitochondria
are a main source of ROS in cells (reviewed in [76]). Thioredoxin (TRX), NADPH,
and thioredoxin reductase (TxR) comprise the thioredoxin system that has multiple
functions in cells including in redox signalling via interactions with other
proteins, in transcriptional regulation, control of the reduced intracellular
redox environment, cell growth, defense against oxidative stress and control of
apoptosis (reviewed in [77]). As outlined in the previous section, the thioredoxin
system is very sensitive to arsenic-based drugs and may well be the basis for
one of the important mechanisms for their actions in inducing cancer cell
death. The TRX system operates as a thiol-disulfide exchange reaction (see
Figure 1).  TRX1 and TRX2 are key
regulatory isozymes that catalyse the reduction of protein disulfide bonds. They
are cofactors of the apoptosis signal-regulating kinase 1 (ASK1) that mediates TNF
cytokine and oxidative stress-induced apoptosis via the mitochondrial dependent
pathway [78].Figure 1
Many vicinal thiol-containing
redox proteins are major mitochondrial targets for binding of arsenic-containing
compounds.  Arsenite and particularly organic arsenites will
disrupt the normal redox systems functioning in the mitochondrial matrix and
intermembrane space by targeting vicinal thiols in proteins and enzymes that
regulate these systems. Such enzymes include Peroxiredoxin III (Prx III), Thioredoxin
2 (Trx2). In addition, several key mitochondrial functions are affected,
including Factor B regulating the ATP synthetase activity, the adenine
nucleotide transporter, ANT, amongst others.
See text for further detail.In
their reduced forms, cytosolic TRX1 and mitochondrial TRX2 each contain two
vicinal thiol groups in their active site sequence as –C–G–P–C–. TRX 1
in the cytosol and TRX2 in mitochondria bind to Cys 250 and Cys 30,
respectively, in the regulatory N-terminal domain of ASK1 and can cooperatively
maintain the enzyme in the inhibited state. However, activation by TNF
resulting in increased production of ROS and leads to the oxidation of the TRX dithiol
group to a disulfide. Under these conditions, the thioredoxins no longer bind
to ASK1 and loss of TRX2
binding to mitochondrial-located ASK1 can lead to apoptosis in a
JNK-independent manner, whereas cytosolic ASK1 upon loss of TRX1 binding then
becomes activated as a MAPKKK resulting in JNK activation, Bid cleavage and Bax
translocation to the mitochondria [78]. Since it is known that the thioredoxins
are major targets of arsenic-containing compounds (see above and [75]), it can
be predicted that arsenite-mediated oxidative binding to thioredoxins will
induce a similar outcome as TNF signalling, leading to the release and
activation of ASK1 and induction of apoptosis.Another
thioredoxin-associated protein of importance in thiol-mediated redox regulation
in mitochondria is thioredoxin peroxidase II (TPX-II, also known as
peroxiredoxin III, Prx-III), an enzyme abundantly expressed in the mitochondria
of cancer cells that protects the cells from oxidative stress [79, 80]. PrxIII
is an important antioxidant that acts in conjunction with TRX2/TXR (Figure 1)
in the mitochondria to remove peroxides such as H2O2 and
offset the apoptosis inducing effects of increased levels of H2O2.
However, PrxIII contains three Cys residues, two of which are involved as
redox-active sites in the formation of a stable intersubunit disulfide-bonded
dimer, which is then reduced by thioredoxin to the monomer.  PrxIII was a more abundantly expressed
arsenic-binding protein when comparing arsenic resistant cells to normal cells
by phenylarsine oxide affinity chromatography [81]. Hence, PrxIII is very
likely to be another protein whose function is inhibited by arsenic-containing
compounds leading to the promotion of apoptosis.Increasingly, it is becoming apparent that dithiols-containing
redox proteins, particularly those present in the mitochondria, act as
controlling sensors during responses to changes in cellular redox. Many thiol
redox proteins contain a vicinal pair or more of reactive thiol groups [82, 83] capable
of binding with arsenic-containing compounds in a similar manner to those in
the thioredoxin system. The most important of these are located in the
mitochondria, a point of extreme sensitivity to arsenic-containing compounds
whose actions culminate in triggering the apoptotic pathways via the induction
of reactive oxygen species, leading to the killing of the cancer cells (Figures
1, 2). For example, two members of the glutaredoxin (GRX) family, including
GRX2 located primarily in the mitochondria, catalyse GSH-dependent TRX-disulfide
redox and protein thiol-disulfide redox reactions, particularly reversible
glutathionylation of protein sulfhydryl groups [84]. Human Grx1 and Grx2
contain C–P–Y–C and C–S–Y–C active sites ; have three and two additional structural
Cys residues, respectively; and are therefore likely to react with arsenic-containing
compounds, although no reports of this could be found.Figure 2
Mitochondrial redox systems
regulating ROS levels via thiol-disulfide exchange/coupling reactions. The mitochondrial form of Thioredoxin (Trx2) is
likely to play the major role in reducing disulfides formed by vicinal thiols
in both the mitochondrial Peroxiredoxin III (Prx III) and other proteins. Prx
III is one of the main ways by which cancer cells can reduce their levels
of H2O2 built up during active respiration. The glutathione redox system comprising
GSH/GSSG, glutathione reductase, glutaredoxin, and glutathione peroxidase,
although present in the mitochondria, is more likely to only become of major
importance during the more extreme conditions of oxidative stress. Both of
these systems are targets for inhibition by arsenic-containing compounds. See
text for further detail.Another mitochondrial protein
targeted by arsenic-containing compounds with a vicinal dithiol group is regulatory
protein “Factor B.” Addition of recombinant factor B back to bovine
submitochondrial particles depleted of this protein restored energy coupling
activity. Thus, reverse electron transfer from succinate to complex I enabling NAD+
reduction, electron transport chain function and oxidative phosphorylation/32Pi-ATP
exchange of the ATP synthetase complex were reactivated [85] as was increased
exchange activity of complex V [86]. Thus, the F0-F1 ATPase activity requires
Factor B coupled to it for full activation. However, factor B contains six
thiols and Cys 92 and Cys 94 in the bovine form were shown to bind phenylarsine
oxide [87] and phenylarsine oxide or arsenite inhibit factor B coupling
activity [88].  From all of these
studies, it is becoming clear that redox changes to vicinal thiols affect the
regulation of mitochondrial function and that these thiols are also major
targets for inhibition by the arsenic-containing compounds (Figures 1, 2).


