Identification of ENO-1 positive extracellular vesicles as a circulating biomarker for monitoring of Ewing sarcoma.

The lack of circulating biomarkers for tumor monitoring is a major problem in Ewing sarcoma management. The development of methods for accurate tumor monitoring is required, considering the high recurrence rate of drug-resistant Ewing sarcoma. Here, we describe a sensitive analytical technique for tumor monitoring of Ewing sarcoma by detecting circulating extracellular vesicles secreted from Ewing sarcoma cells. Proteomic analysis of Ewing sarcoma cell-derived extracellular vesicles identified 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, CD99, SLC1A5, and ENO-1 were identified on extracellular vesicles purified from sera of patients with Ewing sarcoma before treatment but not on extracellular vesicles from those after treatment and healthy individuals. Notably, not only Ewing sarcoma-derived extracellular vesicles but also Ewing sarcoma cells demonstrated proteomic expression of CD99 and ENO-1 on their surface membranes. ENO-1+CD63+ extracellular vesicle detection was reduced after tumor resection while both CD99+CD63+ and ENO-1+CD63+ extracellular vesicles were detected in serum from Ewing sarcoma-bearing mice. Finally, the accuracy of liquid biopsy targeting these candidates was assessed using extracellular vesicles from the sera of patients with Ewing sarcoma. Elevated ENO-1+CD81+ extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals with an area under the curve value of 0.92 (P < 0.001) and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Collectively, circulating ENO-1+CD81+ extracellular vesicle detection could represent a novel tool for tumor monitoring of Ewing sarcoma.

High-quality expert annotations enhance artificial intelligence model accuracy for osteosarcoma X-ray diagnosis.

Primary malignant bone tumors, such as osteosarcoma, significantly affect the pediatric and young adult populations, necessitating early diagnosis for effective treatment. This study developed a high-performance artificial intelligence (AI) model to detect osteosarcoma from X-ray images using highly accurate annotated data to improve diagnostic accuracy at initial consultations. Traditional models trained on unannotated data have shown limited success, with sensitivities of approximately 60%-70%. In contrast, our model used a data-centric approach with annotations from an experienced oncologist, achieving a sensitivity of 95.52%, specificity of 96.21%, and an area under the curve of 0.989. The model was trained using 468 X-ray images from 31 osteosarcoma cases and 378 normal knee images with a strategy to maximize diversity in the training and validation sets. It was evaluated using an independent dataset of 268 osteosarcoma and 554 normal knee images to ensure generalizability. By applying the U-net architecture and advanced image processing techniques such as renormalization and affine transformations, our AI model outperforms existing models, reducing missed diagnoses and enhancing patient outcomes by facilitating earlier treatment. This study highlights the importance of high-quality training data and advocates a shift towards data-centric AI development in medical imaging. These insights can be extended to other rare cancers and diseases, underscoring the potential of AI in transforming diagnostic processes in oncology. The integration of this AI model into clinical workflows could support physicians in early osteosarcoma detection, thereby improving diagnostic accuracy and patient care.

p53-Armed Oncolytic Virotherapy Improves Radiosensitivity in Soft-Tissue Sarcoma by Suppressing BCL-xL Expression.

Soft-tissue sarcoma (STS) is a heterogeneous group of rare tumors originating predominantly from the embryonic mesoderm. Despite the development of combined modalities including radiotherapy, STSs are often refractory to antitumor modalities, and novel strategies that improve the prognosis of STS patients are needed. We previously demonstrated the therapeutic potential of two telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in human STS cells. Here, we demonstrate in vitro and in vivo antitumor effects of OBP-702 in combination with ionizing radiation against human STS cells (HT1080, NMS-2, SYO-1). OBP-702 synergistically promoted the antitumor effect of ionizing radiation in the STS cells by suppressing the expression of B-cell lymphoma-X large (BCL-xL) and enhancing ionizing radiation-induced apoptosis. The in vivo experiments demonstrated that this combination therapy significantly suppressed STS tumors' growth. Our results suggest that OBP-702 is a promising antitumor reagent for promoting the radiosensitivity of STS tumors.

Inhibitory Effect of a Tankyrase Inhibitor on Mechanical Stress-Induced Protease Expression in Human Articular Chondrocytes.

