Figure 5.

HKL-mediated mitochondrial dysfunction potentiates the apoptotic responses of MAPK inhibition. A, A375 cells were treated with DMSO, HKL (40 μm), PLX-4032 (1 μm), or GSK-1120212 (10 nm) for 24 h and then analyzed for changes in OCR using a Seahorse Bioanalyzer. Four basal OCR measurements were made before sequential treatment of oligomycin (1 μm), FCCP (1 μm), and a combination of rotenone and antimycin A (0.5 μm). B–D, kinetic apoptosis assays were performed in A375 cells treated with DMSO, PLX-4032 (10 μm), GSK-1120212 (10 nm), HKL (20 or 40 μm), or in combination. Annexin V-positive events were captured using an IncuCyte Zoom every 2 h over a period of 40 h. Significance was denoted by p < 0.05 (*) by Student's t test. E, colony formation assays were performed using A375 cells plated at a density of 8000 cells/well and treated with DMSO, increasing doses of HKL (5, 10, or 20 μm), PLX-4032 (50, 100, 250, 500, or 1000 nm), or in combination every 2 days. Cells were stained with methylene blue. Representative percentages of colonies are presented. All data are representative of three independent experiments and are reported as the mean ± S.E. F, model summarizing the effects of MAPK inhibitors and HKL on cellular metabolism, mitochondrial shape, cell cycle progression, and cell fate. Inhibition of MAPK signaling causes mitochondrial fusion, increased mitochondrial respiration, cell cycle arrest, and decreased proliferation. HKL inhibits mitochondrial respiration, increases mitochondrial fusion, cell cycle arrest, and apoptosis. Combined HKL and inhibition of MAPK signaling significantly enhances apoptotic responses.