Regulation of the oxidative balance with coenzyme Q10 sensitizes human glioblastoma cells to radiation and temozolomide.

OBJECTIVES: To investigate how the modulation of the oxidative balance affects cytotoxic therapies in glioblastoma, in vitro. MATERIAL AND METHODS: Human glioblastoma U251 and T98 cells and normal astrocytes C8D1A were loaded with coenzyme Q10 (CoQ). Mitochondrial superoxide ion (O2-) and H2O2 were measured by fluorescence microscopy. OXPHOS performance was assessed in U251 cells with an oxytherm Clark-type electrode. Radio- and chemotherapy cytotoxicity was assessed by immunostaining of γH2AX (24 h), annexin V and nuclei morphology, at short (72 h) and long (15 d) time. Hif-1α, SOD1, SOD2 and NQO1 were determined by immunolabeling. Catalase activity was measured by classic enzymatic assay. Glutathione levels and total antioxidant capacity were quantified using commercial kits. RESULTS: CoQ did not affect oxygen consumption but reduced the level of O2- and H2O2 while shifted to a pro-oxidant cell status mainly due to a decrease in catalase activity and SOD2 level. Hif-1α was dampened, echoed by a decrease lactate and several key metabolites involved in glutathione synthesis. CoQ-treated cells were twofold more sensitive than control to radiation-induced DNA damage and apoptosis in short and long-term clonogenic assays, potentiating TMZ-induced cytotoxicity, without affecting non-transformed astrocytes. CONCLUSIONS: CoQ acts as sensitizer for cytotoxic therapies, disarming GBM cells, but not normal astrocytes, against further pro-oxidant injuries, being potentially useful in clinical practice for this fatal pathology.

ß-Cryptoxanthin Inhibits Angiogenesis in Human Umbilical Vein Endothelial Cells Through Retinoic Acid Receptor.

SCOPE: ß-Cryptoxanthin is an abundant carotenoid in fruits and vegetables that can be quantified in human blood serum. Yet, contrary to other carotenoids, its effects on endothelial cells and angiogenesis remain unknown. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) are treated with 0.01, 0.1, or 1 µm of ß-cryptoxanthin. Antioxidant activity is determined by its free radical scavenging and oxygen-radical absorbance capacity. The effect on migration and formation of tubular structures is studied. Additionally, effect on angiogenesis is also analyzed using an in vivo model. ß-Cryptoxanthin exhibits scavenging ability, having an antioxidant effect on HUVEC. Interestingly, ß-cryptoxanthin reduces their migration and angiogenesis, even in the presence of vascular endothelial growth factor (VEGF). Additionally, such carotenoid inhibits in vivo angiogenesis induced by VEGF. In addition, treatment of HUVEC with LE540 (retinoic acid receptor [RAR] panantagonist) inhibits ß-cryptoxanthin antiangiogenic effect on HUVEC. CONCLUSION: ß-Cryptoxanthin inhibits angiogenesis through RAR. Thus, this carotenoid and food containing it may be useful for the prevention and treatment of angiogenic pathologies. That includes tumoral growth and wet macular degeneration associated with aging. To the best of our knowledge, this is the first report of the antioxidant effect and antiangiogenic activity of this carotenoid on HUVEC, both in vitro and in vivo.

Stimulation of in-vitro angiogenesis by low concentrations of risedronate is mitigated by 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3.

The nitrogen-containing or nitrogenous bisphosphonates (N-BF) are currently the main class of drugs used for the treatment of diseases characterized by an increased bone resorption. Preliminary data suggest that N-BF have also direct or indirect anti-tumoral effects, and recent evidence suggests that part of the anti-tumoral activity of N-BF may be attributed to their anti-angiogenic capacity when they are used at high concentrations. On the other hand, an optimal vitamin-D status seems to be necessary to maximize the bone response to N-BF. Our aim has been to evaluate the effect of risedronate, alone or in combination with either 1,25(OH)2D3 or 24,25(OH)2D3 (two main vitamin-D metabolites) on parameters related to the angiogenic capacity of human umbilical-vein endothelial cells (HUVEC). The studies of tube formation through in-vitro angiogenesis assays with Matrigel, chemotaxis and migration in a scratch assay showed that low concentrations of risedronate (0.01 to 1µM) stimulated angiogenesis and cellular migration in vitro. The presence of 1,25(OH)2D3 in the medium inhibited tubular-structure formation and cellular migration. In addition, the presence of 1,25 or 24,25(OH)2D3 in the culture medium also decreased the pro-angiogenic effects of low-concentrations of risedronate. These data show the differential effects of different concentrations of vitamin-D metabolites and risedronate on angiogenesis, thus stressing the importance of an adequate vitamin D status during medical treatment with risedronate. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Risedronate positively affects osteogenic differentiation of human mesenchymal stromal cells.

BACKGROUND AND AIMS: Bisphosphonates are widely used for the treatment of bone pathologies, mainly due to their ability to inhibit osteoclastic activity and thus bone resorption. Yet, their potential effect on bone formation is unclear. Our aim was to determine whether risedronate can affect osteoblastic differentiation of mesenchymal stromal cells (MSC). METHODS: We evaluated the effect of the risedronate, which is a bisphosphonate widely used in clinical settings, on the differentiation of precursor cells of osteoblasts and adipocytes. Thus, MSC from human bone marrow were induced to differentiate into osteoblasts or adipocytes in the presence or absence of two risedronate concentrations (10⁻8 M and 10⁻9 M). RESULTS: Risedronate increased the levels of osteogenic markers including the extracellular matrix mineralization on cells induced to osteoblasts. On the other hand, such bisphosphonates did not have significant effects on the pparγ2 and lpl adipogenic genes or the fat vesicle accumulation on the cells induced into adipocytes. In addition, it increased the expression of early osteogenic differentiation genes like runx2 on MSC not induced to differentiate. CONCLUSIONS: Risedronate concentrations used favor the osteogenic differentiation and the MSC commitment towards osteoblasts. This suggests the possibility of using this bisphosphonate in cyclic bone treatments with anabolic drugs, enhancing the effect of the latter via a previous MSC commitment to preosteoblasts.

Nuclear translocation of ß-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

Wnt/ß-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/ß-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, ß-catenin nuclear translocation, up-regulation of genes related to the Wnt/ß-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/ß-catenin inactivation. Hepatocytes with nuclear translocation of ß-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase ß-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin ß-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/ß-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells.

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.