## 3.1. Inhibitors of Energy Metabolism: Effects on Glycolysis and Oxidative
Phosphorylation

At the
biochemical level, inorganic arsenic in the pentavalent state (As(V), arsenate, AsO4) resembles a
phosphate (PO4) group in structure and it can replace phosphate in many
reactions. The mitochondria is a
major intracellular site where arsenate is metabolised, taken up As(V), rapidly
reducing it, and exporting the As(III) product back into the cytosol [27].  The specific location of the site(s) for
arsenic reduction in the mitochondria has not yet been defined.  However, arsenate can affect oxidative phosphorylation
by binding to the Fo/F1 ATP synthase [28]. Arsenate can be used by the ATP
synthase more efficiently than phosphate depending on the Ca2+ levels [29],
producing ADP-arsenate which unlike ATP becomes rapidly hydrolysed and unable
to form stable high-energy compounds [30]. It is suggested that the structure
and charge similarities of PO43−, AsO43−,
and SO42− result in
indiscriminate binding to at least two sites located in the mitochondrial
matrix [31]. At one site, occupation by any of these three anions results in
protection against uncoupling of the mitochondrial proton gradient by K+;
at the second site, in the Fo/F1 ATP synthase,  AsO43− and SO42− compete for binding against PO43−, leading to the inhibition of ATP production [31].Intriguingly, As2O3,
whilst shown to have no effect on oxidative phosphorylation levels in HeLa [10 μM] and AS-30D [100 μM] hepatoma cancer cells, significantly inhibited
glycolysis, particularly during the exponential growth phase of these cells
when they were actively respiring and producing 70% of their ATP from
mitochondria [32]. Several other studies have also shown that arsenic compounds
do not affect oxidative phosphorylation. Thus, with submitochondrial particles
in the presence of an ATP regenerating system, 20 mM arsenate had no effect on
NADH formation, ATP hydrolysis, and Pi  ↔  H20 exchange [33]. In a related study with
isolated liver mitochondria, As2O3 was again shown to
have no effect on oxygen consumption, or the respiratory control ratio at a concentration
(10 μM) found to be maximally effective in promoting
apoptosis in whole cells [34]. However, at higher concentrations (>50 μM), pyruvate/malate-supported
respiration (via complex I) became blocked, but there was no effect on either
complex II or IV.  The inhibitory effect on complex
I was reversed by the addition of the reducing agent, dithiothreitol,
indicating that direct oxidative damage was involved.  In addition, it was shown that even with the
block in complex I, the cells continued to maintain cellular ATP levels through
glycolysis, and hence, depletion of cellular ATP was not the cause for the
cytotoxicity of As2O3 [34]. Probably the most definitive evidence
for the importance of mitochondria in mediating As2O3 killing
of cancer cells comes from studying cells lacking mitochondrial function [35].
A subclone of mitochondrial respiration deficient cells was derived from the
HL-60 human leukaemia
cell line by growth in the presence of ethidium bromide to mutate the
mitochondrial genome, and these cells are known as HL-60 “ρ0” cells [35]. Due to the lack of
mitochondrial respiration, ρ0cells depend on glycolysis for
their energy source and, as would be expected, produced substantially less
superoxide radicals (∼20% of the control cells). When these ρ0 cells were incubated with ∼10 μM As2O3, they were resistant
to the drug, revealing that mitochondrial respiratory function is required for
the cytotoxic actions of As2O3 [35].The
inhibition of glycolysis by arsenic-based drugs appears unlikely to be a significant
factor involved in the drug-induced killing of cancer cells. Thus, incubating
cells in glucose-deficient medium to block glycolysis had no significant effect
on theAs2O3− [30 uM] mediated levels of cell death in
the Jurkat cell line [36]. However, when glycolysis was blocked and
mitochondrial respiration inhibited using oligomycin A, the cells became very
sensitive to As2O3− mediated cell death.  In this regard, it is also worth noting that studies
of individual glycolytic enzymes analysed in purified form in vitro have shown arsenite
and arsenate to be relatively weak inhibitors. Hence, for hexokinase (IC50:
15 mM for arsenate [37, 38]), phosphofructokinase (IC50 > 5 mM for arsenite; [39])
and  pyruvate dehydrogenase (PDH IC50:
80–120 μM arsenite; [40]), relatively large concentrations
were required to inhibit these enzymes. In fact, arsenate has been shown to stimulate
the activities of the two important glycolytic enzymes, hexokinase [37] and
GAPDH [41], by overcoming product inhibition in these reactions. In the case of
GAPDH, arsenate acts catalytically to promote the oxidation of phosphoglycerate
[42] and the reaction involved the formation of an arsenate analogue of the
phosphate ester as an intermediate which rapidly hydrolysed, helping to drive
the reaction forward [33, 43]. This process has been commonly described in
relation to the effects of arsenate on numerous enzymatic reactions involving
phosphate and has been termed “arsenolysis” [33].Given the relative
insensitivity to the direct effects of arsenic compounds shown by the
glycolytic pathway, it follows that it is unlikely that glycolytic inhibition
results from  direct binding and
modification of the enzymes in this pathway by arsenic-based drugs. More likely,
the inhibition of glycolysis results from an indirect effect, caused by the
actions of arsenic compounds in modifying mitochondrial respiration leading to
production of ROS which then acts to inhibit the glycolytic enzymes. Additional
support for the mitochondrial-mediated ROS involvement in the action of arsenic
to inhibit glycolysis comes from studies where As2O3 was
found to be ∼38 times more potent in cells than in the pure preparation at
inhibiting pyruvate dehydrogenase [40]. Also, inhibiting mitochondrial
respiration suppressed the resulting inhibition of pyruvate dehydrogenase activity
and H2O2 production by this
drug. Furthermore, the inhibition of pyruvate dehydrogenase by As2O3 was shown to require the Fenton reaction occurring via hydroxyl radical intermediates
[40]. The mitochondrial effects of arsenic compounds are detailed later in this
review and to reiterate at this point, the evidence indicates that the actions
of arsenic compounds on glycolysis are not the main cause for the cytotoxic
effects of these drugs at clinically relevant concentrations (1–6 μM) required in plasma for the killing of cancer
cells in APL patients [8, 44].


