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Doxorubicin (adriamycin): A critical

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Pharmac. Ther. Vol. 47, pp. 219-231, 1990 0163-7258/90 $0.00 + 0.50
Printed in Great Britain. All rights reserved 1990 Pergamon Press plc

Associate Editor: D. SHUGAR

DOXORUBICIN (ADRIAMYCIN): A CRITICAL REVIEW OF

FREE RADICAL-DEPENDENT MECHANISMS OF
CYTOTOXICITY

H. G. KEIZER,*~ H. M. PINEDO,t G. J. SCHUURHUISt a n d H. JOENJE*

*Institute of Human Genetics and tDepartment of Oncology, Free University, P.O. Box 7161, 1007 MC
Amsterdam, The Netherlands

Abstract--The antineoplastic drug doxorubicin is capable of generating a variety of free radical species
in subcellular systems and this capacity has been considered critical for its antitumor action. However,
for most tumor cell lines this mechanism of cytotoxicity does not appear to play a major role. Free
radical-independent cytotoxicity mechanisms, taking place in the nuclear compartment of the cell, may
more likely be involved in the antitumor effect of doxorubicin.

CONTENTS
1. Introduction 219
2. Free Radical-Dependent Toxicity Mechanisms of Doxorubicin 220
2.1. Mechanism of free radical production due to redox-cycling 220
2.2. DNA damage due to redox-cycling 220
2.3. Lipid peroxidation induced by redox-cycling 221
2.4. Mechanism of free radical formation by the doxorubicin-iron complex 222
2.5. Doxorubicin-iron complex-mediated DNA damage 222
2.6. Doxorubicin-iron complex-mediated lipid peroxidation 223
3. The Relative Importance of Free Radicals in the Mechanism of Cytotoxicity of Doxorubicin 223
3.1. Free radicals generated by redox-cycling 223
3.2. Free radicals generated by the doxorubicin-iron complex 224
3.3. Doxorubicin-dependent hydroxyl radical formation by intact tumor cells 224
3.4. Effects of radical scavengers on the cytotoxicity of doxorubicin 225
3.5. Role of free radicals in doxorubicin-induced cardiotoxicity 226
3.5.1. Free radical chemistry of doxorubicin in cardiac tissue 226
3.5.2. Free radical damage in cardiac tissue 226
4. Free Radical-Independent Mechanisms of Cytotoxicity 227
5. Summary and Conclusions 228
Acknowledgements 228
References 228

1. I N T R O D U C T I O N by a cumulative dose-related cardiomyopathy that

The anthracycline antibiotic doxorubicin (Adri-
amycin), originally isolated from the fungus
Streptomyces peucetius (Arcamone et al., 1972), is a
chemotherapeutic agent with strong activity against a
wide range of human malignant neoplasms including
acute leukemia, non-Hodgkin lymphomas, breast
cancer, Hodgkin's disease and sarcomas (Young
et al., 1981). Apart from side-effects that are common
to many cancer chemotherapeutics, i.e. hematopoietic
suppression, nausea and vomiting, and alopecia, the
clinical usefulness of doxorubicin is limited largely
++Present address: Department of Toxicology, Duphar
B.V., P.O. Box 900, 1380 DA Weesp, The Netherlands.
To whom correspondence should be addressed.
manifests itself as congestive heart failure (Young
et al., 1981).
Much effort has been devoted to unraveling the
mechanism of antitumor action of doxorubicin.
Currently the belief is widespread that free radical
formation is critically involved in the mechanism
of cytotoxicity of doxorubicin against tumor cells.
However, most of the evidence favoring a free
radical-dependent model has been obtained by study-
ing subcellular fractions, often in combination with
extremely high concentrations of doxorubicin that
are not encountered in clinical practice.
Below we review the mechanism of free radical
formation from doxorubicin and discuss the likeli-
hood of these radicals playing a role in its antitumor
effect. We conclude that, in general, this role must be

219
220
a minor one, even though doxorubicin-induced car-
diotoxicity may well be mediated by free radical
formation. Finally, an attractive alternative mecha-
nism, based on the interference of doxorubicin with
topoisomerase II, is discussed.
2. FREE RADICAL-DEPENDENT TOXICITY
MECHANISMS OF DOXORUBICIN
The chemical structure of doxorubicin is depicted
in Fig. 1. From a 'free radical' point of view both
rings B and C are of special interest. One-electron-
reduction of ring C leads to the formation of a
semiquinone free radical (Fig. 2). This radical is
relatively stable under anoxic conditions, but under
oxic conditions its unpaired electron is donated to
oxygen (02) forming superoxide radicals (Doroshow,
1983a, b; Bachur et al., 1982; Svingen and Powis,
1981). Suitable flavoproteins catalyze the formation
of reduced semiquinone radicals by accepting electrons
from NADH or NADPH and donating them to
doxorubicin. By reducing oxygen to superoxide the
parental doxorubicin molecule is regenerated. This
sequence of reactions, known as 'redox-cycling', is
potentially harmful to cells since relatively little
doxorubicin would suffice to catalyze the formation
of numerous superoxide radicals. Formation of an
oxidized semiquinone in ring B of doxorubicin is
o OH o
1 ~t2 11 to 13
] 4 I] = I * ' %, _."
H3CO O OH O|
2,'N'~ Rg
Anthracydine R1 R2 R3
Adciamycin CH2OH NH 2 OH
Oaun~ubicin CH 3 NH 2 OH
FIG. I. The chemical structure of doxorubicin. Chemically
doxorubicin (adriamycin) consists of a water-insoluble te-
tracycline aglycone (structures ABCD) and a water-soluble
reducing daunosamine sugar. Several parts of the molecule
can be involved in the metabolism of doxorubicin. Ring C
is a quinone group which can be activated into a
semiquinone radical after one-electron-reduction. Ring B is
a hydroquinone which can also be activated into a
semiquinone radical after one-electron-oxidation. The C9
side chain is probably important in the iron-catalyzed
autoxidation of the molecule. Differences in the C9 side
chain form the only difference between doxorubicin and its
analog daunorubicin. The iron-chelation site is probably
formed by the oxygen atoms of CI1 and C12. Iron at this
site can be reduced by the doxorubicin molecule either by
oxidation of the hydroquinone or by oxidation of the C9
side chain. The sugar group is important for the water
solubility of doxorubicin; during anaerobic metabolic reduc-
tion of doxorubicin it is split off and the C7-deoxyaglycone
is formed. From Gianni et al. (1988). (Reproduced with
permission of the authors.)
H.G. KEIZERet al.
known to occur in the presence of iron (Gianni et al.,
1985; Zweier, 1985) when no reducing system is
present. When the oxidized doxorubicin semi-
quinone-iron complex reacts with oxygen, this will
ultimately lead to the formation of a fully oxidized
form of doxorubicin, with formation of oxygen
radicals. Both radical-producing systems are dis-
cussed below.
2.1. MECHANISMOF FREE RADICALPRODUCTIONDUE
TO REDOX-CYCLING
Reductive redox-cycling of doxorubicin was
described by Goodman and Hochstein (1977), who
observed that cytochrome P-450 reductase causes
NADPH-dependent oxygen consumption in the
presence of doxorubicin, in excess of the amount of
drug present. Later studies indicated that not only
NADPH cytochrome P-450 reductase (Pan et al.,
1981), but also NADH dehydrogenase (Doroshow,
1983b; Davies et al., 1983a, b) and xanthine oxidase
(Doroshow, 1983a; Pan and Bachur, 1980), catalyze
the one-electron-reduction of doxorubicin. As a con-
sequence, redox-cycling of doxorubicin can occur in
cytoplasm, mitochondria, endoplasmic reticulum
(Doroshow, 1983a), and nucleus (Bachur et al., 1982).
The nucleus-catalyzed redox-cycling led Bachur et al.
(1978) to postulate the concept of site-specific free
radical formation in the nucleus: doxorubicin binds
selectively to nuclear DNA and can be metabolically
activated in the nucleus to produce free radical-
mediated damage to DNA.
The possible importance of redox-cycling is
stressed by the observation that not only isolated
enzymes, but also intact cells, can support one-
.;~c., electron-reduction of doxorubicin and subsequent
Y' doxorubicin-induced oxygen consumption (Sato
et al., 1977). Furthermore, one-electron-reduction of
3
doxorubicin might also occur in the clinical situation,
since an important metabolite found in patients is
R4 R5 the C7-deoxy-aglycone form of doxorubicin (Mross
et al., 1988), which is a metabolite known to be
H H
formed from doxorubicin semiquinone radicals under
H H anoxic conditions (Guti6rrez et al., 1983; Pan et al.,
1981). In short, doxorubicin may be involved in a
'redox-cycling' process, which is potentially impor-
tant for its cytotoxicity.
2.2. DNA DAMAGEDUE TO REDOx-CYCLING
Redox-cycling is rapid in the presence of oxygen
and slow under hypoxic conditions. Thus, in the
presence of oxygen, large amounts of superoxide may
be formed, whereas in its absence the semiquinone
radical will accumulate. As indicated below, different
toxicity mechanisms might be operative, depending
on the amount of oxygen present.
An oxygen-dependent mechanism of DNA damage
has been described by Berlin and Haseltine (1981),
who observed extensive DNA strand scission in a
system containing NADPH, NADPH cytochrome
P-450 reductase and DNA, and by Gutteridge and
Toeg (1982), who found deoxyribose breakdown in a
system containing deoxyribose, doxorubicin semi-
quinone radicals and iron. The authors proposed that
 Doxorubicin (Adriamycin) 221

_ 9" 9" o. on
Orug-macromolecule DNA,RNA~ * ~ ~ C O R t~;rJ~*,~COR
complexes ' Proteins C H ~ " O H 4 ~ ~ , ~ " O H
so o- o. c's0 6 0H
(CT-Ouinone methide) T (C7-Deoxyaglycone)
/ DNA- nicking

0 OH DNA /
~ C O R /f
\ f \ / - ' ,~j r " o ,
y F~I( %0 o o. 6-s,or ~ ( - - / ~L,p,~,
.,o,.J\, J qo. y\ .,o,00.,o,,o.,,o.
~ "OH ~ "~- 02
CH30 O" OH C)-sugor
(Semiquinone)

Oruo-mocromolecule DNA,RNA ~ j ~ z ~ , , ~ COR

complexes * Proteins ~ L ~ ~ OH
CHso 0 OH
CT-Free rodicol

FIG. 2. Free radical and alkylating intermediates in the metabolism of doxorubicin. Doxorubicin can be
reduced by cellular flavoproteins to form its one-electron reduced semiquinone form. In the presence of
oxygen the semiquinone free radical is oxidized back into the parental quinone form under formation of
superoxide radicals. In the absence of oxygen the semiquinone is unstable; it loses its sugar moiety and
an intermediate C7 free radical can be formed. This radical can bind covalently to cellular macromolecules
or become again reduced forming a relatively stable product, the C7-deoxyaglycone,which is in fact a
doubly reduced doxorubicin molecule that has lost its sugar moiety. A tautomer of C7-deoxyaglyconeis
the C7-quinone-methide, which is a potent DNA alkylating species and potentially toxic for tumor cells.
From Sinha et al. (1984). Reproduced with permission of the authors and the copyright holder, Cancer
Research, Inc., Philadelphia.

