Biological effects of aromatic bis[aminomethylidenebis(phosphonic)] acids in osteoclast precursors in vitro

Article abstract of DOI:10.1111/cbdd.13597

Nitrogen-containing bisphosphonates (N-BPs) inhibit bone resorption by preventing osteoclast activity. Most clinically used BPs are hydroxybisphosphonates with the exception of incadronate, which belongs to the class of aminomethylidenebisphosphonic acids

Full text of DOI:10.1111/cbdd.13597

DR ANNA NASULEWICZ-GOLDEMAN (Orcid ID : 0000-0001-6167-5923)
PROFESSOR JOANNA WIETRZYK (Orcid ID : 0000-0003-4980-6606)
Article type : Research Article
Biological effects of aromatic bis[aminomethylidenebis(phosphonic)] acids in osteoclast
precursors in vitro
Running title: Biological activity of new aminobisphosphonates
Anna Nasulewicz-Goldeman1, Waldemar Goldeman2, Ewa Mrówczyńska1, Joanna Wietrzyk1
1 Department of Experimental Oncology, Hirszfeld Institute of Immunology and
Experimental Therapy, Wrocław, Poland
2 Wrocław University of Technology, Department of Organic Chemistry, Wrocław, Poland
Corresponding Author: Anna Nasulewicz-Goldeman, Hirszfeld Institute of Immunology
and Experimental Therapy, Weigla St. 12, 53-114 Wrocław, Poland. Email: anna.nasulewicz-
goldeman@hirszfeld.pl
This article has been accepted for publication and undergone full peer review but has not
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13597
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Keywords: bisphosphonates, aminomethylidenebisphosphonic acids, osteoclasts, J774E
macrophages, anti-resorptive therapy
Abstract
Nitrogen-containing bisphosphonates (BPs) inhibit bone resorption by preventing osteoclast
activity. Most clinically BPs are hydroxybisphosphonates with the exception of incadronate,
which belongs to the class of aminomethylidenebisphosphonic acids. The aim of this study
was to evaluate the anti-proliferative activity of two previously reported
aminobisphosphonates (WG8185B2 and WG9001B) in combination with doxorubicin and
cisplatin toward J774E cells (a model of osteoclast precursors in vitro). WG8185B2 and
WG9001B BPs enhanced the cytotoxic activity of doxorubicin and cisplatin, especially when
applied 24 hrs prior to cytostatics. The anti-proliferative effect of studied BPs was related to
the changes in cell cycle progression. WG8185B2 leads to significant accumulation of J774E
cells in S phase, whereas WG9001B causes transient arrest in G2/M phase, followed by an
increase in the percentage of cells in S phase. Moreover, WG8185B2 and WG9001B BPs
showed enhanced pro-apoptotic activity in osteoclast precursors, which was manifested by an
increase in caspase-3 activity and percentage of apoptotic cells. In addition, both compounds
influenced the motility of J774E cells. The exact molecular mechanism of action of examined
BPs remains to be determined; however, results show an interesting biological activity of
these compounds, which may be of interest in the context of anti-resorptive therapy.
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Introduction
Excessive bone resorption is observed in many diseases, such as Paget’s disease,
tumor-induced osteolysis, and postmenopausal osteoporosis (Feng & McDonald, 2011).
Approximately 30% of postmenopausal women suffer from the osteoporosis, thus making it a
serious health issue worldwide. Almost half of these women will experience at least one bone
fracture in their lifetime. In addition, despite of the advances in early detection and treatment
of breast cancer, all patients are at risk for disease recurrence, progression, and death. Across
all tumor types, patients with breast cancer have the highest incidence of skeletal
complications. In total, 80% of patients with metastatic breast cancer develop bone lesions
(Coleman, 2009).
Bisphosphonates (BPs) are powerful inhibitors of osteoclast activity, and therefore are
successfully used in the management of postmenopausal osteoporosis and cancer-related
bone diseases, as an adjuvant therapy in patients with osteolytic bone metastases and patients
treated with agents leading to bone loss (for a review see (Drake, Clarke, & Khosla, 2008;
Gnant & Clézardin, 2012)). The molecular mechanism responsible for the cytotoxic activity
of N-containing BPs toward osteoclasts is the inhibition of farnesyl diphosphate synthase
(FPPS), which results in the suppression of mevalonate pathway responsible for the synthesis
of cholesterol and prenyl groups. Post-transcriptional prenylation of small GTPases plays an
important role in protein-protein interactions and anchoring to cell membranes (Shinde &
Maddika, 2018). The inhibition of mevalonate pathway leads to the accumulation of
unprenylated proteins, impairment of a downstream signaling, and in turn to the inhibition of
resorptive activity of osteoclasts and often to apoptosis (K. Iguchi, Tatsuda, Usui, & Hirano,
2010; Miwa et al., 2012; Tsubaki et al., 2013).
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The biological activity of BPs is closely related to their chemical structure, which
enables BPs to chelate calcium ions efficiently as well as to target bone environment
specifically (Ebetino et al., 2011). The character of two substituents R1 and R2, covalently
bound to the carbon atom of the bisphosphonic group (P-C-P), play a crucial role in BP
activity. In most commercial BPs, the R1 substituent is a hydroxyl group that determines the
affinity of BPs to bone mineral surface, while the anti-resorptive activity is dependent
basically upon the chemical structure of R2 chain. Third-generation BPs containing the N-
heterocyclic ring (risedronate, minodronate, and zoledronate) are the most active compounds
among all available commercial BPs, e.g. zoledronic acid is approximately 10,000 times
more active than etidronate (Sharma et al., 2013).
One of the oldest and still commonly used procedure for the
aminomethylidenebisphosphonic acids preparation is based on the three-component reaction
of the primary or secondary amine with trialkyl orthoformate and dialkylphosphonate, and
further hydrolysis of crude tetraalkylesters (Takeuchi et al. 1993). There are some other
methods like reaction of trialkylphosphites with amides in the presence of phosphrous
oxychloride (Olive, Le Moigne, Mercier, Rockenbauer, & Tordo, 1998), oxalyl chloride
(O’Boyle et al., 2011) or triflic anhydride (Wang, Chang, Sun, & Huang, 2015), reaction of
acetals of formamides with dialkylphosphonates (Groß & Costisella, 1968; Prishchenko,
Livantsov, Novikova, Livantsova, & Petrosyan, 2009), phosphonylation of oximes with
trialkylphosphites or dialkylphosphonates via Beckmann Rearrangement (Yokomatsu,
Yoshida, Nakabayashi, & Shibuya, 1994). In contrast to 1-hydroxybisphosphonates,
aminomethylidenebisphosphonates are significantly less investigated and described in the
literature.
Although, the so-called ‘orthoformate method’ is the most often used procedure for
the preparation of the aminobisphosphonic acid it has several disadvantages like elevated
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temperatures (130-150oC), which excludes the use of substrates with labile groups, low or
average yield of the final bisphosphonic acid and problems with the separation of pure esters.
Due to these disadvantages we were looking for another, mild and more efficient method for
the synthesis of this group of compounds.
In 2012, we developed a very convenient and mild procedure to synthesize N-
substituted aminomethylidenebisphosphonates in a reaction of trialkyl phosphites and
isocyanides in the presence of a stoichiometric amount of hydrogen chloride (Goldeman,
Kluczyński, & Soroka, 2012). Next, we used our method for the preparation of a series of
aminomethylidenebisphosphonic acids derived from selected β-arylethylamines (Goldeman
& Nasulewicz-Goldeman, 2015) and a series of diamines (Goldeman & Nasulewicz-
Goldeman, 2014). The preliminary biological evaluation of these new BPs revealed that two
of them, i.e. benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG8185B2) and
naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] acid (WG9001B) (Fig. 1.) showed
high anti-proliferative activity toward J774E mouse macrophages which are a model of
osteoclast precursors in vitro (Goldeman & Nasulewicz-Goldeman, 2014).
The aim of this in vitro study was to evaluate the anti-proliferative activity of above
two aminobisphosphonates in combination with cytostatic drugs toward the well-defined
macrophage cell line J774E cells. The influence on cell motility and cellular mechanisms of
action were also studied.
Materials and methods
General information
All reagents used for the synthesis were of commercial quality and purchased from Sigma-
Aldrich, while solvents were of commercial quality and purchased from a local Supplier
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(POCh). Incadronate and zoledronate were synthesized according to literature procedures
(Singh, Manne, Ray, & Pal, 2008; Takeuchi et al., 1993). Concentrations of WG8185B2 and
WG9001B used for each assay were each time experimentally evaluated and depended on
test conditions, like incubation time, area of wells and density of cells. In each case the
relationship between the concentration of compound and the inhibitory effect was
determined. To avoid the toxicity effect, doses lower than IC40 were used, unless otherwise
stated.
Cell culture
J774E mouse macrophages were obtained from American Type Culture Collection
(Rockville, Maryland, USA) and maintained in the Cell Culture Collection of the Institute of
Immunology and Experimental Therapy (IIET), Polish Academy of Sciences, Wroclaw,
Poland. Cells were maintained in RPMI-1640 GLUTAMAX (Gibco, Scotland, UK) medium
containing 100 U/mL penicillin and 100 µg/mL streptomycin (both from Polfa Tarchomin
S.A., Warsaw, Poland) and supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich,
Darmstadt, Germany).
Combination with cytostatics
Cells were plated in 96-well plates (Sarstedt, Inc. Newton, NC, USA or Corning B.V. New
York, USA) at a density of 1 × 104 cells per well in 100 μL of culture medium without FBS
and antibiotics. Immediately after seeding, cells were pretreated either with doxorubicin (at
serial concentrations of 1000 – 100 – 10 – 1 ng/mL) or cisplatin (at serial concentrations of
10 – 1 – 0.1 – 0.01 µg/mL) or BP at a concentration of 25 µg/mL (54 µM) for WG8185B2
and 2 µg/mL (3.76 µM) for WG9001B. The concentrations of BPs were based on previously
calculated IC50 values and on the kinetics in the wide range of concentrations. Cells treated
with BPs at serial concentrations of 100 – 50 – 25 – 10 – 5 – 2.5 – 1 – 0.1 µg/mL served as
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controls for a calibration curve. After 24 hrs of incubation under standard conditions,
doxorubicin (Sigma-Aldrich), cisplatin (Accord Healthcare Limited, North Harrow, UK), or
BP was applied to obtain the following combinations: I. cytostatic following pretreatment
with BP; II. BP following pretreatment with cytostatic; III. BP and cytostatic used together.
Cells treated with BP or cytostatic alone as well as non-treated cells served as controls. An
SRB assay was performed after 72 hrs, as described by Skehan et al. (Skehan et al., 1990).
The optical densities of the samples were measured by a Synergy H4 Hybrid Reader (BioTek
Instruments, Winooski, VT, USA) at λ = 540 nm.
The results from the SRB assay were calculated with the CalcuSyn program (Biosoft,
Cambridge, United Kingdom) to determine the presence of synergy between BPs and
cytostatics. Based on the method of Chou and Talalay (Chou & Talalay, 1981, 1984), the
CalcuSyn program was used to determine whether the drug combination produced greater
effects together than expected from a simple summation of their individual effects. The
combination index (CI) values obtained from the data reflected the nature of the interaction
between BPs and cisplatin or doxorubicin, i.e. < 1, synergistic activity; = 1, additive; > 1,
antagonism.
GGOH-FOH assay
Cells were plated in 96-well plates at a density of 1 × 104 cells per well in 100 μL of culture
medium with FBS and antibiotics. At 24 hrs after seeding, cells were pretreated for 1 hr with
50 µL of either geranylgeraniol (GGOH, Sigma-Aldrich) or farnesol (FOH, Sigma-Aldrich) at
the concentration 10 or 20 µM. Next, cells were treated with WG8185B2 or WG9001B BP
(at serial concentrations of 100 – 10 – 1 – 0.1 µg/mL) for further 48 hrs. Cells treated with
studied or reference BPs alone as well as non-treated cells served as controls. SRB assay was
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performed as described above, and the IC50 values were calculated based on the inhibition of
proliferation rates using Prolab software (INFORM-TECH, Zabrze, Poland).
Migration and invasion assay
J774E macrophages were seeded at a density of 5  104 cells/mL in the culture medium in
24-well plates, cultured overnight, and treated for 48 hrs with BPs at a concentration of 27.6
μM for WG8185B2 and 3.4 μM for WG9001B. Untreated cells and cells treated with either
zoledronic or incadronic acid served as controls. After incubation cells were non-
enzymatically detached from the wells, centrifuged and counted. For migration assay, 2.5 x
104 of viable cells were suspended in Dulbecco’s Modified Eagle’s medium without FBS and
applied to the upper section of the inserts (Transwell Permeable Supports 6.5 μm Insert,
Corning Incorporated, Kennebunk, USA) coated with type I collagen at a concentration of 10
µg/mL. For invasion assay, 2.5 x 104 of viable cells were suspended in Dulbecco’s Modified
Eagle’s medium without FBS and applied at the top of Matrigel-coated inserts (BioCoat
Matrigel Invasion Chamber, BD Biosciences). Culture medium supplemented with 20% FBS,
applied to the lower section, served as a chemoattractant. Migration and invasion assays were
carried out in standard culture conditions for 6 and 24 hrs, respectively. Thereafter, the cells
on the bottom side of the membrane were fixed, stained with a Diff-Quick Set (Medion
Diagnostics, Düdingen, Switzerland), and counted by light microscopy. Experiment was
repeated at least three times. Statistical analysis was performed using STATISTICA version
10.
Annexin-V assay
J774E macrophages were seeded at a density of 5  104 cells/mL in the culture medium in
24-well plates, cultured overnight, and treated with BPs at a concentration of 27.6 μM for
WG8185B2 and 3.4 μM for WG9001B for 24, 48, and 72 hrs. Untreated cells and cells
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treated with either incadronic or zoledronic acid served as controls (76.7 and 41.4 μM,
respectively). After exposure time, cells were collected and counted. Cells (5  105) were
washed with phosphate-buffered saline (PBS) and resuspended in binding buffer (10 mM
HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). In total, 195 µL of cell suspension
was stained with 5 µL of APC-annexin V (Enzo Lifesciences Ltd., Farmingdale, NY, USA)
for 15 min in dark at room temperature. Then, cells were washed in PBS, centrifuged, and
suspended in binding buffer. In total, 5 µL of DAPI solution (5 mg/mL) was added prior to
FACS analysis. Data analysis was performed by flow cytometry using the Diva Software
program for data acquisition. Data were displayed as two-color dot plot with APC-annexin V
(x-axis) vs DAPI (y-axis). Double-negative cells were live cells, DAPI+/annexin V+/ necrotic
cells, DAPI weak/annexin V+ apoptotic cells, and DAPI -/annexin V+ early apoptotic cells.
Caspase-3 activity
J774E macrophages were seeded at a density of 5  104 cells/mL in the culture medium in
24-well plates, cultured overnight, and treated with BPs at a concentration of 27.6 µM for
WG8185B2 and 3.4 µM for WG9001B for 24, 48, and 72 hrs. Untreated cells and cells
treated with either incadronic or zoledronic acid served as controls (76.7 and 41.4 μM,
respectively). After exposure time, cells were lysed for 30 minutes with 50 µL of lysis buffer
(50 mM HEPES, 10% saccharose, 150 mM NaCl, 5 mM EDTA, 10 mM DTT, 0.1% Triton-X
100, pH 7.5) (IIET). Then, 40 µL of lysate was transferred to a white, 96-well plate (Corning,
NY, USA) and mixed with 160 µL of the reaction buffer (10 µM Ac-DEVD-ACC substrate,
20 mM HEPES, 10% saccharose, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, pH 7.5) (IIET).
Sample fluorescence was continuously recorded at 37°C for 2 hrs using a Biotek Synergy H4
Hybrid Reader (λex = 360 nm, λem = 460 nm). Results were normalized to the protein content
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using the SRB method and expressed as mean relative caspase-3/7 activity compared to the
untreated control sample ± standard deviation (SD).
Cell cycle
J774E macrophages were seeded at a density of 5  104 cells/mL in the culture medium in
24-well plates, cultured overnight, and treated with BPs at a concentration of 14.7 µM for
WG8185B2 and 2.5 µM for WG9001B for 24, 48, and 72 hrs. Untreated cells and cells
treated with either incadronic or zoledronic acid served as controls (52.3 and 27.6 μM,
respectively). After exposure time, cells were collected and counted. The cells (1  106) were
washed twice in cold PBS and fixed for 24 hrs in 70% (v/v) ethanol at 20°C. Then, the cells
were washed twice in PBS and stained for 30 min with DAPI stain solution (DAPI 1 µg/mL,
Triton x100 0.1%, Sigma-Aldrich) on ice. The cellular DNA content was determined using a
BD LSRFortessa instrument (Becton Dickinson, San Jose, CA, USA) and a ModFit LT 3.0
program (Verity Software House, Topsham, ME, USA).
Statistical evaluation
Statistical analysis was performed using GraphPad Prism 7.01 (GraphPad Software Inc., San
Diego, CA, USA). Analysis of variance was determined by Shapiro–Wilk’s normality and
Bartlett’s tests. Tests used for each data analysis are indicated in figure legends. p<0.05 was
considered significant.
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Results
Synthesis
Benzene-1,4-bis[aminomethylidene(bisphosphonic)] acid (WG8185B2) and naphthalene-1,5-
bis[aminomethylidene(bisphosphonic)] acid (WG9001B) were prepared in the reaction of the
appropriate
diisocyanide
(1,4-diisocyanobenzene
and
1,5-diisocyanonaphthalene,
respectively) with triethylphosphite in the presence of hydrochloride and further dealkylation
with bromotrimethylsilane (Fig. 2) according to the authors’ protocol developed in previous
studies (Goldeman et al., 2012; Goldeman & Nasulewicz-Goldeman, 2014).
Anti-proliferative activity of WG8185B2 and WG9001B bisphosphonates in
combination with cytostatics
As previously reported, WG8185B2 and WG9001B BPs show anti-proliferative activity
against J774E macrophages (Goldeman & Nasulewicz-Goldeman, 2014). In these studies, we
showed that WG8185B2 and WG9001B BPs enhance the cytotoxic activity of doxorubicin
and cisplatin. Beneficial effects were observed when cells were pretreated with BPs for 24
hrs prior to cytostatics. For both BPs applied in the study, a strong synergism was recorded,
with CI values ranging from 0.151 to 0.711. Simultaneous treatment of J774E macrophages
with WG8185B2 or WG9001B BP and cytostatics resulted in synergistic effects; however,
antagonism was observed with low concentrations of cytostatics. Similarly, pretreatment with
high concentrations of cytostatics followed by the application of WG8185B2 or WG9001B
BPs led to synergism, whereas pretreatment with low concentrations of cytostatics resulted in
additive or even antagonistic effects (Table 1, Table 2).
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GGOH-FOH assay
To investigate whether the anti-proliferative effect of WG9001B and WG8185B2 BPs is
related to the inhibition of protein prenylation, we used GGOH and FOH, intermediate
metabolites in the mevalonate pathway, which are proven to reverse the effects of
aminobisphosphonates (Benford, Frith, Auriola, Mönkkönen, & Rogers, 1999; Denoyelle,
Hong, Vannier, Soria, & Soria, 2003; Goffinet et al., 2006). J774E cells were pretreated with
either GGOH or FOH at the concentration of 10 or 20 μM for 1 hr and then incubated with
WG9001B or WG8185B2 bisphosphonates for 48 hrs. Cells incubated with zoledronate and
incadronate served as positive controls. Neither GGOH nor FOH significantly influenced the
anti-proliferative activity of WG9001B and WG8185B2 BPs (Fig. 3). IC50 values for
WG9001B and WG8185B2 compounds alone or in combination with GGOH or FOH did not
significantly vary. By contrast, GGOH significantly diminished anti-proliferative effects of
zoledronate and, to a lower degree, of incadronate.
Effect of bisphosphonates on the motility of macrophages
Motility is a very important aspect of osteoclast differentiation and function. Upon
stimulation, osteoclast precursors migrate and attach onto the bone surface, which enables
these cells to differentiate into active resorbing osteoclasts. Thus, we assessed the effect of
WG9001B and WG8185B2 BPs on migratory properties of J774E cells using the transwell
system, both with collagen- and Matrigel-coated wells. In both cases, BPs inhibited the
migration of J774E cells through porous membranes (Fig. 4). However, the effect was much
more pronounced in the case of Matrigel-coated wells. Over 90% inhibition of migration of
J774E cells through Matrigel was observed for WG9001B compound and 80% for
WG8185B2 BP, which was similar for the activity of incadronate (90%) and much higher as
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compared to zoledronate (38% of inhibition). In the case of migration through collagen-
coated wells, the inhibitory effect was much less evident; however, the activity of WG9001B
compound was comparable to that of incadronate and zoledronate.
Pro-apoptotic activity of WG9001B and WG8185B2
All the applied compounds (WG9001B, WG8185B2, incadronate, and zoledronate) caused a
substantial increase in caspase-3 activity in J774E cells as compared to untreated control
(Fig.5). Both WG9001B and WG8185B2 BPs showed enhanced caspase-3 activity as
compared to reference incadronate and zoledronate. Treatment with WG8185B2 BP led to a
significant increase in the caspase-3 activity in a time-dependent manner. Incubation for 24
hrs increased the caspase-3 activity by over twofold, and the enzyme activity increased over
sevenfold after additional 24 hrs in comparison to untreated cells. In the case of WG9001B
BP, the activity of caspase-3 enzyme remained stable upon the time of incubation, with the
mean increase of approximately sixfold. These changes in the caspase-3 activity were
reflected in the percentage of apoptotic cells assessed in annexin-V assay (Fig. 6). Incubation
with BPs led to a substantial increase in the percentage of J774E cells in early and late
apoptosis. Interestingly, the pro-apoptotic effect of WG9001B compound was observed much
earlier as compared to the reference zoledronate and incadronate, and WG8185B2 BP. After
24 hrs of incubation, almost 60% of cells were annexin-V positive and the number of
apoptotic cells increased to over 80% at the time of treatment. Both incadronate and
zoledronate induced apoptosis in J774E macrophages, with the most significant effect
observed after 72 hrs of incubation (58% and 86% of apoptotic cells, respectively). In the
case of WG8185B2 BP, incubation for 72 hrs resulted in a statistically significant increase in
the percentage of apoptotic cells. None of the studied aminobisphosphonates evoked a
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significant necrosis in J774E cells. The percentage of dead cells did not exceed 5% in any of
the incubation time tested and did not differ as compared with control cells.
Effect of bisphosphonates on cell cycle progression
In order to find the mechanism how WG8185B2 and WG9001B BPs affect the cell cycle
progression, J774E cells were incubated with the compounds at concentrations equal to their
IC20 values for 24, 48, and 72 hrs. As shown before, treatment with either zoledronate or
incadronate led to the arrest of cell cycle progression in S phase (T. Iguchi, Miyakawa,
Yamamoto, Kizaki, & Ikeda, 2003; Okamoto et al., 2014). This was manifested most
profoundly after 24 hrs of incubation by the increase in the number of cells in this phase from
37% (control) to 55% and 57% for incadronate- and zoledronate-treated cells, respectively
(Fig. 7). Similarly, use of WG8185B2 compound resulted in significant accumulation of
J774E cells in S phase. However, this effect was also observed after 24 and 48 hrs of
incubation. Interestingly, the WG9001B compound showed different mode of action.
Incubation with WG9001B BP led to slight and transitory accumulation of macrophages in
G2/M phase and tended to the arrest of cell cycle progression in S phase after 72 hrs of
treatment (26% vs 41% of cells).
Discussion
Bisphosphonates are widely used for the prevention and treatment of osteoporosis,
corticosteroid-induced bone loss, and Pagets’s disease. BPs are also used in the management
of cancer-related bone diseases, as an adjuvant therapy in patients with osteolytic bone
metastases, as well as in patients treated with agents leading to bone loss. Numerous clinical
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studies have shown that BPs can reduce the occurrence of bone fractures and pain, thus
leading to improved quality of life. Recently, more and more attention has been paid to the
possible anticancer activity of BPs. It is now postulated that BPs influence directly tumor
growth and progression as well as indirectly by anti-angiogenic (Gao et al., 2017;
Michailidou et al., 2010; Wood et al., 2002) and immunomodulatory effects (Fujimura,
Kambayashi, Furudate, Kakizaki, & Aiba, 2013; Kunzmann et al., 2000; Rossini et al., 2012).
Osteoclasts and macrophages are endocytic cells that are derived from the same
hematopoietic progenitors. They are both very sensitive to the biological effects of BPs. It
was shown that a strong apoptotic activity in J774E macrophages reflects, in general, their
potency for inhibiting bone resorption (M. J. Rogers et al., 1996). BPs were shown not only
to exert pro-apoptotic effect in macrophages, but also to inhibit their migration and invasion
(T. L. Rogers & Holen, 2011). Thus, murine macrophage cell lines, mainly J774E and
RAW264.7, appeared to be an appropriate and convenient model for studies on
pharmacological properties of BPs in vitro.
As we have previously demonstrated, both WG8185B2 and WG9001B BPs show
anti-proliferative activity against J774E macrophages and MCF-7 cancer cells. In former
studies WG8185B2 inhibited the proliferation of J774E and MCF-7, cells with IC50 values of
90.1 and 96.5 M, respectively. The anti-proliferative activity of WG8185B2 against J774E
cells was comparable to zoledronate and six times higher than in the case of incadronate.
WG9001B exhibited a specificity of action against J77E cells manifested by 16 times
stronger anti-proliferative activity in comparison to zoledronic acid (5.8 M vs. 92.4 M)
(Goldeman & Nasulewicz-Goldeman, 2014). These observations point to potential benefits of
the application of WG9001B and WG8185B2 BPs in combined anticancer treatment, not
only as bone protecting agents, but also in the context of tumor–microenvironment
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interactions. This approach is based on the Paget’s hypothesis of “seed” and “soil.” More
than 100 years ago, Stephen Paget suggested that although therapies against the cancer cell
(the “seed”) are important, affecting the microenvironment that supports cancer cell growth
(the “soil”) may be equally valuable. The bone microenvironment provides a supportive
niche for cancer cell survival and tumor growth (Langley & Fidler, 2011). Once metastatic
breast cancer cells reach the bone microenvironment, they start interacting with osteoblast
and osteoclasts to promote bone degradation. In turn, bone-derived growth factors, calcium
ions, and cytokines released during bone resorption attract cancer cells to the bone surface
and facilitate their growth and propagation. This relationship between tumor metastatic cells
and bone resident cells is called “a vicious cycle”. Our results show that the two investigated
BPs may not only inhibit the proliferation of osteoclast precursors, but also influence
beneficially the effects of antitumor treatment by breaking of vicious cycle in bone.
Many studies have reported that the combination of BPs with anticancer agents can
significantly influence antitumor effects both in vitro and in vivo. Thus, based on preliminary
data, we selected two compounds with the highest anti-proliferative activity to examine their
potential influence on cytotoxic activity of cisplatin and doxorubicin, which are commonly
used chemotherapeutics in patients with breast cancer. Our studies show that both
simultaneous and subsequent treatment of J774E macrophages with cytostatics and
WG9001B or WG8185B2 resulted in a synergistic anti-proliferative effect, which is most
pronounced when cells were pretreated with BP. This raises the question of the possible
mechanism of cell sensitization to cytostatics by BPs. Benassi et al. reported that zoledronic
acid may enhance the effectiveness of cisplatin in osteosarcomas expressing wild-type p53
(Benassi et al., 2007). Similarly, zoledronic acid has been shown to augment significantly the
anti-proliferative effects in docetaxel-treated fibrosarcoma (Koto et al., 2010), prostate (Ullén
et al., 2005), or pancreatic cancer cells (M. Zhao et al., 2012) Furthermore, Matsumoto et al.
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showed that zoledronate potentiates the anti-proliferative activity of paclitaxel, etoposide,
cisplatin, irinotecan, and imatinib against SBC-3 human lung cancer cells (Matsumoto et al.,
2005). Thus, BPs when used adjunctively with cytostatics in cancer patients with osteolysis
may prevent skeletal-related events. In addition, many cancer treatments have detrimental
effects on bone and can increase the risk of fracture. The increasing use of aromatase
inhibitors, in particular, results in treatment-induced bone loss, which can be prevented with
BP treatment (Langley & Fidler, 2011).
It is now well accepted that nitrogen-containing BPs exert biological effects through
the inhibition of the mevalonate pathway (Luckman, Hughes, et al., 1998). This results in the
loss of prenylation of small GTPases, which leads to the inhibition of osteoclast
differentiation and apoptosis of cells (Luckman, Coxon, Ebetino, Russell, & Rogers, 1998).
Apoptosis and caspase activation can be prevented by addition of GGOH. This intermediate
of the mevalonate pathway restores protein prenylation and resorptive activity of osteoclasts
(Benford et al., 1999; Halasy-Nagy, Rodan, & Reszka, 2001). However, the effect of FOH
treatment on the mevalonate pathway is not as significant as that of GGOH. Tsubaki et al.
reported that inhibition of osteoclast formation from C7 macrophage-like cells by treatment
with minodronate or alendronate may be successfully re-established by the co-treatment with
20 μM GGOH. In contrast, a combination with FOH did not significantly reverse the effects
of minodronate and alendronate on a generation of TRAP-positive multinucleated cells
(Tsubaki et al., 2014). Here, we present that the addition of GGOH to the culture medium
significantly decreased the anti-proliferative activity of zoledronate in J774E macrophages in
a dose-dependent manner. The value of IC50 increased from 92.4 to 263.1 and 566.6 μM
when cells were simultaneously treated with 10 and 20 μM GGOH for 72 hrs, respectively.
The effect is less remarkable in incadronate-treated cells, with an increase in IC50 value from
380.8 to 682.2 and 840.7 μg/mL in co-culture with 10 and 20 μM GGOH, respectively. By
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contrast, FOH did not significantly influence the anti-proliferative potential of reference BPs.
This finding is in agreement with data previously reported by Iguchi which showed that
pretreatment of myeloma cells with 20 μM GGOH but not with FOH resulted in a significant
reduction of the anti-proliferative effect of incadronate (T. Iguchi et al., 2003). In our studies,
both WG9001B and WG8185B2 compounds inhibited the proliferation of J774E cells, and
WG9001B BP showed approximately fivefold higher activity as compared to reference
zoledronate. Surprisingly, neither GGOH nor FOH affected significantly the anti-proliferative
activity of the WG9001B and WG8185B2 bisphosphonates. These results may suggest that
the mechanism of action of the studied aminobisphosphonates is different from the
mechanism of the reference zoledronate and incadronate. One of the possible explanations
are the differences in the chemical structure of reference and investigated compounds. This
seems probable in the case of zoledronic acid, which belongs to the group of the nitrogen-
containing hydroxybisphosphonates. However, the differences in the mechanism of action
between incadronic acid and investigated BPs are surprising since both studied BPs and
incadronate belong to the same group of BPs-aminomethylidenebisphosphonates. Owing to
the chemical similarity between these compounds, such mechanism of action could be
expected. However, there are two significant structural factors that differentiate the studied
BPs from the control incadronate, which may affect their mechanism of action. WG9001B
and WG8185B2 BPs have two bisphosphonic groups in their structures, and both of studied
compounds contain aromatic ring (1,4-disubstituted phenyl and 1,5-disubstituted naphthyl,
respectively) bounded directly to the nitrogen of the aminomethylidenebisphosphonic group.
The planarity of the phenyl and naphthyl rings makes both molecules more rigid in
comparison to incadronate-containing cycloheptyl ring. Furthermore, their aromatic character
increases hydrophobicity of the investigated BPs and decreases the basicity of the nitrogen
atom of the aminomethylidenebisphosphonate group.
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Next, we examined the cellular effects of WG9001B and WG8185B2 BPs on J774E
macrophages.
Osteoclast precursors migrate to bone resorption sites where they differentiate into
mature osteoclasts. They cross blood vessels walls and migrate through the bone marrow
cavity, which is triggered by the chemokine gradient. Once osteoclast precursors reach the
osteoclast niche, they start to differentiate, fuse, and finally get activated in the resorption
process. Thus, we investigated whether examined BPs influence the migration process in
J774E cells. We showed that treatment with WG9001B and WG8185B2 compounds for 48
hrs led to a significant (90 and 80%, respectively) inhibition of J774E cell migration through
Matrigel-coated pores. The effect was comparable to that observed in incadronate-treated
cells and more pronounced as compared to zoledronate. This inhibitory effect seems to be
related to biochemical changes rather than to anti-proliferative activity of tested BPs, which
were applied at a dose not exceeding IC30. Similarly, Kimachi et al. showed that zoledronic
acid impairs not only the differentiation of RAW264.7 macrophages into osteoclasts, but also
TNF-α-induced migration through collagen type I-coated chambers (Kimachi, Kajiya,
Nakayama, Ikebe, & Okabe, 2011).
As a result of the preventing post-translational prenylation, nitrogen-containing BPs
of the third generation induce apoptosis in a variety of cells, including osteoclasts,
macrophages, and cancer cells (Karabulut et al., 2010; Koto et al., 2010; Luckman, Coxon, et
al., 1998). Interestingly, data obtained from in vitro studies in J774E macrophages showed
that the pro-apoptotic activity of BPs in vitro reflects their anti-resorptive potency in vivo.
According to this, the highest pro-apoptotic activity has been found for zoledronate,
risedronate, and ibandronate (Moreau et al., 2007). In our studies, WG8185B2 and
WG9001B BPs showed a significant pro-apoptotic activity in J774E cells, which was
manifested by a significant increase in caspase-3 activity and the percentage of apoptotic
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cells. However, the pro-apoptotic activity of these aminobisphopshonates seems not to be
related to inhibition of mevalonate pathway.
Cell cycle control is one of the key mechanisms of cell growth and proliferation.
Many therapeutic agents, including BPs, cause cell cycle arrest before inducing apoptosis.
However, the exact molecular mechanism underlying this phenomenon remains to be
revealed, and experimental data concerning the influence of nitrogen-containing BPs on cell
cycle progression are rather conflicting. Results of many studies indicate that nitrogen-
containing BPs, like zoledronate, risedronate, or pamidronate, typically cause the arrest of
cell cycle progression in S phase, with a corresponding decrease in the number of cells in
other phases. This was demonstrated in both normal and cancer cells (Iguchi et al., 2003;
Mansouri, Mirzaei, Lage, Mousavi, & Elahian, 2014; Okamoto et al., 2014; Zhao et al.,
2018). On contrary, it has been also demonstrated that treatment with ibandronate leads to
increased cell cycle arrest in S phase in U-2 osteosarcoma cells and in G2/M phase in MG-63
osteoblast-like cells (Karlic et al., 2017). In addition, Merrel et al. showed that treatment with
zoledronate increased the percentage of MDA-MB-231 cells in G1 phase (Merrell,
Wakchoure, Lehenkari, Harris, & Selander, 2007). The molecular or cellular background of
these discrepancies is unclear. In our studies, the reference BPs, zoledronate and incadronate,
induced the arrest of cell cycle progression of J774E cells in S phase even after 24 hrs of
treatment. Similarly, treatment with WG8185B2 compound significantly increased the
number of cells in S phase and decreased the number of cells in G0/G1 phase. However, the
effect was the most pronounced after 48 hrs of incubation. By contrast, in WG9001B-treated
cells a transient arrest in progression through G2/M-checkpoint followed by an increase in
the percentage of cells in S phase was observed. One of the possible explanations of this
phenomenon may be the differences in molecular structure.
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In summary, BPs are organic compounds that are capable of inhibiting osteoclast-
mediated bone resorption. Nitrogen-containing BPs are widely used for the prevention of
age-related bone loss and as an adjuvant anticancer therapy in patients with bone lesions.
Here, we report that two aminomethylidenebisphosphonates, WG9001B and WG8185B2,
show remarkable anti-proliferative activity toward osteoclast precursors in vitro. They also
significantly augment the anti-proliferative effect of cytostatics. Both BPs influence the
motility of J774E cells. Moreover, they cause cell cycle arrest in osteoclast precursors and
show a potent pro-apoptotic activity. Further studies regarding the molecular mechanism of
action and in vivo activity profile are needed to characterize their therapeutic potential.
Acknowledgments:
This work was supported by National Science Center (Grant No. 2014/13/B/NZ4/01105).
Author contributions:
ANG conceived, planned and performed the experiments, analyzed the data, and wrote the
paper. WG synthesized the studied bisphosphonates, and wrote the paper. EM performed the
experiments with the support of ANG. JW critically read and improved the manuscript.
Conflict of interest:
The authors declare no conflict of interest.
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Data Availability Statement
Author elects to not share data
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