Garcinol prevents oxidative stress-induced bone loss and dysfunction of BMSCs through NRF2-antioxidant signaling.

There are multiple published data showing that excessive oxidative stress contributes to bone loss and even bone tissue damage, and it is also correlated with the pathophysiology of bone degenerative diseases, including osteoporosis (OP). Garcinol, a polyisoprenylated benzophenone derivative, has been recently established as an anti-oxidant agent. However, it remains elusive whether Garcinol protects bone marrow mesenchymal stem cells (BMSCs) and bone tissue from oxidative stress-induced damage. Here, we explored the potential effects of Garcinol supplementation in ameliorating oxidative stimulation-induced dysfunction of BMSCs and bone loss in osteoporotic mice. In this study, we verified that Garcinol exerted potent protective functions in the hydrogen peroxide (H2O2)-induced excessive oxidative stress and dysfunction of BMSCs. Besides, Garcinol was also identified to improve the reduced bone mass and abnormal lineage commitment of BMSCs in the condition of OP by suppressing the oxidative stimulation. Subsequent analysis revealed that nuclear factor erythroid 2-related factor 2 (NRF2) might be a key regulator in the sheltering effects of Garcinol on the H2O2-regulated oxidative stress, and the protective functions of Garcinol was mediated by NRF2-antioxidant signaling. Collectively, Garcinol prevented oxidative stress-related BMSC damage and bone loss through the NRF2-antioxidant signaling, which suggested the promising therapeutic values of Garcinol in the treatment of oxidative stress-related bone loss. Therefore, Garcinol might contribute to treating OP.

Does matching degree matter for proximal femoral intramedullary nail on reoperation rate in intertrochanteric fractures?

INTRODUCTION: Previous articles reported on the tip-apex distance, lag screw placement, fracture pattern, reduction quality, osteoporosis and other factors associated with second surgery. The current study focused on investigating the association of the matching degree between proximal femoral intramedullary nail and femoral medullary cavity on reoperation rate. PATIENTS AND METHODS: A retrospective cohort study was conducted. It included patients with intertrochanteric fracture who were treated with proximal femoral anti-rotatory intramedullary nail (PFNA) between January 2016 and April 2021. The gap between the intramedullary nail and the femoral medullary cavity was equal to the difference in diameter between the two. According to the gap size, all patients were divided into three groups, as follows: high-matching group: gap ≤ 2 mm; middle-matching group: 2 < gap < 4 mm; and low-matching group: gap ≥ 4 mm. The mean gap was measured through standard images. The primary observational index was whether the reoperation was needed, and secondary observational indexes included operative time, length of hospital stay. Patient characteristics were recorded, as follows: age, sex, follow-up time, fracture pattern, reduction grade and length of intramedullary nail. RESULTS: A total of 203 eligible patients were recorded, including 78 males (38.4%) and 125 females (61.6%). They had a mean age of 77.8 ± 9.9 years old and an average follow-up time of 58.1 ± 24.0 weeks. Twenty-seven patients (13.3%) needed a second operation. Coxa varus combined with screw cutting was the most common reason for reoperation (11 cases). Unstable fracture pattern with poor reduction grade tended to contribute to reoperation, whose odds ratio (OR) was 6.61 (95% confidence interval [CI], 1.98-22.09; P = 0.002). The three groups had 11 cases (13.7%), 12 cases (13.8%) and 4 cases (11.1%) of reoperation, respectively, and logistic regression showed no significant association was noted between matching degree of intramedullary nail and reoperation rate. CONCLUSIONS: The matching degree between proximal femoral intramedullary nail and femoral medullary cavity did not seem to be an important factor for reoperation, which offered more options of intramedullary nail size intraoperatively and reduced implants stock from inventory.

Litigation analysis of medical damage after total knee arthroplasty: a case study based on Chinese legal database in the past ten years.

BACKGROUND: The medical damage litigations after knee arthroplasty are on the rise year by year. However, few studies examined the litigation after knee arthroplasty. This study analyzed the litigation of medical damage after knee replacement in the past ten years based on a Chinese database. It synthesized the focus of the dispute in these cases to provide a reference for doctors to reduce the risk of litigation. METHODS: Retrospectively analyzed were medical damage litigations after total knee arthroplasty in the past ten years (June 2011-June 2021) from the "Wolters Kluwer Legal Information Database". The data collected included the characteristics of patients, causes of litigation, the results of litigation and the amount of compensation. RESULTS: A total of 110 litigation cases were analyzed, including 40 male patients (36.3%) and 70 female patients (63.6%). The top cause of litigation was infection (43.6%). The most common factor leading to the doctor losing the case was "complications caused by operational error" (P < 0.05). Complications, such as amputation, postoperative ischemic stroke and infection, were more likely to result in higher compensation. CONCLUSIONS: The prevention of infection and the avoidance of operational errors are very important in avoiding medical litigations. Moreover, avoiding disabling complications or a protracted course of disease could significantly reduce the amount of compensation. In addition, full and reasonable communication, paying full attention to the reaction of patients, and timely diagnosis could also effectively minimize the risk of litigation and loss.

The regulatory roles of miR-26a in the development of fracture and osteoblasts.

Background: MicroRNAs (miRNAs) play a vital role in the bone development and bone regeneration. In this study, we investigated the effects of miR-26a in osteoblasts and fractures. Methods: Human osteoblasts were cultured and used for analysis. To identify differential miRNAs in blood samples from patients with fractures and healthy controls, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed. Human osteoblasts were transfected with miR-26a mimics, miR-26a inhibitor, or their corresponding negative controls (NCs), respectively. MTT assay was performed to identify the effects of miR-26a on the cell viability of osteoblasts. EdU staining was applied to detect the proliferation of osteoblasts. Trypan blue staining was utilized to analyze the effects of miR-26a on the cell death of osteoblasts. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) staining was used to detect apoptotic osteoblasts. Alizarin red S (ARS) staining and qRT-PCR analysis were utilized to measure the mineralized nodule formation to evaluate the bone formation of osteoblasts. Dual luciferase reporter assay and western blot analysis were performed to detect the relationship between miR-26a and its target gene. Results: The results of qRT-PCR analysis identified miR-26a as our miRNA of interest and indicated that miR-26a was significantly decreased in patients with fractures. Overexpression of miR-26a significantly increased the cell viability and proliferation of osteoblasts. An increase in miR-26a reduced the cell death and apoptosis of osteoblasts, and promoted the osteoblastic activity and mineralized nodule formation. Dual luciferase reporter assay, qRT-PCR and western blot analysis showed that miR-26a could negatively regulate the expression of phosphatase and tensin homolog (PTEN). Conclusions: MiR-26a promoted new bone regeneration via regulating the functions of osteoblasts by targeting its target gene PTEN. Therefore, we propose that targeting miR-26a may be a novel therapeutic method for bone regeneration and treating fractures.

Psoralidin prevents caffeine-induced damage and abnormal differentiation of bone marrow mesenchymal stem cells via the classical estrogen receptor pathway.