the DNA damaging species is the hydroxyl radical
formed from the following sequence of reactions:
202- + 2H+ ~H202 + 02
02 + F e 3 + ~ O : + Fe 2+
H202 + Fe 2+ --*'OH + O H - + Fe 3+.
The sum of the latter two reactions
H202 + O~- ~ ' O H + O H - + 02
is known as the iron-catalyzed Haber-Weiss reaction.
That superoxide-generating systems can indeed
damage DNA by this series of reactions was shown
by Rowley and Halliwell (1983), who were able to
inhibit DNA damage in a superoxide-producing
system with superoxide dismutase, catalase, iron
chelators and hydroxyl radical scavengers.
In the absence o f oxygen the semiquinone is
converted to its C7-deoxyaglycone metabolite, which
in itself is not pharmacologically active (Averbuch
et al., 1985). However, its tautomer, the C7-quinone
methide is a DNA-alkylating species (Moore, 1977).
Furthermore, an intermediate in the formation of
C7-deoxyaglycone from the doxorubicin semi-
quinone is the C7 free radical, which can also bind
covalently to DNA (Sinha and Chignell, 1979; Sinha
and Lewis Gregory, 1981; Sinha et al., 1984). The
structures of these reactive species are depicted in
Fig. 2.
Another DNA-damaging mechanism that may
operate at low partial pressure o f oxygen has been
described by Winterbourn and collaborators. They
showed that in the absence of oxygen the semi-
quinone of doxorubicin reacts with H202 causing
breakdown of deoxyribose (Bates and Winterbourn,
1982). This reaction was catalyzed by very low con-
centrations of iron: submicromolar concentrations
are sufficient for a maximal effect (Winterbourn et al.,
1985). Both the low iron requirement of the reaction
and the optimal reaction rate at an oxygen tension of
approximately 4 mm Hg, may mimic the situation in
a tumor cell, since almost all intracellular iron is
believed to be in a complexed form, while oxygen
tensions of 3-6 mm Hg are considered to be common
(Winterbourn et al., 1985). These data underscore the
potential importance of this reaction.
2.3. LIPID PEROXIDATION INDUCED BY
REDOX-CYCLING
Unsaturated fatty acids are peroxidized when
exposed to hydroxyl radicals in the presence of a
metal catalyst (Gutteridge, 1982). This process might
damage cellular membranes and eventually kill cells.
Doxorubicin can cause lipid peroxidation after
metabolic activation by heart, liver or kidney micro-
somes (Glazer et al., 1982; Mimnaugh et al., 1982;
Bachur et al., 1977, 1978), mitochondria (Mimnaugh
222 H.G. KEIZERet al.
et al., 1985b), or nuclei (Mimnaugh et al., 1985a).
In the presence of oxygen, doxorubicin-dependent
lipid peroxidation is generally inhibited by superoxide
dismutase, catalase, glutathione, iron chelators
and hydroxyl radical scavengers (Mimnaugh et al.,
1985a, b, 1983), suggesting that OH radicals gener-
ated by the iron-catalyzed Haber-Weiss reaction are
involved. The rate of lipid peroxidation in the pres-
ence of iron and superoxide, produced by reduced
anthracyclines, was much higher for derivatives of
doxorubicin that bind to microsomes than for non-
binding derivatives (Sterrenberg et al., 1985).
Analogous to D N A damage, membrane lipid per-
oxidation can also be induced in the absence of
oxygen with H202 present (Winterbourn et al., 1985).
Without H:O2 very low concentrations of oxygen are
required for optimal lipid peroxidation, probably to
provide H202 from the dismutation of superoxide,
which is formed by the reaction of 02 with doxoru-
bicin semiquinone radicals. Lipid peroxidation
occurred without added iron, but increased when iron
was added in the 0.1-1pM range. OH radical
scavengers did not inhibit lipid peroxidation in this
system. This is probably due to binding of doxoru-
bicin to the liposomes used in this test, since the
reactivity of "OH is so high that, when generated in
a 'site-specific' way, competition by scavengers is very
inefficient (Winterbourn et al., 1985).
In conclusion, under both hypoxic and normoxic
conditions, lipid peroxidation can be induced by the
reduced doxorubicin semiquinone radical, which in
both cases is probably mediated by the OH radical.
2.4. MECHANISM OF FREE RADICAL FORMATION BY
THE DOXORUBICIN-IRON COMPLEX
The doxorubicin-iron complex can support the
formation of free radicals by two distinct mecha-
nisms, one dependent on the presence of a reducing
system, and another forming radicals from the
doxorubicin-iron complex itself, in the absence of a
reducing system. Both mechanisms are schematically
depicted in Fig. 3.
The upper part of Fig. 3 illustrates free radical
formation in the presence of a reducing system.
Doxorubicin-Fe 3 is reduced to doxorubicin-Fe 2+
either enzymatically by N A D H cytochrome P-450
reductase (Sugioka and Nakano, 1982) or by thiols
like glutathione (GSH) or cysteine (Zweier, 1985).
Doxorubicin-Fe 2 can react with oxygen to form
superoxide radicals (O;-) which dismutate to form
hydrogen peroxide (H202). Hydrogen peroxide can
also react with doxorubicin-Fe 2 and form hydroxyl
radicals (Eliot et aL, 1984; Myers et al., 1982). This
GSH-driven hydroxyl radical formation is a cyclic
process comparable to redox-cycling in that no
doxorubicin metabolites are being formed (Zweier,
1985; Myers et aL, 1982) and, in the presence of
sufficient GSH, the cycling process can proceed in-
definitely.
In the absence of a reducing system, doxoru-
bicin-Fe 3+ can reduce its chelated iron by an intra-
molecular redox reaction, either by oxidation of the
side chain on C9 (Zweier et aL, 1986; Gianni et al.,
1988; see also Fig. l) or the hydroquinone moiety at
ring B (Zweier, 1983; Gianni et al., 1985), forming a
NADP~-.~ 1.,- DOX-Fe ~* ,,~...,-.~GSSG
NADPH - ' / ~'- DOX.Fe ~+ GSH
a+
I:~X-Fe
Doll,g"
OH ~,"~
oox.~=g
[
9-COOH-DOX
FIG. 3. Free radical production from the doxorubicin iron
complex. The doxorubicin-iron II complex (DOX-Fe 2+)
can react with molecular oxygen (O2) or hydrogen peroxide
(H202) leading to the formation of superoxide (O~) or
hydroxyl radicals ('OH), respectively, while the complex is
oxidized to doxorubicin-iron III (DOX-Fe3+). DOX-Fe 3+
can be reduced enzymatically by cytochrome P-450 reduc-
tase (Fp) or nonenzymatically via reaction with reduced
glutathione (GSH), forming back DOX Fe2+, which can
again react with 02 or H202. In the presence of a reducing
system no metabolites are formed and radical production
can proceed indefinitely. In the absence of a reducing
system, DOX-Fe 3+ can reduce its iron intramolecularly by
oxidizing its hydroquinone moiety leading to the formation
of an oxidized doxorubicin semiquinone radical or by
oxidizing its C9 side chain. This intramolecular reduction of
