9-Demethoxy-medicarpin: A potential bone health supplement for the management of protein deficiency-induced bone loss in growing rats.

Human skeleton requires an adequate supply of many different nutritional factors for optimal growth and development. The role of nutrition in bone growth has piqued interest in recent years, especially in relation to maximizing peak bone mass and reducing the risk of osteoporosis. Protein deficiency-induced bone loss was induced in female growing rats. All experimental rodent diets were prepared as per recommendations for growing animals. 9-Demethoxy-medicarpin (DMM) treatment was given to growing Sprague Dawley (SD) rats at 1 mg and 10 mg dose orally for 30 days. Bones were collected for bone mineral density (BMD). Bone marrow cells were isolated from femur for calcium nodule formation. Serum samples were collected for biochemical parameters. We found that DMM treatment speeds up the recovery of musculoskeletal weakness by replenishing nutrients in proven rodent model. DMM supplementation for four weeks showed significantly increased vertebral, femur and tibial BMD compared with the untreated PD group. Albumin levels were significantly enhanced in treatment groups, in which 10 mg dose imparted a better effect. We conclude that DMM treatment led to increased BMD and biochemical parameters in protein deficient condition in growing rats and has potential as a bone growth supplement.

Detection of differential expression of miRNAs in computerized tomography-guided lung biopsy.

Aims: Nonsmall-cell lung carcinoma comprises 85% of lung malignancies and is usually associated with a poor prognosis due to diagnosis at advanced stages. Molecular diagnosis of computerized tomography (CT)-guided biopsy has the potential to identify subtypes of lung carcinoma like adenocarcinoma (AC) and squamous cell carcinoma (SCC) along with its molecular stratification. This approach will help predict the genetic signature of lung cancer in individual patients. Subjects and Methods: Histopathologically proved a CT-guided biopsy sample of lung cancer cases was used to screen for the expression of microRNA (miRNA) earlier quantitated in blood plasma. Primers against hsa-miR2114, hsa-miR2115, hsa-miR2116, hsa-miR2117, hsa-miR449c, and hsa-miR548q with control RNU6 were used to screen 30 AC, 30 SCC, 5 nonspecific granulomatous inflammation, and 8 control samples. Reverse transcription polymerase chain reaction (RT-PCR) data revealed expression of hsa-miR2114 and hsa-miR548q in AC as well as SCC. Results: RT-PCR data revealed that the expression of hsa-miR2116 and hsa-miR449c was found upregulated in AC while hsa-miR2117 was expressed in SCC cases. Bioinformatic analysis revealed that genes, where these miRNAs are located, were also upregulated while targets of these miRNAs were downregulated. Conclusions: miRNAs expression pattern in the CT-guided biopsy samples can be used as a potential tool to differentially diagnose lung cancer subtypes. The expression pattern of miRNAs matches very well in blood plasma and tissue samples, albeit levels were very low in the earlier case than later. This approach can also be used for screening mutations and other molecular markers in a personalized manner for the management of lung cancer patients.

Mitochondrial Stress-Mediated Targeting of Quiescent Cancer Stem Cells in Oral Squamous Cell Carcinoma.

INTRODUCTION: Despite improved therapeutics in oral squamous cell carcinoma (OSCC), tumor cells that are either quiescent and/or endowed with stem cell-like attributes usually survive treatment and recreate tumor load at relapse. Through this study, we aimed strategically to eliminate these stem cell-like cancer cells using a combination drug approach. METHODS: Primary cultures from 15 well-moderately differentiated OSCC were established, and the existence of cancer cells with stem cell-like characteristics using five cancer stem cell (CSC) specific markers - CD44, CD133, CD147, C166, SOX2 and spheroid assay was ascertained. Next, we assessed quiescence in CSCs under normal and growth factor-deprived conditions using Ki67. Among several gene signatures regulating quiescent cellular state, we evaluated the effect of inhibiting Dyrk1b in combination with topoisomerase II and histone deacetylase inhibitors in targeting quiescent CSCs. Multiple drug-effect analysis was carried out with CompuSyn software to determine combination-index values. RESULTS: We observed that CD44+CD133+ showed the highest level of SOX2 expression. CSCs showed varying degrees of quiescence, and inhibition of Dyrk1b decreased quiescence and sensitized CSCs to apoptosis. In the drug-combination study, Dyrk1b inhibitor was combined with topoisomerase II and histone deacetylase inhibitors to target quiescent CSCs. In combination, a synergistic effect was seen even at a 16-fold lower dose than IC50. Furthermore, combined treatment decreased glutathione levels and increased ROS and mitochondrial stress, leading to increased DNA damage and cytochrome c in CSCs. CONCLUSION: We report marker-based identification of CSC subpopulations and synergy of Dyrk1b inhibitor with topoisomerase II and HDAC inhibitors in primary OSCC. The results provide a new therapeutic strategy to minimize quiescence and target oral CSCs simultaneously.