BACKGROUND: Caffeine is broadly present in tea, coffee, and cocoa, and is commonly consumed. The bone microenvironment might be damaged by excessive caffeine, which has been shown to exert negative effects on human health. In this study, we sought to determine whether excessive caffeine could damage the biological functions of bone marrow mesenchymal stem cells (BMSCs) and induce bone loss in mice, and further investigate effective therapeutic methods. METHODS: BMSCs were treated with different concentrations of caffeine (0.01, 0.05, 0.1, 0.5, and 1.0 mM) for 48 h. Cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis were performed to detect the cell viability, proliferation, migration, and pluripotency of BMSCs, respectively. Alizarin red S (ARS) staining, alkaline phosphatase (ALP) staining, oil red O (ORO) staining, and qRT-PCR assay were applied to assess the osteogenic and adipogenic differentiation of BMSCs. BMSCs were treated with caffeine and further exposed to different concentrations of psoralidin (PL) (0.01, 0.1, 1, and 10 µM) for 48 h. Micro-computed tomography (µCT) scanning was used to evaluate the bone mass of mice. 7α-(7-((4,4,5,5,5-Pentafluoropentyl)-sulfiny)nonyl)estra-1,3,5(10)-triene-3,17ß-diol (ICI 182,780, ICI) was applied to examine whether the classical estrogen receptor (ER) pathway was involved. RESULTS: The CCK-8 assay, colony formation assay, wound healing assay, and qRT-PCR analysis indicated that caffeine (0.01, 0.05, 0.1, 0.5, 1.0 mM) attenuated the cell viability, proliferation, migration and pluripotency of BMSCs, respectively, in a concentration-dependent manner. Caffeine treatment inhibited osteogenic differentiation but promoted adipogenic differentiation of BMSCs in a dose-dependent manner. Furthermore, ARS staining, ALP staining, ORO staining, and qRT-PCR assay showed that excessive caffeine induced bone loss and osteoporosis (OP) in mice by regulating the osteogenesis and adipogenesis of BMSCs. Also, PL treatment could reverse the caffeine-induced dysfunctions and aberrant differentiation of BMSCs via the ER pathway. CONCLUSIONS: Our results revealed a novel molecular mechanism for the therapeutic effects of PL in treating excessive caffeine-induced OP, which might shed new light on the clinical application of PL for caffeine-related OP.

Anticancer effects of melatonin via regulating lncRNA JPX-Wnt/ß-catenin signalling pathway in human osteosarcoma cells.

Osteosarcoma (OS) is a type of malignant primary bone cancer, which is highly aggressive and occurs more commonly in children and adolescents. Thus, novel potential drugs and therapeutic methods are urgently needed. In the present study, we aimed to elucidate the effects and mechanism of melatonin on OS cells to provide a potential treatment strategy for OS. The cell survival rate, cell viability, proliferation, migration, invasion and metastasis were examined by trypan blue assay, MTT, colony formation, wound healing, transwell invasion and attachment/detachment assay, respectively. The expression of relevant lncRNAs in OS cells was determined by real-time qPCR analysis. The functional roles of lncRNA JPX in OS cells were further examined by gain and loss of function assays. The protein expression was measured by western blot assay. Melatonin inhibited the cell viability, proliferation, migration, invasion and metastasis of OS cells (Saos-2, MG63 and U2OS) in a dose-dependent manner. Melatonin treatment significantly downregulated the expression of lncRNA JPX in Saos-2, MG63 and U2OS cells. Overexpression of lncRNA JPX into OS cell lines elevated the cell viability and proliferation, which was accompanied by the increased metastasis. We also found that melatonin inhibited the OS progression by suppressing the expression of lncRNA JPX via regulating the Wnt/ß-catenin pathway. Our results suggested that melatonin inhibited the biological functions of OS cells by repressing the expression of lncRNA JPX through regulating the Wnt/ß-catenin signalling pathway, which indicated that melatonin might be applied as a potentially useful and effective natural agent in the treatment of OS.

Hydrogen sulfide inhibits endoplasmic reticulum stress through the GRP78/mTOR pathway in rat chondrocytes subjected to oxidative stress.

The activation of oxidative stress is a primary cause of chondrocyte apoptosis in osteoarthritis (OA). The 78­kDa glucose­regulated protein (GRP78)/mammalian target of rapamycin (mTOR) signaling pathway has been demonstrated to be linked with the endoplasmic reticulum (ER) and autophagy. Hydrogen sulfide (H2S) has been reported to exert antioxidant effects. The present study investigated oxidative stress levels via 2',7'­dichlorofluorescin diacetate and MitoSOX staining, apoptosis rates via flow cytometry and the expression levels of ER stress­related proteins in GYY4137 (donor of H2S)­treated chondrocytes (CHs). CHs were isolated from the bilateral hip joints of male rats to examine mitochondrial permeability transition pore opening­ and mTOR signaling pathway­related proteins. The results demonstrated that tert­Butyl hydroperoxide (TBHP) increased CH apoptosis, and treatment with GYY4137 ameliorated TBHP­mediated the generation of ROS and CH apoptosis. Moreover, TBHP­treated CHs displayed elevated ER stress sensor expression levels and apoptotic rates; however, the TBHP­induced protein expression levels were decreased following GYY4137 treatment. In the present study, treatment with either GYY4137 or transfection with GRP78 siRNA both suppressed the activation of p­P70S6k and p­mTOR. H2S played an important role in regulating ER stress in TBHP­stimulated CHs. GYY4137 promoted autophagy, which was accompanied by the inhibition of ER stress. On the whole, the present study demonstrates that TBHP­induced oxidative stress stimulates ER interactions and CH apoptosis, which are suppressed by exogenous H2S via modulating the GRP78/mTOR signaling pathway.

