(CANCER RESKARCH 5.1. 19-26. January I. IW]
2,2'-Bipyridyl-6-carbothioamide
and Its Ferrous Complex: Their in Vitro
Antitumoral Activity Related to the Inhibition of Ribonucleotide
Reductase R2 Subunit1
Giuseppe Nocentini,2 Federica Federici, l'aimai ¡saFranchetti, and Anna Barzi
Institute of Medinil Pharmacology,
University nf Perugia. Perugia ¡C.N.. r'. F., A. B.I. ami Department of Chemical Sciences. University of Camerino. Camerino IP. F.I. Italy
ABSTRACT
2.2'-Bipyridyl-6-carbothioamide
(BPYTA), a synthetic compound with
antitumoral activity, is characterized In chelating properties hecause of
the N*-N'-S* tridentate ligand system and is therefore comparable to
«•(W)-heterocyclic
carboxaldehyde
thiosemicarbazones
which are potent
inhibitors of ribonucleotide reducÃ-ase (RR).
Klectron paramagnetic resonance studies on the small subunit of mouse
recombinant RR (R2) demonstrated that BPYTA can destroy the R2
tyrosyl radical only if Fe(II) is present (73% destruction at 50 MM,after 20
min of contact). The R2 inhibition was reversible and time dependent.
Studies on tumoral lines confirmed that the main cell target of BPYTA is
RR and demonstrated that the iron-complexed form compared to the
nonchelated form has some difficulty in crossing the cell membrane. Spectrophotometric and electron paramagnetic resonance studies clearly indi
cated that BPYTA chelates iron only when this is reduced and that the
BPYTA-Fe(II) complex is stable in the presence of oxygen.
From reported results we conclude that BPYTA is a powerful RR
inhibitor (R2 subunit) which has a different mechanism of action from
that of Desferal. It has some properties in common with cHW)-hcterocyclic
carboxaldehyde thiosemicarhazones, but they are not identical. It would
be interesting to do further studies on the BPYTA mechanism of action
and evaluate the in vivo antitumoral activity of the preformed complex.
INTRODUCTION
The aim of the present study was to investigate the mechanism of
action of a synthetic compound. BPYTA.1 ( 1) characterized by the
N*-N'-S* tridentate ligand system and therefore comparable to
a-HCATs (2). which are potent RR inhibitors. This enzyme is respon
sible for converting ribonucleoside diphosphates into the deoxyribonucleotide precursors of DNA. and since it is synthesized in small
amounts under tight cellular control the catalyzed reaction is a limiting
factor for DNA synthesis (3-5). RR consists of two nonidentical
subunits. Rl (the larger) and R2 (the smaller), both of which are
necessary for its activity (6. 7). Subunit RI contains nucleotide-binding sites for substrates and allosteric effectors as well as dithioldisulfide groups which participate in the redox reaction (8). Several
synthesized deoxynucleotide analogs are Rl inhibitors. Subunit R2
contains a tyrosyl free radical which is necessary for enzyme activity
and a binuclear ferric iron center (9). The only drug in regular clinical
use for which the primary mechanism of action is believed to be RR
inhibition is HU. but it is not a powerful R2 inhibitor. a-HCATs (e.g..
I-IQ), /V-hydroxy-iV'-aminoguanidine derivatives, HU analogues, and
It has been demonstrated that BPYTA is active in inhibiting tumoral
growth in mice that have received injections of P388 leukemia (I).
Our previous studies demonstrated that BPYTA is highly cytotoxic for
rodent and human tumoral lines (12) and for peripheral blasts from
patients suffering from acute leukemia (13). It can also be active when
it forms a conjugate N*-N*-S* tridentate chelate with transition metals
(12. 14). as the a-HCATs do (15). Other experimental data suggest
that BPYTA has the same cell target as HU and a-HCATs (i.e., the R2
subunit) (16. 17).
We attempted to demonstrate that RR is the main target of BPYTA
and to determine the BPYTA mechanism of R2 inhibition. We com
pared our results with those reported for a-HCATs and DF which are
R2 inhibitors characterized by chelating properties. The simplest ex
planation for the RR inhibition would be that these compounds re
move the R2 iron center. However, this is improbable for various
reasons. First, the binuclear iron center of R2 is "buried" in the core
of the protein (18), suggesting that iron is not available for chelation
except after modification of the protein conformation. Moreover, it
has been demonstrated that a-HCATs (and other molecules with
chelating properties) destroy the R2 radical directly and that the che
lation of iron ions (or other metal ions), from sources other than R2,
potentiates or is essential for causing R2 inactivation (2, 15). It has
therefore been suggested that 1-IQ acts on the enzyme through iron (as
bleomycin does) (19). This hypothesis was confirmed by studies on
cell growth which showed that the addition of iron salts to a-HCATs
potentiates or at least does not affect their cytotoxic activity (15). In
contrast, the cytotoxic activity of DF (like that of other chelators) is
reversed by adding iron to the medium (20). This suggests that DF
inhibits RR activity or intracellular R2 activation by causing iron
starvation. Since iron depletion is life-incompatible, it is improbable
that DF has an in vivo antitumoral activity connected with RR inhi
bition, despite its in vitro antiproliferative activity. Therefore, the
question of whether BPYTA is only a chelator or is also characterized
by the properties of a-HCATs is not only of theoretical interest but
will affect future developments in clinical application.
MATERIALS
Drugs and Chemicals.
BPYTA and its iron (II) complex BPYTA-Fe (mo
lar ratio. 1:1) were synthesized as previously reported ( 1. 14). The commercial
preparations of HU (Oncocarbide; Simes) and DF (Desferal; CIBA-GEIGY)
were used. RPMI 1640. Dulhecco's modified Eagle's medium, sera, and üdditive solutions were purchased from Biochrom-Seromed. ['2'I]-5-Iodo-2'deoxyuridine (35.4 Ci/mmol) was purchased from Amersham. [2-'4C|Cytidine
iron chelators (<-.,?..DF) are also thought to be R2 inhibitors and are
still under study (10. II).
(55.8 mCi/mmol) and the other chemicals were purchased from Sigma.
Drug Solubili/alimi. HU. BPYTA-Fe. and DF were dissolved in hidistilled
water; BPYTA was solubili/ed to a concentration of 18 HIMin DMSO. The
highest DMSO concentration used (0.2^) had no cytotoxic effect with our
testing systems. When necessary, to compare drag permeability. DMSO was
added to the BPYTA-Fe solution at the same DMSO concentration as the one
Received 4/20W2: accepted 10/21/92.
