Apoptosis Induction by SAHA in Cutaneous T-Cell Lymphoma Cells Is Related to Downregulation of c-FLIP and Enhanced TRAIL Signaling Nadya Al-Yacoub, Lothar.

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Apoptosis Induction by SAHA in Cutaneous T-Cell Lymphoma Cells Is Related to Downregulation of c-FLIP and Enhanced TRAIL Signaling Nadya Al-Yacoub, Lothar F. Fecker, Markus Möbs, Michael Plötz, Frank K. Braun, Wolfram Sterry, Jürgen Eberle Journal of Investigative Dermatology Volume 132, Issue 9, Pages 2263-2274 (September 2012) DOI: 10.1038/jid.2012.125 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Proapoptotic effects of suberoylanilide hydroxamic acid (SAHA) on cutaneous T-cell lymphoma (CTCL) cells. (a, b) CTCL cell lines were treated for 48hours with increasing concentrations of SAHA. Induction of apoptosis ((a) flow cytometry after propidium iodide (PI) staining) and of cytotoxicity ((b) lactate dehydrogenase (LDH) release) is shown. For cytotoxicity, triton X-100-treated cells served as positive controls (T=100%). (c) Time kinetic analyses of apoptosis (relative values, apoptosis ELISA) and cytotoxicity are shown for HH (1, 10μM SAHA) and MyLa (2.5, 10μM). (d) Representative histograms of sub-G1 apoptotic cell populations (sG1) are shown in response to SAHA (2.5μM, 48hours), as compared with controls (% of sub-G1 cells indicated). (e) Cell proliferation was determined by the water-soluble tetrazolium (WST)-1 assay in HH and MyLa at 24 and at 48hours of treatment with increasing concentrations of SAHA (C, nontreated controls=100%). Statistical significance of differences is indicated (*P<0.05, Student's t-test). Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Activation of caspases and mitochondria. (a, b) Processing of caspases (Csp) 8, 9, and 3 in response to suberoylanilide hydroxamic acid (SAHA; 1, 2.5, 10μM; 24, 48hours) is demonstrated in cutaneous T-cell lymphoma (CTCL) cells by western blotting (loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 30μg per lane). Caspase activation is seen by specific cleavage products (). (c–e) HH and MyLa were pretreated for 1hour with the pan-caspase inhibitor QVD-OPH (QVD) before SAHA treatment started (2.5μM). Anti-CD95 agonistic antibody (CH-11) was used as control for caspase inhibition by QVD-OPH. After 48hours of treatment, (c) apoptosis (sub-G1 cells), (d) cell survival (calcein staining), and (e) cytotoxicity (lactate dehydrogenase (LDH) release) were determined. (f) Mitochondrial membrane potential (Δψm) was determined by TMRM+ staining in SAHA-treated HH and MyLa (1μM, 24 or 48hours). Representative histograms of treated cells (gray) are shown in overlays with nontreated controls (white graphs). Bars indicate populations with decreased Δψm, and the % of cells with low Δψm is given for 48hours (nontreated/treated). (g) Cytochrome c release in response to SAHA treatment (24hours) is shown in MyLa and HH (Cyto, cytosolic, and Mito, mitochondrial extracts; GAPDH as loading control). Experiments were conducted at least twice, revealing comparable results. Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Activation of caspases and mitochondria. (a, b) Processing of caspases (Csp) 8, 9, and 3 in response to suberoylanilide hydroxamic acid (SAHA; 1, 2.5, 10μM; 24, 48hours) is demonstrated in cutaneous T-cell lymphoma (CTCL) cells by western blotting (loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 30μg per lane). Caspase activation is seen by specific cleavage products (). (c–e) HH and MyLa were pretreated for 1hour with the pan-caspase inhibitor QVD-OPH (QVD) before SAHA treatment started (2.5μM). Anti-CD95 agonistic antibody (CH-11) was used as control for caspase inhibition by QVD-OPH. After 48hours of treatment, (c) apoptosis (sub-G1 cells), (d) cell survival (calcein staining), and (e) cytotoxicity (lactate dehydrogenase (LDH) release) were determined. (f) Mitochondrial membrane potential (Δψm) was determined by TMRM+ staining in SAHA-treated HH and MyLa (1μM, 24 or 48hours). Representative histograms of treated cells (gray) are shown in overlays with nontreated controls (white graphs). Bars indicate populations with decreased Δψm, and the % of cells with low Δψm is given for 48hours (nontreated/treated). (g) Cytochrome c release in response to SAHA treatment (24hours) is shown in MyLa and HH (Cyto, cytosolic, and Mito, mitochondrial extracts; GAPDH as loading control). Experiments were conducted at least twice, revealing comparable results. Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Downregulation of apoptosis regulators. (a) Expression of Bcl-2 family members (Mcl-1, Noxa, Puma) and caspase inhibitors (survivin, XIAP, c-FLIP) is shown in HH and MyLa (western blotting) in response to suberoylanilide hydroxamic acid (SAHA) treatment (2.5, 10μM; 24 and 48hours), and (b) expression of c-FLIP and XIAP is shown in SeAx and HuT-78 at 24hours (loading control: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 30μg per lane). (c) Comparable results were obtained with another set of independent extracts. Expression was quantified by densitometry, and mean values of each two independent experiments (2.5, 10μM SAHA) are shown. Expression in nontreated controls was set to 100%. (d) Apoptotic response to SAHA (sub-G1 cells; 0.5–5μM, 48hours) is shown in MyLa cells that had been stably transduced with a FLIPL retrovirus, as compared with mock-transduced cells. Mean values±SDs of a representative experiment are shown (two independent with triplicates). Statistical significance of reduced apoptosis in FLIPL-transduced cells is indicated (*P<0.005). The proportion of transduced cells (almost 100%) is displayed by green fluorescent protein (GFP) expression, as determined by flow cytometry (shift to the right, insets). (e) FLIPL overexpression is demonstrated by western blotting, as compared with mock-transduced controls (loading control, GAPDH, 30μg per lane). Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Regulation of TNF-related apoptosis-inducing ligand (TRAIL) and DR5. (a) Surface expression of TRAIL (flow cytometry) is shown in cutaneous T-cell lymphoma (CTCL) cells in response to suberoylanilide hydroxamic acid (SAHA; 24 and 48hours; 0.5–5μM; open graphs). Controls: nontreated (0μM) and IgG staining (filled graphs in overlays). (b) Mean values and SDs for TRAIL expression in response to SAHA (2.5μM, 24hours) are given of each three independent experiments. (c) Expression of DR5 is shown in CTCL cells in response to SAHA (1, 2.5, 10μM; western blotting, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control, 40μg per lane). Two experiments with independent extracts showed comparable results. (d) Surface expression of DR5 (flow cytometry) in response to SAHA (24hours; open graphs). Controls: nontreated (0μM) and IgG staining (filled graphs in overlays). (e) Mean values and SDs for DR5 expression in response to SAHA (2.5μM, 24hours) are given of each three independent experiments. Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Enhanced TNF-related apoptosis-inducing ligand (TRAIL)–induced apoptosis in combination with suberoylanilide hydroxamic acid (SAHA). (a) Combination effects of SAHA and TRAIL are shown by induced apoptosis (sub-G1 cells, HH, MyLa) and by decreased cell viability (HH, calcein staining). Cells were pretreated for 24hours with SAHA alone, followed by the combination treatment with TRAIL (10ngml−1) for another 24hours. Mean values±SDs of representative experiments are shown (two independent with triplicates); statistical significance is indicated (*P<0.05). (b) Processing of caspases (Csp) 8 and 3 in HH cells in response to combination treatment was determined by western blotting (30μg per lane, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control). Caspase activation is seen by specific cleavage products (indicated by scissors). Another set of experiments with independent extracts yielded comparable results. (c) The effects of other combinations (5nM bortezomib, Bort, and 100μM methotrexate, MTX) on apoptosis (sub-G1 cells) are shown in HH and MyLa. Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effects of suberoylanilide hydroxamic acid (SAHA) in tumor T cells. (a) Isolated tumor T cells (CD4-selected) of ten Sézary syndrome patients (P1–P10) were treated with SAHA (1μM, 48hours), and apoptosis (sub-G1 cells) and cytotoxicity were determined. (b) Dose-dependent induction of apoptosis (sub-G1 cells, 48hours) was determined in cells of patients (P1, P2, P4) and healthy donors (HD 1–3). (c) Dose-dependent induction of apoptosis (sub-G1 cells) was compared with cytotoxicity (Cytotox) and related to decreased cell proliferation in P10 and HD4. For cytotoxicity, triton X-100-treated cells served as positive control (T=100%). (d) Apoptosis was determined in response to SAHA (2.5μM, 48hours) ±TRAIL (TNF-related apoptosis-inducing ligand; 20ngml−1, last 24hours). (a–d) Mean values±SDs of triplicates. Journal of Investigative Dermatology 2012 132, 2263-2274DOI: (10.1038/jid.2012.125) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions