14 research outputs found

    Data_Sheet_1_Analysis on topological alterations of functional brain networks after acute alcohol intake using resting-state functional magnetic resonance imaging and graph theory.pdf

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    AimsAlcohol consumption could lead to a series of health problems and social issues. In the current study, we investigated the resting-state functional brain networks of healthy volunteers before and after drinking through graph-theory analysis, aiming to ascertain the effects of acute alcohol intake on topology and information processing mode of the functional brain networks.Materials and methodsThirty-three healthy volunteers were enrolled in this experiment. Each volunteer accepted alcohol breathalyzer tests followed by resting-state magnetic resonance imaging at three time points: before drinking, 0.5 h after drinking, and 1 h after drinking. The data obtained were grouped based on scanning time into control group, 0.5-h group and 1-h group, and post-drinking data were regrouped according to breath alcohol concentration (BrAC) into relative low BrAC group (A group; 0.5-h data, n = 17; 1-h data, n = 16) and relative high BrAC group (B group; 0.5-h data, n = 16; 1-h data, n = 17). The graph-theory approach was adopted to construct whole-brain functional networks and identify the differences of network topological properties among all the groups.ResultsThe network topology of most groups was altered after drinking, with the B group presenting the most alterations. For global network measures, B group exhibited increased global efficiency, Synchronization, and decreased local efficiency, clustering coefficient, normalized clustering coefficient, characteristic path length, normalized characteristic path length, as compared to control group. Regarding nodal network measures, nodal clustering coefficient and nodal local efficiency of some nodes were lower in B group than control group. These changes suggested that the network integration ability and synchrony improved, while the segregation ability diminished.ConclusionThis study revealed the effects of acute alcohol intake on the topology and information processing mode of resting-state functional brain networks, providing new perceptions and insights into the effects of alcohol on the brain.</p

    TCN inhibits NF-κB signaling and induces cell cycle arrest.

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    <p>(A) HEK 293T cells were transiently transfected with pNF-κB-Luc plasmids followed by treatment with TCN for 1 h before being stimulated with 25 ng/mL TNF-α for 18 h. (B) Lysates fromcells treated with TCN for 24 h were subjected to western blot analysis with p65, XIAP, cyclin D1 and Bcl-xL antibodies. (C) HepG2 cells were treated with 2.5 µM TCN for 8, 16 and 24 h. Cells were harvested and subjected to cell cycle analysis. The percentage of cells of different phases of cell cycle was analyzed by FlowJo. Experiments were done independently in triplicate, results are reported as means and standard deviations. Statistical significance was analyzed by One-way ANOVA, **p<0.01.</p

    TCN induces apoptosis of NF-κB constitutively activated cancer cells.

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    <p>(A) Chemical structure of trichothecin. (B) Cell cytotoxic effects of trichothecin at successive concentrations. After 48 h treatment, cell viability was determined using MTT assays. (C) Annexin V-FITC/PI analysis of apoptosis in cells treated with TCN for 24 h. (D) HL-60, HepG2, A549 and PANC-1 cells were treated with TCN for 24 h and cell lysates were subjected to western blot analysis with antibodies indicated. β-actin were used as loading controls. Each column represents the mean ± SD of triplicates in three independent experiments.</p

    Trichothecin Induces Cell Death in NF-κB Constitutively Activated Human Cancer Cells via Inhibition of IKKβ Phosphorylation

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    <div><p>Constitutive activation of the transcription factor nuclear factor-κB (NF-κB) is involved in tumorigenesis and chemo-resistance. As the key regulator of NF-κB, IKKβ is a major therapeutic target for various cancers. Trichothecin (TCN) is a metabolite isolated from an endophytic fungus of the herbal plant <i>Maytenus hookeri Loes.</i> In this study, we evaluated the anti-tumor activity of TCN and found that TCN markedly inhibits the growth of cancer cells with constitutively activated NF-κB. TCN induces G0/G1 cell cycle arrest and apoptosis in cancer cells, activating pro-apoptotic proteins, including caspase-3, -8 and PARP-1, and decreasing the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin. Reporter activity assay and target genes expression analysis illustrated that TCN works as a potent inhibitor of the NF-κB signaling pathway. TCN inhibits the phosphorylation and degradation of IκBα and blocks the nuclear translocation of p65, and thus inhibits the expression of NF-κB target genes XIAP, cyclin D1, and Bcl-xL. Though TCN does not directly interfere with IKKβ kinase, it suppresses the phosphorylation of IKKβ. Overexpression of constitutively activated IKKβ aborted TCN induced cancer cell apoptosis, whereas knockdown of endogenous IKKβ with siRNA sensitized cancer cells toward apoptosis induced by TCN. Moreover, TCN showed a markedly weaker effect on normal cells. These findings suggest that TCN may be a potential therapeutic candidate for cancer treatment, targeting NF-κB signaling.</p></div

    TCN inhibits the phosphorylation of IKKβ.

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    <p>(A) IKKβ phosphorylation was detected by phospho-IKKβ antibody in HepG2 cells stimulated with 25 ng/mL TNF-α for 10 min. Cells were fixed, permeabilized, and examined by fluorescence microscope. (B) Western blot analysis showing the inhibition of IKKβ phosphorylation in cells treated with 2.5 µM TCN. Cells were collected after stimulated with 25 ng/mL TNF-α for 10 min. (C) IKKβ kinase activity was analyzed with recombinant IKKβ using the Z’-LYTE™ kinase assay kit. Fluorescence was detected at 400 nm for the excitation wavelengths and 445 nm and 520 nm for the emission wavelengths. Experiments were done independently in triplicate and the results are reported as means and standard deviations.</p

    TCN induced cancer cell apoptosis is mediated by inhibition of IKKβ phosphorylation.

