MEF2C and miR-194-5p: New Players in Triple Negative Breast Cancer Tumorigenesis.

Among breast cancer (BC) subtypes, the most aggressive is triple negative BC (TNBC), which is prone to metastasis. We previously found that microRNA (miR)-194-5p is downregulated at the early stages of TNBC brain metastasis development. Additionally, the transcription factor myocyte enhancer factor 2 (MEF2)C, a bioinformatically predicted miR-194-5p target, was increasingly expressed throughout TNBC brain metastasis formation and disease severity. However, the contributions of these two players to malignant cells' features remain undetermined. This study aimed at disclosing the role of miR-194-5p and MEF2C in TNBC tumorigenesis. The transfection of 4T1 cells with a silencer for MEF2C or with a pre-miRNA for miR-194-5p was employed to study TNBC cells' phenotypic alterations regarding epithelial and mesenchymal markers, as well as migratory capability alterations. MEF2C-silenced cells presented a decline in both vimentin and cytokeratin expression, whereas the overexpression of miR-194-5p promoted an increase in cytokeratin and a reduction in vimentin, reflecting the acquisition of an epithelial phenotype. Both treatments reduced TNBC cells' migration. These results suggest that MEF2C may determine TNBC cells' invasive properties by partially determining the occurrence of epithelial-mesenchymal transition, while the overexpression of miR-194-5p promotes a decline in TNBC cells' aggressive behavior and reinforces this miRNA's role as a tumor suppressor in TNBC.

Therapeutic Options in Neuro-Oncology.

One of the biggest challenges in neuro-oncology is understanding the complexity of central nervous system tumors, such as gliomas, in order to develop suitable therapeutics. Conventional therapies in malignant gliomas reconcile surgery and radiotherapy with the use of chemotherapeutic options such as temozolomide, chloroethyl nitrosoureas and the combination therapy of procarbazine, lomustine and vincristine. With the unraveling of deregulated cancer cell signaling pathways, targeted therapies have been developed. The most affected signaling pathways in glioma cells involve tyrosine kinase receptors and their downstream pathways, such as the phosphatidylinositol 3-kinases (PI3K/AKT/mTOR) and mitogen-activated protein kinase pathways (MAPK). MAPK pathway inhibitors include farnesyl transferase inhibitors, Ras kinase inhibitors and mitogen-activated protein extracellular regulated kinase (MEK) inhibitors, while PI3K/AKT/mTOR pathway inhibitors are divided into pan-inhibitors, PI3K/mTOR dual inhibitors and AKT inhibitors. The relevance of the immune system in carcinogenesis has led to the development of immunotherapy, through vaccination, blocking of immune checkpoints, oncolytic viruses, and adoptive immunotherapy using chimeric antigen receptor T cells. In this article we provide a comprehensive review of the signaling pathways underlying malignant transformation, the therapies currently used in the treatment of malignant gliomas and further explore therapies under development, including several ongoing clinical trials.

In Vitro Study of the Blood-Brain Barrier Transport of Natural Compounds Recovered from Agrifood By-Products and Microalgae.

Agrifood by-products and microalgae represent a low-cost and valuable source of bioactive compounds with neuroprotective properties. However, the neuroprotective effectiveness of therapeutic molecules can be limited by their capacity to cross the blood-brain barrier (BBB) and reach the brain. In this research, various green extracts from Robinia pseudoacacia (ASFE), Cyphomandra betacea (T33), Coffea arabica (PPC1), Olea europaea L., (OL-SS), Citrus sinensis (PLE100) by-products and from the microalgae Dunaliella salina (DS) that have demonstrated in vitro neuroprotective potential were submitted to an in vitro BBB permeability and transport assay based on an immortalized human brain microvascular endothelial cells (HBMEC) model. Toxicity and BBB integrity tests were performed, and the transport of target bioactive molecules across the BBB were evaluated after 2 and 4 h of incubation using gas and liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (GC/LC-Q-TOF-MS). The HBMEC-BBB transport assay revealed a high permeability of representative neuroprotective compounds, such as mono- and sesquiterpenoids, phytosterols and some phenolic compounds. The obtained results from the proposed in vitro BBB cellular model provide further evidence of the neuroprotective potential of the target natural extracts, which represent a promising source of functional ingredients to be transferred into food supplements, food additives, or nutraceuticals with scientifically supported neuroprotective claims.

Brain Metastases and Microenvironment.

The formidable advances in cancer treatment have led to remarkable improvements in patient's survival, so that the major concern shifted from primary tumors to metastatic disease. Brain metastases represent a life-threatening condition with a poor prognosis due to the lack of reliable biomarkers that preclude their timely identification and to the scarce therapeutic possibilities considering that the blood-brain barrier limits the access of most of the drugs to the brain and surgical resection is discouraged in cases of multiple metastases. Moreover, brain metastases have been scarcely investigated, which precludes a comprehensive understanding of the determinants and players, as well as of the complex cross-talk and signaling pathways involved. This chapter summarizes the impressive numbers about cancer and brain metastases and the estimates of progression in the years to come. It also gathers together the relevant concepts about the metastatic cascade, focusing in the extravasation step across the microvascular endothelium that leads to the formation of brain metastases. Moreover, it comprehensively explores the brain tumor microenvironment, detailing on the pre-metastatic niches and their relevance for tumor cell development in the target organ. Additionally, the cellular and acellular components, as well as their interplay, activation status, and acquired phenotypes, are addressed. Collectively, by bringing together historical concepts and state-of-the-art knowledge, this chapter shall contribute to a better understanding of the brain metastasization process, essential for the development of novel approaches to improve patients' life quality and expectancy.

Behind Brain Metastases Formation: Cellular and Molecular Alterations and Blood-Brain Barrier Disruption.

Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells' (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood-brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC-BMEC interaction compromised BBB integrity, as revealed by junctional proteins (ß-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. ß4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.

MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development.

Triple negative breast cancer presents higher mortality and poorer survival rates than other breast cancer (BC) types, due to the proneness to brain metastases formation, which are usually diagnosed at advanced stages. Therefore, the discovery of BC brain metastases (BCBM) biomarkers appears pivotal for a timely intervention. With this work, we aimed to disclose microRNAs (miRNAs) and extracellular vesicles (EVs) in the circulation as biomarkers of BCBM formation. Using a BCBM animal model, we analyzed EVs in plasma by nanoparticle tracking analysis and ascertained their blood-brain barrier (BBB) origin by flow cytometry. We further evaluated circulating miRNAs by RT-qPCR and their brain expression by in situ hybridization. In parallel, a cellular model of BCBM formation, combining triple negative BC cells and BBB endothelial cells, was used to differentiate the origin of biomarkers. Established metastases were associated with an increased content of circulating EVs, particularly of BBB origin. Interestingly, deregulated miRNAs in the circulation were observed prior to BCBM detection, and their brain origin was suggested by matching alterations in brain parenchyma. In vitro studies indicated that miR-194-5p and miR-205-5p are expressed and released by BC cells, endothelial cells and during their interaction. These results highlight miRNAs and EVs as biomarkers of BCBM in early and advanced stages, respectively.

Brain metastasization of breast cancer.

Central nervous system metastases have been reported in 15-25% of breast cancer patients, and the incidence is increasing. Moreover, the survival of these patients is generally poor, with reports of a 1-year survival rate of 20%. Therefore, a better knowledge about the determinants of brain metastasization is essential for the improvement of the clinical outcomes. Here, we summarize the current data about the metastatic cascade, ranging from the output of cancer cells from the primary tumour to their colonization in the brain, which involves the epithelial-mesenchymal transition, invasion of mammary tissue, intravasation into circulation, and homing into and extravasation towards the brain. The phenotypic change in malignant cells, and the importance of the microenvironment in the formation of brain metastases are also inspected. Finally, the importance of genetic and epigenetic changes, and the recently disclosed effects of microRNAs in brain metastasization of breast cancer are highlighted.

The choroid plexus in health and in disease: dialogues into and out of the brain.

