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Indomethacin Sensitizes TRAIL-Resistant Melanoma Cells to TRAIL-Induced Apoptosis through ROS-Mediated Upregulation of Death Receptor 5 and Downregulation.

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Presentation on theme: "Indomethacin Sensitizes TRAIL-Resistant Melanoma Cells to TRAIL-Induced Apoptosis through ROS-Mediated Upregulation of Death Receptor 5 and Downregulation."— Presentation transcript:

1 Indomethacin Sensitizes TRAIL-Resistant Melanoma Cells to TRAIL-Induced Apoptosis through ROS-Mediated Upregulation of Death Receptor 5 and Downregulation of Survivin  Anfernee Kai-Wing Tse, Hui-Hui Cao, Chi-Yan Cheng, Hiu-Yee Kwan, Hua Yu, Wang-Fun Fong, Zhi-Ling Yu  Journal of Investigative Dermatology  Volume 134, Issue 5, Pages (May 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Indomethacin sensitizes TRAIL-induced apoptosis in A375 melanoma cells. (a) Chemical structure of indomethacin. (b) Cell viability was assessed by the MTT assay. (c) Western blotting of lysates from A375 cells pretreated with indomethacin for 24hours followed by treatment with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) for 3hours using the indicated antibodies. (d) Caspase activities in A375 cells that had been treated with indomethacin for 24hours and then TRAIL for 8hours. (e) Cells were stained with PI/Annexin V and then analyzed by FACS. a,b,cP<0.05 with a versus control, b versus indomethacin and c versus TRAIL. (b, d) At least two independent experiments with triplicates revealed comparable results. (c, e) At least two independent experiments revealed largely comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Indomethacin-induced DR5 upregulation is essential for sensitization of TRAIL-mediated apoptosis. (a) Western blotting of DR5 and DR4 in A375 cells. (b) Cell surface expression of death receptor 5 (DR5) in A375 cells treated with 300μM indomethacin for 24hours was measured by flow cytometry analysis. MFI, mean fluorescence intensity. (c) (Top) Reverse transciptase PCR (RT-PCR) product from A375 cells treated as indicated for 18hours. (Bottom) Real-time PCR analysis of DR5 mRNA. *P<0.05 versus control. (d) (Top) Cells were transfected with DR5 small interfering RNAs (siRNAs), and cell extracts were prepared for western blot analysis of DR5 and PARP. (Bottom) After transfection with death receptor 5 (DR5) siRNA, A375 cells viability was assessed by the MTT assay. *P<0.05. (e) siRNA-transfected cells were stained with PI/Annexin V and then analyzed by FACS. (a–e) At least two independent experiments revealed largely comparable results. TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Induction of CHOP is required for indomethacin-induced DR5 expression. (a) (Top) Western blotting of CHOP; (middle) RT-PCR analysis of CHOP; (bottom) western blot analysis of CHOP in A375 cells pretreated with cycloheximide, or actinomycin D before incubation with 300μM indomethacin. (b) Schematic diagram of the deletion mutants and luciferase activities in cells treated with 300μM indomethacin. (c) Luciferase activity of cells transfected with pDR5-310 or pDR5-310ΔCHOP and then treated with 300μM indomethacin. *P<0.05 Compared with indomathacin-treated pDR5-310-transfected cells. (d) (Top left) Cells were transfected with CHOP siRNAs and western blot analysis of DR5 and CHOP; (top right) after transfection with CHOP siRNAs, A375 cell viability was assessed by the MTT assay. *P<0.05; (Bottom) siRNA-transfected cells were stained with PI/Annexin V and then analyzed by FACS. (b,c) At least two independent experiments with triplicates revealed comparable results. (a, d) At least two independent experiments revealed largely comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 ROS mediates indomethacin-induced DR5 and CHOP upregulation. (a) Fluorescence was measured using 6-carboxy-2’,7’-dichlorofluorescein diacetate or dihydroethidium dyes. (b) Western blotting analysis of DR5 and CHOP. (c) A375 cells viability was assessed by the MTT assay. *P<0.05. (d) A375 cells were pretreated with 5mM N-acetyl-L-cysteine (NAC) and 300μM indomethacin for 24hours, and then incubated with 25ngml-1 TRAIL for 3hours. Then, western blotting analysis was performed using anti-PARP and anti-caspase-8 antibodies, respectively. (e) A375 cells were transfected with pDR5-310 together with either empty, catalase, or SOD1 expression vectors and then treated with 300μM indomethacin. After 24hours, cells were lysed and assayed for luciferase activity. *P<0.05 compared with indomethacin-treated empty vector–transfected cells. (a, c, e) At least two independent experiments with triplicates revealed comparable results. (b, d) At least two independent experiments revealed largely comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Downregulation of survivin contributes to the enhancement of TRAIL-induced apoptosis by indomethacin. (a) Western blotting of A375 lysates from cells treated with 300μM indomethacin. (b) Western blotting analysis of transfected A375 cells using anti-PARP and anti-Flag antibodies. (c) Western blotting analysis was performed using the anti-survivin antibody. (d) A375 cells were treated with 300μM indomethacin for 24hours; nuclear and total extracts were isolated as described under Materials and Methods and analyzed for p65, actin, and Sp1 by western blotting. (e) Western blotting analysis of transfected A375 cells using anti-p65, anti-Flag, and anti-survivin antibodies. (f) Luciferase activity of A375 cells transfected with pNF-κB-Luc and treated with 300μM indomethacin in the presence or absence of NAC (5mM) for 24hours. At least two independent experiments with triplicates revealed comparable results. (a–e) At least two independent experiments revealed largely comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Indomethacin sensitizes TRAIL-resistant melanoma cells. (a) Cell viability was assessed by the MTT assay. a,b,cP<0.05 with a versus control, b versus indomethacin and c versus TRAIL. At least two independent experiments with triplicates revealed comparable results. (b) Whole-cell extracts were analyzed for expression of DR5 by western blotting. (c) The cell surface expression levels of DR5. MFI, mean fluorescence intensity. (d) Western blotting of lysates using the indicated antibodies. (e) Cells were stained with propidium iodide (PI)/Annexin V and then analyzed by FACS. (f) Schematic diagram of the mechanism by which indomethacin potentiates TRAIL-induced apoptosis. (b–d) At least two independent experiments revealed largely comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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