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Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.

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Objective: This study examines the relationship between the results of in vitro determinations of carbohydrate digestion rates and the glycemic index. Subjects: Ten healthy non smoking men, aged between 21 and 24-y-old, were selected to participate in the study. Methods: Six different meals with similar lelvels of carbohydrates were assayed at breakfast in ten subjects and blood samples were obtained at 0, 15, 30, 60 and 120 min to determine glucose levels. The Englyst's enzymatic method was used in the in vitro study. The six meals were based in cereals (rice or spaghetti); legumes (lentil soup and beans with spaghetti); and potato (potato stew with meat and vegetables). Results: The meals showed different glycemic indices: rice and spaghetti based meals had lower values (mean value 31.4 and 42, respectiively); the intermediate values corresponded to lentil soup and the bean dish (49.3, and 76.8 respectively) and the higher glycemic index was shown by the potato based meal (82). Conclusions: A significant correlation was observed when the ratio of rapid carbohydrate digestion rate and the lente carbohydrate digestion rate was correlated with the glycemic index of the meals, but not when only the rapid carbohydrate digestion rate was considered. These results demonstrate a useful, simple and inexpensive method to estimate the biological response of high carbohydrate meals. Sponsorship:

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/β-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, β-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of β-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.

Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt‐related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR‐34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin (OPN/SPP1). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next‐generation RNA‐sequencing, protein‐based assays, as well as the loss‐ and gain‐of‐function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell–cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.

The effect of saline stress (NaCl, 40, 80 and 160 mmol L−1 of NaCl) on growth, plant water status and leaf antioxidant enzyme activities was investigated in a commercial cultivar of cherry tomato (Solanum lycopersicum var. cerasiforme L.) and in a wild-related species collected in a salt-affected area of North Chile (Solanum chilense Dun.). Salt stress was applied in a nutrient solution at the vegetative stage during 40 days. The highest NaCl concentration reduced shoot relative growth, fresh and dry weight and leaf area in the cultivated S. lycopersicum but had less impact on S. chilense. Both species were able to efficiently perform osmotic adjustment but S. chilense also exhibited an increase in leaf succulence. The oxidative stress estimated through malondialdehyde quantification was always higher in the cultivated S. lycopersicum, both in the absence and in the presence of salt. Total superoxide dismutase activity (EC 1.15.1.1) increased in response to the highest dose of NaCl in S. chilense but remained constant in S. lycopersicum. Salinity induced an increase in ascorbate peroxidase (EC 1.11.1.11) in S. chilense but reduced it in S. lycopersicum. It is concluded that S. chilense displays efficient strategies to cope with high NaCl doses and that management of the oxidative status is a key mechanism allowing this species to tolerate salinity.

Objective: To examine whether high carbohydrate meals with different carbohydrate digestion rates have an effect on the short-term satiety in normal and obese preschool children. Subjects and setting: In total, 48 children of both gender, 24 normal and 24 obese, according to body mass index, aged between 3 and 6 y, were selected from children who were attending to a day-care center where they consumed three meals in the day. Interventions: Rapid (potato-based meal) and lente (spaghetti-based meal) carbohydrate digestion rate meal were given at lunch, matching energy intake, carbohydrate, protein and fat levels, and then the preschool children's energy intake at the subsequent mealtime was observed. In this last mealtime, the children received varied types of high-acceptability foods in higher quantities than the normal serving. Results: At lunch, a significant higher energy intake, lesser satiation, was observed in both groups, normal-weight and obese children, when they consumed the potato-based meal. In the subsequent mealtime, a significant effect of carbohydrate digestion rate was demonstrated, but only in obese preschool children, being higher in the meal with rapid digestion rate carbohydrates. Conclusions: Rapid carbohydrate digestion rate meal produced a significative lesser satiation in normal-weight and obese children. However, only in obese children a significant lesser satiety was observed after consumption of the rapid carbohydrate digestion rate meal, indicating a decreased capacity of energy regulation in obese children. The finding of the present work could provide dietary strategies required for decreasing prevalence in overweight and obesity in preschool children.