Human MG-63 Research Articles

Hyperthermia effect and antibacterial efficacy of Fe3+ /Co2+ co-substitutions in β-Ca3 (PO4 )2 for bone cancer and defect therapy.

The combined effect of cobalt and iron substitutions in β-Ca3 (PO4 )2 as a potent material for application in hyperthermia and as a bone substitute is presented. Structural analysis reveals the preferential accommodation of Co2+ and Fe3+ at the Ca2+ (5) sites of β-Ca3 (PO4 )2 until the limit of ∼10 mol % and, thereafter, prefer Ca2+ (4) lattice sites. Occupancy of both the Co2+ and Fe3+ ions induces a significant contraction of the β-Ca3 (PO4 )2 unit cell. The Co2+ /Fe3+ co-substitutions in β-Ca3 (PO4 )2 display magnetic characteristics that enhances hyperthermia effect. In addition, the presence of Co2+ in β-Ca3 (PO4 )2 enunciates pronounced antibacterial efficacy against tested microorganisms. Nevertheless, the enhanced level of Co2+ in β-Ca3 (PO4 )2 results to induce significant toxicity. The biocompatibility of the synthesized thermoseeds is verified from the hemolytic tests and cytotoxicity test performed on human sarcoma cell line MG-63. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1317-1328, 2018.

Cytotoxic withanolides from Physalis angulata

A new withanolide (1), physagulide P, together with five known withanolides (2–6), was isolated from the aerial parts of Physalis angulata L. The structure of new compound was elucidated on the basis of extensive spectroscopic techniques, including 1D, 2D NMR and HRESIMS. The activity screening indicated that compound 1 showed significant cytotoxicities against the human osteosarcoma cell line MG-63, HepG-2 hepatoma cells and breast cancer cells MDA-MB-231 with the IC50 value of 3.50, 4.22 and 15.74 μM.

Synthesis and Characterization of β-Tricalcium Phosphate Derived From Haliotis sp. Shells.

To develop a methodology for the synthesis of β-tricalcium phosphate (β-TCP, Ca3(PO4)2) from the shell of Haliotis sp. (abalone shell) and to verify its characterization and biocompatibility. Calcium oxide (CaO) was synthesized from abalone shell by sintering and was suspended in distilled water to prepare calcium hydroxide (Ca(OH)2). For the synthesis of calcium carbonate (CaCO3), carbon dioxide was used to infuse Ca(OH)2 at pH 7.4. CaCO3 was reacted with phosphoric acid at pH 6.0 to obtain dicalcium phosphate (CaHPO4). Subsequently, β-TCP was synthesized by a chemical reaction between CaHPO4 and CaO at 950°C to 1100°C for 3 hours. Fourier transform infrared spectroscopy (FT-IR) and x-ray diffraction (XRD) was performed to verify the physiochemical characteristics of the composite synthesized from abalone shell. FT-IR and XRD results showed that β-TCP was successfully synthesized from abalone shell. The synthesized β-TCP did not affect cell viability of either normal human oral keratinocytes or osteoblastic MG-63 cells. These data indicate that β-TCP synthesized from abalone shell is biologically safe. β-TCP (Ca3(PO4)2) synthesized from abalone shell can be used as a potential source of bone grafting material.

Effect of silver contents in cellulose nanocrystal/silver nanohybrids on PHBV crystallization and property improvements

Ternary nanocomposites including cellulose nanocrystals/silver nanohybrids (CNC-Ag) and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were prepared by using solution casting. The effect of different AgNPs contents in the nanohybrids on crystallization ability and properties of PHBV was comprehensively investigated. Compared to binary PHBV/CNC nanocomposite, the ternary nanocomposites with high AgNPs contents showed larger improvements in the thermal stability, mechanical, barrier, overall migration and antibacterial properties. The property transitions at various AgNPs contents were due to transition of the nanohybrids induced PHBV crystallization ability (from single CNC nucleation to synergistic nucleation of CNC and AgNPs), and increased crystallinity without compromising the amounts of hydrogen bonds. Especially, the PHBV/CNC-Ag-1.7 showed the good barrier property and lower migration levels in food simulants, high antibacterial ratio of 99.9% and biocompatibility to human MG-63 cells, suggesting its potential application in food packaging related materials.

