Enhanced Death Ligand-Induced Apoptosis in Cutaneous SCC Cells by Treatment with Diclofenac/Hyaluronic Acid Correlates with Downregulation of c-FLIP  Lothar F. Fecker, Eggert Stockfleth, Frank K. Braun, Paul M. Rodust, Constanze Schwarz, Anja Köhler, Martin Leverkus, Jürgen Eberle  Journal of Investigative Dermatology  Volume 130, Issue 8, Pages 2098-2109 (August 2010) DOI: 10.1038/jid.2010.40 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Death ligand-mediated apoptosis. (a) Cell lines of cutaneous SCC (SCL-I, SCL-II, SCC-12, SCC-13) were treated for 16hours with CH-11 and TRAIL, or for 24hours with TNF-α, and the relative apoptotic response was determined by DNA fragmentation analysis and compared with untreated controls. Three independent experiments for SCL-II, SCC-12, and SCC-13 and two experiments for SCL-I were performed, each experiment consisting of at least double values, and means±SD of all experiments are given here. Induction of apoptosis by all three death ligands in the four cell lines was statistically significant (*P<0.05). (b) Formation of specific cleavage products of activated caspase-8 (43, 41, 18kDa) and of activated caspase-3 (19, 17, 15kDa) were determined by western blot analysis. Analysis was performed for SCL-I, SCL-II, SCC-12, and SCC-13 cells after treatment with CH-11, TRAIL, or TNF-α. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Caspase-8 activation by diclofenac/HA. SCC cells were treated for 24hours with 0.2% diclofenac/HA (D/HA+) and were compared with untreated controls (-). Formation of specific cleavage products are shown for activated caspase-8 (43, 41, 18kDa) and caspase-3 (17, 15kDa), as determined by western blot analysis. For loading control, blots were incubated with a β-actin antibody. The experiment was performed twice starting from independent cell isolates, which gave highly comparable results. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Enhanced apoptosis by combination of diclofenac/HA and death ligands. (a) Apoptosis levels (DNA fragmentation) of SCL-I, SCL-II, SCC-12, SCC-13, and HaCaT cells are shown without treatment (Control), after treatment with diclofenac/HA (D/HA, 0.2%), after treatment with death ligands (CH-11, TRAIL, TNF-α), and after combined treatment with diclofenac/HA and death ligands. Diclofenac/HA was administered for 20hours and death ligands were applied for 16hours. The relative values were normalized with respect to untreated controls. Three independent experiments for SCL-II, SCC-12, and SCC-13 and two experiments for SCL-I and HaCaT were performed, each experiment consisting of at least double values. The means±SD of all experiments (SCC) or of a representative experiment (HaCaT) are given. The enhancement of death ligand-induced apoptosis by diclofenac/HA was statistically significant in all combinations and all sensitive cell lines (P<0.05), as indicated by asterisks. (b) FACS analyses of propidium iodine-stained SCC-12 cells are shown. Cells were treated with diclofenac/HA alone (0.2%, 20hours), CH-11 alone (1ngml−1, 16hours), or with a combination of both. The percentages of sub-G1 cell populations (apoptotic cells, values indicated) were compared with untreated controls. The experiment was performed twice starting from independent cell isolates, which gave highly similar results. (c) Apoptosis induction in SCC-12 by CH-11 was compared with the highly sensitive T-ALL cell line Jurkat, treated with CH-11 for 16hours. The upper panel shows relative apoptosis values (x-fold change) as determined by ELISA. The lower panel shows the percentages of sub-G1 cells as determined by propidium iodide staining and FACS analysis. The experiments were run in parallel and the experiment has been performed twice resulting in comparable values. Enhancement of apoptosis by CH-11 and by combinations as compared with controls was statistically significant (*P<0.05). (d) Real-time cell analysis monitoring cell confluence (cell index) over time was performed for SCC-12 cells, which were left untreated (Control), treated with diclofenac (90μgml−1), with TRAIL (20ngml−1), or with a combination of both, as indicated in the figure. All treatments started at 22hours. Each value represents the mean of four wells. The cell index curves were normalized to a value of 1 at 1hour before treatment started. The experiment was performed with three different seeding densities (20,000; 10,000; and 5,000 cells per well), of which only the values for 10,000 cells are shown here. The whole experiment was repeated once. The different seeding densities as well as the second experiment gave similar results. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Full activation of the proapoptotic caspase cascade after combined treatment. SCC-12 cells were treated with diclofenac/HA (D/HA), CH-11, TRAIL, or TNF-α alone, or with combinations of diclofenac/HA and death ligands. Specific cleavage products of caspases and of Bid were detected by western blot analysis after treatment, as indicated for caspase-8 (18, 41, 43kDa), caspase-10 (43kDa), Bid (15kDa), caspase-9 (34, 37kDa), caspase-3 (15, 17, 19kDa), and caspase-7 (19, 30kDa). For loading control, blots were incubated with a β-actin antibody. Western blot experiments were performed twice using independent cell isolates, which resulted in highly similar results. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibition of TRAIL and diclofenac/HA-induced apoptosis by caspase inhibitors. SCC 13 cells were treated with TRAIL (20ngml−1, 16hours, a) or with diclofenac/HA (D/HA, 0.3%, 24hours, b). In addition, cells had received treatment with various caspase inhibitors (10μM). The inhibitors were applied 1hour before diclofenac/HA and TRAIL treatment started and were present during the whole period of treatment. The inhibitors used were the pancaspase inhibitor zVAD-fmk (zV) and inhibitors regarded as specific for caspase-1, 2, 3, 4, 6, 8, 9, 10, and 13 (C1–C13). Relative DNA fragmentation values (apoptosis) are shown in relation to untreated cells, which were set to 1 (white bars). Note that apoptosis induced by diclofenac/HA or TRAIL was suppressed by zVAD-fmk as well as by inhibitors of caspase-3, 8, and 10 (black bars). The whole experiment was performed twice with triplicate values both times. Both experiments produced highly similar results. The mean values±SD of one experiment are shown here. Suppression of apoptosis by zVAD-fmk, C3, C8, and C10 was statistically highly significant (P<0.01) as indicated by asterisks on the columns. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Changes at the death receptor level. (a) The effects of two antagonistic TRAIL receptor antibodies directed against TRAIL-R1/DR4 (anti-DR4) and against TRAIL-R2/DR5 (anti-DR5) were investigated in SCC-12 cells, and treated cells were compared with control cells (No antibody). Cells did not receive any extra treatment, or received in addition TRAIL (20ngml−1, 24hours) or diclofenac/HA (0.3%, 24hours), as indicated below. Note that TRAIL-induced apoptosis was blocked by anti-DR4 but not anti-DR5, and diclofenac/HA treatment resulted in proapoptotic antibody effects. The mean values±SD of an experiment with triplicate values are shown. The experiment has been repeated three times with single values each time, which gave similar results. Inhibition of TRAIL-induced apoptosis by TRAIL receptor antibodies as well as enhancement of apoptosis by a combination of diclofenac/HA and antibodies as compared with diclofenac/HA treatment alone were statistically significant (*P<0.05). (b) Surface expression of CD95, TRAIL-R1, and TRAIL-R2 death receptors was monitored by flow cytometry using four SCC cell lines without any extra treatment (thin line) and after treatment with 0.3% diclofenac/HA (D/HA, 24hours; thick line). Isotypic control antibodies are shown as filled graphs. A shift to the right indicates increased surface expression. The experiment has been repeated once using independent cell isolates, giving similar results. (c) Expression of c-FLIPL (55kDa) and c-FLIPS (28kDa) is shown in four SCC cell lines and in HaCaT cells as determined by western blot analysis. Cells treated for 24hours with 0.3% diclofenac/HA (+) were compared with untreated control cells (-). For loading control, blots were incubated with β-actin antibody (SCC cells) and α-tubulin antibody, respectively (HaCaT cells). The experiment was performed three times (SCC cells) and two times (HaCaT), respectively, using independent cell isolates, which gave similar results. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Role of c-FLIP for enhancement of apoptosis by diclofenac/HA. (a) Apoptosis levels (DNA fragmentation) in HaCaT cells stably transduced with a retroviral vector for c-FLIPL overexpression (HaCaT-FLIP) were compared with those in mock-transduced cells (HaCaT-Mock). Both cell populations were treated with death ligands for 16hours (CH-11, TRAIL, TNF-α) or were left untreated (-). In addition, cells received diclofenac/HA (D/HA, 0.3%, for 20hours, black bars) or were left untreated (gray). The relative values were normalized with respect to the untreated HaCaT mock control (=1). The inset shows overexpression of FLIPL in HaCaT-FLIP cells as compared with that in HaCaT-Mock. Two independent experiments were performed with triplicate values both times; the means±SD of one experiment are shown here. Reduction of apoptosis in HaCaT-FLIP cells was statistically significant (P<0.05), as indicated by asterisks. (b) Expression of c-FLIPL (55kDa) and c-FLIPS (28kDa) as determined by western blot analysis is shown in SCC-12 cells after transduction of lentiviral expression vectors for FLIPRNAi and ScrambleRNAi. Non-transduced SCC-12 cells were included as controls (C). Three approaches are shown with different MOIs (10, 5, 2). GAPDH served as loading control. (c) Relative apoptosis (DNA fragmentation) are shown of SCC-12 cells transduced with lentiviral expression vectors for FLIPRNAi or ScrambleRNAi (MOI of 2 and 5). The cells received no additional treatment (-) or were treated with CH-11 or TRAIL for 8hours. The values had been normalized with respect to non-treated SCC-12 cells (=1; not shown). Two independent experiments were performed with triplicate values both times, and the means±SD of one experiment are shown. Enhancement of death ligand-mediated apoptosis in FLIPRNAi-treated cells as compared with that in cells that received ScrambleRNAi was statistically significant (P<0.05), as indicated by asterisks. Journal of Investigative Dermatology 2010 130, 2098-2109DOI: (10.1038/jid.2010.40) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions