Targeting Cancer Cells by an Oxidant-Based Therapy J. Verrax#, H ...

80
Current Molecular Pharmacology, 2008, 1, 80-92

Targeting Cancer Cells by an Oxidant-Based Therapy
J. Verrax#, H. Taper and P. Buc Calderon*
Unité de Pharmacocinétique, Métabolisme, Nutrition et Toxicologie; Département des Sciences Pharmaceutiques, Uni-
versité Catholique de Louvain, Belgium
Abstract: Despite the progress achieved in chemo- and radiotherapy, cancer is still a leading life-threatening pathology.
In that sense, there is a need for novel therapeutic strategies based on our current knowledge of cancer biology. Among
the phenotypical features of cancer cells, two of them are of particular interest: their nearly universal glycolytic phenotype
and their sensitivity towards an oxidative stress, both resulting from the combination of high anabolic needs and hypoxic
growth conditions. By using menadione (vitamin K3) and ascorbate (vitamin C), we took advantage of these features to
develop an original approach that consists in the exposure of cancer cells to an oxidant insult. When used in combination,
these compounds exhibit a synergistic action and are devoid of major toxicity in vivo. Thus, this review is dedicated to the
analysis of the molecular pathways by which this promising combination exerts its antitumoural effect.
Keywords: Ascorbate, menadione, cancer, oxidative stress, glycolysis, cell death.
MAIN FEATURES OF CANCER BIOLOGY
type and a poor antioxidant status. Since they are of major
interest for the rest of this work, they will be extensively
Normal cells perfectly fit their environment and respond
described.
to external signals via tightly regulated pathways that either
trigger or repress growth. Cancer arises when a cell, for a
Glycolytic Phenotype
variety of reasons, escapes the normal brakes placed on its
growth and begins to divide in an uncontrolled fashion. Ac-
The up-regulation of glycolysis is probably the oldest
tually, tumorigenesis appears as a multistep mechanism that
feature of cancer described, since the first observations were
reflects the genetic alterations driving progressively a normal
made almost 80 years ago by Warburg [7]. Nevertheless, this
tissue to malignancy. The best known genes whose muta-
nearly universal phenotype has never been fully investigated,
tions are frequently associated with the arising of cancers are
with the exception of these last years and the widespread
p53, c-myc, erb B or K ras [1]. Nevertheless, despite these
clinical use of 18fluorodeoxyglucose positron-emission to-
classical mutations, no typical cancer cell genotype exists
mography (PETSCAN). PET imaging has now clearly dem-
and each invasive cancer appears as the consequence of a
onstrated that glycolytic rates increase in neoplasms, a fact
particular genetic pathway travelled during carcinogenesis
that can be directly related to tumour aggressiveness and
[2,3]. It is therefore quite surprising to note that the genetic
prognosis [8].
diversity usually presented by cancer cells does not correlate
Different hypotheses are proposed to explain the persis-
with the clinical observations where a common invasive be-
tent metabolism of glucose to lactate even under aerobic
haviour including uncontrolled growth and destruction of
conditions, the so-called “Warburg’s effect”. Among them,
normal tissues are noted. Such a paradox can be explained
hypoxia is frequently suspected. Hypoxia arises from the
by the selective barriers existing within a tumour (hypoxia,
uncontrolled proliferation of cancer cells that leads to the
malnutrition, hormonal fluctuations, attacks of the immune
colonization of areas at increasing distance from blood ves-
system, etc) that lead to the selection of adapted cells [4].
sels. Due to the poor limit of oxygen diffusion (less than 100
Interestingly, this evolutionary process seems to be related to
M), a gradient of oxygen rapidly occurs within the growing
the manifestation of six main alterations in cell physiology,
tumour [9]. This fact, coupled to the increasing metabolic
as nicely described in 2000 by Hanahan and Weinberg: self-
demands of the growing mass of cells provokes a chronic
sufficiency in growth signals, insensitivity to antigrowth
hypoxia, even in tumours of only a few cubic millimetres. In
signals, tissue invasion and metastasis, limitless replicative
addition to chronic hypoxia, the uneven distribution of eryth-
potential, sustained angiogenesis and evading apoptosis [5].
rocytes within the tumour microvasculature can generate a
Supporting this hypothesis, it has been recently shown that a
fluctuant hypoxia. These temporal fluctuations in red cell
simple network of well-defined genetic events is sufficient to
flux occur in the normal microcirculation but are exacerbated
convert a healthy cell into a tumorigenic state [6].
in the tumour microenvironment, due to its particular fea-
The main physiological changes presented above repre-
tures (low pH, low pO2, high vascular permeability…) Cou-
sent novel capabilities and are acquired during tumour de-
pled to the tortuous tumour vasculature, this process results
velopment through multiple mutations. Besides these charac-
in a very unstable blood flow that provokes the appearance
teristics, two other secondary biochemical features are fre-
of areas of fluctuant hypoxia in tumours.
quently presented by cancer cells: a strong glycolytic pheno-
The adaptation of cancer cells to hypoxia notably arises

from the activation of transcription factors such as HIF-1

*Address correspondence to this author at the Unité PMNT 7369, 73, ave-
[10]. HIF-1 is a heterodimer that consists of a constitutively
nue E. Mounier, 1200 Bruxelles, Belgium; Tel: 32-2-764.73.66; Fax: 32-2-
expressed HIF-1 subunit and a tightly regulated HIF-1
764.73.59; E-mail: pedro.buccalderon@uclouvain.be
subunit. The expression of the latter is controlled by the lev-
#Julien Verrax is a FNRS postdoctoral researcher.
els of O2 since its degradation is controlled by O2-dependent


1874-4672/08 $55.00+.00
© 2008 Bentham Science Publishers Ltd.