Different Expression and Localization of Phosphoinositide Specific Phospholipases C in Human Osteoblasts, Osteosarcoma Cell Lines, Ewing Sarcoma and Synovial Sarcoma

Background: Bone hardness and strength depends on mineralization, which involves a complex process in which calcium phosphate, produced by bone-forming cells, was shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the phosphoinositide (PI) pathway and related phospholipase C (PLC) enzymes. Objectives: Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS. Methods: We analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in samples of patients affected with Ewing sarcoma (ES) and synovial sarcoma (SS). Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC δ4 and PLC η subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC ε and PLC η isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines might represent an inappropriate experimental model for studies about the analysis of signal transduction in osteoblasts

Enhancing bioactive properties of silk fibroin with diatom particles for bone tissue engineering applications.

Many studies have highlighted the role of silicon in human bone formation and maintenance. Silicon, in fact, is considered to nucleate the precipitation of hydroxyapatite and to reduce the bone resorption. For this reason, we have combined silk fibroin (SF) with silicon-releasing diatom particles (DPs), as potential material for bone tissue engineering applications. Sponges of fibroin loaded with different amounts and sizes of DPs were prepared by solvent casting-particulate leaching method, and their morphology, porosity and mechanical properties were evaluated. The biological effect of diatom addition was assessed on human osteosarcoma cell line MG63, a suitable osteoblast-like model, through cell adhesion, metabolic activity and proliferation assays. In addition, alkaline phosphatase activity, osterix and collagen type I production in MG63 cell line were assessed as markers of early bone formation to demonstrate a pro-mineralization potential of scaffolds. Results of the studies showed that addition to fibroin of diatoms particles improved the osteogenic properties of osteoblast-like cells compared with the pure SF. Copyright © 2016 John Wiley & Sons, Ltd.

MicroRNA-379 inhibits the proliferation, migration and invasion of human osteosarcoma cells by targetting EIF4G2

Osteosarcoma (OS) is an aggressive malignant mesenchymal neoplasm amongst adolescents. The aim of the present study was to explore the various modes of action that miR-379 has on the proliferation, migration, and invasion of human OS cells. miR-379 achieves this by targetting eukaryotic initiation factor 4GII (EIF4G2). Human OS cell lines U2OS and MG-63 were selected and assigned into blank, miR-379 mimics, miR-379 mimic negative control (NC), miR-379 inhibitors, miR-379 inhibitor NC, EIF4G2 shRNA, control shRNA, and miR-379 inhibitor + EIF4G2 shRNA group. The miR-379 expression and EIF4G2 mRNA expression were detected utilising quantitative real-time PCR (qRT-PCR) and the EIF4G2 protein expression using Western blotting. MTT assay, scratch test, Transwell assay, and flow cytometry were performed to determine the proliferation, migration, invasion, and cell cycle, respectively. In comparison with the miR-379 mimic NC group, the miR-379 mimics group had decreased EIF4G2 expression; the miR-379 inhibitors group indicated an increased EIF4G2 expression. Compared with the control shRNA group, the EIF4G2 expression was lower in the EIF4G2 shRNA group and the miR-379 expression was dropped in the miR-379 inhibitor + EIF4G2 shRNA group. The proliferation, migration, and invasion abilities of OS cells were reduced in the miR-379 mimics and EIF4G2 shRNA groups. The percentage of OS cells at the G0/G1 stage was increased, and the percentage at the S-stage was decreased in the miR-379 mimics and EIF4G2 shRNA groups. miR-379 may inhibit the proliferation, migration and invasion of OS cells through the down-regulation of EIF4G2.

Correlation study of DNA methylation of WNT6 gene with osteosarcoma in children.

The aim of this study was to evaluate the status and explore the impact of DNA methylation of WNT6 gene with osteosarcoma in children. A total of 50 patients with primary osteosarcoma in children were enrolled. The expression of WNT6 protein was determined by immunohistochemical staining. The DNA methylation level of WNT6 gene was evaluated by methylation-specific PCR (MSP). Human MG63 osteosarcoma cells and human normal Hfob1.19 osteoblasts were selected and cultured. Western blot analysis was utilized to measure the expression of WNT6 in the two cell lines. MSP was used to assess the status of DNA methylation of WNT6 gene. The correlation between DNA methylation of WNT6 gene and prognosis of osteosarcoma in children was evaluated by statistical analysis of the clinical and prognostic data. The results of the immunohistochemical assay showed that 84% (42/50) of primary osteosarcoma was WNT6 positive. MSP indicated that DNA methylation of WNT6 gene was found in 9 cases (18%) of primary osteosarcoma. Western blot analysis showed that WNT6 protein expression in MG63 cells was significantly higher than that in normal human Hfob1.19 osteoblasts, whereas, the level of DNA methylation of WNT6 gene in MG63 cells was significantly lower than in Hfob1.19 cells. Analysis of survival, prognosis and their correlation found that the DNA methylation level of WNT6 gene was negatively correlated with the prognosis of children with osteosarcoma. In conclusion, there was a high level of WNT6 gene expression in primary osteosarcoma, which was mainly due to low DNA methylation level of WNT6 gene. The DNA methylation of WNT6 gene was negatively correlated with the prognosis of patients with osteosarcoma in children.