Resveratrol benefits the lineage commitment of bone marrow mesenchymal stem cells into osteoblasts via miR-320c by targeting Runx2.

Bone marrow mesenchymal stem cells (BMSCs) are a potential source of osteoblasts and have been widely used in clinical therapies due to their pluripotency. Recent publications have found that resveratrol (RSVL) played a crucial role in the proliferation and differentiation of BMSCs; however, the underlying molecular mechanism of RSVL-induced BMSCs osteogenic differentiation needs to be fully elucidated. The objective of this study was to explore functions of miRNAs in the RSVL-treated BMSCs and its effects on the differentiation potentials of BMSCs. The findings demonstrated that RSVL enhanced the osteogenesis and suppressed the adipogenesis of BMSCs in a dose-dependent manner. Besides, a novel regulatory axis containing miR-320c, and its target Runx2 was found during the differentiation process of BMSCs under RSVL treatment. Increase of miR-320c reduced the osteogenic potential of BMSCs, while knockdown of miR-320c played a positive role in the osteogenesis of BMSCs. In contrast, overexpression of miR-320c accelerated the adipogenic differentiation, while knockdown of miR-320c restrained the adipogenic differentiation of BMSCs. The results confirmed that Runx2 might be the direct target of miR-320c in RSVL-promoted osteogenic differentiation of BMSCs. This study revealed that RSVL might be used for the treatment of bone loss related diseases and miR-320c could be regarded as a novel and potential target to regulate the biological functions of BMSCs.

MiR-206 regulates the progression of osteoporosis via targeting HDAC4.

BACKGROUND: More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. METHODS: 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3'UTR of HDAC4. RESULTS: Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. CONCLUSIONS: MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

Long noncoding RNA NORAD regulates cancer cell proliferation and migration in human osteosarcoma by endogenously competing with miR-199a-3p.

In this study, we evaluated the expressions and functions of a long noncoding RNA (lncRNA), Noncoding RNA activated by DNA damage (NORAD) in human osteosarcoma. NORAD expressions were evaluated by qRT-PCR in in vitro osteosarcoma cell lines and in vivo clinical specimens. SiRNA-induced NORAD downregulation was conducted in Saos-2 and 143B cells, and the functional effects of NORAD downregulation on osteosarcoma cells were evaluated by CCK-8 proliferation assay, 24-well transwell invasion assay and in vivo tumor explant assay, respectively. The possibility of NORAD endogenously competing microRNA target, hsa-miR-199a-3p was examined by dual-luciferase reporter assay and qRT-PCR. Then, hsa-miR-199a-3p was downregulated in NORAD-inhibited osteosarcoma cells to examine its role in regulating NORAD inhibition induced cancer suppression in osteosarcoma cells. NORAD was found to be significantly overexpressed in both osteosarcoma cells and osteosarcoma tumors. In Saos-2 and 143B cells transfected with NORAD-specific siRNA, their proliferation, invasion, and in vivo explant growth were all markedly suppressed. Hsa-miR-199a-3p was confirmed to be the competing target of NORAD. Its downregulation in Saos-2 and 143B cells inversely augment proliferation and invasion that were initially suppressed by NORAD-downregulation. The results of our study show that NORAD plays an important role in regulating cancer cell functions of osteosarcoma, possibly through endogenously competing with hsa-miR-199a-3p.

Construction and analysis of a ceRNA­ceRNA network reveals two potential prognostic modules regulated by hsa­miR­335­5p in osteosarcoma.

