Journal: BMC Cancer

Article Title: Combination therapy with c-met inhibitor and TRAIL enhances apoptosis in dedifferentiated liposarcoma patient-derived cells

doi: 10.1186/s12885-019-5713-2

Figure Lengend Snippet: The c-Met inhibitor PF enhanced TRAIL-mediated apoptosis in liposarcoma. PF enhances TRAIL-mediated apoptosis in DDLPS cell lines and PDCs. Shown are the cell viabilities of the established cell lines ( a ) LPS224 and LPS246 and the PDCs ( b ) 11GS-013 and 11GS-079 after 48 h of incubation with 5 μM PF and 5 ng/ml rhTRAIL under the following treatment schemes: negative control, PF alone for 48 h; rhTRAIL alone for 48 h; PF for 24 h followed by rhTRAIL for 24 h; rhTRAIL for 24 h followed by PF for 24 h; and concurrent treatment with PF and rhTRAIL for 48 h. We analyzed apoptosis using annexin V and 7-AAD ( c and d )

Article Snippet: Human recombinant TRAIL protein (rhTRAIL) was purchased from R&D Systems (R&D Systems, Minneapolis, MN, USA). c-Met inhibitors (PHA665752: PHA; PF02341066:crizotinib: PF) were bought from Sellekchem (Houston, TX, USA).

Techniques: Incubation, Negative Control

Journal: BMC Cancer

Article Title: Combination therapy with c-met inhibitor and TRAIL enhances apoptosis in dedifferentiated liposarcoma patient-derived cells

doi: 10.1186/s12885-019-5713-2

Figure Lengend Snippet: Efficacy of treatment with rhTRAIL in sarcoma cell lines. Cell viability of ADMSCs ( a ), MFH-ino ( b ), SW872 ( c ), and HT1080 ( d ) after 48 h of incubation with serial dilutions of rhTRAIL protein (0–10 ng/ml)

Article Snippet: Human recombinant TRAIL protein (rhTRAIL) was purchased from R&D Systems (R&D Systems, Minneapolis, MN, USA). c-Met inhibitors (PHA665752: PHA; PF02341066:crizotinib: PF) were bought from Sellekchem (Houston, TX, USA).

Techniques: Incubation

Journal: BMC Cancer

Article Title: Combination therapy with c-met inhibitor and TRAIL enhances apoptosis in dedifferentiated liposarcoma patient-derived cells

doi: 10.1186/s12885-019-5713-2

Figure Lengend Snippet: Efficacy of tumor cell suppression through combined treatment with the c-Met inhibitor PF and rhTRAIL in DDLPS PDCs. Combination treatment with PF and rhTRAIL suppressed cell viability effectively in the DDLPS established cell lines: LPS246 ( a ) and LPS224 ( b ); and in the DDLPS PDCs: 11GS-013 ( c ), 11GS-079 ( d ), 11GS-099 ( e ), 11GS-106 ( f ), 14GS-026 ( g ), and 14GS-076 ( h )

Article Snippet: Human recombinant TRAIL protein (rhTRAIL) was purchased from R&D Systems (R&D Systems, Minneapolis, MN, USA). c-Met inhibitors (PHA665752: PHA; PF02341066:crizotinib: PF) were bought from Sellekchem (Houston, TX, USA).

Techniques:

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 1. rhTRAIL exposure effect alone and in combination with WT HSV-1 virus on HNSCC cell lines and the normal cell line, HaCaT. Treatment was performed with rhTRAIL at 100 ng/mL and in combination with WT HSV-1 virus at MOI 0f. 0.2 and 1.0. Cell viability analysis after 48 (A) and 72 h (B) of exposure. Results are expressed by mean percentage ± standard deviation of cell viability relative to PBS 1X (considering 100% viability). Data represent the average of three independent assays performed in triplicate. The asterisks indicate statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001) in the comparison with the experimental groups by non-normal distribution Kruskal–Wallis test.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Virus, Standard Deviation, Comparison

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 2. Expression analysis of apoptosis-associated proteins in the HCB289 cell line after 24 h of exposure with rhTRAIL ligand, WT HSV-1 and in combination. (A) and (B) detection and quantification of proteins after treatment. Results are expressed as mean percentage ± standard deviation of relative expression of cleaved protein relative to control (considering 100% expression) and normalized by the portion of total protein (non- cleaved). Data represent the average of three independent assays. The asterisks indicate statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001) in the comparison with the experimental groups by one-way ANOVA. Raw blots are presented in Supplementary Data 1.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Expressing, Standard Deviation, Control, Comparison

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 3. Expression analysis of apoptosis-associated proteins in the UD-SCC-2 cell line after 24 h of exposure with rhTRAIL ligand, WT HSV-1 and in combination. (A) and (B) detection and quantification of proteins after treatment. Results are expressed as mean percentage ± standard deviation of relative expression of cleaved protein relative to control (considering 100% expression) and normalized by the portion of total protein (non- cleaved). Data represent the average of three independent assays. The asterisks indicate statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001) in the comparison with the experimental groups by one-way ANOVA. Raw blots are presented in Supplementary Data 2.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Expressing, Standard Deviation, Control, Comparison