A quantitative method to determine osteogenic differentiation aptness of scaffold.

Osteogenic differentiation of Mesenchymal stem cells (MSCs) on scaffold is crucial for bone tissue engineering. Alkaline phosphatase (ALP) assay is an important method of assessing osteogenesis. Here, a very simple and innovative procedure is being described for quantification of osteogenic differentiation of MSCs in presence of scaffold using ALP assay. Different concentrations of the scaffold particles with the same number of MSCs were assayed for alkaline phosphatase activity using p-NPP as substrate for ALP activity. G-bone scaffold was used in concentrations of 5, 20, 60 and 100 mg/ml and same number of MSCs were seeded. Any scaffold which can be grind and weighed may be used. It was found that100 mg/ml G-bone graft was most useful for promoting osteogenesis and addition of growth factors further promoted. So, we were able to ascertain the concentration of scaffold which promotes osteogenesis the most.

Differential diagnosis of non-small cell lung carcinoma by circulating microRNA.

INTRODUCTION: More than 70% of lung cancer comprises nonsmall-cell lung carcinoma and is associated with poor survival outcome owing to late diagnosis. Identification of lung cancer in early stages when no clinical signs or symptoms are evident, can drastically improve the prognosis. To this end, we aimed to evaluate the changes occurring at tissue level by assessing the expression of six microRNAs (miRNAs) in lung adenocarcinoma (AC) and squamous cell carcinoma (SCC). MATERIALS AND METHODS: Peripheral blood of histopathologically proven cases of lung AC and SCC was collected and processed for the isolation of miRNAs using commercially available kit. Primers against mir-2114, mir-2115, mir-2116, mir-2117, mir-449c, and mir-548q with loading control Caenorhabditis elegans were used. Screening was carried out in thirty cases of both AC and SCC, whereas twenty healthy controls were included. RESULTS: Real-time polymerase chain reaction data revealed that the expression of mir-2114 and mir-449c in AC and mir-2115 in SCC was significantly upregulated. The expression of these miRNAs was also confirmed in lung AC cell line. The differential pattern of expression of these miRNAs can be used for precise diagnosis of lung carcinoma. CONCLUSIONS: We have used a noninvasive technique to identify the subtype of lung cancer based on molecular genetic signatures. The results suggest that through molecular profiling of miRNA, we can screen high-risk cases for cancer interception.

Hydroxyapatite-collagen augments osteogenic differentiation of dental pulp stem cells.

The objectives of this study were to isolate and culture dental pulp stem cells (DPSCs) and to investigate their proliferation and osteogenic differentiation on hydroxyapatite-collagen (HA-Col) scaffold. DPSCs were characterized by fluorescence-activated cell sorting (FACS). Cultured cells were CD73+, CD90+, CD105+ and CD31-, CD45-. A commercially available HA-Col scaffold was used for culture of DPSCs. Cell attachment and viability of DPSCs cultured on scaffold was studied by sulforhodamine assay. Osteoblast differentiation capacity was studied by alkaline phosphatase assay and the effects of growth factors such as PDGF, IGF1 and FGF2 were further studied. Scanning electron microscopy (SEM) of cell seeded scaffolds was also performed. We found that DPSCs cultured exhibited the characteristic mesenchymal stem cells (MSCs) morphology and differentiation properties. Scaffold was found to be non-cytotoxic and had good biocompatibility in vitro. Osteoblast differentiation ability was found to increase at higher concentration of scaffold and additive effects were observed with the use of growth factors. In SEM, cells appeared to cover the entire surface of the scaffold forming continuous cell layer and extending filopodial extensions. HA-Col scaffold is apt for MSCs attachment and proliferation in vitro. Their unique self-renewal and multilineage differential potential make them ideal for use in regenerative medicine. The limitations of currently available bone graft materials have led to the emergence of tissue engineering using mesenchymal stem cells (MSCs). Since, HA-Col scaffold potentiated the proliferation and osteogenic differentiation of DPSCs, this biomimetic material may be an ideal one for maxillofacial and alveolar bone regeneration.

