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Tumor-targeted induction of oxystress for cancer therapy
J. FANG1,†, H. NAKAMURA1 & A. K. IYER1,2
1Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Ikeda 4-22-1, Kumamoto
860-0082, Japan, and 2Department of Nanoscience, Faculty of Engineering, Sojo University, Ikeda 4-22-1, Kumamoto
860-0082, Japan
(Received 21 February 2007; accepted 7 June 2007)
AbstractReactive oxygen species (ROS), such as superoxide anion radicals (Oz2
2 ) and hydrogen peroxide (H2O2) are potentiallyharmful by-products of normal cellular metabolism that directly affect cellular functions. ROS is generated by all aerobicorganisms and it seems to be indispensable for signal transduction pathways that regulate cell growth and reduction–oxidation(redox) status. However, overproduction of these highly reactive oxygen metabolites can initiate lethal chain reactions, whichinvolve oxidation and damage to structures that are crucial for cellular integrity and survival. In fact, many antitumor agents,such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin and many othersexhibit antitumor activity via ROS-dependent activation of apoptotic cell death, suggesting potential use of ROS as anantitumor principle. Thus, a unique anticancer strategy named “oxidation therapy” has been developed by inducing cytotoxicoxystress for cancer treatment. This goal could be achieved mainly by two methods, namely, (i) inducing the generation ofROS directly to solid tumors and (ii) inhibiting the antioxidative enzyme (defense) system of tumor cells. Since 1950s, manystrategies have been employed based on the first method, namely, administration of ROS per se (e.g. H2O2) or ROS generatingenzyme to tumor bearing animals. However no successful and practical results were obtained probably because of the lack oftumor selective ROS delivery and hence resulting in subsequent induction of severe side effects. To overcome these obstacles,we developed polyethylene glycol (PEG) conjugated Oz2
2 or H2O2-generating enzymes, xanthine oxidase (XO) and D-aminoacid oxidase (DAO) (PEG–DAO) respectively. More recently, a pegylated (PEG) zinc protoporphyrin (PEG–ZnPP) and ahighly water soluble micellar formulation of ZnPP based on amphiphilic styrene maleic acid (SMA) copolymer, SMA–ZnPP,are prepared, which are potent inhibitors of heme oxygenase-1 (HO-1). HO-1 is a major antioxidative enzyme of tumors, thatis different in mechanism of catalase or superoxide dismutase (SOD). Consequently, both PEG-enzymes and PEG–ZnPPexhibited superior in vivo pharmacokinetics than their parental molecules, particularly in tumor delivery by taking advantageof the EPR effect of macromolecular nature, and thus showed remarkable antitumor effects suggesting the potentials of thisanticancer therapeutic for clinical application. Furthermore, it has been well known that many antioxidative enzymes such ascatalase, SOD are down-regulated in most solid tumors in vivo. On the contrary, HO-1 is highly upregulated and it plays a veryimportant role of antioxidation, because HO-1 generates biliverdin, which being converted to bilirubin exhibits a very potentantioxidative effect, and hence antiapoptosis in tumors. Thus this oxidation therapy, by inhibiting this HO-1 dependentantioxidant (bilirubin) formation by ZnPP, and by enhancing ROS generation, is expected to offer a powerful therapeuticmodality for future anticancer therapy.
Keywords: Reactive oxygen species, EPR effect, D-amino acid oxidase, heme oxygenase-1
Introduction
Reactive oxygen species (ROS) is potentially danger-
ous by-product of cellular metabolism that has direct
effect on cell development, growth, survival, aging,
and on the development of cancer (Davies 1995;
Sundaresan et al. 1995). ROS is a group of free
radicals, which contain molecules with one or more
unpaired electrons. Electron acceptors, primarily
molecular oxygen, react easily with free radicals, to
become oxygen free radicals, named ROS. In aerobic
life, because molecular oxygen is ubiquitous, ROS
became the primary mediators of cellular free radical
reactions which are generated during the production
ISSN 1061-186X print/ISSN 1029-2330 online q 2007 Informa UK Ltd.
DOI: 10.1080/10611860701498286
Correspondence: J. Fang, Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Ikeda 4-22-1,Kumamoto 860-0082, Japan. Tel: 81 96 326 4137. Fax: 81 96 326 5048. E-mail: [email protected]
Journal of Drug Targeting, August–September 2007; 15(7–8): 475–486
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of ATP by aerobic metabolism in mitochondria. The
leakage of electrons from mitochondria during the
electron-transport steps of ATP production generates
the ROS, such as superoxide anion radical (Oz22 ).