This article brings the choroid plexus into the context of health and disease. It is remarkable that the choroid plexus, composed by a monolayer of epithelial cells that lie in a highly vascularized stroma, floating within the brain ventricles, gets so little attention in major physiology and medicine text books and in the scientific literature in general. Consider that it is responsible for producing most of the about 150mL of cerebrospinal fluid that fills the brain ventricles and the subarachnoid space and surrounds the spinal cord in the adult human central nervous system, which is renewed approximately 2-3 times daily. As such, its activity influences brain metabolism and function, which will be addressed. Reflect that it contains an impressive number of receptors and transporters, both in the apical and basolateral sides of the epithelial cells, and as such is a key structure for the communication between the brain and the periphery. This will be highlighted in the context of neonatal jaundice, multiple sclerosis and Alzheimer's disease. Realize that the capillaries that irrigate the choroid plexus stroma do not possess tight junctions and that the blood flow to the choroid plexus is five times higher than that in the brain parenchyma, allowing for a rapid sensing system and delivery of molecules such as nutrients and metals as will be revised. Recognize that certain drugs reach the brain parenchyma solely through the choroid plexus epithelia, which has potential to be manipulated in diseases such as neonatal jaundice and Alzheimer's disease as will be discussed. Without further notice, it must be now clear that understanding the choroid plexus is necessary for comprehending the brain and how the brain is modulated and modulates all other systems, in health and in disease. This review article intends to address current knowledge on the choroid plexus, and to motivate the scientific community to consider it when studying normal brain physiology and diseases of the central nervous system. It will guide the reader through several aspects of the choroid plexus in normal physiology, in diseases characteristic of various periods of life (newborns-kernicterus, young adults-multiple sclerosis and the elder-Alzheimer's disease), and how sex-differences may relate to disease susceptibility.

Endothelial progenitor cells in multiple myeloma neovascularization: a brick to the wall.

Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow that leads to events such as bone destruction, anaemia and renal failure. Despite the several therapeutic options available, there is still no effective cure, and the standard survival is up to 4 years. The evolution from the asymptomatic stage of monoclonal gammopathy of undetermined significance to MM and the progression of the disease itself are related to cellular and molecular alterations in the bone marrow microenvironment, including the development of the vasculature. Post-natal vasculogenesis is characterized by the recruitment to the tumour vasculature of bone marrow progenitors, known as endothelial progenitor cells (EPCs), which incorporate newly forming blood vessels and differentiate into endothelial cells. Several processes related to EPCs, such as recruitment, mobilization, adhesion and differentiation, are tightly controlled by cells and molecules in the bone marrow microenvironment. In this review, the bone marrow microenvironment and the mechanisms associated to the development of the neovasculature promoted by EPCs are discussed in detail in both a non-pathological scenario and in MM. The latest developments in therapy targeting the vasculature and EPCs in MM are also highlighted. The identification and characterization of the pathways relevant to the complex setting of MM are of utter importance to identify not only biomarkers for an early diagnosis and disease progression monitoring, but also to reveal intervention targets for more effective therapy directed to cancer cells and the endothelial mediators relevant to neovasculature development.

Differential expression of aquaporin-3 and aquaporin-5 in pancreatic ductal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Aquaporin-5 (AQP5) and -3 (AQP3) are protein channels that showed to be up-regulated in a variety of tumors. Our goal was to investigate the expression pattern of AQP5 and AQP3 in pancreatic ductal adenocarcinomas (PDA) and correlate with cell proliferation, tumor stage and progression, and clinical significance. METHODS: 35 PDA samples in different stages of differentiation and locations were analyzed by immunohistochemistry for expression of AQP5, AQP3 and several markers of cell proliferation and tumorigenesis. RESULTS: In PDA samples AQP5 was overexpressed in the apical membrane of intercalated and intralobular ductal cells while AQP3 was expressed at the plasma membrane of ductal cells. AQP5 was also found in infiltrative cancer cells in duodenum. Simultaneous overexpression of EGFR, Ki-67, and CK7, with decreased E-cad and increased Vim that characterize epithelial mesenchymal transition, tumor formation and invasion, strongly suggest AQP3 and AQP5 involvement in cell proliferation and transformation. AQP3 overexpression is reinforced in late and more aggressive PDA stages whereas AQP5 is related with tumor differentiation, suggesting it may represent a novel marker for PDA aggressiveness and intestinal infiltration. CONCLUSIONS: These findings suggest AQP3 and AQP5 involvement in PDA development and the usefulness of AQP5 in early PDA diagnosis.

