Identification and Functional Analysis of a Novel CTNNB1 Mutation in Pediatric Medulloblastoma

Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3 CTNNB1 mutations and were immunostained for nuclear β-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of β-catenin Ser37 residue (ΔS37). The ΔS37 β-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type β-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 β-catenin contributed to early medulloblastoma tumorigenesis.

Osteogenic Potential of Magnesium (Mg)-Doped Multicomponent Bioactive Glass: In Vitro and In Vivo Animal Studies

The use of bioactive glasses (BGs) has been quite fruitful in hard tissue engineering due to the capability of these materials to bond to living bone. In this work, a melt-derived magnesium (Mg)-doped BG (composition: 45SiO2–3P2O5–26CaO–15Na2O–7MgO–4K2O (mol.%)) was synthesized for being used in bone reconstruction. The prepared BGs were then manufactured as three-dimensional (3D) scaffolds by using the sponge replica approach. The microstructure of the samples was assessed by X-ray diffraction (XRD) and the surface morphology was observed by using scanning electron microscopy (SEM). The in vitro bioactivity and the release of osteo-stimulatory Mg2+ ions from the prepared samples were investigated over 7 days of incubation in simulated body fluids (SBF). In vitro cellular analyses revealed the compatibility of the Mg-doped BGs with human osteosarcoma cells (MG-63 cell line). Moreover, the Mg-doped BGs could induce bone nodule formation in vitro and improve the migratory ability of human umbilical vein endothelial cells (HUVECs). In vivo osteogenic capacity was further evaluated by implanting the BG-derived scaffolds into surgically-created critical-size bone defects in rats. Histological and immunohistological observations revealed an appropriate bone regeneration in the animals receiving the glass-based scaffolds after 12 weeks of surgery. In conclusion, our study indicates the effectiveness of the Mg-doped BGs in stimulating osteogenesis in both in vitro and in vivo conditions.

Niclosamide Suppresses Migration and Invasion of Human Osteosarcoma Cells by Repressing TGFBI Expression via the ERK Signaling Pathway

Osteosarcoma is a highly common malignant bone tumor. Its highly metastatic properties are the leading cause of mortality for cancer. Niclosamide, a salicylanilide derivative, is an oral antihelminthic drug of known anticancer potential. However, the effect of niclosamide on osteosarcoma cell migration, invasion and the mechanisms underlying have not been fully clarified. Therefore, this study investigated niclosamide’s underlying pathways and antimetastatic effects on osteosarcoma. In this study, U2OS and HOS osteosarcoma cell lines were treated with niclosamide and then subjected to assays for determining cell migration ability. The results indicated that niclosamide, at concentrations of up to 200 nM, inhibited the migration and invasion of human osteosarcoma U2OS and HOS cells and repressed the transforming growth factor beta-induced protein (TGFBI) expression of U2OS cells, without cytotoxicity. After TGFBI knockdown occurred, cellular migration and invasion behaviors of U2OS cells were significantly reduced. Moreover, niclosamide significantly decreased the phosphorylation of ERK1/2 in U2OS cells and the combination treatment of the MEK inhibitor (U0126) and niclosamide resulted in the intensive inhibition of the TGFBI expression and the migratory ability in U2OS cells. Therefore, TGFBI derived from osteosarcoma cells via the ERK pathway contributed to cellular migration and invasion and niclosamide inhibited these processes. These findings indicate that niclosamide may be a powerful preventive agent against the development and metastasis of osteosarcoma.