iron is probably catalyzed by DNA. Electrons from the C9
chain of doxorubicin can keep the iron of the complex
reduced and thus support free radical production until the
side chain is fully oxidized. The end product of such
oxidation is 9-COOH-doxorubicin.
doxorubicin free radical chelate with Fe 2+ (Gianni
et al., 1985; Zweier et al., 1986; Zweier, 1985). The
formation of this doxorubicin-Fe 2+ complex is
probably catalyzed by D N A (Muindi et al., 1985). In
the presence of oxygen this complex is oxidized, with
formation of superoxide radicals (Gianni et al., 1985),
while it can also react with H202 via formation of
hydroxyl radicals (Zweier, 1985; Muindi et al., 1984,
1985). Further oxidation of the side chain will
ultimately lead to the formation of the oxidized meta-
bolite of doxorubicin 9-dehydroxyacetyl-9-carboxyl
doxorubicin (9-COOH-doxorubicin) (Gianni et al.,
1988) (see Fig. 3).
2.5. DOXORUBICIN--IRON COMPLEX-MEDIATED D N A
DAMAGE
Doxorubicin-iron complexes bind tightly to D N A
(Eliot et aL, 1984). However, contrary to intercalated
doxorubicin (Mimnaugh et al., 1985b; Sinha and
Chignell, 1979; Sato et al., 1977; Berlin and Haseltine,
1981; Rowley and Halliwell, 1983), the doxoru-
bicin-iron complex preserves its ability to catalyze the
formation of oxygen free radicals in the presence of
 Doxorubicin (Adriamycin)
double-stranded DNA (Eliot et aL, 1984). Thus, the
doxorubicin-iron complex-driven hydroxyl radical
formation can proceed in close proximity to DNA
and has therefore the potential to damage DNA
efficiently, especially since DNA seems to catalyze
hydroxyl radical formation by this complex (Muindi
et al., 1984, 1985). Hydroxyl radicals are probably
involved in damaging of DNA since the generation of
hydroxyl radicals by the Dox-Fe 3 complex corre-
lates with its ability to cleave DNA (Muindi et al.,
1984) and also since catalase, iron chelators and
hydroxyl radical scavengers are protective in this
system (Eliot et al., 1984). Relatively high concentra-
tions of hydroxyl radical scavengers were required for
protection, indicating that these radicals were indeed
generated in a site-specific way.
2.6. DOXORUBICIN-IRONCOMPLEX-MEDIATEDLIPID
PEROXIDATION
In the presence of GSH, doxorubicin efficiently
damages erythrocyte ghost membranes (Myers et al.,
1982). This reaction was inhibited by superoxide
dismutase, catalase and hydroxyl radical scavengers,
indicating the involvement of the hydroxyl radical.
Even in the absence of reducing agents, doxoru-
bicin-iron complexes appear to have the potential to
damage biomembranes, since such complexes pro-
voke lipid peroxidation (Sugioka and Nakano, 1982;
Gutteridge, 1982, 1983, 1984; Gianni et al., 1988).
In this case, however, superoxide dismutase, catalase
or hydroxyl radical scavengers did not protect
(Sugioka and Nakano, 1982; Gutteridge, 1983;
Gianni et al., 1988). The reduction of iron by the
side chain of doxorubicin was probably involved in
this type of membrane damage, since daunorubicin,
a structural analog of doxorubicin, lacking a readily
oxidizable side chain, was much less efficient (Gianni
et al., 1988). Thus, both in the absence and presence
of reducing agents, the doxorubicin-iron complex
can damage membranes, probably due to a hydroxyl
radical-dependent mechanism.
3. THE RELATIVE IMPORTANCE OF FREE
RADICALS IN THE MECHANISM OF
CYTOTOXICITY OF DOXORUBICIN
As summarized in the previous section, roughly
two mechanisms of doxorubicin-mediated free radi-
cal formation can be distinguished, one dependent on
the formation of semiquinone radicals generated
during flavoprotein-mediated redox-cycling, and the
other dependent on the doxorubicin-iron complex.
Below we discuss the likelihood of such mechanisms
playing a role in the tumoricidal and cardiotoxic
effects of doxorubicin.
3.1. FREE RADICALSGENERATEDBY REDOX-CYCLING
With respect to redox-cycling, Bachur et al. (1978,
1982) have suggested that free radical formation can
occur site-specifically in the nucleus, since isolated
nuclei bind doxorubicin and can also support
semiquinone formation from doxorubicin (Bachur
et al., 1982). However, the situation in intact cells
223
might be different, as in some cell types more than
99.8% of the doxorubicin is present in a DNA-bound
form (Gigli et al., 1988), whereas binding to double
stranded DNA is known to preclude chemical (Sinha
and Chignell, 1979), enzymatic (Berlin and Haseltine,
1981; Rowley and Halliwell, 1983), and microsomal
(Sato et al., 1977) one-electron-reduction of doxoru-
bicin. As a consequence of this, redox-cycling-depen-
dent DNA strand scission (Berlin and Haseltine,
1981), lipid peroxidation (Mimnaugh et al., 1985a)
and generation of superoxide from doxorubicin by
microsomes (Kalyanaraman et al., 1980) is largely
prevented in the presence of double-stranded DNA.
That Bachur et al. (1982) nevertheless found redox
activity of doxorubicin in intact nuclei may be due to
the extremely high concentrations of doxorubicin
( > 500 11M) used.
Even though radical formation by redox-cycling
may not take place in the nucleus, semiquinone or
oxygen radicals may well be formed at some cytoplas-
mic site, e.g. the endoplasmic reticulum (Bachur
et al., 1978), and reach the nucleus by diffusion.
However, the semiquinone radical is relatively un-
stable, with an estimated diffusion radius of 0.6 #m
under anaerobic and 0.1 # m under aerobic conditions
(Svingen and Powis, 1981), while an average cell has
a diameter in the range of 5-20/~m. This suggests that
only a minor fraction of the semiquinone radicals
produced in the cytoplasm may be able to reach
the nucleus. Theoretically, the probability for
semiquinone radicals to reach the nucleus is six-fold
higher under anoxic than under aerobic conditions.
Yet, the sensitivity of cells to doxorubicin is essen-
tially similar under both anoxic and aerobic condi-
tions (Kennedy et al., 1983), which seems to argue
against involvement of semiquinone formation in the
cytotoxicity of doxorubicin.
Alternatively, superoxide and hydrogen peroxide
formed in the cytoplasm by redox-cycling might
diffuse into the nucleus and damage the DNA.
However, such a mechanism seems unlikely, since
increasing the concentration of intracellular SOD and
catalase, either artificially (Cervantes et al., 1988) or
by selection for resistance to hyperoxia (Keizer et al.,
1988), has been shown not to protect against the
cytotoxicity of doxorubicin. Furthermore, a recent
spin-trapping study failed to provide evidence for an
involvement of ESR-observable oxygen-derived radi-
