Inhibition of MDR1 activity and induction of apoptosis by analogues of nifedipine and diltiazem: An in vitro analysis

Article abstract of DOI:10.1007/s10637-009-9340-7

Summary: We report herein the reversal of multidrug resistance-1 (MDR1) in A2780/DX3 cells by the two nifedipine-like compounds 1 and 2 that are part of a library of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modulators bearing in C-4 a different substituted imidazo[2,1-b]thiazole system. By methylthiazol tetrazolium (MTT) assay, cytofluorimetry, and fluorescence microscopy we evaluated their ability to reverse MDR in our cell system. Moreover, together with compound 3 (the diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2- en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability to potentiate the triggering of apoptosis after exposure to doxorubicin, through the nuclear morphological analysis after 4,6-diamidino-2-phenylindole (DAPI), the fluorescein isothiocyanate (FITC)-Annexin-V/propidium iodide (PI) staining and the caspase activity determination. Our results demonstrate that compounds 1 and 2, at concentrations showing a very low (5%) or absent inhibition of cell proliferation, in combination with doxorubicin enhance its antiproliferative activity (from 30% to 54% IC₅₀ reduction) in A2780/DX3 cells through an increase of doxorubicin intracellular accumulation. These compounds together with compound 3, which has already been demonstrated to act as a potent inhibitor of MDR1 function, were also able to significantly potentiate the activation of the apoptosis machinery triggered by the exposure to doxorubicin. In conclusion, our results identify two new molecules structurally related to the calcium-channel blocker nifedipine, but characterized by a very low LTCC blockers activity, able to potentiate the antiproliferative and apoptotic activities of doxorubicin through an increase of its intracellular concentration likely caused by the inhibition of MDR1 function.

Full text of DOI:10.1007/s10637-009-9340-7

Invest New Drugs (2011) 29:98–109
DOI 10.1007/s10637-009-9340-7
PRECLINICAL STUDIES
Inhibition of MDR1 activity and induction of apoptosis
by analogues of nifedipine and diltiazem: an in vitro
analysis
Maurizio Viale & Cinzia Cordazzo & Daniela de Totero & Roberta Budriesi &
Camillo Rosano & Alberto Leoni & Pierfranco Ioan & Cinzia Aiello & Michela Croce &
Aldo Andreani & Mirella Rambaldi & Patrizia Russo & Alberto Chiarini &
Domenico Spinelli
Received: 3 September 2009 /Accepted: 28 September 2009 /Published online: 30 October 2009
#
Springer Science + Business Media, LLC 2009
Summary We report herein the reversal of multidrug
resistance-1 (MDR1) in A2780/DX3 cells by the two
nifedipine-like compounds 1 and 2 that are part of a library
of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modula-
tors bearing in C-4 a different substituted imidazo[2,1-b]
thiazole system. By methylthiazol tetrazolium (MTT) assay,
cytofluorimetry, and fluorescence microscopy we evaluated
their ability to reverse MDR in our cell system. Moreover,
together with compound 3 (the diltiazem-like 8-(4-
chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]
oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability
to potentiate the triggering of apoptosis after exposure to
doxorubicin, through the nuclear morphological analysis after
4′,6-diamidino-2-phenylindole (DAPI), the fluorescein isothio-
cyanate (FITC)-Annexin-V/propidium iodide (PI) staining and
the caspase activity determination. Our results demonstrate
that compounds 1 and 2, at concentrations showing a very low
(5%) or absent inhibition of cell proliferation, in combination
with doxorubicin enhance its antiproliferative activity (from
30% to 54% IC50 reduction) in A2780/DX3 cells through an
increase of doxorubicin intracellular accumulation. These
compounds together with compound 3, which has already
been demonstrated to act as a potent inhibitor of MDR1
function, were also able to significantly potentiate the
activation of the apoptosis machinery triggered by the
exposure to doxorubicin. In conclusion, our results identify
two new molecules structurally related to the calcium-channel
blocker nifedipine, but characterized by a very low LTCC
blockers activity, able to potentiate the antiproliferative and
apoptotic activities of doxorubicin through an increase of its
intracellular concentration likely caused by the inhibition of
MDR1 function.
:
:
:
C. Rosano
M. Viale (*) D. de Totero C. Aiello M. Croce
Istituto Nazionale per la Ricerca sul Cancro,
S.C. Terapia Immunologica,
Istituto Nazionale per la Ricerca sul Cancro,
S.C. Nanobiotecnologie,
L.go R. Benzi 10,
L.go R. Benzi 10,
16132 Genova, Italy
16132 Genova, Italy
e-mail: maurizio.viale@istge.it
P. Russo
C. Cordazzo
Istituto Nazionale per la Ricerca sul Cancro,
S.S. Tumori Polmonari,
L.go R. Benzi 10,
Dipartimento di Chimica Organica “A. Mangini”,
Università degli Studi di Bologna,
Via San Giacomo 11,
16132 Genova, Italy
40126 Bologna, Italy
:
:
:
:
:
R. Budriesi A. Leoni P. Ioan A. Andreani M. Rambaldi
A. Chiarini
D. Spinelli (*)
Dipartimento di Chimica “G. Ciamician”,
Università degli Studi di Bologna,
Via Selmi 2,
Dipartimento di Scienze Farmaceutiche,
Università degli Studi di Bologna,
Via Belmeloro 6,
40126 Bologna, Italy
40126 Bologna, Italy

Invest New Drugs (2011) 29:98–109
99
.
.
