Kainat Khan

A naturally occurring naringenin derivative exerts potent bone anabolic effects by mimicking oestrogen action on osteoblasts

BACKGROUND AND PURPOSE Naringenin and its derivatives have been assessed in bone health for their oestrogen‐‘like’ effects but low bioavailability impedes clinical potential. This study was aimed at finding a potent form of naringenin with osteogenic action.

Thioaryl naphthylmethanone oxime ether analogs as novel anticancer agents

Employing a rational design of thioaryl naphthylmethanone oxime ether analogs containing functional properties of various anticancer drugs, a series of compounds were identified that displayed potent cytotoxicity toward various cancer cells, out of which 4-(methylthio)phenyl)(naphthalen-1-yl)methanone O-2-(diethylamino)ethyl oxime (MND) exhibited the best safety profile. MND induced apoptosis, inhibited migration and invasion, strongly inhibited cancer stem cell population on a par with salinomycin, and demonstrated orally potent tumor regression in mouse MCF-7 xenografts. Mechanistic studies revealed that MND strongly abrogated EGF-induced proliferation, migration, and tyrosine kinase (TK) signaling in breast cancer cells. However, MND failed to directly inhibit EGFR or other related receptor TKs in a cell-free system. Systematic investigation of a putative target upstream of EGFR revealed that the biological effects of MND could be abrogated by pertussis toxin. Together, MND represents a new nonquinazoline potential drug candidate having promising antiproliferative activity with good safety index.

[6]-Gingerol induces bone loss in ovary intact adult mice and augments osteoclast function via the transient receptor potential vanilloid 1 channel.

SCOPE [6]-Gingerol, a major constituent of ginger, is considered to have several health beneficial effects. The effect of 6-gingerol on bone cells and skeleton of mice was investigated. METHODS AND RESULTS The effects of 6-gingerol on mouse bone marrow macrophages and osteoblasts were studied. 6-Gingerol-stimulated osteoclast differentiation of bone marrow macrophages but had no effect on osteoblasts. Capsazepine, an inhibitor of TRPV1 (transient receptor potential vanilloid 1) channel, attenuated the pro-osteoclastogenic effect of 6-gingerol or capsaicin (an agonist of TRPV1). Similar to capsaicin, 6-gingerol stimulated Ca(2) + influx in osteoclasts. The effect of daily feeding of 6-gingerol for 5 wk on the skeleton of adult female Balb/cByJ mice was investigated. Mice treated with capsaicin and ovariectomized (OVx) mice served as controls for osteopenia. 6-Gingerol caused increase in trabecular osteoclast number, microarchitectural erosion at all trabecular sites and loss of vertebral stiffness, and these effects were comparable to capsaicin or OVx group. Osteoclast-specific serum and gene markers of 6-gingerol-treated mice were higher than the OVx group. Bone formation was unaffected by 6-gingerol. CONCLUSION Daily feeding of 6-gingerol to skeletally mature female mice caused trabecular osteopenia, and the mechanism appeared to be activation of osteoclast formation via the TRPV1 channel.

In-Vivo Efficacy of Compliant 3D Nano-Composite in Critical-Size Bone Defect Repair: a Six Month Preclinical Study in Rabbit

Bone defects above critical size do not heal completely by itself and thus represent major clinical challenge to reconstructive surgery. Numerous bone substitutes have already been used to promote bone regeneration, however their use, particularly for critical-sized bone defects along with their long term in vivo safety and efficacy remains a concern. The present study was designed to obtain a complete healing of critical-size defect made in the proximal tibia of New Zealand White rabbit, using nano-hydroxyapatite/gelatin and chemically carboxymethylated chitin (n-HA/gel/CMC) scaffold construct. The bone-implant interfaces and defect site healing was evaluated for a period up to 25 weeks using radiography, micro-computed tomography, fluorescence labeling, and histology and compared with respective SHAM (empty contra lateral control). The viscoelastic porous scaffold construct allows easy surgical insertion and post-operatively facilitate oxygenation and angiogenesis. Radiography of defect treated with scaffold construct suggested expedited healing at defect edges and within the defect site, unlike confined healing at edges of the SHAM sites. The architecture indices analyzed by micro-computed tomography showed a significant increase in percentage of bone volume fraction, resulted in reconciled cortico-trabecular bone formation at n-HA/gel/CMC constructs treated site (15.2% to 52.7%) when compared with respective SHAM (10.2% to 31.8%). Histological examination and fluorescence labeling revealed that the uniformly interconnected porous surface of scaffold construct enhanced osteoblasts’ activity and mineralization. These preclinical data suggest that, n-HA/gel/CMC construct exhibit stimulation of bones innate regenerative capacity, thus underscoring their use in guided bone regeneration.

Prunetin signals via G-protein-coupled receptor, GPR30(GPER1): Stimulation of adenylyl cyclase and cAMP-mediated activation of MAPK signaling induces Runx2 expression in osteoblasts to promote bone regeneration ☆ ☆☆

