Download Activation of clinically used anthracyclines by the formaldehyde

1450
Activation of clinically used anthracyclines by the
formaldehyde-releasing prodrug
pivaloyloxymethyl butyrate
Suzanne M. Cutts,1 Lonnie P. Swift,1
Vinochani Pillay,1 Robert A. Forrest,1
Abraham Nudelman,3 Ada Rephaeli,2
and Don R. Phillips1
1
Department of Biochemistry, La Trobe University, Bundoora,
Victoria, Australia; 2Felsenstein Medical Research Center,
Sackler School of Medicine, Tel Aviv University, Beilinson
Campus, Petach Tikva, Israel; and 3Department of Chemistry,
Bar Ilan University, Ramat Gan, Israel
specificity in sensitive and resistant cells as assessed by
L-exonuclease – based sequencing of A-satellite DNA
extracted from drug-treated cells. Growth inhibition assays
were used to show that doxorubicin, daunorubicin, and
idarubicin were all synergistic in combination with AN-9,
whereas the combination of epirubicin with AN-9 was
additive. Although apoptosis assays indicated a greater
than additive effect for epirubicin/AN-9 combinations, this
effect was much more pronounced for doxorubicin/AN-9
combinations. [Mol Cancer Ther 2007;6(4):1450 – 9]
Abstract
Introduction
The anthracycline group of compounds is extensively used
in current cancer chemotherapy regimens and is classified
as topoisomerase II inhibitor. However, previous work has
shown that doxorubicin can be activated to form DNA
adducts in the presence of formaldehyde-releasing prodrugs and that this leads to apoptosis independently of
topoisomerase II – mediated damage. To determine which
anthracyclines would be useful in combination with
formaldehyde-releasing prodrugs, a series of clinically
relevant anthracyclines (doxorubicin, daunorubicin, idarubicin, and epirubicin) were examined for their capacity to
form DNA adducts in MCF7 and MCF7/Dx (P-glycoprotein
overexpressing) cells in the presence of the formaldehydereleasing drug pivaloyloxymethyl butyrate (AN-9). All
anthracyclines, with the exception of epirubicin, efficiently
yielded adducts in both sensitive and resistant cell lines,
and levels of adducts were similar in mitochondrial and
nuclear genomes. Idarubicin was the most active compound in both sensitive and resistant cell lines, whereas
adducts formed by doxorubicin and daunorubicin were
consistently lower in the resistant compared with sensitive cells. The adducts formed by doxorubicin, daunorubicin, and idarubicin showed the same DNA sequence
Anthracyclines represent a mainstay of treatment for
neoplastic diseases. The major clinically used anthracyclines consist of doxorubicin, daunorubicin, idarubicin, and
epirubicin. Doxorubicin is a broad-spectrum antitumor
antibiotic, and the tumors most commonly responsive to
this drug include breast and esophageal carcinomas,
osteosarcoma, soft tissue sarcoma, Kaposi’s sarcoma, and
Hodgkin’s and non – Hodgkin’s lymphoma. However,
daunorubicin (which differs from doxorubicin only by the
lack of a hydroxyl group; see Fig. 1) is used mainly for the
treatment of acute myelocytic and lymphocytic leukemias.
Idarubicin is used for the treatment of acute myelogenous
leukemias but is also active against multiple myeloma,
non – Hodgkin’s lymphoma, and breast cancer, whereas
epirubicin has found use in advanced breast cancer (1 – 3).
Daunorubicin was the first anthracycline to show antitumor activity and was isolated from the soil bacterium
Streptomyces peucetius. Chemical mutagenesis of this bacterium leads to a variant strain, which produced doxorubicin
(S. peucetius var. caesius; ref. 4). These anthracyclines
proved useful in the treatment of various neoplasms but
suffered from limitations, such as a severe dose-dependent
cardiomyopathy. In a concerted effort to yield new and
improved anthracyclines (an effort that has largely failed),
epirubicin and idarubicin were identified as semisynthetic
derivatives of doxorubicin and daunorubicin, respectively
(5, 6). Epirubicin may offer some advantages over
doxorubicin because it exhibits a lower cardiotoxicity,
and idarubicin is unique because it may be administered
orally due to its increased lipophilicity (1, 7). The structures
of the various clinically used anthracyclines are shown in
Fig. 1.
The anthracyclines are classified as topoisomerase II
inhibitors and can evoke apoptosis via several key pathways (8). In addition, many other possible modes of action
are being increasingly documented (1, 9). It has been
known for several years that anthracyclines, such as
doxorubicin, can also form adducts with DNA. These
adducts have been termed virtual DNA cross-links because
Received 9/7/06; revised 1/31/07; accepted 2/22/07.
Grant support: Australian Research Council (S.M. Cutts and D.R. Phillips)
and Israel Science Foundation grant 542/00-4 (A. Rephaeli and A.
Nudelman).
The costs of publication of this article were defrayed in part 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.
Note: S.M. Cutts and L.P. Swift contributed equally to this work and are
first authors.
Requests for reprints: Suzanne M. Cutts, Department of Biochemistry,
La Trobe University, Bundoora, Victoria 3086, Australia.
Phone: 61-3-94791517; Fax: 61-3-94792467.
E-mail: [email protected]
Copyright C 2007 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-06-0551
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Molecular Cancer Therapeutics
Figure 1. Structures of anthracyclines in current clinical use: doxorubicin (1), idarubicin (2 ), daunorubicin (3 ), and epirubicin (4 ).
dehyde-releasing drug hexamethylenetetramine with doxorubicin also seems promising and is not complicated by
the release of other products with anticancer activity (28).
The capacity of anthracyclines to form DNA adducts, and
how this can be achieved in terms of formaldehyde
activation strategies, has recently been reviewed (29, 30).
If anthracycline/formaldehyde prodrug combinations
eventually prove clinically viable, they may be able to be
used simply as an extension of existing anthracycline
treatment protocols.
To date, the above studies of formaldehyde-releasing
drugs have focused mainly on combination with doxorubicin. In this study, we have attempted to determine the
viability of combining AN-9 with the various clinically used
anthracyclines in terms of efficiency of drug-DNA adduct
formation. The combinations were also compared in
terms of apoptosis and interaction in growth inhibition
assays. Assays were also conducted in P-glycoprotein –
overexpressing cells to determine whether such cell lines
were particularly susceptible to combination treatments.
