Figure 2 from Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

Treatment-mediated hypoxia-promoted VEGF and IL8 signaling influences AR expression and activity, angiogenesis and enzalutamide response. A and B, Effect of 10 μmol/L enzalutamide (Enz) on viability of LNCaP (A) or C4-2B (B) cells under normoxia and hypoxia for 72 hours. Control cells are presented as 100% for either untreated normoxic cells (left y-axis) or cells treated with hypoxia alone (right y-axis). As a control, were treated with an equivalent volume of DMSO. Data shown are mean ± SEM of N = 3 experiments. C, Cell-cycle analysis of 10 μmol/L Enz treated LNCaP and C4–2B cells following 72 hours. Control cells were treated with an equivalent volume of DMSO. Data are mean±SEM of N = 4 experiments. D, Effect of hypoxia on expression of the AR in LNCaP cells (top), and ARFL and AR-V7 expression in 22Rv1 (middle), and CWR-R1 (bottom) cells. Blots shown are representative of N = 3 experiments. Equal loading was assessed using β-Actin. Relative expression was determined by densitometry using Image J software. E, Luciferase reporter assay demonstrating the effect of 2 to 24-hour hypoxia on AR transcriptional activity in LNCaP and 22Rv1 cells. Data are mean±SEM of N = 3 experiments. F, Luciferase reporter assay demonstrating the effect of hypoxia (6 hours) on AR transcriptional activity in LNCaP and 22Rv1 cells. Data are mean±SEM of N = 3 experiments. G, Immunofluorescent analysis of AR distribution in LNCaP cells cultured under normoxia or hypoxia (6 hours). Images present a merged image, DAPI staining (Blue), and AR-related fluorescence (Red). H, qRT-PCR data demonstrating detectable KLK3 (PSA) expression in LNCaP, 22Rv1, and CWRR1 cells. Data shown are mean±SEM of N = 3 experiments. For all experiments, statistical analysis was carried out using a Student two-tailed t test, Mann–Whitney U test, or 2-way ANOVA with Bonferroni post-tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001.