Keywords Antitumor agents Apoptosis
seems to possess interesting characteristics in preclinical
studies, thus having some objective chances to overcome
the MDR phenomena. Nevertheless, also in this case it is
not possible to foresee the possible toxicity of this in
humans, since this drug is also a potent Ca++ antagonist and
might therefore display potential cardiovascular dose-
limiting toxicities.
In this context some time ago we started a series of
experiments to study the ability of different diltiazem-like
compounds, some of which have already demonstrated
good cardiodepressant activities [22, 23], to compete for
excretion with the typical target of Pgp-170 activity,
doxorubicin, causing its accumulation into multidrug-
resistant A2780/DX3 cells [24].
Here we are analyzing the reversal of MDR1 activity by two
nifedipine-like compounds 1 and 2 (Fig. 1), which are again
related to LTCC blockers, but characterized by a very low
LTCC blockers activity (see after) and then would not show
negative cardiovascular effects during the administration.
These two substances are a part of a small library of 1,4-
DHPs and were synthesized and tested for their cardiovascular
profile on isolated guinea pig tissues [25]. All of the 33
examined compounds contained a dimethyl 2,6-dimethyl-4-
[imidazo[2,1-b][1,3]thiazol-5-yl]-1,4-dihydropyridine-3,5-
dicarboxylate scaffold possibly methylated at C-4 and/or
C-5 of the thiazole ring as well variously decorated with
halogens, alkyls, trifluoromethyl group, and differently
substituted aryl groups at C-6 of the imidazole ring.
.
.
1,4-dihydropyridines Multidrug resistance Pgp-170
Introduction
The resistance of many tumors to anticancer drugs has been
frequently linked to the over-expression of the MDR1 gene
[1–3]. This gene encodes for the ATP-dependent transporter
P-glycoprotein-170 (Pgp-170) that is involved in pumping
out of the cells a wide variety of neutral to slightly cationic
hydrophobic xenobiotics [4, 5]. In particular, Pgp-170 is
present in organs such as the placenta and the blood-brain
barrier where it pumps out toxic exogenous compounds
thus protecting, respectively, foetus and brain from toxic
damages [6–8].
Many tumors of epithelial origin display a high cell-
membrane concentration of Pgp-170 that may be involved
in some forms of intrinsic resistance. On the other hand,
different anticancer drugs having unrelated structural
characteristics and mechanisms of action may actively
select cells with high MDR1 copy number thus causing
the onset of an acquired drug resistance characterized by
the unresponsiveness to anthracyclines, taxanes, Vinca
alkaloids, podophyllotoxins, or camptothecins [9, 10].
Many ways to overcome MDR have been pursued in the
two last decades, in particular the inhibition of Pgp-170
activity has been considered as a central field of research
and many novel compounds have been studied for their
ability to block reversibly or irreversibly the pump function
of Pgp-170 [11]. The modified derivatives of the L-type
calcium channel (LTCC) blockers like nifedipine or
diltiazem, represent a group of compounds endowed with
significant features in terms of inhibition of the Pgp-170
activity. Nevertheless until now such compounds [12, 13]
displayed scarce efficacy in clinical tests due to their low
binding affinity, at doses necessary to enable the inhibition
of Pgp-170, while causing the onset of toxic phenomena at
higher doses due to the inhibition of metabolism and
excretion of anticancer drugs [14, 15].
On going from the previously examined diltiazem-like
compounds [24] to 1 and 2 we are again taking into
consideration molecules related to LTCC blockers, but we
are shifting towards different chemical systems showing a
completely different chemical structure with respect to the
In particular, dihydropyridine calcium antagonists such
as nifedipine, amlodipine, nimodipine, and others, are
commonly utilized in adults and pediatric patients as
antihypertensive drugs, having also shown in some cases
interesting properties as fibrinolytic agents [16, 17]. Among
these, nifedipine has been tested at different doses in
clinical trials as an MDR reversing agent without reaching
objective responses and observing the expected dose-
limiting toxicity due to the cardiovascular activity of this
Ca++ antagonist [18, 19]. About the other compounds, in
general, they have not reached the experimentation in
clinical trials. Presently only amlodipine, associated and
delivered together topotecan by stealthy liposomes [20, 21]
Fig. 1 Chemical structures of nifedipine, diltiazem, nifedipine- and
diltiazem-like compounds 1, 2, and 3

100
Invest New Drugs (2011) 29:98–109
series of the diltiazem-like ones. As a matter of fact, we
are moving from 8-aryl-8-hydroxy-5-R’-8H-[1,4]thiazino
[3,4-c]-[1,2,4]oxadiazol-3-ones and/or their acetals [24]
towards dimethyl 2,6-dimethyl-4-[imidazo[2,1-b][1,3]
thiazol-5-yl]-1,4-dihydropyridine-3,5-dicarboxylate op-
portunely substituted.
Our aim was the identification of new molecules able to
inhibit, if only partially, the Pgp-170 activity because of
their binding affinity to the interaction site. The new
examined molecules, for example, show a larger hydro-
philicity and contain atoms (such as the pyridine-nitrogen)
able to give interactions completely different with respect to
the compounds previously examined [24].
In general, compounds 1 and 2 showed a marked
selectivity for the cardiac over the vascular tissue. As a
matter of fact, none of them possesses a vasorelaxant
activity on the K+-depolarized aortic strips greater than
50%, whereas they showed a relaxation activity on K+-
depolarized guinea pig ileum longitudinal smooth muscle
between 51 and 97% [25]. In particular, our compounds 1
and 2 displayed a very low intrinsic activity on all
examined parameters of cardiac functionality, therefore we
selected them as possible interesting candidates to analyze
the results of their interaction with doxorubicin on the Pgp-
170 membrane transporter in doxorubicin sensitive A2780
and MDR1 resistant A2780/DX3 cells.
