In Vitro and In Silico Anti-Glioblastoma Activity of Hydroalcoholic Extracts of Artemisia annua L. and Artemisia vulgaris L.

Glioblastoma, the most aggressive and challenging brain tumor, is a key focus in neuro-oncology due to its rapid growth and poor prognosis. The C6 glioma cell line is often used as a glioblastoma model due to its close simulation of human glioma characteristics, including rapid expansion and invasiveness. Alongside, herbal medicine, particularly Artemisia spp., is gaining attention for its anticancer potential, offering mechanisms like apoptosis induction, cell cycle arrest, and the inhibition of angiogenesis. In this study, we optimized extraction conditions of polyphenols from Artemisia annua L. and Artemisia vulgaris L. herbs and investigated their anticancer effects in silico and in vitro. Molecular docking of the main phenolic compounds of A. annua and A. vulgaris and potential target proteins, including programmed cell death (apoptosis) pathway proteins proapoptotic Bax (PDB ID 6EB6), anti-apoptotic Bcl-2 (PDB ID G5M), and the necroptosis pathway protein (PDB ID 7MON), mixed lineage kinase domain-like protein (MLKL), in complex with receptor-interacting serine/threonine-protein kinase 3 (RIPK3), revealed the high probability of their interactions, highlighting the possible influence of chlorogenic acid in modulating necroptosis processes. The cell viability of rat C6 glioma cell line was assessed using a nuclear fluorescent double-staining assay with Hoechst 33342 and propidium iodide. The extracts from A. annua and A. vulgaris have demonstrated anticancer activity in the glioblastoma model, with the synergistic effects of their combined compounds surpassing the efficacy of any single compound. Our results suggest the potential of these extracts as a basis for developing more effective glioblastoma treatments, emphasizing the importance of further research into their mechanisms of action and therapeutic applications.

Potential Benefits of Dietary Plant Compounds on Normal and Tumor Brain Cells in Humans: In Silico and In Vitro Approaches.

Neuroblastoma can be accessed with compounds of larger sizes and wider polarities, which do not usually cross the blood-brain barrier. Clinical data indicate cases of spontaneous regression of neuroblastoma, suggesting a reversible point in the course of cell brain tumorigenesis. Dual specificity tyrosine-phosphorylation-regulated kinase2 (DYRK2) is a major molecular target in tumorigenesis, while curcumin was revealed to be a strong inhibitor of DYRK2 (PBD ID: 5ZTN). Methods: in silico studies by CLC Drug Discovery Workbench (CLC) and Molegro Virtual Docker (MVD) Software on 20 vegetal compounds from the human diet tested on 5ZTN against the native ligand curcumin, in comparison with anemonin. In vitro studies were conducted on two ethanolic extracts from Anemone nemorosa tested on normal and tumor human brain cell lines NHA and U87, compared with four phenolic acids (caffeic, ferulic, gentisic, and para-aminobenzoic/PABA). Conclusions: in silico studies revealed five dietary compounds (verbascoside, lariciresinol, pinoresinol, medioresinol, matairesinol) acting as stronger inhibitors of 5ZTN compared to the native ligand curcumin. In vitro studies indicated that caffeic acid has certain anti-proliferative effects on U87 and small benefits on NHA viability. A. nemorosa extracts indicated potential benefits on NHA viability, and likely dangerous effects on U87.

Cyanidin inhibits glioma stem cells proliferation through the Wnt signaling pathway.