## 3.2. Interconversion of As(III)↔     As(V) in Cells

Under conditions of
high mitochondrial respiration inside cells, it is possible that trivalent
arsenicals inducing significant production of ROS as superoxide, peroxide, and
hydroxyl radicals can also result in oxidation to produce arsenate (AsO43−)
ionic species [45, 46].  Consequently, the
impact that arsenic compounds
will have on any given cell will most likely depend on the state of
cellular respiration and production of ROS affecting the arsenic speciation and
whether the cell is dependant on glycolysis versus mitochondrial respiration
for its ATP synthesis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the
glycolytic enzyme abundantly found in all cells and especially blood cells and
liver, is a major intracellular arsenate reductase [47] requiring GSH, NAD, and
glycolytic substrate [48, 49]. Given that the levels and specific activity of
GAPDH is much higher in malignant cells than in normal cells [50], this could
contribute to the rapid reduction of As(V) species in their cytosol into more
toxic As(III) forms.In the GAPDH reaction,
As(V) reduction may take place during, or as a consequence of the arsenolytic
cleavage of the thioester bond formed between the enzyme's Cys149 residue and
the 3-phosphoglyceroyl moiety of the substrate. Hydrolysis of
1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of
the normal substrate 1,3-diphosphoglycerate under the same conditions [51].
Hence, GAPDH is proposed as one of the key cellular converting enzymes for
reducing As(V) to As(III). Although purine nucleoside phosphorylase was
proposed to be an arsenate reductase [52], this was later refuted [53].  The other major class of As(V) reductases in
cells are the glutathione S transferases and of these, the omega form or GSTO1
appears to be most important. Thus, GSTO1 can reduce arsenate to arsenite,
MMA(V) to MMA(III), and DMA(V) to DMA(III) and deletion of the GSTO1 gene in
mice reduced the extent of biotransformation by 30–80% in most tissues examined
[54].


