Dose-dependent effects of genistein on bone homeostasis in rats' mandibular subchondral bone

Aim:

To investigate the effect of genistein on bone homeostasis in mandibular subchondral bone of rats.

Methods:

Female SD rats were administered with genistein (10 and 50 mg/kg) or placebo by oral gavage for 6 weeks. Then the animals were sacrificed, and histomorphology and micro-structure of mandibular condyle were examined using HE staining and micro-CT analysis, respectively. The expression levels of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), the receptor activator of nuclear factor κB ligand (RANKL) and estrogen receptors (ERs) in mandibular condyle were detected using real-time PCR. Cultured osteoblasts were prepared from rat mandibular condyle for in in vitro study. The cells were treated with genistein (10⁻⁷ or 10⁻⁴ mol/L) for 48 h. The expression of the bone homeostasis-associated factors and estrogen receptors (ERs) was detected using real-time PCR, and ER silencing was performed.

Results:

At both the low- and high-doses, genistein significantly increased the bone mineral density (BMD) and bone volume, and resulted in thicker subchondral trabecular bone in vivo. In both in vivo and in vitro study, the low-dose genistein significantly increased the expression of ALP, OC and OPG, but decreased the expression of RANKL and the RANKL/OPG ratio. The high-dose genistein decreased the expression of all these bone homeostasis-associated factors. Both the low and high doses of genistein significantly increased the expression of ERβ, while ERα expression was increased by the low dose genistein and decreased by the high dose genistein. ERβ silencing abrogated most of the effects of genistein treatment.

Conclusion:

In rat mandibular condylar subchondral bone, low-dose genistein increases bone formation and inhibit bone resorption, while excess genistein inhibits both bone formation and resorption. The effects of genistein were predominantly mediated through ERβ.     

Involvement of Bcl-2, Src, and ERα in gossypol-mediated growth inhibition and apoptosis in human uterine leiomyoma and myometrial cells

Aim:

To investigate the effect of gossypol on the growth of cultured human uterine leiomyoma and myometrial cells, the level of Bcl-2 and the activity of Src and estrogen receptor (ERα).

Methods:

Human uterine leiomyoma and adjacent normal myometrial cells were cultured in vitro. Both cell types were treated with a graded concentration of gossypol. Cell viability was assayed using CCK-8. Morphological change was observed with optical and electronic microscopy. Apoptosis was evaluated using TUNEL assay. Levels of Bcl-2, ERα and Src were analyzed using Western blotting.

Results:

Gossypol significantly inhibited growth and promoted apoptosis in cultured human uterine leiomyoma cells with the IC₅₀ value and its corresponding 95% confidence intervals (CI) of 6.5 (4.0–10.5), 9.0 (4.9–16.5), and 7.5 (4.0–14.1) μmol/L at 20, 40, and 60 h, respectively. Gossypol exerted inhibitory effects on the myometrial cells with the IC₅₀ value and its 95% CI of 49.1 (28.3–85.0), 14.5 (7.7–27.4), and 2.6 (1.2–5.6) μmol/L at 20, 40, and 60 h, respectively. Compared with control, gossypol 0.1-3.0 μmol/L markedly decreased the protein expression of Bcl-2 (P<0.05) in both leiomyoma and myometrial cells in a concentration-dependent manner, and significantly suppressed the level of phospho-Tyr416Src (P<0.05) in both cell types at 3.0 μmol/L without obvious alteration of c-Src and phospho-Tyr527Src levels (P>0.05). In addition, gossypol markedly reduced both the expression of ERα (P<0.05) at the low concentration of 0.1 μmol/L in the myometrial cells and the level of phospho-ser167ERα (P<0.05) at the high concentration of 3.0 μmol/L in the leiomyoma cells.

Conclusion:

Gossypol inhibits proliferation and induces apoptosis in human uterine leiomyoma and myometrial cells. It is likely that the mechanisms of action involve reducing the protein level of Bcl-2 and the activity of Src and ERα.     

Casticin, a flavonoid isolated from Vitex rotundifolia, inhibits prolactin release in vivo and in vitro

Aim:

To investigate the anti-hyperprolactinemia activity of casticin, a flavonoid isolated from Vitex rotundifolia, and elucidate its molecular mechanism.

Methods:

Hyperprolactinemia (MIHP) was induced by administration of metoclopramide dihydrochloride (50 mg/kg, tid, ip, for 10 d) in SD rats and the primary pituitary cells were prepared from the pituitary glands of the SD rats. Prolactin concentrations were measured using a radioimmunoassay. Cell viability was measured using an MTT assay. The mRNA expression of estrogen receptor alpha and beta in rat pituitary cells was measured using semi-quantitative RT-PCR analysis.

Results:

The level of serum prolactin in the MIHP model group was 2.1 fold higher than that in the untreated control group (P<0.01). Casticin (10, 20, and 40 mg/kg, ip, for 7 d) reduced serum prolactin levels by 33.9%, 54.3%, and 64.7%, respectively (P<0.01). The positive control drug bromocriptine 1 mg/kg decreased the serum prolactin concentration in MIHP rats by 44.9%. 17β-Estradiol (E2) significantly increased the proliferation of pituitary cells and casticin (1 and 10 μmol/L) markedly inhibited E2-induced pituitary cell proliferation by 27.7% and 42.1%, respectively. Stimulation of pituitary cells with E2 increased prolactin secretion into the cell culture supernatants, and casticin (0.1, 1, and 10 μmol/L) significantly inhibited the prolactin release stimulated by E2 in a concentration-dependent manner. Casticin (1 and 10 μmol/L) significantly inhibited ERα mRNA expression in pituitary cells stimulated with E2 (P<0.01) but increased ERβ mRNA expression at a concentration of 10 μmol/L (P<0.01). However, casticin had no effects on proliferation and prolectin release of the unstimulated primary pituitary cells in vitro.

Conclusion:

Casticin inhibited the release of prolactin from pituitary cells of SD rats stimulated with E2 in vivo and in vitro. These effects might be related with inhibiting the ERα mRNA expression and increasing the ERβ mRNA expression.     

Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation

BACKGROUND AND PURPOSE

Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease.

EXPERIMENTAL APPROACH

Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg⁻¹ propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1β, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro.

RESULTS

Propranolol at 0.1 and 5 mg·kg⁻¹ reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg⁻¹ reduced IL-1β, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9.

CONCLUSIONS AND IMPLICATIONS

Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters.     

Effects and mechanism of aromatic aminoketone SY0916 on osteoclastic bone destruction

Aim:

To study the effects and mechanism of aromatic aminoketone (SY0916) on bone destruction in vitro.

Methods:

MC3T3-E1 cells and bone marrow cells were co-cultured to obtain purified osteoclasts. The proliferation of osteoclast-like cells (OCLs) was determined by MTT assay. The number of osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining. The functioning of osteoclasts was determined by measuring the area of bone resorption pits on bone slices. MMP-9 secretion by osteoclasts was measured by an ELISA kit. Osteoclast apoptosis was detected by flow cytometry using an AnnexinV-FITC kit. Gene expression of RANK and MMP-9 in osteoclasts as well as RANKL and OPG in MC3T3-E1 cells was determined by real-time PCR.

Results:

SY0916 significantly inhibited the proliferation of OCLs, decreased both the total and average area of bone resorption pits, and dramatically inhibited the number of osteoclasts between concentrations of 0.01 and 10 μmol/L. Furthermore, SY0916 reversed IL-1β-mediated inhibition of osteoclast apoptosis and shortened osteoclast lifespan. In addition, SY0916 significantly inhibited the mRNA expression of RANK, RANKL, OPG, and MMP-9. However, the inhibition of OPG was weaker than that of RANKL. Accordingly, the ratio of RANKL to OPG mRNA expression in MC3T3-E1 cells was significantly decreased by SY0916. Meanwhile, the expression of MMP-9 protein in osteoclasts was inhibited by SY0916 between 0.01 and 10 μmol/L.

Conclusion:

SY0916 prevents osteoclastic bone destruction by inhibiting the proliferation and function of osteoclasts. The underlying mechanism for this effect involves the regulation of the RANKL-OPG-RANK axis, which determines the direction of bone metabolism.     

Naringenin inhibits the growth of Dictyostelium and MDCK-derived cysts in a TRPP2 (polycystin-2)-dependent manner

Background and Purpose

Identifying and characterizing potential new therapeutic agents to target cell proliferation may provide improved treatments for neoplastic disorders such as cancer and polycystic diseases.

Experimental Approach

We used the simple, tractable biomedical model Dictyostelium to investigate the molecular mechanism of naringenin, a dietary flavonoid with antiproliferative and chemopreventive actions in vitro and in animal models of carcinogenesis. We then translated these results to a mammalian kidney model, Madin-Darby canine kidney (MDCK) tubule cells, grown in culture and as cysts in a collagen matrix.

Key Results

Naringenin inhibited Dictyostelium growth, but not development. Screening of a library of random gene knockout mutants identified a mutant lacking TRPP2 (polycystin-2) that was resistant to the effect of naringenin on growth and random cell movement. TRPP2 is a divalent transient receptor potential cation channel, where mutations in the protein give rise to type 2 autosomal dominant polycystic kidney disease (ADPKD). Naringenin inhibited MDCK cell growth and inhibited cyst growth. Knockdown of TRPP2 levels by siRNA in this model conferred partial resistance to naringenin such that cysts treated with 3 and 10 μM naringenin were larger following TRPP2 knockdown compared with controls. Naringenin did not affect chloride secretion.

Conclusions and Implications

The action of naringenin on cell growth in the phylogenetically diverse systems of Dictyostelium and mammalian kidney cells, suggests a conserved effect mediated by TRPP2 (polycystin-2). Further studies will investigate naringenin as a potential new therapeutic agent in ADPKD.

Linked Articles

This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10     

Influence of food intake on the bioavailability and efficacy of oral calcitonin

AIMS

To investigate the influence of food intake on the bioavailability and pharmacodynamic effects of salmon calcitonin (sCT).

METHODS

A single-blind, randomized, partly placebo-controlled study was conducted in 36 healthy postmenopausal female volunteers aged 62–74 years. The influence of food intake on oral dosing with 0.8 mg of sCT at 22.00 h was evaluated for a (i) predose meal at 18.00 h, (ii) predose meal at 20.00 h, (iii) predose meal at 21.00 h, (iv) postdose meal at 22.10 h, (v) no meal, and (vi) meal at 20.00 h and placebo at 22.00 h. Study biomarkers were plasma sCT levels and changes in the bone resorption marker CTX-I (C-terminal telopeptide of collagen type I).

RESULTS

The predose meal at 18.00 and 21.00 h significantly decreased relative oral bioavailability of sCT to 26% [95% confidence interval (CI) 0.09, 0.73 and 0.09, 0.75, P= 0.009 and P= 0.01]. The meal consumed 10 min after dosing decreased the oral bioavailability of sCT to 59% (95% CI 0.21, 1.68), although nonsignificant (P= 0.48). This decreased bioavailability led to lower relative suppression of serum CTX-I, with an AUC of the 4-h efficacy response of −91%–×–hours for those receiving a meal at 18.00 h, compared with −238%–×–hours for fasting subjects. The Dunnett-adjusted difference between these two treatment sequences was 147%–×–hours (95% CI 68, 225) (P= 0.0003). The AUC was comparable among fasting subjects and those consuming a meal 10 min after dosing.

CONCLUSIONS

Postprandial dosing may limit the bioavailability of orally administered sCT. Maximal benefit can be achieved by dosing at least 10 min prior to meal time.     

Lactoferrin inhibits apoptosis through insulin-like growth factor I in primary rat osteoblasts

Aim： Excessive apoptosis of osteoblasts is the major cause of low bone mass, and bovine lactoferrin （bLF）, an iron-binding glycoprotein, might protect osteoblastic cells from apoptosis induced by serum withdrawal. The aim of this study was to elucidate the mechanisms underlying the anti-apoptotic action of bLF in rat osteoblasts in vitro. Methods： Primary rat osteoblasts were incubated in the presence of varying concentrations of bLF for 24 h. The expression of insulin-like growth factor I （IGF-I） and IGF-I receptor （IGF-IR） was measured uisng RT-PCR and Western blotting. Cell apoptosis was examined with flow cytometry. siRNAs targeting IGF-I was used in this study.

Results： Treatment of bLF （0.1–1000 μg/mL） dose-dependently increased the expression of IGF-I and IGF-IR in the osteoblasts. Treatment with bLF （10, 100 μg/mL） markedly inhibited the osteoblast apoptosis （with the rate of total apoptosis of 70% at 10 μg/mL）, but the high concentration of bLF （1000 μg/mL） significantly promoted the osteoblast apoptosis. Knockdown of the IGF-I gene in osteoblasts with siRNA markedly increased the osteoblast apoptosis.

Conclusion： Lactoferrin （10 and 100 μg/mL） effectively inhibits apoptosis of primary rat osteoblasts by upregulating IGF-I expression.     

Mitogen-activated protein kinase (MAPK) pathway mediates the oestrogen-like activities of ginsenoside Rg1 in human breast cancer (MCF-7) cells

Background and purpose:

The present study was designed to determine how ginsenoside Rg1, an active ingredient in ginseng root, exerts its oestrogenic effects. We hypothesize that Rg1 may exert oestrogen-like actions in MCF-7 cells by activating the mitogen-activated protein kinase (MAPK) pathway in a ligand-independent manner.

Experimental approach:

MCF-7 cells were co-incubated with the MAPK inhibitor PD98059 to determine whether the stimulant effects of Rg1 on cell proliferation, the induction of IGF-IR and pS2, the functional transactivation of oestrogen receptor-α (ERα), as well as ERα phosphorylation are dependent on MAPK. The time-dependent responses of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) to Rg1 in MCF-7 cells were studied. The responses of MEK phosphorylation to Rg1 in oestrogen receptor (ER)-negative HEK293 cells were also determined. The effects of Rg1 on cell proliferation and IGF-IR protein expression were studied in the presence of tyrosine kinase inhibitor genistein to elucidate the involvement of tyrosine kinase in mediating these effects.

Key results:

The oestrogenic effects of Rg1 in MCF-7 cells were abolished in the presence of PD98059. Rg1 could induce MEK protein expression and the phosphorylation level of MEK and ERK significantly in a time- and dose-dependent manner. Rg1 activated MEK phosphorylation in ER-negative HEK293 cells in a time- and dose-dependent manner. Rg1 induction of cell proliferation and IGF-IR protein expression was abolished by co-treatment with genistein.

Conclusions and implications:

Taken together, these results show that the MAPK pathway is involved in mediating the oestrogen-like actions of Rg1 in MCF-7 cells and suggest that Rg1 may activate ERα via MEK/ERK in a ligand-independent manner.     

The natural polyamines spermidine and spermine prevent bone loss through preferential disruption of osteoclastic activation in ovariectomized mice

BACKGROUND AND PURPOSE

Although naturally occurring polyamines are indispensable for a variety of cellular events in eukaryotic cells, little attention has been paid to their physiological and pathological significance in bone remodelling to date. In this study, we evaluated the pharmacological properties of several natural polyamines on the functionality and integrity of bone in both in vitro and in vivo experiments.

EXPERIMENTAL APPROACH

Mice were subjected to ovariectomy (OVX) and subsequent oral supplementation with either spermidine or spermine for determination of the bone volume together with different parameters regarding bone formation and resorption by histomorphometric analyses in vivo. Pre-osteoclasts were cultured with receptor activator of NF-κB ligand (RANKL), with or without spermidine and spermine to determine cellular maturation by tartrate-resistant acid phosphatase (TRAP) staining and cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction in vitro.

KEY RESULTS

Spermidine or spermine, given in drinking water for 28 days, significantly prevented the increased osteoclast surface/bone surface ratio and the reduced bone volume following OVX in mice. Either spermidine or spermine significantly inhibited the increased number of multinucleated TRAP-positive cells in osteoclasts cultured with RANKL in a concentration-dependent manner without affecting cell survival.

CONCLUSIONS AND IMPLICATIONS

The natural polyamines spermidine and spermine prevented OVX-induced bone loss through the disruption of differentiation and maturation of osteoclasts, rather than affecting osteoblasts. The supplementation with these natural polyamines could be beneficial for the prophylaxis as well as therapy of metabolic bone diseases such as post-menopausal osteoporosis.     

Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro

Aim:

Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro.

Methods:

Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression. The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit.

Results:

Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100 μmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 μmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 μmol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L).

Conclusion:

The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.     

Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice

Aim： To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice.
Methods： Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin （2, 4, or 6 mg/kg, ip） 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses.

Results： Plumbagin （2.5–20 μmol/L） concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro （the IC50 value of inhibition of cell viability was 14.7 μmol/L）. Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2–3 weeks and reduced the tumor volume by 44%–74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts.

Conclusion： Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.     

Calcineurin/NFAT pathway mediates wear particleinduced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro

Aim： To investigate the roles of the calcineurin/nuclear factor of activated T cells （NFAT） pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro. Methods： Osteoclasts were induced from mouse bone marrow macrophages （BMMs） in the presence of 100 ng/mL receptor activator of NF-KB ligand （RANKL）. Acridine orange staining and M＇IF assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATcl were examined using RT-PCR and immunofluorescent staining. The production of TNFa was analyzed with ELISA. Results： Titanium （Ti） or polymethylmethacrylate （PMMA） particles （0.1 mg/mL） did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group （96 h）. The expression of NFATcl was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VlVIT peptide （a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 pmol/L） did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VlVlT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group （96 h）. Conclusion： Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VlVlT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.     

2-Methoxystypandrone represses RANKL-mediated osteoclastogenesis by down-regulating formation of TRAF6�CTAK1 signalling complexes

BACKGROUND AND PURPOSE

2-Methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum, a Chinese herb used to treat bone diseases. Here we have determined whether 2-MS antagonised osteoclast development and bone resorption.

EXPERIMENTAL APPROACH

RAW264.7 cells were treated with receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to induce differentiation into osteoclasts. RT-PCR and Western blot were used to analyse osteoclast-associated gene expression and signalling pathways.

KEY RESULTS

The number of multinuclear osteoclasts, actin rings and resorption pit formation were markedly inhibited by 2-MS, targeting osteoclast differentiation at an early stage and without significant cytotoxicity. The anti-resorption effect of 2-MS was accompanied by decreasing dendritic cell-specific transmembrane protein and matrix metalloproteinase-9 (MMP-9) mRNA expression. RANKL-increased MMP-9 gelatinolytic activity was also attenuated by concurrent, but not by subsequent addition of 2-MS. 2-MS markedly inhibited not only the RANKL-triggered nuclear translocations of NF-κB, c-Fos and nuclear factor of activated T cells c1 (NFATc1), but also the subsequent NFATc1 induction. Degradation of IκB and phosphorylation of mitogen-activated protein kinases were also suppressed. RANKL facilitated the formation of singaling complexes of tumour necrosis factor receptor-associated factor 6 and transforming growth factor β-activated kinase 1 (TRAF6–TAK1), important for osteoclastogenesis and formation of such signalling complexes was prevented by 2-MS.