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Targeting Cancer Cells by an Oxidant-Based Therapy
Current Molecular Pharmacology, 2008, Vol. 1, No. 1 81
mechanisms. HIF-1-regulated genes are particularly relevant
[27]. Nevertheless, the overall data from several publications
to cancer and can be divided into four groups: those that en-
support the alteration of the antioxidant status during tumour
code angiogenic factors, glycolytic enzymes, survival factors
progression, a finding that is not really surprising if we con-
and invasion factors. Therefore, the activation of HIF-1 pro-
sider the close relationship existing between the oxi-
vides an explanation for the high levels of some key glyco-
dants/antioxidants and cancer, at several steps. Indeed, at the
lytic proteins found in cancer, such as glucose transporters
beginning of the cancer process, oxidative conditions are
(GLUTs) [11].
often associated with carcinogenicity [28]. During its pro-
gression, some oxygen species like hydrogen peroxide can
The overexpression of the mitochondrial-bound isoforms
mediate signal transduction and promote cell proliferation
of hexokinase (HK-I and HK-II) is another hypothesis that
[29]. Finally, the antioxidant capacity of cancer cells is de-
could explain the glycolytic phenotype exhibited by cancer
scribed to be modified during tumour progression, and influ-
cells [12]. Such an upregulation occurs by epigenetic
ences their resistance to both chemotherapy and radiotherapy
changes (hypomethylation) that allow an open conformation
[30-32].
of the promoter, thus enhancing the binding of transcription
factors. Since the mitochondrial-bound isoforms of
Thus, copper-and zinc-containing superoxide dismutase
hexokinase have an easier access to ATP, they are less sus-
(CuZnSOD) levels as well as those of catalase and
ceptible to inhibition by their product (glucose-6-phosphate)
gluthatione peroxidase appear to be decreased in tumours,
and present a low Km for glucose. They act as a trap mecha-
compared to the tissues of origin [33-37]. Such a decrease
nism for glucose capture and greatly increase the rate of
was even related to cancer progression in oral squamous cell
aerobic glycolysis [13].
carcinoma [38]. The situation is more complex with manga-
nese superoxide dismutase (MnSOD) which appears to be
Finally, it has been proposed that the acquisition of the
frequently overexpressed in human tumours, although its
glycolytic phenotype is the consequence of mitochondrial
overexpression in vitro leads to cell death and cancer regres-
defects, leading to the impairment of oxidative phosphoryla-
sion [39]. In the same way, the recently discovered thiore-
tion. This is likely due to the high sensitivity of the mito-
doxin (Trx) system perfectly reflects this ambiguity since
chondrial genome to mutations. Indeed, the mitochondrial
many human tumours exhibit an overexpression of thiore-
genome lacks histones and relative protective systems, and it
doxins, possibly linked to a resistance to chemotherapy [40].
has no introns as well and is therefore fully required to main-
Nevertheless, thioredoxins have been shown to possess vari-
tain mitochondrial functions [14]. While the occurrence of a
ous cellular activities including growth-stimulating proper-
respiratory defect in cancer cells is still hardly debated [15],
ties and it is difficult to evaluate whether these features are
significantly reduced levels of -F1-ATPase have been ob-
linked or not with their antioxidant capacity. Despite the
served in several types of cancer, linked to an altered bio-
controversial data obtained with some particular enzymes, a
energetic phenotype of mitochondria [16].
poor antioxidant status can generally be considered as an-
Although the up-regulation of glycolysis is usually con-
other classical feature of cancer cells. Up to now, there is no
sidered as the consequence of cancer cell adaptation to ex-
real explanation of the loss of the antioxidant defences by
treme microenvironmental conditions, its role in invasive-
cancer cells. This could reflect a normal process in cancer
ness is now suspected [17,18]. Indeed, the constant release of
progression if it is assumed that this latter process is associ-
acid from the altered tumour metabolism leads to an ex-
ated with a constant adaptation of cancer cells to their envi-
tracellular acidification in the neighbouring cancer cells re-
ronment. Since the cancer environmental growth conditions
sulting in the death of normal cells [19,20]. This is explained
generally lack oxygen (half of all solid tumours have a me-
by the fact that cancer cells are more resistant towards acidi-
dian oxygen concentration less than 10 mm Hg instead of
fication than normal cells, due to mutations in p53 or other
40-60 mm Hg in normal tissues) [9], the expression of anti-
components of the apoptotic pathway [21]. Then, cancer
oxidant enzymes would have no utility and should therefore
cells can progress following the peritumoral acid gradient
be repressed.
and progressively replace the surrounding healthy tissue
where cells are dead. In addition, the acidification promotes
THE ASCORBATE/MENADIONE COMBINATION:
several mechanisms essential for tumour growth like angio-
AN ORIGINAL WAY TO INDUCE AN OXIDATIVE
genesis, through acid-induced release of VEGF and IL8 [22],
STRESS
extracellular matrix degradation by proteolytic enzymes like
cathepsin B [23], or the inhibition of the immune response
Before starting the discussions about the combination of
[24]. Since the acquisition of a glycolytic phenotype repre-
ascorbate and menadione, a brief overview of each com-
sents a key event for both survival and progression of cancer,
pound will be given in the following pages.
the inhibition of glycolysis appears a novel promising target
for cancer therapy [25,26].
Ascorbic Acid
Deficiency in Antioxidant Defences
Vitamin C was first isolated in 1928 by the Hungarian
biochemist Dr. Szent-Gyorgyi who received the Nobel Prize
The antioxidant status of tumours has been a matter of
for its discovery in 1937 [41]. In many species, ascorbic acid
debate for decades. This is mainly due to the difficulties en-
(Fig. (1)) can be generated de novo through the hexuronic
countered in the interpretation of these studies: intratumour
acid pathway of the liver or the kidney. This is due to the
heterogeneity, compartmentalization of the enzymes, ab-
activity of a particular enzyme, the gulonolactone oxidase.
sence of comparison with healthy tissues, artificial condi-
Since humans (as well as other primates, guinea pigs and a
tions of cell culture in vitro, few inhibitors available, etc
few bat species) lack this enzyme, they cannot synthesize