In addition, we analyzed the ability of the two
nifedipine-like compounds to increase the doxorubicin-
driven apoptosis in MDR1 resistant A2780/DX3 cells. The
potentiation of doxorubicin-driven apoptosis was also
tested with the diltiazem-like compound 3 [23] (Fig. 1)
that in our previous work [24] demonstrated a high potency
in terms of inhibition of the MDR1 activity.
doxorubicin. Two-three days before experiments doxorubi-
cin was removed from the medium.
Chemistry
The melting points are uncorrected. Analyses (C, H, N)
were within 0.4% of the theoretical values. TLC was
performed on Bakerflex plates (Silica gel IB2-F); the eluent
was a mixture of petroleum ether 60–80°C/acetone in
various proportions. Kieselgel 60 (VWR, Milano, Italy)
was used for flash chromatography. The IR spectra were
recorded in nujol on a Nicolet Avatar 320 E.S.P.; νmax is
expressed in cm−1. The 1H and 13C NMR spectra were
recorded on a Varian Gemini (300 MHz); the chemical shift
(referenced to solvent signal) is expressed in δ (ppm) and J
in Hz. Abbreviations: th = thiazole, ar = aromatic, py =
pyridine, ex = H linked to N which exchanged with D2O.
ESI-MS were registered on a micromass ZMD Waters
instrument (30 V, 3.2 kV). HRMS were recorded on a
Thermo Finnigan Mat95XP apparatus. The purity degree of
the tested compounds (1–3) was ≥97%.
Synthesis of dimethyl 2,6-dimethyl-4-[6-(3,4,
5-trimethoxyphenyl)imidazo[2,1-b][1,3]thiazol-5-yl]-1,
4-dihydropyridine-3,5-dicarboxylate 1 and of dimethyl 2,
6-dimethyl-4-[2-methyl-6-trifluoromethylimidazo[2,1-b]
[1,3]thiazol-5-yl]-1,4-dihydropyridine-3,5-dicarboxylate 2
The compounds 1 and 2 were synthesized by means of the
versatile reaction developed by Hantzsch [26].
Methylacetoacetate (2 mM) and 30% NH4OH (4 mM)
were added to a stirred solution of 6-(3,4,5-trimethoxyphenyl)
imidazo[2,1-b]thiazole-5-carbaldehyde [25] or of 2-methyl-6-
trifluoromethylimidazo[2,1-b]thiazole-5-carbaldehyde [27]
(1 mM) dissolved in isopropyl alcohol (50 ml). The reaction
mixture was refluxed for 1–4 days (according to a TLC test
acetone/petroleum ether 55–85°C, 1:9 v/v, 2:8 v/v) and added
of methylacetoacetate (4 mM) and 30% NH4OH (2 mM)
every 12 h. After cooling, the mixture was evaporated to
dryness under reduced pressure. The derivatives were purified
by flash chromatography, eluent acetone/petroleum ether,
40–60°C from 1.9 to 4.6 (v/v) with a yield of 10% for 1
and of 18% for 2.
Materials and methods
Chemicals and cells
The clinical form of doxorubicin from Ebewe Italia (Rome,
Italy) was used for experiments and diluted in normal saline
to the opportune concentrations. All of the examined
compounds were firstly dissolved in 100% dimethylsulf-
oxide (DMSO) and then diluted in fetal calf serum (final
concentration DMSO 2%). Nifedipine (purity≥98%, Sigma,
St. Louis, MO, USA) was similarly diluted.
Sensitive to doxorubicin A2780 cells were maintained in
culture in RPMI 1640 medium in the presence of 10% fetal
calf serum, 1% glutamine, and 1% penicillin-streptomycin
(complete medium), while resistant to doxorubicin A2780/
DX3 cells (provided by Dr. YM Rustum and obtained by
exposure to increasing concentrations of doxorubicin) were
maintained in complete medium containing 100 nM
Data for 1 C25H27N3O7S MW 513.567, mp 194–196°C,
1
IR: 3400–3100, 1671, 1250, 1206, 1015. H NMR ([D6]
DMSO): 2.18 (6H, s, CH3), 3.24 (6H, s, COOCH3), 3.70
(3H, s, OCH3), 3.81 (6H, s, OCH3), 5.73 (1H, s, py), 7.18
(2H, s, ar), 7.23 (1H, d, th, J=4.5), 7.27 (1H, d, th, J=4.5),
8.99 (1H, s, NH, ex D2O). 13C NMR ([D6]DMSO): 18.65,
32.21, 51.23, 56.69, 60.91, 98.94, 106.36, 113.81, 119.62,
128.60, 132.23, 137.44, 142.93, 146.15, 147.75, 153.16,
168.03. ESI-MS m/z: 514 [M+ H]+, 536 [M+ Na]+. HRMS
m/z found 513.1567 (calcd. 513.1570). EI-MS m/z (%) 513

Invest New Drugs (2011) 29:98–109
101
(3.2), 454 (26.70), 291 (18.73), 290 (100), 275 (96), 247
(21.38), 232 (23.64), 223 (19.67), 217 (18.93), 192 (31.51),
69 (26.17).
tions of nifedipine or nifedipine-like molecules equal to that
already contained in the wells. By this kind of treatment we
obtained a pre-treatment of 6 h with the experimental
molecules thereafter treated also with the anticancer drug
doxorubicin. After 3 days the MTT assay was applied as
already described.
