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RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells  Anja Berger, Sandra-Annika Quast, Michael Plötz, Nicholas-Frederik Kuhn,

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Presentation on theme: "RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells  Anja Berger, Sandra-Annika Quast, Michael Plötz, Nicholas-Frederik Kuhn,"— Presentation transcript:

1 RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells 
Anja Berger, Sandra-Annika Quast, Michael Plötz, Nicholas-Frederik Kuhn, Uwe Trefzer, Jürgen Eberle  Journal of Investigative Dermatology  Volume 134, Issue 2, Pages (February 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Antiproliferative effects by L-779,450. (a) Levels of phosphorylated extracellular signal-regulated kinase (pERK) and total ERK were determined by western blotting in human melanoma cell lines treated for 24 hours with L-779,450 (0.1–50 μM): A-375, A-375-TS (V600E), and MeWo (BRAF-wild type (WT)). Two independent experiments revealed comparable results; equal loading was proven by glyceraldehyde dehydrogenase (GAPDH). (b) Apoptosis (% of sub-G1 cells) was determined in response to L-779,450 (5–40 μM; 24 hours) in A-375, Mel-HO-TS, Mel-2a, MeWo, and SK-Mel-13. Cytotoxicity (lactate dehydrogenase (LDH) release) was determined in parallel (below). Here, values of non-treated cells were set to 1. (c) Apoptosis was also determined at 48 and 72 hours (L-779,450, 10 μM). (b, c) Means and SDs of triplicates; at least two independent experiments. (d) Cell cycle analyses of human melanoma cell lines: open graphs, L-779,450-treated (10 μM, 24 hours); filled graphs, dimethyl sulfoxide (DMSO)-treated controls. (e) Growth curves determined in real time of melanoma cell lines treated with L-779,450 (5–20 μM) were compared with controls (0). Means and SDs (at 48 hours) of triplicate determinations are shown; two independent experiments were performed. Statistical significance is indicated (*P<0.05), when comparing L-779,450 and DMSO-treated cells. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Sensitization of melanoma cells for tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-induced apoptosis. (a) Percentages of apoptotic cells (sub-G1) were determined in human melanoma cell lines at 24 hours in response to L-779,450 (10–40 μM) and TRAIL. Examples are shown in insets. (b) Cytotoxicity (lactate dehydrogenase (LDH) release) was determined in parallel. Values of non-treated cells are set to 1. (c) Photographs (magnification 1:400) after bisbenzimide (Hoechst 33258) staining: TRAIL-sensitive A-375 and TRAIL-resistant A-375-TS cells treated for 24 hours with 10 μM L-779,450/TRAIL. White arrows show examples of fragmented or condensed nuclei. (d) Quantitative evaluation of cells with fragmented nuclei and/or condensed chromatin. (e) MeWo cells were treated also for 48 and 72 hours (percentages of sub-G1 cells). (f) Growth curves (real time) of A-375 and A-375-TS treated with TRAIL, L-779,450 (5 μM), or the combination were compared with non-treated controls. (g) Percentages of apoptotic cells (sub-G1) were determined in response to 20 μM U0126±TRAIL. (h) Cell cycle analyses of A-375 and MeWo treated with U0126 (20 μM, 24 hours, open graphs), as compared with dimethyl sulfoxide (DMSO) controls (filled graphs). (i) Growth curves of A-375 treated with U0126 (20 μM) and/or TRAIL (means and SDs of triplicate determinations). (j) Percentages of apoptotic cells (sub-G1) were determined in response to PLX4032 (3, 10, and 40 μM)±TRAIL. (k) Cell cycle analyses of A-375, A-375-TS, and Mel-HO treated with PLX4032 (10 μM, 2 hours, open graphs), as compared with DMSO controls (filled graphs). Always, two to three independent experiments revealed highly comparable results. Statistical significance (*P<0.05) is indicated, when comparing L-779,450/TRAIL treatment with TRAIL alone. HOECHST/bisbenzimide staining. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Caspase involvement. (a) Processing of caspase-8, -9, and -3 as well as of Bid was determined by western blotting in A-375- and A-375-TS-treated for 16 hours with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (20 ng ml−1) and/or L-779,450 (10 μM). Cleavage products of caspase-3 and -8 are marked (). (b) Percentages of apoptotic cells (sub-G1) were determined after treatment (24 hours). In addition, the pancaspase inhibitor Q-VD-OPh (10 μM, 1 hour pretreatment, dark gray) was given. Statistical significance of abrogated apoptosis by Q-VD-OPh is indicated (*P<0.05). (a, b) At least two independent experiments revealed comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Activation of mitochondrial (Mito) apoptosis pathways. (a) Mitochondrial membrane potential (Δψm) was determined in A-375 and A-375-TS treated for 24 hours with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (20 ng ml−1) and/or L-779,450 (10 μM). Treated cells (open graphs) were compared with dimethyl sulfoxide (DMSO) controls (gray). (b) Cytosolic extracts (Cyto) of TRAIL-sensitive A-375 and TRAIL-resistant A-375-TS were investigated by western blotting, after treatment for 3 hours with TRAIL±L-779,450 (10 μM). Mitochondrial extracts (Mito) served as controls, and analysis of the mitochondrial protein voltage-dependent anion channels (VDAC) ruled out contaminations of cytosolic extracts with mitochondria. Equal loading was proven by glyceraldehyde dehydrogenase (GAPDH). (c) Percentages of apoptotic cells (sub-G1) were determined in A-375 in response to 10 μM L-779,450±TRAIL (24 hours). Cells with small interfering (si)RNA-mediated Smac knockdown (dark bars) were compared with scramble (Scr)-siRNA-transfected controls (light gray; means and SDs of triplicate values). Smac downregulation is shown by western blotting (inset; 24 hours, GAPDH as control). Statistical significance of abrogated apoptosis by Smac knockdown is indicated (*P<0.05). (a–c) At least two independent experiments revealed comparable results. AIF, apoptosis-inducing factor; Cyt c, cytochrome c; Δψm, mitochondrial membrane potential. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Dependency and activation of Bax. (a, c, d) Percentages of apoptotic cells (sub-G1) were determined in A-375 cells stably transfected with Bcl-2 (A-375-Bcl-2) or mock plasmid (A-375-mock), parental HCT-116 colon carcinoma cells (Bax+, Bak+), and subclones with knockdown for Bax and/or Bak, as well as in A-375 cells with small interfering RNA (siRNA)-mediated knockdown for either Bax or Bak. Treatment was for 24 hours, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-treated cells (dark bars) were compared with cells without TRAIL (light gray). (a, c) L-779,450 was applied at 5 μM, but comparable results were also obtained for 10 μM (data not shown). (d) L-779,450 was applied at 10 μM. Means and SDs of representative experiments (triplicate values) are shown. Expression of Bcl-2, Bax, and Bak is shown in respective insets (24 hours, western blotting, glyceraldehyde dehydrogenase (GAPDH) as loading control). Statistical significance of differences is indicated (*P<0.05), when compared with parental/mock cells. (b) Mitochondrial membrane potential (Δψm) was determined in A-375-Bcl-2 and A-375-mock. L-779,450 (5 μM)/TRAIL treatment (open graphs) was compared with dimethyl sulfoxide (DMSO) controls (gray). (e) A-375 and A-375-TS cells were treated for 2 hours with L-779,450 (10 μM) and/or TRAIL and were analyzed for Bax/Bak conformational changes by flow cytometry (Bax-NT/Bak-NT antibodies). Treated cells (bold lines) were compared with non-treated controls (C, gray) and immunoglobulin G (IgG)–stained cells (thin lines). (a–e) At least two independent experiments revealed comparable results. Scr, scramble. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Induction of Bim and autophagy. (a, b) Expression of BimEL was determined by western blotting in Mel-HO, A-375, and A-375-TS melanoma cells treated with 10 μM L-779,450 and in A-375 and A-375-TS treated with L-779,450±tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Glyceraldehyde dehydrogenase (GAPDH) served as loading control. (c) Percentages of apoptotic cells (sub-G1) were determined in A-375 cells with small interfering RNA (siRNA)-mediated knockdown for Bim (dark bars), as compared with scramble (Scr)-siRNA-transfected controls (light gray). Treatments: 10 μM L-779,450±TRAIL for 24 hours. Means and SDs of representative experiments (triplicate values) are shown. Statistical significance is indicated (*P<0.05), when comparing Bim and scramble siRNA. (Inset) Bim expression is shown by western blotting (24 hours), with GAPDH as loading control. (d) Cell photographs (magnification 1:200) of TRAIL-sensitive A-375 and TRAIL-resistant A-375-TS treated with L-779,450 (10 μM) are shown. (e) Expression of light chain 3-II (LC3-II) was determined by western blotting in A-375 and A-375-TS treated with TRAIL (20 ng ml−1) and/or L-779,450 (10 μM). (a–e) At least two independent experiments revealed comparable results. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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