Diosmetin inhibits cell proliferation and promotes apoptosis through STAT3/c-Myc signaling pathway in human osteosarcoma cells

Background Diosmetin is a bioflavonoid compound naturally abundant in citrus fruits. It is found to perform a variety of activities, while its antitumor property in osteosarcoma, a malignant tumor with unmet clinical treatment, remained unknown. Methods Colony formation assay, cell cycle analysis and apoptosis analysis were conducted respectively to observe the effect of diosmetin on cell proliferation and apoptosis in human osteosarcoma cells. Western blot and immunoprecipitation were used to detect the expression of apoptotic molecules and activation of STAT3/c-Myc pathway in Saos-2 and U2SO cells. Results Diosmetin significantly inhibited cell proliferation, induced cell cycle arrest at G2/M phase and promoted cell apoptosis in both Saos-2 and U2SO cells. Moreover, Diosmetin downregulated the expression of anti-apoptotic protein Bcl-xL while upregulated the levels of pro-apoptotic proteins including cleaved Caspase-3, cleaved-PARP and Bax. Furthermore, diosmetin dose-dependently inhibited STAT3 phosphorylation, reduced the expression of its downstream protein c-Myc and impeded the interaction between STAT3 molecules. Conclusions These results suggest that diosmetin exerts anti-osteosarcoma effects by suppressing cell proliferation and inducing apoptosis via inhibiting the activation of STAT3/c-Myc signaling pathway, which provide the possibility for diosmetin to be a chemotherapeutic candidate for osteosarcoma.

Imperatorin induces autophagy and G0/G1 phase arrest via PTEN-PI3K-AKT-mTOR/p21 signaling pathway in human osteosarcoma cells in vitro and in vivo

Background Osteosarcoma is the third most common cancer in adolescence and the first common primary malignant tumor of bone. The long-term prognosis of osteosarcoma still remains unsatisfactory in the past decades. Therefore, development of novel therapeutic agents which are effective to osteosarcoma and are safe to normal tissue simultaneously is quite essential and urgent. Methods Firstly, MTT assay, cell colony formation assay, cell migration and invasion assays were conducted to evaluate the inhibitory effects of imperatorin towards human osteosarcoma cells. RNA-sequence assay and bioinformatic analysis were then performed to filtrate and assume the potential imperatorin-induced cell death route and signaling pathway. Moreover, quantitative real-time PCR assay, western blot assay and rescue experiments were conducted to confirm the assumptions of bioinformatic analysis. Finally, a subcutaneous tumor-transplanted nude mouse model was established and applied to evaluate the internal effect of imperatorin on osteosarcoma by HE and immunohistochemistry staining. Results Imperatorin triggered time-dependent and dose-dependent inhibition of tumor growth mainly by inducing autophagy promotion and G0/G1 phase arrest in vitro and in vivo. Besides, imperatorin treatment elevated the expression level of PTEN and p21, down-regulated the phosphorylation of AKT and mTOR. In contrast, the inhibition of PTEN using Bpv (HOpic), a potential and selective inhibitor of PTEN, concurrently rescued imperatorin-induced autophagy promotion, cell cycle arrest and inactivation of PTEN-PI3K-AKT-mTOR/p21 pathway. Conclusions This work firstly revealed that imperatorin induced autophagy and cell cycle arrest through PTEN-PI3K-AKT-mTOR/p21 signaling pathway by targeting and up-regulating PTEN in human osteosarcoma cells. Hence, imperatorin is a desirable candidate for clinical treatments of osteosarcoma.

Peiminine Induces G0/G1-Phase Arrest, Apoptosis, and Autophagy via the ROS/JNK Signaling Pathway in Human Osteosarcoma Cells in Vitro and in Vivo