BACKGROUND: Cyanidin has a protective effect on the nervous system and has been reported to treat tumor effectively. However, its impact on glioma stem cells (GSC) is unknown. METHODS: Using seven GSC lines, the anti-tumor effect of cyanidin is tested. The effect of cyanidin on the cell viability in each cell line is evaluated. Wnt signaling pathway-related genes are checked after treatment of cyanidin. Cytoplasmic/nuclear ß-catenin protein levels post cyanidin treatment is detected. Protein levels of c-Myc after cyanidin treatment are determined. Twist1 and Snail1 protein levels after cyanidin treatment are checked as well. RESULTS: Cyanidin significantly reduces the cell viability of all GSCs, and exhibited the most substantial effect in GBM2 but no apparent effect in 293T cells. It can regulate the Wnt signaling pathway of all GSC lines. In the GBM2, GBM7, G166, and G179 cell lines, there is upregulation of WNT1 and MYC genes, while in the G144 and GliNS2 cell line, these two genes are down-regulated after cyanidin treatment. Cytoplasmic and nuclear protein levels of ß-catenin in all cell lines are down-regulated. Cyanidin treatment significantly decreases the protein level for c-Myc in the GBM2 cell line compared with untreated cells, not in G144 or GliNS2 cells. Furthermore, cyanidin strongly reduces the expression of Twist1 and Snail1 in GBM2, G179, and G144 cell lines, while the GliNS2 cells show an opposite change in the cytoplasm and no change in nuclear. CONCLUSION: Cyanidin exerts an anti-tumor effect in glioma stem cell lines, probably through the Wnt signaling pathway.

miR-205-5p/E2F1 signal axis is involved in the regulation of radiosensitivity of glioma cells through suppressing the classical Wnt/β-catenin signaling pathway / 中华放射肿瘤学杂志

Objective:To explore the mechanism of miR-205-5p/E2F1 signal axis in regulating the glioma U251, U87 radiotherapy resistance.Methods:X-ray gradual ascending and intermittent induction method was used to irradiate the glioma U251 cells to establish U251/TR, U87/TR radiation-resistant cell lines. Then, the morphology, migration, invasion and proliferation abilities of cells (U251/TR, U87/TR radiation-resistant cells and U251, U87 radiation-sensitive cells) were analyzed. Luciferase gene detection system and point mutation technique were employed to analyze the mechanism of miR-205-5p and E2F1 gene activity on U251 and U87 radiation-resistant cell lines.Results:Compared with the radiation-sensitive U251 cells, the radiation-resistant cells U251/TR, U87/TR showed increased proliferation activity, enhanced migration and invasion abilities and decreased apoptosis under X-ray irradiation. miR-205-5p mimics transfection could down-regulate the expression of E2F1 factor in U251/TR cells, inhibit cell proliferation, invasion and migration and increase the radiosensitivity of U251/TR cells. miR-205-5p mimics transfection combined with with E2F1 down-regulation exerted anti-tumor effect and decreased cell tolerance by suppressing the Wnt/β-catenin signaling pathway activity.Conclusions:The glioma radiation-resistant cell line U251/TR, U87/TR can be established by X-ray gradual ascending and intermittent induction method. The miR-205-5p/E2F1 signal axis exerts tumor-suppressing effect through the classical Wnt/β-catenin signaling pathway, which can be used as an therapeutic target to increase the radiosensitivity of glioma.

Silencing lncRNA UCA1 affects radiosensitivity of glioma cells by up-regulating miR-873-5p expression / 中华放射肿瘤学杂志

Objective:To investigate the effect of lncRNA UCA1 on the radiosensitivity of in vitro cultured glioma cell lines SHG-44, U87 and U251 by regulating the miR-873-5p expression. Methods:The survival of glioma cells SHG-44, U87 and U251 treated with different radiation intensities (0, 2, 4, 6 and 8 Gy) was detected by colony formation assay. The expression levels of UCA1 in glioma cells SHG-44, U87 and U251 were measured by qRT-PCR. The radiation-resistant glioma cells U87 and U251 were selected for subsequent study. After silencing UCA1 expression and/or over-expressing miR-873-5p, the cell survival rate was detected by colony formation assay, and the cell apoptosis rate was determined by flow cytometry. The dual luciferase reporter gene assay and qRT-PCR were employed to verify the targeting relationship between UCA1 and miR-873-5p.Results:UCA1 was up-regulated in the radiation-resistant U87 and U251 cells. Silencing UCA1 or over-expressing miR-873-5p inhibited the survival of U87 and U251 cells, and promoted the cell apoptosis induced by radiation exposure. miR-873-5p was a target gene of UCA1, and UCA1 negatively regulated the expression of miR-873-5p. The inhibition of miR-873-5p could reverse the effect of silencing UCA1 on the radiosensitivity of glioma cells. Silencing UCA1 increased the inhibitory effect of radiation on the glioma cell U251 xenografts.Conclusion:Silencing UCA1 inhibits the survival of glioma cells and promotes the cell apoptosis by up-regulating the expression of miR-873-5p, thereby increasing the radiosensitivity of glioma cells.

Modulating chitosan-PLGA nanoparticle properties to design a co-delivery platform for glioblastoma therapy intended for nose-to-brain route.

Nose-to-brain delivery is a promising approach to target drugs into the brain, avoiding the blood-brain barrier and other drawbacks related to systemic absorption, and enabling an effective and safer treatment of diseases such as glioblastoma (GBM). Innovative materials and technologies that improve residence time in the nasal cavity and modulate biological interactions represent a great advance in this field. Mucoadhesive nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA) and oligomeric chitosan (OCS) were designed as a rational strategy and potential platform to co-deliver alpha-cyano-4-hydroxycinnamic acid (CHC) and the monoclonal antibody cetuximab (CTX) into the brain, by nasal administration. The influence of formulation and process variables (O/Aq volume ratio, Pluronic concentration, PLGA concentration, and sonication time) on the properties of CHC-loaded NPs (size, zeta potential, PDI and entrapment efficiency) was investigated by a two-level full factorial design (24). Round, stable nano-sized particles (213-875 nm) with high positive surface charge (+ 33.2 to + 58.9 mV) and entrapment efficiency (75.69 to 93.23%) were produced by the emulsification/evaporation technique. Optimal process conditions were rationally selected based on a set of critical NP attributes (258 nm, + 37 mV, and 88% EE) for further conjugation with CTX. The high cytotoxicity of CHC-loaded NPs and conjugated NPs was evidenced for different glioma cell lines (U251 and SW1088). A chicken chorioallantoic membrane assay highlighted the expressive antiangiogenic activity of CHC-loaded NPs, which was enhanced for conjugated NPs. The findings of this work demonstrated the potential of this nanostructured polymeric platform to become a novel therapeutic alternative for GBM treatment. Graphical abstract.

Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma.

Neural cell adhesion molecular L1-like protein (CHL1) is a member of the cell adhesion molecule L1 family and serves an important role in the development and progression of tumors. The cytokine neuregulin 1 (NRG1) has been indicated in the tumorigenesis and promotion of metastasis through the modulation of L1. However, the roles of NRG1 in regulating CHL1 in glioma have not been elucidated. The present study investigated the protein expression levels and roles of CHL1 and the possible correlation between NRG1 and CHL1 protein expression levels in human gliomas, both in vivo and in vitro. Using immunohistochemistry coupled with a human glioma tissue microarray, it was demonstrated that the percentage of CHL1-positive areas was the highest in grade II glioma tissues. Using immunofluorescence staining, a positive correlation was identified between the expression levels of CHL1 and proliferating cell nuclear antigen. In addition, CHL1 downregulation also resulted in increased senescence of U-87 MG human glioblastoma cells. In vitro, administration of NRG1α induced a significant increase in CHL1 protein expression levels in human glioma SHG-44 and U251 cells and in human glioblastoma U-87 MG cells, whereas NRG1ß failed to increase CHL1 expression levels in U251 cells. These findings were further confirmed by the downregulation of NRG1 expression levels using small interfering RNA treatment, which resulted in the reduction of CHL1 protein expression levels in U-87 MG cells. These data indicate that NRG1 can regulate CHL1 protein expression levels in gliomas, that it is correlated with malignancy, and that NRG1 may contribute to malignancy by upregulating CHL1 protein expression levels in glioma/glioblastoma cells.

Flavonoids from the Amazon plant Brosimum acutifolium induce C6 glioma cell line apoptosis by disrupting mitochondrial membrane potential and reducing AKT phosphorylation.

Glioblastoma, which is highly invasive and has a poor patient prognosis, is the most common type of brain tumor. Flavonoids have known antiproliferative and antineoplastic effects, such as apoptosis induction and tumor growth inhibition. We investigated the effects of treatment with three flavonoids (BAS-1, BAS-4, and BAS-6) isolated from the Amazon plant Brosimum acutifolium on the proliferation and migration of the C6 glioma cell line. Cytotoxicity was evaluated by MTT assay, and morphological changes were evaluated by phase-contrast microscopy and by transmission electron microscopy. Apoptosis was determined using Annexin V-FITC-propidium iodide (PI) staining. A hemolysis assay was used to evaluate plasma membrane injury. Antiproliferative effects were assessed by wound migration and colony formation assays. Mitochondrial transmembrane potential (ΔΨm) was determined using JC-1 dye and flow cytometry. To identify the flavonoid targets, western blotting was performed. BAS-1 and BAS-4 reduced C6 cell proliferation in a dose-dependent manner. BAS-6 showed no effect. Due to its high toxicity toward primary glial cells and its high hemolytic index, BAS-1 was not used in the remaining experiments. BAS-4 treatment did not induce cytotoxicity in primary glial cells; however, in glioma cells, it suppressed migration and invasion and led to apoptosis through mitochondrial damage, ΔΨm loss, cell cycle arrest, and reduced AKT phosphorylation, which is a component of the main cell survival pathway. We conclude that BAS-4 showed potential activity against glioma by inducing apoptosis mediated by ΔΨm loss and AKT pathway disruption, and future studies should further evaluate BAS-4 as a promising antineoplastic agent against glioblastoma.

Lidocaine induces protective autophagy in rat C6 glioma cell line.

Malignant glioma is the most common type of brain cancer with poor prognosis. Surgical resection, chemotherapy and radiotherapy are the main therapeutic options; however, in addition to their insufficient efficacy, they are associated with the pain experienced by patients. To relieve pain, local anesthetics, such as lidocaine can be used. In the present study, the effects of lidocaine on the C6 rat glioma cell line were investigated. An MTT assay and Annexin V/propidium iodide analysis indicated the increase in the percentage of apoptotic and necrotic cells in response to lidocaine. Furthermore, light microscopy analysis on the ultrastructural level presented the occurrence of vacuole­like structures associated with autophagy, which was supported by the analysis of autophagy markers (microtubule­associated protein 1A/1B­light chain 3, acridine orange and Beclin­1). Additionally, reorganization of the cytoskeleton was observed following treatment with lidocaine, which serves an important role in the course of autophagy. To determine the nature of autophagy, an inhibitor, bafilomycin A1 was applied. This compound suppressed the fusion of autophagosomes with lysosomes and increased the percentage of apoptotic cells. These results demonstrated that lidocaine may induce cytoprotective autophagy and that manipulation of this process could be an alternative therapeutic strategy in the treatment of cancer.

Dibutyryl cAMP- or Interleukin-6-induced astrocytic differentiation enhances mannose binding lectin (MBL)-associated serine protease (MASP)-1/3 expression in C6 glioma cells.

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

Tannic acid mediated induction of apoptosis in human glioma Hs 683 cells.

Tannic acid (TA), a natural plant compound, is known to induce the death of cancer cells in various types of cancer. The present study was designed with the aim of exploring the effects of tannic acid in vitro on HS 683, a glioma cell line, and to study the mechanism involved in the induction of cytotoxicity and apoptosis by TA. TA exhibited maximum cytotoxic activity against the Hs 683 cell line. Nuclear morphology, 4',6-diamidino-2-phenylindole staining and annexin V/propidium iodide apoptosis assaying of Hs 683 cells confirmed that cell death was due to the induction of apoptosis by TA. Further mechanistic study of TA on Hs 683 cells revealed that it decreased cell growth with increasing TA concentration, that resulted in the activation of pro-caspase 3 and caspase 9 and the cleavage of poly (ADP-ribose) polymerase, implying the induction of apoptosis cascades. Biochemical evidence of apoptosis resulted from the loss of mitochondrial membrane potential and increased intracellular reactive oxygen species production by TA in a dose-dependent manner. Based on this data, TA may be further investigated as a potential anticancer therapeutic lead.

Delivery of bevacizumab by intracranial injection: assessment in glioma model.

