Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Previous studies demonstrated that inhibition of HA suppressed the tumorigenicity of various malignant tumors including breast cancer. 4-methylumbelliferone (MU) has been reported to inhibit HA synthesis in several cell types. However, few studies have focused on the effects of HA inhibition in breast cancer cells by MU, nor the effects on bone metastasis. We hypothesized that MU would suppress the progression of bone metastasis via inhibition of HA synthesis. Here, we investigated the effects of MU on HA expression in MDA-MB-231 breast cancer cell line in addition to their tumorigenicity in vitro and in vivo. HAS2 mRNA expression was downregulated after 6 and 24 hr treatment with MU. Quantitative analysis of HA revealed that MU significantly inhibited the intracellular and cell surface HA. MU significantly inhibited cell growth and induced apoptosis as determined by cell proliferation and TUNEL assays, respectively. Phosphorylation of Akt was suppressed after 12 and 24 hr treatment with MU. MU treatment also inhibited cell motility as well as cell invasiveness. MU also inhibited cell growth and motility in murine fibroblast cell line NIH3T3. In vivo, administration of MU inhibited the expansion of osteolytic lesions on soft X-rays in mouse breast cancer xenograft models. HA accumulation in bone metastatic lesions was perturbed peripherally. These data suggest that MU might be a therapeutic candidate for bone metastasis of breast cancer via suppression of HA synthesis and accumulation.Breast cancer is the most common cancer in women particularly in western countries. 1,2 Approximately 6% of breast cancer patients present to hospital with distant metastasis 2 and about 25% despite neoadjuvant or adjuvant systemic treatment develop distant metastasis. 3 In a retrospective study of patients with metastatic breast cancer, bone was the most common site of metastatic spread and 70% of these patients developed metastases in one or more bones before they died. 4 Bone metastases occasionally result in hypercalcemia, pathologic fractures and spinal cord compression, 5 and substantially reduce patients' quality of life. 6 Hyaluronan (HA) is a high molecular weight linear glycosaminoglycan, the structure of which is composed of repeating disaccharides of D-glucuronic acid and N-acetyl-Dglucosamine. HA is a ubiquitous extracellular and cell surface associated matrix component of connective, epithelial and neural tissues. 7 HA is abundant in surrounding migrating and proliferating cells during morphogenesis and wound healing. 8,9 Increased HA levels are also observed, both in stroma of malignant areas and in a part of tumor parenchyma in some malignant tumors including colorectal, ovarian and breast cancer and liposarcoma. [10][11][12][13][14] Not only the HA accumulation level in the stroma of malignancy 11,15,16 but also the HA level in tumor parenchyma 10,13 has been reported to associate with a poor clinical outcome. In in vitr...

Background:Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma.Methods:We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS).Results:In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 m). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU.Conclusion:These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.

Objective. To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblastlike synoviocytes (FLS) derived from patients with rheumatoid arthritis.Methods. DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively.Results. Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor ␣-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression.Conclusion. Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.

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.

Hyaluronan (HA) oligosaccharides were reported to have suppressive effects on various malignant tumors via disruption of receptor HA interactions. However, no studies have focused on the effects of HA oligosaccharides on bone metastasis of breast cancer. In this study, we clarified the effective size of HA oligosaccharides required to inhibit cell growth in the highly invasive breast cancer cell line, MDA-MB-231 cells. Based on the results of cell growth assay, we subsequently analyzed the effects of HA tetrasaccharides, HA decasaccharides, and high molecular weight HA on the other breast cancer cell behaviors in vitro and breast cancer bone metastasis in vivo. HA decasaccharides significantly inhibited cell growth, motility, and invasion, whereas tetrasaccharides did not. HAS2 mRNA expression was altered after the treatment with both tetrasaccharides and decasaccharides. Phosphorylation of Akt was suppressed after 1 h treatment with HA decasaccharides, and the effect was partially reversed by anti-CD44 monoclonal antibody. In vivo, local application of HA decasaccharides inhibited the expansion of osteolytic lesions in tibia on soft X-rays using mouse bone metastasis model of breast cancer. Histological analysis revealed HA accumulation in bone metastatic lesions was perturbed by decasaccharides. These results suggest that HA oligosaccharides suppressed progression of bone metastasis in breast cancer via interruption of endogenous HA-CD44 interaction, and as such, can be a novel therapeutic candidate to limit bone metastasis of breast cancer.