Osteosarcoma is an aggressive cancer of the skeletal system, which is associated with a poor prognosis due to the high recurrence rate. Although previous studies have revealed that competitive endogenous RNAs (ceRNAs) are involved in various biological processes in the physiology and development of osteosarcoma, the roles of ceRNAs in osteosarcoma recurrence remain largely unexplored. The present study constructed a ceRNA­ceRNA network for osteosarcoma by systematically integrating matched expression profiles for microRNAs (miRNAs/miRs) and mRNAs, and identified two ceRNA­mediated modules that were associated with recurrence in patients with osteosarcoma. A multivariate Cox regression analysis demonstrated that the recurrence­free prognosis associated with the expression of the two modules was independent of other clinical factors. In addition, hsa­miR­335­3p was identified as an upstream regulating factor for both modules. In conclusion, the results of the present study suggested that ceRNAs may act as potential therapeutic biomarkers for predicting the recurrence of osteosarcoma, and may help to identify patients with osteosarcoma at a high risk of recurrence, who may benefit from adjuvant therapy.

Bone marrow stem cells-derived extracellular matrix is a promising material.

The extracellular matrix(ECM), which is primarily composed of collagens and proteoglycans, plays a key role in cell proliferation, differentiation, and migration and interactions between cells. In this study, we produced chitosan/gelatin/bone marrow stem cells-derived extracellular matrix(C/G/BMSCs-dECM) scaffolds via lyophilization and cross-linking, and chitosan/gelatin(C/G) scaffolds were used as controls. For the C/G/BMSCs-dECM scaffolds, the average pore size was 289.17 ± 80.28 µm; the average porosity was 89.25 ± 3.75%; the average compressive modulus was 0.82 ± 0.07 MPa; and the average water uptake ratio was 13.81 ± 1.00. In vitro, the C/G/BMSCs-dECM scaffolds promoted bone marrow stem cells(BMSCs) attachment and proliferation. Moreover, improved osteogenic differentiation was observed for these scaffolds. Thus, C/G/BMSCs-dECM is a promising material for bone tissue engineering.

Tetrahydrocurcumin induces mesenchymal-epithelial transition and suppresses angiogenesis by targeting HIF-1α and autophagy in human osteosarcoma.

Human osteosarcoma is considered a malignant tumor with poor prognosis that readily metastasizes. Tetrahydrocurcumin (THC) has been reported to have anti-tumor activity in numerous tumors. In addition, hypoxia-inducible factor-1α (HIF-1α) has been demonstrated to be associated with tumor metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of THC in osteosarcoma remains uncertain. Therefore, this study aimed to elucidate the potential mechanisms. We found that THC significantly reduced the growth of osteosarcoma cells and suppressed migration and invasion, as tested in a nude mouse lung metastasis model. Additionally, the mesenchymal-epithelial transition (MET) process was facilitated by THC. Mechanistically, our study showed that HIF-1α had a pivotal role in the anti-metastatic effect of THC. Importantly, HIF-1α expression was downregulated by THC by inhibiting Akt/mTOR and p38 MAPK pathways. Moreover, THC exhibited a remarkable inhibitory effect on HIF-1α expression and angiogenesis under hypoxic conditions. Furthermore, THC activated autophagy and induced MET and suppressed angiogenesis in a HIF-1α-related manner. Taken together, our findings suggest that THC suppresses metastasis and invasion and this may be associated with HIF-1α and autophagy, which would potentially provide therapeutic strategies for human osteosarcoma.

Andrographolide Induces Autophagic Cell Death and Inhibits Invasion and Metastasis of Human Osteosarcoma Cells in An Autophagy-Dependent Manner.

