Journal: Oncology reports

Article Title: Andrographolide sensitizes human renal carcinoma cells to TRAIL‑induced apoptosis through upregulation of death receptor 4.

doi: 10.3892/or.2020.7737

Figure Lengend Snippet: Figure 4. Combined treatment of Andro and TRAIL induces caspase‑dependent apoptosis in 786‑0 cells. (A) Cell apoptosis was determined by Annexin V‑FITC after DMSO, TRAIL (50 ng/ml), and/or Andro (5 µM) treatment for 24 h. (B) Indicated protein levels in 786‑0 cells treated with TRAIL (50 ng/ml) and/or Andro (5 µM) for 24 h as detected by immunoblotting. (C) Cells were treated with DMSO, TRAIL (50 ng/ml), and Andro (5 µM), pan‑caspase inhibitor Z‑VAD (0.05 µM), and cell‑necrosis inhibitor necrostatin‑1 (Nec‑1) (0.05 µM). Then cell viability was determined by MTS assay (one‑way ANOVA, Tukey). (D) Images (magnification, x400) show the apoptotic cells following treatment under different conditions. Data are shown as mean ± SD; n.s. (not significant), P>0.05, ***P<0.001, n=3). Andro, andrographolide; TRAIL, tumor necrosis factor‑related apoptosis‑inducing ligand; PI, propidium iodide; PARP1, poly(ADP ribose) polymerase 1; Bax, Bcl‑2 associated X, apoptosis regulator, DR, death receptor.

Article Snippet: Antibodies used in this study were as follows: Phospho-HistoneH2A.X (product #9718), PARP1 (product #9532), DR4 (product #42533), caspase 9 (product #9502), caspase 8 (product #4790), GAPDH (product #51332) (from Cell Signaling Technology, Inc.), Bax (cat. #633601, BioLegend), DR5 (LM11912, Novus, USA), β-actin (ab8227, Abcam), anti-rabbit IgG (product #7054) and anti-mouse IgG (product #7056) (from Cell Signaling Technology, Inc.).

Techniques: Western Blot, MTS Assay

Journal: International journal of oncology

Article Title: Chetomin induces degradation of XIAP and enhances TRAIL sensitivity in urogenital cancer cells.

doi: 10.3892/ijo.2010.874

Figure Lengend Snippet: Figure 2. The combination of chetomin and TRAIL induces caspase-dependent apoptosis in PC-3 cells. (A) PC-3 cells were treated with 200 nM chetomin, 5 ng/ml TRAIL, and/or various caspase inhibitors (20 μM) for 24 h. The sub-G1 population was analyzed by flow cytometry. Columns, means of triplicate data; bars, SD; *P<0.05. C-3, zDEVD-fmk caspase-3 inhibitor; C-8, zIETD-fmk caspase-8 inhibitor; C-9, zLEHD-fmk caspase-9 inhibitor; C-10, zAEVD-fmk caspase-10 inhibitor; VAD, zVAD-fmk pancaspase inhibitor. (B) Western blotting of caspase-3, -8, -9, -10. PC-3 cells were treated with 200 nM chetomin and/or 5 ng/ml TRAIL with or without 20 μM zVAD-fmk for 24 h. ß-actin is a loading control. (C) Western blotting of PARP. (D) PC-3 cells were treated with 200 nM chetomin, 5 ng/ml TRAIL, and/or 1 μg/ml DR5/Fc chimera protein for 24 h. The sub-G1 population was analyzed by flow cytometry. Columns, the means of triplicate data; bars, SD; *P<0.05.

Article Snippet: Rabbit polyclonal antibodies for DR5 and DR4 (Prosci, Poway, CA), caspase-3, cleaved caspase-3, Bcl-xL, cIAP-2 and BAX (Santa Cruz Biotechnology, Santa Cruz, CA), survivin and cIAP-1 (R&D Systems), and mouse monoclonal antibodies for PARP and Bcl-2 (Santa Cruz Biotechnology), XIAP (R&D Systems), caspase-8, -9 and -10 antibodies (MBL, Nagoya, Japan) and ß-actin antibody (Sigma) were used as the primary antibodies.

Techniques: Flow Cytometry, Western Blot, Control

Journal: Cancer letters

Article Title: PI3K/mTOR dual inhibitor NVP-BEZ235 decreases Mcl-1 expression and sensitizes ovarian carcinoma cells to Bcl-xL-targeting strategies, provided that Bim expression is induced.

doi: 10.1016/j.canlet.2014.03.001

Figure Lengend Snippet: Fig. 2. BEZ235 inhibits proliferation of SKOV3 and IGROV1-R10 cell lines without inducing apoptosis. The effect of BEZ235 (10–1000 nM) on proliferation and apoptosis in the SKOV3 cell line (left panel) and in the IGROV1-R10 cell line (right panel) was investigated by studying growth kinetics during 72 h of treatment (A), by studying cell morphology (left column of each panel), DNA content histograms obtained by flow cytometry (middle column of each panel) and nuclear morphology after DAPI staining (right column of each panel) following 24 h of treatment (B), and by studying PARP and caspase 3 cleavages by western-blot after 24 h or 72 h of treatment (C).

Article Snippet: P-Akt (Ser473), P-Akt (Thr308), Akt, P-4E-BP1 (Thr70), 4E-BP1, P-p70S6 K (Thr389), p70S6 K, P-ERK1/2 (Thr202/Tyr204), ERK1/2, caspase 3, PARP, Bcl-xL (54H6), Puma (D30C10) and Bim antibodies were purchased from Cell Signaling (Beverly, MA, USA), Mcl-1 (S19) antibody from Santa–Cruz Biotechnology (Dallas, TX, USA), Noxa antibody from Calbiochem (Darmstadt, Germany), Bcl-2 antibody from DAKO (Glostrup, Denmark), b-actin antibody from Millipore (Billerica, MA, USA) and atubulin antibody from Sigma (Saint–Quentin Fallavier, France).

Techniques: Cytometry, Staining, Western Blot

Journal: Cancer letters

Article Title: PI3K/mTOR dual inhibitor NVP-BEZ235 decreases Mcl-1 expression and sensitizes ovarian carcinoma cells to Bcl-xL-targeting strategies, provided that Bim expression is induced.

doi: 10.1016/j.canlet.2014.03.001

Figure Lengend Snippet: Fig. 4. Combining BEZ235 with Bcl-xL inhibition induces massive cell death in the IGROV1-R10 cell line. IGROV1-R10 cells were transfected with Bcl-xL siRNA or control siRNA and treated 48 h later with BEZ235 250nM or 1000 nM (upper box). The efficiency of Bcl-xL silencing was monitored by western-blot 72 h after transfection (A). The effect of the treatment was investigated 72 h after transfection (24 h after BEZ235 treatment) by studying cell morphology, DNA content histograms obtained by flow cytometry and nuclear morphology after DAPI staining (B), and by analyzing PARP and caspase 3 cleavages by western-blot (C). The impact of the treatment was also studied by real-time cell analysis using the xCELLigence System as described in the ‘‘Materials and methods’’ section. Cell index was recorded every 2 h up to 90 h after transfection (means ± standard deviation of three independent wells). ***p < 0.001 (D). IGROV1-R10 cells were treated with BEZ235 (1000 nM) for 24 h and ABT-737 (5 lM) was then added for an additional 24 h. The effect of the treatment was analyzed at 48 h by studying cell morphology and DNA content histograms obtained by flow cytometry (left panel), and by studying PARP and caspase 3 cleavages by western-blot (right panel) (E). IGROV1-R10 cells were transfected with control siRNA, Bim siRNA or Puma siRNA before being treated 24 h later with the BEZ235/ABT-737 combination according to the protocol described in (E). The efficiency of Bim and Puma silencing was monitored by western-blot 48 h after transfection (right panel). Bim and Puma siRNAs ability to protect against BEZ235/ABT-737-induced apoptosis was evaluated at 72 h by studying cell morphology and DNA content histograms obtained by flow cytometry (left panel) (F). IGROV1-R10 cells were treated with 1000 nM BEZ235 for 24 h and the interaction between Bim and Bcl-xL was evaluated by co-immunoprecipitation. The supernatant and the whole cell lysate were also analyzed by western-blot (G).

Article Snippet: P-Akt (Ser473), P-Akt (Thr308), Akt, P-4E-BP1 (Thr70), 4E-BP1, P-p70S6 K (Thr389), p70S6 K, P-ERK1/2 (Thr202/Tyr204), ERK1/2, caspase 3, PARP, Bcl-xL (54H6), Puma (D30C10) and Bim antibodies were purchased from Cell Signaling (Beverly, MA, USA), Mcl-1 (S19) antibody from Santa–Cruz Biotechnology (Dallas, TX, USA), Noxa antibody from Calbiochem (Darmstadt, Germany), Bcl-2 antibody from DAKO (Glostrup, Denmark), b-actin antibody from Millipore (Billerica, MA, USA) and atubulin antibody from Sigma (Saint–Quentin Fallavier, France).

Techniques: Inhibition, Transfection, Control, Western Blot, Cytometry, Staining, Cell Analysis, Standard Deviation, Immunoprecipitation

Journal: Cancer letters

Article Title: PI3K/mTOR dual inhibitor NVP-BEZ235 decreases Mcl-1 expression and sensitizes ovarian carcinoma cells to Bcl-xL-targeting strategies, provided that Bim expression is induced.

doi: 10.1016/j.canlet.2014.03.001

Figure Lengend Snippet: Fig. 5. Combining BEZ235 with CI-1040-induced Bim expression sensitizes SKOV3 cell line to ABT-737. SKOV3 cells were transfected with Bcl-xL siRNA or control siRNA and treated 48 h later with BEZ235 1000 nM for 24 h. 72 h after transfection (24 h after BEZ235 treatment), the efficiency of Bcl-xL silencing was monitored by western-blot (right upper panel) and the effect of the treatment was investigated by studying cell morphology and DNA content histograms obtained by flow cytometry (left panel), and by analyzing PARP and caspase 3 cleavages by western-blot (right lower panel) (A). SKOV3 cells were treated with BEZ235 (1000 nM) for 24 h and ABT-737 (5 lM) was then added for an additional 24 h. The effect of the treatment was analyzed at 48 h by studying cell morphology and DNA content histograms obtained by flow cytometry (B). SKOV3 cells were treated with BEZ235 (1000 nM) and/or CI-1040 (5 lM) for 24 h. The expression of Phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2 and Bim proteins was analyzed by western-blot at basal level in IGROV1-R10 and SKOV3 cells (upper panel) and in response to treatment in SKOV3 cells (lower panel). The expression of Puma was also analyzed by western-blot in response to treatment in SKOV3 cells (lower panel). The expression levels of P-ERK1/2, ERK1/2 and Bim were quantified using ImageJ

Article Snippet: P-Akt (Ser473), P-Akt (Thr308), Akt, P-4E-BP1 (Thr70), 4E-BP1, P-p70S6 K (Thr389), p70S6 K, P-ERK1/2 (Thr202/Tyr204), ERK1/2, caspase 3, PARP, Bcl-xL (54H6), Puma (D30C10) and Bim antibodies were purchased from Cell Signaling (Beverly, MA, USA), Mcl-1 (S19) antibody from Santa–Cruz Biotechnology (Dallas, TX, USA), Noxa antibody from Calbiochem (Darmstadt, Germany), Bcl-2 antibody from DAKO (Glostrup, Denmark), b-actin antibody from Millipore (Billerica, MA, USA) and atubulin antibody from Sigma (Saint–Quentin Fallavier, France).

Techniques: Expressing, Transfection, Control, Western Blot, Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