IC50s were calculated on the basis of the analysis of
single dose response curves. Each experiment was repeated
4 times.
Data for 2 C18H18F3N3O4S MW 429,417, 270–275°C,
1
IR: 3400–3100, 1702, 1654, 1299, 1149. H NMR ([D6]
DMSO): 2.25 (6H, s, CH3), 2.43 (3H, s, CH3), 3.44 (6H, s,
COOCH3), 5.50 (1H, s, py), 7.21 (1H, s, th), 9.10 (1H, s,
NH, ex D2O). 13C NMR ([D6]DMSO): 14.64, 18.75, 32.34,
51.35, 98.34, 116.38, 125.72, 128.83, 132.51, 147.21,
147.35, 167.50. ESI-MS m/z: 430 [M+ H]+, 452 [M+
Na]+. HRMS m/z found 429.0971 (calcd. 429.0970). EI-
MS m/z (%) 429 (31.29), 371 (19.75), 370 (100), 224
(73.61), 206 (37.30), 165 (15.47), 95 (14.02), 81 (40.04),
69 (73.89), 57 (18.21), 55 (15.62), 41 (21.89).
Cytofluorimetric and microscopic study of intracellular
accumulation of doxorubicin
Sensitive and resistant to doxorubicin cells were plated in
25 cm2 flasks at 1×106 and 1.5×106 cells/flask, respec-
tively, in 10 ml. Twenty-four hours later, when cells
reached about 75–85% confluence, they were treated with
nifedipine or nifedipine-like compounds using concentra-
tions 5X that giving 5% inhibition of cell proliferation, as
calculated in the MTT assay. Doxorubicin was added 2 h
later in a small volume (5.8 or 29 μl) to reach the final
concentrations of 2 and 10 μM in A2780 and A2780/DX3
cells, respectively. Incubation was performed for additional
2 h. Cells were then detached by trypsin at 37°C for 5 min,
rapidly washed twice with cold phosphate buffered saline
(PBS), and fixed for 20 min with 3.7% paraformaldehyde
in PBS containing 2% sucrose. Cells were again washed
twice with PBS containing 2% fetal calf serum, pelleted
and concentrated in the same medium. Untreated cells and
control cells treated with nifedipine and nifedipine-like
compounds were also assayed. The intracellular fluores-
cence intensity of cells was determined by flow cytometry
(FACScan, BD Biosciences, Milano, Italy) using 488 nm
excitation and 575 nm bandpass filter for doxorubicin
detection. Values were expressed in arbitrary units as mean
fluorescence intensity (MFI).
For microscopical analysis A2780 and A2780/DX3 cells
were plated at 1×105 and 0.7×105 cells, respectively, in
chamber slides in a final volume of 1 ml. When they
reached 75–85% confluence they were treated as described
for the cytofluorimetric studies with the only difference
regarding doxorubicin concentration that was 10 μM for
both cell lines. After being washed twice with cold PBS,
cells were fixed for 20 min with 3.7% paraformaldehyde in
PBS containing 2% sucrose and washed twice again in PBS
containing 2% sucrose. Opportune controls (untreated cells
and cells treated with nifedipine and nifedipine-like com-
pounds) were always made. Slides were mounted with a
coverslip using GelMount (Biomeda Inc., CA). Cell
imaging was performed with an Axiovert 200 M epifluor-
escent microscope equipped with the filter set Omega
XF104-2 (Brattleboro, VT USA) using the Axiovision
Software (Carl Zeiss, Jena, Germany).
MTT assay
The resistant A2780/DX3 human ovarian carcinoma cell
line and its sensitive counterpart A2780 were plated at
opportune densities/well into 96-well microtiter plates,
centrifuged for 3 min at 275 g and then incubated for 6–
8 h. In order to evaluate the concentrations of nifedipine
derivatives giving 5% and 0% cell growth inhibition the
compounds were administered in duplicate for a minimum
of 5 concentrations (3-fold serial dilutions, maximal
concentration: 100 μM, maximal volume/well: 200 μl).
Nifedipine was also tested as reference compound. Three
days later 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (50 μl, MTT, Sigma, St. Louis, MO,
USA) solution (2 mg/ml in PBS) was added to the wells
and incubated at 37°C for 4 h. Microplates were then
centrifuged at 275 g for 5 min, culture medium carefully
aspirated and 100% DMSO (100 μl) added. Complete and
homogeneous solubilization of formazan crystals was
achieved after 20 min of incubation and shaking of well
contents by a multichannel pipette. A microculture plate
reader 400 ATC (SLT Labinstruments, Austria) was used
to measure the absorbance at 540 nm [28]. The searched
concentrations and, when possible, the corresponding
IC50s were calculated on the basis of the analysis of single
dose response curves. Each experiment was repeated 3–4
times.
In a second series of experiments cells were immedi-
ately diluted in a medium containing the opportune
concentrations of the various compounds (nifedipine and
nifedipine-like 1 and 2) at the concentrations calculated by
the previous MTT analysis for both A2780 and A2780/
DX3 cells and giving 0% and 5% inhibition of cell
proliferation. The final volume was 180 μl. Plates were
then centrifuged and 6 h later doxorubicin was added at the
opportune concentrations for A2780 (range 1–0.0016 μM)
and A2780/DX3 (range 30–0.048 μM) cells. The added
serial dilutions of doxorubicin also contained concentra-

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Invest New Drugs (2011) 29:98–109
FITC-Annexin-V/PI assay
Results
The induction of apoptosis was evaluated in resistant
A2780/DX3 cells. Cells were plated into 75 cm2 flasks
and treated with the IC50 of doxorubicin and the concen-
trations of 1, 2, and 3 giving 5% inhibition of cell
proliferation. After 72 h culture, floating, and adherent
cells were harvested, washed twice with cold PBS contain-
ing 2% fetal calf serum and apoptosis determined by double
staining with FITC-Annexin-V and PI (rh Annexin-V/FITC
Kit, Bender MedSystem GmbH, Vienna, Austria). Briefly,
0.5×106 cells were resuspended in binding buffer 1X and
stained with 5 μl of FITC-Annexin-V. After 10 min
incubation 10 μl of PI were added and samples incubated
for 5 min. Samples were immediately analyzed using a
Flow Cytometer (FACScan, BD Biosciences, Milano, Italy)
with dedicated software.
