The ortho-quinone metabolite of the anticancer drug etoposide (VP-16) is a potent inhibitor of the topoisomerase II/DNA cleavable complex

Article abstract of DOI:10.1124/mol.53.3.422

Epipodophyllotoxin derivatives, such as etoposide (VP-16), constitute an important class of anticancer agents, the major cytotoxic effects of which are associated with trapping of the topoisomerase II/DNA cleavable complex and formation of protein-DNA cro

Full text of DOI:10.1124/mol.53.3.422

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MOLECULAR PHARMACOLOGY, 53:422–428 (1998).
The Ortho-Quinone Metabolite of the Anticancer
Drug Etoposide (VP-16) Is a Potent Inhibitor of the
Topoisomerase II/DNA Cleavable Complex
TSVETAN G. GANTCHEV and DAREL J. HUNTING
Medical Research Council Group in the Radiation Sciences, Department of Nuclear Medicine and Radiobiology, Faculty of Medicine, Universite´
de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada
Received September 5, 1997; Accepted November 12, 1997
This paper is available online at http://www.molpharm.org
ABSTRACT
Epipodophyllotoxin derivatives, such as etoposide (VP-16), which indicating that VPQ alters the cleavage-reunion equilib-
constitute an important class of anticancer agents, the major rium of topoisomerase II and DNA mainly by noncovalent inter-
cytotoxic effects of which are associated with trapping of the actions, as does the parent compound. However, we observed
topoisomerase II/DNA cleavable complex and formation of pro- several differences between the effects induced by VP-16 and
tein-DNA cross-links and nicked DNA. VP-16, however, can be VPQ, including a strong inhibition of the second DNA strand
metabolized to several highly reactive products, including an religation, which implies the involvement of additional (asym-
ortho-quinone (VPQ). The inhibitory activity of VPQ against metric) mode(s) of interactions of the VPQ, possibly by inter-
purified human topoisomerase II processing of supercoiled ference with ATP binding by the homodimeric enzyme, and/or
DNA was studied and compared with that of the parent com- involving covalent interactions. Reduced or oxidized glutathi-
pound, VP-16. Our results show that VPQ is a powerful inhibitor one prevented trapping of the topoisomerase/DNA cleavable
of topoisomerase II, which prevents DNA strand passage in the complex by VPQ, but not by VP-16, probably by forming co-
presence of ATP. As with VP-16, trapping of the cleavable valent adducts with the former.
complex is highly reversible upon removal of divalent ions,
Type II topoisomerases alter DNA topology by the forma- drugs (e.g., epipodophyllotoxins) exert their therapeutic ac-
tion of a double-stranded break, followed by DNA strand
passage through an ATP-dependent protein clamp (referred
to as a two-gate mechanism) (Liu et al., 1983; Hsieh, 1983;
Roca and Wang, 1994). The eukaryotic enzymes function as
homodimers and exist in two forms: p170 and p180. Both
forms catalyze DNA strand passage, but the reaction mech-
anisms are different: the p170 form relaxes SC DNA in a
highly distributive manner, whereas the p180 form changes
the DNA linking number in a processive way, although the
exact mechanistic differences remain obscure (Drake et al.,
1989). DNA strand passage is preceded by the formation of a
transient DNA-protein (cleavable) complex, anchored by co-
valent phosphotyrosil bonds between the active site of the
homodimeric enzyme and the 5Ј-DNA ends of the cleaved
strands of the gate-segment of DNA (Hsieh, 1990; Wigley,
1995). The final stages of DNA processing by topoisomerases
are the religation of the cleaved strands, followed by release
of the DNA. Most of the topoisomerase-targeting anticancer
tion by a specific and reversible block of the DNA-strand
rejoining steps, resulting in trapping of the covalent topoi-
somerase II/DNA cleavable complex (Chen et al., 1984), ac-
cumulation of protein-linked DNA breaks, late S and/or G2-
phase cell-cycle arrest and, ultimately, cell death (Wozniak
and Ross, 1983; Ross et al., 1984; Markovits et al., 1987;
Gantchev et al., 1996). One of these topoisomerase poisons,
used in the treatment of a number of neoplastic disorders, is the
epipodophyllotoxin VP-16 [4Ј- demethyl-epipodophyllotoxin-9-
(4, 6-O-ethylidene-␤-D-glucopyranoside)]. VP-16, however,
can undergo oxido-reductive transformations in cells. Two
major pathways have been identified: O-demethylation cat-
alyzed by cytochrome P450-dependent monooxygenases (van
Maanen et al., 1987), and one- or two-electron oxidation
mediated by some peroxidases (Haim et al., 1986) and tyrosi-
nase (Gantchev et al., 1994). These enzymatic transforma-
tions affect the pendant dimethoxyphenolic group (E-ring)
of the drug, leading to several products, the major one
being the ortho-quinone derivative VPQ, as summarized in
Fig. 1.
