Sphingomyelin Metabolism Modifies Luminal A Breast Cancer Cell Line under a High Dose of Vitamin C.

The role of sphingomyelin metabolism and vitamin C in cancer has been widely described with conflicting results ranging from a total absence of effect to possible preventive and/or protective effects. The aim of this study was to establish the possible involvement of sphingomyelin metabolism in the changes induced by vitamin C in breast cancer cells. The MCF7 cell line reproducing luminal A breast cancer and the MDA-MB-231 cell line reproducing triple-negative breast cancer were used. Cell phenotype was tested by estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 expression, and proliferation index percentage. Sphingomyelin was localized by an EGFP-NT-Lys fluorescent probe. Sphingomyelin metabolism was analyzed by RT-PCR, Western blotting and UFLC-MS/MS. The results showed that a high dose of vitamin C produced reduced cell viability, modulated cell cycle related genes, and changed the cell phenotype with estrogen receptor downregulation in MCF7 cell. In these cells, the catabolism of sphingomyelin was promoted with a large increase in ceramide content. No changes in viability and molecular expression were observed in MB231 cells. In conclusion, a high dose of vitamin C induces changes in the luminal A cell line involving sphingomyelin metabolism.

Effect of 1α,25(OH)2 Vitamin D3 in Mutant P53 Glioblastoma Cells: Involvement of Neutral Sphingomyelinase1.

Glioblastoma is one the most aggressive primary brain tumors in adults, and, despite the fact that radiation and chemotherapy after surgical approaches have been the treatments increasing the survival rates, the prognosis of patients remains poor. Today, the attention is focused on highlighting complementary treatments that can be helpful in improving the classic therapeutic approaches. It is known that 1α,25(OH)2 vitamin D3, a molecule involved in bone metabolism, has many serendipidy effects in cells. It targets normal and cancer cells via genomic pathway by vitamin D3 receptor or via non-genomic pathways. To interrogate possible functions of 1α,25(OH)2 vitamin D3 in multiforme glioblastoma, we used three cell lines, wild-type p53 GL15 and mutant p53 U251 and LN18 cells. We demonstrated that 1α,25(OH)2 vitamin D3 acts via vitamin D receptor in GL15 cells and via neutral sphingomyelinase1, with an enrichment of ceramide pool, in U251 and LN18 cells. Changes in sphingomyelin/ceramide content were considered to be possibly responsible for the differentiating and antiproliferative effect of 1α,25(OH)2 vitamin D in U251 and LN18 cells, as shown, respectively, in vitro by immunofluorescence and in vivo by experiments of xenotransplantation in eggs. This is the first time 1α,25(OH)2 vitamin D3 is interrogated for the response of multiforme glioblastoma cells in dependence on the p53 mutation, and the results define neutral sphingomyelinase1 as a signaling effector.

Silver ions promote blebs growth in U251 glioblastoma cell by activating nonselective cationic currents.

Glioblastoma (GBM) is the most common and aggressive human brain cancer with low prognosis and therefore the discovery of new anticancer agents is needful. Sulfydryl reagents, such as silver, have been shown to induce membrane vesiculation in several cellular models through a mechanism that has not been yet completely clarified. Using U251 glioblastoma cells, we observed that silver induced irreversible bleb formation of the plasma membrane. This morphological event was anticipated by an increase of intracellular Ca2+ associated to extracellular Ca2+ influx. Accordingly, using patch-clamp whole cell recording during silver ion application, inward current/s (IAg) at -90 mV were detected and cells were permeable to Ca2+ and monovalent ions such as Na+. IAg activation and the intracellular Ca2+ increase promoted by silver ions (Ag+) were prevented by co-application of 20 µM cysteine and 300 µM DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid), suggesting a critical role of thiol groups in the biological effects of silver ions. IAg was partially inhibited by 1 mM Gd3+, an unspecific inhibitor of cationic currents. Cysteine, Gd3+ and extracellular free Ca2+ solution completely abolished blebbing formation promoted by Ag+. Furthermore, extracellular Na+ ion replacement with TEA or an increase of extracellular tonicity by sucrose (100 mM) reduced both size and growth of membrane blebbing. Our data suggest that Ag+ promotes the formation necrotic blebs as consequence of the increase of intracellular Ca2+ and intracellular hydrostatic pressure associated to the activation of cationic currents. Since silver-induced blebs were less evident in benign glial human Müller MIO-M1 cells, silver compounds could represent new adjuvant to anticancer agents to improve GBM therapies.