Superoxide radicals is converted to hydrogen peroxide
(H2O2) by SOD, from which further hydroxyl radicals
(zOH) are generated in a reaction that either depends
on or is catalysed by Fe2þ or Cu2þ ions. Cells have
evolved a series of antioxidative defense systems to
handle these dangerous naturally occuring highly
reactive by-products. These defenses include intra-
cellular superoxide dismutases (SODs), catalase and
glutathione peroxidase that inactivate H2O2, and
other enzymes or compounds that contribute to
scavenging free radicals (e.g. heme oxygenase-1, HO-
1 and glutathione; Figure 1).
Because of their extremely high reactivity, ROS are
potentially harmful, by reacting with and damaging
protein (Davies 1993) and DNA (Lindahl 1993).
Under physiological conditions, damages to oxidisable
molecules caused by ROS are reduced by cellular
antioxidative defenses (e.g., SOD, catalase, HO-1,
glutathione peroxidase) and repair mechanism (Dem-
ple and Harrison 1994). However, over-burden of
ROS, while under pathological conditions such as
acute and chronic inflammation (Oda et al. 1989;
Cerutti and Trump 1991; Maeda and Akaike 1991)
and deficiency of antioxidant defense or repair
systems, will initiate lethal chain reactions that involve
oxidation and damage of cellular integrity and survival
(Davies 1995), by which reversible or irreversible
tissue injury will be induced, consequently leading to
various diseases, including cancer (Maeda and Akaike
1991; Dreher and Junod 1996; McCord 2000).
With regard to ROS and cancer, the prevailing
studies are focused on their roles on carcinogenesis.
In the past ten years, convincing evidence has been
accumulated that ROS is indeed considered an
endogenous class of carcinogens (Guyton and Kensler
1993; Feig et al. 1994; Cerutti 1994). In addition,
ROS is involved in each stage of cancer development,
including initiation, promotion, and progression
(Dreher and Junod 1996). As mentioned above,
overproduction of ROS induces increased damage to
DNA, which further triggers mutagenesis via DNA
base modifications and DNA helix alterations (Dreher
and Junod 1996). Moreover, oxidative stress can
stimulate the expansion of mutated cell clones by
modulating genes related to proliferation and trigger-
ing redox-responsive signaling cascades such as
epidermal growth factor (EGF), tyrosine phosphoryl-
ation and protein kinase C (PKC) (Droge 2002).
In 1990s, Maeda’s group found that during the
course of viral and bacterial infections, nitric oxide
synthase (iNOS) is induced at the site of infection and
inflammation in the same time course as Oz22
generation (either by XO activation and NADPH
oxidation) (Akaike et al. 1990, 2000, 2003; Umezawa
et al. 1997; Fujii et al. 1999). These two reactive
radicals, i.e. NO and Oz22 , react immediately and more
reactive derivative, peroxynitrite (ONOO2) is gener-
ated from nitric oxide (NO) and Oz22 under
pathological conditions and also in cancer. ONOO2
plays important roles on tumor angiogenesis and
metastasis by activating matrix metalloproteinases
(MMPs) as well as damaging RNA (virus) and DNA
(Okamoto et al. 2001; Wu et al. 2001; Sawa et al.
2003). In Maeda’s group, Yoshitake et al. (2004)
showed increase of viral mutation (Sendai virus/mouse
system) under such condition of extensive ONOO2
generation. Antioxidant canalol, a natural product
obtained from crude rape seed oil prevented the
mutation of Salmonella (Ames test) by ONOO2
(Kuwahara et al. 2004).
The effect of ROS on mutation is dose-dependent,
and in a dynamic equilibrium with antioxidant
defenses and cellular repair systems (Dreher and
Junod 1996); their production is a double-edged
sword. While NO is antioxidant per se, ROS makes NO
the most reactive. While low or intermediate levels of
oxidative stress are most effective for triggering
mutagenesis and promoting proliferation of cells,
high levels of oxidative stress not only inhibit cell
proliferation, but it is more damaging and causes
cytotoxic effect (Dreher and Junod 1996).
On the contrary, it is quite intriguing and more
important that the activities of various antioxidative
enzymes, such as catalase, glutathione peroxidase,
SOD, etc. have been found to be greatly reduced in
various tumor cells (Greenstein 1954; Yamanaka and
Deamer 1974; Sato et al. 1992; Hasegawa et al. 2002),
which will increase the vulnerability of tumor cells
Figure 1. ROS metabolism and the antioxidant defense system in
cancer cells. Cancer cells have high levels of metabolism that
generate ROS, and the corresponding antioxidative systems (e.g.
superoxide dismutase, SOD; heme oxygenase, HO-1) are essential
for cancer cells to fight against cytotoxic ROS. Thus, a new
antitumor strategy using potentially toxic ROS was challenged, both
by directly inducing ROS (O22 , H2O2) to tumors, and by targeting
these antioxidative enzymes.
