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Hindawi Publishing Corporation

Metal-Based Drugs
Volume 2008, Article ID 260146, 13 pages
doi:10.1155/2008/260146

Review Article
Arsenic-Based Antineoplastic Drugs and
Their Mechanisms of Action

Stephen John Ralph

School of Medical Sciences, Grith University, Parklands Drive, Southport, Queensland 4215, Australia

Correspondence should be addressed to Stephen John Ralph, s.ralph@grith.edu.au

Received 1 April 2007; Revised 3 July 2007; Accepted 17 August 2007

Recommended by Rafael Moreno-Sanchez

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 selec-
tive 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 eux 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 under-
standing 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.

Copyright 2008 Stephen John Ralph. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. ARSENIC AND ARSENIC-CONTAINING When absorbed at toxic levels, arsenic causes severe
COMPOUNDS IN THE NATURAL ENVIRONMENT health problems, including cancer. Acceptable levels were
OR AS A RESULT OF HUMAN APPLICATION lowered from 50 g/L to 10 g/L in drinking water by the
WHO [4]. Arsenic-containing compounds are applied in
Arsenic is a toxic metalloid [1, 2] that exists throughout na- plant pesticides and insecticides and arsenic environmental
ture in organic and inorganic forms. It is commonly present contamination represents a global health problem, particu-
in soils on average at several mg/kg, as well as in marine larly from leaching into ground water. When the soluble lev-
sediments, and is enriched in mineral deposits as oxides els exceed 50 g/L in drinking water as in many regions of
and sulfides. The basic element, arsenic, exists in either of Bangladesh, arsenic becomes a particular health concern as
three allotropic forms: yellow, black, or grey with the stable, it has recently been associated with increased cancer rates
semimetallic form having a silver/steely-grey colour as a brit- appearing after consumption with a lag time up to 1020
tle, crystalline solid. The semimetallic form oxidises rapidly years [5].
in air, and at high temperatures produces a white cloud of Whilst the carcinogenic aspects of arsenic compounds
arsenic trioxide (As2 O3 ). Arsenic, with its variety of chemi- are not the focus of this review, nevertheless this undesirable
cal forms and oxidation states, is listed in Table 1. The cur- aspect needs to be raised. Thus, arsenic and its methylated
rent IUPAC nomenclature is listed in [3] as well as the more species are known carcinogens but this description is proba-
common names of the arsenic-based compounds which are bly inaccurate as they act more as cocarcinogens by facilitat-
mainly used throughout this review. ing/promoting the induction of tumors of the skin, urinary
2 Metal-Based Drugs

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 = H2 AsHO3 = [AsHO(OH)2 ], for example, PhAsO(OH)2 pheny-
larsonic acid.
Example of alternative names for Arsenic species used
Common name Abbreviation IUPAC Fully systematic additive name Chemical formula
Arsenite, Arsenous acid, Arsorous acid As(III), AsIII Trihydroxidoarsenic H3 AsO3 = [As(OH)3 ]
Arsinious acid As(III), AsIII Dihydrohydroxidoarsenic HAsH2 O = [AsH2 (OH)]
Arsonous acid As(III), AsIII Hydridodihydroxidoarsenic H2 AsHO2 = [AsH(OH)2 ]
Arsenate, Arsenic acid, Arsoric acid As(V), AsV Trihydroxidooxidoarsenic H3 AsO4 = AsO(OH)3
Arsinic acid As(V), AsV Dihydridohydroxidooxidoarsenic HAsH2 O2 = [AsH2 O(OH)]
Arsonic acid As(V), AsV Hydridodihydroxidooxidoarsenic H2 AsHO3 = [AsHO(OH)2 ]
Monomethylarsonic acid MMA(V), MMAV Methanedihydroxidooxidoarsenic CH3 AsO(OH)2
Monomethylarsonous acid MMA(III), MMAIII Methanedihydroxidoarsenic CH3 As(OH)2
Dimethylarsinic acid DMA(V), DMAV Dimethanehydroxidooxidoarsenic (CH3 )2 AsO(OH)
Dimethylarsinous acid DMA(III), DMAIII Dimethanehydroxidoarsenic (CH3 )2 AsOH