Materials and Methods
there is a covalent bond to only one strand of the DNA, and
hydrogen bonding of the drug to the other strand of DNA
endows adducts with cross-link – like characteristics, such
as double-strand stabilization (10 – 14). However, it is only
recently that this knowledge of DNA adduct formation
has been exploited in the form of new drug derivatives
and new approaches to therapy. Because the formation of
DNA adducts requires the involvement of formaldehyde,
derivatives of anthracyclines, which have been preactivated
by formaldehyde (doxoform, daunoform, and epidoxoform), have provided an enormous improvement in activity
against various cell lines. In particular, these drugs have
activity against P-glycoprotein – overexpressing cells because they are less efficient substrates of the pump, and
also, DNA adduct formation renders drugs unavailable for
P-glycoprotein – mediated efflux (15 – 18). To overcome
deleterious effects on normal tissues, these drugs have
been tethered to the molecules 4-hydroxytamoxifen and
cyanonilutamide, which target estrogen receptor – positive
breast cancer cells and androgen-sensitive prostate cancer
cells, respectively (19, 20).
An alternative approach is to combine anthracyclines
with intracellular formaldehyde-releasing drugs. Pivaloyloxymethyl butyrate (AN-9) releases formaldehyde as well
as butyric acid and has been used as a histone deacetylase
inhibitor (21 – 23). The combination of daunorubicin with
pivaloyloxymethyl butyrate is synergistic in various cell
lines and mouse models of leukemia (24, 25). Although
initially thought to be due to an interaction of the butyric
acid with the anthracycline, the synergistic interaction with
doxorubicin has been subsequently found to be primarily
due to formaldehyde release, which results in efficient
DNA adduct formation (26). These DNA adducts can
initiate cell death in the absence of any topoisomerase II –
mediated DNA damage (27). Combination of the formal-
Radiochemicals and Molecular Biology Reagents
QIAamp blood kits for genomic DNA isolation were
obtained from Qiagen (Hilden, Germany). The radionucleotides [a-32P]dCTP and [a-32P]dATP were purchased from
GE Healthcare (Bucks, United Kingdom). Random-primed
labeling kits were from Roche Diagnostics (Basel, Switzerland). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide was from Sigma-Aldrich (St. Louis, MO).
Cell Lines
MCF7 and MCF7/Dx cells were cultured in RPMI 1640
(Invitrogen, San Diego, CA) containing 10% FCS (ThermoTrace, Melbourne, Victoria, Australia). The MCF7/Dx cells
were also supplemented with doxorubicin (300 nmol/L)
during passaging. No antibiotics were used in the growth
medium, and growth of cell lines was maintained at 37jC
in a humidified atmosphere of 5% CO2.
Compounds
Doxorubicin and daunorubicin were provided by Farmitalia Carlo Erba (now Pfizer, New York, NY), and
idarubicin and epirubicin were purchased as the clinical
formulations. Pivaloyloxymethyl butyrate (AN-9) was
synthesized as described previously (21). Z-VAD-FMK
was purchased from Promega (Madison, WI).
Southern-Based Cross-Linking Assay
Preparation of Probes for Southern Hybridization. The
plasmid pBH31R1.8 was provided by Dr. V.A. Bohr
(National Institute of Aging, NIH, Baltimore, MD). A
1.8-kb EcoRI fragment containing exons I and II of the
human DHFR gene (31) was isolated from pBH31R1.8 and
radiolabeled with [a-32P]dCTP using a random-primed
labeling kit. The mitochondrial probe pCRII-H1 was a gift
from Dr. C.A. Filburn (National Institute of Aging). An
EcoRI fragment containing the human mitochondrial probe
(corresponding to nucleotides 652 – 3,226) was also prepared by random priming.
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1452 Activation of Clinical Anthracyclines
Drug Treatment of Cells for Southern Analysis. Cells
were seeded in 10-cm Petri dishes at a density of 2.5 106
per dish. Cells were incubated with differing concentrations of anthracycline (dissolved in H 2O) or AN-9
(dissolved in DMSO) in 10 mL complete medium for 4 h.
The final concentration of DMSO in the medium did not
exceed 0.5%. Cells were harvested and genomic DNA was
isolated as described previously (26). All experiments were
done in duplicate.
Detection of adducts by cross-linking assay. Genomic
DNA was quantitated, restriction digested, extracted, and
ethanol precipitated as described previously (26). Pellets
were resuspended in 10 AL Tris-EDTA and 20 AL loading
buffer containing 90% formamide, 10 mmol/L EDTA,
0.1% xylene cyanol, and 0.1% bromphenol blue (final
formamide concentration of 60%). Samples were denatured
at 60jC for 5 min, quenched on ice, and resolved on 0.5%
agarose gels in 1 Tris-acetic acid-EDTA by overnight
electrophoresis at 30 V.
The DNA was then probed using a Southern hybridization procedure as described previously (26). However,
for detection of the mitochondrial genome, membranes
were hybridized overnight at 65jC in 5 Denhardt’s
solution, 0.5% SDS, 5 saline-sodium phosphate-EDTA,
and 100 Ag/mL salmon sperm DNA. Membranes were
washed in 2 saline-sodium phosphate-EDTA at room
temperature for 5 min and then in 2 saline-sodium
phosphate-EDTA/0.1% SDS at room temperature for
15 min. The 15-min wash was repeated and membranes
were washed in 0.1 saline-sodium phosphate-EDTA/
0.1% SDS at room temperature for 15 min. Then, a final
wash in 0.1 saline-sodium phosphate-EDTA/0.1% SDS
at 60jC for 30 min was done. Membranes were exposed to
phosphor plates and images were captured and the data
were quantitated using a PhosphorImager (model 400B,
Molecular Dynamics, Sunnyvale, CA). The interstrand
DNA cross-linking frequency was calculated from the
zero class of the Poisson distribution as described
previously (32).
Drug Treatment and Isolation of 296-bp A-Satellite
DNA Fragment
On the day before experiments, cells were seeded at a
density of 2.5 106 per 10-cm Petri dish and allowed to
attach overnight. Cells were treated with 4 Amol/L
anthracycline and 500 Amol/L AN-9 for a total of 4 h and
then harvested and washed twice with PBS. Total genomic
DNA was extracted using a QIAamp blood kit, where the
lysis procedure was carried out at 50jC for 30 min to
minimize loss of adducts. DNA was then processed as
described previously to isolate the 296-bp a-satellite DNA
fragment (33). Briefly, DNA was restriction digested with
EcoRI, and the 340-bp a-satellite repeat was isolated by
PAGE. DNA fragments were 3¶ end labeled with [32P]dATP
using the Klenow fragment of DNA polymerase and then
restriction digested using HaeIII. The 296-bp band was
isolated using a 5% nondenaturing polyacrylamide gel.
Samples were exposed to phenol/chloroform extraction,
ethanol precipitated, and resuspended in Tris-EDTA
buffer. The radiolabeled 296-bp fragments were digested
at 37jC for 1.5 h using E-exonuclease to progressively
release 5¶ nucleotides. Maxam-Gilbert G sequencing was
conducted as described previously (34). Samples were
resolved by denaturing electrophoresis through 8% sequencing gels.