Daidzein stimulates osteogenesis facilitating proliferation, differentiation, and antiapoptosis in human osteoblast-like MG-63 cells via estrogen receptor–dependent MEK/ERK and PI3K/Akt activation

Daidzein, a natural soy isoflavone, has a structure similar to estradiol and exhibiting bone-sparing effects against osteoporosis. However, the molecular mechanisms of osteogenesis remain unclear. We hypothesized that daidzein stimulates osteogenesis through estrogen receptor (ER)–dependent signal pathways. To test this hypothesis, we investigated the effects of daidzein compared with 17β-estradiol on proliferation, differentiation, and cisplatin-induced apoptosis in human osteoblast-like MG-63 cells containing 2 ER isoforms. The results showed that daidzein stimulated cell proliferation by altering cell cycle distribution, promoted cell differentiation by increasing the alkaline phosphatase activity and collagen content, and reduced cell apoptosis associated by up-regulating the expression of Bcl-xL. The above actions of daidzein were prevented by cotreatment with the ER antagonist ICI 182780. Using small interfering RNA technology, we further demonstrated that the effects of daidzein on alkaline phosphatase activity, collagen content, and cell apoptosis are mediated by both ERα and ERβ, whereas the effects on cell proliferation are primarily mediated by ERα. However, the effects of 17β-estradiol on osteoblastic proliferation and survival are mediated by both ER isotypes, and the effects on osteoblastic differentiation are primarily mediated by ERα. The use of specific inhibitors indicated that activation of the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) and phosphoinositide 3-kinase/protein kinase B or PKB (PI3K/Akt) signaling pathway at least partially accounts for these effects of daidzein. Taken together, the results indicate that daidzein stimulates osteogenesis through facilitating proliferation, differentiation, and antiapoptosis in human osteoblast-like MG-63 cells via activation of MEK/ERK and PI3K/Akt in an ER-dependent manner.

Role of Hedgehog-Gli1 signaling in the enhanced proliferation and differentiation of MG63 cells enabled by hierarchical micro-/nanotextured topography.

Hedgehog–Gli1 signaling is evolutionarily conserved and plays an essential role in osteoblast proliferation and differentiation as well as bone formation. To evaluate the role of the Hedgehog–Gli1 pathway in the response of osteoblasts to hierarchical biomaterial topographies, human MG63 osteoblasts were seeded onto smooth, microstructured, and micro-/nanotextured topography (MNT) titanium to assess osteoblast proliferation and differentiation in terms of proliferative activity, alkaline phosphatase (ALP) production, and osteogenesis-related gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of Sonic hedgehog (Shh), Smoothened (Smo), and Gli1, and the protein levels were assayed by Western blotting. MG63 cells treated with the Smo inhibitor cyclopamine were seeded onto the titanium specimens, and the cell proliferation and differentiation were studied in the presence or absence of cyclopamine. Our results showed that compared to the smooth and microstructured surfaces, the MNTs induced a higher gene expression and protein production of Shh, Smo, and Gli1 as well as the activation of Hedgehog signaling. The enhanced proliferative activity, ALP production, and expression of the osteogenesis-related genes (bone morphogenetic protein-2, ALP, and runt-related transcription factor 2) enabled by the MNTs were significantly downregulated by the presence of cyclopamine to a similar level as those on the smooth and acid-etched microstructured surfaces in the absence of cyclopamine. This evidence explicitly demonstrates pivotal roles of Hedgehog–Gli1 signaling pathway in mediating the enhanced effect of MNTs on MG63 proliferation and differentiation, which greatly advances our understanding of the mechanism involved in the biological responsiveness of biomaterial topographies. These findings may aid in the optimization of hierarchical biomaterial topographies targeting Hedgehog–Gli1 signaling.