## 3.3. Structure and Reactivity of Arsenic-Containing
Compounds with Reduced Thiols

Arsenic has a high affinity for sulfur
and hence, reactive sulfur-containing molecules such as reduced thiols with an
available sulfur atom have a significant propensity for binding to arsenic [55, 56].
As(III)-containing compounds exist as trigonal pyramidal structures and this is
also the structure formed upon binding of arsenic ions to cellular proteins in
vivo where the sulphur atoms of thiolate groups act as coordinating ligands. The
resulting arsenic-thiol linkages are mainly responsible for the ability of
arsenic to modulate the function of various key molecules, enzymes, and ion
transporters inside cells and this intracellular action of arsenic is discussed
in detail in this section.  Arsenic-containing compounds react with mono- and dithiols,
particularly the latter when two thiols are located in close proximity, acting
to cross-link the thiols together.Some debate exists
about the structures and speciation of arsenic-containing compounds (both
inorganic and organic forms) in solution.
In the absence of sulfide, As(III) hydroxide complexes are the major
arsenic-containing species and these structures probably adopt a trigonal
pyramidal structure with the arsenic atom at the apex [57–59].  This trigonal pyramidal structure provides
the potential for As(III) to coordinate linkages with several proximal thiol
groups.  This is believed to be the case
in bacterial enzyme systems such as the ArsR repressor protein where it is
likely to bind three Cys atoms [60]. Thus, in the more toxic form as the
trivalent state (As(III)) inorganic and organic
(methylated) arsenic reacts with critical thiols in proteins, inhibiting their
function, as is the case with the bacterial ArsR protein. For example, As(III) was shown to target the
reactive sulfhydryl group at the active site of thiolase(s) involved in ketogenesis
from acetyl Coenzyme A [61].The
pentavalent species of inorganic arsenate (AsO43−)
favoured to exist in oxidised environments, as well as organic forms of As(V)
have a different structure with a trigonal-bipyramidal shape where the As(V)
atom is located at the centre, co-ordinating to three equatorial and two polar
atoms [62, 63]. In comparing the two different structural states of As(III) and
As(V), it is not yet clear why the trivalent methylarsenic-containing compounds
show a much higher toxicity than their pentavalent analogues [24, 64]. However,
it could relate to cellular uptake as trivalent organoarsenic compounds are more membrane
permeable than the pentavalent species [65]. Also, trivalent arsenic bonded at
a phenyl ring is able to form much more stable covalent cross-links to cysteine
residues compared to arsenic in small molecules such as arsenious acid or arsenite
[66].  Furthermore, the
organic trivalent arsenic-containing compound, phenylarsine oxide [0.1–0.5 μM], is much more potent than the simple arsenite
[1–10 μM] in its cytotoxic activity in APL cells [67].One major
drawback with phenylarsine oxide as a potential cancer therapy is its high
toxicity in vivo and its nonselectivity for cancer versus normal cells,
resulting in cytotoxicity in normal endothelial cells in the same concentration
range (0.2μM) [68].  Thus, phenylarsine oxide, is precluded from
application in clinical cancer therapy, without being further modified as a
drug. The
greater reactivity of phenylarsene oxide and associated cytotoxicity is in
agreement with the results outlined above [66] where mass spectrometric
analysis of different complexes of peptides and proteins with arsenic-containing
species revealed that inorganic arsenite or arsenates did not interact well
with cysteine or glutathione, whereas the organic phenylarsine (3+) oxide did.
In addition, three different phenyl arsenic acids and dimethylarsinic acid that
all contained As(V) also formed complexes with glutathione [66]. Hence, the bulky hydrophobic groups with
electron withdrawing π  orbitals (in the case of phenyl groups) may
