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.

Microglia and Brain Disorders: The Role of Vitamin D and Its Receptor.

Accounting for 5-20% of the total glial cells present in the adult brain, microglia are involved in several functions: maintenance of the neural environment, response to injury and repair, immunesurveillance, cytokine secretion, regulation of phagocytosis, synaptic pruning, and sculpting postnatal neural circuits. Microglia contribute to some neurodevelopmental disorders, such as Nasu-Hakola disease (NHD), Tourette syndrome (TS), autism spectrum disorder (ASD), and schizophrenia. Moreover, microglial involvement in neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's (PD) diseases, has also been well established. During the last two decades, epidemiological and research studies have demonstrated the involvement of vitamin D3 (VD3) in the brain's pathophysiology. VD3 is a fat-soluble metabolite that is required for the proper regulation of many of the body's systems, as well as for normal human growth and development, and shows neurotrophic and neuroprotective actions and influences on neurotransmission and synaptic plasticity, playing a role in various neurological diseases. In order to better understand the exact mechanisms behind the diverse actions of VD3 in the brain, a large number of studies have been performed on isolated cells or tissues of the central nervous system (CNS). Here, we discuss the involvement of VD3 and microglia on neurodegeneration- and aging-related diseases.

Ceramide releases exosomes with a specific miRNA signature for cell differentiation.

Exosomes are well established effectors of cell-cell communication. Their role on maturation of embryonic cells located in hippocampus, seat of memory, is unknown. Here we show that ceramide facilitates release of exosomes from HN9.10e cells extending information for cell differentiation to neighboring cells. We found only 38 miRNAs differentially expressed in exosomes derived from ceramide-treated cells in comparison with control cells (including 10 up-regulated and 28 down-regulated). Some overexpressed miRNAs (mmu-let-7f-1-3p, mmu-let-7a-1-3p, mmu-let-7b-3p, mmu-let-7b-5p, mmu-miR-330-3p) regulate genes encoding for protein involved in biological, homeostatic, biosynthetic and small molecule metabolic processes, embryo development and cell differentiation, all phenomena relevant for HN9.10e cell differentiation. Notably, the overexpressed mmu-let-7b-5p miRNA appears to be important for our study based on its ability to regulate thirty-five gene targets involved in many processes including sphingolipid metabolism, sphingolipid-related stimulation of cellular functions and neuronal development. Furthermore, we showed that by incubating embryonic cells with exosomes released under ceramide treatment, some cells acquired an astrocytic phenotype and others a neuronal phenotype. We anticipate our study to be a start point for innovative therapeutic strategies to regulate the release of exosomes useful to stimulate delayed brain development in the newborn and to improve the cognitive decline in neurodegenerative disorders.

Sphingomyelin in Human Breast Milk might be Essential for the Hippocampus Maturation.

BACKGROUND: It has been established that sphingomyelin present human breast milk is useful for the brain maturation and cognitive development. At 10 days of breastfeeding the sphingomyelin content is double that present in cow's milk and its content is independent of the maternal diet. The aim of the study was to analyze the content of sphingomyelin in breast milk at 3 months of breastfeeding and to consider the effect of this molecule on synaptic function and nerve conduction through the probable expansion of myelinated axons. METHODS: Therefore, to begin to define and assess this, we performed sphingolipidomic analysis in human breast milk. Then, we cultured embryonic hippocampal cells (HN9.10) in the presence of sphingomyelin at a concentration from 0.6% to 31% of human milk, estimated by considering its bioavailability and its passage into the interstitial fluid. To highlight the effect of sphingomyelin in the cells, cell viability and morphology were evaluated. Analyses of neutral sphingomyelinase gene and protein expression was performed. The entry of sphingomyelin into the cell was studied in immunofluorescence; the expression of heavy neurofilament (NF200) was tested with immunocytochemical technique. RESULTS: We demonstrated that sphingomyelin is able to enter cell nucleus and overexpress the sphingomyelin phosphodiesterase 4 (SMPD4) gene encoding for neutral sphingomyelinase (nSMase), an enzyme useful for its own metabolism. Later, cells displayed changes of the soma and the appearance of neurites supported by NF200 overexpression. CONCLUSIONS: We speculated that the sphingomyelin present in human breast milk is useful in part to regulate nuclear activity and in part to form myelin sheet to facilitate nerve cell maturation. As brain development occurs at 0-3 years, these data open a new avenue of potential intervention to integrate the infant formulas with SM to obtain a product similar to the maternal milk.

Vitamin D3 Enriches Ceramide Content in Exosomes Released by Embryonic Hippocampal Cells.

The release of exosomes can lead to cell-cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.

Human breast milk as source of sphingolipids for newborns: comparison with infant formulas and commercial cow's milk.

BACKGROUND: In the past two decades, sphingolipids have become increasingly appreciated as bioactive molecules playing important roles in a wide array of pathophysiology mechanisms. Despite advances in the field, sphingolipids as nutrients remain little explored. Today the research is starting to move towards the study of the sphingomyelin content in human breast milk, recommended for feeding infants. METHODS: In the present study, we performed a lipidomic analysis in human breast milk in relation with maternal diet during pregnancy, in infant formulas, and in commercial whole and semi-skimmed milks for adults. Mediterranean, carnivorous and vegetarian diets were considered. RESULTS: The results showed that total sphingomyelin, ceramide and dihydroceramide species are independent on the diet. Interestingly, the milk sphingolipid composition is species-specific. In fact, infant formulas and commercial milks for adults have a lower level of total sphingomyelin and ceramide content than human breast milk with very different composition of each sphingolipid species. CONCLUSIONS: We conclude that human breast milk is a better source of sphingolipids than infant formulas for baby nutrition with potential implications for the brain development and cognitive functions.

Relationship between Fatty Acids Composition/Antioxidant Potential of Breast Milk and Maternal Diet: Comparison with Infant Formulas.

The fatty acid composition of human breast milk is relevant for the energy, immunity and eicosanoid production in infants. Additionally, the antioxidant properties of foods are essential for human health. Therefore, in the present study we aimed to investigate the relationship between maternal diet and fatty acids composition as well as the antioxidant potential of breast milk from donors to human milk bank of Perugia's hospital, Italy. Results were compared with infant formulas. We observed increased levels of total fatty acids and, in particular, saturated and monounsaturated fatty acids in milk from mothers fed on a vegetable and fruit-rich diet compared with a Mediterranean diet. In the same milk, a reduced antioxidant potential was found. All infant formulas resulted in richer total fatty acid content than human breast milk. Only some formulas were qualitatively similar to breast milk. Of note, the antioxidant potential of the formulas was higher or lower than the human milk with the exception of one sample. The antioxidant potential of four formulas was very high. Dietary supplementation with antioxidants has been shown to have a teratogenic effect and to increase the formation of metastases in adult. There are no data on the effects of excess antioxidants in the infants, but the possibility that they can be harmful cannot be excluded.