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.

Effects of polyphyllin Ⅵ on the proliferation and apoptosis of glioma cells and potential mechanism / 中国药房

OBJECTIVE To investigate the effects of polyphyllin Ⅵ（PPⅥ） on the proliferation and apoptosis of glioma cells and potential mechanism. METHODS Using human glioma LN229 cells as objects， MTT assay was used to detect the survival rate after treated with different concentrations of PPⅥ [0 （control group）， 1， 2， 4， 8， 16， 32， 64 μmol/L] for different time （24， 48， 72 h）. The clone formation experiments were adopted to detect the number of cell clones and clone formation rate after being treated with different concentrations of PPⅥ [0 （control group）， 2， 4， 8 μmol/L] for 14 days. The flow cytometry and Western blot assay were used to detect the apoptotic rate of cells， the expressions of apoptosis-related protein [B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3]， and the expressions of related proteins of Fas/Fas ligand （FasL） death receptor pathway and protein kinase B （Akt）/glycogen synthesis kinase-3β （GSK-3β） pathway after being treated with different concentrations of PPⅥ [0（control group）， 4， 8 μmol/L] for 24 h. RESULTS Compared with the control group， the survival rate of cells， the number of clones and clone formation rate， the protein expression of Bcl-2， and the phosphorylation levels of Akt and GSK-3β protein were decreased significantly in different concentration groups of PPⅥ （P＜0.05 or P＜0.01）. The apoptotic rate， the protein expressions of Bax， cleaved caspase-3， Fas， FasL and cleaved caspase-8 were increased significantly （P＜0.05 or P＜ 0.01）. CONCLUSIONS PPⅥ can inhibit the proliferation and induce the apoptosis of human glioma LN229 cells， which may be related to the activation of the Fas/FasL death receptor pathway and the inhibition of the Akt/GSK-3β pathway.

Transcriptome sequencing analysis of echovirus 30 infection reveals its potential pathogenesis.

Echovirus 30 (E30) causes various diseases, such as viral encephalitis; aseptic meningitis; hand, foot, and mouth diseases; and acute flaccid paralysis. Related neurological infections are most concerning. However, the molecular mechanisms of E30 pathogenesis are not fully understood. There is a growing research interest in E30 as a cause of neurological disease. The aim of this study was to describe E30 infection, especially the changes in differential factor expressions after infection, in human glioma (U251) cells and mice brains using transcriptome sequencing analysis. Clear changes in the gene expression of factors associated with the defense response to viruses, inflammation-related signaling pathways, and neurological complication-related pathways were observed. Our results suggest that after E30 infection, the genes related to immune response were induced in the human glioma cells and mice brains, whereas genes functioning in the development and function of neural tissue were inhibited. Overall, this study successfully established E30 infection of U251 and mouse brain tissue, profiled the infection-induced changes in cellular and organizational transcriptomes, and revealed the molecular level changes during E30 infection.

MicroRNA-585 inhibits human glioma cell proliferation by directly targeting MDM2.

BACKGROUND: MicroRNAs (miRNAs) are important regulators for cancer cell proliferation. miR-585 has been shown to inhibit the proliferation of several types of cancer, however, little is known about its role in human glioma cells. METHODS: miR-585 levels in human glioma clinical samples and cell lines were examined by quantitative real-time PCR (qRT-PCR) analysis. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) and EdU incorporation assays in vitro. For in vivo investigations, U251 cells were intracranially inoculated in BALB/c nude mice and xenografted tumors were visualized by magnetic resonance imaging (MRI). RESULTS: miR-585 expression is downregulated in human glioma tissues and cell lines compared with non-cancerous counterparts. Additionally, miR-585 overexpression inhibits and its knockdown promotes human glioma cell proliferation in vitro. Moreover, miR-585 overexpression also inhibits the growth of glioma xenografts in vivo, suggesting that miR-585 may act as a tumor suppressor to inhibit the proliferation of human glioma. Furthermore, miR-585 directly targets and decreases the expression of oncoprotein murine double minute 2 (MDM2). More importantly, the restoration of MDM2 via enforced overexpression markedly rescues miR-585 inhibitory effect on human glioma cell proliferation, thus demonstrating that targeting MDM2 is a critical mechanism by which miR-585 inhibits human glioma cell proliferation. CONCLUSIONS: Our study unveils the anti-proliferative role of miR-585 in human glioma cells, and also implicates its potential application in clinical therapy.

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.

Influence of Bufalin on the Growth and Proliferation of Human Glioma Cell Line U251 / 中国药师

Objective:To explore the effects of Bufalin on the growth and proliferation of human glioma cells U251. Methods:Methyl thiazolyl tetrazolium(MTT)assay was used to detect the effect of Bufalin on the proliferation of human glioma cells U251. An in-verted microscope was used to observe the changes of cell number,morphology and activity. AnnexinV/ PI was used to measure the in-duction of cell apoptosis caused by Bufalin. Results:Bufalin at different concentrations(0. 001 - 10. 0μmol·L - 1 )inhibited the pro-liferation of U251 cells in a dose and time-dependent manner. Compared with that of the control group,the apoptosis rate of Bufalin group was increased significantly(P < 0. 01). Conclusion:Bufalin can inhibit the growth and proliferation of U251 cells in a dose and time-dependent manner,and induce the apoptosis of U251 cells.