BACKGROUND: Many reports have indicated that the intravenous administration of bevacizumab produces a number of systemic side effects. Therefore, we investigated the therapeutic effects of intratumoral bevacizumab administration using a glioma animal model. METHODS: The glioma cell lines U251 and U87 that carried luciferase were implanted into the brains of mice to develop glioma models. Glioma-bearing mice were treated with bevacizumab intravenously or intratumorally by Alzet micro-osmotic pumps, and the survival time of mice was monitored. Tumor volumes and location were observed by fluorescence imaging and histological analysis. Levels of microvessel marker, cancer stem cell marker as well as angiogenesis-, invasion-, and inflammation-related factors in tumors were examined by immunohistochemical staining. RESULTS: Mice treated with intratumoral low-dose bevacizumab had smaller tumor volumes, longer survival time, lower microvessel density, and fewer cancer stem cells as compared with untreated and intravenously treated mice. Furthermore, expression levels of inflammation-related factors increased signifiwhereas that of angiogenesis- and invasion-related factors decreased in intratumorally treated animals, compared with intravenously treated mice. CONCLUSION: These results implied bevacizumab delivery by intratumoral injection via Alzet micro-osmotic pumps may be a more effective and safer protocol for treating gliomas.

Effects of LED-Based photodynamic therapy using red and blue lights, with natural hydrophobic photosensitizers on human glioma cell line.

Photodynamic therapy (PDT) has received high attention in cancer treatment due to its minimal side effects, specific cancer-targeting, non-invasion and low cost. It utilizes a specific group of anti-cancer drugs called photosensitizers (PS), which can be only activated under a certain wavelength light illumination and kills cancer cells. To screen the potential of PS and setup of PDT treatment protocol, it is essential to assess the PDT efficacy in vitro. In this study, a light-emitting diode- (LED-) based illumination system at two wavelengths (red & blue) with homogeneous and stable irradiation, and constant temperature conditions in 96-well plates was provided. The photodynamic effect of curcumin (CUR) and methyl ester of 5-aminolevulinic acid (MAL) using LED light on human glioma cell line was investigated. The obtained results indicate that this homemade LED-based illumination system is a favorable light source for in vitro PDT in 96-well plates. The PDT using CUR and MAL was efficient at final concentrations of 25µM and 2mM, and light doses of 60J/cm2 and 40J/cm2 respectively. The blue PDT efficiency was dependent on the light and PS doses. MAL-PDT and CUR-PDT using blue LED significantly decreased cell viability in the treatment groups compared with control groups. Furthermore, MAL-PDT using blue LEDs was more effective in comparison with conventional red LEDs on the human glioma cell line.

Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells.

The paramount problem in the therapy of brain tumors is the inability of most drugs to cross the blood-brain barrier. PLGA nanoparticles overcoated with poloxamer 188 could overcome this problem and enabled a high anti-tumoral effect against the very aggressive intracranial 101.8 glioblastoma in rats that closely resembles human grade IV glioblastomas. The basis for the transport of these particles across the blood-brain barrier appears to be adsorption of blood apolipoproteins (ApoE or ApoA-I) on the nanoparticle surface caused by the poloxamer 188-coating, followed by receptor-mediated transcytosis of the nanoparticles. The objective of the present study is the elucidation of the mechanism by which the poloxamer 188-coated nanoparticles then enter the brain tumor cells. Their intracellular fate, therefore, was investigated using the U87 human glioma cell line. The main mechanism of the PLGA nanoparticle internalization by U87 cells was clathrin-mediated endocytosis. Within 1h free doxorubicin was released from late endosomes and could reach its target site, i.e. the DNA in the nuclei without degradation, whereas the PLGA nanoparticles, which were labeled with Cy5.5, still were observed in the endo-lysosomal compartment. These results demonstrate that the underlying mechanism of action in the brain cells is by diffusive doxorubicin release from the nanoparticles rather than by their intracellular degradation.

Neuroleptic Drugs and PACAP Differentially Affect the mRNA Expression of Genes Encoding PAC1/VPAC Type Receptors.

Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide playing an important role as a neuromodulator. It has been indicated that PACAP is associated with mental diseases, and that regulation of the PACAPergic signals could be a potential target for the treatment of such psychiatric states as schizophrenia. Recent studies have suggested that action of neuroleptic drugs is mediated not only by dopaminergic and serotonergic neurotransmission, but also via neuropeptides which may act both as neurotransmitters and as neuromodulators. The present study examines whether currently-used neuroleptics influence the action of PACAP receptors, whose expression is altered in a schizophrenic patient. Real-time polymerase chain reaction (PCR) was used to examine the effects of haloperidol, olanzapine and amisulpride on the expression of genes coding PAC1/VPAC type receptors in the T98G glioblastoma cell line, as an example of an in vitro model of glial cells. PAC1 mRNA expression fell after 24-h incubation with haloperidol or olanzapine; however the effect was not maintained after 72 h, and haloperidol even up-regulated PAC1 mRNA expression in a dose-dependent manner. All the examined drugs decreased VPAC2 mRNA expression, especially after 72-h incubation. Haloperidol (typical neuroleptic) was distinctly more potent than atypical neuroleptic drugs (olanzapine and amisulpride). In addition, PACAP increased PAC1 and VPAC2 mRNA expression. In conclusion, our findings suggest PACAP receptors may be involved in the mechanism of typical and atypical neuroleptic drugs.

Targetting αvß3 and α5ß1 integrins with Ecballium elaterium (L.) A. Rich. seed oil.

In the present study, the effect of Ecbalium elaterium seed oil on adhesion, migration and proliferation of human brain cancer cell line (U87) was determined. Treatment of U87 cell line with the seed oil resulted in strong inhibition of their adhesion to fibrinogen (Fg), fibronectin (Fn). It also reduced their migration and proliferation in a dose-dependent manner without being cytotoxic. Concomitantly, by using Matrigel™ assays, the oil significantly inhibited angiogenesis. The anti- tumor effect of the oil is specifically mediated by αvß3 and α5ß1 integrins. The presence of integrin antagonists in seed oil from E. elaterium could be used for the development of anticancer drugs with targeted "multi-modal" therapies combining anti-adhesif, antiproliferative, antimetastasic and anti-angiogenic, approaches.

The complete mitochondrial genome sequence of the rat C6 glioma cell line.

In this article we have reported the complete mitochondrial genome sequence of rat C6 glioma cell line for the first time. The total length of the mitogenome was 16,314 bp, with coding 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes. This sequence was deposited in the GenBank (Accession No. KM820837).

Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell.

INTRODUCTION: There is growing evidence that mesenchymal stem cells (MSCs) can be important players in the tumor microenvironment. They can affect the glioma progression through the modulation of different genes. This modulation can be evaluated through a very useful model, treating the tumor cells with MSC-conditioned medium. However, for an accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is a prerequisite. METHODS: We performed a systematic review in an attempt to find a reference gene to use when analyzing gene expression in C6 glioma cells lines. Considering that we were not able to find a reference gene originated by an appropriate validation, in this study we evaluated candidate genes to be used as reference gene in C6 cells under different treatments with adipose-derived stem cells conditioned medium (CM-ADSCs). ß-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine-guanine phosphoribosyltransferase I (HPRT-1); TATA box binding protein (TBP) and beta-2-microglobulin (B2M) were evaluated by real-time reverse transcription PCR (RT-qPCR). The mean Cq, the maximum fold change (MFC) and NormFinder software were used for reference gene evaluation and selection. RESULTS: The GAPDH and ACTB genes have been the most widely used reference genes to normalize among the different investigated genes in our review, however, controversially these genes underwent a substantial variability among the genes evaluated in the present work. Individually, TBP gene was more stable when compared with other genes analyzed and the combination of TBP and HPRT-1 was even more stable. CONCLUSION: These results evidence the importance of appropriate validation of reference genes before performing qPCR experiments. Besides, our data will contribute with researchers that work analyzing the role of ADSCs in glioma microenvironment through gene expression.

MicroRNA-34a induces apoptosis in the human glioma cell line, A172, through enhanced ROS production and NOX2 expression.

BACKGROUND: MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma. METHODS: The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot. RESULTS: MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells. CONCLUSION: MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.

MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.