The costs nf publication of this article were defrayed in pan by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by ML'RST 40%.
- To whom requests for reprints should be addressed, al Istituto di Farmacologia
Medica, Università di Perugia. Via del Giochetto. 06122 Perugia. Italy.
'The abbreviations used are: BPYTA. 2.2'-bipyridyl-6-carbothioamide;
AND METHODS
u-HCATs.
we used for BPYTA.
Cell Culture. The following cell lines were used: P388. murine leukemia;
Bin. murine melanoma; HL-60. human promyelocytic leukemia; LoVo. human
a-(/V)-heterocyclic carboxaldehyde thiosemicarhazones; RR. ribonucleotide reducÃ-ase:
HU. hydroxyurea; 1-IQ. 1-formylisoquinoline thiosemicarba/one: DF. deferoxamine mesylalei BPYTA-Fe. complex BPYTA-Fe synthesized at molar ratio 1:1; DMSO. dimethyl
sulfoxide: EPR. electron paramagnetic resonance: ICW. concentration inhibiting 50%;
RTT. dithiothreitol.
colon adenocarcinoma; TA3, murine mammary adenocarcinoma; TA3H2.
made resistant to HU by treating TA3 (21). TA3 and TA3H2 were kindly
19
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MECHANISM
OF ACTION OF BPYTA
provided by Prof. Lars Thelander (Department of Medical Biochemistry and
Biophysics, University of Umeâ,Sweden), and the other cell lines were pur
chased from the American Type Culture Collection.
Cells were grown exponentially in a humidified atmosphere of 5% CO2 and
95% air at 37°C.TA3 and TA3H2 cell lines were maintained in Dulbecco's
modified Eagle's medium containing antibiotics (penicillin. 100 units/ml;
trations. Every thawing procedure resulted in a signal decrease of 10% in the
control EPR spectra, so no more than two thawings were performed for each
sample. The decrease in tyrosyl-radical concentration caused by the com
streptomycin.
peak heights using a standard mouse R2 protein sample of known tyrosyl free
radical content (7).
The regeneration of tyrosyl free radical R2 after treatment with 200 UM
BPYTA-Fe, in the presence of 200 UMferrous ammonium sulfate, was per
formed as follows. The BPYTA-Fe-treated R2 ( 10 min, 37°C)was passed over
pounds was quantified by comparing the spectra peak heights of the treated
sample and the untreated control and expressing it as a percentage. The
absolute radical concentration (5-7 UM)was quantified by comparing spectra
100 |ag/ml: gentamicin, 50 ug/ml) supplemented with 10% heat-
inactivated horse serum and 3 nui glutamine. TA3H2 cells were continuously
exposed to 2 m.wHU during routine passages, but the drug was omitted at least
4 days prior to experiments. The LoVo cell line was maintained in HAM'S F12
medium supplemented
with 3 HIMglutamine,
1% vitamins, and 10% heat-
inactivated fetal calf serum. The other cell lines were maintained in RPMI 1640
medium containing antibiotics (penicillin. 100 units/ml; streptomycin, 100
ug/ml: gentamicin, 50 ug/ml) and supplemented with 3 HIMglutamine, 10 ITIM
4-(2-hydroxyethyl)-!-piperazineethanesulfonic
acid buffer, and 15% heat-in
activated newborn calf serum (for the P388 cell line) or 10% (for the B16 cell
line) or 15% (for the HL-60 cell line) heat-inactivated fetal calf serum. In
addition, the B16 medium was supplemented with 1% vitamins and 1% nonessential amino acids, and the P388 medium was supplemented with 0.01 mm
2-mercaptoethanol.
Antiproliferative Assay. The assay used to evaluate the BPYTA and BPYTA-Fe antitumoral spectrum was developed for the predictive evaluation of
tumor chemosensitivity (22) and was previously used in in vitro studies of new
compounds with potential antitumor activity (23). Briefly, 4 wells of various
concentrations of each drug were placed with tumor cell suspensions (37°C;
0.2 ml; P388, IO4 cells/well; B16, 2.5 x IO3 cells/well; HL-60, 5 x IO4
cells/well; LoVo, 5 X 10" cells/well) for continuous exposure. Alternatively
(pulse contact), cell suspensions were incubated with various concentrations of
each compound in 4-ml tubes (37°C;1 ml; P388, 5 X IO4 cells/ml; HL-60, 2.5
X 10s cells/ml). After l h of contact, cells were washed twice by cemrifuging
and placed (in quadruplicate) in tissue culture plates (37°C;0.2 ml; P388, IO4
cells/well; HL-60. 5 x IO4 cells/well).
After 48 h of incubation, DNA synthesis was evaluated by adding [l25I]-5iodo-2'-deoxyuridine
(0.1 uCi/well) together with 2'-deoxy-5-fluorouridine
the same way. After freezing and recording the spectra (see above), the reduc
ing agent DTT (final concentration, 5 HIM)was added to the thawed samples.
After incubating in a water bath at 25°Cfor 10 min, the samples were frozen
and the spectra recorded. Then, 30 UMferrous ammonium sulfate, dissolved in
deoxygenated solution [Fe(II)], was added to the thawed samples, and after
freezing the final spectra were recorded.
Evaluation of the free radical content in TA3H2 cells (overexpressing R2)
was performed as follows. Cultures were harvested by scraping when there was
50-60% confluence, washed, and concentrated in physiological Tris solution
(0.5-1 x 10" cells/ml). The suspension of cells was divided into 200-ul
aliquots in EPR tubes to which the drug was then added. A control sample was
always present. The tubes were incubated in a water bath at 37°Cfor the
required time and shaken every 5 min. Before freezing in liquid nitrogen the
suspension of cells was carefully rendered homogeneous. To subtract the
background spectrum from the recorded spectrum the samples were thawed
and treated with 0.1 M HU (IO min, 37°C)to destroy all the free radicals
present, and the spectrum thus obtained was considered the background spec
trum. In the control sample the tyrosyl free radical concentration was equal to
0.08-0.21 UM,varying in each experiment.
The oxidation state of iron in the complex, formed by adding DF or BPYTA
to Fe(II), was determined by EPR spectroscopy at 77°K,with a microwave
power of 10 mW, using a Bruker ESP model 300E and an E-238 cavity.