Nifedipine and nifedipine-like compounds
and cell proliferation
The two nifedipine-like molecules 1 and 2 did not show
significant inhibition of cell proliferation. For both of them
the IC50 for A2780 and A2780/DX3 cells were much
higher than 100 μM. This result is particularly significant if
compared to the mean IC50s calculated for doxorubicin,
which were on average 0.02 0.01 and 1.51 0.16 μM for
A2780 and A2780/DX3 cells (mean resistance index=75),
respectively.
On the basis of the same experiments we also calculated the
concentrations of our molecules giving 0% (IC0) and 5% (IC5)
inhibition of cell proliferation (Table 1) that were then used to
perform all the experiments of co-treatment with doxorubicin.
Visualization of apoptotic cells by DAPI staining
Nifedipine and nifedipine analogues increase intracellular
doxorubicin accumulation
A2780/DX3 cells were plated at 4000/well into 24-well
microtiter plates (flat-bottomed) and treated as described for
the western blot analysis. After 72 h all cells were harvested
and washed twice with cold PBS and then fixed in 70%
ethanol in PBS (100 μl) and maintained at 4°C. Just before
examination of fluorescence at the microscope, 5 μl of a
solution of DAPI (10 μg/ml) in water were added and the
percentage of apoptotic segmented nuclei/cells stained with
the fluorescent dye evaluated.
Nifedipine and compounds 1 and 2 were analyzed for their
ability to counteract the efflux of doxorubicin from A2780
and A2780/DX3 cells. In our experimental conditions
nifedipine caused a percent increase accumulation of
doxorubicin of 160 39% ( SE, n=7), compared to
doxorubicin alone and calculated by the ratio of MFI
values, while the compounds 1 and 2 caused an increase of
intracellular doxorubicin content of 150 37% (n=6) and
128 28% (n=5), respectively. It should also be underlined
that none of the tested substances caused a significant
accumulation of doxorubicin in sensitive A2780 cells (data
not shown). In Fig. 2 representative histograms of flow
cytometric analysis of nifedipine and by comparison of
compounds 1 and 2 are shown.
Determination of caspases’ activation
The activation of caspase complex was evaluated by using the
CaspScreen Flow Cytometric Apoptosis Detection kit (Chem-
icon Int., Temecula, CA). This assay utilizes a substrate that
contains two aspartate residues linked to rhodamine 110
(D2R) that become fluorescent only upon cleavage of the
substrate by cellular caspases. Briefly, cells were treated as
described for the western blot analysis. Once harvested and
washed twice with cold PBS cells were resuspended in 0.3 ml
of incubation buffer at 37°C and DTT (3 μl) plus D2R (1 μl)
reagents added. After 20 min incubation in the dark samples
were analysed by flow cytometry using 488 nm excitation and
530 nm bandpass filter.
Intracellular accumulation of doxorubicin was qualita-
tively confirmed by microscopy analysis of A2780/DX3
cells co-treated with nifedipine and nifedipine-like com-
Table 1 Concentrations (µM) of nifedipine and nifedipine-like
compounds inhibiting 0% and 5% proliferation in sensitive A2780
and resistant to doxorubicin A2780/DX3 cells
A2780
A2780/DX3
0%a
0.9
2.2
0.4
5%
3.3
0%a
12.9
2.0
5%
46.0
4.4
Statistics
Nifedipine
1
2
4.3
1.8
The Wilcoxon signed rank and the Mann-Whitney tests for
non-parametric data were used for statistical analysis
(StatView 4.5 software, Abacus Concepts Inc., Burlington,
MA, USA). The resistance indexes were calculated as the
ratio between the mean IC50 of resistant and that of
sensitive wild-type cells.
0.5
3.1
a
The concentration (µM) giving 0% inhibition of cell growth was
calculated on the basis of concentration-response curves obtained for
all compounds and represents the maximal concentration we can apply
without effect on cell growth

Invest New Drugs (2011) 29:98–109
103
Fig. 2 Representative
histograms of flow cytometric
analysis of nifedipine and
nifedipine-like compounds 1
and 2. All histograms are
superimposed in order to make
evident the shift of doxorubicin
content after combination with
Pgp-170 inhibitors. CTR,
untreated control. In these
experiments A2780/DX3 cells
were treated with the nifedipine,
1, or 2 at concentrations 5-times
higher than that giving 5%
inhibition of cell proliferation.
Doxorubicin (10 μM) was
added 2 h later and cells
incubated for other 2 h. Once
detached by trypsin, washed and
fixed in paraformaldehyde the
doxorubicin fluorescence
intensity of cells was determined
by flow cytometry
pounds. Cells co-treated with compounds 1 or 2 accumulat-
ed more doxorubicin than cells receiving only doxorubicin
(Fig. 3). No apparent drug accumulation was observed
when sensitive A2780 cells were co-treated with nifedipine,
1, and 2 (data not shown).