promote more stable bonds between the arsenic atom and sulfur groups inside
cells, modulating a larger range of enzymes and proteins with important
functions for maintaining cell viability.In
cells, the most common reactive species that are available for interaction with
arsenic are the abundant free thiol moieties in the tripeptide glutathione (γGlu-Cys-Gly,
GSH) and the free amino acid, cysteine.  Thus, arsenic-based drugs can react by
coordinating binding to free (reduced) thiol groups such as those on cysteine,
particularly those of thioredoxin and glutathione as the major intracellular thiol
species important in cellular redox regulation. It was observed early on that arsenite
and phenylarsine oxide in particular, but not arsenate, reacted with vicinal
thiol groups on proteins [69, 70].  Since
demonstrating that phenylarsine oxide was particularly effective at cross-linking
vicinal thiols in the active site of tyrosine phosphatases [71], the range of
proteins -containing vicinal cysteine residues with which phenylarsine oxide reacts
is increasing. Recent examples include the small GTP binding Rho
protein family [72] and the mitochondrial
carnitine/acylcarnitine transporter [73]. Phenylarsine
oxide, as a strong inhibitor of tyrosine phosphatases would increase tyrosine
phosphorylation levels of enzymes in cellular growth signalling pathways.
Whether the inhibition of tyrosine phosphatases and other enzymes contributes
to the toxic effects of arsenic-containing compounds in normal cells is not
clear, but is likely an important contributing factor to its general
cytotoxicity making phenylarsine oxide unsuitable for cancer therapy.Analysis
of the interactions of As(III) with glutathione or cysteine in vitro in aqueous
solutions by equilibrium binding and use of biophysical techniques including
NMR, electronic spectroscopy, and potentiometry revealed that As(III) binds either
of glutathione or cysteine with similar equilibrium constants [74].  However, several analytical studies by mass
spectroscopy have revealed that arsenate and arsenite do not complex readily
with glutathione or cysteine, but prefer to react with the thiols on reduced
thioredoxin molecules [reviewed in [66]]. This was confirmed by analysis of more
biologically relevant samples from the intracellular environment of HeLa cells
where cytosolic thioredoxin 1 (TRX1) and, in particular thioredoxin 2 (TRX2) in
the mitochondria, was shown to be highly reactive with arsenite (10 μM)
whereas little reactivity was detected with cellular GSH/GSSG [75].Studies
with thioredoxin reductase (TxR) purified from mouse liver showed that arsenic-containing
compounds with As(III) and arsinothiols (complexes of As(III) with GSH or L-cysteine)
were extremely potent inhibitors of this enzyme [24]. Methylarsenic(III)oxide
was most potent with a  Ki∼100 nM, as an irreversible competitive inhibitor. The
effects on purified glutathione reductase (GR) showed that the levels of
inhibition were not as marked with inorganic As(III) and As(V) oxides showing
IC50s in the 10–50 mM range, whereas for methylarsinic(III)oxide it was ∼9 μM. Studies
on this enzyme in whole cells as hepatocytes showed the IC50 to be reduced to
∼3 μM
for methylarsinic(III) oxide and for As2O3>100 μM
[24]. Hence, these observations strongly support a role for the components
thioredoxins and the thioredoxin reductase system as providing cellular targets
that are very sensitive to inhibition by arsenic-based drugs in the low
micromolar range. It also explains the importance of studying the chemistry of arsenic-containing
compounds in the context of both purified enzymes as well as whole cells as biological
systems in order to obtain meaningful results. Thus, although the
GSH/glutathione transferase system undoubtedly plays an important role in
arsenic sensitivity of cells
(see below in Section 4 for details), it   would appear that the thioredoxin system might
represent the most immediate point of sensitive reactivity in relation to
cytotoxicity.