Growth Inhibition Assays
On the day before drug addition, cells were seeded into
96-well plates using 5 103 per well. On the following day,
serial dilutions of both AN-9 and various anthracyclines
were done and added to the plates to a final volume of
200 AL. For anthracycline growth inhibition assays, the
maximum concentration was 10 Amol/L and this was
diluted serially in medium to the lowest concentration of
0.316 nmol/L. To determine growth inhibition for AN-9, an
initial concentration of 500 Amol/L was diluted serially to
the lowest concentration of 15.8 nmol/L. Drugs were
added to the plates alone or in combination (anthracycline
to AN-9 ratio of 1:50), and additional medium was added
where required to maintain the same concentrations. Cells
were incubated with drug for 72 h. Cell viability was
assessed by incubating cells with 100 AL of 1 mg/mL
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for 3 h at 37jC, after which all liquid was removed.
DMSO (100 AL) was then added to the plates that were
then shaken for 10 min before absorbance readings at
570 nm. The extent of synergy was analyzed by determining the combination index (CI) as described previously
(26). From this analysis, the combined effects of two drugs
were assessed as either additive (zero) interaction indicated by CI = 1, synergism as indicated by CI < 1, or antagonism as indicated by CI > 1.
Apoptotic Morphology Assay
MCF7 cells were seeded in six-well plates at a density of
1 105/2 mL well. On the following day, drugs were
added at the indicated concentrations and cells were
incubated for 4 h. The medium was then replaced and
cells were incubated for a further 44 h. The medium was
collected, and cells were harvested by trypsinization and
added back to the collected medium. Cells were pelleted,
fixed in 3.7% paraformaldehyde, washed in PBS, and
applied to polylysine-coated coverslips (35). Cells were
permeabilized using 0.1% Triton X-100 and stained using
Hoechst 33258 (1 Ag/mL). Slides were then examined
using an Olympus BX-50 fluorescence microscope (Olympus, Tokyo, Japan). Cells were scored for apoptotic
morphology where at least 200 cells per treatment were
analyzed. Apoptotic cells were scored as those with
hypercondensed and fragmented nuclei.
Analysis of Apoptosis by Flow Cytometry
MCF7/Dx cells were seeded and treated with drugs as
described for MCF7 cells in the apoptotic morphology
assay. Analysis of apoptosis was done by assaying the subG1 cell population as described previously (27). Briefly,
cells were pelleted and fixed by resuspension in 70%
ethanol and incubated at room temperature for 30 min.
Fixed cells were pelleted, washed once with PBS,
and resuspended in DNA staining solution (25 mg/mL
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propidium iodide and 100 mg/mL RNase A in PBS) and
incubated at 37jC for 30 min in the dark. Samples (30,000
events) were analyzed on a FACSCalibur using CellQuest
software (BD Biosciences, San Jose, CA) and gated to
distinguish doublets and small debris.
Results
Southern-Based Cross-Linking Assay
To assess the capacity of the various anthracyclines to
form drug-DNA adducts (or virtual DNA cross-links), a
Southern-based cross-linking assay was used (36). The
basis of this assay is that adduct formation renders DNA
resistant to thermal denaturation in the presence of
formamide, and thus, DNA migrates as a double-strand
band (dsDNA) when detected by Southern hybridization.
On the other hand, non – drug-reacted DNA is susceptible
to denaturation and migrates as ssDNA. The percentage
of dsDNA observed is then used in the Poisson
distribution to calculate the number of DNA adducts per
10 kb. Drug treatment concentrations were established so
that the number of adducts induced by AN-9 – activated
doxorubicin could be reliably quantitated in a linear
region of adduct formation in this assay. Because the peak
of doxorubicin adduct formation begins at 4 h and reaches
a plateau thereafter (26), this time point was chosen for
remaining assays. The other anthracyclines were then
used under these same drug reaction conditions for
comparison with doxorubicin. The result of this study is
presented in Fig. 2A for both MCF7 cells and their
resistant subline MCF7/Dx. Two different gene-specific
probes were used: DHFR to detect adduct formation in
the nuclear genome and a mitochondrial-specific probe to
detect adduct formation in the mitochondrial genome.
Idarubicin and daunorubicin were both efficient at
inducing adducts in the presence of AN-9; however,
epirubicin produced no detectable adducts. Results for
epirubicin are only presented at the highest drug
concentrations used because adducts were also not
detected at lower levels. To further assess this reaction,
a cell-free system was used to compare adduct formation
by doxorubicin and epirubicin. In this assay, a linearized
plasmid was reacted with drug in the presence of
2 mmol/L formaldehyde before being denatured and
characterized by agarose electrophoresis. Adducts
(detected as dsDNA) were detected with doxorubicin at
concentrations as low as 500 nmol/L, whereas adducts
were not detected with epirubicin even up to 20 Amol/L,
the highest concentration used (data not shown).
Quantitation of the results shown in Fig. 2A is presented
in Fig. 2C and D. In the MCF7 cells, both idarubicin and
daunorubicin were superior to doxorubicin in inducing
adducts in both nuclear and mitochondrial genomes, as
these drugs produced f4-fold higher levels of adducts at
equimolar concentrations. In the MCF7/Dx cells, daunorubicin was superior to doxorubicin (f2-fold higher levels
of adducts), whereas idarubicin produced the greatest
levels of adducts (up to 10-fold higher than doxorubicin).
However, because there were large errors apparent in
calculating adduct levels via the Poisson distribution
Figure 2. Southern-based cross-linking assay of anthracyclines in combination with AN-9 in MCF7 and MCF7/Dx cells. A, cells were treated with
anthracyclines doxorubicin (DOX ), idarubicin (IDA ), daunorubicin (DAUNO), and epirubicin (EPI ) at the indicated concentrations (0 – 3 Amol/L in MCF7
cells; 0 – 6 Amol/L in MCF7/Dx cells) in the presence of 200 Amol/L AN-9. Cells were harvested, and DNA was extracted for Southern analysis. Results
from the mitochondrial genome and DHFR gene are shown where migration of drug-stabilized dsDNA (DS ) and non – drug-stabilized ssDNA (SS) is
indicated. B, the anthracyclines indicated (0 – 2 Amol/L) were used to treat cells in the presence of 200 Amol/L AN-9. Cells were then harvested and
processed for Southern analysis. C, dose response of adduct formation. Quantitation of adducts in MCF7 cells (A, left ). Data were quantitated to
calculate the number of drug adducts per 10 kb. Closed symbols, mitochondrial genomes; open symbols, DHFR gene. Doxorubicin results are shown by
dotted lines with the symbols (n) or (5) representing mitochondrial genomes and the DHFR gene, respectively. Idarubicin results are represented by
dashed lines with the symbols (E) or (D), and daunorubicin results are represented by solid lines with the symbols (.) or (o). D, quantitation of adducts
in MCF7/Dx cells (A, right ). E, quantitation of adducts in MCF7 cells (B). F, quantitation of adducts in MCF7/Dx cells (B).