An Ectosteric Inhibitor of Cathepsin K Inhibits Bone Resorption in Ovariectomized Mice.

The potent cathepsin K (CatK) inhibitor, Tanshinone IIA sulfonic sodium (T06), was tested for its in vitro and in vivo antiresorptive activities. T06 binds in an ectosteric site of CatK remote from its active site and selectively inhibits collagen degradation with an IC50 value of 2.7 ± 0.2 µM (CatK:T06 molar ratio of 1:5). However, it does not suppress fluorogenic peptide cleavage and gelatinolysis at a 2500-fold molar excess. Contrary to active site-directed CatK inhibitors, such as odanacatib, T06 suppresses bone resorption in both human and mouse osteoclasts equally well (IC50 value for human and mouse osteoclasts: 237 ± 60 nM and 245 ± 55 nM, respectively) and its antiresorptive activity is fully reversible in both cell types. Moreover, T06 affects neither the metabolic activity of osteoclasts nor osteoclastogenesis. In in vivo studies, 40 mg T06/kg/d given to 12-week-old ovariectomized (OVX) mice for 3 months reduced plasma CTx-1 by 20% and increased osteoblast numbers and plasma P1NP by ∼28% when compared with the OVX control. µCT analysis of T06-treated OVX mice showed a 35% increase in bone mineral density and other femoral trabecular bone parameters when compared with OVX animals. T06 did not alter the number of osteoclasts, had no estrogenic effect on the uterus, did not change plasma estradiol levels, and did not inhibit fibroblast-mediated TGF-ß1 processing or degradation and cognitive functions in OVX mice. This study indicates that the ectosteric inhibitor, T06, is a selective antiresorptive CatK inhibitor that may overcome the shortcomings of side effect-prone active site-directed drugs, which all failed in clinical trials. © 2017 American Society for Bone and Mineral Research.

3-Piperidylethoxypterocarpan: A potential bone anabolic agent that improves bone quality and restores trabecular micro-architecture in ovariectomized osteopenic rats.

A series of new 6H-benzofuro[3, 2-c]chromenes (BFC, pterocarpans) with structure-activity relationships were investigated for their potential use in osteoporosis treatment. One of the BFCs 3-piperidylethoxypterocarpan 20 promotes osteoblast differentiation and mineralization at a dose as low as 1 pM via activation of ER/P38MAPK/BMP-2 pathway. When evaluated for in-vivo osteogenic activity in female Sprague-Dawley rats, BFC 20 increased bone mineral density and new bone formation, compared with control at 1.0 and 10.0 mg/kg/body weight by oral gavage for 30 days. The compound was devoid of any uterotrophic effect and led to the new bone formation in adult ovariectomized osteopenic rats. BFC 20 compound also inhibited bone resorption by reducing Ovx induced increase in urinary CTx, thus exhibiting both bone anabolic and anti-catabolic action. Finally, BFC 20 treatment to Ovx rats led to improved trabecular microarchitectural restoration and exhibited therapeutic potential as a dual acting anti-osteoporotic agent for the management of osteoporosis.

IL-18BP is decreased in osteoporotic women: Prevents Inflammasome mediated IL-18 activation and reduces Th17 differentiation.

IL-18BP is a natural antagonist of pro-inflammatory IL-18 cytokine linked to autoimmune disorders like rheumatoid arthritis. However, its role in post menopausal osteoporosis is still unknown. In this study, we investigated the role of IL-18BP on murine osteoblasts, its effect on osteoblasts-CD4+ T cells and osteoblasts-CD11b+ macrophage co-culture. mIL-18BPd enhances osteoblast differentiation and inhibits the activation of NLRP3 inflammasome and caspase-1 which process IL-18 to its active form. Using estrogen deficient mice, we also determined the effect of mIL-18BP on various immune and skeletal parameters. Ovariectomized mice treated with mIL-18BPd exhibited decrease in Th17/Treg ratio and pro-inflammatory cytokines. mIL-18BPd treatment restored trabecular microarchitecture, preserved cortical bone parameters likely attributed to an increased number of bone lining cells and reduced osteoclastogenesis. Importantly, these results were corroborated in female osteoporotic subjects where decreased serum IL-18BP levels and enhanced serum IL-18 levels were observed. Our study forms a strong basis for using humanized IL-18BP towards the treatment of postmenopausal osteoporosis.