bladder, and lung rather than directly inducing cell trans- ing certain hematological malignancies is covered in the last
formation and oncogenesis [2, 6]. As cocarcinogens, mech- section of this review. However, the mechanisms of action of
anisms include indirect eects such as DNA damaging geno- arsenic-basedcompounds on cells must first be described in
toxicity by altered DNA methylation as well as inducing high detail to provide sucient understanding to enable their se-
levels of oxidative stress leading to altered cell proliferation lective targeting of specific types of cancer cells to be duly
and tumor promotion (detailed in [7]). evaluated.
In terms of chronic toxicity, the 50% lethal dose of Water distributes the majority of inorganic arsenic in
arsenic as arsenic trioxide in mice received by the oral the biosphere either as pentavalent arsenate (As5+ , As(V)) or
route varies from 1548 mg/kg, whereas the acute lethal trivalent arsenic (As3+ , As(III) [2]. In solution, the pH and
dose in humans varies from 13 mg/kg body weight [4]. redox conditions aect the chemical state of arsenic that pre-
Despite its toxicity, arsenic has been applied in Chinese dominates. Thus, in highly oxidised environments, the arsen-
medicines for thousands of years as refined preparations of ate (As5+ ) form predominates as one of four major species of
realgar (As4 S4 ) or orpiment (As2 S3 ) [8] which are two rare arsenic acid; H3 AsO4 , H2 AsO4 , HAsO4 2 , and AsO4 3 [13].
mineralised forms. Realgar, easily discerned by its bright However, mildly reducing conditions such as those generally
red and orpiment with a browny yellow appearance are present inside all mammalian cells [14] will favour the reduc-
ores often occurring close to each other in hydrothermal tion of the pentavalent arsenate (As5+ ) to the trivalent arsenic
veins of precious metal sulfide ores or hot spring deposits (As3+ ). Arsenous acid with the formula As(OH)3 results af-
as volcanic sublimate products (crystallized from gases). ter the slow hydrolysis of arsenic trioxide in water. As the pH
Hence, arsenic is a common waste product from the min- increases, arsenous acid is converted to the arsenic oxide ions
ing of metal ores and the name realgar is possibly de- [AsO(OH)2 ] , [AsO2 (OH)]2 , and [AsO3 ]3 [13].
rived from Arabic words for powder of the mine (rahj al
ghar). 2. METHYLATED FORMS OF ARSENIC SHOW
The long history of medical applications has included INCREASED CYTOTOXICITY INSIDE CELLS
treating many diseases such as cancers administering mi-
cromolar levels in patients and, in particular, arsenic com- Arsenic combines readily with many elements; and bacte-
pounds have proven to be very eective against certain hema- ria have evolved systems to detoxify inorganic arsenic to
tological malignancies (reviewed in [9, 10]). Thus, arsenic form organic arsenic-containing compounds such as methy-
oxides and derivatives have been established as eective treat- lated forms. Plant and insect pesticides have been made us-
ments for acute promyelocytic leukemia (APL), and they are ing organic arsenic derivatives; and monomethylarsonic acid
being tested as therapies for a range of other hematologi- (MMA(V)) and dimethylarsinic acid (DMA(V)) are used
cal cancers including myelodysplastic syndromes, multiple in products for weed control. Also, MMA(V) and DMA(V)
myeloma, and chronic myelogenous leukemia (CML). The are metabolites of inorganic arsenic, formed intracellularly
results of clinical trails using arsenic-based drugs in cancer in mammals, primarily in the liver. The metabolic process
therapy have been extensively reviewed [812]. Hence, this of inorganic arsenic conversion is known as biotransforma-
review mainly concerns those factors that explain the anti- tion and appears to enhance the excretion of arsenic from
neoplastic mechanism of action of arsenic-based drugs, dis- the body, involving formation of methylated compounds of
tinguishing their selectivity for killing certain types of cancer trivalent arsenic as intermediates. The metabolism involves
cells from their potential for toxicity to normal cells in the reduction to a trivalent state and oxidative methylation to
body. The reason for their ecacy and selectivity in treat- a pentavalent state. In addition, reductases present in cells
Stephen John Ralph 3

and other reactions facilitate the reduction of arsenic acid charge similarities of PO4 3 , AsO4 3 , and SO4 2 result in in-
[As(V)] to the arsenous [As(III)] form including methyla- discriminate binding to at least two sites located in the mi-
tion of the arsenous form, mainly via the liver, to produce tochondrial matrix [31]. At one site, occupation by any of
mono-, di-, and trimethylated species [2]. The main en- these three anions results in protection against uncoupling
zyme involved in methylation of As(III) is arsenic methyl- of the mitochondrial proton gradient by K+ ; at the second
transferase (As3MT) that requires glutathione (GSH) to pro- site, in the Fo/F1 ATP synthase, AsO4 3 and SO4 2 compete
mote the reaction and a cycling redox system such as thiore- for binding against PO4 3 , leading to the inhibition of ATP
doxin (reviewed in [15]) to detoxify arsenic-containing com- production [31].
pounds. Many animal species convert inorganic arsenic- Intriguingly, As2 O3 , whilst shown to have no eect on
containing compounds into mono-, di- and tri-methylated oxidative phosphorylation levels in HeLa [10 M] and AS-
arsenic species which are mostly then secreted in the urine 30D [100 M] hepatoma cancer cells, significantly inhibited
[16]. This is because the methylated forms are not as well ab- glycolysis, particularly during the exponential growth phase
sorbed by cells compared to the inorganic forms, although of these cells when they were actively respiring and pro-
they have much greater cytotoxicity if they do enter cells ducing 70% of their ATP from mitochondria [32]. Several
[15, 17]. other studies have also shown that arsenic compounds do
The reductive metabolism of arsenic has an important not aect oxidative phosphorylation. Thus, with submito-
role in its toxicity. The trivalent arsenic-containing com- chondrial particles in the presence of an ATP regenerating
pounds, including the methylated organic forms have much system, 20 mM arsenate had no eect on NADH formation,
greater potency than the pentavalent arsenic-containing ATP hydrolysis, and Pi rightarrow H20 exchange [33]. In
compounds as cytotoxics and carcinogens [1823]. For ex- a related study with isolated liver mitochondria, As2 O3 was
ample, in cytotoxicity assays, the IC50 values for cultures again shown to have no eect on oxygen consumption, or the
of primary hepatocytes, keratinocytes, and epithelial cells respiratory control ratio at a concentration (10 M) found
ranged from 3 M to well over 20 M for trihydroxidoarsenic to be maximally eective in promoting apoptosis in whole
salts whereas for monomethylarsonous acid [MMA(III)], cells [34]. However, at higher concentrations (> 50 M),
the values were consistently much lower at only several pyruvate/malate-supported respiration (via complex I) be-
M for the equivalent normal cell types [2426]. Organic came blocked, but there was no eect on either complex II
arsenic-based ingredients are commonly used as feed ad- or IV. The inhibitory eect on complex I was reversed by
ditives in poultry farming to increase weight gain by pre- the addition of the reducing agent, dithiothreitol, indicat-
venting bacterial and parasitic infections, thereby increas- ing that direct oxidative damage was involved. In addition,
ing feed eciency and improving pigmentation. The three it was shown that even with the block in complex I, the cells
major arsenic-containing compounds used in this man- continued to maintain cellular ATP levels through glycolysis,
ner are arsenilic acid (p-aminophenyl arsonic acid), roxar- and hence, depletion of cellular ATP was not the cause for
sone (4-hydroxy-3-nitrophenylarsonic acid), and nitarsone the cytotoxicity of As2 O3 [34]. Probably the most definitive
(4-nitro-phenylarsonic acid) [22, 23]. The metabolism of evidence for the importance of mitochondria in mediating
these arsenic-containing compounds and waste products As2 O3 killing of cancer cells comes from studying cells lack-
produced by birds and mammals consuming them is still un- ing mitochondrial function [35]. A subclone of mitochon-
certain at present and what the global environmental impact drial respiration deficient cells was derived from the HL-60
might be. human leukaemia cell line by growth in the presence of ethid-
ium bromide to mutate the mitochondrial genome, and these
3. CELLULAR ACTIONS OF ARSENIC-CONTAINING cells are known as HL-60 0 cells [35]. Due to the lack of
COMPOUNDS mitochondrial respiration, 0 cells depend on glycolysis for
their energy source and, as would be expected, produced sub-
3.1. Inhibitors of energy metabolism: effects on stantially less superoxide radicals (20% of the control cells).
glycolysis and oxidative phosphorylation When these 0 cells were incubated with 10 M As2 O3 ,
they were resistant to the drug, revealing that mitochondrial
At the biochemical level, inorganic arsenic in the pentavalent respiratory function is required for the cytotoxic actions of
state (As(V), arsenate, AsO4 ) resembles a phosphate (PO4 ) As2 O3 [35].
group in structure and it can replace phosphate in many re- The inhibition of glycolysis by arsenic-based drugs ap-
actions. The mitochondria is a major intracellular site where pears unlikely to be a significant factor involved in the drug-
arsenate is metabolised, taken up As(V), rapidly reducing it, induced killing of cancer cells. Thus, incubating cells in
and exporting the As(III) product back into the cytosol [27]. glucose-deficient medium to block glycolysis had no signif-
The specific location of the site(s) for arsenic reduction in icant eect on the As2 O3 [30 uM] mediated levels of cell
the mitochondria has not yet been defined. However, arse- death in the Jurkat cell line [36]. However, when glycoly-
nate can aect oxidative phosphorylation by binding to the sis was blocked and mitochondrial respiration inhibited us-
Fo/F1 ATP synthase [28]. Arsenate can be used by the ATP ing oligomycin A, the cells became very sensitive to As2 O3
synthase more eciently than phosphate depending on the mediated cell death. In this regard, it is also worth not-
Ca2+ levels [29], producing ADP-arsenate which unlike ATP ing that studies of individual glycolytic enzymes analysed
becomes rapidly hydrolysed and unable to form stable high- in purified form in vitro have shown arsenite and arsen-
energy compounds [30]. It is suggested that the structure and ate to be relatively weak inhibitors. Hence, for hexokinase
4 Metal-Based Drugs