cals in the antitumor activity of doxorubicin (Alegria
et al., 1989).
In addition to DNA damage, free radical damage
to cellular membranes by lipid peroxidation may also
be involved in the antitumor effect of doxorubicin. In
fact, doxorubicin-induced lipid peroxidaton has been
observed in hepatocytes (Meredith and Reed, 1983;
Babson et al., 1981) and cardiocytes (Julicher et al.,
1985). However, to observe this effect, relatively high
concentrations of doxorubicin (100pM) were re-
quired; in addition, the experiments were done with
glutathione-depleted cells under an atmosphere of
pure oxygen, conditions known to accelerate the
process of lipid peroxidation. Bearing in mind that
hepatocytes and cardiocytes are probably better
suited to support redox-cycling of doxorubicin, com-
pared to tumor cells, such data can not be taken to
indicate that lipid peroxidation is involved in the
224 H.G. KEIZERet al.
antitumor effect of doxorubicin, especially since lipid
peroxidation has not been reported for tumor
cells treated with clinically relevant concentrations of
doxorubicin.
To investigate the role of flavoprotein-mediated
redox-cycling of doxorubicin in its cytotoxicity
against SW-1573 human lung tumor cells, Keizer
et al. (1989a) made use of a set of inhibitors of
flavoprotein-mediated reduction of doxorubicin and
observed that none of these agents protected against
the cytotoxicity of doxorubicin, although the cyto-
toxicity of mitomycin C, which is known to depend
on ftavoprotein activity, was strongly inhibited.
From the evidence presented above we must con-
clude that the situation inside the intact tumor cell is
not favorable for redox-cycling, and that the involve-
ment of redox-cycling in the cytotoxicity of doxoru-
bicin in tumor cells has not been proven.
In the next section we discuss the probability that
doxorubicin-iron complex-dependent free radical
formation is involved in the mechanism of cytotoxic-
ity of doxorubicin.
3.2. FREE RADICALS GENERATED BY THE
DOXORUBICIN-IRON COMPLEX
T h i o l - i n d e p e n d e n t free radical formation is very
unlikely to occur in intact cells, since intracellular
thiols are known to be abundantly present, which is
expected to prevent autoxidation of doxorubicin
(Zweier, 1985). In addition the 9-COOH-doxorubicin
metabolite is not detected in patients treated with
doxorubicin, Although tumor cells in vivo were not
protected by extracellularly added thiols (Freeman et
al., 1980; Doroshow et al., 1981; Yoda et al., 1986),
it cannot be excluded that this mechanism of free
radical formation occurs in extracellular fluids, where
glutathione levels are relatively low.
Thiol-dependent free radical formation from
doxorubicin-iron complexes is a more attractive
model for hydroxyl radical-mediated D N A damage,
since doxorobucin-iron complexes bind efficiently to
DNA, where they can still be reduced by thiols and
produce hydroxyl radicals in a site-specific way at
target. However, thiol depletion does not protect
tumor cells against the cytotoxicity of doxorubicin
(Ramu et al., 1984; Hamilton et al., 1985), nor does
an increased thiol content potentiate its cytotoxicity
(Doroshow et al., 1981). The only system described
thus far, in which thiols increase doxorubicin-
mediated damage, is the microsomal fraction
from kidney, in which thiols potentiate lipid peroxi-
dation (Mimnaugh, 1986). Thus kidney damage due
to doxorubicin may be partially caused by thiol-
dependent free radical formation (Mimnaugh, 1986;
Mimnaugh et al., 1986). For tumor cell killing,
however, the occurrence of this mechanism remains
to be proven.
In conclusion, of all free radical-dependent mecha-
nisms, thiol-dependent free radical formation from
the doxorubicin-iron complex seems to be the most
attractive model for tumor cell killing by doxoru-
bicin, even though it is far from certain whether such
a mechanism actually takes place in the intact tumor
cell, since doxorubicin-iron complexes have not been
demonstrated in intact cells, and may not even exist
in intact cells under clinically relevant conditions
(Gelvan and Samuni, 1988).
3.3. DOXORUBICIN-DEPENDENT HYDROXYL RADICAL
FORMATION BY INTACT TUMOR CELLS
As appears from the data summarized in Section
2.1, in all models for doxorubicin-induced free radical
damage, the ultimate damaging species is presumed
to be the hydroxyl radical. Therefore several groups
of investigators have attempted to detect 'OH
formation in intact tumor cells after treatment with
doxorubicin.
Using spin-trapping, Sinha et al. (1987a, b) de-
tected hydroxyl radical formation in MCF7 human
breast cancer cells; however, the doxorubicin concen-
trations used were three orders of magnitude higher
than that needed to cause 50% inhibition of
growth (IC50). Furthermore, in a doxorubicin-
resistant variant, hydroxyl radical formation was still
undetectable, even at concentrations that were 100-
fold higher than the IC50 value for this variant. This
indicates that hydroxyl radicals measured under such
experimental conditions are not necessarily involved
in the cytotoxic effect of doxorubicin. The observa-
tions that extracellularly added NADPH strongly
stimulated hydroxyl radical formation, whereas both
superoxide dismutase and catalase were inhibitory,
suggest that at least part of the hydroxyl radicals were
generated outside the cells (Sinha et al., 1987a, b; cf.
Alegria et al., 1989). It follows that enzymes at the
outer cell surface might reduce doxorubicin, thus
leading to the formation of superoxide radicals and
hydrogen peroxide through redox-cycling. Since iron
scavengers inhibited hydroxyl radical formation
(Sinha et al., 1987a, b) an iron-catalyzed Haber
Weiss reaction might thus be responsible for hydroxyl
radical formation in this system; alternatively extra-
cellular reduction of doxorubicin-iron complexes
might produce these radicals.
Using methane formation from DMSO as an assay
for hydroxyl radical formation, Doroshow (1986b)
obtained essentially similar data for intact Ehrlich
ascites tumor cells. However, since catalase com-
pletely inhibited hydroxyl radical formation,
Doroshow's results indicate that the hydroxyl radi-
cals detected were generated outside the cells.
Hydroxyl radicals generated at the outer surface of
cells might play a role in the mechanism of tumor cell
killing by immobilized doxorubicin. In this case
doxorubicin is complexed to a synthetic carrier, so
that the drug cannot enter the cells; nevertheless such
a complex is highly cytotoxic (Tritton and Yee, 1982;
Rogers and T6k~s, 1984; Panneerselvam et al., 1987;
Rogers et al., 1983). At sites where the cells make
contact with the immobilized doxorubicin, the local
doxorubicin concentrations may be high enough to
support hydroxyl radical formation after reduction of
doxorubicin by cell surface-bound reducing enzymes.
That redox activity outside the cells, as well as
hydroxyl radical formation, is necessary for the cyto-
toxicity of immobilized doxorubicin, is suggested by
the observations that hydroxyl radical scavengers
together with superoxide dismutase and catalase
inhibited cytotoxicity (Panneerselvam et al., 1987).
Furthermore, immobilized iminodaunorubicin, a
 Doxorubicin (Adriamycin)
derivative of doxorubicin that is less readily reduced
metabolically, was much less cytotoxic than immobi-
lized doxorubicin (Panneerselvam et al., 1987). Such
data underline the possibility that under certain
conditions hydroxyl radicals formed outside the cells
can kill tumor cells. However, this mechanism of
cytotoxicity may be quite distinct from the usual
toxicity mechanism operating against tumor cells,
as is further suggested by the observation that a
pharmacologically inactive analog, 4-demethoxy-7,9-
di-epi-daunorubicin, after coupling to a macromolec-
ular carrier, acquired significant cytostatic activity to
both doxorubicin-resistant and -sensitive L1210 cells
(Rodgers and T6k6s, 1984).
Although OH radical formation has only been
detected at relatively high doxorubicin concentra-
tions at the outer cell surface, we cannot entirely
rule out the possibility that OH radicals are also
formed inside the cells at clinically achievable concen-
trations of doxorubicin, since OH radicals formed
inside the cell might react with cellular constituents
too rapidly to be detected by the relatively insensitive
competitive detection methods that are based on
spin-trapping and reaction with DMSO. To investi-
gate the role of OH radical formation in the cyto-
toxicity of doxorubicin at clinically achievable
concentrations, the effect of several free radical scav-
engers on the cytotoxicity of doxorubicin has been
studied. The findings from these studies are reviewed
in the next section.
3.4. EFFECTSOF RADICALSCAVENGERSON THE
CYTOTOXICITYOF DOXORUBICIN
As indicated in Table 1, extracellularly added
superoxide dismutase or catalase protected MCF7
(Sinha et al., 1987b; Doroshow, 1986a) and Ehrlich
ascites tumor (Doroshow, 1986b) cells against the
cytotoxicity of doxorubicin. However, no protection
by superoxide dismutase and catalase was observed in
A2780 and A2780 AD cells (Cervantes et al., 1988).
Apparently, since superoxide dismutase and catalase
are presumed not to enter cells, MCF7 and Ehrlich
ascites cells are killed to some extent by activated
oxygen species generated outside the cells by a mech-
anism that is probably dependent upon iron, since
iron chelators were also protective, (Ooroshow,
1986a, b; see also Alegria et al., 1989). That A2780
cells are not killed by a similar mechanism, as sug-
gested from the lack of effect of superoxide dismutase
and catalase, indicates that this may not be a univer-
TABLE 1. Effect o f Free Radical Scavengers on the Cytotoxicity o f Doxorubicin
Cell growth in the presence of doxorubicin (%)
Cell type Control + SOD + Catalase
Ehrlich acites 51 72 75
MCF 7 49 93 87
MCF 7 40 80 60
A 2780 31 28 31
A 2780 AD 43-46 46 42
Effect of superoxide dismutase (SOD), catalase, several OH radical scavengers and a synthetic glutathione peroxidase
(PZ 51) on the cytotoxicity of doxorubicin, as reported in the literature. Cells were treated with doxorubicin concentrations
leading to a relative growth of 31-51% compared to untreated controls. The relative growth in the presence of antioxidant
is indicated. ND, not determined.
JPT 47/2--F
225
sal mechanism of cytotoxicity. Furthermore, murine
S180 cells were not protected against doxorubicin by
the iron chelating drug ICRF 187 (Wadler et al.,
1986). An important question now is which cell
model is representative for the most common mecha-
nism of tumor cell killing by doxorubicin.
In this context it is important to note that both
Ehrlich ascites cells and MCF7 cells are exceptional
with respect to the level of their antioxidant de-
fense capacity. Ehrlich ascites cells are known to be
catalase-deficient and to have very low levels of
glutathione-dependent free radical-detoxifying en-
zymes (Bozzi et al., 1981), while MCF7 cells contain
unusually high levels of doxorubicin-reducing en-
zymes (Sinha et al., 1987b), along with a relatively
low glutathione-S-transferase activity (Broxterman et
al., 1989), an important peroxide detoxification en-
zyme. That endogenous antioxidant levels in MCF7
and Ehrlich ascites cells may indeed be limiting for
cellular tolerance to doxorubicin is suggested by the
fact that PZ51, an agent which can be taken up by
cells and function as an intracellular peroxidase in the
presence of glutathione, protects both MCF7 and
Ehrlich ascites cells against the cytotoxicity of dox-
orubicin (Doroshow, 1986a, b). The observation that
doxorubicin-resistant variants of MCF7 cells contain
increased levels of glutathione-S-transferase and glu-
tathione peroxidase (Kramer et al., 1988) further
supports this concept. In several other cell lines
(Ramu et al., 1984; Meijer et al., 1987; Ross et al.,
1988; Keizer et al., 1989b), the level of antioxidant
defense does not increase during selection for doxoru-
bicin resistance, which strongly suggests that in these
cell lines the antioxidant defense does not limit
cellular tolerance to doxorubicin treatment. The same
was concluded for Chinese hamster ovary cells, since
an oxygen-resistant variant having increased levels of
superoxide dismutases, catalase, glutathione and glu-
tathione peroxidase was as sensitive to doxorubicin as
the parental CHO line (Keizer et al., 1988).
In this respect an important observation was made
by Potmesil et al. (1984), who observed that doxoru-
bicin causes two types of DNA strand breaks in
L1210 cells, i.e. protein-associated single-strand
breaks and direct single-strand breaks. Up to a
doxorubicin concentration of 2.8 pM, which kills
more than 99.99% of the cells (Ross and Smith,
1982), only protein-associated strand breaks were
observed (Potmesil et al., 1983). At higher concentra-
tions protein-associated strand breakage decreased,
whereas direct strand breakage increased (Potmesil
et al., 1983). Interestingly, direct strand breakage
'OH scavengers + PZ 51 Reference
87-104 94 Doroshow(1986b)
100 74 Doroshow(1986a)
ND ND Sinha et al. (1987b)
28 70 ND Cervanteset al. (1988)
47-80 ND Cervanteset al. (1988)
226 H.G. KEIZERet al.

occurred only in the presence of oxygen (Potmesil 3.5. ROLE OF FREE RADICALSIN
et al., 1983) and was inhibited by extracellularly DOXORUBICIN-INDUCED CARDIOTOXICITY
added superoxide dismutase or catalase (Potmesil
et al., 1984). These results clearly indicate that free Free radical production in cardiac cells due to
radicals generated at the cell surface have the potency one-electron-reduction of doxorubicin might occur at
to damage the cellular DNA. However, since lethality the nuclear envelope (Bachur et al., 1982), in mito-
of L1210 cells was almost 100% without occurrence chondria (NADH dehydrogenase), cytosol (xanthine
of this type of D N A damage, at doxorubicin concen- oxidase) or sarcoplasmic reticulum (NADPH cyto-
trations which even exceed the clinically achievable chrome P-450 reductase) (Doroshow, 1983a). In this
concentrations in plasma (Potmesil et al., 1984; respect cardiac cells are not exceptional. Neverthe-
Erttmann et al., 1988), a free radical-dependent less, the occurrence of cardiotoxicity is an important
mechanism of toxicity is apparently not very impor- dose-limiting factor in doxorubicin treatment of
tant in the killing of L1210 cells by doxorubicin. cancer patients (Green et al., 1984). The selective
Nevertheless, for cell lines with a relative lack of toxicity of doxorubicin to heart cells might simply
antioxidant enzymes, like Ehrlich ascites and MCF7 reflect an unusually high level of drug accumulation
cells, this type of damage might play a more impor- in these ceils, as reported by several authors
tant role in the cytotoxicity of doxorubicin. (Lampidis et al., 1981; Johnson et al., 1986). In
A rather consistent finding is that scavengers of addition, as discussed below, free radical damage
hydroxyl radicals protect cells against the cytotoxicity might be important for the development of
of doxorubicin (Table 1). However, protection is cardiotoxicity.
typically found only at extremely high scavenger
concentrations, often in the range between 100 and 3.5.1. Free Radical C h e m & t r y o f Doxorubicin in
250 mM (Doroshow, 1986a, b; Cervantes et al., 1988). Cardiac Tissue
Of course, only high "OH scavenger concentrations
are expected to be able to protect against hydroxyl Heart muscle cells are extremely rich in mitochon-
radical damage in view of the extreme reactivity of dria, which might render them particularly vulnerable
this radical. However, in the same concentration to free radicals generated at these organelles. In
range, sodium chloride also protected against the addition, unlike liver microsomes, where doxorubicin
cytotoxicity of doxorubicin (Iliakis and Lazar, 1987). semiquinone radicals react preferentially with molec-
Therefore, there are serious doubts about the specifi- ular oxygen to form relatively harmless superoxide
city of the hydroxyl radical scavengers when used at radicals, semiquinones formed in heart mitochondria
such high concentrations. Thus, although the effects appear to react rather with hydrogen peroxide with
of hydroxyl radical scavengers are consistent with a formation of the highly reactive hydroxyl radical
role for these radicals in the cytotoxicity of doxoru- (Nohl and Jordan, 1983). Compared to liver micro-
bicin, the critical involvement of hydroxyl radicals somes, sarcosomes from heart tissue are relatively
remains unproven. inefficient in reducing doxorubicin to its semiquinone
Not only in vitro but also in vivo effects of radical form (Nohl and Jordan, 1983), probably due to a
scavengers have been investigated on the tumoricidal relative lack of N A D P H cytochrome P-450 reduc-
effect of doxorubicin. Ascorbate (Fujita et al., 1982), tase. However, semiquinones generated at this site
N-acetylcysteine (Freeman et al., 1980; Doroshow et also tend to react preferentially with hydrogen per-
al., 1981 ), cysteine (Freeman et al., 1980), glutathione oxide, even in the presence of oxygen (Thornalley and
(Yoda et al., 1986) and alpha-tocopherol (Myers Dodd, 1985), while in tumor cells this reaction takes
et al., 1977) have been tested in vivo. None of these place only in the absence of oxygen (Bates and
treatments reduced the tumoricidal effect of doxoru- Winterbourn, 1982).
bicin against L1210 (Fujita et al., 1982; Yoda et al., Although intracellular hydroxyl radical forma-
1986), P388 (Freeman et al., 1980; Myers et al., 1977) tion in cardiac tissue could not be demonstrated
or Ehrlich ascites cells (Freeman et al., 1980), while (Rajagopalan et al., 1988), the fact that microscopi-
these treatments did reduce cardiotoxicity in the cally visible cardiac damage starts to occur at the
animals under investigation. Since free radical endoplasmic reticulum and mitochondria (Olson and
damage is likely to be responsible for cardiotoxicity Capen, 1977) suggests that free radical formation
in mice (see below), these data suggest that this type may be involved in the cardiotoxicity of doxorubicin.
of damage is not responsible for the in vivo antitumor
effect of doxorubicin.
3.5.2. Free Radical D a m a g e in Cardiac Tissue
Altogether these results suggest that in the clinical
situation only those cells could be damaged by a free In mice, both with acute (Myers et al., 1977) and
radical-dependent mechanism that are well oxy- chronic (Lazzarino et al., 1987) doxorubicin-induced
genated and exposed to relatively high concentrations cardiotoxicity, lipid peroxidation of heart tissue has
of doxorubicin or have a relative lack of antioxidant been observed, even though this process could not be
defense capacity. Probably this will include only a demonstrated in tumor cells (Myers et al., 1977) or
minor fraction of the tumor cells. However, in heart liver tissue (Lazzarino et al., 1987) of the same mice.
tissue such conditions are met during treatment with This suggests that cardiac tissue is specifically dam-
a single high dose of doxorubicin, and, in fact, acute aged by a free radical-dependent mechanism causing
cardiotoxicity is frequently observed. The possible lipid peroxidation. This is probably not only the
participation of free radicals in the development result of the relative abundance of mitochondria, and
of doxorubicin-induced cardiotoxicity is discussed efficient hydroxyl radical formation in cardiac tissue
below. as discussed above, but also of a relatively poor
 Doxorubicin (Adriamycin)
antioxidant defense, since the heart has rather low
levels of superoxide dismutase and catalase
(Doroshow et al., 1980) and a relatively low rate of
glutathione turnover when compared to e.g. liver
tissue (Griffith and Meister, 1979). Furthermore,
during doxorubicin treatment, glutathione peroxi-
dase tends to become depleted (Doroshow et al.,
1980).
That a relative lack of antioxidant defense is indeed
involved in the development of cardiotoxicity in mice
is suggested by experiments showing that inhibition
of glutathione peroxidase (Doroshow et aL, 1980) or
decreasing glutathione levels in cardiac cells (Olson
et al., 1980) increased the severity of doxorubicin-
induced cardiotoxicity, while agents that protect cells
against free radical damage, including N-acetylcys-
teine (Doroshow et al., 1981; Olson et aL, 1980),
cysteine (Olson et al., 1980), reduced glutathione
(Yoda et al., 1986) and alpha-tocopherol (Myers
et al., 1977), protected against doxorubicin-induced
lipid peroxidation and cardiotoxicity.
In rats, the situation differs from that in mice, since
rat cardiac tissue appears to be unusually resistant to
induction of lipid peroxidation (Mimnaugh et al.,
1981; Julicher et al., 1985). As a consequence, lipid
peroxidation of rat heart tissue is barely detectable
(Porta et al., 1983; Muliawan et al., 1980) and, in
cases where it was detected, did not parallel the
development of cardiotoxicity (Porta et al., 1983).
Furthermore, neither N-acetylcysteine nor alpha-
tocopherol protected rat myocardial cell cultures
in vitro against the cytotoxicity of doxorubicin
(Newman et al., 1981). Therefore, in rats lipid perox-
idation of cardiac tissue is probably not responsible
for the development of cardiotoxicity. On the other
hand, lipid peroxides have been detected in serum of