Methoxyisoflavones formononetin and isoformononetin inhibit the differentiation of Th17 cells and B-cell lymphopoesis to promote osteogenesis in estrogen-deficient bone loss conditions.

OBJECTIVE: Recent studies have shown that immune system plays a major role in pathophysiology of postmenopausal osteoporosis. Previously we have shown that phytoestrogens like daidzein and medicarpin exhibit immunoprotective effects, by virtue of which they alleviate bone loss. With this background, methoxyisoflavones like formononetin (formo) and isoformononetin (isoformo) that have been studied for preventing bone loss in ovariectomized rats were tested for their immunomodulatory effects in estrogen-deficient bone loss mice model. METHODS: Adult Balb/c mice (N = 8/group) were given oral dose of formo and isoformo at 10 mg/kg body weight, post ovariectomy (Ovx) daily for 6 weeks. Animals were autopsied and long bones were harvested to study bone microarchitecture. Peripheral blood mononuclear cells were isolated for fluorescence-activated cell sorting and RNA analysis. Serum was collected for enzyme-linked immunosorbent assay. RESULTS: It was observed that formo and isoformo treatment to Ovx mice led to significant restoration of Ovx-induced deterioration of trabecular microarchitecture. Pro-osteoclastogenic subset Th17 and B cells were decreased in formo/isoformo-treated Ovx mice in comparison with vehicle-treated Ovx group. Formo and isoformo treatment to Ovx mice also led to decreased expression of Th17 diffentiation factors and promoted T-regulatory cell differentiation. Formo was more effective in enhancing the FOXP3 expression compared with isoformo. IL-17A-induced osteoclastogenesis and inhibition of osteoblast apoptosis were also suppressed by formo and isoformo treatment, with formo having a more potent effect. CONCLUSIONS: Our study demonstrates the immunomodulatory activity of methoxyisoflavones, formo, and isoformo, which translate into improved skeletal parameters, thereby preventing Ovx-induced bone loss.

9-Demethoxy-medicarpin promotes peak bone mass achievement and has bone conserving effect in ovariectomized mice: Positively regulates osteoblast functions and suppresses osteoclastogenesis.

We report a new bone anabolic and anti-catabolic pterocarpan 9-demethoxy-medicarpin (DMM) for the management of postmenopausal osteoporosis. DMM promoted osteoblast functions via activation of P38MAPK/BMP-2 pathway and suppressed osteoclastogenesis in bone marrow cells (BMCs). In calvarial osteoblasts, DMM blocked nuclear factor kappaB (NFκB) signaling and inhibited the mRNA levels of pro-inflammatory cytokines. DMM treatment led to increased OPG (osteoprotegrin) and decreased transcript levels of TRAP (tartarate resistant acid phosphatase), RANK (receptor activator of NFκB) and RANKL (RANK ligand) in osteoblast-osteoclast co-cultures. Immature female SD rats administered with DMM exhibited increased bone mineral density, bone biomechanical strength, new bone formation and cortical bone parameters. Ovx mice administered with DMM led to significant restoration of trabecular microarchitecture and had reduced formation of osteoclasts and increased formation of osteoprogenitor cells in BMCs. DMM exhibited no uterine estrogenicity. Overall, these results demonstrate the therapeutic potential of DMM for the management of postmenopausal osteoporosis.

Enhanced immunoprotective effects by anti-IL-17 antibody translates to improved skeletal parameters under estrogen deficiency compared with anti-RANKL and anti-TNF-α antibodies.

