Hydrogen sulphide-releasing diclofenac derivatives inhibit breast cancer-induced osteoclastogenesis in vitro and prevent osteolysis ex vivo

Article abstract of DOI:10.1111/j.1476-5381.2011.01704.x

Background and purpose: Hydrogen sulphide (H₂S) and prostaglandins are both involved in inflammation, cancer and bone turnover, and non-steroidal anti-inflammatory drugs (NSAIDs) and H₂S donors exhibit anti-inflammatory and anti-tumour properties. H₂S-releasing diclofenac (S-DCF) derivatives are a novel class of NSAIDs combining the properties of a H₂S donor with those of a conventional NSAID. Experimental approach: We studied the effects of the S-DCF derivatives ACS15 and ACS32 on osteoclast and osteoblast differentiation and activity in vitro, human and mouse breast cancer cells support for osteoclast formation and signalling in vitro, and osteolysis ex vivo. Key results: The S-diclofenac derivatives ACS15 and ACS32 inhibited the increase in osteoclast formation induced by human MDA-MB-231 and MCF-7 and mouse 4T1 breast cancer cells without affecting breast cancer cell viability. Conditioned media from human MDA-MB-231 cells enhanced II°B phosphorylation and osteoclast formation and these effects were significantly inhibited following treatment by ACS15 and ACS32, whereas the parent compound diclofenac had no effects. ACS15 and ACS32 inhibited receptor activator of NFI°B ligand-induced osteoclast formation and resorption, and caused caspase-3 activation and apoptosis in mature osteoclasts via a mechanism dependent on IKK/NFI°B inhibition. In calvaria organ culture, human MDA-MB-231 cells caused osteolysis, and this effect was completely prevented following treatment with ACS15 and ACS32. Conclusions and implications: S-diclofenac derivatives inhibit osteoclast formation and activity, suppress breast cancer cell support for osteoclastogenesis and prevent osteolysis. This suggests that H₂S-releasing diclofenac derivatives exhibit anti-resorptive properties, which might be of clinical value in the treatment of osteolytic bone disease.

Full text of DOI:10.1111/j.1476-5381.2011.01704.x

DOI:10.1111/j.1476-5381.2011.01704.x
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
Dr Aymen I Idris, Edinburgh
Cancer Research Centre,
RESEARCH PAPERbph_1704 1914..1925
University of Edinburgh, General
Western Hospital, Edinburgh
EH4 2XR, UK. E-mail:
aymen.idris@ed.ac.uk
----------------------------------------------------------------
Hydrogen sulphide-releasing
diclofenac derivatives inhibit
breast cancer-induced
osteoclastogenesis in vitro
and prevent osteolysis
ex vivo
Keywords
diclofenac; NSAID; hydrogen
sulphide; osteoclast; osteoblast;
breast cancer; MDA-MB-231;
MCF7; 4T1
----------------------------------------------------------------
Received
17 June 2011
Revised
4 August 2011
Accepted
30 August 2011
J Frantzias1, JG Logan1,2, P Mollat3, A Sparatore4, P Del Soldato5,
SH Ralston2 and AI Idris1,2
1The Centre for Molecular Medicine, Institute of Genetics and Molecular Medicine, University of
Edinburgh, Western General Hospital, Edinburgh, UK, 2Edinburgh Cancer Research Centre,
Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital,
Edinburgh, UK, 3Galapagos SASU, Romainville, France, 4Department of Pharmaceutical Sciences
‘P. Pratesi’, Università degli Studi di Milano, Milan, Italy, and 5CTG Pharma S.r.l., Milan, Italy
BACKGROUND AND PURPOSE
Hydrogen sulphide (H2S) and prostaglandins are both involved in inflammation, cancer and bone turnover, and non-steroidal
anti-inflammatory drugs (NSAIDs) and H2S donors exhibit anti-inflammatory and anti-tumour properties. H2S-releasing
diclofenac (S-DCF) derivatives are a novel class of NSAIDs combining the properties of a H2S donor with those of a
conventional NSAID.
EXPERIMENTAL APPROACH
We studied the effects of the S-DCF derivatives ACS15 and ACS32 on osteoclast and osteoblast differentiation and activity in
vitro, human and mouse breast cancer cells support for osteoclast formation and signalling in vitro, and osteolysis ex vivo.
KEY RESULTS
The S-diclofenac derivatives ACS15 and ACS32 inhibited the increase in osteoclast formation induced by human MDA-MB-231
and MCF-7 and mouse 4T1 breast cancer cells without affecting breast cancer cell viability. Conditioned media from human
MDA-MB-231 cells enhanced IkB phosphorylation and osteoclast formation and these effects were significantly inhibited
following treatment by ACS15 and ACS32, whereas the parent compound diclofenac had no effects. ACS15 and ACS32
inhibited receptor activator of NFkB ligand-induced osteoclast formation and resorption, and caused caspase-3 activation and
apoptosis in mature osteoclasts via a mechanism dependent on IKK/NFkB inhibition. In calvaria organ culture, human
MDA-MB-231 cells caused osteolysis, and this effect was completely prevented following treatment with ACS15 and ACS32.
CONCLUSIONS AND IMPLICATIONS
S-diclofenac derivatives inhibit osteoclast formation and activity, suppress breast cancer cell support for osteoclastogenesis and
prevent osteolysis. This suggests that H2S-releasing diclofenac derivatives exhibit anti-resorptive properties, which might be of
clinical value in the treatment of osteolytic bone disease.