Aims: Peiminine has been reported to have various pharmacological properties, including anticancer activity. In this study, we investigated the effect of this alkaloid on osteosarcoma and explored the underlying mechanisms.Methods: To evaluate the antiosteosarcoma effects of peiminine in vitro, cell viability was assessed by CCK-8 and live/dead assays; the effects of the drug on apoptosis and the cell cycle were examined by flow cytometry; the effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively, while its effects on autophagy were observed by transmission electron microscopy and an LC3 fluorescent puncta formation assay. The role of autophagy in the peiminine-mediated effects in osteosarcoma cells was evaluated by CCK-8 assay and western blotting after the application of the autophagy inhibitor chloroquine. The effect of peiminine on reactive oxygen species (ROS) production was analyzed using fluorescence confocal microscopy and spectrophotometry. Additionally, peiminine-treated osteosarcoma cells were exposed to SP600125, a JNK inhibitor, and N-acetylcysteine, a ROS scavenger, after which the contribution of the ROS/JNK signaling pathway to osteosarcoma was assessed using cell viability and LC3 fluorescent puncta formation assays, flow cytometry, and western blotting. A xenograft mouse model of osteosarcoma was generated to determine the antitumor effects of peiminine in vivo.Results: Peiminine suppressed proliferation and metastasis and induced cell cycle arrest, apoptosis, and autophagy in osteosarcoma cells. These anticancer effects of peiminine were found to be dependent on intracellular ROS generation and activation of the JNK pathway. In line with these results, peiminine significantly inhibited xenograft tumor growth in vivo.Conclusions: Peiminine induced G0/G1-phase arrest, apoptosis, and autophagy in human osteosarcoma cells via the ROS/JNK signaling pathway both in vitro and in vivo. Our study may provide an experimental basis for the evaluation of peiminine as an alternative drug for the treatment of osteosarcoma.

Calcium Phosphate-Coated Lipid Nanoparticles as a Potential Tool in Bone Diseases Therapy

The treatment of bone diseases (including osteoporosis, osteoarthritis, and bone cancer) often results in reduced efficiency and/or adverse reactions due to the fact that it is not specifically targeted to the site of action. The employment of a suitable carrier should increase drug location to the site of bone disease. The purpose of this study is to prepare and characterize lipid nanoparticles (NPs) coated with calcium phosphate (CaP-NPs). A coating method, to date used only to obtain liposomes covered with CaP, is herein partially-modified to prepare CaP-coated lipid NPs. An extensive physico-chemical characterization was achieved by employing several techniques (DLS, SEM and TEM, and both combined with EDS, XRD, and FTIR) that confirmed the feasibility of the developed coating method. Preliminary uptake studies on human osteosarcoma cells (U-2OS) were performed by entrapping, as a lipid probe, Sudan Red III in NPs. The obtained data provided evidence that CaP-NPs showed higher cell accumulation than uncoated NPs. This result may have important implications for the development of drug loaded CaP-NPs to be tested in vitro with a view of planning future treatment of bone diseases, and indicate that CaP-NPs are potential vehicles for selective drug delivery to bone tissue.

Downregulation of the Proton-Activated Cl- Channel TMEM206 Inhibits Malignant Properties of Human Osteosarcoma Cells

Transmembrane protein 206 (TMEM206), a proton-activated chloride channel, has been implicated in various biochemical processes, including bone metabolism, and has emerged as a novel cancer-related protein in multiple tumor types. However, its role in primary malignant bone tumors, particularly in osteosarcoma (OS), remains unclear. This study is aimed at exploring the effects of TMEM206 gene silencing on the proliferation, migration, invasion, and metastasis of human OS cells in vitro and in vivo using an shRNA-knockdown strategy. We found that TMEM206 is frequently overexpressed and that high levels of TMEM206 correlated with clinical stage and pulmonary metastasis in patients with OS. We provided evidence that TMEM206-silenced OS cancer cells exhibit decreased proliferation, migration, and invasion in vitro. Mechanistically, we identified β-catenin, a key member of Wnt/β-catenin signaling, as a downstream effector of TMEM206. TMEM206 silencing inhibits the Wnt/β-catenin signaling pathway in expression rescue experiments, confirming that TMEM206 silencing attenuates OS cell tumorigenic behavior, at least in part, via the β-catenin mediated downregulation of Wnt/β-catenin signaling. More importantly, TMEM206 knockdown-related phenotype changes were replicated in a xenograft nude mouse model where pulmonary metastases of OS cells were suppressed. Together, our results demonstrate that silencing TMEM206 negatively modulates the Wnt/β-catenin signaling pathway via β-catenin to suppress proliferation, migration, invasion, and metastasis in OS carcinogenesis, suggesting TMEM206 as a potential oncogenic biomarker and a potential target for OS treatment.