Figure 5. Neutrophil elastase activates MAPK signaling and induces MAPK-dependent gene transcription and proliferation in human prostate cancer cells.

A. PC3 cells were serum starved and treated with increasing concentrations of NE for 15 minutes. pERK1/2 and tERK1/2 levels were examined by Western blot. Band densitometry of pERK1/2 to tERK1/2 was performed using ImageJ and normalized to untreated (0). B. PC3 cells were serum starved and treated with NE (2.5µg/mL) for 15 minutes in the presence of increasing concentrations of sivelestat. pERK1/2 and tERK1/2 levels were examined by Western blot. PC3 (C) or C4-2 (D) cells were serum starved and treated with NE (2.5µg/mL) for 15 minutes in the presence of sivelestat (2µM) or vehicle. pERK1/2 and tERK1/2 levels were examined by Western blot, and band densitometry performed using ImageJ and normalized to untreated (UT) samples. Multiple comparisons were performed using row matched one-way ANOVA with Dunnett’s post-hoc testing (n=6 for PC3 and n=3 for C4-2; ***p<0.001, ###p<0.001). E. C4-2 cells were serum starved and treated with NE (2.5µg/mL) for 6 hours in the presence of sivelestat (2µM), PD0325901 (50nM), or vehicle. cFOS mRNA expression was determined using quantitative PCR and normalized to GAPDH. Data were normalized to untreated samples and multiple comparisons were performed using row matched one-way ANOVA with Dunnett’s post-hoc testing (n=4; ****p<0.0001, ##p<0.01, ###p<0.001). F. C4-2 cells were serum starved and treated with NE (2.5µg/mL) for 24 hours in the presence of sivelestat (2µM), PD0325901 (50nM), or vehicle. Proliferation was examined by BrdU incorporation, and data were plotted as percent change in proliferation relative to untreated samples. Multiple comparisons were performed using ordinary one-way ANOVA with Bonferroni’s post-hoc testing (n=9; **p<0.01, ns=not significant).