Design, characterization and in vitro evaluation of novel amphiphilic block sunflower oil-based polyol nanocarrier as a potential delivery system: Raloxifene-hydrochloride as a model

Presently, modern pharmaceuticals, are almost exclusively derived from the arduous refining of petroleum whose supply is inherently unsustainable. In order to address this issue bio-based materials are increasingly being used for chemical synthesis, particularly in drug delivery systems. Biodegradable and biocompatible hyper-branched polyol (an alcohol containing three or more hydroxyl groups) was synthesized via a facile method through the ring-opening and thiol-ene click reactions at room temperature. Due to the bio-based content of the polyol backbone, the synthesized polyol had both excellent biodegradability and low cytotoxicity. Raloxifene hydrochloride, an oral selective estrogen receptor modulator, was used as a hydrophobic drug model to test the potential of polyol as a drug delivery system carrier. Polyol showed an amphiphilic character and could be prepared as a nanoparticle for the sustained delivery of raloxifene hydrochloride, a drug with poor bioavailability in aqueous solution. Raloxifene hydrochloride was readily encapsulated in the lipophilic core of polyol whose branched hydroxyls were on the external part of the prepared nanoparticles. The diameter of the nanoparticles was 94±0.43nm, their drug entrapment efficiency was 93±0.5% and they showed a sustained release profile (17±1.5% after 4weeks). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay showed low toxicity towards human osteoblast MG-63 cells. Based on its good biodegradability and low cytotoxicity, polyol provides a bio-based source for the design new drug delivery systems.

Copper(II) Complexes with Saccharinate and Glutamine as Antitumor Agents: Cytoand Genotoxicity in Human Osteosarcoma Cells.

Copper has shown to be useful in disorders with an inflammation origin such as cancer [1-3]. It has previously shown that Casiopeínas® interact with DNA and promote the disruption by a mechanism related to the increase in the level of free radicals [4-6] which confers antineoplastic potential. Objetive: The aim of the present work was to study the antitumor effects of a series of Cu(II) complexes with saccharinate (sac) and glutamate (gln): [Cu(sac)2(H2O)4].2H2O (Cu-sac), [Cu(gln)2] (Cu-gln) and Na2[Cu(sac)2 (gln)2].H2O (Cu-sac-gln). We have investigated the action of these compounds on cell viability on human osteosarcoma cells MG-63. In particular, we pay special attention to the cyto and genotoxicity actions of these complexes and to the association to oxidative stress. The three complexes: Cu-sac, Cu-gln and Cu-sac-gln caused a decline in cell viability. The half-maximal inhibitory concentration in MG-63 cells for Cu-sac-gln is 170 µM, showing the strongest antiproliferative effect. Moreover, only Cu-sac-gln caused a decrease of the mitochondrial activity from 100 µM. Our results indicate that the copper(II) complexes studied here produce DNA damage and suggest that the rise of reactive oxygen species (ROS) is the central mechanism action. Genotoxicity studied by the Cytokinesis-block micronucleus (MN) assay and the Single cell gel electrophoresis (comet assay) could be observed in MG-63 cells treated with Cu-sac-gln from 100 and 50 µM, respectively. Cu-sac and Cu-gln also induced DNA damage; however their effect was definitively weaker. The generation of reactive oxygen species increased from 50 μM of Cu-sac-gln and Cu-sac and only from 250 µM of Cugln, as well as a reduction of the GSH/GSSG ratio from 50 µM. When cells were treated with several concentrations of the complexes in addition to a combination of 50 μM of vitamin C plus 50 µM of vitamin E, a total recovery in cell survival was obtained for Cu-gln in the whole range of tested concentrations while only a partial viability recovery was obtained from 250 µM of Cu-sac and Cu-sac-gln. Overall, our results point to a differential cyto- and genotoxicity of the three copper(II) complexes and demonstrate that the complexation with both ligands confers the most potent antitumor action in human osteosarcoma cells.

Continuous Fabrication and Assembly of Spatial Cell-Laden Fibers for a Tissue-Like Construct via a Photolithographic-Based Microfluidic Chip

Engineering three-dimensional (3D) scaffolds with in vivo like architecture and function has shown great potential for tissue regeneration. Here we developed a facile microfluidic-based strategy for the continuous fabrication of cell-laden microfibers with hierarchically organized architecture. We show that photolithographically fabricated microfluidic devices offer a simple and reliable way to create anatomically inspired complex structures. Furthermore, the use of photo-cross-linkable methacrylated alginate allows modulation of both the mechanical properties and biological activity of the hydrogels for targeted applications. Via this approach, multilayered hollow microfibers were continuously fabricated, which can be easily assembled in situ, using 3D printing, into a larger, tissue-like construct. Importantly, this biomimetic approach promoted the development of phenotypical functions of the target tissue. As a model to engineer a complex tissue construct, osteon-like fiber was biomimetically engineered, and enhanced vasculogenic and osteogenic expression were observed in the encapsulated human umbilical cord vein endothelial cells and osteoblast-like MG63 cells respectively within the osteon fibers. The capability of this approach to create functional building blocks will be advantageous for bottom-up regeneration of complex, large tissue defects and, more broadly, will benefit a variety of applications in tissue engineering and biomedical research.

Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Estrogen stimulates osteoprotegerin expression via the suppression of miR-145 expression in MG-63 cells.

Osteoprotegerin (OPG) is implicated in the pathogenesis of postmenopausal osteoporosis, and other metabolic bone diseases caused by estrogen deficiency. Previous studies have demonstrated that estrogen may stimulate OPG expression in osteoblast cells at the transcriptional level; however, whether estrogen can regulate OPG expression at a post-transcriptional level remains elusive. The present study aimed to investigate the role of microRNA (miRNA) in estrogen-mediated OPG production in human osteoblast-like MG-63 cells. The results from ELISA, western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirmed that estrogen may upregulate OPG expression. Mechanistic studies indicated that estrogen increased the activity of a luciferase reporter harboring the OPG 3′-untranslated region (3′-UTR). Bioinformatics analysis demonstrated that there is a potential targeting site in the OPG 3′-UTR for miRNA (miR)-145, which is associated with osteoblast differentiation. The results of an RT-qPCR suggested that estrogen suppressed miR-145 expression. In addition, dual-luciferase assay, RT-qPCR and western blot analysis indicated that miR-145 directly targets and negatively regulates OPG expression. Furthermore, transfection of cells with miR-145 mimics was able to partially inhibit the induction of OPG expression by estrogen, thus confirming the role of miR-145 in estrogen-mediated OPG induction. Taken together, the results of the present study demonstrated that estrogen may post-transcriptionally regulate OPG expression through suppression of miR-145 expression.

The response of human osteoblasts, epithelial cells, fibroblasts, macrophages and oral bacteria to nanostructured titanium surfaces: a systematic study.

Nanotopography modification is a major focus of interest in current titanium surface design; however, the influence of the nanostructured surface on human cell/bacterium behavior has rarely been systematically evaluated. In this study, a homogeneous nanofiber structure was prepared on a titanium surface (Nano) by alkali-hydrothermal treatment, and the effects of this Nano surface on the behaviors of human MG-63 osteoblasts, human gingival epithelial cells (HGECs) and human gingival fibroblasts (HGFs) were evaluated in comparison with a smooth titanium surface (Smooth) by polishing and a micro-rough titanium surface (Micro) by sandblasting and acid etching. In addition, the impacts of these different surface morphologies on human THP-1 macrophage polarization and Streptococcus mutans attachment were also assessed. Our findings showed that the nanostructured surface enhanced the osteogenic activity of MG-63 cells (Nano=Micro>Smooth) at the same time that it improved the attachment of HGECs (Nano>Smooth>Micro) and HGFs (Nano=Micro>Smooth). Furthermore, the surface with nanotexture did not affect macrophage polarization (Nano=Micro=Smooth), but did reduce initial bacterial adhesion (Nano<Smooth<Micro). These results suggest that the nanostructured titanium surface may promote bone and soft tissue healing, and thereby increase the success and survival of dental implants.

Homo-aro-cholestane, furostane and spirostane saponins from the tubers of Ophiopogon japonicus

Phytochemical investigation of the tubers of Ophiopogon japonicus led to the isolation of five previously undescribed steroidal saponins, ophiojaponins A–E, together with twelve known ones. The structures of these isolated compounds were elucidated by detailed spectroscopic analyses and chemical methods. Ophiojaponins A–C are rare naturally occurring C29 steroidal glycosides possessing a homo-cholestane skeleton with an aromatized ring E. Ruscogenin 1-O-α-L-rhamnopyranosyl-(1 → 2)-4-O-sulfo-β-D-fucopyranosido-3-O-β-D-glucopyranoside was isolated as single component and its full spectroscopic data was reported for the first time. The isolated steroidal saponins were evaluated for their cytotoxicities against two human tumor cell lines MG-63 and SNU387. Among them, five known spirostane-type glycosides showed cytotoxic activity against both MG-63 and SNU387 cell lines with IC50 values ranging from 0.76 to 27.0 μM.

