Nitric oxide donor doxorubicins accumulate into doxorubicin-resistant human colon cancer cells inducing cytotoxicity

Article abstract of DOI:10.1021/ml100302t

Products 4 and 5, obtained by conjugation of doxorubicin with nitric oxide (NO) donor nitrooxy and phenylsulfonyl furoxan moieties, respectively, accumulate in doxorubicin-resistant human colon cancer cells (HT29-dx), inducing high cytotoxicity. This beha

Full text of DOI:10.1021/ml100302t

LETTER
pubs.acs.org/acsmedchemlett
Nitric Oxide Donor Doxorubicins Accumulate into Doxorubicin-Resistant
Human Colon Cancer Cells Inducing Cytotoxicity
Konstantin Chegaev,† Chiara Riganti,‡ Loretta Lazzarato,† Barbara Rolando,† Stefano Guglielmo,†
Ivana Campia,‡ Roberta Fruttero,*,† Amalia Bosia,*,‡ and Alberto Gasco†
†Department of Drug Science and Technology, University of Turin, Via Pietro Giuria 9, 10125 Torino, Italy
‡Department of Genetics, Biology and Biochemistry, Turin School of Medicine, University of Turin, via Santena 5/bis,
10126 Torino, Italy
S Supporting Information
b
ABSTRACT: Products 4 and 5, obtained by conjugation of doxorubicin with nitric
oxide (NO) donor nitrooxy and phenylsulfonyl furoxan moieties, respectively,
accumulate in doxorubicin-resistant human colon cancer cells (HT29-dx), inducing
high cytotoxicity. This behavior parallels the ability of the compounds to generate
NO, detected as nitrite, in these cells. Preliminary immunoblotting studies suggest
that the mechanism that underlies the cytotoxic effect could involve inhibition of
cellular drug efflux due to nitration of tyrosine residues of the MRP3 protein pump.
KEYWORDS: Multidrug resistance, doxorubicin, nitric oxide, P-glycoprotein
ultidrug resistance (MDR) is one of the major reasons for
the failure of cancer chemotherapy. Among the mechan-
the nitration of tyrosine residues of MRP3 occurred, thus sug-
gesting that nitration of the transporter is a possible mechanism
for resistance inversion. Also, furoxan derivatives (1,2,5-oxadia-
zole 2-oxides), which are known to release NO under the action
of thiol cofactors,8 can inhibit P-gp and MRP1 transporters in
MDK (MadinꢀDarby canine kidney) -MDR1 and -MDRP1 cell
lines, respectively, with an increase of cellular accumulation of
DOXO when coadministered with the antibiotic.9 Experiments
carried out with 3-phenylsulfonylfuroxan derivatives, the most
potent inhibitors of the series, showed that these products were
able to nitrate tyrosine residues of P-gp, which in this form is
probably unable to efflux the antibiotic. Other studies report that
a reduced supply of oxygen could induce MDR in solid cancers
and that hypoxia-induced MDR could be reversed by low con-
centrations of NO mimetics.10,11
Using these bases as our starting point, we decided to syn-
thesize new DOXO semisynthetic derivatives in which the anti-
biotic was joined through an ester linkage to moieties containing
either 3-phenylsulfonylfuroxan or nitrooxy substructures, in view
of the potential ability of these products to trigger anticancer
action combined with a reduced capacity to induce resistance. As
aforementioned, the furoxan system can release NO under the
action of endogenous thiols. By contrast, NO release from
organic nitrates occurs through enzymatic catalysis. A number
of enzymes have been proposed for this conversion; in particular,
the role of mitochondrial aldehyde dehydrogenase (mtALDH)
and P-450 enzyme(s) was emphasized.12ꢀ14 The preliminary
results obtained by studying the products 4 and 5 (NOꢀ
M
isms that underlie this phenomenon, we know the most about
those that increase the capacity of the cancer cells to efflux anti-
cancer drugs, thus limiting their cellular accumulation with a con-
sequent reduction of toxicity; this form of MDR is one of the