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to ROS (Sawa et al. 2000; Fang et al. 2002). Thus,
one can imagine a unique antitumor tactics by
selectively delivering excess oxidative stress to tumor
cells, or preferentially disrupting the antioxidative
defense systems of tumor cells. Indeed, several
challenges were performed and verified the effect by
use of this concept of “oxidation therapy”, by
modulating the redox status in tumor cells (Ben-
Yoseph and Ross 1994; Yoshikawa et al. 1995;
Stegman et al. 1998; Huang et al. 2000). However,
the crucial problem of the concept is that specific and
tumor selective generation of ROS is not clearly
solved. To solve this problem, in Maeda’s laboratory
we developed a series of ROS-generating agents with
tumor-targeting nature, all of which showed potent
and satisfactory in vivo anticancer effects with little
side effect, suggesting a potential for this new
anticancer drug development (Sawa et al. 2000;
Fang et al. 2002, 2003a,b, 2004a,b, 2007). In this
review, the rationales of basic concept, and develop-
ment of tumor selective oxidation therapy, in which
EPR-effect based drug delivery system (DDS), with
high therapeutic efficacy were undertaken. Also, some
candidate agents are introduced.
History and development of oxidation therapy:
ROS generating system
During 1950s–1970s, researchers from different
laboratory tried to use H2O2, a highly cytotoxic and
relative stable ROS, for treating tumors in various
animal tumor models by injecting H2O2 into tumor or
into the circulation (Green and Westrop 1958; Sugiura
1958; Mealey 1965; Kaibara et al. 1971). Even though
no satisfactory antitumor effect was observed, they
became the pioneer of the ROS induced anticancer
therapeutic. This therapy was greatly developed in
1980s by Nathan and Cohn (1981), in which they
utilized glucose oxidase (GO), a H2O2 generating
enzyme, as a ROS donor, resulting in a significant
antitumor effect in a mice tumor model. This GO
method was further optimized and the concept of
oxidation therapy was first introduced by Ben-Yoseph
and Ross (1994). However, these researches were
unable to advance further because of the possible side
effects caused by the systemic generation of ROS, due
to ubiquitous presence of D-glucose.
Strikingly, utilization of D-amino acid oxidase
(DAO) as a ROS (H2O2) generator was a break-
through for this therapy because the production of
ROS can be easily controlled by controlling infusion of
substrate, D-amino acids (Stegman et al. 1998; Fang
et al. 2002, 2007). Besides H2O2, another ROS,
superoxide (Oz22 ) has also attracted considerable
attention. Oz22 generating enzyme xanthine oxidase
(XO) was thus developed and exhibited promising
results as a new anticancer agent (Yoshikawa et al.
1995; Sawa et al. 2000). Meantime, an alternative
strategy by inhibiting the antioxidative defense system
was verified effective by using SOD as a target for
selective killing of cancer cells (Huang et al. 2000).
However, one problem which remained unsolved was
how to target and keep the enzyme in the tumor. This
problem could be best solved by adapting macromol-
ecular drugs, polymeric micelles or nanoparticles
which exhibits EPR-effect in solid tumors.
In our laboratory, we observed that HO-1 is one of
the most potent antioxidative defense in tumor cells,
and thus we developed a series macromolecular
derivatives of HO-1 inhibitor zinc protoporphyrin
(ZnPP) for this goal and found remarkable anticancer
potentials of these agents (Fang et al. 2003a,b; Iyer
et al. 2007). The project for the oxidation therapy by
using polymeric DAO and ZnPP is still ongoing in our
laboratory, and these agents are expected to become
novel anticancer drugs in the future.
Key factors affecting the oxidation therapy:
Targeted generation of ROS in tumor
Choice of ROS-generating system
It is crucial for the oxidation therapy to select the
appropriate ROS and ROS-generating system. As is
well known, there are many ROS with potent
cytotoxicity, such as H2O2 in a broad sense, Oz22 ,
ONOO2 and HClO (both are products of ROS), zOH,
and lipid peroxylradical (LOOz) etc. Among these
ROS, H2O2 is a desirable candidate for this
therapeutic strategy because of its relatively mild
cytotoxic activity, which permits it more stability in
contrast to zOH radicals. Normally, H2O2 readily
crosses cell membrane. Ultimately, it causes oxidative
damage to DNA (Beckman and Ames 1997), proteins
(Berlett and Stadtman 1997), and lipids by direct
oxidation or in the presence of transition metal via
Haber–Weiss reaction to the extremely reactive
hydroxyl radical (Halliwell and Gutteridge 1984).
It was also reported more recently that H2O2 induces
apoptosis of a wide range of tumor cells in vitro
(Bladier et al. 1997; Suhara et al. 1998; Matsura et al.
1999; Yamakawa et al. 2000, Ren et al. 2001) via
activation of the caspase cascade. Of greater import-
ance, many antitumor agents, such as vinblastine,
doxorubicin, camptothecin, and inostamycin, exhibit
antitumor activity via H2O2-dependent activation of
apoptotic cell death, which suggests potential use of
H2O2 as an antitumor principle (Simizu et al. 1998).
However, H2O2 is relatively stable and small water-
soluble molecule, and thus diffuse or travel a long
distance and thus make it difficult to selective delivery
to and retain in tumor tissue. These characteristics
hamper the utility of H2O2 as an antitumor agent.
In fact, H2O2 used alone was ineffective when injected
into tumor or into the circulation (Green and
Westrop 1958; Sugiura 1958; Mealey 1965; Kaibara
et al. 1971), because of the aforesaid reasons. Further
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it will be decomposed by catalase in erythrocytes
which exist in massive levels. Use of an H2O2-
generating enzyme has been proposed as an alternative
approach to developing an H2O2-dependent antitu-
mor system. Nathan and Cohn (1981) and Ben-
Yoseph and Ross (1994) reported that GO, which
generates H2O2 during oxidation of glucose, showed
antitumor activity in solid tumor models. However,
regulation of H2O2 production by exogenously
administered GO in tumor-bearing hosts is proble-
matic because the availability of its both substrates,
oxygen and glucose, cannot be effectively modulated.
Consequently, induction of severe systemic side
effects is anticipated. In fact, GO administration to
produce H2O2 required injection of antioxidants to
minimize systemic toxicity (Nathan and Cohn 1981;
Ben-Yoseph and Ross 1994).
To overcome this drawback, in our laboratory, we
selected another H2O2-generating enzyme, DAO, by
which the generation of H2O2 can be easily modulated
because of the scarcity of its natural substrate, D-
amino acids, in mammalian organisms (Konno and
Yasumura 1992). Therefore, as anticipated, the
preferential production of H2O2 more selectively in
tumor was accomplished, and thus efficient antitumor
activity was successfully achieved with little toxicity.
Besides H2O2, Oz22 is another good candidate for
this therapeutic strategy. It is well known that Oz22
plays an important role in the pathogenesis of various
diseases (McCord 2000; Akaike et al. 1990),
suggesting that it is a relatively active molecule with
highly cytotoxic nature. Accordingly, the Oz22 generat-
ing enzyme XO was also examined for the oxidation
therapy (Yoshikawa et al. 1995; Sawa et al. 2000).
Further, PEG–XO exhibited superior antitumor
activity without much toxicity (Sawa et al. 2000).
Antioxidative conditions of tumor
Another important approach for therapeutic maneu-
ver is to inhibit antioxidative systems, such as catalase,
SOD, glutathione peroxidase, and HO-1 that are
known to serve as the protective role against ROS in
normal cells. It is well known that tumor cells or
tissues adapt to anaerobic energy production system
(Warburg effect), which will lead to lesser oxygen
tension and oxystress in vivo. In any event, the defense
system against oxystress will be potentially protective
for tumor cells. Interestingly and fortunately, com-
pared with normal cells, these antioxidative enzyme
level was found extremely low, perhaps with an
exception of HO-1, instead it was upregulated in most
tumor cells. Thus HO-1 is the key for antioxidant
stress in tumor cells (Greenstein 1954; Yamanaka and
Deamer 1974; Sato et al. 1992; Doi et al. 1999;
Hasegawa et al. 2002; Tanaka et al. 2003). This means
suppression of HO-1 will greatly increase the
vulnerability of tumor cells to ROS. Among these
enzymes, catalase plays a central role in the
antioxidant defense of many healthy organisms, as
do other enzymes, as H2O2 is converted to H2O
(Mates et al. 1999). With a few exception in some
hepatomas (Mochizuki et al. 1971), most tumors has
been reported to show a significantly reduced catalase
activity (Greenstein 1954; Sato et al. 1992). This
might be due to a marked suppression of catalase gene
expression at the level of transcription (Sato et al.
1992). In this context, similar findings were observed
in our recent study by measuring the catalase activities
of three different solid tumors and the corresponding
normal tissues, i.e. liver, kidney and lung, in
comparison with both transplanted and chemically
induced tumor models, namely tumors showed a
decrease of catalase activity to less than 10%
compared to the normal tissues (Fang et al. 2007).
Comparison of the catalase activity between normal
tissues and tumors was summarized in Table I. This
tendency of low catalase level, however, is not
necessarily valid in tumor cells in culture.
On the other hand, many tumor tissues seem to
have extensive infiltration of leukocytes compared
with normal tissues, which might typically lead to
increased production of ROS. These leukocytes
(macrophage) in solid tumors comprises a part of
host defense response, and they generate ROS against
tumor cells. Therefore, the antioxidative systems, e.g.
HO-1, catalase, SOD, become much important in
tumor for defending against ROS in and around
tumor. Along similar context, cancer cells may be
damaged when these antioxidative enzymes are
inhibited. This hypothesis has been validated to be
practicable by Huang et al. (2000) via inhibiting SOD
by certain oestrogen derivatives in tumor cells. We
further verified the antitumor effect by inhibitng HO-
1 (Fang et al. 2003a,b), which will be discussed more
in details later in this review.
