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 ß-catenin, a key member of Wnt/ß-catenin signaling, as a downstream effector of TMEM206. TMEM206 silencing inhibits the Wnt/ß-catenin signaling pathway in expression rescue experiments, confirming that TMEM206 silencing attenuates OS cell tumorigenic behavior, at least in part, via the ß-catenin mediated downregulation of Wnt/ß-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/ß-catenin signaling pathway via ß-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.

NMNAT1 Is a Survival Factor in Actinomycin D-Induced Osteosarcoma Cell Death.

Osteosarcoma is a frequent and extremely aggressive type of pediatric cancer. New therapeutic approaches are needed to improve the overall survival of osteosarcoma patients. Our previous results suggest that NMNAT1, a key enzyme in nuclear NAD+ synthesis, facilitates the survival of cisplatin-treated osteosarcoma cells. A high-throughput cytotoxicity screening was performed to identify novel pathways or compounds linked to the cancer-promoting role of NMNAT1. Nine compounds caused higher toxicity in the NMNAT1 KO U2OS cells compared to their wild type counterparts, and actinomycin D (ActD) was the most potent. ActD-treatment of NMNAT1 KO cells increased caspase activity and secondary necrosis. The reduced NAD+ content in NMNAT1 KO cells was further decreased by ActD, which partially inhibited NAD+-dependent enzymes, including the DNA nick sensor enzyme PARP1 and the NAD+-dependent deacetylase SIRT1. Impaired PARP1 activity increased DNA damage in ActD-treated NMNAT1 knockout cells, while SIRT1 impairment increased acetylation of the p53 protein, causing the upregulation of pro-apoptotic proteins (NOXA, BAX). Proliferation was decreased through both PARP- and SIRT-dependent pathways. On the one hand, PARP inhibitors sensitized wild type but not NMNAT1 KO cells to ActD-induced anti-clonogenic effects; on the other hand, over-acetylated p53 induced the expression of the anti-proliferative p21 protein leading to cell cycle arrest. Based on our results, NMNAT1 acts as a survival factor in ActD-treated osteosarcoma cells. By inhibiting both PARP1- and SIRT1-dependent cellular pathways, NMNAT1 inhibition can be a promising new tool in osteosarcoma chemotherapy.

Safety evaluation of the canine osteosarcoma vaccine, live Listeria vector.

Canine osteosarcoma (OSA) is an aggressive bone tumour in dogs. Standard-of-care treatment typically results in relatively short survival times; thus, alternative treatments are needed to confer a survival advantage. It has been shown that OSA is an immunogenic tumour, suggesting that immune modulation may result in superior outcomes. A cryopreserved, Listeria-based OSA vaccine was recently developed and an initial study in dogs reported prolonged survival for patients receiving the vaccine in conjunction with standard-of-care. The goal of the current observational study was to report on the safety of the lyophilized formulation of this vaccine (the canine OSA vaccine, live Listeria vector [COV-LLV]) in a group of dogs previously diagnosed with OSA. Forty-nine (49) dogs received the COV-LLV and were included for analysis. Adverse events (AEs) noted during and after vaccinations were recorded. The AEs observed were typically mild and self-limiting, with nausea, lethargy and fever being most common. Four dogs (8%) cultured positive for Listeria (three infections including an amputation site abscess, septic stifle joint and bacterial cystitis; and one dog whose lungs cultured Listeria-positive on necropsy within 24 hours of COV-LLV administration). These cases join the previously reported Listeria-positive thoracic abscess that developed in a canine following use of COV-LLV. Although uncommon, it is important to realize this clinically significant AE is possible in patients treated with live therapeutic Listeria vaccines. As Listeria is zoonotic, caution is required not only for the patient receiving the vaccine, but also for the health care workers and family caring for the patient.

MicroRNA-320a inhibits invasion and metastasis in osteosarcoma by targeting cytoplasmic polyadenylation element-binding protein 1.