This work was supported by the Medical Research Council of Canada.
ABBREVIATIONS: VP-16, etoposide; VPQ, ortho-quinone derivative of etoposide; LNR, linear DNA form; NC, nicked circular DNA form; PRLX,
partially relaxed DNA form; RLX, relaxed DNA form; SC, supercoiled DNA form; GSH, reduced glutathione; GSSG, oxidized glutathione; SDS,
sodium dodecyl sulfate.
422

Topoisomerase II Inhibition by VP-16 Ortho-Quinone
423
den). The structure and purity of VPQ were confirmed by high
performance liquid chromatography, IR spectroscopy (carbonyl
bands at 1627, 1661, and 1709 cmϪ1) and mass spectrometry. In all
experiments, SC DNA, pBR 322 (ϳ30 superhelical turns per mole-
cule) was used as a topoisomerase II substrate. Stock DNA was
purchased from Pharmacia Biotech (Uppsala, Sweden), contained an
average of 15–17% nicked circle DNA and was used as supplied after
suitable dilutions.
Topoisomerase II reactions. Topoisomerase II processing of SC
DNA includes the following events: binding to DNA, strand-cleavage,
strand-passage, strand-religation, and enzyme turnover (ATP-de-
pendent). To monitor the effects of VP-16 and VPQ on the overall
reaction of topoisomerase-catalyzed relaxation of SC DNA, standard
assays were performed in 10 mM Tris, pH 7.7, 50 mM NaCl, 50 mM
KCl, 0.1 mM EDTA, 5 mM MgCl2 in the presence, or absence of 0.5
mM ATP. Unless otherwise stated, reduced thiols and bovine serum
albumin, which were expected to affect VPQ activity, were avoided.
The topoisomerase/DNA complex was formed by mixing the enzyme
and DNA on ice and the reactions were incubated for different times
at 37° (unless otherwise stated). Typically, reactions contained 6
units of the enzyme (ϳ13 nM) and 100 ng of pBR 322 per 20-␮l
sample. Special experiments were performed to evaluate the effects
of incubation times and addition order of reagents (topoisomerase,
DNA, and drugs) on the inhibitory efficiency of VP-16 and VPQ.
Cleavage products were trapped with 2 ␮l of 10% SDS, followed by an
additional 5 min incubation at 37° in the presence of 10 mM EDTA
and 200 mM NaCl. Thereafter, samples were treated with proteinase
K (0.8 mg/ml final concentration at 55° for 2 hr). The reversibility of
the drug-trapped cleavable complex was assayed after chelation of
divalent metal ions by the addition of 2 ␮l of 100 mM EDTA before
addition of SDS, while keeping samples at 37°, or at elevated tem-
perature (56–58°) for 5 min. Kinetics of DNA strand religation reac-
tion after topoisomerase-mediated DNA cleavage in the presence of
Ca2ϩ ions and in the presence or absence of drugs were evaluated
essentially as described (Osheroff, 1989).
Samples were subjected to electrophoresis in 1.3% agarose in the
presence of 0.7 ␮g/ml ethidium bromide at 2 V/cm for 18 hr. In the
presence of ethidium bromide, all DNA forms were separated and
migrated as follows: RLX (fastest band), SC, LNR, and finally NC.