Nifedipine and nifedipine-like compounds potentiate
doxorubicin inhibition of cell proliferation
When A2780 and A2780/DX3 cells were co-treated with
scalar concentrations of doxorubicin and with the IC0 and
Fig. 3 Microscopy evaluation of doxorubicin accumulation in A2780/
DX3 cells after co-treatment with nifedipine and nifedipine-like
compounds. Cells were treated as described for the cytofluorimetric
studies in the presence of doxorubicin at 10 μM with or without
previous incubation with Pgp-170 inhibitors. After fixation with
paraformaldehyde the cell imaging was performed with an Axiovert
200 M epifluorescent microscope equipped with the filter set 10 using
the Axiovision Software (Carl Zeiss, Jena, Germany). Successive
images, were rendered using Adobe Photoshop 5.0 software (Adobe
Systems, Mountain View, CA, USA). Bar, 50 μm. Dox, doxorubicin

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Invest New Drugs (2011) 29:98–109
Table 2 Effect of combination of doxorubicin and nifedipine or nifedipine-like molecules in resistant to doxorubicin A2780/DX3 cells
5%a
IC50
0%b
IC50
% IC50 reduction
p valuec
% IC50 reduction
p value
Nifedipine
0.72 0.22d
1.00 0.09
0.06 0.20
−52
−48
−30
0.014
0.021
0.043
1.03 0.13
0.95 0.15
0.76 0.21
−32
−54
−52
0.021
0.021
0.021
1
2
a
Results obtained by the use of combinations of the concentration of nifedipine, 1 and 2 compounds with doxorubicin giving alone 5% growth
inhibition, as calculated by the MTT assay
b
Results obtained by the use of combinations of doxorubicin with the concentration of nifedipine and nifedipine-like compounds giving alone
0% growth inhibition, as calculated by the MTT assay
c
P values were calculated according to the Mann-Whitney test for non-parametric data
d
Data are expressed in µM concentrations
IC5 of 1 and 2, the two nifedipine-like compounds showed
the ability to significantly reduce the IC50 of doxorubicin,
with a percent reduction ranging from 30% to 54%, only in
resistant cells (Table 2), leaving the IC50 of doxorubicin in
A2780 unmodified (doxorubicin alone 18 2 nM vs 22
6 nM and 17 8 nM for 1; vs 20 8 nM and 19 9 nM for 2;
vs 21 4 nM and 18 7 nM for nifedipine). It must be
underlined that the percent reduction for any compound
was calculated on the basis of IC50 values for doxorubicin
alone evaluated for each single experiment.
significant amount of apoptosis compared to controls when
administered individually at the concentration giving 5%
inhibition of cell proliferation (control vs compound; for 1,
12.6 3.1 vs 13.3 3.3; for 2, 12.2 2.2 vs 14.0 2.3; for 3,
10.4 4.3 vs 11.6 3.3).
Determination of apoptosis through morphological analysis
of nuclei after DAPI staining and by Annexin-V/PI assay
To determine whether the observed inhibition of cell
proliferation caused by the co-treatment of the cells with
doxorubicin and the examined compounds was in part due
to apoptosis we sought for the presence of typical markers
of apoptosis. Therefore we analyzed nuclei fragmentation
by DAPI staining, phosphatidylserine externalization by
FITC-Annexin-V labeling and membrane permeability by
PI staining, respectively.
Staining of segmented nuclei with DAPI showed a
significant increase of apoptotic cells, as detected by
fluorescence microscopy analysis, after three days of co-
treatment with doxorubicin and compounds 1, 2, or 3
compared with doxorubicin alone (Fig. 4). Conversely, the
incubation of the cells with the examined compounds alone
did not modify significantly the observed percentage of
segmented nuclei, as compared to the control (1 1% for 1,
2 1% for 2, and 3 2% for 3 vs 2 2% for the control).
FACS analysis of A2780/DX3 cells stained with FITC-
Annexin-V/PI after co-treatment with doxorubicin and the
tested compounds confirmed the increase of apoptosis with
respect to the treatment with doxorubicin alone (Figs. 4 and 5).
In this case the most effective was compound 2 followed by
compounds 3 and 1. None of them was able to induce
Fig. 4 Histograms represent the percent increase of A2780/DX3 cells
showing segmented nuclei (mean SE of 6 data, as detected after
DAPI staining and microscopic observation, ■) and the percent
increase of cells stained with FITC-Annexin-V/PI to make evident
the externalized membrane phosphatidylserine (mean
SE of 5–7
data, □), after co-treatment with doxorubicin and compounds 1, 2, or 3
(used at the concentrations giving 5% inhibition of cell proliferation).
Percentages were calculated in relation to the percentage of apoptotic
cells after doxorubicin alone exposure (100%). P values were
calculated according to the Wilcoxon signed rank test for non-
parametric data considering the number of apoptotic cells recognized
after DAPI staining and the sum of early (Ann-V+/PI−) and late (Ann-
V+/ PI+) apoptotic cells. The absolute number (mean SE) of DAPI
positive apoptotic cells were: for 1 (doxorubicin vs doxorubicin + 1)
6 1 vs 10 2; for 2 (doxorubicin vs doxorubicin + 2) 6 1 vs 9 2; for
3 (doxorubicin vs doxorubicin + 3) 6 1 vs 10 2. The absolute
number (mean SE) of Annexin-V positive early plus late apoptotic
cells were: for 1 (doxorubicin vs doxorubicin + 1) 26.3 3.0 vs 33.6
3.6; for 2 (doxorubicin vs doxorubicin + 2) 18.4 0.4 vs 25.8 3.0; for
3 (doxorubicin vs doxorubicin + 3) 16.5 1.9 vs 22.4 2

Invest New Drugs (2011) 29:98–109
105
Activation of caspases
Discussion
Finally, to assess whether the observed potentiation of
apoptosis by tested compounds was due to an increased
activation of caspases, responsible for triggering this
process, we analyzed in a single confirming experiment
their activation by flow cytometry using a substrate
containing rhodamine linked to two aspartate residues that
become fluorescent only after cleavage by caspases.