Table I. Comparison of catalase activities between tumors and normal tissues.
S180* B16* DMBA induced tumor* AH66† Ac2F† HepG2† Human hepatoma
Relative CAT act (%)‡ 0.95 2.59 6.87 0.80 1.77 3.37 ND{
See reference Fang et al. (2007) and Sato et al. (1992) for details; * S180, mouse sarcoma S180 tumor; B16, mouse B16 molenoma; DMBA
induced tumor, DMBA(7, 12-dimethylbenz (a) anthracene) induced rat breast cancer; † hepatoma cells; ‡ compared with the catalase activity
of mice or rat liver. CAT, catalase; {ND, not detected.
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Tumor-targeted generation of ROS based on EPR effect
Furthermore, in the oxidation therapy, to achieve
satisfactory antitumor effect, targeted delivery of ROS
to tumor tissues is indispensable for this strategy
because nonspecific production of ROS may cause
oxidative stress-induced systemic damage in normal
organs even though they have a high level of
antioxidation defense system such as catalase or
SOD. The concept of the EPR (enhanced per-
meability and retention) effect (Iwai et al. 1984;
Matsumura and Maeda 1986; Maeda 2001; Maeda
et al. 2001; Duncan 2003; Fang et al. 2003a,b; Greish
et al. 2003; Iyer et al. 2006) is also necessary, for the
delivery of ROS. Since the EPR effect is observed only
for macromolecules, polymeric nanoparticles and
lipids in most of solid tumors, ROS generating
macromolecules (i.e. the enzymes conjugated to
polymers) are thus ideal to be utilized in most solid
tumors (Figure 2).
The EPR effect is a complex phenomenon invol-
ving: (i) extensive angiogenesis and hence high
vascular density; (ii) extensive extravasation (vascular
permeability) induced by various vascular mediators,
such as bradykinin, NO, vascular permeability factor
(VPF)/vascular endothelial growth factor (VEGF),
prostaglandins involving cyclooxygenases and matrix
metalloproteinases (MMPs/collagenases); (iii) defec-
tive vascular architecture; and (iv) impaired lymphatic
clearance from the interstitial space of tumor tissues
(Iwai et al. 1984; Matsumura and Maeda 1986;
Maeda 2001; Maeda et al. 2001; Fang et al. 2003a,b;
Iyer et al. 2006). As a result of EPR effect,
biocompatible macromolecules and lipids preferen-
tially and spontaneously leak out of tumor vessels, and
remain there at high concentration for a long period of
time. This means it is more than passive targeting
because retention plays a crucial role. Further, the
molecular size-dependency of EPR effect is applicable
for the molecular size above 40 kDa (Matsumura and
Maeda 1986; Seymour et al. 1995; Noguchi et al.
1998). This dependency exhibits a reverse correlation
to the renal clearance of the compounds (Maeda et al.
2001). Therefore, EPR effect is observed for any
biocompatible macromolecule with a molecular size
larger than the renal excretion threshold. EPR effect
occurring in solid tumors is a universal phenomenon,
which has now been considered a “gold standard” in
the field of drug design for new anticancer drug
development (Muggia 1999; Maeda et al. 2001;
Duncan 2003; Vicent and Duncan 2006).
Poly(ethylene glycol) (PEG), one of the best
biocompatible polymers, is widely used for polymeric
drug preparation because of the favourable properties
such as increased solubility both in organic and
aqueous media, increased in vivo half-life, and
virtually no toxicity and immunogenicity, non-
biodegradability and easy excretion from body
Figure 2. Scheme of enhanced permeability and retention (EPR)-effect of macromolecules in solid tumor. For low molecular weight agents,
they distribute indiscriminately in normal and tumor tissues, and the concentration in each tissue is paralleled with the concentration in
circulation. Namely, the drug concentration in tumor drops quickly in parallel to the clearance of the drugs from blood. For high molecular
weight agents, they cannot enter normal tissues because of the big size, but will distribute in tumor tissue due to the anatomic and pathological
difference of tumor blood vessels. Moreover, they will retain in tumor tissues for long time because of the lack of lymphatic function in tumor
tissue. See text for details.
Tumor-targeted induction of oxystress for cancer therapy 479
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(Zalipsky 1995). Thus, it has been used as a covalent
modifier of a wide range of proteins/enzymes and
other drugs, to produce PEG conjugates (Harris
1992; Pasut et al. 2004). PEG-ademasew and PEG-
aspargasew are used for therapy of the severe
combined immunodeficiency disease and leukemia,
respectively (Duncan 2003; Iyer et al. 2006).
