Kunihiro Ikuta

CTNNB1 S45F mutation predicts poor efficacy of meloxicam treatment for desmoid tumors: a pilot study.

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.

EMMPRIN co-expressed with matrix metalloproteinases predicts poor prognosis in patients with osteosarcoma.

Several studies have focused on the relationships between the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and the prognosis of patients with malignant tumors. However, few of these have investigated the expression of EMMPRIN in osteosarcoma. We examined expression levels of EMMPRIN immunohistochemically in 53 cases of high-grade osteosarcoma of the extremities and analyzed the correlation of its expression with patient prognosis. The correlation between matrix metalloproteinases (MMPs) and EMMPRIN expression and the prognostic value of co-expression were also analyzed. Staining positivity for EMMPRIN was negative in 7 cases, low in 17, moderate in 19, and strong in 10. The overall and disease-free survivals (OS and DFS) in patients with higher EMMPRIN expression (strong-moderate) were significantly lower than those in the lower (weak-negative) group (0.037 and 0.024, respectively). In multivariate analysis, age (P = 0.004), location (P = 0.046), and EMMPRIN expression (P = 0.038) were significant prognostic factors for overall survival. EMMPRIN expression (P = 0.024) was also a significant prognostic factor for disease-free survival. Co-expression analyses of EMMPRIN and MMPs revealed that strong co-expression of EMMPRIN and membrane-type 1 (MT1)-MMP had a poor prognostic value (P = 0.056 for DFS, P = 0.006 for OS). EMMPRIN expression and co-expression with MMPs well predict the prognosis of patients with extremity osteosarcoma, making EMMPRIN a possible therapeutic target in these patients.

Nuclear expression of β-catenin predicts the efficacy of meloxicam treatment for patients with sporadic desmoid tumors.

This study aimed to determine the prevalence of β-catenin nuclear positivity as a prognostic factor in patients with desmoid tumors (DTs) treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive 31 patients with extraabdominal, sporadic DTs were prospectively treated with meloxicam as a systemic medical therapy. Immunohistochemistry was performed on formalin-fixed material to quantify the nuclear expression of β-catenin and Ki-67, and cytoplasmic expression of COX-2. All clinicopathological characteristics including the intensity of immunohistochemical staining were analyzed with respect to their prognostic value for meloxicam treatment. Of the 31 patients with meloxicam treatment, there was 1 with complete remission (CR), 7 with partial remission (PR), 12 with stable disease (SD), and 11 with progressive disease (PD). Higher nuclear expression of β-catenin was significantly associated with a poor response (PD/SD) (p = 0.017). The positivity of COX-2 and Ki-67 and none of the other clinical variables were associated with prognosis. The nuclear expression of β-catenin can predict the efficacy of meloxicam treatment for patients with sporadic DTs.

In vivo heat-stimulus-triggered osteogenesis

Abstract Several studies have reported that heat stress stimulates the activity of osteoblastic cells in vitro. However, few have addressed the effects of heat stress on osteogenesis in vivo, nor have the optimal temperatures for bone formation been determined. The aim of the present study was to investigate the effects of hyperthermia treatment on osteogenesis in a rat tibial defect model. Forty-four Sprague Dawley rats were divided into two groups with or without hyperthermia treatment. A 3-mm circular defect in the proximal tibia filled with magnetite cationic liposomes embedded in alginate beads was subjected to hyperthermia treatment (43–46 °C). Radiological assessment at 2 weeks after the treatment showed that significantly stimulated osteogenesis was observed in the hyperthermia group as compared to the control group (p = 0.003). Histomorphometrical analysis at 2 weeks revealed a significant increase of newly formed bone in the hyperthermia group, compared with the control group (p < 0.001). Area of newly formed bone in each hyperthermia group was significantly increased as compared with the control group (43 °C; p = 0.005, 44 °C; p = 0.019, 45 °C; p = 0.003, and 46 °C; p = 0.003, respectively). Alkaline phosphatase was overexpressed at the surfaces of newly formed bone adjacent to magnetite cationic liposome implantation. Our results demonstrate for the first time that heat stimulus accelerates osteogenesis in vivo, and may thus be of interest as a novel and promising tool to induce osteogenesis clinically as well.

