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.

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.

Heat Shock Protein 90 Involvement in the Development of Idiopathic Epiretinal Membranes.

Purpose: This work was aimed to further characterize cells of idiopathic epiretinal membranes (iERMs). We wanted to determine the contribution of 90-kDa heat shock protein (HSP90) to sustain the transforming growth factor-ß (TGF-ß)-mediated signal transduction pathway in iERM. Methods: Immunofluorescence and confocal microscopy were carried out on deplasticized sections from 36 epiretinal membranes processed for electron microscopy and on frozen sections from five additional samples with antibodies against α-smooth muscle actin (αSMA), vimentin, glial fibrillary acidic protein (GFAP), SMAD2, HSP90α, type-II TGF-ß1 receptor (TßRII), type-I collagen, and type-IV collagen. In addition, Müller MIO-M1 cells were transfected with HSP90 and challenged with TGF-ß1. Results: Double and triple labeling experiments showed that a variable number of TßRII+ cells were present in 94.1% of tested iERMs and they were mostly GFAP-/αSMA+/vimentin+/HSP90α+. In almost half of the cases these cells contained type-I collagen, suggesting their involvement in matrix deposition. HSP90 overexpressing MIO-M1 cells challenged with TGF-ß1 showed increased levels of TßRII, SMAD2, SMAD3, and phosphor-SMAD2. Nuclear SMAD2 staining could be observed in HSP90α+ cells on frozen sections of iERMs. Conclusions: Cells in iERMs that express TßRII are also HSP90α+ and show the antigenic profile of myofibroblast-like cells as they are GFAP-/αSMA+/vimentin+. HSP90α-overexpressing MIO-M1 cells challenged with TGF-ß1 showed an increased activation of the SMAD pathway implying that HSP90α might play a role in sustaining the TGF-ß1-induced fibrotic response of iERM cells.

Reductive stress in striated muscle cells.

Reductive stress is defined as a condition of sustained increase in cellular glutathione/glutathione disulfide and NADH/NAD+ ratios. Reductive stress is emerging as an important pathophysiological event in several diseased states, being as detrimental as is oxidative stress. Occurrence of reductive stress has been documented in several cardiomyopathies and is an important pathophysiological factor particularly in coronary artery disease and myocardial infarction. Excess activation of the transcription factor, Nrf2-the master regulator of the antioxidant response-, consequent in most cases to defective autophagy, can lead to reductive stress. In addition, hyperglycemia-induced activation of the polyol pathway can lead to increased NADH/NAD+ ratio, which might translate into increased levels of hydrogen sulfide-via enhanced activity of cystathionine ß-synthase-that would fuel reductive stress through inhibition of mitochondrial complex I. Reductive stress may be either a potential weapon against cancer priming tumor cells to apoptosis or a cancer's ally promoting tumor cell proliferation and making tumor cells resistant to reactive oxygen species-inducing drugs. In non-cancer pathological states reductive stress is definitely harmful paradoxically leading to reactive oxygen species overproduction via excess NADPH oxidase 4 activity. In face of the documented occurrence of reductive stress in several heart diseases, there is much less information about the occurrence and effects of reductive stress in skeletal muscle tissue. In the present review we describe relevant results emerged from studies of reductive stress in the heart and review skeletal muscle conditions in which reductive stress has been experimentally documented and those in which reductive stress might have an as yet unrecognized pathophysiological role. Establishing whether reductive stress has a (patho)physiological role in skeletal muscle will hopefully contribute to answer the question whether antioxidant supplementation to the general population, athletes, and a large cohort of patients (e.g. heart, sarcopenic, dystrophic, myopathic, cancer, and bronco-pulmonary patients) is harmless or detrimental.

S100 proteins in obesity: liaisons dangereuses.

Obesity is an endemic pathophysiological condition and a comorbidity associated with hypercholesterolemia, hypertension, cardiovascular disease, type 2 diabetes mellitus, and cancer. The adipose tissue of obese subjects shows hypertrophic adipocytes, adipocyte hyperplasia, and chronic low-grade inflammation. S100 proteins are Ca2+-binding proteins exclusively expressed in vertebrates in a cell-specific manner. They have been implicated in the regulation of a variety of functions acting as intracellular Ca2+ sensors transducing the Ca2+ signal and extracellular factors affecting cellular activity via ligation of a battery of membrane receptors. Certain S100 proteins, namely S100A4, the S100A8/S100A9 heterodimer and S100B, have been implicated in the pathophysiology of obesity-promoting macrophage-based inflammation via toll-like receptor 4 and/or receptor for advanced glycation end-products ligation. Also, serum levels of S100A4, S100A8/S100A9, S100A12, and S100B correlate with insulin resistance/type 2 diabetes, metabolic risk score, and fat cell size. Yet, secreted S100B appears to exert neurotrophic effects on sympathetic fibers in brown adipose tissue contributing to the larger sympathetic innervation of this latter relative to white adipose tissue. In the present review we first briefly introduce S100 proteins and then critically examine their role(s) in adipose tissue and obesity.

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.

Niemann-Pick Type A Disease: Behavior of Neutral Sphingomyelinase and Vitamin D Receptor.

Sphingomyelinase (SMase) is responsible for the breakdown of sphingomyelin (SM) with production of ceramide. The absence of acid sphingomyelinase (aSMase) causes abnormal synapse formation in Niemann-Pick type A (NPA) disease. Because high levels of ceramide in the NPA brain were demonstrated, the involvement of other SMases were supposed. In the present study we focused the attention on the neurogenic niches in the hippocampal gyrus dentatus (GD), a brain structure essential for forming cohesive memory. We demonstrated for the first time the increase of (Sex determining region Y)-box 2 (SOX2), and the down-regulation of glial fibrillary acidic protein (GFAP) NPA mice GD. Moreover, we found that the expression of Toll like receptors (TLRs), was increased in NPA mice, particularly TLR2, TLR7, TLR8 and TLR9 members. Although no significant change in neutral sphingomyelinase (nSMase) gene expression was detected in the NPA mice hippocampus of, protein levels were enhanced, probably because of the slower protein degradation rate in this area. Many studies demonstrated that vitamin D receptor (VDR) is expressed in the hippocampus GD. Unexpectedly, we showed that NPA mice exhibited VDR gene and protein expression up-regulation. In summary, our study suggests a relation between hippocampal cell differentiation defect, nSMase and VDR increase in NPA mice.

Parenchymal and non-parenchymal immune cells in the brain: A critical role in regulating CNS functions.

The presence of immune cells in the central nervous system has long been the subject of research to find out their role. For a long time it was believed that the CNS was a privileged area from an immunological point of view, due to the presence of the blood-brain barrier (BBB), as circulating immune cells were unable to penetrate the brain parenchyma, at least until the integrity of the BBB was preserved. For this reason the study of the CNS immune system has focused on the functions of microglia, the immunocompetent resident element of the brain parenchyma that retain the ability to divide and self-renew during lifespan without any significant contribution from circulating blood cells. More recently, the presence of lymphatic vessels in the dural sinuses has been demonstrated with accompanying lymphocytes, monocytes and other immune cells. Moreover, meningeal macrophages, that is macrophages along the blood vessels and in the choroid plexus (CP), are also present. These non-parenchymal immune cells, together with microglia, can affect multiple CNS functions. Here, we discuss the functional role of parenchymal and non-parenchymal immune cells and their contribution to the regulation of neurogenesis.

Neutral sphingomyelinase increases and delocalizes in the absence of Toll-Like Receptor 4: A new insight for MPTP neurotoxicity.

Both sphingomyelinase and Toll-Like Receptor 4 (TLR4) are implicated in neurodegenerative diseases. However, the relationship between the two molecules remains unclear. In this study, using WT and TLR4-deficient mice, treated or not with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we aimed to investigate the relation between TLR4 and neutral sphingomyelinase (nSMase) in the midbrain. We found that the lack of TLR4 caused increase in nSMase protein expression and enzyme activity in the midbrain, as well as a marked delocalization from the cell membranes. This provoked a decrease in sphingomyelin (SM) species and an increase in ceramide levels. We found that exposure of TLR4-deficient mice to MPTP reduces unsaturated SM species by increasing saturated/unsaturated SM ratio. Saturated fatty acid make SM more rigid and could contribute to reducing neural plasticity. In this study we showed that the absence of TLR4 also induced reduction of both heavy neurofilaments and glial fibrillary acidic protein (GFAP) and mice exhibited higher sensitivity to MPTP administration. We speculated about the possible association between nSMase-TLR4 complex and MPTP midbrain damage. Taken together, our findings provide for the first time indications about the role of TLR4 in change of SM metabolism in MPTP neurotoxicity.

Solid Dispersion of Resveratrol Supported on Magnesium DiHydroxide (Resv@MDH) Microparticles Improves Oral Bioavailability.