Under these conditions, topoisomers of intermediate superhelicity
(PRLX, observed also in the absence of ethidium) were also resolved
when the average change of the linking number was between 10 and
20%. Negatives of gel photographs (Polaroid type 55 film; Polaroid,
Cambridge, MA) were scanned and DNA bands were quantified
using NIH Image (version 1.58) together with appropriate calibra-
tion curves. Numerical data for drug induced effects were expressed
as percent difference from control: % DNA form ϭ 100 ϫ (C/T Ϫ
Co/To)/(Co/To), where C is the quantity of a given DNA form and T is
the total DNA in a sample, and Co and To are the corresponding
values for the control sample.
Fig. 1. Major oxido-reductive pathways of metabolic transformations of
etoposide (VP-16) mediated by P450 monooxygenases and/or peroxidases.
These transformations alter the pendant dimethoxyphenolic group (E-
ring): (1) VP-16; (2) phenoxyl free radical; (3) catechol; (4) semiquinone
free radical; (5) VPQ.
In addition to any inhibitory effects resulting from nonco-
valent interactions, the cytotoxicity of the VP-16 ortho-qui-
none might also result from its nucleophilic character (i.e., its
ability to form covalent adducts with -SH and -NH2 groups of
enzymes) and/or its conversion to a toxic semiquinone radical
(Kalyanaraman et al., 1987). Earlier studies have also shown
that VPQ can directly induce damage to DNA (e.g., by form-
ing adducts in alkaline solutions) and that glutathione pro-
tected DNA from inactivation (Mans et al., 1991; Mans et al.,
1992). Other studies have correlated the antioxidant poten-
tial of cells (i.e. suppression/enhancement of VP-16 metabolic
transformations) with VP-16 toxicity (Gantchev et al., 1994;
Yokomizo et al., 1995; Yalowich et al., 1996; Gantchev and
Hunting, 1997a). Analysis of the effects of the ortho-quinone
VP-16 derivative on topoisomerase II processing of SC DNA
is the subject of the present study.
Materials and Methods
Reagents. Purified human type II topoisomerase (p170 form, 2
units/␮l) was obtained from TopoGene (Columbus, OH). Activity was
measured according to the unit definition of the supplier: 1 unit of
topoisomerase II decatenates 0.2 ␮g of catenated DNA in 30 min at
Results
Effect of drug concentration and addition order of
reagents. The gel photographs presented in Fig. 2 show the
37°. No nuclease or topoisomerase I contamination was detected. effect of different concentrations of VP-16 and VPQ on topoi-
VP-16 was purchased from Sigma (St. Louis, MO) and stock solu-
tions in dimethylsulfoxide were kept at Ϫ20°. The ortho-quinone
derivative VPQ was synthesized by controlled potential electrolysis
of VP-16 at a Pt-gauze electrode under nitrogen atmosphere as
described previously (Holthuis et al., 1985). Reaction rates were
followed by optical spectroscopy (broad absorption band between 300
and 400 nm, characteristic for the quinone and centered at ␭ ϭ 356
nm). Purification of the product from unreacted starting material
and impurities (mainly the aromatized derivative of VP-16) was
performed by chloroform extraction followed by semipreparative liq-
somerase II processing of SC pBR 322 DNA. As is well known
for VP-16 (Chen et al., 1984; Ross et al., 1984), and demon-
strated here for VPQ, the two drugs have a strong effect on
the topoisomerase II/DNA cleavage/religation equilibrium,
as shown by the increased formation of nicked DNA forms
(LNR and NC). Quantitative analysis performed by measur-
ing band intensities on photographic negatives reveals the
rapid accumulation of linear DNA with drug doses up to
about 25 ␮M for both VP-16 and VPQ (Fig. 3). Thereafter, the
uid chromatography on LH-50 Sephadex (Pharmacia, Uppsala, Swe- amounts of linear DNA change only slightly with increasing

424
Gantchev and Hunting
Fig. 3. Accumulation of linear (Œ, ‚) and relaxed (F, E) DNA forms at
the end of the reaction in the presence of different concentrations of
VP-16 (filled symbols) and VPQ (open symbols). Reaction conditions as in
Fig. 2. Averaged data (mean Ϯ standard deviation) from 3 independent
experiments. ء, DNA is only partially relaxed in the presence of increas-
ing VPQ concentrations.
Fig. 2. Topoisomerase II-catalyzed reaction products during pBR322 re-
laxation in the absence or in the presence of different concentrations of
VP-16 (A) and VPQ (B). Lane 1, 100 ng of pBR 322; lane 2, 6 units of
topoisomerase/100 ng of pBR 322 (no drugs, control); lanes 3–9, topoi-
somerase II/DNA in the presence of 0.5 ␮M (lane 3), 1.25 ␮M (lane 4), 2.5
␮M (lane 5), 12.5 ␮M (lane 6), 25 ␮M (lane 7), 62.5 ␮M (lane 8), and 125 ␮M
(lane 9) drugs. Reaction conditions: drugs were mixed with the enzyme in
cleavage buffer containing 0.5 mM ATP and incubated on ice for 10 min;
10 ␮l of the enzyme solutions were mixed with 10 ␮l DNA in the same
buffer and further incubated for 8 min at 37°; the reactions were termi-
nated by SDS. Positions of RLX, SC, NC, and PRLX are indicated.
product formation (nicked and relaxed DNA forms). We ex-
plored all three possibilities: preincubation of drugs with the
purified enzyme or with DNA before mixing to form the
complex, and incubation of the already formed complex with
the drugs. Typical gel photographs are shown in Fig. 4, A and
concentration of VP-16 or VPQ (Fig. 3). Formation of linear B, for VP-16 and VPQ, respectively. These gels also show the
DNA was accompanied by a monotonous increase in the reversal of the trapped complexes induced by elevated tem-
levels of NC DNA within the entire range of drug concentra- perature in the presence of EDTA (see below). Band intensi-
tions used (not shown). Trapping of the topoisomerase/DNA ties were measured and data are presented as percent differ-
cleavable complex in the presence of either VP-16 or VPQ ence from control (topoisomerase/DNA in the absence of
resulted in a decreased formation of RLX DNA (Fig. 3). How- drugs, Fig. 5). The levels of trapped topoisomerase/DNA com-
ever, there was a marked difference in the rate of this con- plexes, which generated linear DNA after digestion with
version in the presence of VP-16 versus VPQ as seen in Figs. proteinase K, were highest when drugs were added directly
2 and 3. In the case of VP-16, the concentration dependence to the already existing complex (stronger for VPQ), followed
of the formation of LNR DNA and the inhibition of DNA by the case when drugs were first preincubated with DNA.
relaxation followed similar trends [e.g., steep slopes below 20 Changes in the levels of RLX and SC DNA forms for the
␮M and gentle slopes (plateau) up to 125 ␮M]. In contrast, individual drugs were less pronounced when drug addition
inhibition of DNA relaxation by VPQ was much stronger and order was changed. Incubation of VPQ with DNA alone, even
at concentrations Պ25 ␮M, no RLX DNA was formed; instead, at elevated temperatures, did not result in any detectable
PRLX (topoisomers with intermediate superhelicity, Fig. 2B) changes in the DNA migration pattern (not shown).
were observed, and at higher VPQ concentrations, neither
Reversibility of the trapped topoisomerase/DNA
PRLX nor RLX DNA were formed (Fig. 2B and Fig. 3). The complexes and rates of DNA strand religation. Enzyme-
above results indicate that although VP-16 and VPQ exhibit mediated DNA breaks resulting from trapping of the cleav-
similarities in their mode of action as inhibitors of the topoi- able complex by VP-16 have been shown to be reversible [i.e.,
somerase/DNA cleavable complex, the mechanism(s) may be cleaved DNA strands can be religated upon addition of salt,
different and/or VPQ may possess more than one mode of EDTA, or by dilution or heating (Hsiang and Liu, 1989;
action. In addition, our previous results showed that VPQ Osheroff, 1989; Robinson and Osheroff, 1991)]. Experiments
does not inhibit the enzyme before its binding to DNA; were performed to determine whether the cleavable complex
rather, it suppresses topoisomerase processing of DNA after trapped by VPQ could also be reversed under different con-
the complex has been formed (Gantchev and Hunting, ditions.