Osteosarcoma is a primary malignant bone tumor, which affects children, adolescents, and young adults commonly. MicroRNAs (miRNAs) have been proved to be dysregulated in different cancers, including osteosarcoma. Although miR-320a has been implicated in many types of malignancies, little is known about the role of miR-320a in osteosarcoma. In this study, we show that the overexpression of miR-320a or knockdown of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) inhibited osteosarcoma cell migration and invasion. miR-320a downregulates CPEB1 expression by directly targeting the CPEB1 3'-UTR. Furthermore, CPEB1 reintroduction reversed the antiproliferation, antimigration, and antiinvasion roles of miR-320a, indicating that miR-320a might function as a tumor suppressor in osteosarcoma through CPEB1. In conclusion, our study demonstrates that miR-320a plays a critical role in osteosarcoma progression and may provide a potential therapeutic target for osteosarcoma.

LncRNA MEG3 inhibits proliferation and promotes apoptosis of osteosarcoma cells through regulating Notch signaling pathway.

OBJECTIVE: To explore the effect of long non-coding ribonucleic acid (lncRNA)-maternally expressed gene 3 (MEG3) on the Notch signaling pathway, and its influences on the proliferation and apoptosis of osteosarcoma MG-63 cells. MATERIALS AND METHODS: LncRNA MEG3 was overexpressed in osteosarcoma MG-63 cells, and the cells were divided into Blank group, Len-con group, and Len-MEG3 group. The expression level of MEG3 in each group was detected via quantitative Polymerase Chain Reaction (qPCR), the cell proliferation level in each group was detected via Cell Counting Kit-8 (CCK-8) assay, and the apoptosis in each group was detected via Hoechst 33258 staining. Moreover, the content of the inflammatory factors in each group was determined using the Enzyme-Linked Immunosorbent Assay (ELISA), and the expression levels of apoptosis-related proteins and Notch signaling pathway-related proteins were determined through Western blotting. RESULTS: The expression level of lncRNA MEG3 in Len-MEG3 group was significantly higher than that in the Blank group and Len-con group (p<0.01). The overexpression of lncRNA MEG3 could significantly weaken the proliferation (p<0.01) and enhance the apoptosis of osteosarcoma cells (p<0.01). The overexpression of lncRNA MEG3 could significantly increase the content of the inflammatory factor interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (p<0.01), and remarkably decrease the content of the anti-inflammatory factor IL-10 in osteosarcoma cells (p<0.01). Besides, the overexpression of lncRNA MEG3 could evidently raise the expression of Caspase3 (p<0.01) and reduce the Bcl-2/Bax expression in osteosarcoma cells (p<0.01). Finally, the overexpression of lncRNA MEG3 could remarkably reduce the protein expressions of Jagged1, Notch1, and NICD1 in osteosarcoma cells (p<0.01). CONCLUSIONS: The overexpression of lncRNA MEG3 can inhibit the proliferation and promote the apoptosis of osteosarcoma MG-63 cells by suppressing the Notch signaling pathway.

Vaccine strain Listeria monocytogenes abscess in a dog: a case report.

BACKGROUND: Listeria monocytogenes is a promising therapeutic vaccine vector for cancer immunotherapy. Although highly attenuated, three cases of systemic listeriosis have been reported in people following treatment with Listeria-based therapeutic vaccines. This complication has thus far not been reported in canine patients. CASE PRESENTATION: A dog previously diagnosed with osteoblastic osteosarcoma was presented for care following administration of three doses of the Canine Osteosarcoma Vaccine-Live Listeria Vector. On routine staging chest radiographs, mild sternal lymphadenopathy and a right caudoventral thoracic mass effect were noted. Further evaluation of the mass effect with computed tomography and ultrasound revealed a cavitated mass associated with the 7th right rib. Aspirates of the mass cultured positive for Listeria monocytogenes. The mass and associated ribs were surgically removed. Histopathology was consistent with metastatic osteoblastic osteosarcoma. Treatment was continued with doxorubicin chemotherapy and at the time of publication, the dog was alive over 1 year following diagnosis with no evidence of further disease progression. Genotyping of the abscess-derived L. monocytogenes was consistent with the vaccine strain. CONCLUSIONS: This case represents the first veterinary case to describe development of a Listeria abscess following administration of a Listeria-based therapeutic vaccine.

ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells.

Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stem cells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapy-resistant osteosarcoma stem cells. SIGNIFICANCE: These findings suggest that induction of trans-terminal differentiation through regulation of actin dynamics is a potential novel therapeutic approach for targeting chemoresistant stem-like tumor cells.

Cytotoxic Tolerance of Healthy and Cancerous Bone Cells to Anti-microbial Phenolic Compounds Depend on Culture Conditions.

Carnosol and carnosic acid are polyphenolic compounds found in rosemary and sage with known anti-oxidant, anti-inflammatory, and anti-microbial properties. Here, we addressed the potential use of carnosol and carnosic acid for in vitro bone tissue engineering applications, specifically depending on their cytotoxic effects on bone marrow stromal and stem cells, and osteosarcoma cells in monolayer and 3D cultures. Carnosol and carnosic acid displayed a bacteriostatic effect on Gram-positive bacteria, especially on S. aureus. The viability results indicated that bone marrow stromal cells and bone marrow stem cells were more tolerant to the presence of carnosol compared to osteosarcoma cells. 3D culture conditions increased this tolerance further for healthy cells, while not affecting the cytotoxic potential of carnosol for osteosarcoma cells. Carnosic acid was found to be more cytotoxic for all cell types used in the study. Results suggest that phenolic compounds might have potential use as anti-microbial and anti-carcinogenic agents for bone tissue engineering with further optimization for controlled release.

MicroRNA-1294 targets HOXA9 and has a tumor suppressive role in osteosarcoma.

OBJECTIVE: MicroRNA-1294 (miR-1294) was reported to act as a tumor suppressor in several cancers. However, the biological function of miR-1294 in osteosarcoma (OS) has not been investigated. We, therefore, investigated the clinical significance and underlying mechanisms of miR-1294 in OS. PATIENTS AND METHODS: Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) was conducted to detect the levels of miR-1294. Targets of miR-1294 were validated by luciferase reporter assay and Western blot. In vitro functional assays were performed to investigate the effects of miR-217 on cell proliferation and invasion. RESULTS: We found miR-1294 was downregulated in OS tissues and cell lines. Downregulation of miR-1294 has a significant negative impact on the overall survival of OS patients. Overexpression of miR-1294 suppresses OS cell proliferation and invasion in vitro. Then, luciferase reporter assay validated Homeobox A9 (HOXA9) was a downstream target of miR-1294. Expression patterns of miR-1294 were inversely correlated with HOXA9 in OS tissues, strengthening the findings from the luciferase reporter assay. Further functional assays revealed that overexpression of HOXA9 could reverse the inhibition effects of miR-1294 on cell proliferation and invasion. CONCLUSIONS: These results suggested miR-1294 functions as a tumor suppressor in OS progression by targeting HOXA9.

IL-6 and CXCL8 mediate osteosarcoma-lung interactions critical to metastasis.

Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.

Integrated bioinformatics analysis of miRNA expression in osteosarcoma.

The expression of miRNA influencing the pathogenesis of OS have been reported previously, however, different samples selection and sequencing platforms made obvious differences in miRNA expression analysis. We aim to identify reliable prognostic and treatment biomarkers for OS by systematic analysis of miRNAs expression data sets from biased data set. Seven miRNA data sets were selected from corresponding articles. Collectively, two miRNAs, hsa-miR-19-3p and hsa-miR106b-3p, and transcription factor SIX3 were identified and may be reliable markers for prognostic and treatment of osteosarcoma.

Galangin suppresses human osteosarcoma cells: An exploration of its underlying mechanism.

