Journal: Life

Article Title: Santin (5,7-Dihydroxy-3,6,4′-Trimetoxy-Flavone) Enhances TRAIL-Mediated Apoptosis in Colon Cancer Cells

doi: 10.3390/life13020592

Figure Lengend Snippet: Santin enhanced TRAIL-induced apoptosis in colon cancer cells. SW480 and SW620 cells were incubated for 48 h with rhsTRAIL at the concentrations of 25–100 ng/mL and/or with 25–100 μM santin. The percentage of apoptotic cells was determined by flow cytometry using annexin V-FITC staining. The values represent mean ± SD of three independent experiments performed fourfold ( n = 3) (*** p < 0.001 compared with control, +++ p < 0.001 compared with santin, ### p < 0.001 compared with TRAIL).

Article Snippet: Soluble recombinant human TRAIL (rhsTRAIL) was bought from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Incubation, Flow Cytometry, Staining, Control

Journal: Life

Article Title: Santin (5,7-Dihydroxy-3,6,4′-Trimetoxy-Flavone) Enhances TRAIL-Mediated Apoptosis in Colon Cancer Cells

doi: 10.3390/life13020592

Figure Lengend Snippet: The effects of TRAIL combined with santin on the mitochondrial membrane potential (ΔΨm) in colon cancer cells. SW480 and SW620 cells were subject to incubation for 48 h with rhsTRAIL (concentration of 25–100 ng/mL) and/or with santin (25–100 μM). The fluorescent microscopic analysis of DePsipher staining was used to assess the ΔΨm loss in cancer cells (*** p < 0.001 compared with control, +++ p < 0.001 compared with santin, ### p < 0.001 compared with TRAIL).

Article Snippet: Soluble recombinant human TRAIL (rhsTRAIL) was bought from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Membrane, Incubation, Concentration Assay, Staining, Control

Journal: Cancers

Article Title: Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis

doi: 10.3390/cancers15030967

Figure Lengend Snippet: TRAIL RNA and protein levels are upregulated by IFNα in cancer cells. ( A ) TRAIL RNA expression increased upon IFNα and IFNγ treatment but not upon TNFα or IL-6 treatment in THP-1. Cells were treated with 1 μg/mL TNFα, 0.8 μg/mL IFNα2, 1 μg/mL IFNγ or 100 ng/mL IL-6 for 6 or 24 h, and TRAIL levels were quantified using qPCR. ANOVA followed by Dunnett’s multiple comparison test was performed (** p < 0.01 and **** p < 0.0001). ( B ) TRAIL protein levels on the cell surface increased upon IFNα treatment in THP-1. Cells were treated with 0.8 μg/mL IFNα2 for 24 h, and membrane TRAIL protein was quantified in fresh cells using flow cytometry. Two-tailed t -test was performed (* p < 0.05). ( C ) TRAIL RNA expression increased upon IFNα treatment but not upon TNFα treatment in cancer cell lines. Breast cancer cell lines (MCF7 and BT549) and lung cancer cells (A549) were treated with 1 μg/mL TNFα or 0.8 μg/mL IFNα2 for 6 or 24 h, and TRAIL levels were quantified using qPCR. ANOVA followed by Dunnett’s multiple comparison test was performed (** p < 0.01 and *** p < 0.001). ( D ) TRAIL protein levels increased upon IFNα treatment in cancer cell lines. Cells were treated with 0.8 μg/mL IFNα2 for 24 h (A549) or 48 h (MCF7 and BT549), and total TRAIL protein was quantified using immunofluorescence. TRAIL signal is shown in green. Nuclei were stained with DAPI (blue). Representative images are shown. Two-tailed t -test was performed (* p < 0.05 and ** p < 0.01). MFI: mean fluorescence intensity.

Article Snippet: For cytokine treatment, Recombinant Human IFNα2 (Biolegend, San Diego, CA, USA), Recombinant Human soluble TRAIL (PeproTech, Cranbury, NJ, USA), Recombinant Human IFNγ (Gibco), Recombinant Human Tumor Necrosis Factor α (TNFα, PeproTech) or Recombinant Human Interleukin-6 (IL-6, PeproTech) dissolved in PBS was added to the cell media at the concentrations indicated in the figures.

Techniques: RNA Expression, Flow Cytometry, Two Tailed Test, Immunofluorescence, Staining, Fluorescence

Journal: Cancers

Article Title: Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis

doi: 10.3390/cancers15030967

Figure Lengend Snippet: Enhancer clusters mediate TRAIL upregulation by IFNα. ( A ) TRAIL is associated with enhancer clusters in specific cancer cell lines. Publicly available H3K27ac ChIP-seq data for several cell lines from breast cancer (MCF7, MDAMB468, T47D, ZR-75-1 and BT549) and other solid cancers (A549, HCT116, HepG2, PC-9 and SK-N-SH) were analyzed. Peaks of H3K27ac signal represent accessible chromatin and were found upstream and downstream of TRAIL (TNFSF10), which is transcribed from the opposite strand (right to left). Distal H3K27ac peaks overlapping with DNase I hypersensitive sites (a mark of open regulatory DNA) indicate accessible open DNA regions carrying putative regulatory elements. Distal DNase I hypersensitive sites, called regulatory elements 1 to 8 and shown in red in the diagram, were identified through DNase I hypersensitivity assays in 95 cell lines from ENCODE. Only distal DNase I hypersensitive regions that were found in more than 20 out of the 95 cell lines were considered as putative regulatory elements in this analysis. Regulatory elements 1 to 5 cluster together upstream of TRAIL (to the right in the figure), whereas regulatory elements 6 to 8 cluster together downstream of TRAIL (to the left in the figure). Upstream and downstream enhancer clusters are represented as pink and green lines, respectively. As observed in the H3K27ac data, MCF7, MDAMB468 and A549 cell lines show an upstream enhancer cluster, while only A549 shows a downstream enhancer cluster. ( B ) Chromatin accessibility at distal enhancer regions upstream and downstream of TRAIL-coding DNA correlates with TRAIL RNA levels in pan-cancer tumors. Scatter plots show correlation between TRAIL RNA-seq expression and ATAC-seq data on chromatin accessibility for 4 open DNA regions (regulatory elements 2, 4, 7 and 8) from 404 pan-cancer tumors from TCGA dataset. Pearson (r) and Spearman (ρ) correlation coefficients and p -values are shown for each plot. ( C ) Blocking BRD4 binding to highly acetylated enhancer regions with BET inhibitors reduced TRAIL RNA expression in MCF7 and A549, but not in BT549. Cells were treated with vehicle (DMSO), 1 μM JQ1 or 1 μM I-BET151 for 6 h. After treatment, changes in TRAIL RNA levels were analyzed using qPCR. ANOVA followed by Dunnett’s multiple comparison test was performed (**** p < 0.0001). ( D ) Enhancer clusters enhanced TRAIL upregulation by IFNα. Cells were pre-treated with vehicle (DMSO), 1 μM JQ1 or 1 μM I-BET151 for 3 h, followed by stimulation with 0.8 μg/mL IFNα2 for 6 h. After treatment, changes in TRAIL RNA levels were analyzed using qPCR. ANOVA followed by Dunnett’s multiple comparison test was performed (**** p < 0.0001).

Article Snippet: For cytokine treatment, Recombinant Human IFNα2 (Biolegend, San Diego, CA, USA), Recombinant Human soluble TRAIL (PeproTech, Cranbury, NJ, USA), Recombinant Human IFNγ (Gibco), Recombinant Human Tumor Necrosis Factor α (TNFα, PeproTech) or Recombinant Human Interleukin-6 (IL-6, PeproTech) dissolved in PBS was added to the cell media at the concentrations indicated in the figures.