Aquaporin-5: from structure to function and dysfunction in cancer.

Aquaporins, a highly conserved group of membrane proteins, are involved in the bidirectional transfer of water and small solutes across cell membranes taking part in many biological functions all over the human body. In view of the wide range of cancer malignancies in which aquaporin-5 (AQP5) has been detected, an increasing interest in its implication in carcinogenesis has emerged. Recent publications suggest that this isoform may enhance cancer cell proliferation, migration and survival in a variety of malignancies, with strong evidences pointing to AQP5 as a promising drug target and as a novel biomarker for cancer aggressiveness with high translational potential for therapeutics and diagnostics. This review addresses the structural and functional features of AQP5, detailing its tissue distribution and functions in human body, its expression pattern in a variety of tumors, and highlighting the underlying mechanisms involved in carcinogenesis. Finally, the actual progress of AQP5 research, implications in cancer biology and potential for cancer detection and prognosis are discussed.

Deconstructing breast cancer cell biology and the mechanisms of multidrug resistance.

Cancer complexity constantly challenges the way that clinicians manage breast cancer therapy. Tumor heterogeneity and intratumoral stroma characteristics allow cells with different phenotypes and deregulated apoptotic, proliferative and migration abilities to co-exist contributing to a disappointing therapeutic response. While new approaches are being associated with conventional chemotherapy, such as hormonal therapy or target monoclonal antibodies, recurrence and metastasization are still observed. Membrane transporters are the cell's first line of contact with anticancer drugs having a major role in multidrug resistance events. This structural-based activity enables the cell to be drug-resistant by decreasing drug intracellular concentration through an efflux-transport mechanism, mainly associated with overexpression of ATP-binding cassette (ABC) proteins. This review focuses on some of the important structural and biological properties of the malignant cell and tumor microenvironment, addressing the role of the membrane ABC transporters in therapeutic outcomes, and highlighting related molecular pathways that may represent meaningful target therapies.

Circulating low-molecular-weight (poly)phenol metabolites in the brain: unveiling in vitro and in vivo blood-brain barrier transport.

Circulating metabolites resulting from colonic metabolism of dietary (poly)phenols are highly abundant in the bloodstream, though still marginally explored, particularly concerning their brain accessibility. Our goal is to disclose (poly)phenol metabolites' blood-brain barrier (BBB) transport, in vivo and in vitro, as well as their role at BBB level. For three selected metabolites, benzene-1,2-diol-3-sulfate/benzene-1,3-diol-2-sulfate (pyrogallol-sulfate - Pyr-sulf), benzene-1,3-diol-6-sulfate (phloroglucinol-sulfate - Phlo-sulf), and phenol-3-sulfate (resorcinol-sulfate - Res-sulf), BBB transport was assessed in human brain microvascular endothelial cells (HBMEC). Their potential in modulating in vitro BBB properties at circulating concentrations was also studied. Metabolites' fate towards the brain, liver, kidney, urine, and blood was disclosed in Wistar rats upon injection. Transport kinetics in HBMEC highlighted different BBB permeability rates, where Pyr-sulf emerged as the most in vitro BBB permeable metabolite. Pyr-sulf was also the most potent regarding BBB properties improvement, namely increased beta(ß)-catenin membrane expression and reduction of zonula occludens-1 membrane gaps. Whereas no differences were observed for transferrin, increased expression of caveolin-1 upon Pyr-sulf and Res-sulf treatments was found. Pyr-sulf was also capable of modulating gene and protein expression of some solute carrier transporters. Notably, each of the injected metabolites exhibited a unique tissue distribution in vivo, with the remarkable ability to almost immediately reach the brain.

Tricellulin expression in brain endothelial and neural cells.