Expression of colony-stimulating factor 1 is associated with occurrence of osteochondral change in pigmented villonodular synovitis

Pigmented villonodular synovitis (PVNS) is a benign, translocation-derived neoplasm. Because of its high local recurrence rate after surgery and occurrence of osteochondral destruction, a novel therapeutic target is required. The present study aimed to evaluate the significance of protein expression possibly associated with the pathogenesis during the clinical course of PVNS. In 40 cases of PVNS, positivity of colony-stimulated factor 1 (CSF1), its receptor (CSF1R), and receptor activator of nuclear factor kappa-B ligand (RANKL) were immunohistochemically determined. The relationship between the positivity and clinical outcomes was investigated. High positivity of CSF1 staining intensity was associated with an increased incidence of osteochondral lesions (bone erosion and osteoarthritis) (p = 0.009), but not with the rate of local recurrence. Positivity of CSF1R and RANKL staining was not associated with any clinical variables. The number of giant cells was not correlated with positivity of any of the three proteins, or with the clinical outcome. Focusing on knee cases, CSF1 positivity was also associated with the incidence of osteochondal change (p = 0.02). CSF1R positivity was high in cases which had local recurrence, but not significantly so (p = 0.129). Determination of CSF1 and CSF1R expression may be useful as a prognosticator of the clinical course and/or outcomes of PVNS.

Antitumor effects of 4-methylumbelliferone, a hyaluronan synthesis inhibitor, on malignant peripheral nerve sheath tumor.

Hyaluronan (HA) has been shown to play important roles in the growth, invasion and metastasis of malignant tumors. Our previous study showing that high HA expression in malignant peripheral nerve sheath tumors (MPNST) is predictive of poor patient prognosis, prompted us to speculate that inhibition of HA synthesis in MPNST might suppress the tumorigenicity. The aim of our study was to investigate the antitumor effects of 4‐methylumbelliferone (MU), an HA synthesis inhibitor, on human MPNST cells and tissues. The effects of MU on HA accumulation and tumorigenicity in MPNST cells were analyzed in the presence or absence of MU in an in vitro as well as in vivo xenograft model using human MPNST cell lines, sNF96.2 (primary recurrent) and sNF02.2 (metastatic). MU significantly inhibited cell proliferation, migration and invasion in both MPNST cell lines. HA binding protein (HABP) staining, particle exclusion assay and quantification of HA revealed that MU significantly decreased HA accumulation in the cytoplasms and pericellular matrices in both MPNST cell lines. The expression levels of HA synthase2 (HAS2) and HA synthase3 (HAS3) mRNA were downregulated after treatment with MU. MU induced apoptosis of sNF96.2 cells, but not sNF02.2 cells. MU administration significantly inhibited the tumor growth of sNF96.2 cells in the mouse xenograft model. To the best of our knowledge, our study demonstrates for the first time the antitumor effects of MU on human MPNST mediated by inhibition of HA synthesis. Our results suggest that MU may be a promising agent with novel antitumor mechanisms for MPNST.

Characteristics of cultured desmoid cells with different CTNNB1 mutation status

Desmoid tumors are benign mesenchymal neoplasms with a locally aggressive nature. The mutational status of β‐catenin gene (CTNNB1) is presumed to affect the tumorous activity of the cells. In this study, we isolated three kinds of desmoid cell with different CTNNB1 status, and compared their characteristics. Cells were isolated from three patients with abdominal wall desmoid during surgery, all of which were resistant to meloxicam treatment. The mutational status of the CTNNB1 exon 3 was determined for both parental tumor tissues and isolated cultured cells. β‐catenin expression was determined with immunohistochemistry. Responsiveness to meloxicam was investigated with MTS assay together with COX‐2 immunostaining. mRNA expressions of downstream molecules of Wnt/β‐catenin pathway were determined with real‐time RT‐PCR. Three kinds of cell isolated from desmoid tumors harboring different CTNNB1 mutation status (wild type, T41A, and S45F), all exhibited a spindle shape. These isolated cells could be cultured until the 20th passage with unchanged proliferative activity. Nuclear accumulation of β‐catenin was observed in all cultured cells, particularly in those with S45F. Proliferating activity was significantly suppressed by meloxicam (25 μmol/L, P < 0.007) in all three cell cultures, of which parental desmoid was resistant to meloxicam clinically. The mRNA expressions of Axin2, c‐Myc, and Cyclin D1 differently increased in the three cultured cell types as compared with those in human skin fibroblast cells (HDF). Inhibitors of Wnt/β‐catenin pathway downregulated Axin2, c‐Myc, and Cyclin D1 significantly. Isolated and cultured desmoid tumor cells harboring any one of the CTNNB1 mutation status had unique characteristics, and could be useful to investigate desmoid tumors with different mutation status of CTNNB1.

Conditional knockdown of hyaluronidase 2 in articular cartilage stimulates osteoarthritic progression in a mice model

The catabolism of hyaluronan in articular cartilage remains unclear. The aims of this study were to investigate the effects of hyaluronidase 2 (Hyal2) knockdown in articular cartilage on the development of osteoarthritis (OA) using genetic manipulated mice. Destabilization of the medial meniscus (DMM) model of Col2a promoter specific conditional Hyal2 knockout (Hyal−/−) mice was established and examined. Age related and DMM induced alterations of articular cartilage of knee joint were evaluated with modified Mankin score and immunohistochemical staining of MMP-13, ADAMTS-5, KIAA11199, and biotinylated- hyaluronan binding protein staining in addition to histomorphometrical analyses. Effects of Hyal2 suppression were also analyzed using explant culture of an IL-1α induced articular cartilage degradation model. The amount and size of hyaluronan in articular cartilage were higher in Hyal2−/− mice. Hyal2−/− mice exhibited aggravated cartilage degradation in age-related and DMM induced mice. MMP-13 and ADAMTS-5 positive chondrocytes were significantly higher in Hyal2−/− mice. Articular cartilage was more degraded in explant cultures obtained from Hyal2−/− mice. Knockdown of Hyal2 in articular cartilage induced OA development and progression possibly mediated by an imbalance of HA metabolism. This suggests that Hyal2 knockdown exhibits mucopolysaccharidosis-like OA change in articular cartilage similar to Hyal1 knockdown.

Short-range ultraviolet irradiation with LED device effectively increases serum levels of 25(OH)D

Impairment of the activities of daily living (ADL) by osteoporosis is an important concern in developed countries with a super-aging population. Vitamin D, which is a crucial molecule in bone metabolism and mainly produced endogenously with ultraviolet (UV) light exposure, is known to be insufficient in the elderly population. We used an UV Light-Emitting Diode (UV-LED) instrument generating a narrow-range wavelength to analyze the efficacy of endogenous vitamin D production. The primary purpose of this study was to examine the effects of UV irradiation at various narrow-range wavelengths using UV-LED on vitamin D supplementation. The second one was to clarify the short-term effects of UV irradiation on bone morphology in mice. Vitamin D-starved C57BL/6 female mice (n=7 per group) were UV-irradiated (268nm, 282nm, 290nm, 305nm, and 316nm) with 1kJ/m2 twice a week for 4weeks. UV irradiation using UV-LED had significant effects on increasing serum 25(OH)D levels in all wavelength groups (P<0.001, all groups) as compared to a control group. Among irradiated groups, wavelength of 316nm had a less marked effect on 25(OH)D production compared with other wavelengths at 1week of UV irradiation (P<0.05). Levels of 1,25(OH)2D were significantly increased after 4weeks irradiation with UV-B or UV-C irradiation (P<0.05). mRNA levels of vitamin D 25-hydroxylase were increased with UV-B or UV-C irradiation (268nm-305nm), significantly. Micro-CT examination revealed that short-term (4weeks) UV-irradiation did not induce morphological change of mice in any group. This study provides essential information that narrow-range UV irradiation with LED can increase the endogenous production of vitamin D, and mRNA levels of the responsible enzyme. Although bone morphology was not altered by short-term UV irradiation in this study, an increase of serum vitamin D might improve bone morphology with long-term irradiation.

Suppression of hyaluronan synthesis attenuates the tumorigenicity of low-grade chondrosarcoma

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low‐grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of an HA synthesis inhibitor, 4‐methylumbelliferone (MU), on ECM formation as well as antitumor effects in chondrosarcoma. We investigated the effects of MU on rat chondrosarcoma (RCS) cells with a grade I histological malignancy in vitro and in vivo grafted model. HA binding protein (HABP) stainability on and around the RCS cells was effectively reduced with treatment of MU. ECM formation was markedly suppressed by MU at a dose of 1.0 mM. Cell proliferation was significantly reduced by MU at 24 h. Cell motility and invasion were suppressed in a dose‐dependent manner by MU. No significant changes in mRNA expression of Has1−3 were observed. Furthermore, MU inhibited the growth of grafted tumors in vivo. Histologically, chondrosarcoma cells of control tumors showed a cell‐clustering structure. HABP stainability was markedly decreased in the MU‐treated group. These results suggest that MU exhibits antitumor effects on low‐grade chondrosarcoma, via inhibition of HA accumulation and ECM formation. MU, which is an approved drug in bile therapy, could be a new off‐label medication for chondrosarcomas.