## 3.4. The Major Mechanism of Action of Arsenic-Containing Compounds: Modifying
Mitochondrial Function and Redox Regulation of the Production of Reactive Oxygen Species

Mitochondria
are a main source of ROS in cells (reviewed in [76]). Thioredoxin (TRX), NADPH,
and thioredoxin reductase (TxR) comprise the thioredoxin system that has multiple
functions in cells including in redox signalling via interactions with other
proteins, in transcriptional regulation, control of the reduced intracellular
redox environment, cell growth, defense against oxidative stress and control of
apoptosis (reviewed in [77]). As outlined in the previous section, the thioredoxin
system is very sensitive to arsenic-based drugs and may well be the basis for
one of the important mechanisms for their actions in inducing cancer cell
death. The TRX system operates as a thiol-disulfide exchange reaction (see
Figure 1).  TRX1 and TRX2 are key
regulatory isozymes that catalyse the reduction of protein disulfide bonds. They
are cofactors of the apoptosis signal-regulating kinase 1 (ASK1) that mediates TNF
cytokine and oxidative stress-induced apoptosis via the mitochondrial dependent
pathway [78].Figure 1
Many vicinal thiol-containing
redox proteins are major mitochondrial targets for binding of arsenic-containing
compounds.  Arsenite and particularly organic arsenites will
disrupt the normal redox systems functioning in the mitochondrial matrix and
intermembrane space by targeting vicinal thiols in proteins and enzymes that
regulate these systems. Such enzymes include Peroxiredoxin III (Prx III), Thioredoxin
2 (Trx2). In addition, several key mitochondrial functions are affected,
including Factor B regulating the ATP synthetase activity, the adenine
nucleotide transporter, ANT, amongst others.
See text for further detail.In
their reduced forms, cytosolic TRX1 and mitochondrial TRX2 each contain two
vicinal thiol groups in their active site sequence as –C–G–P–C–. TRX 1
in the cytosol and TRX2 in mitochondria bind to Cys 250 and Cys 30,
respectively, in the regulatory N-terminal domain of ASK1 and can cooperatively
maintain the enzyme in the inhibited state. However, activation by TNF
resulting in increased production of ROS and leads to the oxidation of the TRX dithiol
group to a disulfide. Under these conditions, the thioredoxins no longer bind
to ASK1 and loss of TRX2
binding to mitochondrial-located ASK1 can lead to apoptosis in a
JNK-independent manner, whereas cytosolic ASK1 upon loss of TRX1 binding then
becomes activated as a MAPKKK resulting in JNK activation, Bid cleavage and Bax
translocation to the mitochondria [78]. Since it is known that the thioredoxins
are major targets of arsenic-containing compounds (see above and [75]), it can
be predicted that arsenite-mediated oxidative binding to thioredoxins will
induce a similar outcome as TNF signalling, leading to the release and
activation of ASK1 and induction of apoptosis.Another
thioredoxin-associated protein of importance in thiol-mediated redox regulation
in mitochondria is thioredoxin peroxidase II (TPX-II, also known as
peroxiredoxin III, Prx-III), an enzyme abundantly expressed in the mitochondria
of cancer cells that protects the cells from oxidative stress [79, 80]. PrxIII
is an important antioxidant that acts in conjunction with TRX2/TXR (Figure 1)
in the mitochondria to remove peroxides such as H2O2 and
offset the apoptosis inducing effects of increased levels of H2O2.
However, PrxIII contains three Cys residues, two of which are involved as
redox-active sites in the formation of a stable intersubunit disulfide-bonded
dimer, which is then reduced by thioredoxin to the monomer.  PrxIII was a more abundantly expressed
arsenic-binding protein when comparing arsenic resistant cells to normal cells
by phenylarsine oxide affinity chromatography [81]. Hence, PrxIII is very
likely to be another protein whose function is inhibited by arsenic-containing
compounds leading to the promotion of apoptosis.Increasingly, it is becoming apparent that dithiols-containing
redox proteins, particularly those present in the mitochondria, act as
controlling sensors during responses to changes in cellular redox. Many thiol
redox proteins contain a vicinal pair or more of reactive thiol groups [82, 83] capable
of binding with arsenic-containing compounds in a similar manner to those in
the thioredoxin system. The most important of these are located in the
mitochondria, a point of extreme sensitivity to arsenic-containing compounds
whose actions culminate in triggering the apoptotic pathways via the induction
of reactive oxygen species, leading to the killing of the cancer cells (Figures
1, 2). For example, two members of the glutaredoxin (GRX) family, including
GRX2 located primarily in the mitochondria, catalyse GSH-dependent TRX-disulfide
redox and protein thiol-disulfide redox reactions, particularly reversible
glutathionylation of protein sulfhydryl groups [84]. Human Grx1 and Grx2
contain C–P–Y–C and C–S–Y–C active sites ; have three and two additional structural
Cys residues, respectively; and are therefore likely to react with arsenic-containing
compounds, although no reports of this could be found.Figure 2
Mitochondrial redox systems
regulating ROS levels via thiol-disulfide exchange/coupling reactions. The mitochondrial form of Thioredoxin (Trx2) is
likely to play the major role in reducing disulfides formed by vicinal thiols
in both the mitochondrial Peroxiredoxin III (Prx III) and other proteins. Prx
III is one of the main ways by which cancer cells can reduce their levels
of H2O2 built up during active respiration. The glutathione redox system comprising
GSH/GSSG, glutathione reductase, glutaredoxin, and glutathione peroxidase,
although present in the mitochondria, is more likely to only become of major
importance during the more extreme conditions of oxidative stress. Both of
these systems are targets for inhibition by arsenic-containing compounds. See
text for further detail.Another mitochondrial protein
targeted by arsenic-containing compounds with a vicinal dithiol group is regulatory
protein “Factor B.” Addition of recombinant factor B back to bovine
submitochondrial particles depleted of this protein restored energy coupling
activity. Thus, reverse electron transfer from succinate to complex I enabling NAD+
reduction, electron transport chain function and oxidative phosphorylation/32Pi-ATP
exchange of the ATP synthetase complex were reactivated [85] as was increased
exchange activity of complex V [86]. Thus, the F0-F1 ATPase activity requires
Factor B coupled to it for full activation. However, factor B contains six
thiols and Cys 92 and Cys 94 in the bovine form were shown to bind phenylarsine
oxide [87] and phenylarsine oxide or arsenite inhibit factor B coupling
activity [88].  From all of these
studies, it is becoming clear that redox changes to vicinal thiols affect the
regulation of mitochondrial function and that these thiols are also major
targets for inhibition by the arsenic-containing compounds (Figures 1, 2).