most studied in cancer cell models.1,2 Indeed, cancer cells are able
to overexpress ATP binding cassette (ABC) transporter proteins,
which are the products of a family of 49 genes. ABC B1, better
known as P-glycoprotein (P-gp) or MDR1, and ABC C1ꢀ6,
better known as MDR-associated proteins MRP1ꢀ6, are the
most representative pumps involved in MDR.3 One of the strat-
egies followed to reverse MDR is the coadministration of an
anticancer agent with an inhibitor of these efflux pumps. Elacri-
dar, tariquidar, and laniquidar are examples of third-generation
inhibitors that have been studied in clinical trials in association
with a number of antitumor drugs.4,5 The identification of new
MDR-reversing agents selectively targeting drug-resistance cells
is a field of active investigation.6
In doxorubicin (DOXO)-resistant human epithelial colon cell
line HT29-dx, there is a relationship between the reduced endo-
genous nitric oxide (NO) production and the resistance of these
cells to the antibiotic.7 Classic NO donors, such as S-nitrosope-
nicillamine (SNAP), sodium nitroprusside (SNP), and S-nitro-
soglutathione (GSNO), were able to reduce the efflux of DOXO
from HT29-dx cells.7 This effect was neither inhibited by ODQ
(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a well-known
inhibitor of the soluble guanylate cyclase (sGC), or by the protein-G
kinase inhibitor 8-bromoguanosine-30,50-cyclic monophosphorothio-
ate (Rp-8-Br-cGMPS), nor was it mimicked by 8-bromoguanosine-
30,50-cyclic monophosphate (8-Br-cGMP), thus indicating inde-
pendence from guanosine-30,50-cyclic monophosphate (cGMP).
Experiments carried out with SNAP showed that in HT29-dx cells
Received: December 20, 2010
Accepted: April 4, 2011
Published: April 04, 2011
r
2011 American Chemical Society
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Chart 1
Figure 1. Dose-dependent accumulation of DOXO and NOꢀDOXOs
in HT29-dx cells. Cells were incubated for 24 h in the absence or
presence of different concentrations (0.5, 1, 2.5, 5, and 25 μM) of
DOXO (open columns), 4 (hatched columns), or 5 (solid columns),
then lysed, and checked for the intracellular drug content, as reported in
the Supporting Information. Data are presented as means ( SDs (n = 3)
vs untreated cells (0): *p < 0.005.
Figure 2. Efflux kinetics of DOXO and NOꢀDOXOs from HT29-dx
cells. HT29-dx cells were loaded in PBS buffer for 10 min at 37 °C with
different amounts (1ꢀ200 μM) of DOXO (open circle), 4 (open
square), or 5 (solid square) and then washed. One aliquot of each
sample was checked immediately for the content of drug (time 0),
whereas a second aliquot was further incubated in fresh PBS for an
additional 10 min, washed, and tested for the drug content as previously
described (for details, see the Supporting Information). The rate of
DOXO efflux was calculated from the difference between the drug
concentration at time 0 and at 10 min, respectively. Measurements
(n = 3) were done in duplicate, and data are presented (means ( SDs) as
the rate of efflux vs the intracellular drug concentration at time 0. The
significance of 4 vs DOXO: *p < 0.01; significance of 5 vs DOXO,
°p < 0.002; significance of 5 vs 4, )p < 0.001.
DOXO) (Chart 1) on HT29-dx cell populations are reported
here and discussed.
Compounds 4 and 5 were prepared from a mixture of 14-
bromo and 14-chlorodaunorubicine hydrobromide 1 by reaction
with 4-(2,3-dinitrooxypropyl)benzoic acid 2 and 3-[(3-phenyl-
sulfonyl)furoxan-4-yloxy]propanoic acid 3 in acetone, respec-
tively. After purification by flash chromatography, the products
were isolated as hydrochlorides. The simple methyl esters 6 and
7, used for a comparison, were prepared treating 2 and 3 in
refluxing methanol, in the presence of H2SO4 (see the Support-
ing Information).