(0.01 ug/well) to the cultured cells for an additional 18 h. During this time the
cells continued to grow exponentially. Harvesting was performed by a multiple
suction filtration apparatus (Mash II) on a fiberglass filter (Whittaker Co.).
Paper disks containing the aspirate cells were read in a gamma scintillation
counter. For each concentration of the compounds tested, cell growth inhibition
was expressed as the percentage of inhibited radioisotope incorporation in the
treated cultures compared with the untreated controls.
A different assay was used to evaluate the cytotoxic activity of BPYTA and
BPYTA-Fe against the TA3H2 cell line (24). Logarithmically growing cells
were treated with trypsin-EDTA. and the cell suspension was plated in 6-cm
dishes (10s cells/dish). After the cells had attached (6 h later) various concen
Spectrophotometric
Studies. Absorption spectra were taken in a Gary
spectrophotometer (model 4). Digitized spectra were taken at I-min intervals
and stored for later retrieval and analysis. All the experiments were carried out
at a controlled temperature of 25°C. Drugs were dissolved in a Tris-KCl
solution (50 tn.MTris-HCl buffer, pH 7.6, plus 100 ITLMKCI).
Inhibition of Ribonucleotide Reductase Activity in Intact Cells. The
following method, developed in our laboratory,4 was used to evaluate RR
inhibition. Briefly, exponentially growing cells (murine leukemia, P388) were
incubated (at 37°C)in a fresh medium (3 X IO6 cells/ml, 4 ml/sample) with
various concentrations of each compound. After 90 min, 0.32 uCi [ 14C]cytidine
trations of each drug were added (5 ml medium/dish, in total). After 66 h, to
determine the effects on growth, the dishes were washed twice and the cells
were treated with 2 ml of 0.1 M sodium hydroxide for 15 min at 50°C.
Absorbance at 260 nm was considered a quantitative measure of cell density
(25). Results are expressed as the percentage ratio of A2N, nm in the treated
cultures versus untreated controls.
EPR Spectroscopy. Tyrosyl free radical concentration in mouse recombi
nant R2 preparations (7) or in highly concentrated cell suspensions was de
termined by EPR spectroscopy at 77°K,with a microwave power of 20 mW,
was added to each sample (0.08 uCi/ml) for a further 30 min. Then the cells
were rapidly washed twice in ice-cold saline solution, put in Eppendorf tubes
(IO7 cell/tube), disrupted with ice-cold 5% trichloroacetic acid for 20 min, and
centrifuged (3000 x g, 4 min) at 4°C.Both the supernatant (acid-soluble
material) and pellet (nucleic acids) were kept and treated separately. When the
TA3H2 cell line (adhering cells) was used, the cells were placed in 50-ml flasks
(4 X 10s cells/flask), incubated for 48 h to allow attachment and logarithmic
growth, and treated as reported for P388. Before disruption with trichloroacetic
acid, the cells were detached from the flasks by 0.25% trypsin (10 min, 37°C)
and put in Eppendorf tubes (1.5 x IO6 cells/tube).
using a Bruker ESP 300E model and an E-238 cavity.
Experiments with recombinant R2 preparations were performed as follows.
Frozen R2 apoprotein (a generous gift from Prof. Thelander) was concentrated,
desalted, and dissolved in a solution of 50 m,MTris-HCl (pH 7.5) and 100 mM
KCI. It was then reactivated with iron, as reported (7, 26), passed over a
Sephadex G-25 column, balanced with the same R2 buffer (column:sample
volume, 10:1 ) to remove the nonincorporated iron, and then stored at -70°C at
The supernatant was neutralized and processed as previously described by
Steeper and Steuart (27) and subsequently modified by Cory et al. (28), to
separate [l4C]deoxycytidine and its nucleotides from [l4C]cytidine and its
nucleotides.
The pellet was resuspended in 800 ul ice-cold 80 ITIMTris (pH 8) to which
DNase-free RNase (250 ug/ml) had been added. The final pH was 7.8. After
2 h of incubation at 37°C,the solution was cooled in ice, and 100 ul 50%
trichloroacetic acid were added. The solution was left for 15 min at 4°C,and
a concentration of 15 UM.Immediately before the experiment it was rapidly
thawed, aliquoted in EPR tubes (200 ul), and maintained in ice. After the
addition of a few microliters of buffer or drug solution, the tubes were incu
bated in a water bath at 37°Cand, after the required interval, were frozen in
the trichloroacetic acid-precipitable material was obtained by centrifuging
(4000 X g, 3 min, 4°C).The supernatant was transferred to the vials and read
in a beta counter to evaluate the RNA-incorporated [14C]cytidine, after the
liquid nitrogen. After recording the tyrosyl radical concentration by EPR
measurements, the samples were rapidly thawed, transferred to ice. and again
incubated in the water bath at 37°C.After the required interval, they were
refrozen and stored in liquid nitrogen, until remeasurement
a Sephadex G-25 column balanced with the same R2 buffer (column:sample
volume, 10:1 ) to remove the drug. A drug-free control sample was treated in
4G. Nocentini. F. Federici, and A. Barzi. A new assay for testing ribonucleotide
of radical concen
reducÃ-aseactivity in proliferating cells, manuscript in preparation.
20
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
MECHANISM
OF ACTION
OF BPYTA
addition of 15 ml of scintillation fluid (Insta-gel). The pellet was washed four
times in ice-cold 5% trichloroacetic acid (5000 x #, 3 min. 4°C)and dissolved
Table 2 Destruction of the murine recombinant R2 radical evaluated by
EPR spectra
in 400 pi 1.25 M NaOH overnight. The sample was prepared for scintillation
counting by mixing it with 19 nil scintillation fluid to evaluate the DNAincorporated |'4C|cytidine.
RR activity was the sum of the | '4C]cytidine cpm incorporated in DNA and
the [l4C]deoxycytidine (plus its nucleotides) cpm. For each concentration of
[BPYTA-Fe]''000105050100200200200400800200
rdcII221024654583867
[Enzyme]"4.5554.51.54.51.51.544.554.54.51.51.5551.556.5IBPYTAl''2
the compound tested the residual en/ymatic activity was expressed as a per
centage ratio of the RR activity in the treated sample versus the control sample.