Abbreviations
ALP, alkaline phosphatase; ALZ, alizarin red; BM, bone marrow; BV, bone volume; COX, cyclo-oxygenase; DAPI,
4,6-diamidino-2-phenylindole; GI, gastrointestinal; H2S, hydrogen sulphide; M-CSF, mouse macrophage colony
stimulating factor; NSAID, non-steroidal anti-inflammatory drugs; RANKL, receptor activator of NFkB ligand;
S-diclofenac, H2S-releasing diclofenac; S-NSAID, H2S-releasing NSAID; TRAcP, tartrate-resistant acid phosphatase
© 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
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S-NSAIDs inhibit osteoclast–tumour cell interaction
BJP
Introduction
Hydrogen sulphide (H2S) is involved in the regulation of
vasodilatation, neuromodulation, cardiovascular homeosta-
sis and inflammation (Lefer, 2007; Li et al., 2009). A number
of preclinical studies reported that H2S-releasing compounds
such as garlic-related products exert anti-tumour effects. For
example, natural components of garlic such as diallyl sul-
phide, diallyl disulphide and diallyl trisulphide inhibit pro-
liferation and induce apoptosis of colon, breast, lung and
pancreatic tumour cells in vitro and attenuate tumour
growth in animal models of cancer (Dipaolo and Carruth-
ers, 1960; Hui et al., 2008; Lei et al., 2008; Oommen et al.,
2004; Seki et al., 2008; Sriram et al., 2008; Gayathri et al.,
2009; Kim and Kwon, 2009). Studies also showed that
S-allylmercapto-L-cysteine, a by-product of naturally occur-
ring garlic derivatives, inhibited growth and induced apop-
tosis of human colon and prostate cells in vitro and in vivo
and suppressed tumorigenesis in
a xenograft model of
gastric cancer (Pinto et al., 2000; Shirin et al., 2001; Lee
et al., 2011). The beneficial effect of these compounds is
mostly attributed to H2S production due to catabolism of
the polysulphide group in allicin, the major biologically
active component of garlic (Seki et al., 2008). There is also
evidence to suggest that H2S donors affect bone remodel-
ling. Garlic extract inhibited serum markers of bone turn-
over and significantly reduced bone loss in rodent models
of sex hormone deficiency (Mukherjee et al., 2004; 2006).
Whilst these studies suggest that H2S donors may exhibit
anti-resorptive and anti-tumour properties, very little
research has been conducted on the direct effects of these
agents on bone and breast cancer cell proliferation, survival,
activity and crosstalk.
H2S-releasing non-steroidal anti-inflammatory drugs
(S-NSAIDs) are a novel class of NSAID in which the core
structure of the parent compound was modified by the addi-
tion of a H2S-releasing moiety (reviewed in Li et al., 2009;
Fiorucci and Santucci, 2011). The rationale behind the devel-
opment of S-NSAIDs is based on the notion that the vasodi-
lator effects of the H2S moiety are likely to counteract the
gastrointestinal (GI) toxicity associated with inhibition of
cyclo-oxygenase (COX-1) activity and prostaglandin produc-
tion (Lefer, 2007; Li et al., 2007; 2009; Wallace, 2007; Fiorucci
and Santucci, 2011). A number of studies demonstrated that
S-NSAIDs retain the desired anti-inflammatory and analgesic
properties of conventional NSAIDs, but it has yet to be estab-
lished whether these agents are less likely to cause GI toxicity
(Lefer, 2007; Wallace, 2007; Li et al., 2009; Fiorucci and San-
tucci, 2011).
Figure 1
Chemical structure of the NSAID diclofenac and the S-diclofenac
derivatives, ACS15 and ACS32.
studies also showed that the S-diclofenac derivative ACS15
(Figure 1) inhibited prostaglandin production and cell pro-
liferation in cultured lung cancer cells in vitro and reduced
tumour growth in a xenograft model of lung cancer (Moody
et al., 2010). As H2S and prostaglandins are known to exert
modulatory effects on both tumorigenesis and bone re-
modelling, we hypothesized that S-diclofenac derivatives
may exert an inhibitory effects on breast cancer-induced
osteoclastogenesis and osteolysis.
H2S-releasing diclofenac (S-diclofenac) derivatives,
such
as
2-[(2,6-dichlorophenyl)amino]
benzeneacetic
In this study, we showed that the S-diclofenac derivatives
ACS15 and ACS32 inhibited the increase in osteoclast forma-
tion induced by human and mouse breast cancer cells in vitro
and prevented MDA-MB-231-induced osteolysis ex vivo.
ACS-15 and ACS-32 inhibited NFkB activity in mature osteo-
clasts and their precursors, significantly suppressed osteoclast
formation and bone resorption and caused mature osteoclast
apoptosis. This suggests that H2S-releasing diclofenac deriva-
tives may be of clinical value for the prevention and treat-
ment of skeletal complications associated with osteolytic
bone disease.
acid 4-(3-thioxo-3H-1,2-dithiol-5-yl) phenyl ester (ACS15)
and [3-(2,6-dichloro-phenylamino)-phenyl]-acetic acid 2-
methanesulphonylsulphanyl-ethyl ester (ACS32) (Figure 1),
combine the properties of a H2S donor with those of the
parent compound. S-diclofenac derivatives slowly release H2S
in vitro and in vivo, protect from oxidative damage, inhibit
NFkB activation in vitro and in vivo and reduce production of
pro-inflammatory cytokines in animal models of arthritis
through a mechanism that involves H2S production (Li et al.,
2007; Sidhapuriwala et al., 2007; Moody et al., 2010). Recent
British Journal of Pharmacology (2012) 165 1914–1925 1915

J Frantzias et al.
BJP
TRAcP staining
Methods
TRAcP staining was used to identify multi-nucleated osteo-
clasts. At the end of the culture, osteoclast cultures were fixed
in 4% paraformaldehyde, washed with PBS and incubated
with naphthol-ASBI-phosphate, pararosanilin and tartrate in
acetate buffer (30 mM) at 37°C for 45 min as previously
described (van’t Hof, 2003). TRAcP positive cells with three or
more nuclei were considered to be osteoclasts and manually
counted on a Zeiss Axiovert light microscope using a 10¥
objective lens.
Synthesis of ACS32
A
1N solution of dicyclohexylcarbodiimide (2.8 mL) in
dichloromethane was added to a solution of diclofenac
(758 mg; 2.56 mmol), methanethiosulphonic acid S-(2-
hydroxyethyl) ester (400 mg, 2.56 mmol) and dimethylami-
nopyridine (15 mg) in dichloromethane (40 mL) and the
mixture was stirred at room temperature, under nitrogen for
1.5 h (Foong et al., 1997). After filtration of the dicyclohexy-
lurea, the solution was extracted with a saturated solution of
NaHCO3 and then with cold water. The organic phase was
dried on anhydrous sodium sulphate and evaporated to
dryness. The residue was purified by flash chromatography
on silica gel (cyclohexane/ethylacetate; 80:20) and crystal-
Quantification of resorption area
Resorption pits were visualized by reflected light microscopy
and the area resorbed was quantified by image analysis using
custom software developed using Aphelion ActiveX objects
(ADCIS, 14280 Saint-Contest, France) as previously described
(van’t Hof, 2003).
ֺ
lized from ether. Yield was 51.5%. Melting point (Buchi appa-
ratus) was 41–44°C. 1H NMR (Varian Mercuri 300VX
spectrometer (Milan, Italy); CDCl3): d 7.30 (d, 2H); 7.20 (t,
2h); 7.10 (t, 1H); 6.90–7.05 (q, 2H), 6.65 (s, 1H, collapses with
D2O); 4.40 (t, 2H), 3.80 (s, 2H); 3.40 (t, 2H); 3.30 (s, 3H).
Osteoblast culture
Primary osteoblasts were isolated from the calvarial bones of
2 day-old mice by sequential collagenase digestion as previ-
ously described (Bakker and Klein-Nulend, 1988). For bone
nodule assay, osteoblasts were seeded into 12-well plates
(10 ¥ 105 cells per well) in standard aMEM supplemented
with b-glycerol phosphate (10 mM) and L-ascorbic acid
(50 mg·mL-1) for up to 21 days. Osteoblast number, differen-
tiation and bone nodule formation were determined by
Alamar Blue assay, alkaline phosphatase (ALP) assay and
alizarin red (ALZ) staining respectively.
RANKL and M-CSF mouse osteoclast culture
All animal care and experimental procedures complied with
UK Home Office regulations and were approved by the ethical
review board of the University of Edinburgh. Osteoclast for-
mation, apoptosis and resorption were studied using RANKL
and M-CSF-generated mouse osteoclasts, as described in
detail by Idris et al. (2010). Briefly, female C57BL/6 mice
(Harlan Laboratories, UK), 3 to 5 months old, were killed by
cervical dislocation and bone marrow cells were flushed from
the long bones. These bone marrow cells were plated into
Petri dishes and incubated for 48 h in standard aMEM
(aMEM supplemented with 10% fetal calf serum, penicillin
and streptomycin) and mouse M-CSF (100 ng·mL-1). For
osteoclast generation, the resulting M-CSF-dependent osteo-
clast precursor cells were plated into tissue culture plates
(96-well plates, 15 ¥ 103 cells per well; 12-well plates, 150 ¥
103 cells per well) in standard aMEM supplemented with
M-CSF (25 ng·mL-1) and RANKL (100 ng·mL-1). The cultures
were terminated by fixation in 4% paraformaldehyde and
stained for tartrate-resistant acid phosphatase (TRAcP).
Alamar Blue assay
Alamar Blue assay was used to measure the number of osteo-
blast and breast cancer cells (Nakayama et al., 1997). At the
end of the culture period, Alamar Blue reagent (10%, v/v) was
added to each well, cells were incubated for a further 3 h and
then fluorescence was measured (excitation, 530 nm, emis-
sion 590 nm) using a Biotek Synergy HT plate reader (Bed-
fordshire, UK).
ALP assay
ALP activity was used to assess the differentiation of mouse
calvarial osteoblasts (Bakker and Klein-Nulend, 1988). Osteo-
blasts were homogenized in ALP lysis buffer [1 M diethano-
lamine, 1 mM MgCl2, 0.05% Triton X-100 (Sigma Aldrich,
Dorset, UK)] and cell lysate was mixed with an equal volume
of p-nitrophenol phosphate (20 mM). Absorbance was mea-
sured at 414 nm, and ALP activity was normalized to cell
number as determined by the Alamar Blue assay.
Bone marrow–breast cancer cell co-culture
Mouse M-CSF-dependent osteoclast precursors generated as
described above were plated into 96-well plates (10 ¥ 103 cells
per well) in standard aMEM supplemented with M-CSF
(25 ng·mL-1) and RANKL (100 ng·mL-1) for 6 h before addi-
tion of human MDA-MB-231, human MCF-7 or mouse 4T1
breast cancer cells (300 cells per well). For studies involving
conditioned media, human MDA-MB-231 breast cancer cells
were cultured in standard aMEM and allowed to grow to 80%
confluence. Media were removed and replaced with serum
free aMEM and then the cells were allowed to grow for a
further 24 h. The conditioned media from these cultures were
removed, filtered (0.2 mm filter diameter) and then added to
osteoclast cultures at a concentration of 10% (v/v) in stan-
dard aMEM supplemented with M-CSF (25 ng·mL-1) and
RANKL (100 ng·mL-1). The cultures were terminated by fixa-
tion in 4% paraformaldehyde and stained for TRAcP.
ALZ staining
ALZ staining was used to assess bone nodule formation in
calvarial osteoblast cultures. Briefly, osteoblast cultures were
fixed in 70% cold ethanol for 1 h and immersed in 40 mM
ALZ solution (pH 4.2) for 20 min at room temperature on an
orbital rotator. Cultures were incubated in destaining solu-
tion (10% (w/v) cetylpyridinium chloride in 10 mM sodium
phosphate) for 15 min. Absorbance of the extracted stain was
then measured at 562 nm using a Biotek Synergy HT plate
reader and compared with an ALZ standard curve. The values
were normalized to cell number as determined by the Alamar
Blue assay.
1916 British Journal of Pharmacology (2012) 165 1914–1925

S-NSAIDs inhibit osteoclast–tumour cell interaction
BJP
the end of the culture period, both adherent and non-
adherent cells were collected, fixed with 4% paraformalde-
hyde, cytospun into glass slides and immersed in DAPI
solution for 10 min at room temperature. An average of six
microscopic fields per treatment group were analysed at 10 ¥
magnification. Number of apoptotic osteoclasts was quanti-
fied and expressed as percentage of total osteoclast number as
identified by TRAcP staining.
Human breast cancer cell–mouse
calvarial co-culture
Neonatal mouse calvarias were isolated from 7 day-old mice
and incubated in standard aMEM for 24 h as previously
described (Garrett, 2003). Each mouse calvaria was divided
into two halves along the median sagittal suture. Each half
was placed in organ culture as free-floating bones on stainless
steel rafts in 48-well plates containing standard media
(Figure 6A, top) and treated for 7 days as follows: group 1, one
half treated with DMSO (veh1) and the other half treated
with veh1 in the presence of MDA-MB-231 cells (10 ¥ 103 cells
per well) (veh2); group 2, one half treated with 0.1% DMSO
in the presence of MDA-MB-231 cells (10 ¥ 103 cells per well)
and the other half treated with diclofenac (10 mM) in the
presence of MDA-MB-231 cells (10 ¥ 103 cells per well); group
3, one half treated with 0.1% DMSO in the presence of MDA-
MB-231 cells (10 ¥ 103 cells per well) and the other half
treated with ACS15 (10 mM) in the presence of MDA-MB-231
cells (10 ¥ 103 cells per well); group 4, one half treated with
0.1% DMSO in the presence of MDA-MB-231 cells (10 ¥ 103
cells per well) and the other half treated with ACS32 (10 mM)
in the presence of MDA-MB-231 cells (10 ¥ 103 cells per well).
Osteolysis was assessed by measuring bone volume using
micro-computed tomography (Skyscan 1172 scanner,
Skyscan, Antwerp, Belgium) at a resolution of 5 mm.
Measurement of NFkB activity
Osteoclasts were generated in six-well plates as described
earlier and treated with test compounds for the desired
period of time. Following the incubation period, nuclear
extracts were prepared using a nuclear extract kit (Active
Motif, Rixensart, Belgium) and DNA binding was measured
using TransAM® Transcription Factor ELISA for p65 NFkB
(Active Motif, Rixensart, Belgium), according to the manu-
facturer’s instructions.
Statistical analysis
Differences between groups were assessed by one-way ANOVA
followed by Bonferroni post-test using SPSS for Windows
version 11 (Chicago, IL, USA). A P-value value of 0.05 or
below was considered statistically significant. The concentra-
tion that produced 50% of response IC50 was calculated using
GraphPad Prism 4 for Windows (GraphPad Software, La Jolla,
CA, USA).