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82 Current Molecular Pharmacology, 2008, Vol. 1, No. 1
Verrax et al.
ascorbic acid and must satisfy a high requirement in foods,
and K2. Vitamin K is an essential nutrient for the production
notably in fruits and vegetables [42]. That is the reason why
of clotting factors II, VII, IX, and X in mammals by acting
ascorbic acid is considered as a vitamin. Ascorbic acid is a
as a cofactor in the enzymatic carboxylation by gamma-
potent water-soluble antioxidant in biological fluids, and also
glutamyl-carboxylase of glutamic acid residues forming
possesses a variety of other functions. It acts as a cofactor in
gamma-carboxyglutamic acid in plasma proteins [59]. Actu-
a number of hydroxylation reactions, by transferring elec-
ally, menadione acts rather as a provitamin since it has to be
trons to enzymes that provide reducing equivalents. Thus, it
converted into active homologues by the liver [60,61]. It is
is notably required for the conversion of certain proline and
generally considered now that menadione is ineffective for
lysine residues in procollagen to hydroxyproline and hy-
the treatment of excessive anticoagulation [62].
droxylysine in the course of collagen synthesis, a role that
O
explains its trivial name of “ascorbic acid” which designates
its function in preventing scurvy.
CH3
OH
O
HO
O
O

Fig. (2). Menadione
HO
OH
Fig. (1). Ascorbic acid
As for vitamin C, a sizeable amount of research has been
done on vitamin K and cancer (for review, see Lamson et al.,
Ascorbic acid is transported by sodium-dependent trans-
2003) [61]. Actually, most of these studies have focused on
porters SVCT1 and 2 [43]. SVCT1 is largely confined to the
menadione. Indeed, menadione clearly presents a greater
bulk transporting epithelial systems (intestine, kidney, liver)
toxicity than its congeners, both in vitro and in tumour-
and other epithelial tissues (lung, epididymus and lacrymal
bearing mice models [63,64]. In vitro, menadione is effective
gland), whereas SVCT2 is widely expressed. Despite the
against a wide variety of tumour cells at concentrations that
existence of these transporters for ascorbic acid, several tis-
are clinically achievable (IC50 values usually ranging from
sues (e.g. the erythrocytes) prefer the transport of the oxi-
10 to 50 M) [65,66]. Basically, two phenomena explain the
dized form of ascorbate, dehydroascorbate (DHA), by the
toxicity of menadione towards cancer cells: oxidative stress
glucose transporters (GLUTs) [44]. DHA is then rapidly
and covalent binding (also known as arylation process). Oxi-
reduced on the internal side of the plasma membrane, thus
dative stress is considered to be a mechanism of action of
preventing its efflux and allowing the accumulation of
quinoid compounds because toxic oxygen species can be
ascorbate against a concentration gradient. Since tumours
generated during redox cycling involving the quinoid struc-
frequently overexpress GLUT, this mechanism explains why
tures. In the case of menadione, the oxidative stress gener-
they present high amounts of vitamin C by comparison to
ated appears to be dose-dependent and capable to induce
their tissues of origin [11, 45-47].
both apoptosis and necrosis, depending on the doses used
With the exception of scurvy prevention, vitamin C pos-
[67]. A second mechanism of toxicity for menadione and
sesses few pharmacological actions. Nevertheless, an exten-
related quinones is the direct arylation of cellular thiols re-
sive literature exists on the use of this vitamin in a wide vari-
sulting in glutathione (GSH) depletion. Once GSH is de-
ety of diseases, notably for cancer, where it has a controver-
pleted, cellular macromolecules start to be alkylated, result-
sial history (for review, see Gonzalez et al., 2005) [48]. In
ing in their inactivation [68]. This is particularly true for
1954, W.J. McCormick, a Canadian physician, observed that
sulfhydryl-dependent proteins such as Cdc25 phosphatases.
the generalized stromal changes of scurvy were identical
These latter enzymes are the key regulators of the cell cycle
with the local stromal changes observed in the immediate
since they control the phosphorylation of the cyclin-
vicinity of invading neoplastic cells. Following his observa-
dependent kinases, thereby regulating their activity and cell
tions, he formulated the hypothesis that cancer is a collagen
cycle progression. Menadione, as well as other vitamin K
disease, secondary to a vitamin C deficiency [49]. Twenty
derivatives, was notably described to covalently bind to the
years after McCormick, Linus Pauling and Ewan Cameron
catalytic domain of Cdc25A phosphatase, leading to the
proposed the use of vitamin C supplementation in large
formation of an inactive hyperphosphorylated Cdk1 [69]. In
doses for the prevention and treatment of cancer [50] but the
addition, menadione was reported to also inhibit cyclin E
attempts to duplicate the amazing results obtained by Cam-
expression at the late G1 phase and cyclin A expression at
eron and Pauling showed that high-dose vitamin C therapy
the G1/S transition. Since Cdc25A phosphatase regulates
(10 g per day orally) was not effective against advanced ma-
both G1/S and G2/M cell-cycle transitions [70], its inhibition
lignant disease [51,52]. Although new pharmacokinetic data
by vitamin K derivatives explains how these compounds can
claim that i.v. administration of vitamin C could act as a new
inhibit cancer cell growth [71,72]. These mechanisms were
non-toxic adjuvant therapy by achieving higher plasma con-
particularly well studied in the case of hepatoma cell lines, a
centrations than those reached orally, the use of only vitamin
kind of tumour for which menadione and other vitamin K
C in cancer therapy remains controversial and is yet to be
derivatives seem to be particularly effective [73,74]. The
proven [53-58].
encouraging results of these in vitro investigations have led
to different human trials in patients suffering from gastroin-
Menadione
testinal and lung cancer. These phase I and II trials were
conducted in the 90’s and established the maximum tolerated
Menadione (also known as vitamin K3) (Fig. (2)) is a
dose of menadione at 2.5 g/m2 as a continuous intravenous
synthetic derivative of the naturally occurring vitamins K1