HT29-dx cells were incubated in RPMI 1640-medium with
DOXO and NOꢀDOXO (4 and 5) at different concentrations
(0.5ꢀ25 μM) for 24 h. At the end of this period, the antibiotics
were dosed on the cell lysate, using a spectrofluorimeter method
with λ emission 553 nm and λ excitation 475 nm. At these
wavelengths, the spectra of DOXO, 4, and 5 were superimpo-
sable (data not shown). The results, collected in Figure 1, show
that NOꢀDOXOs accumulate into the cells in a concentration-
dependent manner, unlike DOXO, whose intracellular concen-
tration does not significantly increase, except at the highest
concentration (25 μM). This effect was more accentuated for
furoxan derivative 5 than for nitrooxy derivative 4 and was
reversed (Figure 3A) by coincubation with 2-phenyl-4,4,5,
5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) and red blood
cells, chosen as effective NO scavengers (Figure 3C). The
pretreatment with phenylsulfonylfuroxan derivatives 3 or 7
increased the intracellular accumulation of DOXO (see the
Supporting Information). The ester 7 was more potent than
the corresponding simple acid 3, most likely because of the
different permeability of the compounds. Furthermore, coincu-
bation of 3 and 7 with DOXO increased the accumulation of the
antibiotic into the cells (see the Supporting Information). Also,
these experiments clearly indicate that the phenomenon is NO-
dependent.
Similarly NOꢀDOXOs increase their accumulation in a time-
dependent manner: Elevation of the intracellular content of 5 μM
antibiotics became significant after 1 h for 5 and after 3 h for
4 and was never significant between 1 and 24 h for DOXO (data not
shown). This behavior parallels the drug efflux kinetics from HT29-
dx cells. Indeed, when cell monolayers were loaded with different
amounts of the three drugs (1ꢀ200 μM solutions) for 10 min and
then the antibiotic intracellular content was examined spectrofluor-
imetrically on cell lysates collected after 10 and 20 min, the results
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Figure 5. Nitration of drug efflux pumps by DOXO and NOꢀDOXOs
in HT29-dx cells. HT29-dx cells were incubated for 24 h with 5 μM
DOXO, 4, or 5. Afterward, cells were lysed, and the whole cellular lysate
was immunoprecipitated with an antinitrotyrosine polyclonal antibody.
The immunoprecipitated proteins were subjected to Western blotting,
using an anti-Pgp or an anti-MRP3 antibody (see the Supporting
Information). An aliquot of cells lysate before immunoprecipita-
tion was analyzed for total P-gp and MRP3. The expression of the
housekeeping protein GAPDH (glyceraldehydes-3-phosphate dehydro-
genase) was measured as a control of equal loading. The experiment is
representative of three similar experiments.
Figure 3. Effects of NO scavengers on DOXO and NOꢀDOXOs
accumulation and toxicities. HT29-dx cells were incubated in the
absence (ꢀ) or presence (CTRL) of 5 μM DOXO, 4, or 5 for 24 h;
when indicated, 100 μM PTIO (PT) or 10 μL/mL of packed red blood
cells was coincubated. The intracellular drug content (A), the extra-
cellular activity of LDH (B), and the nitrite amount in the culture
supernatant (C) were measured in duplicate as described in the
Supporting Information (n = 3). Significance for A: vs CTRL DOXO,
*p < 0.002; vs DOXO or 4 or 5, °p < 0.05. For B and C: vs CTRL DOXO,
*p < 0.05; vs DOXO or 4 or 5, °p < 0.05.
when incubated with 5 μM solutions of NOꢀDOXOs, the cells
exhibited a significant increase in the release of lactate dehydro-
genase (LDH), a sensitive index of a drug's toxicity, with respect
to DOXO alone (Figure 3B). The level of LDH was higher for
the furoxan derivative 5 than for the nitrooxy-substituted product
4. In both cases, the toxicity was reduced in the presence of the
NO scavengers PTIO and red blood cells. By contrast, no signi-
ficant increase in LDH levels was observed with the simple acids
2 and 3, used to obtain the two codrugs 4 and 5, and the related
methyl esters 6 and 7. This finding indicates that the cytotoxic
effect is due to the anthracycline pharmacophore rather than to
the NO-releasing moieties.