Osteosarcoma is the most common malignant bone tumor that frequently affects adolescents. Osteosarcoma cells tend to proliferate and invade other tissues such as those of the lungs. Currently, neoadjuvant chemotherapy is the primary strategy to prevent tumor progression. However, its adverse effects result in poor long-term outcomes. Previous research has shown that galangin exhibits antitumor properties on several types of cancer cells; however its effect on osteosarcoma cells is yet unknown. The aims of this study were to evaluate the effects of galangin on the proliferation, apoptosis, migration, and invasion of osteosarcoma cells and to explore the underlying mechanisms. We found that the proliferation of MG63 and U20S osteosarcoma cells decreased significantly, while the apoptosis of MG63 cells accelerated significantly after exposure to galangin. In addition, the migration and invasion of MG63 cells were significantly inhibited by galangin. Moreover, phosphoinositide 3-kinase (PI3K) and Aktp-Thr308 expression levels were found to be significantly lower in galangin-treated MG63 cells than in the control cells, and the protein expression levels of their downstream regulators cyclin D1 and matrix metalloproteinase 2/9 were also downregulated in galangin-treated groups, while those of p27Kip1, caspase-3, and caspase-8 were upregulated. These findings suggest that galangin suppresses osteosarcoma cells by inhibiting their proliferation and invasion and accelerating their apoptosis, and the mechanism may be associated with the inhibition of PI3K and its downstream signaling pathway.

Itinerário terapêutico de adolescentes com osteossarcoma: implicações para o diagnóstico precoce / Therapeutic study of teens with osteosarcoma: implications for early diagnosis / Itinerario terapéutico de adolescentes con osteosarcoma: implicaciones para el diagnóstico temprano

Vários fatores interferem na demora do diagnóstico e tratamento de adolescentes portadores de osteossarcoma. O objetivo deste artigo foi descrever o itinerário terapêutico de adolescentes portadores de osteossarcoma. Trata-se de estudo descritivo, do tipo relato de caso, realizado numa instituição pública especializada em Oncologia no estado do Rio de Janeiro. Foram realizados três estudos de casos de adolescentes portadores de osteossarcoma amputados com a descrição do itinerário terapêutico. Foram identificadas questões relacionadas a dificuldades do diagnóstico precoce, como a relação familiar com o adolescente, a fragilidade de uma rede de saúde adequada ao adolescente e a dificuldade de um diagnóstico de câncer infantil. O diagnóstico precoce deve ser divulgado por meio de um sistema de saúde integrado da atenção básica com o atendimento especializado. É fundamental, para que ocorra a detecção precoce do osteossarcoma nos adolescentes, um sistema de saúde integrado, da atenção básica com o atendimento especializado, a fim de evitar a descoberta de novos casos mais tardiamente.

Several factors interfere with the delay of diagnosis and treatment of adolescents suffering from osteosarcoma. The purpose of this article wasto describe the therapeutic itinerary of adolescents suffering from osteosarcoma. This is a descriptive study of type case report, carried out in apublic institution specialized in Oncology in the State of Rio de Janeiro. Three case studies of adolescents suffering from osteosarcoma amputees with the description of the itinerary were conducted. Related issues have identified the difficulties of early diagnosis, as the family relationship with the teenager; the fragility of a health network suitable for the teenager and the difficulty of a diagnosis of childhood cancer. Early diagnosis shouldbe disclosed through a system of integrated health basic attention with the specialized care. It is essential for early detection of osteosarcoma in adolescents, an integrated health system, basic care with specialized care, in order to prevent the discovery of new cases later.