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Targeting Cancer Cells by an Oxidant-Based Therapy
Current Molecular Pharmacology, 2008, Vol. 1, No. 1 83
infusion [75-78]. It must be noted that the administration of
ergism was investigated between ascorbate/menadione and
high doses of menadione (4 and 8 g/m2) was associated with
radiotherapy [88]. Mice with intramuscularly transplanted
hemolysis, despite the presence of red blood cell glucose-6-
solid liver tumours were orally and parenterally pretreated
phosphate dehydrogenase. Interestingly, no resulting coagu-
with combined ascorbate and menadione before being locally
lopathy was recorded, a fact already observed in clinical tri-
irradiated with single doses of X-rays (ranging from 20 to 40
als conducted with vitamin K1 [79].
Gy). For all the X-ray doses tested, a synergism with the
combination was described. Finally, an inhibition of the de-
The Ascorbate/Menadione Combination
velopment of metastases was reported in 2004 [89]. In this
article, Taper investigated the effect of a dietary ascor-
The first description of a synergistic antitumoural effect
bate/menadione combination on the metastasis of mouse
developed by the combination of ascorbate and menadione
liver tumour (TLT) cells implanted in C3H mice. Reductions
was made nearly forty years ago. In 1969, a team from the
of 50 % and 63 % for lung and lymph node metastases, re-
National Cancer Institute observed that menadione at a very
spectively, were recorded, demonstrating that the oral ascor-
low dose (1 ppm) was able to greatly potentiate the cytotox-
bate/menadione combination significantly inhibited the me-
icity of ascorbate towards Ehrlich ascites carcinoma cells in
tastases of TLT tumours in C3H mice.
vitro [80]. The authors already postulated that the cytolytic
effect was due to the intracellular production of hydrogen
Three major conclusions arise from the several studies
peroxide. These observations are remarkable since they are
conducted by Taper during these last twenty years. First, the
still the basis of many recent studies dealing with the toxicity
synergism presented by the ascorbate/menadione combina-
of ascorbate [53, 58]. At the same time, the reports of Daoust
tion seems to be related to the production of hydrogen perox-
and collaborators described the deficiency of nuclease activ-
ide. Second, the potentiation of both radio-and chemotherapy
ity in both animal and human tumours [81]. Since this de-
suggests a non-specific process. This is further confirmed by
crease was associated with cancer progression, several at-
the fact that various chemotherapeutic agents (acting through
tempts were made in order to reactivate these enzymes. In-
different pathways) can be potentiated in a similar way. The
deed it was thought that such a reactivation could lead to
third conclusion (and maybe the most important) is the fact
cancer cell necrosis as well as cancer regression. A few years
that the ascorbate/menadione combination appeared to be
later, Taper discovered that vitamins C (ascorbate) and K3
selective for cancer cells. Indeed, histopathological examina-
(menadione) were able to reactivate alkaline (DNase I) and
tions did not indicate any sign of toxicity in normal organs
acid DNase (DNase II) respectively [82].
and tissues of ascorbate/menadione-treated mice. It should
In 1987, Taper et al. reported a non-toxic potentiation of
be noted that cumulative evidence suggests that this “appar-
cancer chemotherapy in TLT (Transplantable Liver Tumour)
ent selectivity” is more a “differential sensitivity” between
bearing mice by combined sodium ascorbate and menadione
healthy and cancer cells, as described by Zhang et al. [90].
pre-treatment [83]. This potentiation appeared to be non-
Actually, both vitamins are able to induce either apoptosis or
specific for a particular chemotherapy. Indeed alkylating
necrosis depending on the dose, the incubation time, and the
agents (cyclophosphamide, procarbazine) as well as an anti-
cell type utilized [91]. Nevertheless, at the doses at which
metabolite (5-fluorouracil), an enzyme (asparaginase), a mi-
they were used in our in vitro and in vivo studies, neither
totic inhibitor (vinblastine) or an anthracycline derivative
menadione nor ascorbate alone exhibit cytotoxicity. Only the
(adriamycin), all appeared to be potentiated by the ascor-
ascorbate/menadione combination was toxic for cancer cells.
bate/menadione association. It should be noted that a similar
In our studies we have sought to examine the mecha-
potentiation of gemcitabine (an antimetabolite) was recently
nisms that could explain the interesting results obtained by
reported, both in in vitro and in vivo experiments in which
Taper et al. As further described, the synergistic anticancer
human urothelial tumours were used [84]. The first results
effect of the ascorbate/menadione combination is likely ex-
obtained by Taper highlighted an anticancer effect produced
plained by the redox-cycling that occurs between these com-
by the combination itself, a fact that was later confirmed by
pounds. This generates reactive oxygen species and leads to
the demonstration that the ascorbate/menadione combination
an oxidative stress particularly deleterious for cancer cells
completely suppressed the tumour cell growth of three dif-
since they exhibit a poor antioxidant status. Among the dif-
ferent cell lines (namely MCF-7, a breast carcinoma; KB, an
ferent cellular pathways that can be impaired, glycolysis is
oral epidermoid carcinoma and AN3-CA, an endometrial
well-known to be rapidly inhibited by an oxidative stress
adenocarcinoma) [85]. In this in vitro study, Taper and col-
[92]. Since cancer cells have a high energetic dependence on
leagues reported a synergistic inhibition of cell growth at 10
glycolysis, we therefore postulate that this fact, coupled to
to 50 times lower vitamin concentrations than that required
the poor antioxidant status, is the rationale that explains the
when either agent was used alone. A few years later, using
preferential killing of cancer cells by the ascor-
the same cell lines, a synergism was also described between
bate/menadione combination, this latter appearing as an in-
ascorbate/ menadione and various chemotherapeutic drugs
teresting strategy that takes advantage of the biochemical
[86], a fact already described in vivo [83]. Although the pre-
features of cancer cells.
cise mechanism explaining this synergy remained obscure, a
hydrogen peroxide-mediated oxidative stress was suggested
It should be noted that the salt form of vitamin C, known
since the addition of catalase to the culture medium sup-
as sodium ascorbate, was always used in our studies (in vitro
pressed the antitumour effect [85]. The sensitisation of can-
as well as in vivo), in order to avoid any pH interference.
cer chemotherapy by ascorbate/menadione pre-treatment in a
Menadione was used in the form of sodium bisulphite salt
mouse tumour resistant to Oncovin (vincristin) was also de-
because of the poor solubility of menadione.
scribed in 1992 [87]. As for chemotherapy, a potential syn-

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84 Current Molecular Pharmacology, 2008, Vol. 1, No. 1
Verrax et al.
THE RATIONALE THAT SUPPORTS THE ASCOR-
After having defined redox cycling as the mechanism
BATE/MENADIONE ANTITUMOURAL EFFECT
supporting the cytolytic activity of the ascorbate/menadione
combination, the involved oxidizing agent remains to be
A Redox-Cycling to Explain the Synergistic Cytotoxicity
determined. Among the various ROS that can be generated,
the role of hydrogen peroxide was early evoked due to the
Menadione, like the other quinoid compounds, may be
fact that the addition of catalase completely suppressed cell
cytotoxic through either electrophilic arylation or redox-
death [85]. Supporting this hypothesis, our results show a
cycling. Menadione redox-cycling is greatly increased by the
key role of hydrogen peroxide. Firstly, among different anti-
addition of ascorbate; a fact that explains the synergistic an-
oxidants tested on several cell lines, only catalase and NAC
titumour activity of the ascorbate/menadione combination.
were able to suppress cytolysis [94,95]. Secondly, the ab-
Actually, menadione is non-enzymatically reduced by ascor-
sence of lipid peroxidation is consistent with a mechanism of
bate to form semidehydroascorbate and a semiquinone free
toxicity based primarily on the generation of hydrogen per-
radical (reaction 1). Such a semiquinone is rapidly reoxi-
oxide since this latter agent is ineffective in oxidizing lipids.
dized to its quinone form by molecular oxygen thus generat-
Thirdly, since hydrogen peroxide is detoxified by the en-
ing ROS (reaction 2). It is noted that the one-electron reduc-
zyme catalase, we observed that co-incubation in the pres-
tion of menadione driven by ascorbate is not favoured ther-
ence of aminotriazole (a potent catalase inhibitor) leads to
modynamically. Indeed, it occurs because the semiquinone
the potentiation of the ascorbate/menadione-driven cytotox-
product is continuously removed by autooxidation [93].
icity, as evidenced by the increased release of lactate dehy-
AscH- + Q
SQ.- + Asc.- + H+


(1)
drogenase into the incubation medium (Fig. (3)) [96, 99].
Taken together these data definitely confirm the key role
SQ.- + O2
Q + O2.-



(2)
played by hydrogen peroxide. Since cancer cells exhibit a
Various compounds without arylation sites (such as 2,3-
poor antioxidant status, this fact explains their preferential
dimethoxy-naphtoquinone and dichlone) were able to pro-
killing by the ascorbate/menadione combination.

duce the same profile of cytotoxicity as observed for the

ascorbate/menadione combination, underlining the key role
of redox cycling in the cytolytic process [94-95]. It should be

noted that we cannot refute the possible occurrence of some

arylations within ascorbate/quinone-treated cells. However,
the occurrence of such a pathway, if any, cannot be a worth-

while explanation for the cytotoxicity developed by these

associations. Supporting the importance of the oxidative

stress, we were able to detect the intracellular presence of
ROS in K562 ascorbate/menadione-treated cells [96]. This

was further confirmed by the inhibitory effect exhibited by

the antioxidant N-acetylcysteine (NAC) that abolishes both
the formation of ROS and cell death [95].

Since the higher redox potential of quinone molecules

has been correlated with enhanced cellular deleterious ef-

fects, we studied the ability of the association of ascorbate

with several quinone derivatives to cause cell death. Besides

its interest by providing us with new mechanistic insights,

this study demonstrated that menadione can be easily re-
Fig. (3). K562 cells were incubated for 18 h at 37 °C in the absence
placed. Indeed, several quinoid compounds are able to pro-
(control) or in the presence of a mixture of sodium ascorbate and
duce a synergistic cytotoxic effect in the presence of ascor-
menadione (Asc/Men, 2 mM and 10 mM, respectively). When pre-
bate, depending on their half-redox potentials. We observed
sent, aminotriazole (ATA) was used at 5 mM and preincubated for
that only quinones having half-redox potentials between –
1 h. The viability of cells was estimated by measuring the activity
250 and +50 mV were able to be reduced by ascorbate, thus
of lactate dehydrogenase (LDH), according to the procedure of
generating a redox cycling and the formation of ROS [95].
Wrobleski and Ladue [99] both in the culture medium and in the
Such associations resulted in decreased levels of both ATP
cell pellet obtained after centrifugation. The results are expressed as
and GSH, in vitro cell death and decreased tumour cell pro-
100 minus the ratio of released activity to the total activity, a per-
liferation in TLT-bearing mice. As for menadione, all tested
centage that reflects the loss of cell viability. In ascor-
quinones were devoid of toxic effects by themselves, at least
bate/menadione-treated cells, the addition of ATA reinforces sig-
at the concentrations we used. Interestingly, we noted that
nificantly the loss of cell survival, underlining the key role played
the type of cell death driven by these combinations between
by H
ascorbate and quinone was sharing the same pattern as de-
2O2 in the cytolytic effect developed by ascorbate/menadione.
The results represent the mean ± S.E.M of at least three independ-
scribed for ascorbate/menadione (necrotic-like, caspase-
ent experiments. The effects of ascorbate/menadione and aminotri-
independent). This is in agreement with many previous stud-
azole + ascorbate/menadione were significantly different (p <
ies reporting a correlation between quinone potential, reac-
0.001, using one-way ANOVA) as well as compared to the control
tivity with ascorbate and antitumoural action [97,98].
group (p < 0.001, using one-way ANOVA).

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Targeting Cancer Cells by an Oxidant-Based Therapy
Current Molecular Pharmacology, 2008, Vol. 1, No. 1 85
What are the Intracellular Targets of the Ascorbate/
GAPDH. The NAD+ depletion is likely related to a strong
Menadione Combination?
poly(ADP-ribose) polymerase (PARP) activation, as evi-
denced by the rapid appearance of poly(ADPribosylated)
The involvement of H2O2 in the cytolytic process driven
proteins (PAR) following the exposure to ascor-
by the ascorbate/menadione combination is no longer a mat-
bate/menadione combination (Fig. (4)). PARP activation is
ter of discussion. Since the exposure to an oxidative stress
likely provoked by the DNA damage occurring within ascor-
leads to the activation of MAP kinases, their role in the
bate/menadione-treated cells, as indicated by the phosphory-
ascorbate/menadione-induced cell death has been investi-
lation of the histone H2AX (the phosphorylated form being
gated. Since no activation was observed, the MAP kinase
known as -H2AX), a marker of DNA strand breaks (Fig.
pathway probably does not play a major role in the mecha-
(4)) [107]. Due to the glycolysis arrest, the ATP depletion
nisms underlying the cytotoxicity of ascorbate/menadione
will follow, culminating in cell death.
[100].

Another classical feature that generally appears after an
exposure to H

2O2 is the activation of the transcription factor
NF- B [101]. However, our results showed that the combi-

nation of menadione and ascorbate is inhibiting rather than
activating NF- B, that is the opposite of the expected effect

[102]. This observation is likely explained by the fact that

most studies performed on H2O2 use a bolus instead of a con-

tinuous production, as in the case of the ascor-
bate/menadione combination. Since these two ways of pro-

duction are known to have completely different effects, this

result is finally not surprising [103]. Whatever the cause of
this inhibition, it could be an interesting mechanism since

cancer cells often exhibit a constitutive activation of NF- B

that contributes to their aggressiveness [104].

Interestingly, we observed that sodium orthovanadate, a
well-known inhibitor of protein tyrosine phosphatases, com-

pletely suppresses the cytotoxicity induced by the combina-

tion of menadione and ascorbate, while other inhibitors of
protein kinases and phosphatases failed [105]. The mecha-

nism underlying the suppressive effect of vanadate remains

unclear but three different explanations have been postu-
lated: the first one involves a potential interfering reaction by

vanadate in redox-cycling. However, this is very unlikely
Fig. (4). Effect of ascorbate/menadione on protein
because vanadate did not modify the oxygen uptake ob-
poly(ADP)ribosylation in K562 cells. K562 cells were incubated in
served during the ascorbate/menadione reaction (unpublished
the absence (control) or in the presence of 2 mM sodium ascorbate,
data). The second is related to the formation of peroxova-
10 mM menadione or a mixture of both compounds. Cells were
nadate, a possible reaction of vanadate with H2O2. Such a
harvested after 90 minutes and immunoblotting was performed
reaction might consume most (or all) of H2O2, thus avoiding
using antibodies against poly(ADP-ribose) (PAR), ß-actin and -
its interaction with the intracellular putative targets. Again,
H2AX.
this mechanism is very unlikely since a suppressive effect on
Although the activation of PARP is obvious, it does not
cytotoxicity was also recorded by the addition of potassium
entirely explain the cytotoxicity triggered by the ascor-
bisperoxo(1,10-phenanthroline)oxovanadate, an oxidized
bate/menadione combination since PARP inhibitors only
form of vanadate unable to react with H2O2 (unpublished
decrease cell death by 60% [106]. This indicates that other
data). Finally, the third possibility is that vanadate, by inhib-
critical events are likely occurring upstream in the cascade
iting tyrosine phosphatases, modifies the phosphorylation
leading to cell death, as evidenced by the suppressive effect
state of some critical proteins. The results obtained with an-
of vanadate. These results prompted us to suggest that PARP
other tyrosine phosphatase inhibitor, namely Et-3,4-
activation is a major but probably not the only intracellular
dephostatin, confirms that the protective effect by vanadate
event related to cell death. The way by which PARP is acti-
may be attributed to an inhibition of tyrosine phosphatases
vated is also a matter of discussion. PARP activation occurs
[105]. Nevertheless, the specific proteins that are involved in
in response to DNA damage, but the way by which this latter
this process have not been yet identified.
event occurs remains to be discovered. One might expect
Since lactate and ATP are severely and rapidly depressed
that DNA damage induced by the ascorbate/menadione com-
following the exposure of cancer cells to ascor-
bination is due to transition metal ions (iron and/or copper)
bate/menadione, we suggest that the major intracellular event
which catalyze the formation of hydroxyl radicals in the vi-
is related to the impairment of glycolysis. The arrest of gly-
cinity of DNA. Since the addition of mannitol (an hydroxyl
colysis occurs at the step catalyzed by glyceraldehyde-3-
radical scavenger) failed to protect against the ascor-
phosphate dehydrogenase (GAPDH) and appears as the re-
bate/menadione cytotoxicity, this latter pathway is of interest
sult of NAD+ depletion [106]. Indeed, we observed that
but remains to be demonstrated [95].
NAD+ is rapidly consumed, leading to the inhibition of

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86 Current Molecular Pharmacology, 2008, Vol. 1, No. 1
Verrax et al.
A Particular Necrotic Cell Death
pathway. Actually, the absence of apoptosis in ATP-depleted
cells (such as in the case of ascorbate/menadione-treated
Previous reports of ascorbate/menadione driven cancer
cells) can be explained by the ATP-dependence of some
cell death postulated the occurrence of a new kind of cell
apoptotic steps such as the apoptosome formation [114-116].
death, namely the autoschizis [108]. Indeed, most of ascor-
bate/menadione-treated cancer cells are characterized by a
The exposure of cancer cells to the ascorbate/menadione
particular morphology, as nicely reviewed by Jamison et al.
combination was reported to induce DNA strand breaks [96].
[109]. These cells exhibit exaggerated membrane damage
This damage did not correspond to an oligosomal DNA
that drives progressive loss of organelle-free pieces of cyto-
fragmentation since the electrophoretic profile exhibited a
plasm through a series of self-excisions. Based on this par-
smear pattern [105, 117-122]. Taken together, the absence of
ticular morphology, such a cell death was called autoschizis
caspase activity and the DNA fragmentation profile suggest
by Gilloteaux et al. [108]. During the process, the nucleus
a necrotic outcome for cancer cells treated by the ascor-
becomes smaller and most of organelles relocate around it.
bate/menadione association. This profile was further con-
At the extreme, cells can be reduced to a perikaryon that
firmed by flow cytometry (double staining with Annexin-V
consists of an apparently intact nucleus surrounded by a thin
and propidium iodide) [96, 108, 121]. Other experiments of
rim of cytoplasm containing damaged organelles. At this
flow cytometry performed on human bladder tumour cells
step, the final cell volume is decreased by at least one-third
exposed to ascorbate/menadione also revealed a growth ar-
to one-half the original cell size and karyolysis occurs. Con-
rested population and a population undergoing cell death.
trary to apoptosis, such a process is accompanied by the re-
Cells in G1 arrested in G1 while those in S phase progressed
lease of intracellular constituents, thus provoking in vivo
through S phase and arrested in G2/M [121].
inflammatory response [109]. Although this cell death was
In conclusion, based on the biochemical features of auto-
essentially described from a morphological point of view,
schizis as well as on its particular morphology, we suggest
several biochemical markers were also used in order to char-
that this cell demise induced by ascorbate/menadione must
acterize the cell death pathways induced by the ascor-
be considered as a subtype of necrotic cell death, according
bate/menadione combination [110-112].
to the recommendation of the Nomenclature Committee on
The absence of caspase activity was reported at any dos-
Cell Death (NCDD) [123]. The analysis of the cytoskeletal
age and in different cell lines. This was further confirmed by
modifications that occur following the exposure to ascor-
both the use of caspase inhibitors (Fig. (5)) and the analysis
bate/menadione (cleavage of cytoskeletal proteins, actin po-
of typical caspase substrates [95,96,105]. Such a lack of ac-
lymerisation, etc), as well as the study of other parameters
tivity is likely the reflection of the absence of activation
(intracellular Ca2+ level, involvement of lysosomal proteases)
since no pro-caspase processing was observed [96, 105].
could be helpful in the future to better define this concept of
Interestingly, a weak release of cytochrome c was generally
autoschizis.
observed upon ascorbate/menadione exposure, coupled to a
loss of mitochondrial membrane potential [96, 113]. Since
Additional Mechanisms to Explain the Antitumoural
cytochrome c was released from mitochondria without
Effect
caspase activation, we postulated that apoptotic signals, if
In vitro, the oxidative stress is the main mechanism by
any, are being blocked upstream in the caspase activation
which ascorbate/menadione exerts its cytolytic action. Nev-
ertheless, we cannot exclude the involvement of other path-
ways in the in vivo activity of this combination. In that sense,

several studies were performed, including studies on a puta-
tive effect on the immune response, the destabilization of

oncogenic proteins and the influence of the association on
the angiogenic and metastatic processes. The rationale for

studies on the immune response arises from the fact that an
association of ascorbate with a benzoquinone has been re-

ported to enhance the immune function [124]. Thus, we ex-
plored a putative stimulation of the immune system by the

ascorbate/menadione combination on the cytolytic T lym-
phocyte (CTL) response of mice immunized with tumour-

specific peptides model, a model described by Silla et al. in
1999 [125]. However, no influence of this combination was

noted, suggesting that stimulation of the immune system is
unlikely [100].
Fig. (5). K562 cells were incubated for 24 hours either in the pres-
ence or the absence of the ascorbate/menadione combination (used
Another pathway that could explain the antitumoural ef-
at 2 mM and 10 M, respectively). Z-VAD-FMK was used at 10
fect exhibited by ascorbate/menadione involves the degrada-
M without preincubation. Cellular viability was estimated by
tion of key oncogenic proteins. Indeed, the exposure of K562
measuring the activity of lactate dehydrogenase (LDH), both in the
cells (a human leukaemia cell line) to this association leads
culture medium and in the cell pellet obtained after centrifugation.
to the degradation of Bcr-Abl, a critical protein for K562 cell
The results are expressed as a ratio of released activity to the total
survival (unpublished data). Actually, we postulate that the
activity. The results represent the mean ± S.E.M of at least three
ATP depletion caused by oxidative stress provokes the disas-
independent experiments.
sembling of stabilizing complexes between some heat shock

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Targeting Cancer Cells by an Oxidant-Based Therapy
Current Molecular Pharmacology, 2008, Vol. 1, No. 1 87
proteins (such as Hsp 90) and Bcr-Abl, resulting in the pro-
ticipate in the antitumoural effect exhibited in vivo by this
teolysis of the latter. Since Bcr-Abl is required for cell sur-
association, as well as in the inhibition of metastasis de-
vival, its degradation could be an interesting antitumoural
scribed by Taper et al. [89].
mechanism.
As our knowledge of the ascorbate/menadione combina-
The last mechanism investigated was a putative anti-
tion growths, it is likely that multiple mechanisms explain its
angiogenic effect. Indeed, our results showed that culture
antitumoural activity in vivo (Fig. (6)). In that sense, many of
media in which tumour cells were incubated in the presence
these pathways are still to be discovered and the involvement
of the ascorbate/menadione combination develop a strong
of those described in vitro remains to be confirmed in vivo.
inhibitory effect in one model reflecting the angiogenic ca-
pacity (tube formation, unpublished data). This inhibition is
THE FIRST STEPS TOWARDS A CLINICAL APPLI-
not linked to a cytotoxic effect on endothelial cells by the
CATION?
combination itself but rather appears to be mediated by the
leakage of an anti-angiogenic compound. The identity of this
The ultimate question for every compound expected to
latter agent remains to be determined, notably by the use of
possess an antitumoural activity is that of its clinical rele-
screening methods such as protein arrays. A putative anti-
vance, taking into account its efficacy as well as its safety.
angiogenic effect is of major importance since it could par-
This is usually done through a multistep process that follows



















Fig. (6). Schematic illustration of the supposed mechanism of action of the ascorbate/menadione combination. The redox-cyling that occurs
between ascorbate and menadione leads to the production of intracellular hydrogen peroxide that triggers a variety of damage. PARP be-
comes activated in response to DNA damage, leading to NAD+ depletion and subsequent glycolysis arrest. Since cancer cells rely on glyco-
lysis for their ATP production, this provokes an energetic crisis that kills the cell. Dying cells exhibit a necrotic profile reported as auto-
schizis and characterized by huge membrane deformations. Since cancer cells have a poor antioxidant status and frequently over-express
glucose transporters, they are particularly sensitive to the action of ascorbate/menadione, thus explaining the efficacy of the combination
against a broad range of tumours.

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88 Current Molecular Pharmacology, 2008, Vol. 1, No. 1
Verrax et al.
the guidelines published by international agencies such as
cokinetic data suggest that is not appropriate for the admini-
the European Agency for the Evaluation of Medicinal Prod-
stration of high doses of ascorbate in humans [54]. Indeed,
ucts (EMEA) or the Food and Drug Administration (FDA).
complete plasma saturation occurs at the daily oral dose of
The problem with compounds such as ascorbate and me-
1000 mg of ascorbate [55,56]. This dose was associated with
nadione is that they are primarily perceived as vitamins, a
a plasma concentration of approximately 100 M, whereas
term associated with the idea of low-toxic products that
our in vitro conditions use 2 mM ascorbate that is twentyfold
therefore do not require any pre-clinical evaluation before
higher. Therefore, the only possibility to reach such “phar-
use in clinics. Nevertheless, as illustrated by Pauling’s stud-
macologic” concentrations is the i.v. administration [53,57,
ies, this lack of early-phase research leads to a bad evalua-
58]. As highlighted in this section, considerable work has
tion of some critical parameters (doses, route of administra-
already been carried out on the ascorbate/menadione combi-
tion, optimal schedule) resulting in mixed results and con-
nation but an optimised protocol remains to be discovered
troversy [48]. Since ascorbate and menadione were already
(i.e. route of administration, doses, schedule) and the puta-
registered as drugs, many data dealing with their pharma-
tive clinical applications are still to be explored as well.
codynamic, pharmacokinetic and toxicological properties are
already available. In addition to these data, a lot of knowl-

edge has been acquired about the combination itself.

The usual objectives of pre-clinical studies are the inves-

tigation of the mechanism(s) of action, resistance, schedule
dependencies as well as the anti-tumour activity of the prod-

ucts in vivo. Concerning the mechanism(s) of action, we

have clearly identified the redox-cycling of the ascor-

bate/menadione combination and the subsequent oxidative
stress generated as the explanation for the synergistic anti-

tumoural effect exhibited by this combination. This oxidative

stress, mainly observed in vitro, is responsible for the strong
cytolytic effect of ascorbate/menadione, as observed in sev-

eral cancer cell lines (for review, see Buc Calderon et al.,

2002) [94]. According to this mechanism of action, we can
anticipate that cancer cell resistance could arise from the

overexpression of antioxidant enzymes. In that sense, tu-

mours such as mesothelioma that contain simultaneous over-
expression of several antioxidant enzymes and related pro-
Fig. (7). Effect of ascorbate/menadione on the survival of mice
teins with antioxidant capacity could be resistant towards
bearing the ascitic TLT tumour. Approximately 106 of Transplant-
ascorbate/menadione [27].
able Liver Tumor (TLT) cells were intraperitoneally (i.p.) injected
into male NMRI mice. Forty-eight hours after implantation, ascor-
The results obtained in tumour-bearing mice support the
bate/menadione-treated group was injected i.p. with a mixture of
effectiveness of the ascorbate/menadione combination in
sodium ascorbate and menadione sodium bisulfite (1 g/kg and 10
vitro (Fig. (7)). Nevertheless, these studies were conducted
mg/kg, respectively). The mean survival time was of 15 days for the
in a few models (murine hepatoma TLT cells, human
control group whereas it reached 29 days for the group treated by
erythroleukaemia K562 cells and UMUC-14 urothelial car-
ascorbate/menadione (p<0.001, using Student’s t-test).
cinoma cells) and remain to be carried out at a larger scale
[83,84,87-89,96]. Indeed, these studies have highlighted the
CONCLUSION
in vivo antitumoural activity of the ascorbate/menadione
combination but did not provide us with a typical schedule of
The combination of ascorbate and menadione could be of
treatment. Most of the work performed in rodents was using
particular interest in cancer therapy for several reasons. First,
i.p. injections of 1g/kg and 10mg/kg of ascorbate and me-
since many tumour cells exhibit defects in the apoptotic sig-
nadione respectively. Similar doses have already been used
nalling pathways, there is a need for additional death mecha-
in humans with each compound separately, and these ap-
nisms. Second, since cancer cells are often deficient in anti-
peared to be relatively safe [57, 75]. Nevertheless, since the
oxidant defenses and accumulate ascorbate through the over-
ascorbate/menadione combination exerts a synergistic effect,
expression of glucose transporters, they are expected to be
this prompts us to recommend a preliminary phase I study in
more sensitive towards an oxidative stress induced by the
order to determine the maximal tolerated doses for the asso-
ascorbate/menadione combination than healthy cells. Third,
ciation itself. This could be achieved by using the classical
due to the high energetic dependence of cancer cells on gly-
ratio of 100 to 1 (for ascorbate and menadione, respectively),
colysis, the impairment of such an essential pathway may
a ratio that was previously demonstrated as the most effec-
explain the effectiveness of this combination on many tu-
tive one [85]. Hence a safe dose will be defined, thus avoid-
mour cells. Taken together, our data suggest that the associa-
ing a misevaluation of the antitumoural effect of the combi-
tion of ascorbate/menadione could represent a new interest-
nation.
ing auxiliary cancer therapy. Since both compounds are low-
The route of administration is another critical parameter
toxic and cheap, they could be, indeed after a correct pre-
that remained to be assessed. Although oral administration
clinical evaluation, easily included in the classical anticancer
was effective in vivo and appeared to be the easiest, pharma-
protocols without any supplementary risk for the patients.

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Targeting Cancer Cells by an Oxidant-Based Therapy
Current Molecular Pharmacology, 2008, Vol. 1, No. 1 89
ACKNOWLEDGEMENTS
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= Cytolytic T lymphocyte
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DHA =
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GSH =
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PET =
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