To determine if NOꢀDOXOs are able to restore NO produc-
tion in HT29-dx cells, solutions of 4 and 5 of different concentra-
tion (0.5ꢀ25 μM) were incubated with cell monolayers for 24 h,
and nitrite in the culture medium was then quantified by Griess
reaction. The results reported in Figure 4 show that nitrite pro-
duction is concentration-dependent and that it is maximized
for furoxan derivative 5. The nitrite release was time-dependent:
Using a 5 μM solution of the antibiotics, a significant increase
of nitrite was detected after 1 h with 5 and after 3 h with 4 but
remained not significant between 1 and 24 h with DOXO (data
not shown).
It is known that drug efflux pumps P-gp and MRP3 are
overexpressed in HT29-dx cells.7 To check whether the capacity
of 4 and 5 to accumulate in these cells could be due to the
nitration of tyrosine residues of these efflux pumps, following
NO release by the products, HT29-dx cells were incubated for 24
h with the two antibiotics or with DOXO. The whole cellular
lysate was immunoprecipitated with a specific antinitrotyrosine
antibody, and the immunoprecipitated proteins were subjected
to Western blotting, using anti-P-gp and anti-MRP3 antibodies
(Figure 5). The presence of nitrotyrosine residues was detectable
on MRP3 protein in HT29-dx cells incubated with 4 or 5 but not
Figure 4. Dose-dependent production of nitrite in HT29-dx cells
treated with DOXO and NOꢀDOXOs. Cells were incubated for 24 h
in the absence or presence of different concentrations (0.5, 1, 2.5, 5, and
25 μM) of DOXO (open columns), 4 (hatched columns), or 5 (solid
columns), and then, the levels of nitrite in the cell medium were
evaluated by the Griess method. Data are presented as means ( SDs
(n = 3) vs untreated cells (0): *p < 0.001.
outlined in Figure 2 were obtained. The measured Vmax values rank
the order DOXO (8.38 ( 0.38 nmol of the drug transported/min/
mg prot) > 4 (4.23 ( 0.18 nmol of the drug transported/min/mg
prot) > 5 (2.65 ( 0.09 nmol of the drug transported/min/mg prot),
showing that the efflux of NOꢀDOXOs decreases dramatically with
respect to the simple antibiotic.
As a consequence of this behavior, the toxicity of NOꢀ
DOXOs was definitively higher than that of DOXO. Indeed,
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LETTER
with DOXO alone. A number of reactive nitrogen species,
generated under different conditions and environments, can
mediate this nitration. The amount of nitro tyrosine produced
by 5 appeared greater than that produced by 4. This result is in
keeping with the higher levels of nitrite detected with 5. Also, the
intracellular accumulation of DOXO with 5 was higher than with
4. It is known that DOXO up-regulates the inducible isoform of
NO synthase (iNOS),15 thereby increasing the endogenous syn-
thesis of NO in mammalian cells. Thus, it is possible that the
nitration of MRP3 elicited by 4 and 5 is due in part to the release
of NO by the products and in part to the increased production of
endogenous NO via the induction of the iNOS enzyme. No
nitration occurred in P-gp protein. Both P-gp and MRP3 are
involved in DOXO efflux, depending on the different tissues and
on the relative abundance of the transporters in tumor cells.3
Because the treatment with the NO donor compounds 3 and 7
did not modify the activity of P-gp in HT29-dx cells (see the
rhodamine 123 test in the Supporting Information), we have to
conclude that this transporter does not play a primary role in the
effect exerted by NO on DOXO retention. This is most likely due
to the lower number of tyrosine residues of the transporter with
respect to MRP3 and/or to its lower expression.7,16
(3) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in
cancer: Role of ATP-dependent transporters. Nat. Rev. Cancer 2002,
2, 48–58.
(4) Colabufo, N. A.; Berardi, F.; Cantore, M.; Contino, M.; Inglese,
C.; Niso, M.; Perrone, R. Perspectives of P-glycoprotein modulating
agents in oncology and neurodegenerative diseases: Pharmaceutical,
biological, and diagnostic potentials. J. Med. Chem. 2010, 53, 1883–
1897 and references therein.
(5) www.clinicaltrials.gov (NCT00028873).
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Gottesman, M. M.; Szakꢀacs, G. Identification of compounds selectively
killing multidrug-resistant cancer cells. Cancer Res. 2009, 69, 8293–8301.