Western blotting
Western blot analysis was used to detect protein expression
and phosphorylation in cultured bone and breast cancer
cells. Briefly, cells were seeded in 12-well plates and main-
tained in standard aMEM until confluent. Before stimulation
with test agents or vehicle, cells were incubated in serum-free
aMEM medium for 60 min. Test agents or vehicle were pre-
pared in serum free aMEM medium and were then added for
the desired period of time. The cells were then gently scraped
in standard lysis buffer [0.1% (w/v) SDS, 0.5% (w/v) sodium
deoxycholate, 1% Triton X-100, 1 mM EDTA, 2% (v/v) pro-
tease inhibitor cocktail, 10 mM of sodium fluoride and 2%
(v/v) phosphatase inhibitor cocktail]. The lysate was incu-
bated on ice for 10 min and centrifuged at 14 000¥ g at 4°C
for 5 min. The supernatant was collected and protein con-
centration was determined using BCA assay (Thermo Fisher
Scientific, Northumberland, UK). Total protein (30–100 mg)
was resolved by SDS-PAGE on 12% polyacrylamide SDS gels,
transferred onto PVDF membranes (BioRAD, Herts, UK) and
immunoblotted with antibodies according to manufacturer’s
instructions. The immuno-complexes were visualized by an
enhanced chemiluminescence detection kit (Pierce, USA)
using horseradish peroxidase-conjugated secondary antibody
(Jackson ImmunoResearch Laboratories, West Grove, PA,
USA) and then visualized using chemiluminescence (Amer-
sham, Buckinghamshire, UK) on a Syngene GeneGnome
imaging system.
Materials
Diclofenac was purchased from Sigma Aldrich (Dorset, UK).
The diclofenac derivative ACS15 (Figure 1) was prepared as
previously described (Isenberg et al., 2007), and ACS32
(Figure 1) was prepared as described above. Human MDA-MB-
231 and MCF-7 and mouse 4T1 breast cancer cell lines were
purchased from ATCC (Manassas, VA, USA). Mouse macroph-
age colony stimulating factor (M-CSF) was obtained from
R&D Systems (Abingdon, UK) and receptor activator of NFkB
ligand (RANKL) was a gift from Patrick Mollat (Galapagos
SASU, Romainville, France) and was prepared as previously
described (Idris et al., 2010). All primary antibodies were pur-
chased from Cell Signalling Biotechnology (Danvers, MA,
USA), unless otherwise stated. Tissue culture medium
(Minimum Essential Medium Alpha; aMEM) was obtained
from Invitrogen (Paisley, UK). All other reagents were pur-
chased from Sigma-Aldrich unless otherwise stated.
Results
The S-diclofenac derivatives ACS15 and
ACS32 inhibit human and mouse breast
cancer cell support for osteoclast formation
We first investigated the effects of the S-diclofenac derivatives
ACS15 and ACS32 (Figure 1) on osteoclast formation in bone
marrow–breast cancer cell co-cultures. As shown in Figure 2,
the human MDA-MB-231 (Figure 2A) and MCF-7 (Figure 2C)
and mouse 4T1 (Figure 2D) breast cancer cells increased
osteoclast number, and these effects were significantly inhib-
ited by ACS15 and ACS32 with the maximum response
Morphological assessment of apoptosis
Apoptosis in mature osteoclast cultures was detected by the
characteristic changes in nuclear morphology following 4,6-
diamidino-2-phenylindole (DAPI) staining. Briefly, osteo-
clasts were generated in 48-well plates as described earlier and
then treated with test agents for the desired period of time. At
British Journal of Pharmacology (2012) 165 1914–1925 1917

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BJP
Figure 2
ACS15 and ACS32 inhibit osteoclast formation induced by mouse and human breast cancer cells. (A, C, D) Number of mouse osteoclasts following
treatment with vehicle (veh; 0.1% DMSO) or test compounds at the indicated concentrations for 48 h in the presence and absence of (A) human
MDA-MB-231, (C) human MCF-7 or (D) mouse 4T1 cells. Osteoclast numbers were assessed by counting TRAcP-positive multi-nucleated cells.
Representative photomicrographs from osteoclast–MDA-MB-231 cells co-cultures are shown in panel B. +P < 0.05 from vehicle treated osteoclast
– cancer cell co-cultures and *P < 0.05 from vehicle treated osteoclast cultures.
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S-NSAIDs inhibit osteoclast–tumour cell interaction
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Figure 3
ACS15 and ACS32 inhibit osteoclast formation and IkB phosphorylation induced by human MDA-MB-231 breast cancer cell-derived factors. (A)
Number of mouse osteoclasts following treatment with vehicle (veh; 0.1% DMSO) or test compounds at the indicated concentrations for 48 h
in the presence and absence of MDA-MB-231 conditioned media (10% v/v in aMEM). Osteoclast numbers were assessed by counting
TRAcP-positive multinucleated cells. (B) Western blot of IkB phosphorylation in mouse M-CSF-dependent osteoclast precursors treated with vehicle
(veh; 0.1% DMSO) or test compounds (ACS15; ACS32; diclofenac, DCF) at the indicated concentrations for 3 h prior stimulation with
MDA-MB-231 conditioned media (20% v/v in aMEM) for 15 min. The results shown are representative of three independent experiments. (C)
Quantification of phospho-IkBa from blots in (B) as a ratio to total IkBa. #P < 0.05 from vehicle treated osteoclast–MDA-MB-231 conditioned
media cultures; *P < 0.05 from vehicle treated osteoclast cultures; +P < 0.05 from DCF treated osteoclast cultures.
observed at concentrations equal to and greater than 10 mM
(Figure 2A–D). The parent compound diclofenac had no
effect on osteoclast formation at concentration up to 30 mM
(Figure 2A, C and D). None of the compounds tested had any
significant effects on human breast cancer cell viability or
growth at concentrations that inhibited osteoclast formation
(Figure S1).
nism of action by which S-diclofenac derivatives inhibit
osteoclast formation, we examined the effects of ACS15 and
ACS32 (30 mM) on NFkB activity, a common pathway affected
by H2S, diclofenac, S-diclofenac and RANKL (Feng, 2005; Li
et al., 2007; Zhi et al., 2007; Karakawa et al., 2009). As shown
in Figure 3B and C, ACS15 and ACS32 (30 mM) suppressed IkB
phosphorylation induced by conditioned media from human
MDA-MB-231 cells. The parent compound diclofenac had no
significant effects on MDA-MB-231-induced osteoclast forma-
tion or IkB phosphorylation at concentrations up to 30 mM
(Figure 3A–C).
ACS15 and ACS32 inhibit IkB
phosphorylation and osteoclast formation
induced by human breast cancer cell
ACS15 and ACS32 inhibit RANKL-induced
osteoclast formation and resorption
derived factors
To gain an insight into the mechanism by which these
S-diclofenac derivatives suppressed breast cancer cells support
for osteoclastogenesis, we next examined the effects of ACS15
and ACS32 on osteoclast formation and signalling induced by
factors derived from breast cancer cells. As shown in
Figure 3A, ACS15 and ACS32 inhibited the increase in osteo-
clast number induced by conditioned media from human
MDA-MB-231 breast cancer cells. To investigate the mecha-
We next examined the effects of ACS15 and ACS32 in RANKL
and M-CSF-induced osteoclast formation and activity. As
shown in Figure 4A, ACS15 (3–30 mM) and ACS32 (1–30 mM)
strongly inhibited RANKL-stimulated osteoclast formation in
a concentration-dependent manner. The mean (ϮSD) concen-
tration of these compounds, which half maximally inhibited
osteoclast formation (IC50), was 4.2 Ϯ 0.7 mM for ACS15; 2.9 Ϯ
British Journal of Pharmacology (2012) 165 1914–1925 1919

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Figure 4
ACS15 and ACS32 inhibit RANKL-induced osteoclast formation and
bone resorption. (A) Number of osteoclasts cultured in M-CSF
(25 ng·mL-1) and RANKL (100 ng·mL-1) in presence and absence of
vehicle (veh; 0.1% DMSO) or test compounds (ACS15; ACS32;
diclofenac, DCF) for 48 h at the indicated concentrations. Osteoclast
numbers were assessed by counting TRAcP-positive multinucleated
cells. Representative photomicrographs from mouse osteoclast cul-
tures are shown in (B). (C) Resorbed area in mouse osteoclasts
cultured for 6–8 days on dentine slices and treated with vehicle (veh;
0.1% DMSO) or test compounds at the indicated concentrations for
4 days. Resorbed area was measured using reflective microscopy.
Data are mean Ϯ SD of three independent experiments. *P < 0.05,
significantly different from vehicle control; +P < 0.05, significantly
$
different from DCF-treated cultures; P < 0.05, significantly different
from ACS15-treated cultures.
᭣
of osteoclast number (30 mM; 17% Ϯ 1.4, P < 0.05) and resorp-
tion (30 mM; 18.7% Ϯ 11.2, P < 0.05) (Figure 4A and C).
ACS15 and ACS32 inhibit NFkB nuclear
translocation and induce apoptosis in
mature osteoclasts
To further assess the mechanisms responsible for osteoclast
inhibition, we investigated the effects of diclofenac, ACS15
and ACS32 on apoptosis in mature osteoclasts cultured in
RANKL and M-CSF for 6–7 days prior to drug treatment. ACS15
and ACS32 induced activation of caspase-3 within 18 h
(Figure 5B) and caused apoptosis in a dose-dependent manner
after 32 h of continuous treatment (Figure 5A; IC50 = 22.1 Ϯ
2.1 mM for ACS15 and 5.3 Ϯ 0.5 mM for ACS32). A small
increase in the number of apoptotic osteoclasts was observed
following treatment with the parent compound diclofenac at
concentration of 30 mM (Figure 5A). In order to investigate the
mechanisms by which these compounds induce osteoclast
apoptosis, we studied the effects of diclofenac, ACS15 and
ACS32 on RANKL-induced IkB phosphorylation and NFkB
DNA binding, key signalling events for osteoclast survival and
activity (Feng, 2005). ACS15 and ACS32 inhibited RANKL-
induced IkB phosphorylation (Figure 5C) and NFkB DNA
binding (Figure 5D), indicating a strong inhibitory effect on
NFkB activity in osteoclasts. ACS15 and ACS32 (30 mM) also
showed a non-significant trend towards enhancing M-CSF-
induced ERK1/2 phosphorylation in osteoclast cultures (4% Ϯ
3 for ACS15 and 6% Ϯ 4 for ACS32, P < 0.05) (Figure S2). The
parent compound diclofenac had no significant effects on
NFkB or ERK1/2 activity in mature osteoclasts at concentra-
tions up to 30 mM (Figure 5C and D, Figure S2).
ACS15 and ACS32 protect against osteolysis
ex vivo
We next studied the effects of diclofenac, ACS15 and ACS32
on osteolysis using the human MDA-MB-231–mouse calvarial
co-culture system (Figure 6A, top). As shown in Figure 6A and
B, human MDA-MB-231 breast cancer cells caused osteolysis
characterized by a significant loss in bone volume (P < 0.05)
when co-cultured with mouse calvaria for 7 days, and these
effects were completely prevented by ACS15 and ACS32
(10 mM). In fact, treatment with ACS15 and ACS32 (10 mM)
1.2 mM for ACS32. ACS15 and ACS32 also inhibited RANKL-
induced bone resorption at concentrations that suppressed
osteoclast formation (10 mM; P < 0.05) (Figure 4C). In contrast,
the parent compound diclofenac exerted only weak inhibition
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S-NSAIDs inhibit osteoclast–tumour cell interaction
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Figure 5
ACS15 and ACS32 inhibit NFkB activation and cause apoptosis in mature osteoclasts. (A) Number of apoptotic mouse osteoclasts following
treatment with vehicle (0.1% DMSO) or test compounds (ACS15; ACS32; diclofenac, DCF) at the indicated concentrations for 32 h (A). Apoptotic
osteoclasts were visualized by DAPI staining. Values in the graphs are means Ϯ SD and are obtained from three independent experiments.
**P < 0.01, significantly different from vehicle (0.1% DMSO)-treated cultures. (B) Western blot analysis of caspase-3 activation in mature mouse
osteoclasts exposed to test compounds at the concentrations indicated for 18 h. The results shown are representative of three independent
experiments. (C) Western blot analysis of IkBa phosphorylation in mouse osteoclasts exposed to test compounds at the concentrations indicated
for 180 min prior stimulation with vehicle (veh2; 0.3% BSA) or RANKL (150 ng·mL-1) for 5 min. (D) NFkB DNA binding in mouse osteoclasts
exposed to test compounds at the concentrations indicated for 60 min prior stimulation with vehicle (veh1; 0.3% BSA) or RANKL for 30 min.
Nuclear extracts were prepared and NFkB DNA-binding activity analysed using a TRANSAM active motif ELISA kit. Data are mean Ϯ SD of three
independent experiments. *P < 0.05, significantly different from vehicle-treated (veh2; 0.1% DMSO) and +P < 0.05, significantly different from
diclofenac-treated (DCF, 30 mM).
caused a significant gain in bone volume in comparison with
Diclofenac, ACS15 and ACS32 inhibit
differentiation and bone nodule formation of
calvarial osteoblasts
vehicle-treated cultures (P < 0.05; Figure 6A). A small reduc-
tion in osteolytic bone loss was observed in co-cultures
treated with the parent compound diclofenac (10 mM)
(P < 0.05). None of the compounds tested affected cell
number or caspase-3 activity in human MDA-MB-231 breast
cancer cells (Figure 6C and D).
We next investigated the effects of diclofenac, ACS15 and
ACS32 on osteoblast differentiation and activity in primary
mouse calvarial osteoblast cultures using ALP and bone
British Journal of Pharmacology (2012) 165 1914–1925 1921

J Frantzias et al.
BJP
Figure 6
ACS15 and ACS32 prevent MDA-MB-231-induced osteolysis in mouse calvaria organ cultures. (A, upper part) Diagram of the human MDA-MB-
231–mouse calvaria co-culture system. (A, lower part) Bone volume in neonatal mouse calvarias isolated from 7 day-old mice and then cultured
in standard tissue culture medium for 24 h prior exposure to vehicle (veh; 0.1% DMSO) or test compounds at the indicated concentrations for
7 days. Calvarias were analysed using microCT scanning and changes in bone volume were normalized to vehicle-treated calvarial half that were
unexposed to MDA-MB-23, and values were expressed as percent change. Values in each panel are means Ϯ SD from seven calvarias. *P < 0.05,
significantly different from vehicle-treated, +P < 0.05 , significantly different from vehicle-treated MDA-MB-231–calvarial organ cultures and
#P < 0.05 , significantly different from diclofenac-treated MDA-MB-231–calvarial organ cultures. (B) Representative photomicrographs of mouse
calvarias from the experiment described earlier following microCT analysis. Red arrows denote focal osteolysis. (C) MDA-MB-231 cell number in
co-cultures described earlier as assessed by Alamar Blue assay. (D) Caspase-3 activity in human MDA-MB-231 breast cancer cells from the
MDA-MB-231–mouse calvaria co-cultures described earlier. The results shown are representative of three independent experiments.
1922 British Journal of Pharmacology (2012) 165 1914–1925

S-NSAIDs inhibit osteoclast–tumour cell interaction
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Figure 7
Diclofenac (DCF), ACS15 and ACS32 inhibit osteoblast differentiation and bone nodule formation in calvarial osteoblast cultures. (A) Alkaline
phosphatase (ALP) levels in mouse calvarial osteoblast cells cultured in osteogenic medium for 21 days in the presence of vehicle (veh; 0.1%
DMSO) or test compounds at the concentrations indicated. Osteoblast differentiation was assessed by alkaline phosphatase assay. (B) Quantifi-
cation of bone nodule formation in mouse osteoblasts cultured and treated as described earlier. Bone nodule formation was assessed by alizarin
red assay. (C) Osteoblast number in mouse osteoblasts cultured and treated as described earlier. Osteoblast number was measured using Alamar
Blue assay. Values in the graphs are mean Ϯ SD *P < 0.05 and **P < 0.01 , significantly different from vehicle (veh; 0.1% DMSO)-treated cultures.
Representative photomicrographs from mouse calvarial osteoblast cultures are shown in (D).
nodule assays respectively. At concentrations that inhi-
bited MDA-MB-231-induced osteoclast formation in vitro
(Figure 2A) and osteolysis ex vivo (Figure 6A), none of the
compound tested affected calvarial osteoblast number, differ-
entiation or bone nodule formation (Figure 7A–C). At higher
concentrations (30 mM), diclofenac, ACS15 and ACS32
reduced ALP activity (Figure 7A) and bone nodule formation
(Figure 7B) despite causing a significant increase in osteoblast
number (Figure 7C).
tion induced by human and mouse breast cancer cells. We
excluded non-specific effects of these compounds on breast
cancer cell growth and death by studying the effects of
diclofenac, ACS15 and ACS32 on breast cancer cell number
and apoptosis. In these studies, none of the compounds
tested exerted any significant effects on MDA-MB-231 growth
or caspase-3 activation at concentrations that inhibited
osteoclast formation. ACS15 and ACS32 inhibited IkB phos-
phorylation and osteoclast formation induced by condi-
tioned media from human breast cancer cells. This indicates
that ACS15 and ACS32 inhibit osteoclast formation via a
mechanism dependent on IKK/NFkB inhibition in osteo-
clasts. The possibility that suppression of COX-1/2 activity
may contribute to these effects was excluded since the parent
compound diclofenac was found to be completely ineffective
in inhibiting breast cancer-induced IkB phosphorylation and
osteoclast formation despite the fact that it is a more potent
inhibitor of prostaglandin production than its H2S-releasing
derivatives (Rossoni et al., 2008). Previous studies showed
that S-NSAIDs, including ACS15, release H2S in vitro and in
vivo (Li et al., 2007; Sidhapuriwala et al., 2007; Moody et al.,
Discussion and conclusions
Non-steroidal anti-inflammatory drugs and H2S donors
exhibit anti-inflammatory, anti-resorptive and anti-tumour
properties. These observations provided the rationale for the
present study in which we investigated the effects of the
S-diclofenac derivatives ACS15 and ACS32 on bone and
breast cancer cell proliferation, activity and crosstalk. Studies
in vitro using BM–breast cancer cell co-cultures showed that
ACS15 and ACS32 inhibited the increase in osteoclast forma-
British Journal of Pharmacology (2012) 165 1914–1925 1923

J Frantzias et al.
BJP
2010). Taken together, these findings imply that ACS15 and
ACS32 exert their anti-resorptive effects by a mechanism
mediated by H2S production. However, further studies are
needed to determine if the amount of H2S released by these
compounds in vitro and in vivo is sufficient to influence osteo-
clastogenesis.
in vivo studies will need to be performed to investigate this
possibility. Nonetheless, the data presented in this study
suggest that H2S-releasing NSAIDs may be of value in the
prevention and treatment of skeletal complications associ-
ated with osteolytic bone disease.
Studies in osteoclast cultures showed that ACS15 and
ACS32 inhibited RANKL- and M-CSF-induced osteoclast for-
mation and caused osteoclast apoptosis in mature osteoclasts.
This confirms that these agents inhibit osteoclast formation
and survival acting directly on osteoclasts and their precur-
sors. ACS15 and ACS32 also inhibited bone resorption, but
these effects were likely to be due to induction of apoptosis
and cell death rather than a specific effect on osteoclast func-
tion. Further mechanistic studies in mature osteoclasts
showed that the mechanism of action by which ACS15 and
ACS32 exerted their anti-resorptive effects involved inhibi-
tion of RANKL-induced NFkB nuclear translocation and DNA
binding, an essential pathway for osteoclast formation and
survival (Feng, 2005). Non-specific effects in M-CSF signalling
were excluded by the fact that ACS15 and ACS32 did not
inhibit M-CSF-induced ERK1/2 MAPK activation (Figure S2)
or viability of M-CSF-dependent osteoclast precursors (data
not shown). Interestingly, the parent compound diclofenac
caused a weak inhibitory effect on osteoclast formation in
RANKL- and M-CSF-stimulated cultures, even though it failed
to inhibit RANKL- or M-CSF-induced signalling. We speculate
that this effect was probably due to inhibition of COX activ-
ity and prostaglandin production, which are both known to
regulate osteoclast formation (Li et al., 2000; Okada et al.,
2000).
Acknowledgements
These studies were supported in part by a ECTS/AMGEN
fellowship grant to Dr. A.I.Idris.
Conflicts of interest
Piero Del Soldato is a shareholder of CTG Pharma S.r.l., Viale
Gran Sasso 17, 20131 Milano, Italy. Patrick Mollat is an
employee of Galapagos SASU (102 Avenue Gaston Roussel,
93230 Romainville, France).
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Figure S1 Diclofenac and S-diclofenac derivatives ACS15
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cancer cell viability. Panel A shows the number of human
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cated for 48 h. Cell number was measured using Alamar Blue
assay. Values in the graphs are mean Ϯ SD and are obtained
from three independent experiments. Representative photo-
micrographs from MDA-MB-231 cultures are shown in
panel B.
Figure S2 ACS15 and ACS32 enhance M-CSF-induced
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