Data Analysis. The lC-,t]was calculated using Chou's median effect equa
tion (29)
I-/
where C represents the concentration of drug which produces a determined
fractional inhibition on the system. f: represents the fractional inhibition value,
and HIrepresents the Hill-type coefficient. This coefficient indicates the degree
of sigmoid shape in the dose-effect curve. If the correlation coefficient for the
regression line is greater than 0.9. the equation can be considered to tit the
dose-effect relationship. In our analyses the value of the correlation coefficient
was always greater than 0.95.
RESULTS
" Radical concentration
<HM)of the nontreated
protein evaluated by EPR spectra
measurement and compared with a standard sample.
'' Compound concentration (UM)kept in contact with the protein for 5 min at 37°C.The
Fe(II) was ferrous ammonium sulfale. BPYTA-Fe is the preformed complex (molar ratio.
1:1).
' Percentage destruction of the radical in the treated sample with respect to the
In Vitro Antiproliferative Activity. The antiproliferative activity
of BPYTA. BPYTA-Fe. and a ferrous salt are presented in Table I.
ICJ() values were obtained after two different periods of contact be
tween cells and compounds (i.e., 66 h and 1 h plus recovery). The
results indicate that BPYTA was more active than BPYTA-Fe with all
the cell lines tested. The FeSO4 cytotoxicity was insignificant with
respect to that of BPYTA-Fe.
Ability of BPYTA Iron Complex to Destroy R2 Radical. To
evaluate the effect of BPYTA and BPYTA-Fe on R2 we used recom
binant mouse R2 recently obtained by Mann et al. (7). Since the R2
radical is essential for RR activity and the concentration of the tyrosyl
free radical is proportional to the EPR peak, we studied the BPYTA
action on the R2 subunit by recording the EPR spectra (19. 24).
Table 2 gives the relative destruction of the radical caused by
BPYTA. BPYTA-Fe, or BPYTA plus different Fe(II) concentrations.
The complex BPYTA-Fe was very active in destroying the R2 radical.
Results obtained with 200 JJMBPYTA plus 50, 100, and 200 JJMFe(II)
(radical concentration, 1.5 UM)demonstrated that radical-destroying
activity increases as Fe(II) is added, up to a molar ratio of 2:1
[BPYTA:Fe(II)j. When this molar ratio is reached, the amount of
radical destroyed is identical to that obtained with the complex BPY
TA-Fe, if the same radical concentration is considered. A further
untreated sample.
BPYTA+ Fe(ll)
100
200
BO
c
_o
t)
200
10
50
60
V
•¿o
15
40
T3
S
M
20
10
15
20
Time (minutes)
increase in Fe(II) concentration neither increased nor decreased R2
inhibition.
When evaluating BPYTA activity, without the addition of iron, one
must take into account that, although the buffer solution was made
iron free after the reactivation of R2 by iron, slight traces of the metal
were probably still present. So the very low activity of BPYTA can be
explained by a presumed low Fe(II) concentration present in the
Fig. I. Destruction of murine recombinanl R2 radical treated with BPYTA and its iron
complex after 5. 10, and 20 min of contact at 37°C.Symbols linked by lines refer to the
percentage ratio between EPR spectra peak heights for each sample (treated, frozen,
thawed, retreated) and the respective control (warmed, frozen, thawed, warmed up).
BPYTA (200 JIM)plus ferrous ammonium sulfate (2(X) UM)(•)or BPYTA (200 UM)(T)
were also treated directly for 20 min. The free radical concentration of control samples
was 4.5-6.5 UMin the different experiments, at the first evaluation, but every thawingfreezing procedure caused a 10-I.SCf decrease in the signal. Similar inhibition, obtained
after 20 min of contact or 5 plus 5 plus IO min treatment with 2(K)MMBPYTA plus Fe(II).
molar ratio 1:1. demonstrated that the thaw ing-freezing procedure caused no modification
in relative inhibition values.
Table I 1Cw for l
l ¡deo.\yuridine incorporation in tumor cell lines
after pulse and continuous compound-cell contact
ICso" after pulse
cell-compound contaci
(I h. 65 h recovery)
IC^o" after continuous
cell-compound contact (66 h)
Cell
lineBPYTA
BPYTA-Fe
>2(KX)
1435
>IOO>2(KX)
28.0
24.7
33.9
>2000P3886.8
>2000HL-607.8
>2000B166.61607LoVo6.3
FeSO4P388844 >2000HL-60588
" IC-so values refer to the concentration (JJM) which inhibits incorporation of the
radioactive isotope by 50^ in the sample treated with respect to control. Each IC^, value
is the mean of at least 3 runs, analyzed separately.
medium, and it seems reasonable to conclude that BPYTA can destroy
the R2 radical only if iron is present.
Fig. 1 shows the percentage radical destruction as a function of the
time of BPYTA-R2 contact. In the samples treated with BPYTA plus
Fe(II) (molar ratio, I : I ) R2 inhibition was increased by increasing the
concentration of BPYTA plus iron and/or the time of contact. In
particular, the R2 inhibition value of 73% was obtained after 5 min of
contact with 200 (JMBPYTA plus iron and after 20 min with 50 ^M,
indicating that the same R2 inhibition value can be obtained by
21
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MECHANISM
OF ACTION
OF BPYTA
varying the concentration (C) and the time of contact (T) in an in
versely proportional manner (fitting of the C X T law). In the sample
treated with BPYTA (without added iron) the relatively effective
inhibition after 10 or 20 min was independent of the BPYTA concen
tration. On the contrary, it was well correlated to the uncontrolled
increase of iron concentration in the buffer during the time of contact.
In fact. R2 is not completely stable at 37°C,and denatured R2 can
release its iron. The same can happen after the thawing procedure
(every thawing-freezing procedure caused a 10-15% decrease in the
radical signal of the control sample). So, the limiting factor in the
inhibition curve caused by BPYTA seems to be iron rather than
BPYTA. which would confirm that BPYTA can destroy the R2 radical
only if iron is present.
The reversibility of R2 inhibition caused by BPYTA-Fe was dem
onstrated through EPR measurements (see Ret. 19). Fig. 2 gives the
recorded EPR spectra of a R2 sample treated with 200 UMBPYTA-Fe
and in the presence of 200 UMferrous ammonium sulfate, after re
moval of the drug (Fig. 2, spectrum b) and after subsequent treatment
with the reducing agent DTT (5 niM) (Fig. 2, spectrum c). The EPR
spectrum a was recorded from a control sample treated with only
Fe(II), passed over the column, and treated with DTT. It is reported for
comparison with spectrum c. After the passage over the column and
before DTT treatment the control spectrum, comparable with spec
trum b, was just higher (110%) than spectrum a. This would mean that
DTT treatment does not increase R2 radical concentration in untreated
samples. Fig. 2 shows that the R2 free radical concentration was equal
to 0 after removal of the drug but equal to 62% of the untreated sample
after DTT treatment. Addition of Fe(II) to the sample did not signif
icantly increase the reactivation value (65%) (spectrum not shown).
State of the "Active" Drug. The results reported above clearly
indicate that BPYTA is active on R2 when iron is present in the
solution. To evaluate whether BPYTA can form an iron complex and
to study the nature and stability of the complex, spectrophotometric
and EPR tests were performed.
Fig. 2. EPR spectra at 77°Kof the murine recombinant R2 subunit of ribonucleotide
reducÃ-ase. Microwave power = 20 mW. a. untreated sample after passage over the
Sephadex G-25 column and 5 imi DTT treatment: b. BPYTA-Fe-trealed sample, after
removal of the compound, on the Sephadex G-25 column; c. BPYTA-Fe-treated sample
after removal of compound and reactivation with 5 IHMDTT.
700
250
390
450
550
650
300
400
500
eoo
WAVELENGTH (NM)
Fig. 3. UV-visible absorption spectra, a, 100 UMBPYTA (
), 100 UMBPYTA-Fe
). h. 1 [TIMBPYTA in association with I mM ferrous ammonium sulfate (
) or
with 1 HIMferric chloride (
). Addition of 10 HIMsodium diihk late to BPYTA and
subsequent addition of ferrous ammonium sulfate gave rise to a spec rum identical to that
described hv /-'»<•
/// (
-). f. I mM DF in association with I HIMf( rie chloride or 1 mM
ferrous ammonium sulfate (
); I mM DF plus 10 trot sodium dilh onate in association
with I mM ferrous ammonium sulfate (
K 1 mM DF plus IO m sodium dithionale
(
). BPYTA and DF were dissolved in a Tris-KCI solution (pH 7.6) and kept al 25°C.
Spectra a were recorded in a 1-cm path cuvet; spectra b and <•
were recorded in a 2-mm
path cuvet. IO min after mixing solutions.
The optical spectra of BPYTA and BPYTA-Fe dissolved in a TrisKCI solution (pH 7.6) are given in Fig. 3«(Lines I and //, respec
tively). There was no change in the BPYTA spectrum even several
days after dissolution of the compound (25°C),thus demonstrating
that BPYTA is stable in solution. The BPYTA-Fe spectrum had only
minimal modifications when observed after 6 h following the solubilization in DMSO-water and was stable for a week in a water solution.
The addition of an equimolar concentration of ferrous ammonium
sulfate, dissolved in an oxygen-free buffer, to BPYTA caused the
sudden appearance of the BPYTA-Fe spectrum (identical to Fig. 3a,
Line II). The same modification occurred on adding other Fe(II) salts,
dissolved in an oxygen-free buffer, or on adding a small amount of 0.1
MFe(II) salts dissolved in a 0. l MHC1 solution. On the other hand, the
addition of Fe(IlI) salts caused the sudden appearance of a spectrum
similar to the BPYTA spectrum, which can be explained as being the
sum of the BPYTA spectrum and the Fe(III) salt spectrum (not
shown).
Because of the tendency of Fe(III) to quickly form an extremely
insoluble hydroxide salt at basic pH and the tendency of Fe(II) to be
oxidized in air, further experiments were performed to characterize the
BPYTA-Fe complex. Since it is known that the presence of other
chelators (e.g., DF) can accelerate the oxidation of Fe(II) to Fe(III)
and can form a complex with the latter (30). we compared the effects
of BPYTA and DF on Fe(II) solutions. In Fig. 3 (b and c) the spectrum
obtained by first associating BPYTA with a powerful reducing agent
(sodium dithionite) and then with ferrous ammonium sulfate is com
pared with that obtained by associating DF with the same reducing
agent and the same iron salt. The experiments were repeated twice.
The addition of Fe(II) to BPYTA (Fig. 3b) caused the sudden appear
ance of a 580 nm peak, independent of the presence of sodium
dithionite. In the case of DF, sodium dithionite did not allow rapid
oxidation of Fe(II), and the 430 nm peak was very low (Fig. \c, line
VI). These experiments strongly suggest that BPYTA binds iron in the
Fe(II) form, in contrast to the Fe(III)-DF complex. Further evidence
for this is provided in Fig. 4. which gives the EPR spectra of iron
chelated by BPYTA and by DF. Fe(III) in rhombic complexes is
characterized by a quantitative low temperature EPR spectrum, as
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
MHCHANISM
OF ACTION
OK BPYTA
inhibition is rapid, carried out in a way similar to that of HU but
different from that of DF. Comparison of the results obtained using
BPYTA and BPYTA-Fe gave the same information as the EPR studies
on cells: BPYTA is more active than BPYTA-Fe in inhibiting RR
activity in cells.
In other cell growth inhibition studies (Table 3), it was sufficient to
add an equimolar concentration of FeSO4 to render DF completely
inactive (even if iron was added 6 h after the beginning of contact
between DF and cells), whereas BPYTA was only partially inactivated
by FeSO4. and this effect was a function of the timing of addition of
iron and not a function of the iron concentration. Moreover, the
activity of BPYTA-Fe did not change when there was an excess of
iron. The reported results demonstrate conclusively that the BPYTA
40»r
mechanism of action is not due to the depletion of free iron and
Fig. 4. Comparison of EPR spectra at 77"K of iron-chelated compounds in a Tris-KCI
suggest that the different permeability of cell membranes to BPYTA
solulion (pH 7.6). Microwave power = 10 mW. a. 4 imi BPYTA plus 2 nisi ferrous
and BPYTA-Fe could explain their different cytotoxic activity.
ammonium sulfate: b. 4 imi DF plus 4 nisi ferrous ammonium sulfate.
Further studies on TA3H2 demonstrated that the resistance of these
cells to BPYTA and BPYTA-Fe (Table 4) was markedly lower if
shown here tor the DF complex (Fig. 4, Line b), whereas Fe(II) is
compared with their resistance to HU. The addition of Fe(II) to the
generally not visible by EPR. These spectra show conclusively that
medium together with BPYTÀ-Fe did not modify the IC,() and the
BPYTA forms a complex with Fe(II). which remains in this oxidation
IC9() values of either the sensitive or resistant cell lines (data not
state. This is in contrast to the DF complex, which has Fe(III) as its
shown), suggesting that iron starvation was not the cause of the low
stable form. We conclude that BPYTA immediately forms a stable
cross-resistance. In our opinion this could mean that BPYTA inhibits
complex with Fe(II), and this gives rise to the 580-nm absorption
R2 in a different way with respect to HU or that it recognizes other
peak.
(less important) cell targets.
When different concentrations of Fe(Il) were added to 100 UM
The evaluation of RR inhibition in intact TA3H2 cells ([I4C|BPYTA, absorption spectra, qualitatively and quantitatively identical
cytidine method) supports the last hypothesis. The highest BPYTA
to that described by Line II (Fig. 3«).were obtained with molar ratios
concentration used (200 UM)was not able to inhibit RR activity in the
1:1 and 1:2 of Fe(II):BPYTA. A further decrease in the ratio ( 1:3, 1:4)
TA3H2 cell line overproducing R2. Since the concentration able to
resulted in a peak at the visible wavelengths similar to, but lower than,
inhibit cell growth by 50% on TA3H2 (Table 4) was much lower than
that described by Line II. The quantitative analysis of the results is in
the concentration unable to inhibit RR activity, the results were com
good agreement with the hypothesis that, in the presence of an excess
pletely different from those obtained for the wild-type P388 cells and
of ligand, the complex present in solution is BPYTA-Fe (2:1). This
reported in Fig. 5.
again contrasts with DF. which forms a 1:1 complex with iron.
Antiproliferative Activity of BPYTA-Fe and BPYTA Related to
Direct RR Inhibition. To verity whether BPYTA can destroy the DISCUSSION
tyrosyl free radical also when RR is in a physiological condition in the
BPYTA is a compound which exercises a powerful inhibitory ac
cells, the free radical concentration in intact R2 overproducing cells
tivity on tumoral cell growth (12) (see also Tables 1 and 4) and could
(TA3H2) was evaluated by recording the EPR spectra. This cell line
was used because it is characteri/.ed by a powerful EPR signal from
Table 3 IC^ifor I'^^lldetixyitridmi' itian-juirÃ-tÃ-ñm
in tilt' nnirint' Iftikt'tnit celt line
the tyrosyl radical and it is possible to obtain good quantitative in
P3KK after addition of FeSO4
Inhibition was evaluated after 66 h of compound-cell contact. Reponed dala refer to
formation using a relatively low number of cells, even after treatment
one experiment. Two other similar experiments confirmed the reported results.
with drugs which partially destroy the R2 radical. The decrease of the
EPR peaks, caused by BPYTA (with respect to the untreated sample
peaks), was equal to 5l<7r or 28%. respectively, when IO7 or 2 X IO7
cells were kept in contact with 10 UMBPYTA for 20 min in 200 ul.
This demonstrates that BPYTA can destroy the R2 radical in the cells.
Under the same conditions BPYTA-Fe caused no inhibition, but after
the breakage of cell membranes, by freezing and thawing, it became
active while BPYTA did not increase its activity. This result might
suggest that BPYTA-Fe does not easily penetrate the cell membranes,
whereas BPYTA on its own does.
To verify whether RR is the main cell target of the studied com
pound, en/.ymatic inhibition assays were carried out using [I4C]cytidine on P388 murine leukemia. RR activity in intact cells was
measured after 90 min of cell-compound contact by adding [I4C]cytidine for an additional 30 min. In Fig. 5 the enzyme inhibition due
to BPYTA, BPYTA-Fe. HU, and DF is compared with the inhibition
of cell proliferation due to the same molecules (after 66 h of cellcompound contact, evaluated by the ['-5I]-5-iodo-2'-deoxyuridine
in
corporation method in the same cell line). The ratio 1C.,,,growth: cell
ICS()enzyme activity was > 1 for BPYTA, BPYTA-Fe. and HU and
< 1 for DF. This means that cell growth inhibition caused by BPYTA
and its iron complex can be explained by RR activity inhibition. The
Timing of
additionNo
iron
molar
ratio1:1
(MM)26.0825.01
(control)Simultaneous6
addition
1:41:11:4BPYTA(UM)6.3422.31
23.289.7212.531C,,,BPYTA-Fe
26.8425.9327.25DF(MM)68.58>1000
>IOOO>1000
h laterCompoundiFe
>1(XX)
Table 4 Concent mtion.\ inhii>itini>cell t>rowtfì
f//w) h\ 50% ami 90{7t in tumor cell
lines sensitive ÕTA3HT) or 40-fold resistan! to HU (TA3H2)
Inhibition was evaluated after 66 h ot compound-cell contact by measuring the de
crease in the absorbance at 260 nm.
WT0.86
BPYTABPYTA-Fe1C«,
1C*,1C,«
2.2011.05
8.5232.
3.92.98.5
11
1C«,TA3
42.51TA3H22.38 360.04R"2.8
" Resistance index, which is expressed as the ratio between the IC^o (or 1C«*))
of the
tested compound in the resistance cell line and that ot" the sensitive cell line.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
MECHANISM
OF ACTION
BPYTA to which no iron had been added. This again demonstrates
that BPYTA has different properties with respect to DF.
The above results suggest that BPYTA (and its iron complex) is an
RR inhibitor with a mechanism of action similar to that of HU and
I-IQ (i.e.. direct destruction of the R2 tyrosyl radical). As reported for
HU and a-HCATs (9, 19, 31). the R2 inhibition is reversible.