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    <p>(A) HEK 293T cells were transiently transfected with IKKβ CA or empty vector for 12 h, then pretreated with 2.5 µM TCN and stimulated with 25 ng/mL TNF-α for 18 h. Cells subjected to analysis of luciferase activity. (B) HepG2 cells were transiently transfected with IKKβ CA or empty vector for 12 h, and then treated with 2.5 µM TCN for 24 h. Cells subjected to apoptosis analysis. (C) HepG2 cells were transfected with IKKβ CA or empty vector for 12 h, and then treated with 2.5 µM TCN for 24 h. Cells subjected to western blot analysis for the expression of indicated proteins. (D) HepG2 cells were transiently transfected with a scrambled siRNA or IKKβ-siRNA for 48 h, treated with 2.5 µM TCN for 24 h and subjected to apoptosis analysis. (E) HepG2 cells transiently transfected with scrambled siRNA or IKKβ-siRNA were treated with 2.5 µM TCN for 24 h. Cell lysates were collected and subjected to western blot analysis with the specified antibodies. Experiments were done independently in triplicate and the results are reported as means and standard deviations. Statistical analysis was perform with Student’s t-test, *p<0.05.</p

    Structure-activity relationship of Henryin.

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    <p>(A) Structures of henryin and its analogues. (B) Effects of henryin analogues on Topflash activity in HEK293T cells. Cells were transfected with Wnt1 and ST-Luc for 3h and then incubated with henryin and its analogues of different dosages for 24h and afterward luciferase activities were measured. (C) The median growth inhibition (GI<sub>50,</sub> 72h) and ST-Luc inhibition (IC<sub>50,</sub> 24h) values of henryin and its analogues in SW480 cells are shown. (D) Effects of henryin analogues on the growth of various cell lines (48hours). Data shown is the mean ± SD from three independent experiments.</p

    Henryin selectively inhibits the growth of colon cancer cells and induces G1 phase arrest.

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    <p>(A) The structures of henryin and ent-kaurane are shown. (B) Growth inhibition of henryin on various cell lines, including colorectal cancer cell lines (SW480, HCT116 and HT29), normal colonic epithelial cell line CCD-CoN-841, lung cancer cell line A549, and normal lung epithelial cell line Beas-2B, determined by MTS assays with cisplatin used as a control (C). Cells were treated with henryin up to 72h at various concentrations. The absorbance was measured at 490 nm. All sampling was done in triplicates and the data are presented as mean ± SD. (D) The median growth inhibition concentration (GI<sub>50,</sub> 72h) values of henryin and cisplatin for various cell lines. (E) Representative histograms depicting cell-cycle distribution as analyzed by flow cytometry in HCT116 cells treated with 4µM of henryin for 0h, 12h and 24 h respectively. Counts of G1 phase cells increased remarkably in the treated cells in a time-dependent manner.</p

    Henryin inhibits endogenous Wnt target gene expression.

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    <p>(A) Henryin down-regulates the expression of Wnt target genes in microarray assay. (B–C) Henryin inhibits Cyclin D1 and C-myc expression in wnt1 transfected HEK293T cells and SW480 cells. The cells were treated with indicated dosages of henryin for 12hours. The levels of Cyclin D1 and C-myc mRNAs were determined by real-time PCR and normalized to β-actin. (D) The effects of henryin on the expression of Axin2, CyclinD1 and Survivin proteins in wnt1 transfected HEK293T cells. The cells were transfected with wnt1 3 hours before henryin treatment. (E) The effects of henryin on the expression of Axin2, CyclinD1 and Survivin proteins in SW480 cells. (F) The effects of henryin on the expression of CyclinD1, Survivin, C-myc and P21 proteins in HCT116 cells. The cells were treated with henryin for 6, 12, 24h and cell extracts were assayed with western blotting of the indicated proteins. β-actin was used as a loading control. *P<0.05, **P<0.01, relative to vehicle control.</p

    Henryin interferes with the association of β-catenin with TCF4.

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    <p>(A) Henryin does not affect the amount of total β-catenin and pho-β-catenin in SW480 cells. (B) Henryin does not affect the distribution of β-catenin in cytosol and nucleus fractions in SW480 cells. SW480 cells were treated with the indicated dosages of henryin for 24h. The cell extracts or fractions were prepared and analyzed by western blotting. Lamin A/C and β-actin were used as loading controls of nuclei and cytoplasm proteins, respectively. (C) Henryin antagonizes the Wnt signaling stimulated with LiCl or β-catenin. HEK293T cells were transiently transfected with ST-Luc and β-catenin or constitutively active β-catenin (S37A) plasmids, respectively, or treated with LiCl (20mM). Around 3h after transfection or treatment, henryin was added and cells were incubated for an additional 24h. Each bar is the mean ± SD from three independent experiments. (D) Henryin, but not enmenol and minheryin C, reduced the TCF4 levels associated with β-catenin in SW480 cells in a dose dependent manner. Cells were treated with the indicated dosages of henryin and its analogs enmenol and minheryin C for 12h and immune-precipitation was performed by β-catenin antibody with mouse IgG as a control, and then TCF4 was detected by western blotting. (E) Henryin directly disrupts the interaction of β-catenin with TCF4 in the in vitro binding assay. Human recombinant β-catenin (0.8µg) and TCF4 (0.5µg) were mixed, incubated with different concentrations of henryin and its analogs enmenol and minheryin C, and the mixture was subjected to co-immunoprecipitation with β-catenin antibody and western blotting analysis with TCF4 antibody, with mouse IgG used as control. (F) Quantification of the western blots shown in D and E. The software ImageJ was used to analyze the intensities of bands. Data was presented as mean ± SD from three experiments. Hen, henryin, En, enmenol, Min, minheryin C.</p
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