Oxidative stress-induced S100B accumulation converts myoblasts into brown adipocytes via an NF-κB/YY1/miR-133 axis and NF-κB/YY1/BMP-7 axis.

Muscles of sarcopenic people show hypotrophic myofibers and infiltration with adipose and, at later stages, fibrotic tissue. The origin of infiltrating adipocytes resides in fibro-adipogenic precursors and nonmyogenic mesenchymal progenitor cells, and in satellite cells, the adult stem cells of skeletal muscles. Myoblasts and brown adipocytes share a common Myf5+ progenitor cell: the cell fate depends on levels of bone morphogenetic protein 7 (BMP-7), a TGF-ß family member. S100B, a Ca2+-binding protein of the EF-hand type, is expressed at relatively high levels in myoblasts from sarcopenic humans and exerts anti-myogenic effects via NF-κB-dependent inhibition of MyoD, a myogenic transcription factor acting upstream of the essential myogenic factor, myogenin. Adipogenesis requires high levels of ROS, and myoblasts of sarcopenic subjects show elevated ROS levels. Here we show that: (1) ROS overproduction in myoblasts results in upregulation of S100B levels via NF-κB activation; and (2) ROS/NF-κB-induced accumulation of S100B causes myoblast transition into brown adipocytes. S100B activates an NF-κB/Ying Yang 1 axis that negatively regulates the promyogenic and anti-adipogenic miR-133 with resultant accumulation of the brown adipogenic transcription regulator, PRDM-16. S100B also upregulates BMP-7 via NF-κB/Ying Yang 1 with resultant BMP-7 autocrine activity. Interestingly, myoblasts from sarcopenic humans show features of brown adipocytes. We also show that S100B levels and NF-κB activity are elevated in brown adipocytes obtained by culturing myoblasts in adipocyte differentiation medium and that S100B knockdown or NF-κB inhibition in myoblast-derived brown adipocytes reconverts them into fusion-competent myoblasts. At last, interstitial cells and, unexpectedly, a subpopulation of myofibers in muscles of geriatric but not young mice co-express S100B and the brown adipocyte marker, uncoupling protein-1. These results suggest that S100B is an important intracellular molecular signal regulating Myf5+ progenitor cell differentiation into fusion-competent myoblasts or brown adipocytes depending on its levels.

S100B protein in myoblasts modulates myogenic differentiation via NF-kappaB-dependent inhibition of MyoD expression.

S100B, a Ca(2+)-binding protein of the EF-hand type, is expressed in myoblasts, the precursors of skeletal myofibers, and muscle satellite cells (this work). S100B has been shown to participate in the regulation of several intracellular processes including cell cycle progression and differentiation. We investigated regulatory activities of S100B within myoblasts by stable overexpression of S100B and by inhibition of S100B expression. Overexpression of S100B in myoblast cell lines and primary myoblasts resulted in inhibition of myogenic differentiation, evidenced by lack of expression of myogenin and myosin heavy chain (MyHC) and absence of myotube formation. S100B-overexpressing myoblasts showed reduced MyoD expression levels and unchanged Myf5 expression levels, compared with control myoblasts, and transient transfection of S100B-overexpressing myoblasts with MyoD, but not Myf5, restored differentiation and fusion in part. The transcriptional activity of NF-kappaB, a negative regulator of MyoD expression, was enhanced in S100B-overexpressing myoblasts, and blocking NF-kappaB activity resulted in reversal of S100B's inhibitory effects. Yin Yang1, a transcriptional repressor that is induced by NF-kappaB (p65) and mediates NF-kappaB inhibitory effects on several myofibrillary genes, also was upregulated in S100B-overexpressing myoblasts. Conversely, silencing S100B expression in myoblast cell lines by RNA interference resulted in reduced NF-kappaB activity and enhanced MyoD, myogenin and MyHC expression and myotube formation. Thus, intracellular S100B might modulate myoblast differentiation by interfering with MyoD expression in an NF-kappaB-dependent manner.