Tricellulin is a tight junction (TJ) protein, which is not only concentrated at tricellular contacts but also present at bicellular contacts between epithelial tissues. We scrutinized the brain for tricellulin expression in endothelial and neural cells by using real-time polymerase chain reaction, Western blot and immunohistochemical and immunocytochemical analysis of cultured brain cells and paraffin sections of brain. Tricellulin mRNA was detected in primary cultures and in a cell line of human brain microvascular endothelial cells. Protein expression was confirmed by Western blot and immunofluorescence analysis, which further highlighted the localization of tricellulin in the cell membrane at tricellular and along bicellular contacts, and in the nucleus and perinuclear region. Compared with the well-studied TJ protein, zonula occludens-1, tricellulin expression was less marked at the cell membrane but more evident in the nuclear and perinuclear regions. The presence of tricellulin in cultured endothelial cells was corroborated by immunohistochemical and immunofluorescence staining in brain blood vessels, where it was colocalized with another TJ protein, claudin-5. Tricellulin mRNA was detected in neurons and astrocytes, whereas protein expression was observed in astrocytes but not in neurons, as shown by immunofluorescence analysis. This study reveals the presence and subcellular distribution of tricellulin in brain endothelial cells, both in vitro and in situ and its colocalization with other relevant TJ proteins. Moreover, it demonstrates the expression of the protein in astrocytes opening new avenues for future research to establish the biological significance of tricellulin expression in glial cells.

Abrogating Metastatic Properties of Triple-Negative Breast Cancer Cells by EGFR and PI3K Dual Inhibitors.

Triple-negative breast cancer (TNBC) is a devastating BC subtype. Its aggressiveness, allied to the lack of well-defined molecular targets, usually culminates in the appearance of metastases that account for poor prognosis, particularly when they develop in the brain. Nevertheless, TNBC has been associated with epidermal growth factor receptor (EGFR) overexpression, leading to downstream phosphoinositide 3-kinase (PI3K) signaling activation. We aimed to unravel novel drug candidates for TNBC treatment based on EGFR and/or PI3K inhibition. Using a highly metastatic TNBC cell line with brain tropism (MDA-MB-231 Br4) and a library of 27 drug candidates in silico predicted to inhibit EGFR, PI3K, or EGFR plus PI3K, and to cross the blood-brain barrier, we evaluated the effects on cell viability. The half maximal inhibitory concentration (IC50) of the most cytotoxic ones was established, and cell cycle and death, as well as migration and EGFR pathway intervenient, were further evaluated. Two dual inhibitors emerged as the most promising drugs, with the ability to modulate cell cycle, death, migration and proliferation, morphology, and PI3K/AKT cascade players such as myocyte enhancer factor 2C (MEF2C) and forkhead box P1 (FOXP1). This work revealed EGFR/PI3K dual inhibitors as strong candidates to tackle brain metastatic TNBC cells.

Costunolide and parthenolide: Novel blood-brain barrier permeable sesquiterpene lactones to improve barrier tightness.

Sesquiterpene lactones - such as those found in chicory - are considered promising bioactive compounds. These small molecules have shown several health benefits for various diseases, including brain disorders. However, it is unknown whether these compounds can cross the blood-brain barrier (BBB), and which could be the effects on brain microvascular endothelial cells. We show that six sesquiterpene lactones evaluated in an in vitro model of the BBB have different capacities to be transported through the barrier. Costunolide presented more than 20 % of transport while lactucin, 11ß-13-dihydrolactucin, 11ß-13-dihydrolactucopicrin, and parthenolide presented between 10 % and 20 %, whilst almost no transport was detected for lactucopicrin. Furthermore, costunolide and parthenolide reduced P-gp ABC transporter expression alongside an increase in caveolin-1, the main protein of caveolae. Remarkably, these two compounds improved barrier tightness by increasing the expression of both tight and adherens junctions. These findings open a new avenue to explore costunolide and parthenolide as promising compounds for brain therapies.

Breast Cancer Brain Metastases: Implementation and Characterization of a Mouse Model Relying on Malignant Cells Inoculation in the Carotid Artery.