DAPT suppresses the proliferation of human glioma cell line SHG-44.

OBJECTIVE: To explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism. METHODS: The SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133(+) were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcriptase-polymerase chain reaction and western blotting. RESULTS: DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133(+) was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses. CONCLUSIONS: DAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.

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.

DAPT suppresses the proliferation of human glioma cell line SHG-44

OBJECTIVE@#To explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.@*METHODS@#The SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133(+) were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcriptase-polymerase chain reaction and western blotting.@*RESULTS@#DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133(+) was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.@*CONCLUSIONS@#DAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.

Invasion ability and collagenolytic activity of human glioma cells in vitro / 吉林大学学报(医学版)

Objective To investigate the invasion ability and collagenolytic activity of human glioma cells in vitro.Methods Boyden chamber invasion assay was employed to evaluate the cell migration ability of human malignant glioma cell line U87MG in vitro and the effect of conditioned medium of U87MG cells on matrix collagenolytic activity was tested by agar-gelatin gel.Results The number of U87MG glioma cells migrating through the Matrigel-coated membrane was more than that of addition of EDTA or anti-MMP-2 antibody in U87MG glioma cells(P0.05).EDTA or anti-MMP-2 antibody markedly inhibited the number of the migrated cells,the inhibitory rates were 79.2% and 77.1%,respectively. The conditioned medium collected from the U87MG cells showed an increase in the transparent ring area on agar-gelatin gel.Inhibition of MMP-2 enzymatic activity by EDTA or anti-MMP-2 antibody reduced the transparent ring area on agar-gelatin gel.Conclusion Both invasion ability and collagenolytic activity of U87MG cells are depended on MMP-2,suggesting that MMP-2 plays an important role in glioma invasiveness.

Effect of curcumin on the activity of ATPase in U-251 cells / 重庆医科大学学报

Objective:To study the effects of curcumin on the activity of ATPase and the mechanism of apoptosis in U-251 cell.Methods:U-251 cells were treated with 20,40,80,100?mol/L curcumin for 24 h and the growth inhibition rates of U-251 cells were measured with MTT method.Cell apoptosis was inspected with flow cytometry(FCM).The activities of ATPase were determined by colorimetry method.Results:Curcumin inhibited the proliferation of U-251 cells and induced apoptosis of U-251 cells.level of ATPase in U-251 plasma Membrane was low remarkably.Conclusion:Curcumin induced apoptosis and inhibited proliferation of U-251 cell via inhibition of activation of plasma Membrane ATPase.

Curcumin induces U251 cell apoptosis via alteration of expression of FAK and activation of caspase / 中国药理学通报

Aim To study the mechanism of curcumin induced U251 cell apoptosis. Methods U251 cells were treated with 20～100?mol?L~ -1 curcumin for 24 h and the growth inhibition rates of U251 cells were measured with MTT method. MTT method was used to measure the caspase inhibitors effect on curcumin too. Cell apoptosis was inspected with flow cytometry (FCM) and observed using electron microscope. The protein levels of FAK in U251 cells were observed by SP immunocytochemistry. The activity of caspase-3 was measured by spectrofluorometry. Results Curcumin inhibited the proliferation of U251 cells,induced apoptosis of U251 cells. At the same time the protein levels of FAK in U251 cells decreased and the activity of caspase-3 increased significantly. The apoptosis of U251 cells was partially reversed by caspase inhibitors. Conclusion Curcumin induced apoptosis via inhibition of expression of FAK and activation of caspase-3.

IMMUNOFLUORESCENCE STUDY OF THE CYTOSKELETON OF A HUMAN GLIOMA CELL LINE BT_(325) / 解剖学报

The cytoskeleton of BT_(325), a human glioma cell line, was studied by immunofluorescence microscopy. The effects of different fixatives and buffers on microtubules, microfilaments and intermediate filaments were also compared. It was observed that besides microtubules and microfilaments all cells expressed vimentin. However, only a small fraction of the cells were GFAP positive. We conclude that vimentin is the main component of the intermediate filaments in BT_(325) cells. It was also observed that methanol fixation and formaldehyde fixation followed by acetone or Triton X-100 treatment gave rise to satisfactory results for microtubule immune-staining, and methanol or acidic alcohol fixation resulted in bright staining of intermediate filaments. Formaldehyde fixation also resulted in excellent staining for mierotubnles, but weaker staining for intermediate filaments.If the cells were immune-stained after treatment with Triton X-100, the composition of the buffers had profound effects on microtubules and intermediate filaments, but less effects on microfilaments.It was also observed that if Triton-treated ceils were fixed with formaldehyde before immunostaining with monoclonal antibodies to vimentin, the staining was very weak. However, if the ceils were immunostained first and then fixed with for-maldehyde the staing was quite bright. We conclude that for-maldehyd e fixation may "mask" or destroy certain epitopes on vimentin molecules.