CONCLUSIONS AND IMPLICATIONS

The anti-osteoclastogenic effects of 2-MS could reflect the block of RANKL-induced association of TRAF6–TAK1 complexes with consequent decrease of IκB-mediated NF-κB and mitogen-activated protein kinases-mediated c-Fos activation pathways and suppression of NFATc1 and other gene expression, essential for bone resorption.     

Synergistic enhancement of parasiticidal activity of amphotericin B using copaiba oil in nanoemulsified carrier for oral delivery: an approach for non-toxic chemotherapy

Background and Purpose

The aim of this study was to devise a nanoemulsified carrier system (CopNEC) to improve the oral delivery of amphotericin B (AmB) by increasing its oral bioavailability and synergistically enhance its antileishmanial activity with copaiba oil (Cop).

Experimental Approach

The AmB encapsulated NEC (CopNEC-AmB) comprised of Cop, d-α-tocopheryl polyethylene glycol 1000 succinate and phosphatidylcholine was prepared by high-pressure homogenization method. Stability study of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. The CopNEC-AmB and plain AmB were compared as regards their in vitro antileishmanial activity, pharmacokinetics, organ distribution and toxicity.

Key Results

The optimal CopNEC-AmB had a small globule size, low polydispersity index, high ζ potential and encapsulation efficiency. The high resolution transmission electron microscopy illustrated spherical particle geometry with homogeny in their sizes. The optimal CopNEC-AmB was found to be stable in gastrointestinal fluids showing insignificant changes in globule size and encapsulation efficiency. The AUC_0–48 value of CopNEC-AmB in rats was significantly improved showing 7.2-fold higher oral bioavailability than free drug. The in vitro antileishmanial activity of CopNEC-AmB was significantly higher than that of the free drug as Cop synergistically enhanced the antileishmanial effect of AmB by causing drastic changes in the morphology of Leishmania parasite and rupturing its plasma membrane. The CopNEC-AmB showed significantly less haemolytic toxicity and cytotoxicity and did not change the histopathology of kidney tissues as compared with AmB alone.

Conclusions and Implications

This prototype CopNEC formulation showed improved bioavailability and had a non-toxic synergistic effect on the antileishmanial activity of AmB.     

17β-Estradiol inhibition of PPARγ-induced adipogenesis and adipocyte-specific gene expression

Aim:

To investigate the molecular interaction of peroxisome proliferator-activated receptor γ (PPARγ) with 17β-estradiol (E) in the regulation of adipogenesis.

Methods:

Female ovariectomized (OVX) mice and differentiated 3T3-L1 adipocytes were treated with combinations of the PPARγ agonist troglitazone or E, and the variables and determinants of adipogenesis were measured using in vivo and in vitro approaches.

Results:

Troglitazone (250 mg·kg⁻¹·d⁻¹ for 13 weeks) decreased the size of adipocytes without the change in white adipose tissue (WAT) mass and increased the expression of adipocyte-specific genes, such as PPARγ, adipocyte fatty acid binding protein, and lipoprotein lipase, compared with OVX control mice. E (0.05 mg/pellet, sc implanted) significantly reduced WAT mass, adipocyte size, and adipose marker gene expression. When mice were concomitantly treated with troglitazone and E, E blunted the effects of troglitazone on WAT mass, adipocyte size, and adipose PPARγ target gene expression. Consistent with the in vivo data, E (10 μmol/L) treatment inhibited lipid accumulation and the expression of adipocyte-specific genes caused by troglitazone (10 μmol/L) in 3T3-L1 cells. E (10 μmol/L) also decreased troglitazone-induced PPARγ reporter activity through both estrogen receptor (ER) α and ERβ. Mechanistic studies indicated that E (0.1 μmol/L) decreased the DNA binding of PPARγ induced by troglitazone (1 μmol/L) and inhibited the recruitment of the PPARγ coactivator CREB-binding protein.