Breast cancer (BC) brain metastases (BCBM) is a severe condition frequently occurring in the triple-negative subtype. The study of BCBM pathogenesis and treatment has been hampered by the difficulty in establishing a reliable animal model that faithfully recapitulates the preferential formation of brain metastases. The injection of BC cells in the carotid artery of mice has been proposed but the procedure is challenging, with the metastatic pattern being scarcely characterized. In this work, we thoroughly describe an improved procedure, highlighting the tricks and challenges of the process, and providing a characterization of the brain and peripheral metastatic pattern at the cellular and molecular level. Triple-negative BC (4T1) cells were inoculated in the common carotid artery of BALB/c mice. Brains and peripheral organs were harvested at 7-14 days for the histological characterization of the metastases' pattern and the immunofluorescence analysis of specific markers. With our surgical procedure, both mouse death and procedure-associated weight loss were negligible. Brain metastases mostly occurred in the hippocampus, while sparse peripheral lesions were only detected in the lungs. Brain-colonizing BC cells presented proliferative (Ki-67) and epithelial (pan-cytokeratin and tomato lectin) features, which account for metastases' establishment. The presented surgical approach constitutes an important and reliable tool for BCBM studies.

A Drug Screening Reveals Minocycline Hydrochloride as a Therapeutic Option to Prevent Breast Cancer Cells Extravasation across the Blood-Brain Barrier.

Among breast cancer (BC) patients, 15-25% develop BC brain metastases (BCBM), a severe condition due to the limited therapeutic options, which points to the need for preventive strategies. We aimed to find a drug able to boost blood-brain barrier (BBB) properties and prevent BC cells (BCCs) extravasation, among PI3K, HSP90, and EGFR inhibitors and approved drugs. We used BCCs (4T1) and BBB endothelial cells (b.End5) to identify molecules with toxicity to 4T1 cells and safe for b.End5 cells. Moreover, we used those cells in mixed cultures to perform a high-throughput microscopy screening of drugs' ability to ameliorate BBB properties and prevent BCCs adhesion and migration across the endothelium, as well as to analyse miRNAs expression and release profiles. KW-2478, buparlisib, and minocycline hydrochloride (MH) promoted maximal expression of the junctional protein ß-catenin and induced 4T1 cells nucleus changes. Buparlisib and MH further decreased 4T1 adhesion. MH was the most promising in preventing 4T1 migration and BBB disruption, tumour and endothelial cytoskeleton-associated proteins modifications, and miRNA deregulation. Our data revealed MH's ability to improve BBB properties, while compromising BCCs viability and interaction with BBB endothelial cells, besides restoring miRNAs' homeostasis, paving the way for MH repurposing for BCBM prevention.

Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood-Brain Barrier and Target EGFR/PI3Kp110ß Signaling.

The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110ß signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood-brain barrier (BBB) limits drugs' brain penetration. We aimed to discover EGFR/PI3Kp110ß pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure-activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110ß, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110ß, and two dual inhibitors) decreased cell viability by 40-99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets' downregulation and increased apoptosis (15-85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110ß inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.

Astrocyte reactivity to unconjugated bilirubin requires TNF-α and IL-1ß receptor signaling pathways.

Jaundice and sepsis are common neonatal conditions that can lead to neurodevelopment sequelae, namely if present at the same time. We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1ß are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. The present study was designed to evaluate the role of TNF-α and IL-1ß signaling on astrocyte reactivity to UCB in rat cortical astrocytes. Exposure of astrocytes to UCB increased the expression of both TNF-α receptor (TNFR)1 and IL-1ß receptor (IL-1R)1, but not TNFR2, as well as their activation, observed by augmented binding of receptors' molecular adaptors, TRAF2 and TRAF6, respectively. Silencing of TNFR1, using siRNA technology, or blockade of IL-1ß cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κB activation. Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, although an increase was observed after extended exposure; in contrast, inhibition of IL-1ß cascade produced a sustained blockade of astrocyte injury by UCB. Together, our data show that inflammatory pathways are activated during in vitro exposure of rat cortical astrocytes to UCB and that this activation is prolonged in time. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets.