Inflammatory Effects of Co-Exposure to Heavy Metals (Cd and Pb) and Respiratory Viruses (229E, OC43, and H3N2) on IL-6 and IL-8 Responses in U87 Glioblastoma Cells

Ethnopharmacological relevance: Dapivirine is one of reverse transcriptase inhibitors (RTIs). It is the prototype of diarylpyrimidines (DAPY), formerly known as TMC120 or DAPY R147681 (IUPAC name: 4- [[4-(2, 4, 6-trimethylphenyl) amino]-2-pyrimidinyl] amino]-benzonitrile; CAS no.244767-67-7).Aim: The purpose of this study is to investigate the antitumor activity of dapivirine, one of the RTIs, on U87 glioblastoma (GBM) cells in vitro and in vivo.Materials and Methods: U87 GBM cells were cultured and treated with or without dapivirine. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis was analyzed by flow cytometry; cell migration was evaluated by Boyden Chamber assay; Western blotting was performed to detect proteins related to apoptosis, epithelial-to-mesenchymal transition and autophagy. PathScan intracellular signaling array kit was used to detect important and well-characterized signaling molecules. Tumor xenograft model in nude mice was used to evaluate the antitumorigenic effect in vivo.Results: Dapivirine weakened proliferation of glioma cells and induced the apoptosis of U87 glioblastoma cells. Furthermore, dapivirine regulated autophagy and induced Akt, Bad and SAPK/JNK activations. Moreover, the inhibition of glioma cell growth by dapivirine was also observed in nude mice in vivo.Conclusion: In summary, in our study dapivirine exposure induces stress, resulting in JNK and PI3K/Akt pathway activation through diminished inhibition of the apoptosis and autophagy cascade in U87 GBM cells, which inhibits cell growth in vitro and in vivo.

Infections, Immunity & their Effects on Asthma

Gold nanoparticles (GNPs) have demonstrated significant dose enhancement with kilovoltage (kV) X-rays; however, recent studies have shown inconsistent findings with megavoltage (MV) X-rays. We propose to evaluate the radiosensitization effect on U87 glioblastoma (GBM) cells in the presence of 42 nm GNPs and irradiated with a clinical 6 MV photon beam. Cytotoxicity and radiosensitization were measured using MTS and clonogenic cellular radiation sensitivity assays, respectively. The sensitization enhancement ratio was calculated for 2 Gy (SER2Gy) with GNP (100 μg/mL). Dark field and MTS assays revealed high co-localization and good biocompatibility of the GNPs with GBM cells. A significant sensitization enhancement of 1.45 (p = 0.001) was observed with GNP 100 μg/mL. Similarly, at 6 Gy, there was significant difference in the survival fraction between the GBM alone group (mean (M) = 0.26, standard deviation (SD) = 0.008) and the GBM plus GNP group (M = 0.07, SD = 0.05, p = 0.03). GNPs enabled radiosensitization in U87 GBM cells at 2 Gy when irradiated using a clinical platform. In addition to the potential clinical utility of GNPs, these studies demonstrate the effectiveness of a robust and easy to standardize an in-vitro model that can be employed for future studies involving metal nanoparticle plus irradiation.

ANO1, a Ca2+-activated chloride channel, is highly expressed in glioblastoma cells and its surface expression is involved in their migration and invasion. However, the regulation of ANO1 surface expression in glioblastoma cells is largely unknown. In this study, we found that Ca2+/Calmodulin-dependent protein kinase II (CaMKII) β specifically enhances the surface expression and channel activity of ANO1 in U251 glioblastoma cells. When KN-93, a CaMKII inhibitor, was used to treat U251 cells, the surface expression and channel activity of ANO1 were significantly reduced. Only CaMKIIβ, among the four CaMKII isoforms, increased the surface expression and channel activity of ANO1 in a heterologous expression system. Additionally, gene silencing of CaMKIIβ suppressed the surface expression and channel activity of ANO1 in U251 cells. Moreover, gene silencing of CaMKIIβ or ANO1 prominently reduced the migration and invasion of U251 and U87 MG glioblastoma cells. We thus conclude that CaMKIIβ plays a specific role in the surface expression of ANO1 and in the ANO1-mediated tumorigenic properties of glioblastoma cells, such as migration and invasion.

Antimicrobial peptide tilapia piscidin 4 (TP4) from Oreochromis niloticus exhibits potent bactericidal and anti-tumorigenic effects. In a variety of cancers, the mutation status of p53 is a decisive factor for therapeutic sensitivity. Therefore, we investigated the impact of p53 status on TP4-induced cytotoxicity in glioblastoma cell lines and the molecular mechanisms that govern cytotoxic effects. Both U87MG (wild-type/WT p53) and U251 (mutant p53) glioblastoma cell lines were sensitive to TP4-induced cytotoxicity. The necrosis inhibitors Necrostatin-1 and GSK’872 attenuated TP4-induced cytotoxicity, and TP4 treatment induced the release of cyclophilin A, a biomarker of necrosis. Moreover, TP4 induced mitochondrial hyperpolarization and dysfunction, which preceded the elevation of intracellular reactive oxygen species, DNA damage, and necrotic cell death in both U87MG and U251 glioblastoma cells. p38 was also activated by TP4, but did not contribute to cytotoxicity. SB202190, a specific p38 inhibitor, enhanced TP4-induced oxidative stress, mitochondrial dysfunction, and cytotoxicity, suggesting a protective role of p38. Furthermore, TP4-induced cytotoxicity, oxidative stress, phosphorylation of p38, and DNA damage were all attenuated by the mitochondrial-targeted reactive oxygen species (ROS) scavenger MitoTEMPO, or the reactive oxygen species scavenger N-acetyl-L-cysteine. Based on these data, we conclude that TP4 induces necrosis in both WT and mutant p53 glioblastoma cells through a mitochondrial ROS-dependent pathway.

This study aimed to investigate β-Caryophyllene (BCA) pharmacokinetics as well as the potential antitumor activity and mechanism of action of BCA and eugenol (EU), alone or in combination, in U87 glioblastoma (GB) cells. The BCA pharmacokinetic was studied by evaluating its concentration profiles in rat blood and cerebrospinal fluid after oral and intravenous administration. EU and BCA antitumor mechanisms were assessed by comparing their effects in U87 GB cells and non-tumoral HMC3 cells. Cell death, cell cycle regulation and mitochondrial membrane potential (MMP) were evaluated using flow cytometry. mRNA levels of target genes were evaluated by qPCR. Secreted cytokines were measured by Luminex®. BCA, as well as EU, permeates the brain. EU and BCA affected the viability and proliferation of U87 cells (up to 50%, p < 0.001) but not HMC3 cells and showed a synergistic effect. BCA and EU induced G0/G1 cell cycle arrest, increasing apoptosis/necrosis. EU and BCA induced the downregulation of mRNAs encoding for key proteins involved in GB angiogenesis (VEGFA decreased op to 60%, p < 0.01), proliferation and progression, and showed anti-inflammatory activity (IL-4 significantly decreased, p < 0.001). EU and BCA demonstrated strong and multitarget antitumor activity in U87 cells. Our results provide a strong rationale for the further evaluation of EU and BCA as possible therapeutic molecules in GB management.

&lt;div&gt;Abstract&lt;p&gt;Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of &lt;i&gt;IDH1&lt;/i&gt; mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized &lt;sup&gt;13&lt;/sup&gt;C-magnetic resonance spectroscopy (&lt;sup&gt;13&lt;/sup&gt;C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-&lt;sup&gt;13&lt;/sup&gt;C-pyruvate to 5-&lt;sup&gt;13&lt;/sup&gt;C-glutamate, relative to cells expressing wild-type IDH1. &lt;sup&gt;13&lt;/sup&gt;C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-&lt;sup&gt;13&lt;/sup&gt;C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by &lt;i&gt;IDH1&lt;/i&gt; mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the &lt;i&gt;IDH1&lt;/i&gt; mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. &lt;i&gt;Cancer Res; 75(15); 2999–3009. ©2015 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of &lt;i&gt;IDH1&lt;/i&gt; mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized &lt;sup&gt;13&lt;/sup&gt;C-magnetic resonance spectroscopy (&lt;sup&gt;13&lt;/sup&gt;C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-&lt;sup&gt;13&lt;/sup&gt;C-pyruvate to 5-&lt;sup&gt;13&lt;/sup&gt;C-glutamate, relative to cells expressing wild-type IDH1. &lt;sup&gt;13&lt;/sup&gt;C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-&lt;sup&gt;13&lt;/sup&gt;C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by &lt;i&gt;IDH1&lt;/i&gt; mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the &lt;i&gt;IDH1&lt;/i&gt; mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. &lt;i&gt;Cancer Res; 75(15); 2999–3009. ©2015 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications.

