Adenosine A2A Receptor Activation Enhances Blood-Tumor Barrier Permeability in a Rodent Glioma Model.

The blood-tumor barrier (BTB) limits the entry of effective chemotherapeutic agents into the brain for treatment of malignant tumors like glioblastoma. Poor drug entry across the BTB allows infiltrative glioma stem cells to evade therapy and develop treatment resistance. Regadenoson, an FDA-approved adenosine A2A receptor (A2AR) agonist, has been shown to increase drug delivery across the blood-brain barrier in non-tumor-bearing rodents without a defined mechanism of enhancing BTB permeability. Here, we characterize the time-dependent impact of regadenoson on brain endothelial cell interactions and paracellular transport, using mouse and rat brain endothelial cells and tumor models. In vitro, A2AR activation leads to disorganization of cytoskeletal actin filaments by 30 minutes, downregulation of junctional protein expression by 4 hours, and reestablishment of endothelial cell integrity by 8 hours. In rats bearing intracranial gliomas, regadenoson treatment results in increase of intratumoral temozolomide concentrations, yet no increased survival noted with combined temozolomide therapy. These findings demonstrate regadenoson's ability to induce brain endothelial structural changes among glioma to increase BTB permeability. The use of vasoactive mediators, like regadenoson, which transiently influences paracellular transport, should further be explored to evaluate their potential to enhance central nervous system treatment delivery to aggressive brain tumors. IMPLICATIONS: This study provides insight on the use of a vasoactive agent to increase exposure of the BTB to chemotherapy with intention to improve glioma treatment efficacy.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma.

BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. RESULTS: We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. CONCLUSION: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Model systems for studying the blood-brain barrier: Applications and challenges.

The blood-brain barrier (BBB) poses a serious impediment to the delivery of effective therapies to the central nervous system (CNS). Over time, various model systems have been crafted and used to evaluate the complexities of the BBB, which includes an impermeable physical barrier and a series of energy-dependent efflux pumps. Models of the BBB have mainly sought to assess changes in endothelial cell permeability, the role of ATP-dependent efflux transporters in drug disposition, and alterations in communication between BBB cells and the microenvironment. In the context of disease, various animal models have been utilized to examine real time BBB drug permeability, CNS dynamic changes, and overall treatment response. In this review, we outline the use of these in vitro and in vivo blood-brain barrier model systems to study normal physiology and diseased states. These current models each have their own advantages and disadvantages for studying the response of biologic processes to physiological and pathological conditions. Additional models are needed to mimic more closely the dynamic quality of the BBB, with the goal focused on potential clinical applications.

Cerebrovascular Angiogenic Reprogramming upon LRP1 Repression: Impact on Sphingosine-1-Phosphate-Mediated Signaling in Brain Endothelial Cell Chemotactism.

Switches in sphingolipid metabolism have recently been associated with oncogenic transformation, and a role for the low-density lipoprotein receptor-related protein 1 (LRP1) in sphingosine-1-phosphate (S1P) proangiogenic signaling inferred. S1P signaling crosstalk with LRP1 in brain microvascular endothelial cells (HBMEC) is however unclear. Transient in vitro siLRP1 gene silencing was compared to stable shLRP1 knockdown. We observed decreased expression of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor for which multiple binding sites are found within the promoter sequences of all five S1P receptor members, upon stable but not transient LRP1 repression. Chemotactic migration of brain EC isolated from Lrp1(EC)-/- mice and of stable shLRP1 HBMEC became unresponsive to S1P, partly due to altered ERK and p38 MAPK pathways, whereas chemotactism remained unaltered following transient in vitro siLRP1 repression. Diminished S1P1, S1P3, and S1P5 expression were observed in stable shLRP1 HBMEC and in brain EC isolated from Lrp1(EC)-/- mice. Overexpression of LRP1 cluster IV rescued S1P-mediated cell migration through increased S1P3 transcription in shLRP1 HBMEC. Our study highlights an adaptive signaling crosstalk between LRP1 and specific S1P receptors which may regulate the angiogenic response of brain EC and be targeted at the blood-brain barrier in future therapeutic strategies.

Isotope-labeled differential profiling of metabolites using N-benzoyloxysuccinimide derivatization coupled to liquid chromatography/high-resolution tandem mass spectrometry.

RATIONALE: An isotopic labeling strategy based on derivatizing amine-containing metabolites has been developed using light ((12) C6 ) and heavy ((13) C6 ) N-benzoyloxysuccinimide reagents for semi-targeted metabolomic applications. METHODS: Differentially labeled samples were combined and analyzed simultaneously by liquid chromatography/high-resolution tandem mass spectrometry (LC/HR-MS/MS) to compare relative amounts of amine-containing metabolites. The selectivity of the reaction was determined with model metabolites and was shown to also be applicable to thiol and phenol moieties. The potential for relative quantitation was evaluated in cell extracts and the method was then applied to quantify metabolic perturbations occurring in human cultured cells under normal vs. oxidative stress conditions. RESULTS: A total of 279 derivatized features were detected in HL60 cell extracts, 77 of which yielded significant concentration changes upon oxidative stress treatment. Based on accurate mass measurements and MS/MS spectral matching with reference standard solutions, 10 metabolites were clearly identified. Derivatized compounds were found to have diagnostic fragment ions from the reagent itself, as well as structurally informative ions useful for metabolite identification. CONCLUSIONS: This simple derivatization reaction can be applied to the relative quantitation of amine-, thiol- and phenol-containing compounds, with improved sensitivity and chromatographic peak shapes due to the increased hydrophobicity of polar metabolites not readily amenable to reversed-phase LC/MS analysis.

Mesenchymal stromal cell ciliogenesis is abrogated in response to tumor necrosis factor-α and requires NF-κB signaling.

The primary cilium is a cell surface-anchored sensory organelle which expression is lost in hypoxic cancer cells and during mesenchymal stromal cells (MSC) adaptation to low oxygen levels. Since pro-inflammatory cues are among the early events which promote tumor angiogenesis, we tested the inflammatory cytokine tumor necrosis factor (TNF)-α and found that it triggered a dose-dependent loss of the primary cilia in MSC. This loss was independent of IFT88 expression, was abrogated by progranulin, an antagonist of the TNF receptor and required the NF-κB signaling intermediates IκB kinase α, ß, and γ, as well as NF-κB p65. These findings strengthen the concept that the primary cilium may serve as a biomarker reflecting the tumor-supporting potential of MSC and their capacity to adapt to hypoxic and pro-inflammatory cues.

EGCG targeting efficacy of NF-κB downstream gene products is dictated by the monocytic/macrophagic differentiation status of promyelocytic leukemia cells.

Central nervous system infiltration by circulating leukemic cells and enhanced in vitro transendothelial migration of promyelocytic leukemia HL-60-derived macrophages through a blood-brain barrier model was recently demonstrated. The intrinsic molecular and signaling mechanisms involved are, however, poorly documented. Drug targeting of such translocation event performed by circulating microbes and immune cells may prevent secondary cerebral infections and development of brain pathologies. In this study, we specifically investigated the in vitro targeting efficacy of the chemopreventive and dietary-derived epigallocatechin-3-gallate (EGCG) molecule on the NF-κB-mediated transcriptional regulation of a panel of 89 biomarkers associated with promyelocytic HL-60 differentiation into macrophages. NF-κB-mediated signaling during HL-60 macrophage differentiation was reversed by EGCG, in part through reduced IκB phosphorylation and led to the inhibition of moderately to highly expressed NF-κB gene targets among which the matrix metalloproteinase (MMP)-9 and the cyclooxygenase (COX)-2. In contrast, EGCG exhibited low efficacy in reversing NF-κB-regulated genes and showed selective antagonism toward COX-2 expression while that of MMP-9 remained high in terminally differentiated macrophages. Decreased expression of the 67-kDa non-integrin Laminin Receptor in terminally differentiated macrophages may explain such differential EGCG efficacy. Our results suggest that terminally differentiated macrophage transendothelial migration associated with neuroinflammation may not be pharmacologically affected by such a specific class of flavonoid. The differentiation status of a given in vitro cell model must therefore be carefully considered for optimized assessment of therapeutic drugs.

Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent.

The occurrence of a functional relationship between the release of metalloproteinases (MMPs) and the expression of cyclooxygenase (COX)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB), increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC) were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I-IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I-III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA), a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.