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 4. Expression analysis of apoptosis-associated proteins in the HCB289 cell line after 48 h of exposure with rhTRAIL ligand, WT HSV-1 and in combination. (A) and (B) detection and quantification of proteins after treatment. Results are expressed as mean percentage ± standard deviation of relative expression of cleaved protein relative to control (considering 100% expression) and normalized by the portion of total protein (non- cleaved). Data represent the average of two independent assays. The asterisks indicate statistical significance (*P < 0.05) in the comparison with the experimental groups by one-way ANOVA. Raw blots are presented in Supplementary Data 3.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Expressing, Standard Deviation, Control, Comparison

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 5. Expression analysis of apoptosis-associated proteins in the UD-SCC-2 cell line after 48 h of exposure with rhTRAIL ligand, WT HSV-1 and in combination. (A) and (B) detection and quantification of proteins after treatment. Results are expressed as mean percentage ± standard deviation of relative expression of cleaved protein relative to control (considering 100% expression) and normalized by the portion of total protein (non- cleaved). Data represent the average of two independent assays. The asterisks indicate statistical significance (*P < 0.05) in the comparison with the experimental groups by one-way ANOVA. Raw blots are presented in Supplementary Data 4.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Expressing, Standard Deviation, Control, Comparison

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 6. Flow cytometry apoptosis analysis after treatment with rhTRAIL ligand, WT HSV-1 and in combination. A and B) Analysis of HCB289 cell line after 24 and 48 h of treatment exposure. C and D) Analysis of UD-SCC-2 cell line after 24 and 48 h of treatment exposure. Results are expressed as mean percentage ± standard deviation of the cell death rate. Data represent the average of three independent assays. The asterisks indicate statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001) in the comparison with the experimental groups by one-way ANOVA.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Flow Cytometry, Standard Deviation, Comparison

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 7. DR-5 expression analysis in the HCB289 cell line after treatment with rhTRAIL ligand, WT-HSV-1 and combination. (A) and (B) Detection and quantification of the receptor after 24 h of exposure treatment. (C) and (D) Detection and quantification of the receptor after 48 h of exposure treatment. Results are expressed as mean percentage ± standard deviation of relative expression of cleaved protein relative to control (considering 100% expression) and normalized by the protein α-tubulin. Data represent the average of two independent. Raw blots are presented in Supplementary Data 5.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Expressing, Standard Deviation, Control

Journal: Scientific reports

Article Title: Combined effect of the pro-apoptotic rhTRAIL protein and HSV-1 virus in head and neck cancer cell lines.

doi: 10.1038/s41598-023-44888-9

Figure Lengend Snippet: Figure 8. DR-5 expression analysis in the UD-SCC-2 cell line after treatment with rhTRAIL ligand, WT-HSV-1 and combination. (A) and (B) Detection and quantification of the receptor after 24 h of exposure treatment. (C) and (D) Detection and quantification of the receptor after 48 h of exposure treatment. Results are expressed as mean percentage ± standard deviation of relative expression of cleaved protein relative to control (considering 100% expression) and normalized by the protein α-tubulin. Data represent the average of two independent assays. Raw blots are presented in Supplementary Data 6.

Article Snippet: :(0123456789) Scientific Reports | (2023) 13:18023 | https://doi.org/10.1038/s41598-023-44888-9 treated with rhTRAIL (#375-TL, R&D Systems, Minneapolis, MN, EUA) at 100 ng/mL, HSV-1 MOI 0.2 and/ or HSV-1 MOI 1.

Techniques: Expressing, Standard Deviation, Control

Journal: Cancer biology & therapy

Article Title: TRAIL induces endocytosis of its death receptors in MDA-MB-231 breast cancer cells.

doi: 10.4161/cbt.8.10.8141

Figure Lengend Snippet: Figure 1. TRAIL undergoes internalization in MDA-MB-231 but not BT474 breast cancer cells. (A) Cells ( 1 x 106 cells/mL) were first incubated in complete medium supplemented with or without 100 ng/mL of rhTRAIL at 4°C for 10 min, followed by incubation with 10 μg/mL of anti-TRAIL-PE for an additional 10 min. The mixtures were then incubated at 37°C for the indicated times (0–60 min). After extensive washes as described in Material and Methods, cells were resuspended in 0.5 mL cold PBS and analyzed by flow cytometry. The left panel shows a histogram for selected times during a kinetic analysis of TRAIL internalization, and the right panel shows the time-dependent increase in the mean fluorescence intensity. (B) MDA-MB-231 cells were grown on glass chamber slides, treated as in (A), fixed, and analyzed by confocal microscopy. Nuclei were visualized by Hoechst33342 staining (blue). (C) Cell surface expression of TRAIL receptors was assessed by FACS analysis after staining with PE-conjugated monoclonal antibodies to DR4, DR5, DcR1 and DcR2, respectively. Shadowed his- tograms represent control cells stained with isotype-matched control IgG. Right-shift indicates the pres- ence of receptors on cell surface. Data shown are representative of three independent experiments.