Pharmakokinetic superiority of PEG-interferon over
native interferon has almost replaced the use of native
interferon therapy (Duncan 2003; Vicent and Duncan
2006). Moreover, PEG–doxorubicin and PEG–
camptothecin have been reported to exhibit better
pharmacokinetics, tumor accumulation and in vivo
half-life compared with their native counterparts.
Further, reduction of antigenicity of the native agents
is possible (Harris and Chess 2003).
Similarly, for targeted delivery of ROS generating
system to tumor, we modified DAO and XO with
PEG, and conjugated a potent HO-1 inhibitor, ZnPP
to PEG. All these PEG conjugates showed superior
pharmacological characteristics to native proteins or
drugs (Sawa et al. 2000; Fang et al. 2002; Sahoo et al.
2002; Fang et al. 2003a,b). For example, pegylation
of ZnPP greatly increased the water-solubility of ZnPP,
which makes the clinical application of ZnPP possible.
In addition it exhibited macromolecular nature, thus
EPR-effect for tumor targeting could be anticipated.
Further significant extension of plasma half-life (t1/2)
and area under the concentration time curve (AUC)
could be achieved (Figure 3 A,B; Fang et al. 2003a,b).
Likewise, PEG–DAO exhibited tumor-selective
accumulation and increased plasma t1/2 after i.v.
injection to solid tumor-bearing mice (Figure 3C;
Fang et al. 2002). We also anticipate PEG-interferon
(Pegasus, MW 58 kDa) would show the EPR-effect in
solid tumor, but it is unknown in our knowledges.
Examples of oxystress induced anticancer
therapeutics
Poly(ethylene glycol) conjugated xanthine oxidase
(PEG–XO)
XO is an iron-containing metalloflavoprotien that
catalyzes the oxidation of purines, in which ROS
including superoxide anion radical (O22 ) and hydrogen
Figure 3. Pharmacokinetics of PEG–ZnPP and PEG–DAO. A and B show the plasma level and tumor accumulation of PEG–ZnPP
respectively; C shows the body distribution of PEG–DAO. Results are expressed as means; bars, ^ SE (n ¼ 3–4). *P , 0.01, PEG–ZnPP vs.
native ZnPP. See references for details (Fang et al. 2002; Fang et al. 2003a,b).
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peroxide (H2O2) are generated. Thus XO was
expected as a good candidate for oxystress induced
anticancer therapeutic and was reported in 1995
(Yoshikawa et al. 1995). However, the high binding
affinity of native XO to the wall of blood vessels would
cause systemic vascular damage and hence limits the
use of native XO in clinical settings (Yoshikawa et al.
1995). To overcome this drawback, and to achieve
selective delivery of the drug to tumor by EPR effect,
we converted XO to macromolecular nature with high
biocompatibility by conjugating with PEG (PEG–
XO) (Sawa et al. 2000). The 1-amino groups of lysine
residues of XO, which play a crucial role in its binding
to the vascular wall, was modified by PEG, in this case
49% of free amino groups were modified with PEG.
Pharmacokinetic study by using 125I-labeled deriva-
tives showed a 2.8-fold higher accumulation of PEG–
XO in solid tumor than that of native XO at 24 h after
administration, whereas only a slight or negligible
increase in accumulation of PEG–XO was found in
normal organs. Moreover, 24 h after i.v. injection of
PEG–XO to tumor-bearing mice, the highest PEG–
XO enzyme activity was detected in tumor compared
with normal organs expect blood; enzyme activity in
tumor was 5.0, 3.9, and 9.4 times higher than that in
liver, kidney, and spleen, respectively, and the
accumulation of PEG–XO in tumor retained for at
least 96 h. Administration of hypoxanthine, a sub-
strate of XO, via i.p. route 12 h after the injection of
PEG–XO resulted in significant suppression of tumor
growth, with no tumor growth even after 52 days
(Figure 4A). No or very little side effect, if any at all,
was observed after this treatment. These findings
suggest the validity of PEG–XO as a tumor tropic
novel anticancer agent.
Poly(ethylene glycol) conjugated D-amino acid oxidase
(PEG–DAO)
Along with PEG–XO, we explored another ROS-
generating enzyme, DAO, by PEG conjugation (Fang
et al. 2002). DAO is a flavoprotein that catalyzes the
stereoselective oxidative deamination of D-amino
acids to the corresponding a-keto acids. During this
oxidation reaction, molecular oxygen (O2) is used as
an electron acceptor, and H2O2 is generated (Yagi
Figure 4. Antitumor effect of PEG–XO, PEG–DAO, PEG–ZnPP, and PEG–ZnPP plus PEG–DAO in the S-180 tumor model. S-180 cells
(2 £ 106 cells) were implanted s.c. in ddY mice. Six to ten days after inoculation, mice were treated by each agents as indicated. A, PEG–XO
treatment; B, PEG–DAO treatment; C, PEG–ZnPP treatment; D, PEG–ZnPP plus PEG–DAO treatment. Data are means (n ¼ 4–8); bars,
SE. *P , 0.001. See references for details (Sawa et al. 2000; Fang et al. 2002, 2003a,b, 2004b).