BACKGROUND/AIMS: Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue. Although treatment effectiveness has improved, the OS survival rate has fluctuated in recent years. Andrographolide (AG) has been reported to have antitumor activity against a variety of tumors. Our aim was to investigate the effects and potential mechanisms of AG in human osteosarcoma. METHODS: Cell viability and morphological changes were assessed by MTT and live/dead assays. Apoptosis was detected using Annexin V-FITC/PI double staining, DAPI, and caspase-3 assays. Autophagy was detected with mRFP-GFP-LC3 adenovirus transfection and western blot. Cell migration and invasion were detected by wound healing assay and Transwell® experiments. RESULTS: AG dose-dependently reduced the viability of osteosarcoma cells. No increase in apoptosis was detected in AG-treated human OS MG-63 and U-2OS cells, and the pan-caspase inhibitor z-VAD did not attenuate AG-induced cell death. However, AG induced autophagy by suppressing PI3K/Akt/mTOR and enhancing JNK signaling pathways. 3-MA and Beclin-1 siRNA could reverse the cytotoxic effects of AG. In addition, AG inhibited the invasion and metastasis of OS, and this effect could be reversed with Beclin-1 siRNA. CONCLUSION: AG inhibits viability and induces autophagic death in OS cells. AG-induced autophagy inhibits the invasion and metastasis of OS.

Deoxyelephantopin Induces Reactive Oxygen Species-Mediated Apoptosis and Autophagy in Human Osteosarcoma Cells.

BACKGROUND/AIMS: Osteosarcoma is the predominant form of primary bone malignancy. Although the combinational application of neoadjuvant chemotherapy and surgical resection significantly increases the survival rate, the therapeutic outcome remains unsatisfactory. Deoxyelephantopin (DET), an active ingredient of Elephantopus scaber, has been reported to have an anti-tumor effect in recent publications. This study aimed to investigate whether DET has antineoplastic effects on osteosarcoma cells and its underlying mechanism. METHODS: Cell viability and morphological changes were assessed by MTT and Live/dead assays. Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected utilizing Annexin V-FITC/PI double staining, DCFH-DA and JC-1 probes, respectively. Autophagy was detected by mRFP-GFP-LC3 adenovirus transfection and western blot. RESULTS: DET dose-dependently reduced the viability of osteosarcoma cells following the increase in intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) reversed this effect. Furthermore, DET induced mitochondrial apoptosis. Depolarized cells were increased, and apoptosis-related proteins, such as Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved ploy ADP-ribose polymerase, were activated. Additionally, we found that DET could induce autophagy in osteosarcoma cells, but autophagy inhibition did not affect the decrease in cell viability. CONCLUSION: DET induced apoptosis in osteosarcoma cells through ROS generation, mitochondrial dysfunction and caspase activation; in addition, autophagy was involved in the effects of DET on osteosarcoma cells.

Preparation and Characterization of a Chitosan/Gelatin/Extracellular Matrix Scaffold and Its Application in Tissue Engineering.

Previous studies have demonstrated that extracellular matrix (ECM) can be used in tissue engineering due to its bioactivity. However, adipose-derived ECM (A-dECM) has never been applied in bone tissue engineering, and it is unknown whether it would be beneficial to the growth of bone marrow mesenchymal stem cells (BMSCs). In this study, we produced chitosan/gelatin/A-dECM (C/G/A-dECM) scaffolds via lyophilization and crosslinking; chitosan/gelatin (C/G) scaffolds were used as controls. For the C/G/A-dECM scaffolds, the average pore size was 285.93 ± 85.39 µm; the average porosity was 90.62 ± 3.65%; the average compressive modulus was 0.87 ± 0.05 kPa; and the average water uptake ratio was 13.73 ± 1.16. In vitro, A-dECM scaffolds could promote the attachment and proliferation of BMSCs. In the same osteogenic-inducing reagent, better osteogenic differentiation could be observed for the C/G/A-dECM scaffolds than for the C/G scaffolds. Thus, we conclude that A-dECM is a promising material and that C/G/A-dECM scaffolds are a candidate for bone tissue engineering.

A New Perspective for Osteosarcoma Therapy: Proteasome Inhibition by MLN9708/2238 Successfully Induces Apoptosis and Cell Cycle Arrest and Attenuates the Invasion Ability of Osteosarcoma Cells in Vitro.

