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Sensitization of Melanoma Cells for Death Ligand TRAIL Is Based on Cell Cycle Arrest, ROS Production, and Activation of Proapoptotic Bcl-2 Proteins  Sandra-Annika.

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Presentation on theme: "Sensitization of Melanoma Cells for Death Ligand TRAIL Is Based on Cell Cycle Arrest, ROS Production, and Activation of Proapoptotic Bcl-2 Proteins  Sandra-Annika."— Presentation transcript:

1 Sensitization of Melanoma Cells for Death Ligand TRAIL Is Based on Cell Cycle Arrest, ROS Production, and Activation of Proapoptotic Bcl-2 Proteins  Sandra-Annika Quast, Katja Steinhorst, Michael Plötz, Jürgen Eberle  Journal of Investigative Dermatology  Volume 135, Issue 11, Pages (November 2015) DOI: /jid Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Induced cell cycle arrest correlates with enhanced TRAIL sensitivity. (a and d) Apoptosis (% of sub-G1 cells) was determined in seven melanoma cell lines in response to TRAIL (20 ng ml-1). Cells were seeded in low (LD) or high density (HD), as well as with or without fetal calf serum (FCS). Means and SDs are of three independent experiments, each consisting of triplicates. Statistical significance (*P<0.005) is indicated. (b and e) Corresponding histograms of cell cycle analyses and % of cells in G1/G2 (mean values). (c and f) Quantitative cell cycle analyses show % of cells in G1/G2, corresponding to b and e. Significant changes are indicated by asterisks (P<0.05). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Activation of caspases and mitochondria. (a) Processing of caspases-3, -9, and -8 in response to TRAIL (20 ng ml-1) was monitored by Western blotting in cells seeded in low density/high density (LD/HD; loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)). Two independent experiments revealed comparable results. (b) Apoptosis (% of sub-G1 cells) in response to TRAIL and the pancaspase inhibitor Q-VD-OPh (QVD, 10 μM, 1 hour pretreatment). (c) Time kinetic analyses of apoptosis induced by TRAIL (20 ng ml-1) in A-375 at LD/HD. (d) Mitochondrial membrane potential (Δψm) was determined by TMRM+ staining. (f) Bax activation was determined by Bax-NT antibody in cells at LD/HD±FCS (fetal calf serum). (e and g) Quantification of Δψm and activated Bax (each two independent experiments=6 values, means±SD). Rel, relative. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Role of reactive oxygen species (ROS) production. (a and b) ROS levels were determined by CM-H2DCFDA staining in cells seeded in low density/high density (LD/HD)±FCS (fetal calf serum)±20 ng ml-1 TRAIL. Higher fluorescence corresponds to increased ROS levels. (c and d) A-375 and Mel-HO cells grown at LD/HD also received the antioxidant N-acetylcysteine (NAC, 800 μM, 6 hours). (e) Apoptosis (% of sub-G1 cells) was determined in cells grown at LD/HD. Treatment was for 24 hours (TRAIL) and for 26 hours (NAC, 800 μM, 2 hours pretreatment). (b, d and e) Means and SDs of at least two independent experiments (6 values) are shown; statistical significance is indicated (*P<0.005). Rel, relative. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 CDK 4/6 inhibitor enhances TRAIL sensitivity. (a) Apoptotic cells (sub-G1, in %) were determined by cell cycle analysis after treatment with TRAIL (20 ng ml-1, 24 hours) and PD (2 μM, 48 hours, preincubation for 24 hours). Means and SDs of at least two independent experiments (6 values) are shown; statistical significance is indicated (*P<0.005). (b) Corresponding histograms of cell cycle analyses and % of cells in G1/G2 (mean values). (c, d and e) A-375 cells treated with PD (2 μM, 4 hours) are compared with non-treated controls. Right panels, quantification of each two independent experiments (6 values, means±SD) (c) Δψm was determined by TMRM+ staining. (d) Bax activation was determined by Bax-NT antibody. (e) Reactive oxygen species (ROS) levels were determined by CM-H2DCFDA staining. Rel, relative. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Effects of Bcl-2 and Mcl-1. (a) TRAIL-induced apoptosis (sub-G1 cells, %) was investigated in A-375 stably transfected with Bcl-2 (A-375-Bcl-2) and in mock-transfected controls (A-375-pIRES) seeded in low density/high density (LD/HD). Statistical significance is indicated (*P<0.005). (b) Quantification of cell cycle analyses (% of cells in G1/G2, mean values). (c) Impact of Mcl-1 on TRAIL sensitivity (20 ng ml-1, 24 hours) was determined in cells after small interfering RNA-mediated knockdown and compared with respective mock controls. (d) Mitochondrial membrane potential (Δψm) was determined by TMRM+ staining. (e) ROS levels were determined by CM-H2DCFDA staining. (f) Δψm and (g) ROS levels were quantified (each two independent experiments, 6 values; *P<0.05). FCS, fetal calf serum; Rel, relative. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Regulation of Bcl-2 proteins. (a) Expression of Bcl-2 proteins in cells grown at low density/high density (LD/HD; Western blotting, loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)). Mel-HO cells were cultivated with/without FCS whereas all other cultures contained FCS. Two independent experiments showed comparable results. (b) Downregulation of Puma (25 kDa) by two distinct small interfering RNA (siRNA) sequences (siPu 1/2) as compared with controls transfected with scrambled RNA (Scr; A-375, Western blotting; loading control: GAPDH). (c) Apoptosis (sub-G1 cells, %) in response to TRAIL (20 ng ml-1, 24 hours) in cells grown at LD/HD, after siRNA-mediated Puma knockdown (siPu 1/2), as compared with mock controls transfected with scrambled RNA (Scr; mean values±SDs of two independent experiments, 6 values). Statistical significance of suppressed apoptosis is indicated (*P<0.005). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

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