Conclusion:

These results suggest that in vivo and in vitro treatment of E interferes with the actions of PPARγ on adipogenesis by down-regulating adipogenesis-related genes, which are mediated through the inhibition of PPARγ coactivator recruitment. In addition, it is likely that the activities of PPARγ activators may be enhanced in estrogen-deficient states.     

Thiocolchicoside suppresses osteoclastogenesis induced by RANKL and cancer cells through inhibition of inflammatory pathways: a new use for an old drug

BACKGROUND AND PURPOSE

Most patients with cancer die not because of the tumour in the primary site, but because it has spread to other sites. Common tumours, such as breast, multiple myeloma, and prostate tumours, frequently metastasize to the bone. To search for an inhibitor of cancer-induced bone loss, we investigated the effect of thiocolchicoside, a semi-synthetic colchicoside derived from the plant Gloriosa superba and clinically used as a muscle relaxant, on osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL) and tumour cells.

EXPERIMENTAL APPROACH

We used RAW 264.7 (murine macrophage) cells, a well-established system for osteoclastogenesis, and evaluated the effect of thiocolchicoside on RANKL-induced NF-κB signalling and osteoclastogenesis as well as on osteoclastogenesis induced by tumour cells.

KEY RESULTS

Thiocolchicoside suppressed osteoclastogenesis induced by RANKL, and by breast cancer and multiple myeloma cells. Inhibition of the NF-κB pathway was responsible for this effect since the colchicoside inhibited RANKL-induced NF-κB activation, activation of IκB kinase (IKK) and suppressed inhibitor of NF-κBα (IκBα) phosphorylation and degradation, an inhibitor of NF-κB. Furthermore, an inhibitor of the IκBα kinase γ or NF-κB essential modulator, the regulatory component of the IKK complex, demonstrated that the NF-κB signalling pathway is mandatory for osteoclastogenesis induced by RANKL.

CONCLUSIONS AND IMPLICATIONS

Together, these data suggest that thiocolchicoside significantly suppressed osteoclastogenesis induced by RANKL and tumour cells via the NF-κB signalling pathway. Thus, thiocolchicoside, a drug that has been used for almost half a century to treat muscle pain, may also be considered as a new treatment for bone loss.

LINKED ARTICLE

This article is commented on by Micheau et al., pp. 2124–2126 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01792.x     

Pleiotropic effects of statins on the treatment of chronic periodontitis – a systematic review

Aim

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and are an important group of hypolipidaemic drugs, widely used in the treatment of hypercholesterolaemia and cardiovascular disease. Some studies have shown that statins are able to modulate inflammation and alveolar bone loss.

Methods

In order to evaluate whether statins could influence periodontal treatment, improving the clinical and radiographic parameters in chronic periodontitis, a systematic review was conducted in the databases PUBMED and BIREME, searching for articles in English and Portuguese, published between the years 2004 and 2014, using the combined keywords statin, periodontal disease, periodontitis and alveolar bone. Studies regarding the treatment of chronic periodontitis in humans, blind or double-blind, retrospective cohort or randomized controlled trials that used statins topically or systemically were selected.

Results

Statins have important anti-inflammatory and immune effects, reducing levels of C-reactive protein and matrix metalloproteinases and their intermediate products, such as tumour necrosis factor-α, and are also able to inhibit the adhesion and extravasation of leukocytes, which block the co-stimulation of T cells. Statins reduce bone resorption by inhibiting osteoclast formation and lead to increased apoptosis of these cells. The effect of statins on bone formation is related to the increased gene expression of bone morphogenetic protein in osteoblasts.

Conclusion

Although we found biological mechanisms and clinical results that show lower alveolar bone loss and reduction of clinical signs of inflammation, further studies are needed to evaluate the clinical applicability of statins in the routine treatment of chronic periodontitis.