The BPYTA molecular structure and its strong tendency to form a
complex with iron suggest that BPYTA and a-HCATs could share
some properties. Several authors have demonstrated that the iron
chelates of a-HCATs are more effective than free a-HCATs, both in
inhibiting ribonucleotide reduction (32) and in prolonging the survival
time of leukemia-bearing mice (33). It has been proved that the active
form of a-HCATs is a preformed iron chelate ( 15) or, as Thelander and
Gräslundhave more recently demonstrated for 1-IQ (19), they act on
the enzyme through iron. Results reported in Table 2 (relative tyrosyl
free radical concentration in treated R2 protein) clearly suggest that
BPYTA can destroy the R2 radical and inhibit RR only in the presence
of Fe(ll). So, in this respect also, our compound is very similar to
a-HCATs. However, some differences between BPYTA and a-HCATs
\m
BPYTABPYTA-
o•
FeHUDF•o
;i
g
! o0•
5
10
50
100
500
ic50 OHM)
Fig. 5. Anliproliterative
activity (|':5I]-5-iodo-2'-deoxyuridine
OF BPYTA
method) (O) of the
reported molecules compared to their inhibition activity on RR evaluated in proliferating
cells (| MC)c>lidme method) <•).The tumoral line P3SS was used in hoth cases, and
results are expressedas IC^o- Results regarding ribonucleolide reducÃ-ase
inhibition are the
mean of 2 different tests, analy/ed separately. The cpm of control were, respectively.
10257 and 8S4I. Al the highest concentration tested (50 UMBPYTA. 150 MMBPYTA-Fe.
2(XK)UMHU and DF) none of the compounds inhibited RNA synthesis, evaluated by the
same method. Results regarding anliproliferative activity are the mean of al least 3 tests.
anal>/ed separately.
consequently be of some use in the treatment of cancer. We demon
strated that the BPYTA iron complex |BPYTA-Fe(II)] has a high
affinity for the R2 subunit of RR and destroys the tyrosyl free radical
of this subunil (Table 2). In addition, the EPR test, which evaluated the
tyrosyl free radical concentration in R2-overproducing cells (TA3H2).
showed that BPYTA can destroy the R2 radical also in intact cells, and
the results obtained with the P388 cell line, shown in Fig. 5. demon
strated that R2 is the main cell target of the studied compound.
Since BPYTA has metal chelating properties we compared it with
DF to investigate whether it is characterized by the same mechanism
of action. Several results refute this hypothesis. First, the much higher
activity of BPYTA-Fe with respect to BPYTA in inhibiting R2 (EPR
were revealed. It was demonstrated that the concentrations of the
a-HCAT iron chelates inhibiting cell growth were lower than those of
the free ligand (34). Despite the fact that BPYTA-Fe was the only
species inhibiting the R2 subunit, BPYTA was more active than
BPYTA-Fe in inhibiting both cell growth (Tables 1 and 4) and enzyme
activity when tested in intact cells (Fig. 5). We suggest that a slower
cellular uptake of BPYTA-Fe is responsible for the lower degree of
activity in the complex. This hypothesis could also explain the results
shown in Table 3. Equimolar concentrations of FeSO4 added to
BPYTA solution, before the addition of cells, partially decreased
BPYTA activity (IC5() increased =4-fold and was very similar to that
of BPYTA-Fe), while the activity was almost similar to that of
BPYTA when FeSO4 was added after 6 h. It is possible that in the first
case BPYTA rapidly chelated the iron present in the solution (as
demonstrated by spectrophotometric studies) and then had difficulty
in crossing the cell membrane. In the second case BPYTA crossed the
cell membrane, and the addition of FeSO4 did not modify the cyto
toxic activity.
The study of the optical and EPR spectra of BPYTA-Fe suggests
that BPYTA is characterized by a particular iron complex. In fact
a-HCATs can chelate both Fe(II) and Fe(III), and in the presence of
oxygen the complex a-HCATs-Fe(II) is oxidized in a few minutes (32,
35). On the contrary, BPYTA can only form the BPYTA-Fe(II) com
plex (Fig. 3a line II). The fact that the 580 nm absorbing species
spectrum did not change, even a week after dissolution in water,
means that iron retains its reduced form after the BPYTA-Fe(II)
complex is formed, even in the presence of oxygen.
Although it was possible to synthesize both 1:1 and 2:1 BPYTA-Fe
complexes ( 14), several results seem to suggest that the only species
present in solution is the 2:1 complex. In fact, the BPYTA spectrum
in the presence of an equimolar concentration of Fe(II) is identical to
that recorded in the presence of a Fe(II) concentration one-half that of
BPYTA (molar ratio, 2:1 BPYTA-Fe(II)). In addition, radical destruc
tion, caused by a fixed concentration of BPYTA, increased with the
increase of Fe(II) concentration until the molar ratio 2:1 was reached,
and a further increase of iron did not increase R2 inhibition (see Table
2). This not only means that the complex BPYTA-Fe(II) 2:1 is the iron
complex in solution (even in the presence of an excess of iron) but that
this complex is the active form of BPYTA.
On the basis of the present study we can propose a hypothesis about
the interaction of BPYTA and its complex with the cells. BPYTA-Fe(II) 2:1 is the active form of BPYTA and destroys the R2 tyrosyl free
radical. The complex cannot cross cell membranes easily but, once it
has entered the cell, there is no reason to suppose its disassociation.
experiments on recombinant R2 protein: Table 2) suggests that
BPYTA does not remove the iron center from R2. On the contrary,
iron must be present in the medium for it to carry out R2 inhibition.
These findings were supported by studies on RR activity in intact
cells. Since the R2 subunit is relatively unstable (21) and needs to be
continuously replaced with new iron-activated R2, it is possible to
hypothesize that the iron starvation caused by chelators inhibits cell
growth by inhibiting the formation of the iron center of R2. So,
chelators need a longer period of cell-compound contact than that
corresponding to the half-life of R2 (3^ h) in order to inhibit RR
activity. On the contrary. BPYTA inhibited the EPR signal by 40%
after only 20 min of molecule-cell contact (TA3H2 cell line), sug
gesting that its action is not due to iron starvation. The same conclu
sion can be drawn from the results shown in Fig. 5 (RR inhibition in
intact cells, evaluated through labeled cytidine. compared to the antiproliferative activity). HU, BPYTA. and BPYTA-Fe, "directly" ac
tive on the enzyme, had lower IC^oS on the enzyme compared to the
respective ICsoS on cell growth. In contrast. DF had a very high IC50
on the enzyme (with respect the IC5() on cell growth and in absolute
value). In our opinion DF behavior can be explained by the brief time
of cell-compound contact in the tirsi test (2 h) compared to that in the
second (66 h). In fact after 2 h, the enzyme inhibition caused by DF
is very low because part of the R2 subunit, activated before iron
starvation, is still active and DF cannot inactivate RR directly. On the
contrary the antiproliferative activity can be explained by prolonged
iron starvation. Furthermore, inhibition of RR activity and DNA syn
thesis observed at such a high concentration of DF could be correlated
to other effects on the cells and could be aspecific.