rats treated with doxorubicin (Thayer, 1984); these
possibly originate from the liver (Mimnaugh et al.,
1981), since this organ is very well suited to support
free radical formation from doxorubicin. Hypotheti-
cally, these peroxides might contribute to cardiotoxi-
city, since heart cells are known to be very sensitive
to exposure to lipid peroxides (Noronha-Dutra and
Steen, 1982). However, this possibility awaits further
verification.
4. FREE R A D I C A L - I N D E P E N D E N T
M E C H A N I S M S OF TOXICITY
As doxorubicin is active against a wide variety of
cancers, while many tumor cell types, as pointed out
above, are not likely to be killed via a free radical-
dependent mechanism of cytotoxicity, efficient
radical-independent mechanisms of cytotoxicity of
doxorubicin must exist. The most widely studied
nonradical-dependent mechanisms of cytotoxicity are
based on the capacity of this drug to intercalate into
double-stranded DNA. It has been known for 20
years that anthracyclines can give rise to an intercala-
tion complex with D N A (for review see Aubel-
Sadron and Londos-Gagliardi, 1984). Possibly as a
consequence of this property, doxorubicin inhibits
both RNA and D N A synthesis, a process which
might be involved in its antitumor effect (Aubel-
Sadron and Londos-Gagliardi, 1984). Non-DNA-
227
binding derivatives of doxorubicin, like AD-32, may
directly interact with the RNA polymerase to inhibit
transcription (Aubel-Sadron and Londos-Gagliardi,
1984).
Recently, a novel mechanism by which doxoru-
bicin, as well as other intercalating drugs, may kill
tumor cells has been described; it is based on interfer-
ence with the D N A breakage-reunion reaction
mediated by topoisomerase II (Tewey et al., 1984a).
This mechanism would explain, why in tumor cells
treated with clinically relevant doxorubicin concen-
trations, both single- and double-strand protein-
associated D N A breaks are observed (Potmesil et al.,
1983, 1984). A major question now is whether this
reaction plays a dominant role in the antitumor effect
of doxorubicin.
Evidence favoring this possibility is derived from a
comparison of treatments that inhibit topoisomerase
II-mediated D N A damage and affect doxorubicin
cytotoxicity. Three conditions are known to inhibit
topoisomerase II in vitro : low temperature (Liu et al.,
1983), high salt concentrations (Tewey et al., 1984b;
Liu et al., 1983) and doxorubicin concentrations
exceeding a critical value between 0.5 and 2.5 #M
(Tewey et al., 1984b). For all three conditions there
is evidence for a similar effect on doxorubicin cyto-
toxicity: (1) Incubation of cells at 0C totally abol-
ished doxorubicin cytotoxicity (Lane et al., 1987); (2)
high salt dramatically protected against doxorubicin
cytotoxicity at concentrations very similar to those
inhibiting topoisomerase II-dependent D N A damage
(Table 2; Liu et al., 1983; Iliakis and Lazar, 1987); (3)
high concentrations of doxorubicin are known to
inhibit the induction of protein-associated D N A
strand breaks (Potmesil et aL, 1983) which is expected
to inhibit its own cytotoxicity. Indeed, clonal survival
curves usually level off at relatively high concentra-
tions of doxorubicin (Barranco, 1984), possibly due
to this phenomenon. Together these data seem to be
consistent with a major role for interference with the
breakage-reunion reaction of topoisomerase II in the
mechanism of cytotoxicity of doxorubicin.
Such a mechanism is further supported by the
observation that topoisomerase II-dependent D N A
damage is inhibited by ouabain (an inhibitor of
cellular Na/K ATPase), and that this inhibition
was closely correlated with decreased cytotoxicity of
doxorubicin in the presence of ouabain (Lawrence,
1988). Interestingly, a decreased endogenous Na/
TABLE 2. Effect of Medium Tonicity on Doxorubicin Cyto-
toxicity and Topoisomerase I1 Activity
Interference with
Medium % Clonal survival topoisomerase II
tonicity after treatment activity
(mOsm) with doxorubicin (arbitrary units)
300 0.1 --
500 5.0 ++
1000 70.0 +++*
1300 100.0 ND
The tonicity of the medium can interfere with both
the cytotoxicity of doxorubicin (Liu et al., 1983) and the
activity of topoisomerase II (Iliakis and Lazar, 1987).
Tonicity was increased by adding NaCI. The asterisk
indicates that interference was still incomplete. ND, not
determined.
228 H. G. KEIZER et al.
K + ATPase activity was observed in doxorubicin-
resistant L5178Y cells (Sugimoto et al., 1981). Fur-
thermore, cell types found to be low in topoisomerase
lI activity were relatively resistant to doxorubicin
(Potmesil et al., 1988). In a doxorubicin-resistant
variant of P388 cells, reduced topoisomerase II
activity was observed (Defile et al., 1989). Further a
cell line selected for resistance to VP-16-213, another
anticancer drug that is considered to have topoiso-
merase II as an important target (Ross et al., 1984),
had a 10-fold lower topoisomerase II activity and was
cross-resistant to doxorubicin (Yalowich et al., 1987).
In addition, cells hypersensitive to doxorubicin
contained an elevated activity of topoisomerase II
(Davies et al., 1988). Lastly, nuclei of doxorubicin-
resistant P388 cells showed less anthracycline-
induced D N A breaks after exposure to doxorubicin
than nuclei from sensitive cells, probably as a
consequence of reduced topoisomerase II activity
(Capranico et al., 1987).
In conclusion, interference with topoisomerase II
activity is an attractive mechanism in the tumoricidal
action of doxorubicin, but its relative importance
remains to be established.
5. S U M M A R Y AND CONCLUSIONS
Doxorubicin or doxorubicin-iron complexes can
initiate the formation of hydroxyl radicals after re-
duction of free doxorubicin or after reduction or auto-
oxidation of the doxorubicin-iron complex. These
hydroxyl radicals can damage cellular membranes by
lipid peroxidation or damage cellular D N A . How-
ever, the relative importance of such free radical
damage in the mechanism of killing tumor cells by
doxorubicin is not clear, because double-stranded
D N A , to which almost all intracellular doxorubicin
is bound, inhibits free radical formation from dox-
orubicin while doxorubicin-iron complexes have
never been detected in cells treated with doxorubicin.
To investigate the possible involvement of hy-
droxyl radicals in the mechanism of cytotoxicity of
doxorubicin, two approaches have been chosen. The
first is to try to detect O H radical formation under
conditions that kill the tumor cells and to investigate
whether O H radical formation correlates with the
cytotoxicity of doxorubicin under various conditions.
The second is to investigate the effect of free radical
scavengers on the cytotoxicity of doxorubicin. Under
conditions that kill tumor cells efficiently, no intra-
cellular hydroxyl radical formation can be detected.
Only at relatively high doxorubicin concentrations
was extracellular O H radical formation detectable.
Experiments with free radical scavengers show that
certain cell types, including normal cardiac cells and
some tumor cell lines with a relative deficiency in their
antioxidant defense capacity, are killed by these
extracellularly generated radicals. However, for the
majority of tumor cells lines, free radical involvement
could not be demonstrated in the mechanism of
cytotoxicity of doxorubicin.
As doxorubicin is active against a variety of solid
tumors and leukemias, while many tumor cell types
are not killed by a free radical-dependent mechanism
of cytotoxicity, efficient nonradical-dependent mech-
anisms of cytotoxicity must also exist. Since doxoru-
bicin at very low concentrations has been shown to
interfere with the activity of topoisomerase II, result-
ing in D N A strand breakage, while both D N A strand
breakage and topoisomerase II activity are correlated
with the cytotoxicity of doxorubicin, interference
with topoisomerase II forms an attractive alternative
for free radical-dependent mechanisms of doxoru-
bicin cytotoxicity against tumor cells.
Acknowledgements--Research in the authors' laboratories is
supported by the Dutch Cancer Society.
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