BACKGROUND: The proteasome exists in all eukaryotic cells and provides the main route of intracellular proteins degradation involved in cell growth and apoptosis. Proteasome inhibition could block protein degradation pathways and disturb regulatory networks, possibly leading to profound effects on cell growth, particularly in cancer cells. A proteasome inhibitor with an appropriate toxicity index for malignant cells rather than normal cells would be an attractive anticancer therapy. METHODS: The human osteosarcoma (OS) cell lines MG-63 and Saos-2 and normal osteoblast cells were used to study the antitumour activity of the proteasome inhibitor MLN9708/2238. RESULTS: MLN2238 inhibited cell growth, induced cell cycle arrest and apoptosis, and attenuated the invasion abilities of MG-63 and Saos-2 cells, with little cytotoxicity to normal cells. In addition, MLN2238 promoted antitumour mechanisms including the accumulation of E2F1, P53, P21 and other negative G2/M checkpoint proteins; up-regulated the relative expression ratio of BAX/BCL-2, APAF-1 and pro-apoptotic proteins of the BCL-2 family; triggered mitochondrial outer membrane permeabilization (MOMP); down-regulated BCL-2 and XIAP; activated caspase3/8/9; and suppressed MMP2/9 expression and secretion levels. CONCLUSIONS: The proteasome may be a novel biochemical target for OS treatment in vitro. Our study provides a promising mechanistic framework for MLN9708/2238 in OS treatment, supporting its clinical development.

Synthesis and evaluation of benzylisoquinoline derivatives for their inhibition on pancreatic lipase and preadipocyte proliferation.

The present study was designed to synthesize and evaluate a series of benzylisoquinoline derivatives. These compounds were synthesized by Bischler-Napieralski cyclization to yield 1-benzyl-3,4-dihydroisoquinolines, and the products were obtained by reductions. All these compounds were identified by MS, (1)H NMR and (13)C NMR. The inhibitory activities on pancreatic lipase and preadipocyte proliferation for the synthesized compounds and alkaloids from Nulembo nucifera were assessed in vitro. Most of the compounds showed inhibitory activities on both pancreatic lipase and preadipocyte proliferation. Particularly, compounds 7p-7u and 9d-9f exhibited significant inhibitory activity on pancreatic lipase while compounds 7c, 7d, 7f, 7g, 7i, and 7j potently inhibited the proliferation of 3T3-L1 preadipocytes. Our results provided a basis for future evaluation and development of these compounds as leads for therapeutics for human diseases.

Long noncoding RNA FGFR3-AS1 promotes osteosarcoma growth through regulating its natural antisense transcript FGFR3.

Long noncoding RNAs (lncRNAs), a new class of RNAs with no protein-coding potential, have been reported to have crucial roles in the regulation of a variety of tumors. However, the functions and molecular mechanisms of lncRNAs to osteosarcoma are still largely unknown. The purpose of this study is to examine the expression, functions and molecular mechanisms of a new lncRNA FGFR3 antisense transcript 1 (FGFR3-AS1) in osteosarcoma. The expression of FGFR3-AS1 was examined by real-time quantitative PCR. The regulation of FGFR3 by FGFR3-AS1 was examined by RNase protection assay, real-time quantitative PCR, western blotting, and luciferase reporter assay. The effects of FGFR3-AS1 on osteosarcoma cell proliferation and cell cycle were determined by Cell Counting Kit-8, Ethynyl deoxyuridine incorporation assay and flow cytometry. FGFR3-AS1 was upregulated in osteosarcoma. Increased FGFR3-AS1 expression correlates with large tumor size, advanced Enneking stage, metastasis and poor survival. Through antisense pairing with FGFR3 3'UTR, FGFR3-AS1 increases FGFR3 mRNA stability and upregulates FGFR3 expression. The expression of FGFR3-AS1 and FGFR3 is positively correlated in osteosarcoma tissues. Knockdown of FGFR3-AS1 inhibits the proliferation and cell cycle progression of osteosarcoma cells in vitro. Moreover, knockdown of FGFR3-AS1 inhibits xenograft tumor growth of osteosarcoma cells in vivo. These data demonstrate the mechanisms of how antisense noncoding RNA regulate the expression of sense genes, and show the pivotal functions of FGFR3-AS1 in osteosarcoma.