Luteolin triggers global changes in the microglial transcriptome leading to a unique anti-inflammatory and neuroprotective phenotype

<p>Abstract</p> <p>Background</p> <p>Luteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. Luteolin also shows potent anti-inflammatory activities by inhibiting nuclear factor kappa B (NFkB) signaling in immune cells. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization.</p> <p>Methods</p> <p>Resting and LPS-activated BV-2 microglia were treated with luteolin in various concentrations and mRNA levels of pro-inflammatory markers were determined. DNA microarray experiments and bioinformatic data mining were performed to capture global transcriptomic changes following luteolin stimulation of microglia. Extensive qRT-PCR analyses were carried out for an independent confirmation of newly identified luteolin-regulated transcripts. The activation state of luteolin-treated microglia was assessed by morphological characterization. Microglia-mediated neurotoxicity was assessed by quantifying secreted nitric oxide levels and apoptosis of 661W photoreceptors cultured in microglia-conditioned medium.</p> <p>Results</p> <p>Luteolin dose-dependently suppressed pro-inflammatory marker expression in LPS-activated microglia and triggered global changes in the microglial transcriptome with more than 50 differentially expressed transcripts. Pro-inflammatory and pro-apoptotic gene expression was effectively blocked by luteolin. In contrast, mRNA levels of genes related to anti-oxidant metabolism, phagocytic uptake, ramification, and chemotaxis were significantly induced. Luteolin treatment had a major effect on microglial morphology leading to ramification of formerly amoeboid cells associated with the formation of long filopodia. When co-incubated with luteolin, LPS-activated microglia showed strongly reduced NO secretion and significantly decreased neurotoxicity on 661W photoreceptor cultures.</p> <p>Conclusions</p> <p>Our findings confirm the inhibitory effects of luteolin on pro-inflammatory cytokine expression in microglia. Moreover, our transcriptomic data suggest that this flavonoid is a potent modulator of microglial activation and affects several signaling pathways leading to a unique phenotype with anti-inflammatory, anti-oxidative, and neuroprotective characteristics. With the identification of several novel luteolin-regulated genes, our findings provide a molecular basis to understand the versatile effects of luteolin on microglial homeostasis. The data also suggest that luteolin could be a promising candidate to develop immuno-modulatory and neuroprotective therapies for the treatment of neurodegenerative disorders.</p

Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming

Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions

Agonists and knockdown of estrogen receptor β differentially affect invasion of triple-negative breast cancer cells in vitro

Background: Estrogen receptor beta (ER beta) is expressed in the majority of invasive breast cancer cases, irrespective of their subtype, including triple-negative breast cancer (TNBC). Thus, ER beta might be a potential target for therapy of this challenging cancer type. In this in vitro study, we examined the role of ER beta in invasion of two triple-negative breast cancer cell lines. Methods: MDA-MB-231 and HS578T breast cancer cells were treated with the specific ER beta agonists ERB-041, WAY200070, Liquiritigenin and 3 beta-Adiol. Knockdown of ER beta expression was performed by means of siRNA transfection. Effects on cellular invasion were assessed in vitro by means of a modified Boyden chamber assay. Transcriptome analyses were performed using Affymetrix Human Gene 1.0 ST microarrays. Pathway and gene network analyses were performed by means of Genomatix and Ingenuity Pathway Analysis software. Results: Invasiveness of MBA-MB-231 and HS578T breast cancer cells decreased after treatment with ER beta agonists ERB-041 and WAY200070. Agonists Liquiritigenin and 3 beta-Adiol only reduced invasion of MDA-MB-231 cells. Knockdown of ER beta expression increased invasiveness of MDA-MB-231 cells about 3-fold. Transcriptome and pathway analyses revealed that ER beta knockdown led to activation of TGF beta signalling and induced expression of a network of genes with functions in extracellular matrix, tumor cell invasion and vitamin D3 metabolism. Conclusions: Our data suggest that ER beta suppresses invasiveness of triple-negative breast cancer cells in vitro. Whether ER beta agonists might be useful drugs in the treatment of triple-negative breast cancer, has to be evaluated in further animal and clinical studies

Stanniocalcin 1 is overexpressed in multipotent mesenchymal stromal cells from acute myeloid leukemia patients

Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD). Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated. Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis. Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis. Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease

Molecular Biological Comparison of Dental Pulp- and Apical Papilla-Derived Stem Cells

Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering

Curcumin is a potent modulator of microglial gene expression and migration

Background Microglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin. Methods Resting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. Results Curcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures. Conclusions Collectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders

Effect of estrogen receptor β agonists on proliferation and gene expression of ovarian cancer cells

Abstract Background Estrogen receptor (ER) β has been suggested to affect ovarian carcinogenesis. We examined the effects of four ERβ agonists on proliferation and gene expression of two ovarian cancer cell lines. Methods OVCAR-3 and OAW-42 ovarian cancer cells were treated with the ERβ agonists ERB-041, WAY200070, Liquiritigenin and 3β-Adiol and cell growth was measured by means of the Cell Titer Blue Assay (Promega). ERβ expression was knocked down by transfection with specific siRNA. Additionally, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. To confirm the results of DNA microarray analysis, Western blot experiments were performed. Results All ERβ agonists tested significantly decreased proliferation of OVCAR-3 and OAW-42 cells at a concentration of 10 nM. Maximum antiproliferative effects were induced by flavonoid Liquiritigenin, which inhibited growth of OVCAR-3 cells by 31.2% after 5 days of treatment, and ERB-041 suppressing proliferation of the same cell line by 29.1%. In OAW-42 cells, maximum effects were observed after treatment with the ERβ agonist WAY200070, inhibiting cell growth by 26.8%, whereas ERB-041 decreased proliferation by 24.4%. In turn, knockdown of ERβ with specific siRNA increased cell growth of OAW-42 cells about 1.9-fold. Transcriptome analyses revealed a set of genes regulated by ERβ agonists including ND6, LCN1 and PTCH2, providing possible molecular mechanisms underlying the observed antiproliferative effects. Conclusion In conclusion, the observed growth-inhibitory effects of all ERβ agonists on ovarian cancer cell lines in vitro encourage further studies to test their possible use in the clinical setting