Prunetin is found in red clover and fruit of Prunus avium (red cherry). The effect of prunetin on osteoblast function, its mode of action and bone regeneration in vivo were investigated. Cultures of primary osteoblasts, osteoblastic cell line and HEK293T cells were used for various in vitro studies. Adult female rats received drill-hole injury at the femur diaphysis to assess the bone regenerative effect of prunetin. Prunetin at 10nM significantly (a) increased proliferation and differentiation of primary cultures of osteoblasts harvested from rats and (b) promoted formation of mineralized nodules by bone marrow stromal/osteoprogenitor cells. At this concentration, prunetin did not activate any of the two nuclear estrogen receptors (α and β). However, prunetin triggered signaling via a G-protein-coupled receptor, GPR30/GPER1, and enhanced cAMP levels in osteoblasts. G15, a selective GPR30 antagonist, abolished prunetin-induced increases in osteoblast proliferation, differentiation and intracellular cAMP. In osteoblasts, prunetin up-regulated runt-related transcription factor 2 (Runx2) protein through cAMP-dependent Erk/MAP kinase activation that ultimately resulted in the up-regulation of GPR30. Administration of prunetin at 0.25mg/kg given to rats stimulated bone regeneration at the site of drill hole and up-regulated Runx2 expression in the fractured callus and the effect was comparable to human parathyroid hormone, the only clinically used osteogenic therapy. We conclude that prunetin promotes osteoinduction in vivo and the mechanism is defined by signaling through GPR30 resulting in the up-regulation of the key osteogenic gene Runx2 that in turn up-regulates GPR30.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before.

Developmental Exposure to As, Cd, and Pb Mixture Diminishes Skeletal Growth and Causes Osteopenia at Maturity via Osteoblast and Chondrocyte Malfunctioning in Female Rats

We studied the effect of metal mixture (MM), comprising As, Cd, and Pb, in developing female rat skeleton from gestation day 5 until postnatal day 60 (P-60). MM resulted in synergistic inhibition in viability and differentiation of osteoblasts in vitro, likely induced by reactive oxygen species. MM, administered at their most frequently occurring concentrations present in the groundwater of India, i.e., As: 0.38 ppm, Pb: 0.22 ppm, and Cd: 0.098 ppm or 10× of the ratio to developing rats, exhibited a synergistic decrease in ex vivo mineralization of bone marrow stromal (osteoprogenitor) cells. MM group showed a dose-dependent attenuation in weight and axial lengths and shortening of tibias at P-60. Furthermore, the growth plate was shortened, which was associated with shorter proliferative and hypertrophic zones, decreased parathyroid hormone-related protein and Indian hedgehog expression in the chondrocytes, reduced primary and secondary spongiosa, and hypomineralized osteoids-a major characteristic of osteomalacia. In addition, compared with the control, MM-treated rats were clearly osteopenic based on bone mineral density, microarchitecture, biomechanical strength, and particularly the biochemical profile, that suggested high turnover bone loss. Finally, in comparison to the control, the fracture-healing ability of MM group was delayed and accompanied by inferior quality of the healed bone. Together, these data demonstrated that the mixture of As, Cd, and Pb induced synergistic toxicity to developing skeleton, thereby diminishing modeling-directed bone accrual, inducing osteopenia and dampening fracture healing.

Greater Skeletal Gains in Ovary Intact Rats at Maturity Are Achieved by Supplementing a Standardized Extract of Butea monosperma Stem Bark that Confers Better Bone Conserving Effect following Ovariectomy and Concurrent Treatment Withdrawal

With a longitudinally designed study, we tested whether an acetone soluble fraction (ASF) from the stem bark of Butea monosperma resulted in maximizing bone gain in rats during growth and maturation and thus protected against osteopenia following ovariectomy (OVx) with concomitant treatment withdrawal. Female rats at weaning were given ASF (100 mg/kg/d) or vehicle for 12 weeks, and baseline skeletal parameters (micro-CT) and total plasma antioxidant status (TAS) were measured. At this stage, one group was OVx and the other group was sham operated. Vehicle group (untreated) after OVx was given E2 or continued with vehicle (OVx control). ASF group after OVx was given vehicle (ASF withdrawn, ASFW). After another 12 weeks, all groups were killed and various skeletal parameters were determined. ASF resulted in substantially better skeletal parameters and higher plasma TAS over control at maturity. Rats treated with ASF before OVx had reduced rates of bone loss compared to OVx control. Twelve weeks after OVx, the ASFW group exhibited better trabecular microarchitectural preservation, bone turnover profiles, increased cortical deposition, and biomechanical strength over the OVx control, and the effects were comparable to OVx + E2 group. ASF supplementation during skeletal growth could maximize bone accrual and could confer increased resistance to post-OVx osteopenia despite treatment withdrawal.

Orally Active Osteoanabolic Agent GTDF Binds to Adiponectin Receptors, With a Preference for AdipoR1, Induces Adiponectin-Associated Signaling, and Improves Metabolic Health in a Rodent Model of Diabetes

Adiponectin is an adipocytokine that signals through plasma membrane–bound adiponectin receptors 1 and 2 (AdipoR1 and -2). Plasma adiponectin depletion is associated with type 2 diabetes, obesity, and cardiovascular diseases. Adiponectin therapy, however, is yet unavailable owing to its large size, complex multimerization, and functional differences of the multimers. We report discovery and characterization of 6-C-β-d-glucopyranosyl-(2S,3S)-(+)-5,7,3′,4′-tetrahydroxydihydroflavonol (GTDF) as an orally active adiponectin mimetic. GTDF interacted with both AdipoRs, with a preference for AdipoR1. It induced adiponectin-associated signaling and enhanced glucose uptake and fatty acid oxidation in vitro, which were augmented or abolished by AdipoR1 overexpression or silencing, respectively. GTDF improved metabolic health, characterized by elevated glucose clearance, β-cell survival, reduced steatohepatitis, browning of white adipose tissue, and improved lipid profile in an AdipoR1-expressing but not an AdipoR1-depleted strain of diabetic mice. The discovery of GTDF as an adiponectin mimetic provides a promising therapeutic tool for the treatment of metabolic diseases.

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.