1997b).
As shown in Fig. 4, if the reaction in the presence of either
We next compared the effect of the addition order of re- VP-16 or VPQ was stopped by EDTA and heating before
agents (topoisomerase, DNA, and inhibitors) on the rate of addition of SDS, double-stranded DNA breaks were reversed

Topoisomerase II Inhibition by VP-16 Ortho-Quinone
425
Fig. 5. Numerical presentation of the results obtained following differ-
ent addition orders of reagents, as shown in Fig. 4, lanes 2–5. Results are
presented as percent difference from control (topoisomerase/DNA with-
out drugs). Bar groups: 1, drugs preincubated with the enzyme; 2, drugs
preincubated with DNA; and 3, drugs added to the existing complex. DNA
forms as indicated. Averaged data from three independent experiments
(mean Ϯ standard deviation). p, RLX; s, SLC; z, LNR.
Fig. 4. Effect of addition order of reagents on topoisomerase II process-
ing of supercoiled pBR 322 DNA in the presence of VP-16 (A) and VPQ (B)
and the effect of Mg2ϩ removal by EDTA at elevated temperature. Lane
1, 100 ng of DNA; lane 2, 6 units of topoisomerase II/100 ng of DNA (no
drugs); lane 3, drugs were preincubated with the enzyme alone for 10 min
at 0°; lane 4, drugs (25 ␮M) were preincubated with DNA for 10 min at 0°;
lane 5, drugs were added to the mixture of topoisomerase/DNA and
incubated for 10 min at 0°. After preincubation on ice the full component
reactions were incubated for 6 min at 37°. Reactions in lanes 2–5 were
terminated normally by addition of SDS. Reactions in lanes 6–8, where
samples were treated identically to lanes 3–5, were additionally incu-
bated for 5 min at 56° in the presence of EDTA/NaCl and terminated
thereafter by addition of SDS.
(i.e., religation of one or both of the cleaved DNA strands
occurred). Reversal of the double-stranded DNA breaks of the
drugs trapped complex also took place when the reaction was
stopped only by EDTA/NaCl at 37°, or when the reaction was
performed in the absence of ATP, conditions that prevent
enzyme turnover (Fig. 6). However, there were several im-
portant differences between VP-16 and VPQ, concerning both ence of 6 units of topoisomerase II performed in a cleavage buffer con-
the effect of the two drugs on the product yields as well as the
absence or presence of ATP. One of the major features during
Fig. 6. Reaction products after cleavage of 100 ng of DNA in the pres-
taining Mg2ϩ and VP-16 or VPQ, but no ATP, and the effect of removal of
Mg2ϩ ions by EDTA at 37° before protein denaturing by SDS. Lane 1,
DNA; lanes 2–3, topoisomerase II/DNA; lanes 4–5, enzyme/DNA com-
the reversal of the topoisomerase/DNA complex trapped by
VPQ was the appearance of additional slow-moving bands
when the reaction was performed in the presence of ATP (Fig.
4, *), but not when this cofactor was absent (Fig. 6). These
new bands could not be eliminated by altering the digestion
plexes trapped by 25 ␮M VP-16; lanes 6–7, enzyme/DNA complexes
trapped by 25 ␮M VPQ. Drugs were added to already existing complexes
and the reactions were incubated for 30 min at 37°. Lanes 2, 4, and 6, SDS
was added before EDTA/NaCl; lanes 3, 5, and 7, EDTA/NaCl was added
before SDS.
conditions with proteinase K. They probably represent par- nicked DNA products formed in the presence of VP-16 and
tially digested DNA/(poly)peptide complexes containing VPQ (LNR and NC) change in a similar manner (i.e., LNR
VPQ(poly)peptide covalent adducts. Fig. 7 compares the DNA decreases by 70–75%, and NC increases; the effects are
changes in product yields after the EDTA/NaCl reversal pro- more pronounced in the case of VPQ) (Fig. 7B). In all cases
cedure of the cleavable complex trapped by either of the two (topoisomerase, topoisomerase/VP-16, and topoisomerase/
drugs and in the presence or absence of ATP. Data are plotted VPQ), reversal of the cleavable complex enhances the levels
as percent difference from control, where the controls are of SC DNA, as expected, indicating that under these condi-
levels of each DNA form when the reaction was stopped by tions (no ATP), complexes were mainly trapped before the
SDS, before the addition of EDTA/NaCl. Note that in the strand-passage event. In the presence of ATP, however, the
absence of ATP, no measurable levels of RLX DNA were effects of the two drugs were significantly different (Fig. 7A).
formed in the presence of drugs (Fig. 6) (only minor quanti- When the complex was trapped by VPQ, the reversibility
ties of PRLX were observed). In the absence of ATP, the procedure resulted in a significant increase in SC DNA (i.e.,

426
Gantchev and Hunting
appearance of LNR DNA) in the absence of inhibitors (topoi-
somerase II only), or in the presence of VP-16 was fast and
kinetically unresolved. A gradual disappearance of LNR
DNA within about 50 sec after initiation of religation by
Mg2ϩ was observed when the cleavable complex was trapped
by VPQ (Fig. 8). This result indicates that VPQ decreases
substantially the rate of first-strand religation compared
with the enzyme alone or in the presence of VP-16. It has
been shown previously that the religation rate of the second
cleaved strand is about an order of magnitude lower than
that of the first strand (Osheroff, 1989; Robinson and Osher-
off, 1991). In this case, the kinetics were well resolved and we
were able to monitor the time dependence of the changes of
the levels of NC DNA (Fig. 8) in the absence or presence of
drugs. The rate of religation of the second strand was about
2.8-fold lower in the presence of VP-16 compared with the
enzyme alone. In contrast, when the complex was trapped by
VPQ, instead of a time-dependent decrease in the levels of
NC DNA, an initial (within the first 20–30 sec) increase in
the amount of NC DNA was observed, followed by a plateau.
This result is interpreted as evidence of a strong inhibition of
the religation of the second DNA strand by VPQ.
The role of GSH and GSSG. It has previously been
shown that GSH can form covalent adducts with VPQ, but
not with VP-16 (Mans et al., 1992). In the present work, we
examined the effects of GSH and GSSG on the efficiency of
the two drugs to trap the topoisomerase/DNA cleavable com-
plex. Because glutathione and other reduced thiols are usu-
ally added to the enzyme assays to prevent oxidative inacti-
vation of the topoisomerase, we performed the experiments
so that the controls (topoisomerase/DNA without drugs) were
in the presence or absence of thiols. Drugs (25 ␮M) were
Fig. 7. Comparison of the effects of the complexing of Mg2ϩ ions by
EDTA on the levels of different DNA forms produced by topoisomerase II
(6 U) during cleavage in the presence, or absence of drugs, and in the
presence (A), or absence (B) of ATP. Reaction conditions as in Fig. 6. Data
presented as % difference from controls (samples where the reaction was
terminated by SDS before the addition of EDTA). Averaged data from
triplicate experiments. p, RLX; s, SLC; z, LNR; `, NC.
inhibition of DNA strand passage) and a stronger increase in
NC DNA (2–3 times higher than in the case of VP-16). This
latter difference prompted us to perform experiments de-
signed especially to monitor strand religation kinetics. Pre-
vious kinetic analyses revealed that topoisomerase II reli-
gates cleaved DNA by a sequential two-step mechanism
(Robinson and Osheroff, 1991), and the apparent rate con-
stant for first- and second-strand religation is reduced ap-
proximately 3-fold in the presence of VP-16 (Osheroff, 1989).
In our experiments, DNA cleavage was performed in the
presence of Ca2ϩ ions, the complexes were trapped with
EDTA, and religation was initiated by the addition of Mg2ϩ
ions (Osheroff and Zechiedrich, 1987). Under these experi-
Fig. 8. Effects of VP-16 and VPQ on DNA strand religation reaction of
topoisomerase II. The religation of the first strand (i.e., conversion of
LNR DNA to NC) was kinetically resolved only in the case of the VPQ
trapped complex (). Transformation of the second strand (i.e., conver-
sion of NC DNA to negatively supercoiled form) was resolved for topoi-
somerase II without drugs (F); for topoisomerase II/DNA complex
mental conditions, religation of the first cleaved strand (dis- trapped by VP-16 (f), and by VPQ (Œ).

Topoisomerase II Inhibition by VP-16 Ortho-Quinone
427
preincubated with 3 mM GSH or GSSG for 15 min before enzyme alone. However, our results point out several differ-
mixing with topoisomerase/DNA in cleavage buffer, contain- ences between the mode of action of the two drugs. VPQ is
ing ATP, followed by incubation for 6 min at 37°. As seen in obviously a stronger inhibitor and traps the complex predom-
Fig. 9, the presence of GSH or GSSG does not induce changes inantly before the strand-passage event (in the presence of
in the trapping of the cleavable complex by VP-16 (i.e., LNR ATP). Previously, it has been shown that VP-16 inhibits the
DNA formation); however, virtually no linear DNA is formed complex, both before and after strand passage (Robinson and
in the presence of VPQ and either GSH or GSSG. Although Osheroff, 1991), which is consistent with our experimental
no further analysis of the interaction of VPQ with both thiol data. Thus, in the reversibility experiments performed in the
compounds was performed in this study, it can be suggested presence of ATP, trapping of the complex by VPQ resulted in
that VPQ, as a nucleophile, may attack the -SH groups of the an increased formation of SC and NC DNA compared with
reduced glutathione as well as the amino-terminal groups of the corresponding controls and in contrast to VP-16, where
both peptides (Kalyanaraman et al., 1987). In addition, it is reversal resulted in an increase in RLX and a decrease in SC
clear that the VPQ-glutathione conjugates are not able to DNA. These data indicate that, although VPQ may interfere
trap the topoisomerase/DNA cleavable complex.
with the topo II-DNA cleavage reaction, it predominantly
interacts with the cleavable complex and affects the strand-
passage event. The involvement of multi-step conformational
rearrangements is a prerequisite for topoisomerase process-
ing of DNA (Roca and Wang, 1994). Consequently, it is not
surprising that VPQ may exhibit additional mode(s) of action
against the topoisomerase/DNA complex in interactions that
depend on the stage of the enzyme cycle. Apart from the
residues that are important for ATP binding (Lys 103 and
Lys 337), the ATPase carboxyl-terminal regions of DNA gy-
rase and topoisomerase II are unusually enriched with argi-
nine residues (i.e., potential targets for nucleophilic attack)
(Wigley et al., 1991; Berger et al., 1996). Therefore, possible
interference of VPQ with ATP binding and/or modification of
the DNA strand-passage channel of the complex might be
expected. Different interactions are conceivable, including
coordination with metal ions and/or blocking of essential
amino acid side-chain groups by VPQ. The question of
whether VPQ undergoes covalent chemical reactions with
the complex cannot be answered unequivocally, but it is
likely that covalent adducts are also formed, as evidenced by
the appearance of new slow-migrating DNA bands observed
only after removal of divalent ions by EDTA and in the
presence of ATP before digestion with proteinase K. It has
been shown that quinolone compounds cause asymmetric
conformational changes in the gyrase/DNA complex (Or-
phanides and Maxwell, 1994). This may be also the case with
VPQ and may explain the results obtained in the religation
experiments, namely the strong (asymmetric) inhibition of
the second strand rejoining step in the presence of this drug.