Son varios los factores que interfieren en la demora del diagnóstico y tratamiento de adolescentes con osteosarcoma. El propósito de este artículoera describir el itinerario terapéutico de adolescentes con osteosarcoma. Se trata de un estudio descriptivo de caso llevado a cabo en una institución pública especializada en oncología en el estado de Río de Janeiro. Se realizaron tres estudios de caso de adolescentes con osteosarcoma amputados con la descripción del itinerario terapéutico. Se identificaron problemas relacionados con las dificultades del diagnóstico temprano, tales como larelación familiar con el adolescente; la fragilidad de la red de salud adecuada para el adolescente y la dificultad del diagnóstico de cáncer infantil.El diagnóstico temprano debe ser revelado a través de un sistema de atención básica de la salud integrado con atención especializada. Para la detección temprana del osteosarcoma en adolescentes es fundamental que el sistema de salud sea integrado, de atención básica con atención especializada, para evitar el descubrimiento de nuevos casos más tarde.

3-Hydroxyflavone inhibits human osteosarcoma U2OS and 143B cells metastasis by affecting EMT and repressing u-PA/MMP-2 via FAK-Src to MEK/ERK and RhoA/MLC2 pathways and reduces 143B tumor growth in vivo.

Many natural flavonoids have cytostatic and apoptotic properties; however, we little know whether the effect of synthetic 3-hydroxyflavone on metastasis and tumor growth of human osteosarcoma. Here, we tested the hypothesis that 3-hydroxyflavone suppresses human osteosarcoma cells metastasis and tumor growth. 3-hydroxyflavone, up to 50 µM without cytotoxicity, inhibited U2OS and 143B cells motility, invasiveness and migration by reducing matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) and also impaired cell adhesion to gelatin. 3-hydroxyflavone significantly reduced p-focal adhesion kinase (FAK) Tyr397, p-FAK Tyr925, p-steroid receptor coactivator (Src), p-mitogen/extracellular signal-regulated kinase (MEK)1/2, p-myosin light chain (MLC)2 Ser19, epithelial cell adhesion molecule, Ras homolog gene family (Rho)A and fibronectin expressions. 3-hydroxyflavone also affected the epithelial-mesenchymal transition (EMT) by down-regulating expressions of Vimentin and α-catenin with activation of the transcription factor Slug. In nude mice xenograft model and tail vein injection model showed that 3-hydroxyflavone reduced 143B tumor growth and lung metastasis. 3-hydroxyflavone possesses the anti-metastatic activity of U2OS and 143B cells by affecting EMT and repressing u-PA/MMP-2 via FAK-Src to MEK/ERK and RhoA/MLC2 pathways and suppresses 143B tumor growth in vivo. This may lead to clinical trials of osteosarcoma chemotherapy to confirm the promising result in the future.

Lentivirus-Mediated Knockdown of CTHRC1 Inhibits Osteosarcoma Cell Proliferation and Migration.

Collagen triple helix repeat containing-1 (CTHRC1), a secreted protein, is transiently expressed in the arterial wall in response to injury, indicating that it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration. Recent studies showed that it is aberrantly upregulated in most human solid tumors, yet its role in osteosarcoma remains unclear. In this study, the authors investigated the role of CTHRC1 in human osteosarcoma tumorigenesis. The authors used lentivirus-mediated short hairpin RNA (shRNA) against CTHRC1 to limit its endogenous expression in U2OS and SW1353 cells. Interestingly, they found that depletion of CTHRC1 significantly inhibited cell proliferation and colony formation in U2OS and SW1353 cells. Flow cytometry assay showed that knockdown of CTHRC1 increased the cell percentage of G0/G1 phase, resulting in cell cycle arrest in U2OS cells. Moreover, CTHRC1 silencing induced the cell cycle arrest by a decrease in the cell percentage in G0/G1 phase and increased in G2/M phase in SW1353 cells. In addition, crystal violet staining suggested CTHRC1 silencing inhibited migration of U2OS and SW1353 cells. These results demonstrated that CTHRC1 might play an important role in osteosarcoma progression.

A monoclonal antibody against the Wnt signaling inhibitor dickkopf-1 inhibits osteosarcoma metastasis in a preclinical model.