Techniques: ChIP-sequencing, RNA Sequencing Assay, Expressing, Blocking Assay, Binding Assay, RNA Expression

Journal: Cancers

Article Title: Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis

doi: 10.3390/cancers15030967

Figure Lengend Snippet: IFNα-induced TRAIL protein accumulates intracellularly in epithelial cancer cells. ( A ) Soluble TRAIL protein levels increased upon IFNα stimulation in THP-1. Cells were treated with 0.8 μg/mL IFNα2 for 24 h, and TRAIL protein in the culture media was quantified using ELISA. Two-tailed t -test was performed (** p < 0.01). ( B ) TRAIL protein levels on the cell surface did not increase upon IFNα stimulation in epithelial cancer cells. MCF7 and A549 were treated with 0.8 μg/mL IFNα2 for 48 h, and membrane TRAIL protein was quantified in fresh cells using flow cytometry. Two-tailed t -test was performed (* p < 0.05). ( C ) Total TRAIL protein levels increased upon IFNα stimulation in epithelial and immune cancer cells. MCF7, A549 and THP-1 were treated with 0.8 μg/mL IFNα2 for 48 h, and total TRAIL protein was quantified in fixed and permeabilized cells using flow cytometry. Two-tailed t -test was performed (* p < 0.05 and ** p < 0.01). ( D ) IFNα-induced TRAIL was detected intracellularly in epithelial cancer cells. Cells were treated with 0.8 μg/mL IFNα2 for 24 h (A549) or 48 h (MCF7 and BT549), and total TRAIL protein was quantified using immunofluorescence. TRAIL signal is shown in green. Nuclei were stained with DAPI (blue). Red arrows mark punctuated cytoplasmic TRAIL staining. White arrows mark TRAIL staining in a big compartment next to the cell nucleus. Representative immunofluorescence images are shown. MFI: mean fluorescence intensity.

Article Snippet: For cytokine treatment, Recombinant Human IFNα2 (Biolegend, San Diego, CA, USA), Recombinant Human soluble TRAIL (PeproTech, Cranbury, NJ, USA), Recombinant Human IFNγ (Gibco), Recombinant Human Tumor Necrosis Factor α (TNFα, PeproTech) or Recombinant Human Interleukin-6 (IL-6, PeproTech) dissolved in PBS was added to the cell media at the concentrations indicated in the figures.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Flow Cytometry, Immunofluorescence, Staining, Fluorescence

Journal: Cancers

Article Title: Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis

doi: 10.3390/cancers15030967

Figure Lengend Snippet: TRAIL upregulation by IFNα in epithelial cancer cells is not enough to induce apoptosis. ( A ) TRAIL upregulation by IFNα did not induce apoptosis in most cancer cells. Breast cancer cells MCF7 and BT549 and lung cancer cells A549 were treated with 0.8 μg/mL IFNα2 or 0.5 μg/mL TRAIL. After 24 or 48 h, apoptosis was quantified with Annexin V staining using flow cytometry. ANOVA followed by Dunnett’s multiple comparison test was performed (** p < 0.01 and **** p < 0.0001). ( B ) TRAIL upregulation by IFNα did not induce cytotoxicity in cancer cells. Breast cancer cells MCF7 and BT549 and lung cancer cells A549 were treated with 0.8 μg/mL IFNα2 or 0.5 μg/mL TRAIL. After 24 or 48 h, cytotoxicity was quantified using the lactate dehydrogenase cytotoxicity assay. ANOVA followed by Dunnett’s multiple comparison test was performed (** p < 0.01, *** p < 0.001 and **** p < 0.0001). ( C ) TRAIL upregulation by IFNα in epithelial cancer cells did not induce cell death in a paracrine manner. THP-1 and A549 were treated with 0.8 μg/mL IFNα2 or control. After 48 h, the supernatants were collected, centrifuged and supplemented with 1 μM BV6 and 1 μg/mL PI. Cell death induction by the conditioned media was monitored in real time for 24 h using HeLa as target cells. Cell death is represented as the percentage of PI-positive cells.

Article Snippet: For cytokine treatment, Recombinant Human IFNα2 (Biolegend, San Diego, CA, USA), Recombinant Human soluble TRAIL (PeproTech, Cranbury, NJ, USA), Recombinant Human IFNγ (Gibco), Recombinant Human Tumor Necrosis Factor α (TNFα, PeproTech) or Recombinant Human Interleukin-6 (IL-6, PeproTech) dissolved in PBS was added to the cell media at the concentrations indicated in the figures.

Techniques: Staining, Flow Cytometry, Cytotoxicity Assay

Journal: Journal of Biomedicine and Biotechnology

Article Title: Exploration of the Lysis Mechanisms of Leukaemic Blasts by Chimaeric T-Cells

doi: 10.1155/2010/234540

Figure Lengend Snippet: Apoptosis induced on Jurkat, SupB15, or Raji cell lines and B-ALL blasts by agonist antibody anti-Fas (clone CH11) or recombinant human TRAIL (killerTRAIL) measured after 4 hours and 18 hours exposition. Isotype control (Ig) is indicated.

Article Snippet: The sensitivity of cells to Fas- and TRAIL-mediated apoptosis was investigated using 500 ng/mL agonistic anti-CD95 (IgM clone CH11; Alexis Biochemicals, San Diego, CA) and 500 ng/mL recombinant human soluble TRAIL (KillerTRAIL; Alexis Biochemicals).

Techniques: Recombinant

Journal: PLoS ONE

Article Title: MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

doi: 10.1371/journal.pone.0035345

Figure Lengend Snippet: Cells were transfected with miR-133b alone or together with a control antimiR (ctrl αmiR) or a specific miR-133b inhibitor (αmiR-133b). After 48 h, cells were either left untreated (Unstim) or stimulated for 4 h with 20 ng/ml tumor necrosis factor-alpha (TNFα), 100 ng/ml of a cross-linking activating antiFas antibody (αFas/CD95) or 20 ng/ml recombinant human TRAIL (rhTRAIL). (A) Treated cells were harvested, stained and scanned by flow cytometry for the presence of cleaved active caspase 8 (upper graph) and 3 (lower graph). 7-Amino-actinomycin D (7-AAD) served for exclusion of cells with compromised membrane integrity from the caspase activation quantification assay. Cells transfected with ctrl miR alone were used as reference. (B) Western blot analysis of poly [ADP ribose] polymerase (PARP-1) in transfected, unstimulated cells (upper panel) and TNFα-, αFas/CD95- or rhTRAIL-treated cells (lower panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. Graphs are representative of at least three independent experiments. Asterisk represents p<0.01. Errors bars indicate standard deviation.

Article Snippet: For stimulation and apoptosis induction experiments recombinant human TNFα from Active Bioscience (Hamburg, Germany) was used; human activating antiFas/CD95 (Clone CH11) (αFas/CD95) was from Upstate (Temecula, CA, USA); soluble human recombinant TRAIL was purchased from Alexis Biochemicals (Farmingdale, NY, USA).

Techniques: Transfection, Recombinant, Staining, Flow Cytometry, Activation Assay, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

doi: 10.1371/journal.pone.0035345

Figure Lengend Snippet: Cells were transfected with miR-133b (red) or a control miR (ctrl miR, black), as reference. After 48 h, cells were either left untreated (Unstim) or stimulated for 4 h with 20 ng/ml tumor necrosis factor-alpha (TNFα), 100 ng/ml of a cross-linking activating antiFas antibody (αFas/CD95) or 20 ng/ml recombinant human TRAIL (rhTRAIL). Treated cells were harvested, stained and scanned by flow cytometry for the presence of cleaved active caspase 8 (left) and caspase 3 (right). 7-Amino-actinomycin D (7-AAD) was used for exclusion of cells with compromised membrane integrity from the caspase activation quantification assay.

Article Snippet: For stimulation and apoptosis induction experiments recombinant human TNFα from Active Bioscience (Hamburg, Germany) was used; human activating antiFas/CD95 (Clone CH11) (αFas/CD95) was from Upstate (Temecula, CA, USA); soluble human recombinant TRAIL was purchased from Alexis Biochemicals (Farmingdale, NY, USA).

Techniques: Transfection, Recombinant, Staining, Flow Cytometry, Activation Assay