Elucidation of the possible involvement of these additional
modes of action will require further experimental work. It
can be anticipated that when the interactions of VPQ with
the topoisomerase/DNA complex are better understood, this
drug may be a useful tool for mechanistic studies of topoi-
somerase II.
Discussion
The present study shows that the o-demethylated meta-
bolic product of the anticancer drug etoposide, VP-16 ortho-
quinone (VPQ), is a potent inhibitor of the topoisomerase
II/DNA cleavable complex. This is an important finding in
view of structure-activity relationships and implies that sub-
stitution of one of the methoxy groups and the hydroxyl
group on the VP-16 E-ring by keto groups does not adversely
affect trapping of the topoisomerase/DNA cleavable complex.
Several mechanisms of VPQ action against the topoisomer-
ase/DNA cleavable complex are possible and may include
noncovalent (Burden et al., 1996) or covalent interactions, or
may involve slow transformations to a toxic semiquinone
radical (Kalyanaraman et al., 1987). The ortho-quinone
might also act as a metal ion chelator in Fenton-type oxida-
tive reactions (Sakurai et al., 1991). The fact that the topoi-
somerase-mediated double-stranded DNA breaks were
highly reversible indicates that, as for VP-16, the ortho-
quinone derivative inhibits DNA-strand rejoining mainly by
forming a noncovalent complex with the topoisomerase ho-
modimer/DNA duplex and does not inactivate the purified
In this study, we have also shown that GSH and GSSG
prevent the inhibition of the topoisomerase/DNA complex by
VPQ but not by VP-16. In this case, as has been shown
previously (Mans et al. 1992), the process is expected to
involve formation of covalent conjugates between SH- and/or
terminal NH2-groups of the peptides and VPQ. Interestingly,
VPQ did not deactivate the topoisomerase itself, indicating
that there are no accessible and sensitive amino acid groups
in the protein in the absence of DNA. As hypothesized above,
such groups might become exposed to drug attack after con-
formational rearrangements following the formation of the
topoisomerase/DNA complex. The prevention of the topoi-
somerase/DNA complex inactivation by VPQ in the presence
Fig. 9. Effects of thiols (GSH and GSSG) on the efficiency of VP-16 and
VPQ inhibition of topoisomerase II/DNA complex. Data presented as
percent difference from control (topoisomerase II/DNA without drugs, Ϯ
3 mM thiols). DNA forms as indicated. Averaged data from triplicate
experiments. p, RLX; s, SLC; z, LNR.

428
Gantchev and Hunting
Hsieh T-S (1990) Mechanistic aspects of type-II DNA topoisomerases, in DNA To-
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of GSH (or by other protein and nonprotein thiols) can be
expected to play an important role in the cellular toxicity of
this VP-16 metabolite (Yokomizo et al., 1995; Gantchev and
Hunting, 1997a). In agreement with previously reported cy-
totoxicity data (Sinha et al., 1990), our experiments with cells
in culture under similar conditions to those described in
Gantchev and Hunting (1997a) showed that when drugs were
incubated in RPMI 1640 medium, containing 1 mM GSH and
10% fetal bovine serum, VPQ was about 50% less toxic than
its parent compound, VP-16 (results not shown). However,
when the incubation was performed in phosphate buffered
saline instead of medium, both drugs were equally toxic and
promoted formation of DNA breaks at similar efficiencies.
These results imply that VPQ itself probably cannot be ad-
ministered as an anticancer drug, because it will exert its
toxic potential primarily when formed directly in cells, where
one of its effects will be to act as a powerful topoisomerase/
DNA cleavable complex inhibitor. It can be also anticipated
that the levels and distribution of intracellular VP-16 metab-
olizing enzymes (cytochrome P450 monooxygenases and per-
oxidases), as well as the antioxidant potential of a given cell
type, will significantly alter the cytotoxic potency of VP-16.
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Send reprint requests to: Dr. Tsvetan G. Gantchev, Department of Nuclear
Medicine and Radiobiology, Faculty of Medicine, Universite´ de Sherbrooke,
Sherbrooke, QC, J1H 5N4, Canada. E-mail: gantchev@courrier.usherb.ca