## 4. The Adenine Nucleotide Transporter (ANT): A Critical Target of Arsenic-Containing Compounds in the Mitochondria

A channel formed by the association of two
proteins, the voltage dependent anion channel (VDAC) in the outer mitochondrial
membrane and the adenine nucleotide transporter (ANT) in the inner mitochondrial
membrane (Figure1), is a complex involved in the induction of apoptosis
activated via the mitochondrial pathway (see [89, Figure 3]). The two
components of this complex form part of the mitochondrial permeability
transition pore (MPTP), a megachannel mediating release of molecules from the
mitochondria activating apoptosis. A rapidly increased permeability of the inner
mitochondrial membrane (mitochondrial permeability transition) leads to apoptosis
that is mediated by the MPTP.Arsenite induces apoptosis by a
direct effect on the MPTP [90, 91] and VDAC has been shown to play an essential
role in opening of the permeability pore and cytochrome c release induced by
arsenic trioxide, which also caused VDAC to homodimerise [92]. In addition, the
thiol-reactive compound 4,4’-diisothiocyanostilbene-2,2’-disulfonate (DIDS) has
been shown to block the VDAC channel [93] and inhibit ROS-mediated cytochrome c
release by VDAC [94]. Hence, the data indicates that VDAC contains critical Cys
residues that can undergo intermolecular cross-links mediated by reaction with
arsenic.Analysis of ion channel activity of
purified ANT-containing lipid bilayers also revealed that As2O3 treatment [30μM] altered the ANT channel electrophysiological properties
[36]. Interestingly, glutathione depletion leading to increased ROS may play an
important role in the action of arsenic trioxide [91]. Whereas in both normal
and cancer cells, glutathione S transferase (GST) is found to interact with ANT,
during the induction phase of apoptosis, GST dissociates from ANT suggesting
that GST/GSH may act as a repressor of MPTP and ANT pore opening [91, 95]. This
is supported by the observation that increasing the expression of the enzyme
GSTP1 in Jurkat and Raji leukemic cells renders them more resistant to arsenic
trioxide-induced apoptosis at clinically relevant levels [1-2 μM].  GSTP1
expression in these cells is also accompanied by accumulation of lower levels
of H2O2 production  [95].Both of the
mammalian proteins, VDAC and ANT, contain two or more Cys residues in their
structure thereby providing reactive thiol groups whose modification  affects their function. It has been
established that the redox state of thiol reactive groups are important for
activation of the mitochondrial permeability transition
[94, 96, 97]. Consequently,
thiol cross-linkers such as DIDS, diamide, and phenylarsine oxide [20–100 μM] affect VDAC and ANT
channel function and activation of mitochondrial permeability transition [93, 94, 96–98].
Many published results, such as cross-linking experiments, protein/inhibitor
stoichiometry, chimeric dimers, analytical ultracentrifugation, or neutron
scattering, indicate that the ANT carrier acts as a dimer and this or higher
oligomers are involved in membrane permeability transition [99, 100].Facing out into the
mitochondrial matrix, ANT has three exposed loop regions containing a conserved
repeat structure with one Cys residue in each loop. These Cys residues are
important to the process of ANT dimerisation, but it is not clear how this
operates and whether the Cys residues form intermolecular disulfide bonds or
not [101]. Nevertheless, copper-o-phenanthroline is able to dimerise ANT by
intermolecular cross-linking of Cys 56 (in the first matrix loop) [98]. In
addition, phenylarsine oxide, eosin 5-maleimide, and diamide form
intramolecular cross-links between Cys 160 and Cys 257 on the other two matrix
loops, restricting ANT in the open conformation, promoting mitochondrial
permeability transition [98]. Arsenic trioxide is much weaker than
phenylarsine oxide at binding to the ANT Cys residues [96, 97] and this may
explain the greater sensitivity exhibited by APL cells to phenylarsine oxide
[IC50: 0.1 μM] than to As2O3 [IC50: 4 μM] [67].Single thiol interacting compounds such
as N-ethylmaleimide (NEM) can inhibit the mitochondrial permeability transition
and this could be either the result of direct interaction with the key Cys
residues on the matrix loops of ANT or indirectly via reaction with GSH and
thereby preventing GSH from being oxidized and catalysing disulfide bridging
between the adjacent thiol groups in the ANT loops [98]. NEM or monobromobimane,
in the 25–50 μM range, preferentially
react with GSH, leading to its modification in mitochondria and thereby
prevents GSH from being oxidised. As a result, NEM inhibits mitochondrial
permeability transition activation by the thiol reactive compounds, diamide or
t-butylhydroperoxide, implying a role for GSSG in the action of these agents on
the permeability transition
[96, 97]. Arsenites, albeit that
much higher
concentrations would be required given their lower affinity for glutathione
interaction, could have a similar action. Thus, high levels of arsenites could
modify and inhibit glutathione redox control such that glutathione-based
enzymes are unable to function, as well as directly mediating disulfide
cross-linking of ANT, leading to increases in cellular ROS production, MPTP,
and apoptosis. However, given their low reactivity with glutathione systems,
this appears to be unlikely as opposed to the indirect action via the
mitochondrial effects leading to increased ROS production which then reduces
cellular GSH levels.The multidrug resistance (MDR) protein
MRP1/ABCC1 has been shown to transport AsIII out of cells as a tri-GSH
conjugate (As-(GSH)3), and glutathione S-transferase (GST) probably facilitates
the process [102]. This is likely to be part of the normal cell and cancer cell
resistance mechanisms against the cytotoxic effects of arsenic-based compounds.
GSH-depleted cells are more sensitive to killing by arsenic-containing compounds
[103] and transfection of cells to express glutathione S transferase protects
them from arsenite inducing death by promoting arsenite transport from the
cells and decreasing ROS levels [104, 105].
In support of this proposal, the long-term exposure of cells to arsenic-containing
compounds induced increased expression of glutathione S transferase and MRPs [106].
Arsenic levels do not attain very high
levels in blood plasma of patients, rapidly becoming eliminated [8, 44, 107].
This removal probably results from efficient uptake of As-(GSH)3 via MRP2 in the
proximal tubules of the kidneys as part of the detoxification process during
the excretion of arsenic-based drugs predominantly into the urine [108, 109]. The
remainder is mostly removed via uptake in the liver and secretion as bile [110–112].
As-(GSH)3 may be an important part of
the metabolic process for converting inorganic As(III) to methylated species
during detoxification in the liver by ASMT1/Cyt19 [113].