Activated T cell has a key role in the interaction between bone and immune system. T cells produce proinflammatory cytokines, including receptor activator of NF-κB ligand (RANKL), tumor necrosis factor α (TNF-α), and interleukin 17 (IL-17), all of which augment osteoclastogenesis. RANKL and TNF-α are targeted by inhibitors such as denosumab, a human monoclonal RANKL antibody, and infliximab, which neutralizes TNF-α. IL-17 is also an important mediator of bone loss, and an antibody against IL-17 is undergoing phase II clinical trial for rheumatoid arthritis. Although there are a few studies showing suppression of Th17 cell differentiation and induction of regulatory T cells (Tregs) by infliximab, the effect of denosumab remains poorly understood. In this study, we investigated the effects of anti-TNF-α, anti-RANKL, or anti-IL-17 antibody administration to estrogen-deficient mice on CD4(+) T-cell proliferation, CD28 loss, Th17/Treg balance and B lymphopoesis, and finally, the translation of these immunomodulatory effects on skeletal parameters. Adult Balb/c mice were treated with anti-RANKL/-TNF-α/-IL-17 subcutaneously, twice a week, postovariectomy (Ovx) for 4 weeks. Animals were then autopsied; bone marrow cells were collected for FACS and RNA analysis and serum collected for ELISA. Bones were dissected for static and dynamic histomorphometry studies. We observed that although anti-RANKL and anti-TNF-α therapies had no effect on Ovx-induced CD4(+) T-cell proliferation and B lymphopoesis, anti-IL-17 effectively suppressed both events with concomitant reversal of CD28 loss. Anti-IL-17 antibody reduced proinflammatory cytokine production and induced Tregs. All three antibodies restored trabecular microarchitecture with comparable efficacy; however, cortical bone parameters, bone biomechanical properties, and histomorphometry were best preserved by anti-IL-17 antibody, likely attributable to its inhibitory effect on osteoblast apoptosis and increased number of bone lining cells and Wnt10b expression. Based on the superior immunoprotective effects of anti-IL-17, which appears to translate to a better skeletal preservation, we propose beginning clinical trials using a humanized antibody against IL-17 for treatment of postmenopausal osteoporosis.

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.

Estrogen deficiency induces the differentiation of IL-17 secreting Th17 cells: a new candidate in the pathogenesis of osteoporosis.

Th17 cells produce IL-17, and the latter promotes bone loss in collagen-induced arthritis in mice. Blocking IL-17 action in mouse model of rheumatoid arthritis reduces disease symptoms. These observations suggest that Th17 cells may be involved in the pathogenesis of bone loss. However, the role of Th17 cell in estrogen (E2) deficiency-induced bone loss is still not very clear. We investigated the effect of E2 on Th17 differentiation in vivo and IL-17 mediated regulation of osteoclast and osteoblast differentiation. Additionally, effect of IL-17 functional block under E2 deficiency-induced bone loss was studied. In murine bone marrow cells, E2 suppressed IL-17 mediated osteoclast differentiation. IL-17 inhibited formation of mineralized nodules in osteoblasts and this effect was suppressed by E2. E2 treatment to mouse calvarial osteoblasts inhibited the IL-17-induced production of osteoclastogenic cytokines and NF-kB translocation. In ovariectomized mice, there was increase in the number of Th17 cells, transcription factors promoting Th17 cell differentiation and circulating IL-17 levels. These effects were reversed by E2 supplementation. Treatment of neutralizing IL-17 monoclonal antibody to Ovx mice mitigated the E2 deficiency-induced trabecular bone loss and reversed the decreased osteoprotegerin-to-receptor activator of nuclear factor kappa B ligand (RANKL) transcript levels in long bones, increased osteoclast differentiation from the bone marrow precursor cells and decreased osteoblast differentiation from the bone marrow stromal cells. Our findings indicate that E2 deficiency leads to increased differentiation of Th17 cells with attendant up regulation of STAT3, ROR-γt and ROR-α and downregulation of Foxp3 which antagonizes Th17 cell differentiation. Increased IL-17 production in turn induces bone loss by increasing pro-osteoclastogenic cytokines including TNF-α, IL-6 and RANKL from osteoblasts and functional block of IL-17 prevents bone loss. IL-17 thus plays a critical causal role in Ovx-induced bone loss and may be considered a potential therapeutic target in pathogenesis of post menopausal osteoporosis.

Formononetin reverses established osteopenia in adult ovariectomized rats.

