Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Olaparib and TRAIL synergize in cancer cells independently of BRCA2 status. A-B Proliferation assays of the effects of olaparib, TRAIL, or their combination on BRCA2 deficient CAPAN1 ( A ), and the respective syngenic BRCA2/CIN cell line ( B ) complemented to express wild-type BRCA2. C-D Proliferation assays of the effects of olaparib, TRAIL or their combination on BRCA2 deficient HCT116 ( C ), and BRCA2 proficient SW620 ( D ). E-F Proliferation assays of the effects of olaparib, TRAIL or their combination on wild-type DLD1 cells ( E ) vs. the homozygous BRCA2 knockout colorectal cancer cell line DLD1 termed DLD1 BRCA2 KO ( F ). Cells were treated for 6 days with the indicated agents and subsequently analyzed via SYBR green proliferation assay. All experiments were performed in triplicate with error bars representing SEM from three independent experiments. Drug interactions were analyzed using the Chou–Talalay method with a combination index (CI) of <1, 1, and >1 indicating synergistic, additive, and antagonistic drug effects, respectively. In the combination index graphs, dots depict the CI at the respective fraction of cancer cells affected (x-axis, 0.0 = no cells dead, 1.0 = all cells dead).

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Knock-Out, SYBR Green Assay, Proliferation Assay

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Combined olaparib and the clinically tested TRAIL targeting compound dulanermin synergize in pancreatic cancer cells independently of BRCA2 status. Proliferation assays of the effects of olaparib, dulanermin, or their combination on BRCA2 deficient CAPAN1 ( A ) and BRCA2/CIN ( B ). Cells were treated for 6 days with the indicated agents and subsequently analyzed via SYBR green proliferation assay. All experiments were performed in triplicate with error bars representing SEM from three independent experiments. Drug interactions were analyzed using the Chou–Talalay method with a combination index (CI) of <1, 1, and >1 indicating synergistic, additive, and antagonistic drug effects, respectively. In the combination index graphs, dots depict the CI at the respective fraction of cancer cells affected (x-axis, 0.0 = no cells dead, 1.0 = all cells dead).

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: SYBR Green Assay, Proliferation Assay

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Combined olaparib and TRAIL enhance S/G2 phase cell cycle arrest and apoptosis. CAPAN1 ( A – C ), and the wild-type BRCA2 expressing BRCA2/CIN treated with various doses of olaparib alone ( A , D ) or in combination with TRAIL (10 ng/mL) ( B , C , E , F ) for 48 h. A,B,D,E depict the percentage of cells in the respective cell cycle phase from all viable cells, i.e., non-sub-G1 cells. C,F depict the percentage of cells in sub-G1 from all cells. Figure ( G ): Representative flow cytometry histograms of CAPAN1 and BRCA2/CIN cells after incubation with control, olaparib (10 µM), TRAIL (10 mg/mL), or its combination. Error bars represent mean ± SEM from at least three experiments. * = statistical significance, p < 0.05.

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Expressing, Flow Cytometry, Incubation, Control

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Olaparib influences death receptor expression and caspase activation. ( A – C ): Western blot analysis to assess the expression levels of the indicated regulators of apoptosis in BRCA2 deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with various doses of olaparib alone or in combination with TRAIL (10 ng/mL) for 48 h. Data show representative results from three experiments. GAPDH served as a loading control. ( D ): Flow cytometry of TRAIL surface receptor 2 (death receptor 5, DR5) in BRCA2 deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with 1 μM olaparib for 6 days. Error bars represent mean ± SEM from at least three experiments. * = statistical significance, p < 0.05, NS = non-significant, MFI = median fluorescence intensity.

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Expressing, Activation Assay, Western Blot, Control, Flow Cytometry, Fluorescence

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Influence of Olaparib and TRAIL on DNA damage repair. ( A , B ): Western blot analysis to assess the expression levels of the indicated DNA damage proteins in BRCA2 -deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with various doses of olaparib alone or in combination with TRAIL for 48 h. Data show representative results from three experiments. GAPDH served as a loading control. ( C , D ): 53BP1 immunostaining of BRCA2 deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with 10 μM olaparib +/− 10 ng/mL TRAIL or dimethyl sulfoxide (DMSO) control for 48 h. Quantification of 53BP1 foci per nucleus was based on at least 10 nuclei per sample. Error bars represent mean ± SD. * = statistical significance, p < 0.05.

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Western Blot, Expressing, Control, Immunostaining

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in cancer cell survival under chronic extracellular acidosis. A549 (A) or HCT116 (B) cells were cultured in pH 6.7 (6.7EXT) or pH 7.4 (7.4EXT) buffered medium for about 3 months. Cells were then cultured in pH 6.7 or 7.4-buffered media for 48 h with/without 1 mM IPTG and/or 30 μM A484954, before lysis and Western blot analysis. A549 (C) or HCT116 (D, G) cells were also subjected to flow cytometry to determine subG1 population. (E) HCT116 cells were cultured at pH 7.4 in the presence or absence of 1 mM IPTG and 30 μM A484954 for 48 h, and G1 cell size was then determined by flow cytometry. Results are shown as forward scatter-height (FSC-H). (F) HCT116 cells were cultured as described for panel E and then subjected to flow cytometry analysis; the percentage of sub-G1 population is shown. (H) HCT116 cells cultured at pH 6.7 (6.7EXT) or 7.4 (7.4EXT) for approximately 3 months were transfected with GFP-spectrin alone or GFP-spectrin plus FLAG-tagged eEF2K as indicated. Cells were lysed 8 h after transfection, and eEF2K levels were analyzed by Western blotting. Data are expressed as mean ± SE from 3 independent experiments. P values were obtained by two-way ANOVA. *, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001. For panels A and B, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Cell Culture, Lysis, Western Blot, Flow Cytometry, Transfection

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Acidosis activates eEF2K in cancer cells. (A) Validation of the P-eEF2 Thr56 antibody for immunohistochemistry. eEF2K WT and knockout (KO) MEFs were stained with P-eEF2 Thr56. Scale bar, 50 μm. (B) Representative sequential sections of human lung cancer to support expression of NHE-1, GLUT-1, and P-eEF2 Thr56. Nuclei were counterstained with hematoxylin. Ten lung cancer carcinoma samples were stained in total, 7 of which showed strong areas of GLUT-1 expression; of these, 6 codisplayed NHE-1 and P-eEF2 Thr56. Scale bar, 100 μm. A549 (C) or HCT116 (D) cells were cultured in medium buffered at pH 6.4, 6.8, or 7.4 for the indicated times. IPTG (1 μM) was added to A549 (E) or HCT116 (F) cells 5 days before the experiment. Cells were then incubated in medium buffered at different pH values for 48 h with/without 1 mM IPTG, 30 μM A484954, or 25 μM etoposide, followed by lysis and Western blot analysis. (G) A549 cells were treated as described for panel E, and Western blot analysis was then performed to study the phosphorylation of p70S6K1 at Thr389 and S6 at Ser240/Ser244. For panels C to G, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Biomarker Discovery, Immunohistochemistry, Knock-Out, Staining, Expressing, Cell Culture, Incubation, Lysis, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Activation of eEF2K is important in cancer cell survival under acute acidic conditions. A549 (A) or HCT116 (B) cells were treated as described for Fig. 9E and ​andF,F, respectively, and then subjected to cell ATP assay using the CellTitre-Glo kit. A549 (C) or HCT116 (E) cells were treated as described for panels A and B, respectively, and then subjected to cytotoxicity assay using CellTox Green kit. A549 (D) or HCT116 (F) cells were cultured as described for panels A and B, respectively, and then subjected to flow cytometry analysis. The percentage of sub-G1 population is presented. For panels A, B, C, and E, results are expressed as means ± SE from 3 independent experiments in duplicate. For panel D, results are means ± SE from 3 independent experiments. For panel E, results are means ± SE from 2 independent experiments and a third one in duplicate. P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001).

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Activation Assay, ATP Assay, Cytotoxicity Assay, CellTox Assay, Cell Culture, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in protein synthesis under acidosis. (A) IPTG (1 μM) was added to A549 cells 5 days before experiment. A549 cells were then transferred to media at different pH values for 1 h with/without 1 μM IPTG. Rates of protein synthesis were determined, and results are presented as counts per minute (cpm) per microgram protein and expressed as means ± SE from 4 independent experiments. (B) Quantification of IPTG-treated cells as described for panel A expressed as a percentage of control (no IPTG treatment). For panels A and B, P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001). (C) A549 cells were incubated at pH 6.7 or 7.4 for 1 h. Lysates were fractionated on sucrose density gradients. Positions of the 40S, 60S, and 80S ribosomal particles and polysome fractions are shown. Absorbance values (254 nm) are in arbitrary units and on the same scale for each panel. Representative data from 4 independent experiments are shown.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Control, Incubation