(IC50: 15 mM for arsenate [37, 38]), phosphofructokinase rapid reduction of As(V) species in their cytosol into more
(IC50 > 5 mM for arsenite; [39]) and pyruvate dehydroge- toxic As(III) forms.
nase (PDH IC50: 80120 M arsenite; [40]), relatively large In the GAPDH reaction, As(V) reduction may take place
concentrations were required to inhibit these enzymes. In during, or as a consequence of the arsenolytic cleavage of
fact, arsenate has been shown to stimulate the activities of the thioester bond formed between the enzymes Cys149
the two important glycolytic enzymes, hexokinase [37] and residue and the 3-phosphoglyceroyl moiety of the substrate.
GAPDH [41], by overcoming product inhibition in these re- Hydrolysis of 1-arseno-3-phosphoglycerate is at least 2000
actions. In the case of GAPDH, arsenate acts catalytically to times faster than hydrolysis of the normal substrate 1,3-
promote the oxidation of phosphoglycerate [42] and the re- diphosphoglycerate under the same conditions [51]. Hence,
action involved the formation of an arsenate analogue of the GAPDH is proposed as one of the key cellular converting en-
phosphate ester as an intermediate which rapidly hydrolysed, zymes for reducing As(V) to As(III). Although purine nu-
helping to drive the reaction forward [33, 43]. This process cleoside phosphorylase was proposed to be an arsenate re-
has been commonly described in relation to the eects of ductase [52], this was later refuted [53]. The other major
arsenate on numerous enzymatic reactions involving phos- class of As(V) reductases in cells are the glutathione S trans-
phate and has been termed arsenolysis [33]. ferases and of these, the omega form or GSTO1 appears to
Given the relative insensitivity to the direct eects of ar- be most important. Thus, GSTO1 can reduce arsenate to ar-
senic compounds shown by the glycolytic pathway, it follows senite, MMA(V) to MMA(III), and DMA(V) to DMA(III)
that it is unlikely that glycolytic inhibition results from di- and deletion of the GSTO1 gene in mice reduced the ex-
rect binding and modification of the enzymes in this path- tent of biotransformation by 3080% in most tissues exam-
way by arsenic-based drugs. More likely, the inhibition of ined [54].
glycolysis results from an indirect eect, caused by the ac-
tions of arsenic compounds in modifying mitochondrial res- 3.3. Structure and reactivity of arsenic-containing
piration leading to production of ROS which then acts to compounds with reduced thiols
inhibit the glycolytic enzymes. Additional support for the Arsenic has a high anity for sulfur and hence, reactive
mitochondrial-mediated ROS involvement in the action of sulfur-containing molecules such as reduced thiols with an
arsenic to inhibit glycolysis comes from studies where As2 O3 available sulfur atom have a significant propensity for bind-
was found to be 38 times more potent in cells than in ing to arsenic [55, 56]. As(III)-containing compounds exist
the pure preparation at inhibiting pyruvate dehydrogenase as trigonal pyramidal structures and this is also the structure
[40]. Also, inhibiting mitochondrial respiration suppressed formed upon binding of arsenic ions to cellular proteins in
the resulting inhibition of pyruvate dehydrogenase activity vivo where the sulphur atoms of thiolate groups act as co-
and H2 O2 production by this drug. Furthermore, the inhibi- ordinating ligands. The resulting arsenic-thiol linkages are
tion of pyruvate dehydrogenase by As2 O3 was shown to re- mainly responsible for the ability of arsenic to modulate the
quire the Fenton reaction occurring via hydroxyl radical in- function of various key molecules, enzymes, and ion trans-
termediates [40]. The mitochondrial eects of arsenic com- porters inside cells and this intracellular action of arsenic is
pounds are detailed later in this review and to reiterate at this discussed in detail in this section. Arsenic-containing com-
point, the evidence indicates that the actions of arsenic com- pounds react with mono- and dithiols, particularly the lat-
pounds on glycolysis are not the main cause for the cytotoxic ter when two thiols are located in close proximity, acting to
eects of these drugs at clinically relevant concentrations (1 cross-link the thiols together.
6 M) required in plasma for the killing of cancer cells in APL Some debate exists about the structures and speciation of
patients [8, 44]. arsenic-containing compounds (both inorganic and organic
forms) in solution. In the absence of sulfide, As(III) hydrox-
3.2. Interconversion of As(III) rightarrow As(V) in cells ide complexes are the major arsenic-containing species and
these structures probably adopt a trigonal pyramidal struc-
Under conditions of high mitochondrial respiration inside ture with the arsenic atom at the apex [5759]. This trigonal
cells, it is possible that trivalent arsenicals inducing signif- pyramidal structure provides the potential for As(III) to co-
icant production of ROS as superoxide, peroxide, and hy- ordinate linkages with several proximal thiol groups. This is
droxyl radicals can also result in oxidation to produce arsen- believed to be the case in bacterial enzyme systems such as
ate (AsO4 3 ) ionic species [45, 46]. Consequently, the impact the ArsR repressor protein where it is likely to bind three Cys
that arsenic compounds will have on any given cell will most atoms [60]. Thus, in the more toxic form as the trivalent state
likely depend on the state of cellular respiration and pro- (As(III)) inorganic and organic (methylated) arsenic reacts
duction of ROS aecting the arsenic speciation and whether with critical thiols in proteins, inhibiting their function, as is
the cell is dependant on glycolysis versus mitochondrial res- the case with the bacterial ArsR protein. For example, As(III)
piration for its ATP synthesis. Glyceraldehyde-3-phosphate was shown to target the reactive sulfhydryl group at the active
dehydrogenase (GAPDH), the glycolytic enzyme abundantly site of thiolase(s) involved in ketogenesis from acetyl Coen-
found in all cells and especially blood cells and liver, is a zyme A [61].
major intracellular arsenate reductase [47] requiring GSH, The pentavalent species of inorganic arsenate (AsO4 3 )
NAD, and glycolytic substrate [48, 49]. Given that the levels favoured to exist in oxidised environments, as well as organic
and specific activity of GAPDH is much higher in malignant forms of As(V) have a dierent structure with a trigonal-
cells than in normal cells [50], this could contribute to the bipyramidal shape where the As(V) atom is located at the
Stephen John Ralph 5