Wilms' tumor gene 1 silencing inhibits proliferation of human osteosarcoma MG-63 cell line by cell cycle arrest and apoptosis activation.

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. A high WT1 expression level was described in various types of human bone and soft-tissue sarcomas, including osteosarcoma (OS), but its function in carcinogenesis is not yet well understood. This study investigated WT1 both in human OS tissues and in human OS MG-63 cell line in which WT1 gene is up-regulated. The results demonstrated that WT1 is expressed in 50% of human OS cases. WT1-silenced MG-63 cells showed deregulation of proteins of cell cycle and down-regulation of PI3K/AKT pathway. Induction of apoptotic programme was also established by activation of caspase-3 and increase of Bax/Bcl2 ratio and p53 protein. This study provided new findings on role of WT1 and indicated an association between WT1 expression, cell cycle and apoptotic machinery. In conclusion, WT1 acts as a tumour promoter in osteosarcoma and it could be a potential therapeutic target.

Ethnomedicine based evaluation of osteoprotective properties of Tinospora cordifolia on in vitro and in vivo model systems.

Indian ethnomedicine acclaims the use of Tinospora cordifolia (TC) in the treatment of bone fractures and vat rakta (gout). The objective of the study is to investigate the effects of alcoholic extract of Tinospora cordifolia on bone remodeling (involving osteoblastic and osteoclastic actions) in vitro and protect against ovariectomy-induced bone loss in vivo. Human osteoblast-like cells MG-63 and primary osteoblast cells isolated from rat femur were used as osteoblast models and RAW macrophage cell line 264.7 induced to take up osteoclastic lineage using RANK ligand were used as osteoclast models in the current study. Sirius red staining, quantification of osteocalcin, cytopathological analysis by Hematoxylin/eosin staining and semiquantitative reverse transcription PCR (RT-PCR) was carried out to ascertain the effects of T. cordifolia extract on osteoblast cells. MTT assay was perfomed to understand the influence of T. cordifolia extract on osteoclast cells. Adult female Sprague-Dawley rats were used as in vivo models to study the effect of T. cordifolia on ovariectomy induced bone loss. Radiological (DEXA analysis), Biochemical (markers of bone formation and resorption), histopathological (Hematoxylin/eosin staining) and histomorphometric analysis of the bone was carried out. Treatment with T. cordifolia extract resulted in enhanced collagen deposition, increased levels of osteocalcin, increased expression of osteogenic genes all indicative of favourable osteoblastogenesis. Treatment with T. cordifolia extract did not exert any significant influence on the proliferation of osteoclasts. Pretreatment with T. cordifolia extract at a dose of 50mg/kg body wt/day orally for 21days followed by treatment for 12 weeks post ovariectomy was able to prevent ovariectomy-induced bone loss in vivo. Results of the study support the use of T. cordifolia in Indian ethnomedicine for the treatment of bone diseases and fractures.

High glucose-induced LIF suppresses osteoblast differentiation via regulating STAT3/SOCS3 signaling

High glucose (HG) is conceived to regulate bone metabolism in patients with diabetic mellitus (DM). In the present study, we examined the level of leukemia inhibitory factor (LIF), a pleiotropic cytokine in interleukin (IL)-6 family, in T2DM patients and investigated the regulation by HG on the induction of LIF/signal transducer and activator of transcription 3 (STAT3) signaling. Then we determined the regulation of HG and LIF on the osteoblast differentiation via measuring the ALP activity, matrix mineralization, and the expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), Osteocalcin (OCN) and osteopontin (OPN) in human osteoblast MG-63 cells. In addition, we evaluated the dependence of suppressor of cytokine signaling 3 (SOCS3)/STAT3 signaling in the progress. Results indicated significantly higher serum levels of high-sensitivity C-reactive protein (hsCRP), IL-1β, IL-6 and LIF in T2DM patients. HG induced markedly higher levels of these cytokines in vitro. Furthermore, either HG or LIF reduced the expression of ALP, OCN and RUNX2 in both mRNA and protein levels. In addition, LIF markedly promoted the expression of SOCS3, significantly upregulated the phosphorylation of STAT3 in MG-63 cells; and the downregulation of the four osteogenic differentiation-associated markers were restored by 50 or 100nM STAT3 inhibitor, JSI-124. In summary, this study has shown that LIF is implicated in the HG-mediated inhibition of osteoblast differentiation, via promoting STAT3/SOCS3 signaling. This study may provide insights into the signal pathway of HG-induced bone loss or delayed injured joint healing.