macrophage cells using MTT assay. The results of these experiments
are elaborated in this communication.
micro-architecture under estrogen-deficient condition. However,
compared to ovx mice group, treatment with 26a (at 1 and 5 mg-kg-1
body weight dose) and raloxifene, (RAL, at 1mg-kg-1 body weight
dose) significantly increased BV/TV, Tb.N, and Tb.Th with
concurrent reduction in Tb.Sp, and Tb.Pf which were compromised
under estrogen-deficient condition in Ovx mice (Fig. 7). For in-vivo
analysis, we compared statistics between Ovx and rest of treated
groups using prism software version (6.2) with one-way ANOVA
Alkaline phosphatase (ALP) present on the surface of osteoblasts,
plays an important role in osteogenesis and therefore it has been
considered as an important bone marker to follow the osteogenesis.29
Thus, bone-forming activity of target compounds (19a-i, 20a-b, 24a-
b, 25a-c, and 26a-c) was assessed by measuring ALP activity
spectrophotometrically using MCOs. Results showed that 19c, 19g,
20c, 24b, 25a, 25c, and 26a significantly increased ALP activity
within the range of 1pM - 1nM concentrations (Fig. 3). While 19c
and 25c increased ALP activity at 1nM and 100pM respectively,
19g, 20c, 24b, 25a, and 26a increased ALP at 1pM concentration.
The observed osteogenic activity of compounds indicated that unlike
phytoestrogens, these compounds might have biphasic response.
Amongst all, 26a was found to be the most active and was selected
for further biological evaluation. Initially, the observed ALP activity
of 26a was validated through qPCR analysis of marker gene which
showed increased mRNA expression of ALP gene and confirmed its
activity (Fig. 5).
±
(Data are mean SEM; n = 3; ***P<0.001; **P<0.01). The in-vivo
study showed significant bone restorative effect of 26a at 1 and 5
mg-kg-1 body weight dose. Of note, 26a presented pronounced
osteogenic effect at 5mg-kg-1 body weight dose which was
comparable with the sham and RAL treated mice. These results
validated the observed in-vitro osteogenic effect of 26a.
As mentioned above, the role of estrogen and its receptor(s) in
differentiation and activity of osteoblast cells is well established.11-14
It is reported that expression of ER-α is prominent in the osteoblast
cells of cortical bones, whereas ER-β in the osteoblast cells of
cancellous bones.15b In view of these observations, we evaluated 26a
for its possible estrogen receptor-mediated osteogenic effect and
estrogen receptor subtype selectivity. For this purpose, MCOs were
treated with 26a for 48h and lysates were prepared. The prepared
lysates were electrophoresed on SDS-PAGE gel and probed with
antibodies against ER-α and ER-β. The result showed that 26a
enhanced the protein levels of both ER-α and ER-β with significant
selectivity towards ER-β which indicated that 26a exhibited estrogen
receptor-mediated osteogenic effect preferentially via ER-β.
During bone mineralization, apatite crystals viz Ca10(PO4)6(OH)2,
Ca10(PO4)6F2 and Ca10(PO4)6Cl2 are formed on nano-composite
structures of bone (osteoid) formed by osteoblast cells. It is known
that ALP is involved in the deposition of calcium phosphate during
bone formation.30 Therefore, mineralization of osteoblasts is an
imperative indicator of osteoblast differentiation. Following ALP
activity of 26a at 1pM, 26a was evaluated for its effect on bone
mineralization. Results showed that 26a had ability to induce the
formation of mineralized nodules significantly as compared with
control untreated cells (Fig. 4).
In order to validate the observed ER subtype selectivity, the possible
interactions of 26a with ER-α and ER-β were studied using molecular
docking analysis. The structures of ER-α and ER-β were retrieved from
protein data bank (ER-α: 3ERT and ER-β: 2FSZ) and docking
experiments were performed using BioSolveIT, FlexX v2.1.8 package.
Results showed that 26a occupied ligand-binding pocket of ER-α and
ER-β and had interactions with good docking scores (Table 1 & 2).
However, 26a had preferential affinity for ER-β than ER-α (docking
score -12.21 and -11.01 respectively). In these experiments, 17β-estradiol
(1) was taken as control for comparison. These in-silico observations had
good correlation with results obtained through western blot analysis
which further supported our assumptions.
