Figure 3: MEK inhibitor reduces ERK phosphorylation while digitoxin depolarizes the plasma membrane and the combination additively or synergistically promotes cell death.
From: Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma
(a,b) Patient-derived xenografts were treated 4 days in vivo with MEK inhibitor and/or digitoxin then surgically excised, fixed and sectioned. (a) Cell death by TUNEL (n=3–5 mice per treatment per melanoma; each melanoma tested in independent experiments). (b) Melanomas (M481, M214) infected with BCL2 or control construct (turbo RFP) that had formed tumours in the flanks of NSG mice, were treated 4 days with digitoxin plus MEK inhibitor and stained for activated caspase-3+ (n=4 mice per treatment; each melanoma tested in independent experiments; statistical significance was assessed with one-way analysis of variance followed by Dunnett’s multiple comparisons test). (c,d) Mice were subcutaneously transplanted with gelatin sponges containing M214 melanoma cells or immortalized melanocytes (hiMEL) in opposite flanks then treated for four days with digitoxin plus MEK inhibitor (n=14 sponges per treatment from three independent experiments; statistical significance was assessed by unpaired two-tailed t-tests). (e–i) Mice engrafted with human (HLA+) hUCB cells were treated for 4–10 days with digitoxin plus MEK inhibitor (n=7 mice per treatment from two independent experiments; statistical significance was assessed by unpaired two-tailed t-tests). (j) Western blot analyses of xenografts treated for 4 days in vivo with MEK inhibitor and/or digitoxin (full blot in Supplementary Fig. 8). (k) Relative DiBAC4(3) fluorescence, measuring plasma membrane potential in live melanoma cells acutely dissociated from tumours treated for 4 days in vivo (n=4–5 mice per treatment per melanoma; each melanoma tested in independent experiments; statistical significance was assessed by one-way analysis of variance followed by Dunnett’s multiple comparisons test). Dissociated cells treated with 150 mM KCl were a positive control for depolarization. (l) Plasma membrane potential of haematopoietic cells in the blood of xenografted NSG mice treated with digitoxin plus MEK inhibitor for 10 days (n=5 mice per treatment; statistical significance was assessed by unpaired two-tailed t-tests). (m) Plasma membrane potential of hiMEL melanocytes or M214 melanoma cells in gelatin sponges from opposing flanks of mice treated with digitoxin plus MEK inhibitor for 4 days (n=4 mice per treatment; statistical significance assessed by two-tailed t-tests). All data represent mean±s.d. For all statistical tests, comparisons with control (NS, not significant; *P<0.05; **P<0.01; ***P<0.001) or the combination compared with MEK inhibitor alone (#P<0.05; ###P<0.001) or the combination compared with digitoxin alone (†P<0.05; ††P<0.01; †††P<0.001).