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1454 Activation of Clinical Anthracyclines
Table 1. Summary table showing concentrations (Mmol/L) required to achieve adduct levels of 0.2 and 0.5 adduct per 10 kb from
various anthracyclines in nuclear and mitochondrial genomes
DHFR gene
Drug (Amol/L)
0.2 adduct/10 kb
Doxorubicin
Idarubicin
Daunorubicin
Mitochondrial genome
0.5 adduct/10 kb
0.2 adduct/10 kb
0.5 adduct/10 kb
MCF7
MCF7/Dx
MCF7
MCF7/Dx
MCF7
MCF7/Dx
MCF7
MCF7/Dx
1.5
0.24
0.37
4.5
0.44
2.0
2.6
0.44
0.50
NA
0.40
4.6
1.7
0.34
0.31
6.0
0.60
3.6
2.9
0.61
0.46
NA
1.1
NA
NOTE: Data were generated from Fig. 2.
(where cross-link levels approach 100%), drug treatment
levels were decreased so that adduct levels would be in the
linear detectable region of the Poisson distribution. These
results are presented in Fig. 2B, and quantitation is
presented in Fig. 2E and F. Daunorubicin and idarubicin
produced adducts at a similar efficiency in the MCF7 cells,
and unlike the other anthracyclines used, idarubicin was
only slightly less efficient in forming adducts in the MCF7/
Dx cells compared with the MCF7 cells. To provide a
rigorous comparison of these drugs and produce an
efficiency ranking, drug concentrations required to achieve
adduct levels of 0.2 and 0.5 adduct per 10 kb are presented
in Table 1. Several conclusions can be drawn from the
values in this table. (a) Slightly higher levels of anthracycline are generally needed to form adducts in the
mitochondrial genome versus the DHFR gene. (b) Although idarubicin and daunorubicin have similar adductforming capacity in MCF7 cells, idarubicin is by far the
more effective compound in the MCF7/Dx cells. For
instance, the concentration required to form 0.5 adduct/
10 kb in the DHFR gene in the MCF7 versus MCF7/Dx
cells is similar, and in other examples, although the
concentration of idarubicin required to form similar adduct
levels in the MCF7 versus MCF7/Dx cells is higher for the
MCF7/Dx cells, this value is always less than a 2-fold
difference in concentration. (c) Both doxorubicin and
daunorubicin are less effective in MCF7/Dx cells. (d)
Doxorubicin is the least efficient compound overall (of the
compounds where adducts can be detected); however,
daunorubicin yields the greatest difference in adductforming ability when the MCF7 cells are compared with
the MCF7/Dx cells, and therefore, it seems that the adducts
formed by this agent are most affected by P-glycoprotein
overexpression.
Sequence-Specific Drug Binding Assay
To analyze the DNA sequence specificity of various
anthracyclines in comparison with doxorubicin, the 296-bp
a-satellite fragment was isolated from MCF7 and MCF7/
Dx cells treated with each of the anthracyclines and AN-9
and was subjected to E-exonuclease digestion. Adducts
cause direct blockages to the stepwise digestion of
E-exonuclease, and the remaining lengths of radiolabeled
fragment therefore reveal the location of adduct binding
sites. The digestion patterns of E-exonuclease are shown in
Fig. 3A. Doxorubicin produced a characteristic pattern of
digestion by the exonuclease as the truncated digestion
products occurred at GC and GG sequences as previously
observed (33). The same sequence specificity was also
observed for doxorubicin in the resistant cells, albeit at a
reduced frequency. Idarubicin and daunorubicin also
produced a very similar sequence specificity pattern to
doxorubicin; however, the level of idarubicin adducts was
not significantly reduced in the resistant cells, whereas
daunorubicin adduct levels were lowered. The epirubicintreated cells exhibited a completely different pattern of
sequence specificity, and this pattern was also reduced in
the resistant cells. However, this same pattern of specificity
was also present in control lanes (AN-9 only) at a lowered
frequency. Replicate experiments revealed that these blockages were often observed at a higher frequency in control
lanes or not produced significantly in epirubicin-treated
lanes (see Fig. 3B for example). A qualitative comparison
was made between the sequences modified by doxorubicin
compared with daunorubicin and idarubicin (Supplementary Data 1),4 where it is apparent that all three drugs yield
a virtually identical pattern of adduct formation (mainly at
5¶-GG and GC sequences); however, idarubicin was more
selective for GG sequences because the two high-intensity
blockages before 5¶-GC sequences (at positions 241 and 273)
observed for doxorubicin and daunorubicin were absent in
idarubicin-reacted samples.
To establish whether the drug-DNA adducts formed by
the anthracyclines exhibited similar characteristics, their
melting profile was assessed. Doxorubicin is known to
form thermally labile, sequence-specific adducts as
evidenced in Fig. 3B by a decline with increasing
temperature. Idarubicin and daunorubicin also exhibited
a similar melting profile. The band at 120 was quantitated
to permit a comparison of melting profiles at a single
adduct site corresponding to midpoint melting temperatures of 74.4jC, 70.2jC, and 69.2jC for doxorubicin,
idarubicin, and daunorubicin, respectively. The nonspecific
blockages produced in epirubicin-reacted samples and
control lanes (also in the 80jC lanes of doxorubicin- and
4
Supplementary materials for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
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idarubicin-reacted samples after thermal denaturation of
adducts) persisted after high temperature treatment,
thus distinguishing them from anthracycline adducts.
Interestingly, epirubicin-reacted samples exhibited three
low-intensity blockage sites, which were lost at higher
temperature (at locations 120, 190, and 225 and indicated
by arrows in Fig. 3B), implying a minor degree of adduct
formation compared with the other anthracyclines. These
blockages were all before GG sequences, although a GC
was also present at the blockage at position 120.
Drug Interaction in Growth Inhibition Assays
It is known that the combination of doxorubicin and
AN-9 is synergistic in several cell lines, including MCF7.
To compare the various anthracyclines in combination,
and to establish if this synergy was maintained or improved in resistant cells, the combinations were used in
growth inhibition assays. The results of this study are
presented in Table 2. The anthracycline values for growth
inhibition in the MCF7 cells were similar; however, the
drugs were less efficient in the MCF7/Dx cells as expected.
The resistance indices obtained for doxorubicin, daunorubicin, and epirubicin were all f30. Although idarubicin
was less active against the MCF7/Dx cells, the resistance
index was reduced to 6.8. AN-9 was similarly active against
both sensitive and resistant cell lines. When used in
combination, it is apparent that epirubicin was the only
drug that was not synergistic in combination with AN-9 in
either the sensitive or resistant cells. Although combinations involving doxorubicin, idarubicin, and daunorubicin
were all synergistic in the resistant cells, anthracycline activity was never restored to the equivalent degree observed
seen in the MCF7 cells, indicating that the combinations
would not fully overcome the resistance phenotype.
Because doxorubicin adduct levels were optimal at 4 h,
the growth inhibition assay was repeated for doxorubicin
and epirubicin in MCF7 cells using this short-term
4-h treatment, and then, cells were incubated in drug-free
medium for the remainder of the assay. These conditions
have been previously shown to reduce the effectiveness of
doxorubicin as a single agent presumably because topoisomerase II – mediated damage can be minimized or more
effectively repaired (27). The results of this short-term
treatment are presented in Table 3, where it is apparent that
indeed doxorubicin was markedly less effective as a single
agent compared with the 72-h drug incubation presented in
Table 2 and that synergy between doxorubicin and AN-9
was more striking under these conditions. However, even
under these conditions, epirubicin remained ineffective in
combination with AN-9.
Apoptosis Assays
The combination of doxorubicin and AN-9 is a potent
inducer of apoptosis in HL60 cells (27). The extent of
apoptosis in doxorubicin/AN-9 versus epirubicin/AN-9
treatments was monitored to test if synergistic growth
inhibition was mediated through apoptosis in the MCF7
cells. MCF7 cells do not have functional caspase-3; hence,
apoptosis cannot be easily assessed by DNA fragmentation
or caspase-3 activity assays. Therefore, simple morphology
assays were used to measure apoptosis. The drug
concentrations chosen for doxorubicin and AN-9 were
based on their IC50 values (Table 3). These same concentrations were then used for epirubicin to maximize any
possible interaction with AN-9. The apoptosis morphology
data are presented in Fig. 4A, where the doxorubicin
combination resulted in very high levels of apoptosis
compared with minimal levels with either agent alone.
Figure 3.
DNA sequence specificity of anthracyclines. A, the 296-bp
fragment was isolated from MCF7
and MCF7/Dx cells treated for
4 h with 4 Amol/L doxorubicin (A ),
idarubicin (I ), daunorubicin (D ), or
epirubicin (E ) in combination with
500 Amol/L AN-9 or treated with
AN-9 alone (C ). Radiolabeled samples were digested with E-exonuclease and resolved using an 8%
denaturing polyacrylamide sequencing gel. G, Maxam-Gilbert G
sequencing lanes. B, thermal denaturation of site-specific anthracycline
adducts. The radiolabeled 296-bp
fragment from MCF7 cells was then
exposed to temperatures ranging
from 50jC to 80jC (as shown) for
10 min. Samples were digested with
E-exonuclease and characterized
electrophoretically. Control undigested samples (C, A, I, D , and E ) and
Maxam-Gilbert G sequencing lanes
(G ) are also indicated.
Mol Cancer Ther 2007;6(4). April 2007
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1456 Activation of Clinical Anthracyclines
Table 2. Activity of anthracyclines and their interaction with AN-9 in MCF7 and MCF7/Dx cells
Treatment
MCF7
MCF7/Dx
CI
IC50
Doxorubicin
Idarubicin
Daunorubicin
Epirubicin
AN-9
AN-9 + doxorubicin
AN-9 + idarubicin
AN-9 + daunorubicin
AN-9 + epirubicin
329
141
223
328
35.5
142
53.2
212
308
F 75
F 39
F 31
F 78
F 12.3*
F 61
F16
F 41
F 45
0.74
0.62
0.9
1.04
F
F
F
F
RI
IC50
9,184
964
6,849
9,709
53.0
566
360
507
1,495
0.23
0.11
0.17
0.24
F
F
F
F
F
F
F
F
F
1,375
283
2,719
546
12.8*
164
76
56
302
CI
27.9
6.8
30.7
29.6
0.79
0.69
0.7
1.2
F
F
F
F
0.09
0.09
0.18
0.09
4
6.8
2.4
4.8
NOTE: Cells were treated as described in Materials and Methods. Growth inhibition values are shown in nmol/L, except for AN-9 values, which are shown in
Amol/L (*), and where combined treatments are described, only the anthracycline growth inhibition value is shown (the corresponding AN-9 concentration is
50-fold higher). Growth inhibition of 50% (IC50) of cells and their combination index values are presented. The figures obtained represent the average of three
to five independent experiments, each consisting of four replicates. Errors represent SE. The resistance index is a measure of the difference of activity of the
indicated anthracycline against MCF7 versus MCF7/Dx cells.
Abbreviations: CI, combination index; RI, resistance index.
Epirubicin as a single agent induced f5-fold higher levels
of apoptosis than doxorubicin (as expected based on its
greater inhibitory effect presented in Table 3), whereas the
epirubicin/AN-9 combination seemed to induce a greater
than additive effect. Although the epirubicin combination
was greater than additive, the degree of epirubicin
potentiation by AN-9 was not as pronounced as the degree
of doxorubicin potentiation by AN-9 (3.2-fold compared
with 40-fold, respectively). Z-VAD-FMK is a broad-spectrum
inhibitor of caspases and was used to verify that the
morphology assay was indeed detecting apoptosis and not
other forms of cell death. The failure of apoptosis to revert to
background levels was addressed by using higher concentrations of Z-VAD-FMK, but this did not decrease the level
of apoptosis further (data not shown). It is probable that this
level of apoptosis reflects apoptosis that has already
progressed during the initial 4-h drug incubation.
The extent of apoptosis was also examined in MCF7/Dx
cells. The apoptosis morphology assay was initially used to
measure apoptosis; however, MCF7/Dx cells undergo far
more extensive nuclear fragmentation than MCF7 cells,
and because nuclei became highly fragmented, it was not
possible to attribute these dispersed fragments to individual
cells. Flow cytometry was therefore used as an alternative
procedure to measure apoptosis in these cells. Because such
different methods were used to assess apoptosis in the two
different cell lines, absolute levels of apoptosis cannot be
compared between the two methods. Regardless, as shown
in Fig. 4B, the doxorubicin-resistant cells were very similar
to MCF7 cells in their pattern of response to the various
drugs with the doxorubicin/AN-9 combination, again
leading to the highest levels of apoptosis.
Discussion
Formation of Adducts by Anthracyclines
The studies relating to efficiency of adduct formation
revealed a clear hierarchy among the four clinical anthra-
cyclines, where daunorubicin and idarubicin were the best
adduct-forming compounds in drug-sensitive cells, whereas idarubicin was by far the most effective in resistant cells.
It is unlikely that differing affinities for cellular DNA are
responsible for these effects. In previous studies of adduct
formation by these anthracyclines in a cell-free system,
doxorubicin, daunorubicin, and idarubicin displayed similar reaction kinetics (28). Furthermore, the relative DNA
binding of daunorubicin, idarubicin, and doxorubicin
using isolated calf thymus DNA does not correlate with
our results (37). However, these results may be explained in
terms of the higher lipophilicity of both daunorubicin and
idarubicin in comparison with doxorubicin. The lipophilicity, as determined by octanol/aqueous partition coefficients (fluorescence of octanol phase divided by
fluorescence of NaCl/phosphate buffer phase), was 0.8
for doxorubicin, 3.5 for daunorubicin, and 15 for idarubicin
(38), potentially indicative of a higher cellular uptake of
Table 3. Activity of anthracyclines and their interaction with
AN-9 in MCF7 cells after short-term drug treatment
Treatment
Doxorubicin
Epirubicin
AN-9
AN-9 + doxorubicin
AN-9 + epirubicin
IC50
CI
810 F 220
370 F 100
93 F 1*
166 F 1
304 F 83
0.3
0.98
NOTE: Cells were treated as described in Materials and Methods, except
that drug treatment was for 4 h, and then, cells were incubated in drug-free
medium for the remainder of the assay. Growth inhibition values are shown
in nmol/L, except for AN-9 values, which are shown in Amol/L (*), and
where combined treatments are described, only the anthracycline growth
inhibition value is shown (the corresponding AN-9 concentration is 50-fold
higher). Growth inhibition of 50% (IC50) of cells and their combination index
values are presented. The figures obtained represent the average of two
independent experiments, each consisting of eight replicates. Errors
represent the SD.
Mol Cancer Ther 2007;6(4). April 2007
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Molecular Cancer Therapeutics
Figure 4. Apoptosis induced by doxorubicin/AN-9 and epirubicin/AN-9
combinations in MCF7 and MCF7/Dx cells. A, MCF7 cells were treated
under the following conditions: untreated cells (Con ), 100 Amol/L AN-9,
1 Amol/L doxorubicin (Dox), AN-9 plus doxorubicin (Dox +), AN-9 plus
doxorubicin and 20 Amol/L Z-VAD-FMK (Z-Vad ), 1 Amol/L epirubicin (Epi ),
and AN-9 plus epirubicin (Epi +). After drug treatment for 4 h, the medium
was replaced and cells were incubated for a further 44 h before assessing
apoptotic morphology. For the Z-VAD-FMK – treated cells, inhibitor was
added after the initial AN-9/doxorubicin treatment. B, MCF7/Dx cells were
treated as described above and assessed by flow cytometry for percentage
sub-G1 events. Columns, average of three independent experiments;
bars, SE.
daunorubicin and idarubicin in the sensitive MCF7 cells.
However, the vast improvement in adduct formation
displayed by idarubicin in MCF7/Dx cells may be
explained in terms of a reduced propensity of this drug
to be removed effectively by P-glycoprotein – overexpressing cells, and there have been many reports in the literature
describing the greater effectiveness of idarubicin versus
other anthracyclines, such as daunorubicin and doxorubicin, in P-glycoprotein – overexpressing cells. However,
rather than representing a poor substrate for P-glycoprotein
efflux, most of these studies have concluded that idarubicin
is in fact a good substrate for P-glycoprotein and its
increased effectiveness against these cells is due to
favorable cell uptake kinetics (39 – 42). When a study was
conducted on a group of 98 acute myelogenous leukemia
patients to assess why idarubicin may be superior to
daunorubicin in acute myelogenous leukemia treatment, no
correlation of P-glycoprotein with improved response rate
was found (43).
In clear contrast to the other anthracyclines studied,
epirubicin showed little evidence of adduct formation. This
agrees with a previous study in a cell-free system where
epirubicin showed only background levels of adduct
formation in comparison with other anthracyclines where
the formaldehyde was provided using the pH-dependent
formaldehyde-releasing compound hexamethylenetetramine (28). However, in other studies, epirubicin has been
observed to form DNA adducts (16, 17, 44, 45). It is
important to consider the different experimental conditions
used and the characteristics of the observed adducts to
attempt to reconcile this discrepancy. The adducts formed
by epidoxoform and epirubicin plus formaldehyde were
analyzed by high-performance liquid chromatography
detection of drug bound to (GC)4 oligonucleotides. Five
structurally different adducts were found (16). In a
subsequent study, the cellular retention of doxoform and
epidoxoform fluorescence was used to calculate adduct
half-lives. It was concluded that drug was released slower
from virtual cross-linking sites in the case of doxoform
compared with epidoxoform due to a decreased stability of
epidoxoform adducts (17). The crystal structure of the
epirubicin-formaldehyde virtual cross-link of DNA has
been solved (45), and in this structure, it was shown that
(as observed with other anthracycline-DNA complexes) the
aglycone of epirubicin intercalates at CpG, the D ring
reaches into the major groove, and the sugar moiety lies in
the minor groove (12). However, compared with the
daunorubicin-formaldehyde virtual cross-link, there were
differences in hydrogen bonding. Overall, these differences
suggest that daunorubicin is bound more effectively to the
covalently bonded strand, whereas epirubicin is held more
tightly to the opposing strand (17). Overall, there are
several differences between epirubicin and doxorubicin/
daunorubicin adducts that may explain the apparent
discrepancy with our observation. (a) If the epirubicin
adduct exhibits an altered structure and is less stable, it
may be less amenable to the rigorous isolation conditions
required in the current study to do the Southern and
sequence-specific analyses. These conditions require DNA
isolation at 50jC, phenol/chloroform extraction, and
ethanol precipitation. Furthermore, for Southern analyses,
DNA must be denatured in formamide, and for the
sequence-specific analyses, isolation of the DNA required
takes over 2 days where the DNA is subjected to extended
incubations at 4jC. Although these conditions suit the
isolation of doxorubicin adducts, this may not be appropriate for epirubicin. (b) The initial reaction of doxorubicin
and daunorubicin with DNA may be more efficient when
an initial cyclization to a five-membered mono-oxazolidine
ring forms (16). However, this cannot occur in the case of
epirubicin due to the equatorial position of the hydroxyl
Mol Cancer Ther 2007;6(4). April 2007
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1457
1458 Activation of Clinical Anthracyclines
group at C-4¶ (Fig. 5). The different structures of the
anthracycline prodrugs daunoform and doxoform, in
comparison with epidoxoform, illustrate this point (16).
(c) Epidoxoform exhibits an f70-fold lower potency
against MCF7 and MCF7/ADR cells compared with doxoform, and this may at least in part be due to reduced
efficiency of adduct formation or altered structure of the
DNA adducts.
Drug Interactions
In previous studies, doxorubicin and daunorubicin are
synergistic in combination with AN-9 (24 – 26). The combination is synergistic when doxorubicin treatment is
simultaneous with (or before) AN-9; however, it is
antagonistic when AN-9 treatment precedes doxorubicin.
These observations correlate well with the levels of DNA
adduct formation (i.e., levels of DNA adducts are much
higher under synergistic conditions). The critical involvement of formaldehyde has been established by sequestration of formaldehyde with semicarbazide, which ablated
both adduct formation and cell sensitivity to the combination treatment (26). Furthermore, a series of AN-9 derivatives were used to confirm that formaldehyde was
absolutely required for these effects (26, 46). In the present
study, we have assessed the suitability of various clinically
used anthracyclines to achieve these types of effects in
combination with AN-9. It is apparent that doxorubicin,
daunorubicin, and idarubicin all exhibit synergy in
combination with AN-9, and this is accompanied by adduct
formation. However, epirubicin does not display this
synergy, and this is consistent with a lack of observed
adduct formation. Idarubicin seems particularly useful in
combination with AN-9, and its enhanced cellular uptake
may render it more effective against anthracycline-resistant
populations of cells. It is significant that the other
anthracyclines used were all more effective against the
resistant cells in combination with AN-9. This would be
expected if the ability of the drugs to bind to cellular
molecules via a formaldehyde-mediated linkage rendered
them less available for P-glycoprotein – mediated cellular
efflux. The mode of cell death induced by the combinations
seems to be apoptosis as indicated using the apoptosis
morphology assay and the Z-VAD-FMK apoptosis inhibitor. This is consistent with our previous observations in
HL60 cells where cell death was blocked by Bcl-2 overexpression (27). We are currently investigating the DNA
damage response pathways that initiate this apoptotic
response.
In summary, we have identified combinations of anthracyclines and AN-9 that synergistically inhibit the growth of
sensitive and resistant tumor cells. These combinations
represent conditions where adduct formation can be
achieved, and this is consistent with our previous studies
of doxorubicin and AN-9 where adduct formation always
accompanies the synergy observed. This now extends the
repertoire of drugs that can be used to achieve such effects.
The current results suggest that idarubicin would seem to
be the most favorable drug to use in combination with
formaldehyde-releasing drugs in anthracycline-sensitive
and anthracycline-resistant tumors. Daunorubicin and
doxorubicin are also useful alternatives, but the use of
epirubicin in such circumstances would be predicted to be
less effective. Confirmation of these recommendations
would require testing in suitable animal models.
Acknowledgments
We thank Dr. Rosanna Supino (Istituto Nazionale per lo Studio e la Cura dei
Tumori, Milan, Italy) for the MCF7 and MCF7/Dx cell lines.
References
1. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines:
molecular advances and pharmacologic developments in antitumor activity
and cardiotoxicity. Pharmacol Rev 2004;3:185 – 229.
2. DeVita VT, Hellman S, Rosenberg SA, editors. Principles and practice
of oncology. 6th ed. Philadelphia: Lippincott Williams and Wilkins; 2001.
3. Hortobagyi GN. Anthracyclines in the treatment of cancer. An
overview. Drugs 1997;54 Suppl 4:1 – 7.
4. Arcamone F, Cassinelli G, Fantini G, et al. Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius .
Biotechnol Bioeng 2000;67:704 – 13.
5. Arcamone F. Adriamycin and its analogs. Tumori 1984;70:113 – 9.
6. Weiss RB. The anthracyclines: will we ever find a better doxorubicin?
Semin Oncol 1992;19:670 – 86.
7. Bakker M, van der Graaf WTA, Groen HJM, Smit EF, De Vries EGE.
Anthracyclines—pharmacology and resistance, a review. Curr Pharmaceut
Design 1995;1:133 – 44.
8. Sordet O, Khan QA, Kohn KW, Pommier Y. Apoptosis induced by
topoisomerase inhibitors. Curr Med Chem Anti-Cancer Agents 2003;3:
271 – 90.
9. Gewirtz DA. A critical evaluation of the mechanisms of action proposed
for the antitumor effects of the anthracycline antibiotics adriamycin and
daunorubicin. Biochem Pharmacol 1999;57:727 – 41.
10. Phillips DR, Cullinane C. Adriamycin. In: Creighton TE, editor.
Encyclopedia of molecular biology, vol. 1. New York: John Wiley and
Sons; 1999. p. 68 – 72.
Figure 5.
Model of anthracycline reactivity with free formaldehyde and
subsequent DNA reaction. A, the axial position of the hydroxyl group (as
in doxorubicin, idarubicin, and daunorubicin) allows for formation of the
mono-oxazolidine as a precursor to reaction with the N2 of guanine. B, the
equatorial position of the hydroxyl group (as in epirubicin) does not allow
for stabilization by mono-oxazolidine formation.
11. Taatjes DJ, Guadiano G, Resing K, Koch TH. Redox pathway leading
to the alkylation of DNA by the anthracycline, antitumor drugs adriamycin
and daunomycin. J Med Chem 1997;40:1276 – 86.
12. Wang AH, Gao YG, Liaw YC, Li YK. Formaldehyde cross-links
daunorubicin and DNA efficiently: HPLC and X-ray diffraction studies.
Biochemistry 1991;30:3812 – 5.
Mol Cancer Ther 2007;6(4). April 2007
Downloaded from mct.aacrjournals.org on May 10, 2017. © 2007 American Association for Cancer Research.
Molecular Cancer Therapeutics
13. Zeman SM, Phillips DR, Crothers DM. Characterization of covalent adriamycin-DNA adducts. Proc Natl Acad Sci U S A 1998;95:
11561 – 5.
14. Cutts SM, Parker BS, Swift LP, Kimura K-I, Phillips DR. Structural
requirements for the formation of anthracycline-DNA adducts. Anticancer
Drug Design 2000;15:373 – 86.
30. Cutts SM, Nudelman A, Rephaeli A, Phillips DR. The power and
potential of doxorubicin-DNA adducts. IUBMB Life 2005;57:73 – 81.
31. Zhen W, Evans MK, Haggerty CM, Bohr VA. Deficient gene specific
repair of cisplatin-induced lesions in xeroderma pigmentosum and
Fanconi’s anemia cell lines. Carcinogenesis 1993;14:919 – 24.
15. Fenick DJ, Taatjes DJ, Koch TH. Doxoform and daunoform:
anthracycline-formaldehyde conjugates toxic to resistant tumor cells.
J Med Chem 1997;40:2452 – 61.
32. Vos JM. Analysis of psoralen monoadducts and interstrand crosslinks
in defined genomic sequences. In: Friedberg EC, Hanawalt PC, editors.
DNA repair: a laboratory manual of research procedures, vol. 3. New York:
Marcel Dekker; 1988. p. 367 – 98.
16. Taatjes DJ, Fenick DJ, Koch TH. Epidoxoform: a hydrolytically more
stable anthracycline-formaldehyde conjugate toxic to resistant tumor cells.
J Med Chem 1998;41:1306 – 14.
33. Cutts SM, Swift LP, Rephaeli A, Nudelman A, Phillips DR. Sequence
specificity of adriamycin-DNA adducts in human tumor cells. Mol Cancer
Ther 2003;2:661 – 70.