The osteogenic marker genes such as Bone morphogenetic proteins
(BMPs) and Runt-related transcription factor-2 (RUNX-2) are the
key regulators of early osteoblast differentiation and have impact on
ALP activity, collagen synthesis and skeletal morphogenesis.31 It has
been shown that RUNX-2 is responsible for inducing the multipotent
mesenchymal cells into immature osteoblast cells and has
unregulated expression in immature osteoblast cells.32 Similarly,
BMP-2 is most potent regulator among the local factors related to
osteoblast differentiation which is necessary component of the
signaling cascade that governs osteogenesis.33 Considering the
importance of these markers, mRNA expression levels of these
markers were evaluated in presence of 26a for confirmation of its
bone-forming efficacy. For this purpose, osteoblast cells were treated
with 26a at 1 pM and 100pM concentrations for 48h and total RNA
was isolated. Following this, cDNA was synthesized and used as a
template in real-time quantitative PCR. In these experiments,
housekeeping gene GAPDH was used as the internal control. These
experiments showed that 26a significantly increased the transcript
levels of BMP-2 and RUNX-2 at 1pM and 100pM concentrations as
compared with untreated cells (Fig. 5). Together, ALP activity,
mineralization of osteoblasts and qPCR data showed that 26a had
osteogenic activity. For in-vitro analyses of compounds, we
compared statistics between control and rest of treated groups using
prism software version (6.2) with one-way ANOVA (Data are mean
It is desirable from an osteogenic agent to have protective effect against
hormone-dependent breast cancer. In view of anticipated mixed estrogen
agonistic and anti-estrogenic activity of designed molecules, it was
worthwhile to evaluate the effect of these compounds on estrogen
receptor-positive (ER+) cancer cells (MCF-7 cells) since estrogens are
known to proliferate breast cancer cells. Therefore, compounds 19a-i,
20a-b, 24a-b, 25a-c, and 26a-c were evaluated for their cytotoxicity
against MCF-7 cells at 30 µg/mL using MTT assay. Result showed that
19b, 19g, 19i, 20a, 20c, 24a-b, 25b, and 26a-c presented 30-48% growth
inhibition of cancer cells (Fig. 8). The maximum growth inhibition was
observed with 24a (48.4%) whereas 26a inhibited 32.1% growth of
cancer cells.
Once the bone-forming and antiproliferative activities of 26a
were determined, it was important to evaluate 26a for its
inherent toxicity in healthy cells. Therefore, 26a was evaluated
for its toxicity in healthy peritoneal macrophage cells using
MTT assay at 5, 10 and 20µM concentrations. Result showed
that 26a was devoid of any toxicity within 5-20µM concentrations.
±
SEM; n = 3; ***P<0.001; **P<0.01).
Following in-vitro osteogenic activity, 26a was evaluated for its
effect against deterioration of trabecular bone in ovariectomized
(Ovx) balb/c mice. For this purpose, three groups of ovx mice were
treated with 26a (at 1 and 5 mg-kg-1 body weight dose) and
raloxifene, (RAL, at 1 mg-kg-1 body weight dose) through oral
gavage for one month, whereas groups of Ovx mice and sham mice
were treated with vehicle only. In this experiment, RAL was taken as
positive control. The quality of bone micro-architecture was
analyzed in terms of bone volume density (BV/TV), trabecular
number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing
(Tb.Sp), and trabecular pattern factor (Tb.Pf) using µCT. Significant
reduction in the BV/TV, Tb.N, and Tb.Th with concurrent increase
in Tb.Sp, and Tb.Pf was observed in Ovx mice group compared with
sham mice group (Fig. 7). This indicated deterioration of bone
In this study, two subsets of stilbene derivatives viz 19a-i, 20a-b, and
24a-b, 25a-c, 26a-c were synthesized in anticipation of their possible
ER-β mediated osteogenic activity. These subsets differed with each
other mainly with respect to the central core of the molecules. Total
sixteen amide derivatives were synthesized incorporating alkylamines
and heteroaryl (picolyl) amines connected to stilbene core through spacer
of different lengths. The biological activity results showed that in
general, molecular thickness around the core of the molecule was
optimal for observed osteogenic activity. As reported in the literature, in
case of stilbenes interacting with ER, molecular thickness around the