We investigated the effects of a Tankyrase (TNKS-1/2) inhibitor on mechanical stress-induced gene expression in human chondrocytes and examined TNKS-1/2 expression in human osteoarthritis (OA) cartilage. Cells were seeded onto stretch chambers and incubated with or without a TNKS-1/2 inhibitor (XAV939) for 12 h. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 8% elongation, 30 min) was applied and the gene expression of type II collagen a1 chain (COL2A1), aggrecan (ACAN), SRY-box9 (SOX9), TNKS-1/2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and matrix metalloproteinase-13 (MMP-13) were examined by real-time PCR. The expression of ADAMTS-5, MMP-13, nuclear translocation of nuclear factor-κB (NF-κB), and ß-catenin were examined by immunocytochemistry and Western blotting. The concentration of IL-1ß in the supernatant was examined by enzyme-linked immunosorbent assay (ELISA). TNKS-1/2 expression was assessed by immunohistochemistry in human OA cartilage obtained at the total knee arthroplasty. TNKS-1/2 expression was increased after CTS. The expression of anabolic factors were decreased by CTS, however, these declines were abrogated by XAV939. XAV939 suppressed the CTS-induced expression of catabolic factors, the release of IL-1ß, as well as the nuclear translocation of NF-κB and ß-catenin. TNKS-1/2 expression increased in mild and moderate OA cartilage. Our results demonstrated that XAV939 suppressed mechanical stress-induced expression of catabolic proteases by the inhibition of NF-κB and activation of ß-catenin, indicating that TNKS-1/2 expression might be associated with OA pathogenesis.

Expression of Acetabular Labral Vascular Endothelial Growth Factor and Nerve Growth Factor Is Directly Associated with Hip Osteoarthritis Pain: Investigation by Immunohistochemical Staining.

The acetabular labrum enhances hip joint stability and plays a key role in osteoarthritis (OA) progression. Labral nerve endings contribute to hip OA pain. Moreover, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) are associated with pain. Consequently, we analysed VEGF and NGF expression levels in the labrum and their roles in OA. Labra obtained from OA patients were stained immunohistochemically, and labral cells were cultured and subjected to a reverse transcription (RT)-polymerase chain reaction (PCR) to analyse VEGF and NGF mRNA expression. VEGF and NGF expression were compared in each region of the labrum. Correlations between VEGF and NGF expression and age, body mass index, Kellgren-Lawrence grade, Harris Hip Score, the visual analogue scale (VAS), and Krenn score were analysed, and the RT-PCR confirmed the findings. VEGF and NGF expression were high on the labral articular side, negatively correlated with the Krenn score, and positively correlated with the VAS in early OA. VEGF and NGF mRNA expression increased significantly in patients with severe pain and decreased significantly in severely degenerated labra. In early OA, VEGF and NGF expression in the acetabular labrum was associated with the occurrence of hip pain; therefore, these factors could be effective targets for pain management.

CD30-targeted therapy induces apoptosis of inflammatory cytokine-stimulated synovial fibroblasts and ameliorates collagen antibody-induced arthritis in mice.

OBJECTIVE: It has been reported that levels of soluble CD30 in serum and joint fluid are significantly elevated in patients with rheumatoid arthritis (RA). This study aimed to investigate whether CD30 could be a therapeutic target for RA. METHODS: The expression and localization of CD30 were examined by immunohistochemical and double immunofluorescence staining on synovial tissue samples obtained from patients with RA or osteoarthritis (OA) during surgery. Changes in CD30 expression of fibroblast-like synoviocytes (FLS) from RA patients with or without TNFα and IL-1ß stimulation were examined by the polymerase chain reaction (PCR) and flow cytometry. Collagen antibody-induced arthritis (CAIA) was created in DBA/1 mice, and the therapeutic effect of brentuximab vedotin (BV) was examined by clinical score, histological findings and measurement of serum levels of SAA, IL-6, and TNFα. RESULTS: CD30 expression was significantly higher in samples from patients with RA than from those with OA. Double immunofluorescence showed a low rate of co-localization of CD30 with CD20 or CD90, but a high rate of co-localization of CD30 and CD138. CD30 mRNA expression was upregulated 11.7-fold in FLS following stimulation by inflammatory cytokines. The clinical scores of CAIA mice were significantly lower following both BV treatments, however, the histological scores of CAIA mice were significantly lower only following treatment with high dose BV (70 mg/kg). CONCLUSIONS: CD30 was expressed on immunocompetent cells in synovial tissue from RA patients and in cytokine-stimulated FLS in vitro. High dose BV (70 mg/kg) showed significant therapeutic effects in ameliorating inflammation and joint destruction in CAIA mice, but low dose BV (30 mg/kg) was insufficient.