17. Taatjes DJ, Koch TH. Nuclear targeting and retention of anthracycline
antitumor drugs in sensitive and resistant tumor cells. Curr Med Chem
2001;8:15 – 29.
18. Dernell WS, Powers BE, Taatjes DJ, Cogan P, Gaudiano G, Koch
TH. Evaluation of the epidoxorubicin-formaldehyde conjugate, epidoxoform, in a mouse mammary carcinoma model. Cancer Invest 2002;20:
713 – 24.
34. Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl
Acad Sci U S A 1977;74:560 – 4.
19. Burke PJ, Koch TH. Design, synthesis, and biological evaluation of
doxorubicin-formaldehyde conjugates targeted to breast cancer cells.
J Med Chem 2004;47:1193 – 206.
20. Cogan PS, Koch TH. Rational design and synthesis of androgen
receptor-targeted nonsteroidal anti-androgen ligands for the tumor-specific
delivery of a doxorubicin-formaldehyde conjugate. J Med Chem 2003;46:
5258 – 70.
21. Nudelman A, Ruse M, Aviram A, et al. Novel anticancer prodrugs of
butyric acid 2. J Med Chem 1992;35:687 – 94.
35. Malorni W, Fais S, Fiorentini C. Morphological aspects of apoptosis.
In: Watson J, editor. Purdue Cytometry CD-ROM Series, vol. 4. West
Lafayette: Purdue University Cytometry Laboratories; 1997.
36. Cullinane C, Cutts SM, Panousis C, Phillips DR. Interstrand crosslinking by adriamycin in nuclear and mitochondrial DNA of MCF-7 cells.
Nucleic Acids Res 2000;28:1019 – 25.
37. Binaschi M, Capranico G, Dal Bo L, Zunino F. Relationship between
lethal effects and topoisomerase II-mediated double-stranded DNA breaks
produced by anthracyclines with different sequence specificity. Mol
Pharmacol 1997;51:1053 – 9.
38. Wielinga PR, Westerhoff HV, Lankelma J. The relative importance of
passive and P-glycoprotein mediated anthracycline efflux from multidrugresistant cells. Eur J Biochem 2000;267:649 – 57.
22. Rephaeli A, Rabizadeh E, Aviram A, Shaklai M, Ruse M, Nudelman A.
Derivatives of butyric acid as potential anti-neoplastic agents. Int J Cancer
1991;49:66 – 72.
39. Mulder HS, Dekker H, Pinedo HM, Lankelma J. The P-glycoproteinmediated relative decrease in cytosolic free drug concentration is similar
for several anthracyclines with varying lipophilicity. Biochem Pharmacol
1995;50:967 – 74.
23. Patnaik A, Rowinsky EK, Villalona MA, et al. A phase I study of
pivaloyloxymethyl butyrate, a prodrug of the differentiating agent butyric
acid, in patients with advanced solid malignancies. Clin Cancer Res 2002;
8:2142 – 8.
40. Garnier-Suillerot A, Marbeuf-Gueye C, Salerno M, et al. Analysis of
drug transport kinetics in multidrug-resistant cells: implications for drug
action. Curr Med Chem 2001;8:51 – 64.
24. Kasukabe T, Rephaeli A, Honma Y. An anti-cancer derivative of
butyric acid (pivalyloxmethyl buterate) and daunorubicin cooperatively
prolong survival of mice inoculated with monocytic leukaemia cells. Br
J Cancer 1997;75:850 – 4.
25. Niitsu N, Kasukabe T, Yokoyama A, et al. Anticancer derivative of
butyric acid (pivalyloxymethyl butyrate) specifically potentiates the
cytotoxicity of doxorubicin and daunorubicin through the suppression of
microsomal glycosidic activity. Mol Pharmacol 2000;58:27 – 36.
26. Cutts SM, Rephaeli A, Nudelman A, Hmelnitsky I, Phillips DR.
Molecular basis for the synergistic interaction of adriamycin with the
formaldehyde-releasing prodrug pivaloyloxymethyl butyrate (AN-9). Cancer Res 2001;61:8194 – 202.
27. Swift LP, Rephaeli A, Nudelman A, Phillips DR, Cutts SM. Doxorubicin-DNA adducts induce a non-topoisomerase II-mediated form of cell
death. Cancer Res 2006;66:4863 – 71.
28. Swift LP, Cutts SM, Rephaeli A, Nudelman A, Phillips DR. Activation
of adriamycin by the pH-dependent formaldehyde-releasing prodrug
hexamethylenetetramine. Mol Cancer Ther 2003;2:189 – 98.
29. Cutts SM, Swift LP, Rephaeli A, Nudelman A, Phillips DR. Recent
advances in understanding and exploiting the activation of anthracyclines by formaldehyde. Curr Med Chem Anti-Canc Agents 2005;5:
431 – 47.
41. Bennis S, Faure P, Chapey C, et al. Cellular pharmacology of lipophilic
anthracyclines in human tumor cells in culture selected for resistance to
doxorubicin. Anticancer Drugs 1997;8:610 – 7.
42. Bogush T, Robert J. Comparative evaluation of the intracellular
accumulation and DNA binding of idarubicin and daunorubicin in sensitive
and multidrug-resistant human leukaemia K562 cells. Anticancer Res
1996;16:365 – 8.
43. Broxterman HJ, Sonneveld P, van Putten WJ, et al. P-glycoprotein
in primary acute myeloid leukemia and treatment outcome of idarubicin/
cytosine arabinoside-based induction therapy. Leukemia 2000;14:
1018 – 24.
44. Taatjes DJ, Fenick DJ, Koch TH. Nuclear targeting and nuclear
retention of anthracycline-formaldehyde conjugates implicates DNA
covalent bonding in the cytotoxic mechanism of anthracyclines. Chem
Res Toxicol 1999;12:588 – 96.
45. Podell ER, Harrington DJ, Taatjes DJ, Koch TH. Crystal structure of
epidoxorubicin-formaldehyde virtual crosslink of DNA and evidence for its
formation in human breast-cancer cells. Acta Crystallogr D Biol Crystallogr
1999;55:1516 – 23.
46. Cutts SM, Nudelman A, Pillay V, et al. Formaldehyde-releasing
prodrugs in combination with doxorubicin can overcome cellular drug
resistance. Oncol Res 2005;15:199 – 213.
Mol Cancer Ther 2007;6(4). April 2007
Downloaded from mct.aacrjournals.org on May 10, 2017. © 2007 American Association for Cancer Research.
1459
Activation of clinically used anthracyclines by the
formaldehyde-releasing prodrug pivaloyloxymethyl butyrate
Suzanne M. Cutts, Lonnie P. Swift, Vinochani Pillay, et al.
Mol Cancer Ther 2007;6:1450-1459.
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