Results reported in Table 3 reproduce in our system those described
by Becton and Roberts (20). demonstrating that equimolar concentra
tions of FeSO4 are sufficient to completely nullify the cell growth
inhibition caused by DF. Under all conditions, on the other hand.
BPYTA and BPYTA-Fe were at least in part active. When FeSO4 was
added after 6 h. the cytotoxic activity of BPYTA was similar to that of
24
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
MECHANISM
OF ACTION
()l
HHYIA
sity of Umeâand Goran Wikander. Department of Physical Chemistry. Uni
versity of Umeâ.for access to EPR facilities.
since it remains unmodified in a solution with oxygen present. In
contrast. BPYTA can easily cross cell membranes. It cannot chelate
iron buried in the R2 but can chelate intraccllular Fe(II) supplied by
cellular proteins which contain reducing groups. Once complexed
with iron, BPYTA (originally iron-free) can destroy the R2 radical in
the same way as demonstrated for BPYTA-Fe(II) 2:1. So, in our
opinion, the higher potency of BPYTA with respect to BPYTA-Fe in
inhibiting cell growth is correlated with higher intracellular concen
tration due to an easy crossing of the cell membrane. Furthermore, it
is possible to exclude the possibility that BPYTA cytotoxicity is
caused by the depletion of free iron in the system.
On the basis of previous studies on a-HCAT IQ-1 ( 19) and other
drugs involving iron (36, 37). the most probable hypothesis about the
mechanism by which BPYTA-Fe(Il) destroys the R2 radical is that the
iron complexed with BPYTA may be oxidized, and during this reac
tion very reactive oxygen radicals, which destroy the tyrosyl radical of
R2. are formed. Studies are being carried out to verify whether this
mechanism of action could apply to BPYTA.
Why TA3H2 (resistant to HU) has a low cross-resistance to BPYTA
and BPYTA-Fe (Table 4) is an open question. Since an excess of iron
in the medium did not increase the resistance value of BPYTA-Fe it
seems that the low cross-resistance is not due to iron starvation caused
by BPYTA. The lack of cross-resistance in HU-resistant cell lines was
also found for a-HCATs (38). and so it is probably a common property
of molecules characterized by the N*-N*-S* tridentate ligand system.
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aclivily of melai (II) chclales of 2,2'-bipyridyl-6-carbothioamide:
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Since BPYTA forms a complex with iron before acting, the lack of a
good cross-resistance could be explained in part by the work of
McClarty et al. (39). which supports the conclusion that resistance to
H U leads to alterations in the expression of the gene for the iron
storage protein ferritin. Moreover, the result discussed above suggests
that BPYTA could have different cell targets. While in wild-type cell
lines the R2 inhibition is critical in causing cell growth inhibition, in
HU-resistant cell lines (R2-overproducing) the inhibition of different
cell targets could occur at concentrations lower than those inhibiting
RR enzymatic activity, and this could be critical for determining cell
growth inhibition. This hypothesis could explain why a high BPYTA
concentration (200 UM)did not utfee t the enzymatic activity in TA3H2
(|'4C)cytidine method), while the growth of the same cells was in
M. Nieolini (ed.). Platinum and Other .Melai Coordinaron Compounds in Cancer
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evaluation of 2,2'-bipyridyl-6-carbothioamide
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invesligalion of 2.2'-bipyridyl-6-carbolhioamide
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The R2-overproducing activity of TA3H2 cells could also explain the
discrepancy between the lack of enzyme inhibition ([l4C|cytidine
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17. Nocentini. G.. Federici. F., Armellini. R.. Franchelli. P.. and Barzi. A. Isolalion of Iwo
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19. Thelander. L., and Gráslund. A. Mechanism of inhibition of mammalian ribonucle
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21. Eriksson. S., Graslund. A., Skog. S.. Thelander, L.. and Tribukait. B. Cell cycledependent regulation of mammalian ribonucleotide reducÃ-ase.The S phase-correlated
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23. Cristalli. G.. Franchelli, P., Grifanlini, M., Noeenlini, G.. and Viuori, S. 3.7-Dideazapurine nucleosides. Synlhesis and anliiumor aclivily of 3-deazotubercidin and
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method) after treatment with 200 MMBPYTA (2 h) and the EPR
spectrum decrease (4()9r) in the same cell line after treatment with 10
UMBPYTA (20 min). Probably the overexpression of R2 is so high
(40-fold) (40) that it allows sufficient synthesis of deoxyribonucleotides even after the inuctivution of a great part of R2 detected by the
EPR signal.
In conclusion, it is possible that BPYTA acts on other cell targets,
probably by the same mechanism used to destroy the R2 radical, as
has been suggested for a-HCATs (41, 42). These targets are less
important than the R2. since several results reported here and the
cross-resistance of cell lines made resistant to BPYTA (16, 17) clearly
demonstrate that R2 is the main target in non-R2-overproducing cells.
It would be interesting to investigate the in vivo antitumoral activity
of BPYTA-Fe because of some of its properties (e.g., solubility and
stability).
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ACKNOWLEDGMENTS
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26. Ochiai, E.. Mann. G. J.. Gráslund.A., and Thelander, L. Tyrosyl free radical formation
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The authors wish to express their gratitude to Prof. Astrid Gráslund and
Prof. Lars Thelander. Department of Medical Biochemistry and Biophysics.
University of Umeâ.for providing technical facilities, helpful suggestions, and
critical comments. G. N. wishes to thank Margherela Thelander and Susan
Nyholm for their excellent technical assistance during his work at the Univer
25
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MECHANISM
OF ACTION Ol
28. Cory. J. G.. Mansell. M. M.. George, C. B.. and Wilkinson. D. S. Inhibition of nucleicacid synthesis in Ehrlich tumor cells by periodale-oxidized adenosine and adenylic
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systems. J. Theor. Biol.. (¡5:345-356, 1977.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
2,2′-Bipyridyl-6-carbothioamide and Its Ferrous Complex: Their
in Vitro Antitumoral Activity Related to the Inhibition of
Ribonucleotide Reductase R2 Subunit
Giuseppe Nocentini, Federica Federici, Palmarisa Franchetti, et al.
Cancer Res 1993;53:19-26.
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