Telomerase-specific oncolytic immunotherapy for promoting efficacy of PD-1 blockade in osteosarcoma.

Immune checkpoint inhibitors including anti-programmed cell death 1 (PD-1) antibody have recently improved clinical outcome in certain cancer patients; however, osteosarcoma (OS) patients are refractory to PD-1 blockade. Oncolytic virotherapy has emerged as novel immunogenic therapy to augment antitumor immune response. We developed a telomerase-specific replication-competent oncolytic adenovirus OBP-502 that induces lytic cell death via binding to integrins. In this study, we assessed the combined effect of PD-1 blockade and OBP-502 in OS cells. The expression of coxsackie and adenovirus receptor (CAR), integrins αvß3 and αvß5, and programmed cell death ligand 1 (PD-L1) was analyzed in two murine OS cells (K7M2, NHOS). The cytopathic activity of OBP-502 in both cells was analyzed using the XTT assay. OBP-502-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). Subcutaneous tumor models for K7M2 and NHOS cells were used to evaluate the antitumor effect and number of tumor-infiltrating CD8+ cells in combination therapy. K7M2 and NHOS cells showed high expression of integrins αvß3 and αvß5, but not CAR. OBP-502 significantly suppressed the viability of both cells, in which PD-L1 expression and the release of ATP and HMGB1 were significantly increased. Intratumoral injection of OBP-502 significantly augmented the efficacy of PD-1 blockade on subcutaneous K2M2 and NHOS tumor models via enhancement of tumor-infiltrating CD8+ T cells. Our results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the efficacy of PD-1 blockade in OS.

Histological Analysis of Repaired Tissue after Pullout Repair of a Medial Meniscus Posterior Root Tear.

A 65-year-old man presented with a left medial meniscus (MM) posterior root tear (PRT). Unicompartmental knee arthroplasty was performed 12 months after transtibial pullout repair of the MMPRT. Repaired MM posterior root tissue was subjected to histological analysis. Immunostaining and picrosirius red staining showed sufficient deposition of type I collagen, and hematoxylin-eosin staining using a polarized microscope showed well-aligned fiber orientation in the repaired tissue. The repaired posterior root (post-transtibial pullout repair) showed mature and well-aligned ligament-like tissue. Preserving the MM posterior root remnant to mimic the original posterior root tissue might be useful when performing pullout repair.

Adipose-Derived Extract Suppresses IL-1ß-Induced Inflammatory Signaling Pathways in Human Chondrocytes and Ameliorates the Cartilage Destruction of Experimental Osteoarthritis in Rats.

We investigated the effects of adipose-derived extract (AE) on cultured chondrocytes and in vivo cartilage destruction. AE was prepared from human adipose tissues using a nonenzymatic approach. Cultured human chondrocytes were stimulated with interleukin-1 beta (IL-1ß) with or without different concentrations of AE. The effects of co-treatment with AE on intracellular signaling pathways and their downstream gene and protein expressions were examined using real-time PCR, Western blotting, and immunofluorescence staining. Rat AE prepared from inguinal adipose tissues was intra-articularly delivered to the knee joints of rats with experimental osteoarthritis (OA), and the effect of AE on cartilage destruction was evaluated histologically. In vitro, co-treatment with IL-1ß combined with AE reduced activation of the p38 and ERK mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB), and subsequently downregulated the expressions of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, IL-6, and IL-8, whereas it markedly upregulated the expression of IL-1 receptor type 2 (IL-1R2) in chondrocytes. Intra-articular injection of homologous AE significantly ameliorated cartilage destruction six weeks postoperatively in the rat OA model. These results suggested that AE may exert a chondroprotective effect, at least in part, through modulation of the IL-1ß-induced inflammatory signaling pathway by upregulation of IL-1R2 expression.

Metformin induces CD11b+-cell-mediated growth inhibition of an osteosarcoma: implications for metabolic reprogramming of myeloid cells and anti-tumor effects.

CD11b+ myeloid subpopulations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), play crucial roles in the suppression of T-cell-mediated anti-tumor immunity. Regulation of these cell types is a primary goal for achieving efficient cancer immunotherapy. We found that metformin (Met) induces CD11b+-cell-mediated growth inhibition of a K7M2neo osteosarcoma independent of T cells, as growth inhibition of K7M2neo was still observed in wild-type (WT) mice depleted of T cells by antibodies and in SCID; this contrasted with the effect of Met on Meth A fibrosarcoma, which was entirely T-cell-dependent. Moreover, the inhibitory effect seen in SCID was abrogated by anti-CD11b antibody injection. PMN-MDSCs were significantly reduced in both spleens and tumors following Met treatment. In TAMs, production of IL-12 and TNF-α, but not IL-10, became apparent, and elevation of MHC class II with reduction of CD206 was observed, indicating a shift from an M2- to M1-like phenotype via Met administration. Metabolically, Met treatment decreased basal respiration and the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio of CD11b+ cells in tumors, but not in the spleen. In addition, decreased reactive oxygen species (ROS) production and proton leakage in MDSCs and TAMs were consistently observed in tumors. Uptake of both 2-deoxy-2-d-glucose (2-NBDG) and BODIPY® decreased in MDSCs, but only BODIPY® incorporation was decreased in TAMs. Overall, our results suggest that Met redirects the metabolism of CD11b+ cells to lower oxidative phosphorylation (OXPHOS) while elevating glycolysis, thereby pushing the microenvironment to a state that inhibits the growth of certain tumors.

Analysis of Adverse Reactions Caused by Potentially Inappropriate Prescriptions and Related Medical Costs That Are Avoidable Using the Beers Criteria: The Japanese Version and Guidelines for Medical Treatment and Its Safety in the Elderly 2015.

We conducted a retrospective study to investigate adverse drug reactions and associated medical costs among elderly individuals that could be avoided if pharmacotherapy was performed in accordance with the Beers Criteria: the Japanese Version (BCJV) and Guidelines for Medical Treatment and Its Safety in the Elderly 2015 (GL2015). Patients aged at least 65 years who were either hospitalized at Gifu Municipal Hospital between October 1 and November 30, 2014 (n = 1236) or had outpatient examinations at Gifu Municipal Hospital on October 1-2, 2014 (n = 980) were included in the study. The outcomes measured were usage rates of drugs listed in the BCJV and GL2015, incidence rates of adverse drug reactions, and additional costs incurred per patient due to adverse reactions. Among the inpatients, usage rates of drugs listed in the BCJV and GL2015 were 24.0 and 72.4%, respectively, and adverse reactions to these drugs occurred at rates of 3.0 and 8.2%, respectively. Among the outpatients, while the usage rates were 26.2% (BCJV) and 59.9% (GL2015), the incidence rates of adverse reactions were 4.7% (BCJV) and 3.9% (GL2015). The additional costs incurred due to adverse drug reactions ranged from 12713-163925 yen per patient. Our results demonstrate that appropriate use of drugs based on the BCJV and GL2015 can help prevent adverse reactions; this would reduce the overall medical costs.

Mini-open excision of osteoid osteoma using intraoperative O-arm/Stealth navigation.

BACKGROUND: Although osteoid osteomas have traditionally been treated by surgical excision, radiofrequency ablation (RFA) has gained favor as a less invasive procedure. However, RFA is contraindicated for osteoid osteomas close to the skin or crucial neurovascular structures, and is not covered by national health insurance in Japan. The aim of the present study was to evaluate the efficacy of surgical excision of osteoid osteomas using intraoperative navigation. METHODS: We performed a retrospective review of five patients with osteoid osteoma who underwent a mini-open excision using O-arm/Stealth navigation at our institution. The osteoid osteomas were excised using a cannulated cutter or curetted out with the assistance of navigation. RESULTS: Complete excision was achieved in all patients, which was confirmed by pathological examination. The mean skin incision was 2.1 cm (range, 1.5 to 3.0 cm) and the mean duration required for setup three-dimensional image was 15 min (range, 12 to 20 min). Although the mean visual analog scale score was 7 (range, 4 to 8) before surgery, all patients experienced relief from their characteristic pain immediately after surgery, with the mean scores of 2.2 (range, 1 to 3) and 0 at 2 days and 4 weeks after surgery, respectively. There was no intra-operative complication related to the navigation and no recurrence was observed during the mean follow-up period of 25 months (range, 13 to 33 months). CONCLUSIONS: Mini-open excision using intraoperative O-arm/Stealth navigation is a safe and accurate procedure for patients with osteoid osteoma, which could cover the limitation of RFA.

A simple method for exploring adverse drug events in patients with different primary diseases using spontaneous reporting system.

BACKGROUND: Patient background (e.g. age, sex, and primary disease) is an important factor to consider when monitoring adverse drug events (ADEs) for the purpose of pharmacovigilance. However, in disproportionality methods, when additional factors are considered, the number of combinations that have to be computed increases, and it becomes very difficult to explore the whole spontaneous reporting system (SRS). Since the signals need to be detected quickly in pharmacovigilance, a simple exploration method is required. Although association rule mining (AR) is commonly used for the analysis of large data, its application to pharmacovigilance is rare and there are almost no studies comparing AR with conventional signal detection methods. METHODS: In this study, in order to establish a simple method to explore ADEs in patients with kidney or liver injury as a background disease, the AR and proportional reporting ratio (PRR) signal detection methods were compared. We used oral medicine SRS data from the Japanese Adverse Drug Event Report database (JADER), and used AR as the proposed search method and PRR as the conventional method for comparison. "Rule count ≥ 3", "min lift value > 1", and "min conviction value > 1" were used as the AR detection criteria, and the PRR detection criteria were "Rule count ≥3", "PRR ≥ 2", and "χ2 ≥ 4". RESULTS: In patients with kidney injury, the AR method had a sensitivity of 99.58%, specificity of 94.99%, and Youden's index of 0.946, while in patients with liver injury, the sensitivity, specificity, and Youden's index were 99.57%, 94.87%, and 0.944, respectively. Additionally, the lift value and the strength of the signal were positively correlated. CONCLUSIONS: It was suggested that computation using AR might be simple with the detection power equivalent to that of the conventional signal detection method as PRR. In addition, AR can theoretically be applicable to SRS other than JADER. Therefore, complicated conditions (patient's background etc.) that must take factors other than the ADE into consideration can be easily explored by selecting the AR as the first screening for ADE exploration in pharmacovigilance using SRS.

Clinical and Functional Significance of Intracellular and Extracellular microRNA-25-3p in Osteosarcoma.

Although there is considerable evidence indicating that the dysregulation of microRNAs (miRNAs) in malignant tumors plays a role in tumor development, the overall function of miRNAs and their clinicopathological significance are not well understood. In this retrospective analysis of 45 biopsy specimens from osteosarcoma (OS) patients, we investigated the functional and clinical significance of miR-25-3p in OS, which we previously identified as a highly expressed miRNA in OS patients' serum. We observed that miR-25-3p dysregulation in human OS tissues was negatively correlated with the clinical prognosis, whereas the expression level of its target gene, Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3), was positively correlated with the clinical prognosis. Endogenous miR-25-3p upregulation promoted tumor growth, invasion, and drug resistance, which was consistent with DKK3 silencing in OS cells. In addition, secretory miR-25-3p was embedded in tumor-derived exosomes, where it promoted capillary formation and the invasion of vascular endothelial cells. Overall, our results show that miR-25-3p has intracellular and extracellular oncogenic functions as well as clinicopathological relevance in OS, indicating its potential as a novel diagnostic and therapeutic tool for the clinical management of this disease.

Medical and economic factors influencing generic drug use in the Japanese public health system: Influencing factors in different populations.

Factors influencing generic drug use must be considered when new drug policies are established and initiatives are implemented to promote generic drug use. This study was conducted to elucidate medical and economic factors that influence generic drug use in the Japanese public health system by evaluating the degree of generic drug use via a multivariate analysis. We conducted a retrospective study of medications administered to inpatients at Gifu Municipal Hospital (Japan) from November 1 to 14, 2014. Details of inpatients (age, sex, and type of medical insurance) and the drugs administered (prescribing institution, dispensing pharmacy, price, and class) were assessed. A total of 1409 drugs (original, 639; generic, 770) were analyzed. Multivariate analysis showed significant differences in out-of-pocket medical fees [odds ratio (OR), 0.595], drugs prescribed at Gifu Municipal Hospital (OR, 1.811), drugs prepared at a health insurance pharmacy (OR, 1.541), drugs containing the same active substances as in the generic drugs used at Gifu Municipal Hospital (OR, 3.712), and drugs costing ≥30 yen and containing the same active substance/having the same specifications (OR, 0.516). Drugs prescribed at a large key hospital in the community with high adoption rates of generic drugs, drugs containing the same active substances as the generic drugs adopted by the hospital, and drugs prepared at health insurance pharmacies contributed to a more frequent use of generic drugs. By contrast, out-of-pocket medical fees and being prescribed expensive drugs contributed to the less frequent use of generic drugs.

Intraoperative O-arm-navigated resection in musculoskeletal tumors.

BACKGROUND: Although emerging evidence has suggested that computer-assisted navigation allows surgeons to plan the optimal level of resection without compromising the surgical margins, the precise accuracy of the procedures has been unclear. The aim of this study was to investigate the accuracy and safety of the musculoskeletal tumor resection using O-arm/Stealth intraoperative navigation assistance. METHODS: A retrospective study of six patients with bone and soft tissue tumors who underwent surgical resection using O-arm/Stealth navigation system was performed. The histological diagnosis was osteosarcoma, metastatic bone tumor, leiomyosarcoma, undifferentiated sarcoma, and synovial sarcoma, respectively. Tumor resection was performed according to planned osteotomy planes determined on O-arm/Stealth three-dimensional intraoperative images. The resection accuracy, length of time for the procedures, surgical margins, and perioperative complications were evaluated. RESULTS: The distances between the entry and exit points for the planned and actual cuts were 1.5 ± 0.3 mm and 2.3 ± 0.3 mm, respectively, and the mean discrepancy of the osteotomy angle was 2.8 ± 1.2°. The mean length of time required for navigation was 14 min. A histological examination revealed clear margins in all patients. There were no complications related to navigation, and no patients developed local recurrence during a mean follow-up of 30.6 months. CONCLUSIONS: The O-arm/Stealth intraoperative CT navigation system provides safe and accurate osteotomy in musculoskeletal tumor resections. However, surgeons should keep in mind and be careful of minimal errors during osteotomy, which are around 2 mm from the planned line.

Role of zoledronic acid in oncolytic virotherapy: Promotion of antitumor effect and prevention of bone destruction.

Osteosarcoma is an aggressive malignant bone tumor that causes bone destruction. Although tumor-specific replicating oncolytic adenovirus OBP-301 induces an antitumor effect in an osteosarcoma tumor, it cannot prevent bone destruction. Zoledronic acid (ZOL) is a clinically available agent that inhibits bone destruction. In this study, we investigated the potential of combination therapy with OBP-301 and ZOL against osteosarcomas with bone destruction. The antitumor activity of OBP-301 and ZOL in monotherapy or combination therapy was assessed using three human osteosarcoma cell lines (143B, MNNG/HOS, SaOS-2). The cytotoxic effect of OBP-301 and/or ZOL was measured by assay of cell apoptosis. The effect of OBP-301 and ZOL on osteoclast activation was investigated. The potential of combination therapy against tumor growth and bone destruction was analyzed using an orthotopic 143B osteosarcoma xenograft tumor model. OBP-301 and ZOL decreased the viability of human osteosarcoma cells. Combination therapy with OBP-301 and ZOL displayed a synergistic antitumor effect, in which OBP-301 promoted apoptosis through suppression of anti-apoptotic myeloid cell leukemia 1 (MCL1). Combination therapy significantly inhibited tumor-mediated osteoclast activation, tumor growth and bone destruction compared to monotherapy. These results suggest that combination therapy of OBP-301 and ZOL suppresses osteosarcoma progression via suppression of MCL1 and osteoclast activation.

Unique mutation portraits and frequent COL2A1 gene alteration in chondrosarcoma.