The outcome of patients with metastatic osteosarcoma has not improved since the introduction of chemotherapy in the 1970s. Development of therapies targeting the metastatic cascade is a tremendous unmet medical need. The Wnt signaling pathway has been the focus of intense investigation in osteosarcoma because of its role in normal bone development. Although the role of Wnt signaling in the pathogenesis of osteosarcoma is controversial, there are several reports of dickkopf-1 (DKK-1), a Wnt signaling antagonist, possibly playing a pro-tumorigenic role. In this work we investigated the effect of anti-DKK-1 antibodies on the growth and metastasis of patient-derived osteosarcoma xenografts. We were able to detect human DKK-1 in the blood of tumor-bearing mice and found a correlation between DKK-1 level and tumor proliferation. Treatment with the anti-DKK-1 antibody, BHQ880, slowed the growth of orthotopically implanted patient-derived osteosarcoma xenografts and inhibited metastasis. This effect was correlated with increased nuclear beta-catenin staining and increased expression of the bone differentiation marker osteopontin. These findings suggest that Wnt signaling is anti-tumorigenic in osteosarcoma, and support the targeting of DKK-1 as an anti-metastatic strategy for patients with osteosarcoma.

Analysis of Selenium Levels in Osteosarcoma Patients and the Effects of Se-Methylselenocysteine on Osteosarcoma Cells In Vitro.

The form of selenium appears to be important for preventing cancer in humans. Here, we evaluated selenium levels in the serum and bone tissue samples from osteosarcoma patients using atomic absorption spectrometry. The in vitro effects of Se-methylselenocysteine (Se-MSC) on growth, cell cycle status, and apoptosis of osteosarcoma cells were assessed using the WST-1 assay, Hoechst 33342/propidium iodide staining, and flow cytometry, respectively. In osteosarcoma cases, the mean serum selenium levels in osteosarcoma tissue and normal bone were 0.08 mg/kg and 0.03 mg/kg, respectively (P < 0.05). Serum selenium levels in osteosarcoma and non-osteosarcoma cases were 0.09 mg/L and 0.08 mg/L, respectively (P > 0.05). Se-MSC-treated MG63 cells showed altered cellular morphology, decreased viability in a time- and dose-dependent manner, and an increase in the sub-G1 cell population. Se-MSC also downregulated Bcl-2 expression and upregulated Bax. Se-MSC inhibited the proliferation of the drug-resistant osteosarcoma cell line Saos-2/MTX300 and enhanced the inhibitory effect of pirarubicin on MG63 cells. Our data demonstrate that selenium levels are significantly higher in osteosarcoma tissue than normal bone tissue in osteosarcoma patients. The results also support the anticancer effects of Se-MSC in osteosarcoma. Further development of Se-MSC as an ancillary chemotherapy agent in osteosarcoma is warranted.

Acting on black box warnings requires a GRADE evidence table and an implementation guide: the case of teriparatide.

OBJECTIVES: To assess how well do the black box warnings present and communicate evidence in a way that is consistent with evidence-based patient-centered practice, through evaluating the boxed warning on teriparatide-induced osteosarcoma. STUDY DESIGN AND SETTING: We critically appraised teriparatide black box warning for osteosarcoma by reviewing human and animal studies that were used as basis for the warning. We also evaluated the quality of the evidence using the Grading of Recommendations Assessment, Development and Evaluation framework. RESULTS: We found that this warning was based on very low-quality evidence that was derived primarily from animal studies. The quality of evidence was rated down because of high risk of bias in addition to inconsistency, indirectness, and imprecision of the estimates. The warning does not provide sufficient guidance for practice like an implementation tool or an evidence profile to clarify the limitations of the evidence. CONCLUSION: Black box warning for teriparatide-associated osteosarcoma does not explicitly present the quality of evidence, and therefore, it could be of limited use in evidence-based practice. We propose that black box warnings should include an evidence profile and an implementation guide to be more useful in evidence-based patient-centered practice.