By this analysis we observed a moderate activation of
caspases after co-treatment with doxorubicin and com-
pounds 1, 2 with respect to doxorubicin alone (Fig. 6). In
particular, the most effective compound was 3 followed by
2 and 1 (185%, 136%, and 122%, respectively, expressed as
percent increase compared to doxorubicin alone treatment).
Moreover, the treatment of the cells with the individual
compounds alone did not involve the activation of caspases
(data not shown).
The main purpose of this study was to investigate the
results of interaction of doxorubicin and the now examined
nifedipine-like compounds 1 and 2 on Pgp-170 membrane
transporter in doxorubicin sensitive A2780 and MDR1
resistant A2780/DX3 cells [29]. These compounds showed a
different chemical structure with respect to the series of the
diltiazem-like ones [24], being shifted towards different
chemical systems, as reported in Introduction. In fact, for
example, they show a lower log P and contain pyridine-
nitrogen atoms that could cause different chemical interac-
tions as compared to the diltiazem-like compounds previously
studied [24].
In particular, the choice of studying these two new
molecules was also taken on the basis of our previous
results indicating a low activity for these compounds on
cardiac functionality (for their cardiovascular activity, see
Fig. 5 Representative dotplots of A2780/DX3 treated with doxorubicin
plus compounds 1, 2 and 3 and stained with FITC-Annexin-V/PI.
Dotplots of single compounds 1, 2 and 3 were omitted for simplicity.
Mean data pertinent to these dotplots are reported in the paragraph
“Determination of apoptosis through morphological analysis of nuclei
after DAPI staining and by Annexin-V/PI assay” of the “Results” section

106
Invest New Drugs (2011) 29:98–109
data in Table 3). In fact the observation that nifedipine-like
compounds 1 and 2 possess a low intrinsic activity on all of
the examined parameters of cardiac functionality let us
think that they could have also low toxic effect (particularly
at cardiac level) once assayed as MDR1 reversing agents in
specifically designed in vivo experimental models.
10–15 times more active than nifedipine itself on a molar
basis. These data, together with the observation that
compounds 1 and 2 at equitoxic concentrations affect
doxorubicin antiproliferative activity similarly to the parent
nifedipine or to the active diltiazem-like compounds [24],
may imply the existence of other cytotoxic mechanisms of
action different from the inhibition of Pgp-170, still to be
determined, and possibly acting sinergistically with the
mechanism here studied. On the other hand the fact that
both 1 and 2 do not increase the doxorubicin activity
reaching that observed in A2780 cells, in terms of MTT
activity and drug accumulation, is in part counteracted by the
observation that also nifedipine, an active compound that has
reached the clinical phase studies [18, 19], has a similar
activity in our experimental conditions.
The membrane expression of Pgp-170, studied by the
anti-MDR1 MM4.17 monoclonal antibody (kindly provid-
ed by Dr. M. Cianfriglia), was not down-regulated in the
presence of active concentration of both nifedipine-like
compounds (data not shown), thus confirming that the
intracellular accumulation of doxorubicin was not a
consequence of the Pgp-170 down-regulation.
Finally, concerning the evaluation of hydrophobicity of 1
and 2, we calculated their log P (ChemBioDraw 11.0
software), as 1.75 and 1.72, respectively. It is of note that
an appropriate value of log P (in the 3–5 range) together
with the presence of one or more coplanar rings have been
proposed as important characteristics of chemosensitizers
acting as competitive inhibitor of Pgp-170 [6, 30, 31]. Our
results seem to indicate that notwithstanding their more
hydrophilic characters also compounds 1 and 2 with a
Both 1 and 2, when tested for the inhibition of cell
proliferation, showed an extremely low effect, with IC50s
much higher than 100 μM on both sensitive A2780 and
resistant A2780/DX3 cells and well different from the
respective IC50 values for doxorubicin. When co-
administered with doxorubicin at the inactive or slightly active
concentrations giving 0% and 5% inhibition of cell prolifer-
ation both 1 and 2 were able to potentiate by a significant 30–
54% doxorubicin activity, as evaluated considering the IC50
parameter. The inhibition of Pgp-170 activity was also
demonstrated by cytofluorimetric and microscopic analyses
that showed, in the presence of nifedipine-like compounds 1
and 2, a +150% and +128% accumulation of co-administered
doxorubicin, respectively, similar to that observed using a
comparable nifedipine concentration (+160%). In this context
it is also worth of note that no higher accumulation was
observed when we used A2780 sensitive cells as target, that
express membrane Pgp-170 at extremely low levels.
On the base of these results our nifedipine-like compounds
could appear less active in terms of doxorubicin accumulation
(about 40% of doxorubicin accumulation in sensitive A2780
cells) than some of our previously described diltiazem-like
compounds [24], other than in general. Nevertheless it must
be underlined that both molecules 1 and 2 are active 5 to 7
more times than the considered diltiazem-like molecules and
Fig. 6 Histograms of flow
cytometric analysis of activation
of caspase complex obtained
using the CaspScreen Flow
Cytometric Apoptosis Detection
kit in A2780/DX3 cells. Only
histograms obtained after
treatment with doxorubicin
alone and after co-treatment
with doxorubicin and 1, 2, or 3
were reported. Nifedipine—and
diltiazem-like compounds 1, 2,
and 3 showed histograms
superimposed to those obtained
for untreated controls while
doxorubicin showed about 93%
increase of MFI compared to
controls (data not shown)

Invest New Drugs (2011) 29:98–109
107
Table 3 Cardiovacular activity of compounds 1–3, of nifedipine, and of diltiazema,b
% decrease (M SEM)
EC50 of inotropic
neg. activity
EC30 of chronotropic
neg. activity
Vasorelaxant activity
e
f
e
Negative
inotropic
activityc
Negative
EC50
(µM)
95% conf.
EC30
(µM)
95% conf.
Activityf
(M SEM)
IC50
95% conf.
lim. (×10−6
)
chronotropic
lim. (×10−6
)
lim. (× 10−6
)
(µM)
activityd
Nifedipinea
1a
2a
Diltiazemb
3b
97.0 2.0g
42.0 0.2
46.0 1.3
78.0 3.5h
67.0 2.4i
85.0 4.2h
25.0 1.5j
36.0 1.7g
94.0 5.6k
5.0 0.2
0.26
0.19–0.36
0.025
0.07
0.019–0.031
0.064–0.075
82.0 1.3k
44.0 3.7
28.0 1.5i
88.0 2.3
10.0 0.8i
0.009
0.003–0.02
2.2–3.1
0.79
1.31
0.70–0.85
0.97–1.43
2.6
a
From reference [25]
From reference [23]
b
c
Activity: decrease in developed tension in isolated guinea-pig left atrium at 106 M, expressed as percent changes from the control (n=4–6). The
left atria were driven at 1 Hz. The 10−6 M concentration gave the maximum effect for most compounds
d
Activity: decrease in atrial rate in guinea-pig spontaneously beating isolated right atria at 5×10−5 M, expressed as percent changes from the
control (n=6–8). Pretreatment heart rate ranged from 170 to 195 beats/min. The 5×10−5 M concentration gave the maximum effect for most
compounds
e
Calculated from log concentration-response curves [Probit analysis according to Litchfield and Wilcoxon (Tallarida, R. J.; Murray, R. B. Manual
of Pharmacologic Calculation with Computer Programs, 2nd ed.; Springer-Verlag: New York; 1987)] with n=6–8. When the maximum effect was
<50%, the EC50 inotropic, EC30 chronotropic and IC50 values were not calculated
f
Activity: percent inhibition of calcium-induced contraction on K+ -depolarized guinea-pig aortic strip at 10−4 M. The 10−4 M concentration gave
the maximum effect for most compounds
g
At 10−5
M
h
i
At 5×10−7
M
M
At 10−5
At 5×10−6
M
j
k
At 10−6
M
dihydropyridine scaffold decorated by 4-substituted imi-
dazo[2,1-b]thiazole may act as inhibitors of Pgp-170.
Finally, in spite of the relatively recent controversial
opinions on the real role of apoptosis in clinical setting [32]
the concept that this process may play a crucial role for the
response to anticancer chemotherapy [33–35] has become a
milestone for the anticancer drug research and for new
chemotherapy-based therapeutic procedures. It is well
known that apoptosis represents an important physiological
pathway of cell death other than one of the possible main
modalities of cell death after chemotherapy, the other one
being necrosis. As many other anticancer drugs, doxorubi-
cin is able to trigger apoptosis in tumor and normal tissues
both in vitro and in vivo [36–38]. This is the reason why we
decided to study this important mechanism of cell death in
vitro when the topoisomerase inhibitor was co-administered
together with the tested nifedipine or diltiazem-like com-
pounds. This was also important to demonstrate that both 1
and 2 and also the diltiazem-like compound 3 do not
represent possible inhibitors of this important mechanism.
On the whole, our results indicate that apoptotic triggering
of doxorubicin, as detected by FITC-Annexin-V/PI assay and
DAPI staining, is significantly potentiated by the addition of
slightly active concentrations of the diltiazem-like 3 and of
the nifedipine-like 1 and 2. At these concentrations no one
of our compounds was able to induce apoptosis in a
significant extent compared to untreated controls. These
results were confirmed, although in a small extent, by the
cytofluorimetric analysis of caspases’ activity.
In conclusion our data show that nifedipine-like 1 and 2
increase the intracellular concentration of doxorubicin,
likely by competing for the efflux mediated by the Pgp-
170 transporter, and together with the diltiazem-like
compound 3 potentiate its antiproliferative and pro-
apoptotic activity. Although the activity of these com-
pounds may not seem to be particularly high it must be
underlined that in term of doxorubicin accumulation and
increase of doxorubicin antiproliferative activity their effi-
ciency was similar to that of nifedipine while it is highly
predictable a very lower cardiac activity. All these properties,
in particular their low activity on cardiac functionality, may
have potential therapeutic relevance prompting us for future
modifications of chemical structures in order to obtain more
active substances and in vivo studies.

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Invest New Drugs (2011) 29:98–109
Acknowledgements This research was supported by funds from
“Ministero dell’Istruzione, dell’Università e della Ricerca” (FIRB
2001, PRIN-2005034305_001, PRIN-20078J9L2A_005) and from the
University of Bologna. Dr. M. Croce is recipient of a fellowship by
“Fondazione italiana per la lotta al neuroblastoma”.
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