(7) Riganti, C.; Miraglia, E.; Viarisio, D.; Costamagna, C.; Pescar-
mona, G.; Ghigo, D.; Bosia, A. Nitric oxide reverts the resistance to
doxorubicin in human colon cancer cells by inhibiting the drug efflux.
Cancer Res. 2005, 65, 516–525.
(8) Gasco, A.; Schoenafinger, K. The NO-releasing heterocycles. In
Nitric Oxide Donors; Wang, P. G., Cai, T. B., Taniguchi, N., Eds.; Wiley-
VCH Verlag GmbH & Co KGaA: Weinheim, 2005; pp 131ꢀ175.
(9) Fruttero, R.; Crosetti, M.; Chegaev, K.; Guglielmo, S.; Gasco, A.;
Berardi, F.; Niso, M.; Perrone, R.; Panaro, M. A.; Colabufo, N. A.
Phenylsulfonylfuroxans as modulators of multidrug-resistance asso-
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Graham, C. H. Nitric oxide-mediated regulation of chemosensitivity in
cancer cells. J. Natl. Cancer I 2001, 93, 1879–1885.
The preliminary results disclosed in the present work show
that the design of hybrid NO donor antitumor drugs could be a
useful strategy to address the problem of MDR in cancer therapy.
(11) Frederiksen, L. J.; Siemens, D. R.; Heaton, J. P.; Maxwell, L. R.;
Adams, M. A.; Graham, C. H. Hypoxia induced resistance to doxorubicin
in prostate cancer cells is inhibited by low concentrations of glyceryl
trinitrate. J. Urol. 2003, 170, 1003–1007.
’ ASSOCIATED CONTENT
(12) Mayer, B.; Beretta, M. The enigma of nitroglycerin bioactiva-
tion and nitrate tolerance: News, views and troubles. Br. J. Pharmacol.
2008, 155, 170–184.
(13) Daiber, A.; Wenzel, P.; Oelze, M.; Munzel, T. New insights into
bioactivation of organic nitrates, nitrate tolerance and cross-tolerance.
Clin. Res. Cardiol. 2008, 97, 12–20.
S
Supporting Information. Syntheses and characterization
b
data for the compounds and procedures for biological assays.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Grossi, L. Nitrite anion: The key intermediate in alkyl nitrates
degradative mechanism. J. Med. Chem. 2008, 51, 3318–3321.
(15) Aldieri, E.; Bergandi, L.; Riganti, C.; Costamagna, C.; Bosia, A.;
Ghigo, D. Doxorubicin induces an increase of nitric oxide synthesis in rat
cardiac cells that is inhibited by iron supplementation. Toxicol. Appl.
Pharmacol. 2002, 185, 85–90.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ39 011 6707850. Fax: þ39 011 6707286. E-mail: roberta.
fruttero@unito.it.
(16) Doublier, S.; Riganti, C.; Voena, C.; Costamagna, C.; Aldieri,
E.; Pescarmona, G.; Ghigo, D.; Bosia, A. RhoA silencing reverts the
resistance to doxorubicin in human colon cancer cells. Mol. Cancer Res.
2008, 6, 1607–1620.
’ ACKNOWLEDGMENT
We are indebted to Istituto Zooprofilattico Sperimentale del
Piemonte, Liguria e Valle d'Aosta, for mass spectral data.
’ ABBREVIATIONS
DOXO, doxorubicin; NO, nitric oxide; MDR, multidrug resistance;
P-gp, P-glycoprotein; ABC, ATP binding cassette; SNAP, S-nitroso-
penicillamine; SNP, sodium nitroprusside; GSNO, S-nitrosoglu-
tathione; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; sGC,
soluble guanylate cyclase; Rp-8-Br-cGMPS, 8-bromoguanosine-30,50-
cyclic monophosphorothioate; 8-Br-cGMP, 8-bromoguanosine cyclic
monophosphate;cGMP, guanosine-30,50-cyclicmonophosphate;
MDCK cells, MadinꢀDarby canine kidney cells; mtALDH, mi-
tochondrial aldehyde dehydrogenase
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