Article Snippet: Recombinant human TRAIL (rhTRAIL) expressed and purified as homotrimeric protein from E. coli, and monoclonal antibodies specific to TRAIL death receptor-4 (AF347) and -5 (MAB6312) and their phycoerythrin (PE)-conjugated forms were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Flow Cytometry, Fluorescence, Confocal Microscopy, Staining, Expressing, Bioprocessing, Control

Journal: Cancer biology & therapy

Article Title: TRAIL induces endocytosis of its death receptors in MDA-MB-231 breast cancer cells.

doi: 10.4161/cbt.8.10.8141

Figure Lengend Snippet: Figure 2. TRAIL downregulates cell surface expression of DR4 and DR5 in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with TRAIL (100 ng/mL) at 37°C for 0, 10 or 30 min, and analyzed by FACS for the remaining surface DR4, DR5, Her-2, epidermal growth factor recep- tor (EGFR), respectively, as described in 1C. (B) The percent of receptors remaining on cell surface was estimated by the median fluorescence intensities at the indicated times (Ft) relative to their basal levels (F0); receptor remaining % = (Ft - FIgG)/(F0 - FIgG), where FIgG is the median fluorescence of cells stained with control IgG-PE (shaded histogram). (C) MDA-MB-231 cells were transiently transfected with a plasmid for GFP fusion protein of DR4 (GFP-DR4). After 24 h post-transfection, cells were treated with rhTRAIL (100 ng/mL) for the indicated times at 37°C, counterstained with CellTracker red, fixed and analyzed by confocal microscope.

Article Snippet: Recombinant human TRAIL (rhTRAIL) expressed and purified as homotrimeric protein from E. coli, and monoclonal antibodies specific to TRAIL death receptor-4 (AF347) and -5 (MAB6312) and their phycoerythrin (PE)-conjugated forms were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Fluorescence, Staining, Control, Transfection, Plasmid Preparation, Microscopy

Journal: Cancer biology & therapy

Article Title: TRAIL induces endocytosis of its death receptors in MDA-MB-231 breast cancer cells.

doi: 10.4161/cbt.8.10.8141

Figure Lengend Snippet: Figure 3. DR4 is cleaved after internalization. (A) TRAIL induces proteolytic cleavage of endogenous DR4, but not DR5. Cells were treated with TRAIL (20 ng/mL) for the indicated times and analyzed by west- ern blotting. RhoGDI, a caspase-resistant protein,26 was used as a loading control. Caspase activation is indicated by the cleavage of pro-caspase 8 (C-8) and caspase-3 (C-3) and the appearance of their frag- ments p43/41, p18 and p20/17. (B) The percentage of remaining intact receptor (%) and caspase activities were quantified by densitometry analysis of the bands in (A) and are expressed relative to untreated cells (Time 0). (C) Cleavage of DR4 is inhibited by caspase inhibitor. Cells were pretreated for 1 h with or without 50 μg/mL of cell permeable inhibitors for caspase-3 (Z-DEVD-fmk), caspase-8 (Z-IETD-fmk), or a general caspase inhibitor Z-VAD-fmk, followed by treatments with 20 ng/mL TRAIL for an additional 4 h. Shown are western blots for the indicated proteins. (D) DR4 cleav- age is not inhibited by lysosome or proteasome inhibi- tors. Cells were pretreated for 1 h with Z-VAD-fmk (50 μg/mL), Bortezomib (20 nM) or chloroquine (50 μM), followed by treatments with 20 ng/mL TRAIL for an additional 4 h. DR4 and DR5 proteins were detected as in (C). Caspase activity is indicated by the cleav- age of PARP. (E) Purified recombinant DR4 and DR5 protein were incubated for the indicated times at 37°C with recombinant active caspase-3 or caspase-8 at a final concentration of 5 ng/ μl in a caspase reaction buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% sucrose and 10 mM DTT. The resulting mixtures were analyzed by immunoblots using anti-DR4 or anti-DR5 antibodies.

Article Snippet: Recombinant human TRAIL (rhTRAIL) expressed and purified as homotrimeric protein from E. coli, and monoclonal antibodies specific to TRAIL death receptor-4 (AF347) and -5 (MAB6312) and their phycoerythrin (PE)-conjugated forms were purchased from R&D Systems (Minneapolis, MN).

Techniques: Control, Activation Assay, Western Blot, Activity Assay, Purification, Recombinant, Incubation, Concentration Assay