centre, co-ordinating to three equatorial and two polar atoms factor to its general cytotoxicity making phenylarsine oxide
[62, 63]. In comparing the two dierent structural states unsuitable for cancer therapy.
of As(III) and As(V), it is not yet clear why the trivalent Analysis of the interactions of As(III) with glutathione or
methylarsenic-containing compounds show a much higher cysteine in vitro in aqueous solutions by equilibrium binding
toxicity than their pentavalent analogues [24, 64]. However, and use of biophysical techniques including NMR, electronic
it could relate to cellular uptake as trivalent organoarsenic spectroscopy, and potentiometry revealed that As(III) binds
compounds are more membrane permeable than the pen- either of glutathione or cysteine with similar equilibrium
tavalent species [65]. Also, trivalent arsenic bonded at a constants [74]. However, several analytical studies by mass
phenyl ring is able to form much more stable covalent spectroscopy have revealed that arsenate and arsenite do not
cross-links to cysteine residues compared to arsenic in small complex readily with glutathione or cysteine, but prefer to
molecules such as arsenious acid or arsenite [66]. Further- react with the thiols on reduced thioredoxin molecules [re-
more, the organic trivalent arsenic-containing compound, viewed in [66]]. This was confirmed by analysis of more bio-
phenylarsine oxide [0.10.5 M], is much more potent than logically relevant samples from the intracellular environment
the simple arsenite [110 M] in its cytotoxic activity in APL of HeLa cells where cytosolic thioredoxin 1 (TRX1) and, in
cells [67]. particular thioredoxin 2 (TRX2) in the mitochondria, was
One major drawback with phenylarsine oxide as a poten- shown to be highly reactive with arsenite (10 M) whereas
tial cancer therapy is its high toxicity in vivo and its nonse- little reactivity was detected with cellular GSH/GSSG [75].
lectivity for cancer versus normal cells, resulting in cytotox- Studies with thioredoxin reductase (TxR) purified from
icity in normal endothelial cells in the same concentration mouse liver showed that arsenic-containing compounds with
range (0.2 M) [68]. Thus, phenylarsine oxide, is precluded As(III) and arsinothiols (complexes of As(III) with GSH
from application in clinical cancer therapy, without being or L-cysteine) were extremely potent inhibitors of this en-
further modified as a drug. The greater reactivity of pheny- zyme [24]. Methylarsenic(III)oxide was most potent with a
larsene oxide and associated cytotoxicity is in agreement Ki100 nM, as an irreversible competitive inhibitor. The ef-
with the results outlined above [66] where mass spectromet- fects on purified glutathione reductase (GR) showed that
ric analysis of dierent complexes of peptides and proteins the levels of inhibition were not as marked with inorganic
with arsenic-containing species revealed that inorganic ar- As(III) and As(V) oxides showing IC50s in the 1050 mM
senite or arsenates did not interact well with cysteine or glu- range, whereas for methylarsinic(III)oxide it was 9 M.
tathione, whereas the organic phenylarsine (3+) oxide did. In Studies on this enzyme in whole cells as hepatocytes showed
addition, three dierent phenyl arsenic acids and dimethy- the IC50 to be reduced to 3 M for methylarsinic(III) ox-
larsinic acid that all contained As(V) also formed complexes ide and for As2 O3 > 100 M [24]. Hence, these observations
with glutathione [66]. Hence, the bulky hydrophobic groups strongly support a role for the components thioredoxins and
with electron withdrawing orbitals (in the case of phenyl the thioredoxin reductase system as providing cellular targets
groups) may promote more stable bonds between the ar- that are very sensitive to inhibition by arsenic-based drugs in
senic atom and sulfur groups inside cells, modulating a larger the low micromolar range. It also explains the importance of
range of enzymes and proteins with important functions for studying the chemistry of arsenic-containing compounds in
maintaining cell viability. the context of both purified enzymes as well as whole cells
In cells, the most common reactive species that are avail- as biological systems in order to obtain meaningful results.
able for interaction with arsenic are the abundant free thiol Thus, although the GSH/glutathione transferase system un-
moieties in the tripeptide glutathione (Glu-Cys-Gly, GSH) doubtedly plays an important role in arsenic sensitivity of
and the free amino acid, cysteine. Thus, arsenic-based drugs cells (see below in Section 4 for details), it would appear that
can react by coordinating binding to free (reduced) thiol the thioredoxin system might represent the most immediate
groups such as those on cysteine, particularly those of thiore- point of sensitive reactivity in relation to cytotoxicity.
doxin and glutathione as the major intracellular thiol species
important in cellular redox regulation. It was observed early 3.4. The major mechanism of action of
on that arsenite and phenylarsine oxide in particular, but arsenic-containing compounds: modifying
not arsenate, reacted with vicinal thiol groups on proteins mitochondrial function and redox regulation
[69, 70]. Since demonstrating that phenylarsine oxide was of the production of reactive oxygen species
particularly eective at cross-linking vicinal thiols in the ac-
tive site of tyrosine phosphatases [71], the range of proteins Mitochondria are a main source of ROS in cells (reviewed in
-containing vicinal cysteine residues with which phenylar- [76]). Thioredoxin (TRX), NADPH, and thioredoxin reduc-
sine oxide reacts is increasing. Recent examples include the tase (TxR) comprise the thioredoxin system that has mul-
small GTP binding Rho protein family [72] and the mito- tiple functions in cells including in redox signalling via in-
chondrial carnitine/acylcarnitine transporter [73]. Phenylar- teractions with other proteins, in transcriptional regulation,
sine oxide, as a strong inhibitor of tyrosine phosphatases control of the reduced intracellular redox environment, cell
would increase tyrosine phosphorylation levels of enzymes growth, defense against oxidative stress and control of apop-
in cellular growth signalling pathways. Whether the inhibi- tosis (reviewed in [77]). As outlined in the previous sec-
tion of tyrosine phosphatases and other enzymes contributes tion, the thioredoxin system is very sensitive to arsenic-based
to the toxic eects of arsenic-containing compounds in nor- drugs and may well be the basis for one of the important
mal cells is not clear, but is likely an important contributing mechanisms for their actions in inducing cancer cell death.
6 Metal-Based Drugs

Trx1
Cytosol

As III

SH
As
III
SH

Outer membrane

VDAC VDAC

As
III
H+ H+ H+
Electron transport chain H+ H+
H+ H+

C III
CI e
UbQ C II e Inner membrane
C IV
ANT ANT
NADH
SH SH Fac ATP synthase
SH SH NAD+ Fumarate tor
Succinate B

2
O
/2
+1 SH
H2 O
As

Trx2
III

SH
H+

ASK1 ADP + Pi ATP


Prx III
2
SH SH
SH

H+

As
SH Mitochondrial matrix III

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 aected, including Factor B regulating the ATP synthetase activity, the adenine
nucleotide transporter, ANT, amongst others. See text for further detail.

The TRX system operates as a thiol-disulfide exchange re- Another thioredoxin-associated protein of importance in
action (see Figure 1). TRX1 and TRX2 are key regulatory thiol-mediated redox regulation in mitochondria is thiore-
isozymes that catalyse the reduction of protein disulfide doxin peroxidase II (TPX-II, also known as peroxiredoxin
bonds. They are cofactors of the apoptosis signal-regulating III, Prx-III), an enzyme abundantly expressed in the mito-
kinase 1 (ASK1) that mediates TNF cytokine and oxidative chondria of cancer cells that protects the cells from oxida-
stress-induced apoptosis via the mitochondrial dependent tive stress [79, 80]. PrxIII is an important antioxidant that
pathway [78]. acts in conjunction with TRX2/TXR (Figure 1) in the mito-
In their reduced forms, cytosolic TRX1 and mitochon- chondria to remove peroxides such as H2 O2 and oset the
drial TRX2 each contain two vicinal thiol groups in their apoptosis inducing eects of increased levels of H2 O2 . How-
active site sequence as CGPC. TRX 1 in the cytosol ever, PrxIII contains three Cys residues, two of which are in-
and TRX2 in mitochondria bind to Cys 250 and Cys 30, re- volved as redox-active sites in the formation of a stable in-
spectively, in the regulatory N-terminal domain of ASK1 and tersubunit disulfide-bonded dimer, which is then reduced
can cooperatively maintain the enzyme in the inhibited state. by thioredoxin to the monomer. PrxIII was a more abun-
However, activation by TNF resulting in increased produc- dantly expressed arsenic-binding protein when comparing
tion of ROS and leads to the oxidation of the TRX dithiol arsenic resistant cells to normal cells by phenylarsine oxide
group to a disulfide. Under these conditions, the thioredox- anity chromatography [81]. Hence, PrxIII is very likely to
ins no longer bind to ASK1 and loss of TRX2 binding to be another protein whose function is inhibited by arsenic-
mitochondrial-located ASK1 can lead to apoptosis in a JNK- containing compounds leading to the promotion of apopto-
independent manner, whereas cytosolic ASK1 upon loss of sis.
TRX1 binding then becomes activated as a MAPKKK result- Increasingly, it is becoming apparent that dithiols-
ing in JNK activation, Bid cleavage and Bax translocation to containing redox proteins, particularly those present in the
the mitochondria [78]. Since it is known that the thioredox- mitochondria, act as controlling sensors during responses to
ins are major targets of arsenic-containing compounds (see changes in cellular redox. Many thiol redox proteins con-
above and [75]), it can be predicted that arsenite-mediated tain a vicinal pair or more of reactive thiol groups [82, 83]
oxidative binding to thioredoxins will induce a similar out- capable of binding with arsenic-containing compounds in
come as TNF signalling, leading to the release and activation a similar manner to those in the thioredoxin system. The
of ASK1 and induction of apoptosis. most important of these are located in the mitochondria,
Stephen John Ralph 7

4. THE ADENINE NUCLEOTIDE TRANSPORTER (ANT):


As A CRITICAL TARGET OF ARSENIC-CONTAINING
As
III As COMPOUNDS IN THE MITOCHONDRIA
Trx2 -(SH)2 III H2 O2 2GSH
NADP+ III
TrxR2 Prx III GPx
Trx2 -S-S- 2H2 O GS-SG A channel formed by the association of two proteins, the
NADPH
2GSH
GR
-S-S protein ox
voltage dependent anion channel (VDAC) in the outer mito-
Grx2 chondrial membrane and the adenine nucleotide transporter
GS-SG (ANT) in the inner mitochondrial membrane (Figure 1), is
Protein red a complex involved in the induction of apoptosis activated
-SH
-SH

As via the mitochondrial pathway (see [89, Figure 3]). The two
III components of this complex form part of the mitochondrial
As
III permeability transition pore (MPTP), a megachannel medi-
ating release of molecules from the mitochondria activating
Figure 2: Mitochondrial redox systems regulating ROS levels via
apoptosis. A rapidly increased permeability of the inner mi-
thiol-disulfide exchange/coupling reactions. The mitochondrial form
of Thioredoxin (Trx2) is likely to play the major role in reducing
tochondrial membrane (mitochondrial permeability transi-
disulfides formed by vicinal thiols in both the mitochondrial Perox- tion) leads to apoptosis that is mediated by the MPTP.
iredoxin III (Prx III) and other proteins. Prx III is one of the main Arsenite induces apoptosis by a direct eect on the MPTP
ways by which cancer cells can reduce their levels of H2 O2 built up [90, 91] and VDAC has been shown to play an essential role
during active respiration. The glutathione redox system comprising in opening of the permeability pore and cytochrome c re-
GSH/GSSG, glutathione reductase, glutaredoxin, and glutathione lease induced by arsenic trioxide, which also caused VDAC
peroxidase, although present in the mitochondria, is more likely to to homodimerise [92]. In addition, the thiol-reactive com-
only become of major importance during the more extreme condi- pound 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS)
tions of oxidative stress. Both of these systems are targets for inhibi- has been shown to block the VDAC channel [93] and inhibit
tion by arsenic-containing compounds. See text for further detail. 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 re-
a point of extreme sensitivity to arsenic-containing com- action with arsenic.
pounds whose actions culminate in triggering the apoptotic Analysis of ion channel activity of purified ANT-
pathways via the induction of reactive oxygen species, leading containing lipid bilayers also revealed that As2O3 treatment
to the killing of the cancer cells (Figures 1, 2). For example, [30 M] altered the ANT channel electrophysiological prop-
two members of the glutaredoxin (GRX) family, including erties [36]. Interestingly, glutathione depletion leading to in-
GRX2 located primarily in the mitochondria, catalyse GSH- creased ROS may play an important role in the action of
dependent TRX-disulfide redox and protein thiol-disulfide arsenic trioxide [91]. Whereas in both normal and cancer
redox reactions, particularly reversible glutathionylation of cells, glutathione S transferase (GST) is found to interact
protein sulfhydryl groups [84]. Human Grx1 and Grx2 con- with ANT, during the induction phase of apoptosis, GST dis-
tain CPYC and CSYC active sites ; have three and sociates from ANT suggesting that GST/GSH may act as a
two additional structural Cys residues, respectively; and are repressor of MPTP and ANT pore opening [91, 95]. This is
therefore likely to react with arsenic-containing compounds, supported by the observation that increasing the expression
although no reports of this could be found. of the enzyme GSTP1 in Jurkat and Raji leukemic cells ren-
Another mitochondrial protein targeted by arsenic- ders them more resistant to arsenic trioxide-induced apop-
containing compounds with a vicinal dithiol group is reg- tosis at clinically relevant levels [1-2 M]. GSTP1 expression
ulatory protein Factor B. Addition of recombinant factor in these cells is also accompanied by accumulation of lower
B back to bovine submitochondrial particles depleted of this levels of H2 O2 production [95].
protein restored energy coupling activity. Thus, reverse elec- Both of the mammalian proteins, VDAC and ANT, con-
tron transfer from succinate to complex I enabling NAD+ tain two or more Cys residues in their structure thereby pro-
reduction, electron transport chain function and oxidative viding reactive thiol groups whose modification aects their
phosphorylation/32 Pi-ATP exchange of the ATP synthetase function. It has been established that the redox state of thiol
complex were reactivated [85] as was increased exchange ac- reactive groups are important for activation of the mitochon-
tivity of complex V [86]. Thus, the F0-F1 ATPase activity re- drial permeability transition [94, 96, 97]. Consequently, thiol
quires Factor B coupled to it for full activation. However, cross-linkers such as DIDS, diamide, and phenylarsine ox-
factor B contains six thiols and Cys 92 and Cys 94 in the ide [20100 M] aect VDAC and ANT channel function
bovine form were shown to bind phenylarsine oxide [87] and and activation of mitochondrial permeability transition [93,
phenylarsine oxide or arsenite inhibit factor B coupling ac- 94, 9698]. Many published results, such as cross-linking ex-
tivity [88]. From all of these studies, it is becoming clear that periments, protein/inhibitor stoichiometry, chimeric dimers,
redox changes to vicinal thiols aect the regulation of mito- analytical ultracentrifugation, or neutron scattering, indi-
chondrial function and that these thiols are also major targets cate that the ANT carrier acts as a dimer and this or higher
for inhibition by the arsenic-containing compounds (Figures oligomers are involved in membrane permeability transition
1, 2). [99, 100].
8 Metal-Based Drugs

Facing out into the mitochondrial matrix, ANT has three sults from ecient uptake of As-(GSH)3 via MRP2 in the
exposed loop regions containing a conserved repeat structure proximal tubules of the kidneys as part of the detoxification
with one Cys residue in each loop. These Cys residues are process during the excretion of arsenic-based drugs predom-
important to the process of ANT dimerisation, but it is not inantly into the urine [108, 109]. The remainder is mostly re-
clear how this operates and whether the Cys residues form moved via uptake in the liver and secretion as bile [110112].
intermolecular disulfide bonds or not [101]. Nevertheless, As-(GSH)3 may be an important part of the metabolic pro-
copper-o-phenanthroline is able to dimerise ANT by inter- cess for converting inorganic As(III) to methylated species
molecular cross-linking of Cys 56 (in the first matrix loop) during detoxification in the liver by ASMT1/Cyt19 [113].
[98]. In addition, phenylarsine oxide, eosin 5-maleimide,
and diamide form intramolecular cross-links between Cys 5. MODIFIED ORGANIC ARSENIC
160 and Cys 257 on the other two matrix loops, restricting STRUCTURES WITH INCREASED POTENCY
ANT in the open conformation, promoting mitochondrial AS ANTINEOPLASTIC AGENTS
permeability transition [98]. Arsenic trioxide is much weaker
than phenylarsine oxide at binding to the ANT Cys residues Arsenic-containing compounds substituted with organic
[96, 97] and this may explain the greater sensitivity exhib- groups such as modified phenylarsine oxides have been syn-
ited by APL cells to phenylarsine oxide [IC50: 0.1 M] than thesized and examined for their cytotoxic eect on human
to As2 O3 [IC50: 4 M] [67]. leukemic cells and breast cancer cells in culture. Some of
Single thiol interacting compounds such as N- these compounds were found to exhibit potent cytotoxic an-
ethylmaleimide (NEM) can inhibit the mitochondrial ticancer activity, particularly against human breast cancer
permeability transition and this could be either the result of and leukemic cell lines, including primary leukemia cells,
direct interaction with the key Cys residues on the matrix at micromolar concentrations. One of these compounds,
loops of ANT or indirectly via reaction with GSH and the novel glutathionyl peptide trivalent arsenic-containing
thereby preventing GSH from being oxidized and catalysing compound para 4-[N-(S-glutathionylacetyl)amino]pheny-
disulfide bridging between the adjacent thiol groups in larsenoxide (p-GSAO) shows promise as a novel antineoplas-
the ANT loops [98]. NEM or monobromobimane, in the tic drug and is now in clinical trials. P-GSAO, like pheny-
2550 M range, preferentially react with GSH, leading larsine oxide, inactivates ANT-mediated ATP/ADP trans-
to its modification in mitochondria and thereby prevents port and triggers Ca2+ -dependent MPTP opening by cross-
GSH from being oxidised. As a result, NEM inhibits mi- linking the critical Cys residues of ANT. This leads to in-
tochondrial permeability transition activation by the thiol creased production of cellular ROS, ATP depletion, mito-
reactive compounds, diamide or t-butylhydroperoxide, chondrial depolarization, and apoptosis of angiogenic en-
implying a role for GSSG in the action of these agents on the dothelial cells and inhibition of tumor growth in mice with
permeability transition [96, 97]. Arsenites, albeit that much no apparent toxicity [114]. However, the action of p-GSAO
higher concentrations would be required given their lower was indirect, and did not appear to be as a result of selective
anity for glutathione interaction, could have a similar tumor cell toxicity. Rather, p-GSAO inhibited the proliferat-
action. Thus, high levels of arsenites could modify and in- ing, but not growth-quiescent endothelial cells in vitro and
hibit glutathione redox control such that glutathione-based angiogenesis in vivo and thus acted to eliminate tumors by
enzymes are unable to function, as well as directly mediating blocking their blood supply [114, 115]. The trivalent arsenic-
disulfide cross-linking of ANT, leading to increases in cellu- containing moiety of p-GSAO was shown to cross-link the
lar ROS production, MPTP, and apoptosis. However, given matrix facing Cys160 and Cys257 thiols of ANT [114] and ef-
their low reactivity with glutathione systems, this appears fectively locks ANT into the open configuration. Inactivation
to be unlikely as opposed to the indirect action via the of ANT by p-GSAO causes an increase in superoxide levels,
mitochondrial eects leading to increased ROS production proliferation arrest, ATP depletion, mitochondrial depolar-
which then reduces cellular GSH levels. ization, and apoptosis in the dividing endothelial cells.
The multidrug resistance (MDR) protein MRP1/ABCC1 It is likely that the arsenic-containing moiety of p-GSAO
has been shown to transport AsIII out of cells as a tri-GSH reacts similarly as does arsenite (see above) with one or two
conjugate (As-(GSH)3), and glutathione S-transferase (GST) molecules of glutathione before it is removed from the cell
probably facilitates the process [102]. This is likely to be part by MRPs [116]. Tumor cells export p-GSAO much more e-
of the normal cell and cancer cell resistance mechanisms ciently than endothelial cells because they have higher MRP1
against the cytotoxic eects of arsenic-based compounds. or MRP2 activity and cellular glutathione levels [116] and
GSH-depleted cells are more sensitive to killing by arsenic- this may explain why p-GSAO is not highly eective at in-
containing compounds [103] and transfection of cells to ex- hibiting tumor cell growth in vivo. In addition, the greater
press glutathione S transferase protects them from arsenite water soluble properties of p-GSAO than other arsenic-
inducing death by promoting arsenite transport from the containing compounds, particularly organic species should
cells and decreasing ROS levels [104, 105]. In support of help to retain p-GSAO in the intravascular system where it is
this proposal, the long-term exposure of cells to arsenic- more likely to aect endothelial cells and inhibit tumor an-
containing compounds induced increased expression of glu- giogenesis.
tathione S transferase and MRPs [106]. Arsenic levels do not Interestingly, although the para form of GSAO revealed
attain very high levels in blood plasma of patients, rapidly no apparent toxicity in treated animals and inhibited tumor
becoming eliminated [8, 44, 107]. This removal probably re- growth leading to phase I clinical trials as an anticancer agent
Stephen John Ralph 9