Resveratrol, because of its low solubility in water and its high membrane permeability, is collocated in the second class of the biopharmaceutical classification system, with limited bioavailability due to its dissolution rate. Solid dispersion of resveratrol supported on Magnesium DiHydroxide (Resv@MDH) was evaluated to improve solubility and increase bioavailability of resveratrol. Fluorimetric microscopy analysis displays three types of microparticles with similar size: Type 1 that emitted preferably fluorescence at 445 nm with bandwidth of 50 nm, type 2 that emitted preferably fluorescence at 605 nm with bandwidth of 70 nm and type 3 that is non-fluorescent. Micronized pure resveratrol displays only microparticles type 1 whereas type 3 are associated to pure magnesium dihydroxide. Dissolution test in simulated gastric environment resveratrol derived from Resv@MDH in comparison to resveratrol alone displayed better solubility. A 3-fold increase of resveratrol bioavailability was observed after oral administration of 50 mg/kg of resveratrol from Resv@MDH in rabbits. We hypothesize that type 2 microparticles represent magnesium dihydroxide microparticles with a resveratrol shell and that they are responsible for the improved resveratrol solubility and bioavailability of Resv@MDH.

Cellular and molecular mechanisms of sarcopenia: the S100B perspective.

Primary sarcopenia is a condition of reduced skeletal muscle mass and strength, reduced agility, and increased fatigability and risk of bone fractures characteristic of aged, otherwise healthy people. The pathogenesis of primary sarcopenia is not completely understood. Herein, we review the essentials of the cellular and molecular mechanisms of skeletal mass maintenance; the alterations of myofiber metabolism and deranged properties of muscle satellite cells (the adult stem cells of skeletal muscles) that underpin the pathophysiology of primary sarcopenia; the role of the Ca2+ -sensor protein, S100B, as an intracellular factor and an extracellular signal regulating cell functions; and the functional role of S100B in muscle tissue. Lastly, building on recent results pointing to S100B as to a molecular determinant of myoblast-brown adipocyte transition, we propose S100B as a transducer of the deleterious effects of accumulation of reactive oxygen species in myoblasts and, potentially, myofibers concurring to the pathophysiology of sarcopenia.

Nuclear Lipid Microdomains Regulate Daunorubicin Resistance in Hepatoma Cells.

Daunorubicin is an anticancer drug, and cholesterol is involved in cancer progression, but their relationship has not been defined. In this study, we developed a novel experimental model that utilizes daunorubicin, cholesterol, and daunorubicin plus cholesterol in the same cells (H35) to search for the role of nuclear lipid microdomains, rich in cholesterol and sphingomyelin, in drug resistance. We find that the daunorubicin induces perturbation of nuclear lipid microdomains, localized in the inner nuclear membrane, where active chromatin is anchored. As changes of sphingomyelin species in nuclear lipid microdomains depend on neutral sphingomyelinase activity, we extended our studies to investigate whether the enzyme is modulated by daunorubicin. Indeed the drug stimulated the sphingomyelinase activity that induced reduction of saturated long chain fatty acid sphingomyelin species in nuclear lipid microdomains. Incubation of untreated-drug cells with high levels of cholesterol resulted in the inhibition of sphingomyelinase activity with increased saturated fatty acid sphingomyelin species. In daunodubicin-treated cells, incubation with cholesterol reversed the action of the drug by acting via neutral sphingomyelinase. In conclusion, we suggest that cholesterol and sphingomyelin-forming nuclear lipid microdomains are involved in the drug resistance.

Probing Internalization Effects and Biocompatibility of Ultrasmall Zirconium Metal-Organic Frameworks UiO-66 NP in U251 Glioblastoma Cancer Cells.

The synthesis of ultrasmall UiO-66 nanoparticles (NPs) with an average size of 25 nm, determined by X-ray powder diffraction and electron microscopies analysis, is reported. The NPs were stabilized in water by dialyzing the NP from the DMF used for the synthesis. DLS measurements confirmed the presence of particles of 100 nm, which are spherical aggregates of smaller particles of 20⁻30 nm size. The NP have a BET surface area of 700 m²/g with an external surface area of 300 m²/g. UiO-66_N (UiO-66 nanoparticles) were loaded with acridine orange as fluorescent probe. UV-vis spectroscopy analysis revealed no acridine loss after 48 h of agitation in simulated body fluid. The biocompatibility of UiO-66_N was evaluated in human glioblastoma (GBM) cell line U251, the most malignant (IV grade of WHO classification) among brain tumors. In U251 cells, UiO-66_N are inert since they do not alter the cell cycle, the viability, migration properties, and the expression of kinases involved in cancer cell growth. The internalization process was evident after a few hours of incubation. After 24 h, UiO-66_N@Acr (UiO-66_N loaded with acridine orange) were detectable around the nuclei of the cells. These data suggest that small UiO-66 are biocompatible NP and could represent a potential carrier for drug delivery in glioblastoma therapies.