## 5. Modified Organic Arsenic Structures with Increased Potency as Antineoplastic Agents

Arsenic-containing
compounds substituted with organic groups such as modified phenylarsine oxides have
been synthesized and examined for their cytotoxic effect on human leukemic
cells and breast cancer cells in culture. Some of these compounds were found to
exhibit potent cytotoxic anticancer activity, particularly against human breast
cancer and leukemic cell lines, including primary leukemia cells, at micromolar
concentrations.  One of these
compounds, the novel glutathionyl peptide trivalent arsenic-containing compound
para 4-[N-(S-glutathionylacetyl) amino]phenylarsenoxide (p-GSAO) shows promise
as a novel antineoplastic drug and is now in clinical trials. P-GSAO, like
phenylarsine oxide, inactivates ANT-mediated ATP/ADP transport and triggers Ca2+-dependent
MPTP opening by cross-linking the critical Cys residues of ANT. This leads to increased production of cellular
ROS, ATP depletion, mitochondrial depolarization, and apoptosis of angiogenic endothelial
cells and inhibition of tumor growth in mice with no apparent toxicity [114].
However, the action of p-GSAO was indirect, and did not appear to be as a
result of selective tumor cell toxicity. Rather, p-GSAO inhibited the
proliferating, but not growth-quiescent endothelial cells in vitro and angiogenesis
in vivo and thus acted to eliminate tumors by blocking their blood supply [114, 115].
The trivalent arsenic-containing moiety of p-GSAO was shown to cross-link the matrix
facing Cys160 and Cys257 thiols of ANT [114] and effectively locks ANT into the
open configuration. Inactivation of ANT by p-GSAO causes an increase in
superoxide levels, proliferation arrest, ATP depletion, mitochondrial
depolarization, and apoptosis in the dividing endothelial cells.It is likely that the arsenic-containing moiety of p-GSAO reacts
similarly as does arsenite (see above) with one or two molecules of glutathione
before it is removed from the cell by MRPs [116]. Tumor cells export p-GSAO much
more efficiently than endothelial cells because they have higher MRP1 or MRP2
activity and cellular glutathione levels [116] and this may explain why p-GSAO
is not highly effective at inhibiting tumor cell growth in vivo. In addition,
the greater water soluble properties of p-GSAO than other arsenic-containing compounds,
particularly organic species should help to retain p-GSAO in the intravascular
system where it is more likely to affect endothelial cells and inhibit tumor
angiogenesis.Interestingly, although the para form of
GSAO revealed no apparent toxicity in treated animals and inhibited tumor
growth leading to phase I clinical trials as an anticancer agent [114], the
ortho form (o-GSAO) was toxic and this was proposed to result from increased
accumulation of the drug in cells, including normal cells due to loss of multidrug
resistance efflux [116]. Consequently, o-GSAO is unlikely to be of much further
interest as an antineoplastic agent, whereas the clinical efficacy of p-GSAO is
eagerly awaited.


## 6. Targeting of Cancer Cells: Selective Uptake and Delivery into Specific Types of Cancer Cells

As(III)
as the anhydrous form of As(OH)3 (Trisenox, Cell Therapeutics, Seattle, Wash,
USA) received
FDA approval in 2000 as a chemotherapeutic agent for the treatment of APL [117].
Acute promyelocytic leukemia (APL) is associated with reciprocal and balanced
chromosomal translocations always involving the retinoic acid receptor alpha
(RARalpha) gene on chromosome 17 and variable partner genes on distinct
chromosomes. RARalpha fuses to the promyeloctyic leukemia (PML) gene in the
majority of APL cases (reviewed in [118]). Arsenic trioxide is particularly
effective at killing APL cells and this was proposed to be the direct result of
its ability to induce the relocalization and degradation of the nuclear body
protein PML, as well as the degradation of PML-RARalpha
in APL cells [119–122]. However,
this seems unlikely to be the main mechanism of action for arsenic trioxide
given that no differences in sensitivity to growth inhibition and killing by
apoptosis have been observed between wild-type and PML-/-
cells [123].Arsenic
trioxide as a single agent has provided 86% complete hematologic remission with
minimal toxicity in APL patients [124], equal to any of the current standards
of care for treating APL, including the combination of all-trans retinoic acid
(ATRA) plus chemotherapy [125].  This
raises the question why APL cells are very sensitive to arsenic-containing
compounds like arsenic trioxide. It would appear that the reason is because APL
cells express the transmembrane transporter protein, aquaglyceroporin 9 (AQP9)
involved in arsenic uptake [126]  at much
higher levels in APL cells than in other leukemic cell types and that
correlates with arsenite sensitivity [127].  In this regard, it is worth noting that
aquaglyceroporins AQP7 and AQP9 are present in normal cell types.
Interestingly, AQP9 is primarily expressed
in human lung, liver, and leukocytes [128] and this may help explain arsenic
toxicity, given that liver is one of the main organs affected. The fact that AQP9 provides
APL cancer cell specificity with high response rates suggests that if arsenic-containing
compounds could be targeted for specific delivery into cancer cells, then they
would represent outstanding agents for killing these cells. However, further
modifications will be required to provide suitable drug targeting for improved
delivery of arsenic-containing compounds to cancer cells.


## 7. Conclusions

Vicinal thiols
located in key enzymes and proteins provide targets for reaction with arsenic-containing
compounds, particularly organic derivatives such as phenylarsenic-containing
compounds that favour intramolecular cross-linking between adjacent thiols. Intriguingly,
most of the key intracellular targets for this reaction have been identified to
include the main REDOX regulatory systems in the mitochondria, including thioredoxin
and peroxiredoxin systems and the adenine nucleotide transporter, all of whose
function is adversely affected. The net result is the activation of several
independent pathways including ROS production to facilitate the induction of
apoptosis. One main pathway operates via the opening of the MPTP, the other via
activation of ASK1 kinase, and the JNK/Bid/Bax pathway of channel formation in
MOM.  In the case of APL, cell
selectivity for sensitive responses to these drugs is facilitated by selective
transport systems such as provided by the AQP9 transporter. Low MDR levels
present in dividing endothelial cells also provides selective targeting by
specially substituted phenylarsenic-containing compounds like p-GSAO, leading
to decreased blood supply into tumors, with some toxicity to cancer cells, but
little toxicity on normal cells. Hence, a combination of selective delivery and
retention provides the necessary targeting of arsenic-containing compounds to
tumors and provides scope for additional modifications to be made to enhance
the antineoplastic activity of arsenic-containing compounds, given their range
of actions and efficiency in killing cancer cells.



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*Source: 260146-2008-04-09.xml*