[114], the ortho form (o-GSAO) was toxic and this was pro- ing compounds that favour intramolecular cross-linking be-
posed to result from increased accumulation of the drug in tween adjacent thiols. Intriguingly, most of the key intra-
cells, including normal cells due to loss of multidrug resis- cellular targets for this reaction have been identified to in-
tance eux [116]. Consequently, o-GSAO is unlikely to be of clude the main REDOX regulatory systems in the mitochon-
much further interest as an antineoplastic agent, whereas the dria, including thioredoxin and peroxiredoxin systems and
clinical ecacy of p-GSAO is eagerly awaited. the adenine nucleotide transporter, all of whose function is
adversely aected. The net result is the activation of sev-
6. TARGETING OF CANCER CELLS: SELECTIVE eral independent pathways including ROS production to fa-
UPTAKE AND DELIVERY INTO SPECIFIC TYPES cilitate the induction of apoptosis. One main pathway op-
OF CANCER CELLS erates via the opening of the MPTP, the other via activa-
tion of ASK1 kinase, and the JNK/Bid/Bax pathway of chan-
As(III) as the anhydrous form of As(OH)3 (Trisenox, Cell nel formation in MOM. In the case of APL, cell selectiv-
Therapeutics, Seattle, Wash, USA) received FDA approval ity for sensitive responses to these drugs is facilitated by
in 2000 as a chemotherapeutic agent for the treatment of selective transport systems such as provided by the AQP9
APL [117]. Acute promyelocytic leukemia (APL) is associ- transporter. Low MDR levels present in dividing endothelial
ated with reciprocal and balanced chromosomal transloca- cells also provides selective targeting by specially substituted
tions always involving the retinoic acid receptor alpha (RAR- phenylarsenic-containing compounds like p-GSAO, leading
alpha) gene on chromosome 17 and variable partner genes to decreased blood supply into tumors, with some toxicity to
on distinct chromosomes. RARalpha fuses to the promyeloc- cancer cells, but little toxicity on normal cells. Hence, a com-
tyic leukemia (PML) gene in the majority of APL cases (re- bination of selective delivery and retention provides the nec-
viewed in [118]). Arsenic trioxide is particularly eective at essary targeting of arsenic-containing compounds to tumors
killing APL cells and this was proposed to be the direct result and provides scope for additional modifications to be made
of its ability to induce the relocalization and degradation of to enhance the antineoplastic activity of arsenic-containing
the nuclear body protein PML, as well as the degradation of compounds, given their range of actions and eciency in
PML-RARalpha in APL cells [119122]. However, this seems killing cancer cells.
unlikely to be the main mechanism of action for arsenic tri-
oxide given that no dierences in sensitivity to growth inhi-
ACKNOWLEDGMENT
bition and killing by apoptosis have been observed between
wild-type and PML/ cells [123].
Arsenic trioxide as a single agent has provided 86% com- The author would like to thank Professor R. K. Ralph for
plete hematologic remission with minimal toxicity in APL helpful comments and editing of the manuscript.
patients [124], equal to any of the current standards of
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