Chondrosarcoma is the second most frequent malignant bone tumor. However, the etiological background of chondrosarcomagenesis remains largely unknown, along with details on molecular alterations and potential therapeutic targets. Massively parallel paired-end sequencing of whole genomes of 10 primary chondrosarcomas revealed that the process of accumulation of somatic mutations is homogeneous irrespective of the pathological subtype or the presence of IDH1 mutations, is unique among a range of cancer types, and shares significant commonalities with that of prostate cancer. Clusters of structural alterations localized within a single chromosome were observed in four cases. Combined with targeted resequencing of additional cartilaginous tumor cohorts, we identified somatic alterations of the COL2A1 gene, which encodes an essential extracellular matrix protein in chondroskeletal development, in 19.3% of chondrosarcoma and 31.7% of enchondroma cases. Epigenetic regulators (IDH1 and YEATS2) and an activin/BMP signal component (ACVR2A) were recurrently altered. Furthermore, a novel FN1-ACVR2A fusion transcript was observed in both chondrosarcoma and osteochondromatosis cases. With the characteristic accumulative process of somatic changes as a background, molecular defects in chondrogenesis and aberrant epigenetic control are primarily causative of both benign and malignant cartilaginous tumors.

Inhibitory effect of JAK inhibitor on mechanical stress-induced protease expression by human articular chondrocytes.

OBJECTIVE: To investigate whether janus kinase (JAK) inhibitor exhibits a chondro-protective effect against mechanical stress-induced expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinase (MMPs) in human chondrocytes. MATERIALS AND METHODS: Normal human articular chondrocytes were seeded onto stretch chambers and incubated with or without tofacitinib (1000 nM) for 12 h before mechanical stimulation or cytokine stimulation. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 10% elongation, 30 min) was applied and the gene expression levels of type II collagen α1 chain (COL2A1), aggrecan (ACAN), ADAMTS4, ADAMTS5, MMP13, and runt-related transcription factor 2 (RUNX-2) were examined by real-time polymerase chain reaction. Nuclear translocation of RUNX-2 and nuclear factor-κB (NF-κB) was examined by immunocytochemistry, and phosphorylation of mitogen-activated protein kinase (MAPK) and signaling transducer and activator of transcription (STAT) 3 was examined by western blotting. The concentration of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α in the supernatant was examined by enzyme-linked immunosorbent assay. RESULTS: COL2A1 and ACAN gene expression levels were decreased by CTS, but these catabolic effects were canceled by tofacitinib. Tofacitinib significantly down-regulated CTS-induced expression of ADAMTS4, ADAMTS5, MMP13, and RUNX2, and the release of IL-6 in supernatant by chondrocytes. Tofacitinib also reduced CTS-induced nuclear translocation of RUNX-2 and NF-κB, and phosphorylation of MAPK and STAT3. CONCLUSION: Tofacitinib suppressed mechanical stress-induced expression of ADAMTS4, ADAMTS5, and MMP13 by human chondrocytes through inhibition of the JAK/STAT and MAPK cascades.

Hyaluronan suppresses mechanical stress-induced expression of catabolic enzymes by human chondrocytes via inhibition of IL-1ß production and subsequent NF-κB activation.

OBJECTIVE: To investigate the inhibitory effect of hyaluronan (HA) on mechanical stress- induced expression of a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS)-4, -5 and matrix metalloproteinase (MMP)-13 by human chondrocytes. MATERIALS AND METHODS: Normal human articular chondrocytes were pre-incubated with or without 1.0 mg/mL HA (2700 kDa) for 12 h at 37 °C in stretch chambers, then they were exposed to uni-axial cyclic tensile strain (CTS, 0.5 Hz, 10% elongation). The expression of ADAMTS-4, -5, and MMP-13 were analyzed by real-time polymerase chain reaction and Immunocytochemistry. The concentration of IL-1ß in the supernatant was measured using enzyme-linked immunosorbent assay (ELISA). The nuclear translocation of runt-related transcription factor 2 (RUNX-2) and nuclear factor-κB (NF-κB) was examined by ELISA and immunocytochemistry, and phosphorylation of NF-κB was examined by western blotting. RESULTS: HA inhibited mRNA expression of ADAMTS-4, -5, and MMP13 after 24 h CTS via inhibition of IL-1ß secretion and NF-κB activation. However, HA failed to inhibit CTS-induced RUNX-2 expression and subsequent expression of ADAMTS-5 and MMP-13 1 h after CTS. CONCLUSIONS: Our results demonstrated that HA significantly suppressed mechanical stress-induced expression of catabolic proteases by inhibition of the NF-κB-IL-1ß pathway, but did not suppress mechanical stress-induced RUNX-2 signaling.