Precision medicine against ALK-positive non-small cell lung cancer: beyond crizotinib.

Anaplastic lymphoma kinase (ALK) rearrangements represent the molecular driver of a subset of non-small cell lung cancers (NSCLCs). Despite the initial response, virtually all ALK-positive patients develop an acquired resistance to the ALK inhibitor crizotinib, usually within 12 months. Several next-generation ALK inhibitors have been developed in order to overcome crizotinib limitation, providing an unprecedented survival for this subset of patients. The aim of this review to summarize the current knowledge on ALK tyrosine kinase inhibitors (TKIs) in the treatment of advanced ALK-positive NSCLC, focusing on the role of novel ALK inhibitors in this setting. In addition, we will discuss their role in the pharmacological management of ALK-positive brain metastasis. Next-generation ALK inhibitors showed an impressive clinical activity in ALK-positive NSCLC, also against the sanctuary site of CNS. Sequential therapy with ALK TKIs appears to be effective in patients who fail a first ALK TKI and translates in clinically meaningful benefit. However, these agents display different activity profiles against crizotinib resistance mutation; therefore re-genotyping the disease at progression in order to administer the right TKI to the right patient is going to be necessary to correctly tailor the treatment. To avoid repeated invasive procedure, noninvasive methods to detect and monitor ALK rearrangement are under clinical investigation.

PP242 Counteracts Glioblastoma Cell Proliferation, Migration, Invasiveness and Stemness Properties by Inhibiting mTORC2/AKT.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and is associated with poor prognosis due to its thorny localization, lack of efficacious therapies and complex biology. Among the numerous pathways driving GBM biology studied so far, PTEN/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling plays a pivotal role, as it controls cell survival, proliferation and metabolism and is involved in stem cell maintenance. In front of recent and numerous evidences highlighting mTOR upregulation in GBM, all the strategies developed to inhibit this pathway have been substantially unsuccessful. Our study focused on mTOR complex 2 (mTORC2) to understand its involvement in GBM cell growth, proliferation, migration and invasiveness. We utilized an in vitro model, characterized by various genetic alterations (i.e., GL15, U257, U87MG and U118MG cell lines) in order to achieve the clonal heterogeneity observed in vivo. Additionally, being the U87MG cell line endowed with glioblastoma stem cells (GSCs), we also investigated the role of the PTEN/PI3K/AKT/mTOR pathway in this specific cell population, which is responsible for GBM relapse. We provide further insights that explain the reasons for the failure of numerous clinical trials conducted to date targeting PI3K or mTOR complex 1 (mTORC1) with rapamycin and its analogs. Additionally, we show that mTORC2 might represent a potential clinically valuable target for GBM treatment, as proliferation, migration and GSC maintenance appear to be mTORC2-dependent. In this context, we demonstrate that the novel ATP-competitive mTOR inhibitor PP242 effectively targets both mTORC1 and mTORC2 activation and counteracts cell proliferation via the induction of high autophagy levels, besides reducing cell migration, invasiveness and stemness properties.

Effect of Vitamin D in HN9.10e Embryonic Hippocampal Cells and in Hippocampus from MPTP-Induced Parkinson's Disease Mouse Model.

It has long been proven that neurogenesis continues in the adult brains of mammals in the dentatus gyrus of the hippocampus due to the presence of neural stem cells. Although a large number of studies have been carried out to highlight the localization of vitamin D receptor in hippocampus, the expression of vitamin D receptor in neurogenic dentatus gyrus of hippocampus in Parkinson's disease (PD) and the molecular mechanisms triggered by vitamin D underlying the production of differentiated neurons from embryonic cells remain unknown. Thus, we performed a preclinical in vivo study by inducing PD in mice with MPTP and showed a reduction of glial fibrillary acidic protein (GFAP) and vitamin D receptor in the dentatus gyrus of hippocampus. Then, we performed an in vitro study by inducing embryonic hippocampal cell differentiation with vitamin D. Interestingly, vitamin D stimulates the expression of its receptor. Vitamin D receptor is a transcription factor that probably is responsible for the upregulation of microtubule associated protein 2 and neurofilament heavy polypeptide genes. The latter increases heavy neurofilament protein expression, essential for neurofilament growth. Notably N-cadherin, implicated in activity for dendritic outgrowth, is upregulated by vitamin D.