Figure 4: Digitoxin and MEK inhibitor additively or synergistically reduced intracellular pH in melanoma cells by synergistically inhibiting the NHE Na⁺/H⁺ exchanger.

(a) Intracellular pH in live melanoma cells acutely dissociated from tumours treated for 4 days in vivo (each melanoma was tested in two independent experiments). (b) Intracellular pH of human and mouse cells in the blood of NSG mice xenografted with hUCB cells and treated with digitoxin plus MEK inhibitor for 10 days. (c) Intracellular pH of immortalized melanocytes (hiMEL) and M214 melanoma cells grown subcutaneously in gelatin sponges in NSG mice that were treated with digitoxin plus MEK inhibitor for four days (n=2 independent experiments). (d) NHE activity in dissociated tumour cells isolated from mice treated 4 days in vivo with digitoxin and/or MEK inhibitor was measured based on their rate of recovery from sodium propionate-induced acute intracellular acidification (each melanoma was tested in 2–3 independent experiments). (e,f) Intracellular pH in dissociated cells (e) and activated caspase-3⁺ cells in sections (f) from melanomas (M481 and M214) obtained from mice treated for 4 days with amiloride (NHE inhibitor) or digitoxin plus MEK inhibitor (n=4–9 mice per treatment; each melanoma was tested in 1–2 independent experiments). In these experiments, an earlier passage of M214 cells was used, with a higher intracellular pH, compared with the M214 cells shown in a and g. (g,h) Intracellular pH in dissociated tumour cells (g) and activated caspase-3⁺ cells in sections (h) of melanoma xenografts (M481 and M214) expressing vector (tRFP control) or NHE1 and treated 4 days with digitoxin plus MEK inhibitor (each melanoma was tested in an independent experiment). (i) Intracellular pH in dissociated tumour cells from M481 xenografts that were uninfected, expressing vector (tRFP control) or BCL2 and treated 4 days with digitoxin plus MEK inhibitor. Statistical significance was assessed by one-way analysis of variance followed by Dunnett’s multiple comparisons test. All data represent mean±s.d. In each panel the number of mice per treatment is written on the bars.