Glioblastoma (GBM) is the most common primary intracranial malignancy. GBM still exhibits high recurrence and mortality rates even following combined treatment with surgery, radiotherapy and chemotherapy, Therefore, the identification of novel therapeutic targets is urgent. Previous research has shown that nicotinamide phosphoribosyltransferase (NAMPT) plays a key role in cell metabolism and is closely related to the occurrence and development of many tumor types; yet, little is known concerning its relationship with GBM. Oncomine database analysis showed that the expression of NAMPT in GBM was higher than that in normal tissues; this finding was further confirmed by immunohistochemical staining of a tissue microarray. Data analysis with the R2 platform showed that patients with higher expression of NAMPT had worse prognoses than those with lower NAMPT expression. Using the GBM data in TCGA, four pathways enriched in the high NAMPT expression group were identified by gene set enrichment analysis (GSEA). NAMPT expression was knocked down in U87 and U251 GBM cells by lentiviral vectors carrying a small hairpin RNA (shRNA) targeting NAMPT. CCK-8, colony formation, wound healing, Transwell and apoptosis assays were carried out. The results showed that NAMPT knockdown decreased cell proliferation, migration, and invasion and promoted apoptosis. U87 GBM cells were used in a model of subcutaneous tumorigenesis in nude mice. The results showed that NAMPT knockdown slowed the growth of tumors in vivo. Therefore, we speculate that NAMPT may be a potential prognostic and therapeutic biomarker for glioblastoma.

Molecular basis of the therapeutic effect of butyrate in glioblastoma revealed by in vitro and in silico approach.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

BackgroundOroscomucoid 3 (ORMDL3) has been linked to susceptibility of childhood asthma and respiratory viral infection. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of viral double-stranded RNA, a toll-like receptor 3 (TLR3) ligand and mimic of viral infection.MethodsTo investigate the functional role of ORMDL3 in the poly I:C-induced inflammatory response in airway epithelial cells, ORMDL3 knockdown and over-expression models were established in human A549 epithelial cells and primary normal human bronchial epithelial (NHBE) cells. The cells were stimulated with poly I:C or the Th17 cytokine IL-17A. IL-6 and IL-8 levels in supernatants, mRNA levels of genes in the TLR3 pathway and inflammatory response from cell pellets were measured. ORMDL3 knockdown models in A549 and BEAS-2B epithelial cells were then infected with live human rhinovirus (HRV16) followed by IL-6 and IL-8 measurement.ResultsORMDL3 knockdown and over-expression had little influence on the transcript levels of TLR3 in airway epithelial cells. Time course studies showed that ORMDL3-deficient A549 and NHBE cells had an attenuated IL-6 and IL-8 response to poly I:C stimulation. A549 and NHBE cells over-expressing ORMDL3 released relatively more IL-6 and IL-8 following poly I:C stimulation. IL-17A exhibited a similar inflammatory response in ORMDL3 knockdown and over-expressing cells, but co-stimulation of poly I:C and IL-17A did not significantly enhance the IL-6 and IL-8 response. Transcript abundance of IFNB following poly I:C stimulation was not significantly altered by ORMDL3 knockdown or over-expression. Dampening of the IL-6 response by ORMDL3 knockdown was confirmed in HRV16 infected BEAS-2B and A549 cells.ConclusionsORMDL3 regulates the viral inflammatory response in airway epithelial cells via mechanisms independent of the TLR3 pathway.

Unravelling synergistic immune interactions between respiratory virus infections and allergic airway inflammation.

Cytokine responses in cord blood predict the severity of later respiratory syncytial virus infection