doi: 10.1158/1078-0432.CCR-13-0901

Figure Lengend Snippet: EWS-FLI1 drives expression of WRN, an activity which is blocked by trabectedin treatment. A, Mean (+/− SEM) fold change in WRN expression as a function of GAPDH (2ddCT) as measured by qPCR for treatment with solvent control (S)(1.2 +/− 0.1), 10 nM(0.03 +/− 0.01), 7.5 nM (0.08 +/− 0.007) or 5 nM trabectedin ((0.23 +/− 0.02) for 12 hours P < 0.0001. Data representative of 3 independent experiments. B, Western blot time course experiment showing expression of WRN as a function of trabectedin treatment over time (hours) relative to NR0B1, ID2, PARP cleavage and ACTB (loading control). C, Western blot showing expression of WRN as a function of 10 nM trabectedin for 18 hours (ET) or solvent control (SC) in 7 different ES cell lines. Western blots representative of 3 independent experiments (time course) or 2–3 different experiments in 7 cell lines (cell line panel). D, Western blot showing expression of EWS-FLI1, WRN, the EWS-FLI1 target gene ID2, or other DNA damage genes XRCC4, ATM, KU80 and PRKDC for the medium control (C), lipid control (L) or siRNA silencing with a non-targeting siRNA (−) or siRNA targeting EWS-FLI1 (siEF) or SP1 (siSP1) in TC32 ES cellsor two other ES cell lines TC71 or EW8. Data representative of 2 (EW8) or 3 independent experiments (TC32 and TC71).

Article Snippet: Immunoblot analysis Immunoblotting was performed as previously described ( 39 ) using the following antibodies: mouse monoclonal anti-FLI1 (1.5 μg/mL; Becton Dickinson (BD), Franklin Lakes, NJ), anti-WRN (1:1000, Cell Signaling, Danvers, MA), anti-ATM (1:1000, Cell Signaling), anti-XRCC4 (1:1000, Abcam, Cambridge, UK), rabbit monoclonal anti-ID2 (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-NR0B1 (1:1000 dilution; Abcam), mouse monoclonal anti-PARP (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-ACTB (1:10,000 dilution, Abcam), mouse monoclonal anti-phospho-histone H2A.X (ser 139)(γH2AX)(1:1000 dilution, Millipore, Billerica, MA).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

doi: 10.1158/1078-0432.CCR-13-0901

Figure Lengend Snippet: Trabectedin and SN38 cooperate to increase DNA double strand breaks and augment the suppression of EWS-FLI1 downstream targets relative to either agent alone. A, Single cell imaging showing DNA double strand breaks as measured by the formation and immunostaining of γH2AX foci (green) with DAPI stained nuclei for reference (blue) following treatment of TC32 cells with 5 nM trabectedin, SN38 or the combination for 6 hours. B, Populations of TC32 cells showing the increased penetrance of γH2AX in the combination therapy relative to each agent alone. White arrows highlight unaffected cells. Quantitation of γH2AX foci in 50 to 65 cells from 8 different images in each of the treatment groups, Control (C), Trabectedin (T), SN38 (SN) or the combination (T&SN). C, Normalized bioluminescence of stable NR0B1 promoter construct (EWS-FLI1 reporter) treated with solvent control (S) or the designated concentration of trabectedin (Trabec) or SN38 (* & ^ denote comparisons for P< 0.0005). Data represents the mean value normalized to the solvent control for 8 replicates from two independent experiments for each treatment group. D, Western blot showing the effect of SN38 treatment (concentrations above the blot) +/− 5 or 1 nM trabectedin (T) on PARP cleavage, EWS-FLI1 downstream target expression (ID2) and γH2AX expression. Identical results obtained with three independent experiments. (*) Highlights specific concentrations of trabectedin, SN38 and the combination for comparison.

Article Snippet: Immunoblot analysis Immunoblotting was performed as previously described ( 39 ) using the following antibodies: mouse monoclonal anti-FLI1 (1.5 μg/mL; Becton Dickinson (BD), Franklin Lakes, NJ), anti-WRN (1:1000, Cell Signaling, Danvers, MA), anti-ATM (1:1000, Cell Signaling), anti-XRCC4 (1:1000, Abcam, Cambridge, UK), rabbit monoclonal anti-ID2 (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-NR0B1 (1:1000 dilution; Abcam), mouse monoclonal anti-PARP (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-ACTB (1:10,000 dilution, Abcam), mouse monoclonal anti-phospho-histone H2A.X (ser 139)(γH2AX)(1:1000 dilution, Millipore, Billerica, MA).

Techniques: Imaging, Immunostaining, Staining, Quantitation Assay, Construct, Concentration Assay, Western Blot, Expressing