OBJECTIVE: Formononetin (Formo) prevents ovariectomy (Ovx)-induced bone loss in rats. However, there are no reports on the curative effects of Formo. The objective of this study was to investigate the ability of Formo in restoring trabecular microarchitecture and promoting new bone formation in osteopenic rats. METHODS: Adult Sprague-Dawley rats were ovariectomized and left for 90 days for osteopenia to develop. After 90 days, Formo (10.0 mg kg d) was given orally for the next 12 weeks to Ovx rats in a therapeutic protocol. Sham-operated, Ovx + vehicle, and Ovx + parathyroid hormone (PTH) groups served as controls. Trabecular microarchitecture, osteoid formation, bone turnover/resorption markers, and bone osteoprotegerin-to-receptor activator for nuclear κB ligand ratio were studied. One-way analysis of variance was used to test significance of effects. RESULTS: Formo treatment significantly restored the lost trabecular microarchitecture in the femurs and tibia of osteopenic Ovx rats and promoted new bone formation. Formo was devoid of any uterine estrogenicity. Serum levels of type I collagen N-terminal propeptide, which is a reliable marker of bone formation, were increased in Ovx rats treated with Formo compared with Ovx + vehicle group, and the levels were comparable with those in the sham group. Formo prevented the Ovx-induced increase in bone turnover markers, including serum osteocalcin and urinary type I collagen degradation product. Furthermore, Formo-treated Ovx rats had an increased bone osteoprotegerin-to-receptor activator for nuclear κB ligand ratio compared with the Ovx + vehicle group. CONCLUSIONS: Daily oral administration of Formo for 12 weeks has a substantial anabolic effect, thus raising the possibility of its use in postmenopausal osteoporosis.

Daidzein prevents the increase in CD4+CD28null T cells and B lymphopoesis in ovariectomized mice: a key mechanism for anti-osteoclastogenic effect.

Estrogen deficiency leads to an upregulation of TNF-α producing T cells and B-lymphopoesis which augments osteoclastogenesis. Estrogen deficiency also increases the population of premature senescent CD4⁺ CD28null T cells which secrete a higher amount of TNF-α thus leading to enhanced osteoclastogenesis. Isoflavonoids like daidzein and genistein are found mostly in soybeans, legumes, and peas. These share structural similarity with 17ß-stradiol (E2) and have osteoprotective role. This study explores the effect of daidzein (Daid) on the proliferation of TNF-α producing T cells, premature senescent T cells and B cell lymphopoesis under estrogen deficient conditions. For this study adult Balb/c mice were treated with Daid at 10 mg/kg body weight dose by oral gavage daily post ovariectomy (Ovx). After six weeks animals were autopsied and bone marrow and spleen cells were collected for FACS analysis. Blood serum was collected for ELISA. It was observed that Ovx mice treated with Daid for six weeks show reduction in Ovx induced expansion of CD4⁺ T cells in bone marrow and spleen when analysed by flow cytometry. Estrogen deficiency led to increased prevalence of TNF-α secreting CD4⁺CD28null T cells, however, treatment with Daid increased the percentage of CD4⁺CD28⁺ T cells. Co-culture of CD4⁺CD28null T cells and bone marrow resulted in enhanced osteoclastogenesis as evident by increased tartarate resistant acid phosphatase (TRAP) expression, an osteoclast marker. However, treatment with Daid resulted in reduced osteoclastogenesis in CD4⁺CD28null T cells and bone marrow cell co-culture. Daid also regulated B lymphopoesis and decreased mRNA levels of RANKL in B220⁺ cells. Taken together, we propose that one of the mechanisms by which Daid prevents bone loss is by reversing the detrimental immune changes as a result of estrogen deficiency.

Synthetic analogs of daidzein, having more potent osteoblast stimulating effect.

A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and 3c were several folds more potent than daidzein in stimulating differentiation and mineralization of osteoblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the mineralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidzein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein analogs at significantly lower concentrations than daidzein.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats.

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.

Neo-tanshinlactone inspired synthesis, in vitro evaluation of novel substituted benzocoumarin derivatives as potent anti-breast cancer agents.

A small library of novel benzocoumarin derivatives based on naturally occurring neo-tanshinlactone scaffold was constructed and their antiproliferative activities against breast cancer cells MCF-7 and MDA-MB-231 were evaluated. A number of derivatives showed good anti-breast cancer activity, in some cases higher to that of the reference compound tamoxifen. In particular, benzocoumarins Bc-5, Bc-8 and Bc-9 strongly inhibited the proliferation of MCF-7 cancer cell line with the IC(50) values of 3.8, 7.9 and 6.5 µM, respectively. The compounds were capable of inducing nuclear fragmentation, cell cycle arrest and caspase dependent apoptosis in MCF-7 cell lines. In addition, these derivatives were devoid of cytotoxic effect against normal osteoblast cells. These synthetic benzocoumarins hold promises for developing safer alternative to the existing anti-breast cancer agents.