Tumor-targeted induction of oxystress for cancer therapy 481
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1971). The natural substrate of DAO is D-amino
acids, with the exception of D-aspartic acid and
D-glutamic acid. Since D-amino acids do not usually
exist in mammalian organisms to a significant level
(Konno and Yasumura 1992), it is thus reasonable
that H2O2 generation can be regulated by the level of
exogenous administration of D-amino acids, thus
avoiding the possible induction or severe systemic side
effects because of systemic generation of H2O2.
However, DAO is relatively small protein (Mw
39 kDa), and it may be excreted gradually as
previously observed for other small proteins or
polymer drugs smaller than 40 kDa (Matsumura and
Maeda 1986; Seymour et al. 1995; Fang et al.
2003a,b). Pegylation of DAO resulted in a 63 kDa
molecule by calculating the number of amino groups
of DAO conjugated with PEG (Fang et al. 2002).
However, the apparent molecular size of PEG–DAO
in solution was determined to be 120–150 kDa by
SDS-PAGE analysis, while it existed as a micellar
form in aqueous solution with a mean hydrodynamic
diameter of 119 nm as determined by dynamic light
scattering studies (Fang et al. 2007). Accordingly, the
pharmacokinetic parameters after i.v. injection was
significantly improved in mice compared with the
native DAO; namely, its plasma t1/2 and AUC
increased 2.6- and 2.9-fold, respectively. Further-
more, PEG conjugated DAO dramatically improved
intratumoral accumulation as well as plasma level, i.e.
3.2-fold relative to native one and 7.4-fold to
untreated control in tumor (Figure 3C). In contrast,
PEG–DAO injection showed no increase on the
enzyme activity in normal organs and tissues.
Experiments in mice showed that administration of
PEG–DAO followed by administration of its substrate
D-proline, significantly suppressed tumor growth.
Growth suppression continued for at least 27 days
after tumor implantation (Figure 4B). In contrast, no
significant antitumor effect was observed in mice
treated with native DAO plus D-proline. In addition,
oxidative metabolites were significantly increased in
solid tumor by administration of PEG–DAO followed
by D-proline, as evidenced by thiobarbituric acid-
reactive substance (TBARS) assay, whereas it was not
increased in the liver and the kidney (Fang et al.
2002). These results strongly indicate the antitumor
potential of PEG–DAO, by selective generation of
H2O2 in tumor warrants further investigation towards
clinical application.
PEG conjugated zinc protoporphyrin IX (PEG–ZnPP)
Second method of oxidation therapy is a disruption of
the defense system against oxidation in tumor. For this
purpose we developed PEG conjugates of ZnPP,
which is a strong inhibitor of HO (Sahoo et al. 2002).
HO plays a key role in heme degradation: oxidation of
the porphyrin ring results in formation of biliverdin,
which is subsequently reduced by biliverdin reductase
yielding bilirubin, carbon monoxide, and free iron. All
three of them showed potent antioxidative effects
(Fang et al. 2004a). To date, three isoforms of
mammalian HO have been identified: HO-1, HO-2,
and HO-3, of which HO-1 is belongs to the family of
heat shock proteins (i.e. HSP32) and its expression is
triggered by diverse stress-inducing stimuli (Maines
et al 1988; Fang et al. 2004a; Figure 5).
HO-1 is suggested to serve as a key biological
molecule in the adaptation to and/or defense against
oxidative stress and cellular stress. More important,
it is interesting to note that several tumors, including
renal cell carcinoma (Goodman et al. 1997) and
Figure 5. Schematic representation of role of HO-1 in tumor growth and mechanism of PEG–ZnPP induced antitumor effect.
J. Fang et al.482
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prostate tumors (Maines and Abrahamsson 1996) in
humans, express a high level of HO-1. The high
HO-1 expression was also found in experimental
solid tumors, i.e. the rat hepatoma AH136B (Doi
et al. 1999) and the mouse sarcoma S180 (Sahoo
et al. 2002; Fang et al. 2003a,b). Administration of
the HO inhibitor zinc protoporphyrin (ZnPP), via
tumor-feeding artery significantly suppressed the
growth of AH136B tumors, which suggests a vital
role for HO-1 in tumor growth (Doi et al. 1999;
Tanaka et al. 2003). These findings also indicate a
potentially beneficial role of HO inhibitor, e.g. ZnPP
as a novel anticancer agent (Figure 5). However,
ZnPP is sparsely soluble in water, but may be
dissolved in non-physiological alkaline solution at
high pH, which limits its practical use as drug. To
solve this problem and to obtain better tumor-
targeting effect, PEG conjugation was carried out to
modified ZnPP, resulting in a highly water-soluble
compound PEG–ZnPP (Sahoo et al. 2002). More
importantly, pegylation of ZnPP greatly increased
the molecular size of ZnPP, from about 600 Da of
free ZnPP to more than 70 kDa of the PEG-
conjugates in aqueous media (Sahoo et al. 2002).
In vivo pharmacokinetic analysis revealed that PEG–
ZnPP administered i.v. had a circulation time that
was 40 times longer than that for nonconjugated
ZnPP (Figure 3A). More important, PEG–ZnPP
preferentially accumulated in solid tumor tissue in a
murine model (Figure 3B). In vivo treatment with
PEG–ZnPP remarkably suppressed the growth of
S180 tumors implanted in the dorsal skin of ddY
mice without any apparent side effects (Figure 4C).
In addition, this PEG–ZnPP treatment
produced tumor-selective suppression of HO activity
as well as induction of apoptosis (Fang et al.
2003a,b). These findings suggest that tumor-
targeted inhibition of HO by PEG–ZnPP will lead
to apoptosis in solid tumors, probably through
increased oxidative stress.
Furthermore, when PEG–ZnPP is combined with
ROS or ROS generating agents, additive or synergis-
tic effect on ROS-induced tumor cell killing is
envisaged. For instance, PEG–ZnPP treated
SW480 cells became vulnerable to insults caused by
various oxidative cytotoxic agents; the 50% inhibition
does (IC50) were reduced to 75, 61, 17 and 39% for
H2O2, t-butyl hydroperoxide, camptothecin and
doxorubicin, respectively. Cells treated with PEG–
ZnPP plus cytotoxic oxidants exhibited marked
production of intracellular ROS, which paralleled
the incidence of apoptosis. In vivo, PEG–ZnPP
pretreatment significantly suppressed the tumor
growth in mice receiving PEG–DAO/D-proline
compared to no PEG–ZnPP pretreatment
(Figure 4D; Fang et al. 2004b). These findings
suggest a definit potential of HO-1 be an attractive
target for chemotherapeutic intervention, and thus
the potential of PEG–ZnPP as a novel anticancer
drug by itself or in combination therapy.
Similar to PEG–ZnPP, we successfully prepared a
highly water-soluble micellar form of ZnPP recently,
by the use of amphiphilic styrene-maleic acid
copolymer (SMA), named SMA–ZnPP. SMA–
ZnPP existed as nanoparticles in aqueous solution
having a average hydrodynamic diameter of 176.5 nm
with relatively narrow size distribution profile. Static
light scattering and size exclusion chromatography
revealed that SMA–ZnPP micelles were associated to
form macromolecular complexes. The molecular size
of the SMA–ZnPP micelles was of the order of
144 kDa as determined by Sephacryl S-200 gel
chromatography. Thus the macromolecular nature of
these micelles may take advantage of EPR effect to
target tumor tissues selectively. SMA–ZnPP exhibited
similar HO inhibitory activity as that of native ZnPP
indicating its potential as a potent HO-1 inhibitor with
tumor targeting characteristics (Iyer et al. 2007).
From the preliminary in vitro and in vivo studies, we
found that SMA–ZnPP revealed a potent antitumor
effect, but without any apparent side effects even at
the dose of 200 mg/kg when injected i.v. in mice
(Iyer et al. 2007; Greish et al. unpublished data).
Thus we anticipate a pronounced therapeutic benefit
of PEG–ZnPP and SMA–ZnPP against tumor,
especially in combination with ROS-generating
systems including traditional antitumor agents.
Conclusion
ROS, a group of highly reactive molecules which
exhibit very important, yet paradoxical role on tumor.
On one hand, it can damage DNA, and thus initiate
and promote cancer development, yet on the other
hand it can serve as a tumor terminator if it is present
in excessive concentration due to its cytotoxic effect.
Moreover, although being not described here, the
highly elevated level of NO production in tumor will
further facilitate the antitumor effects of ROS by
generating more reactive species such as ONOO2,
which will be a combined effector in this anticancer
tactics. Our aim is to develop strategies for tumor-
selective generation of ROS. This could be achieved by
conjugating macromolecular carriers (polymers) to
appropriate enzymes or antioxidant inhibitors as
shown in this work and our previous works. Alternately
micellar formulations and nanonoparticles can also be
utilized to selectively deliver the drug to tumor site,
thereby successful antitumor effect can be achieved
without apparent side effects by taking advantage of
the EPR effect of macromolecules in solid tumor
(Figure 6). Another important rationale of this study is
that majority of tumor cells frequently possess very
little antioxidative enzymes, e.g. catalase, which makes
them very vulnerable to oxidative stresses. Thus, the
unique antitumor strategy ‘oxidation therapy’, might
Tumor-targeted induction of oxystress for cancer therapy 483
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be a promising approach for clinical anticancer trials in
the future, and warrants further investigation.
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