Figure 3.

Enrichment of UGT2B isoforms may provide a mechanism for loss of potency to ralaniten in LNCaP-RAL^R cells. (A) Analysis of global gene expression identified genes which were specifically induced by R1881 (LNCaP = 243; LNCaP-RAL^R = 260). The Venn diagrams show the percentage of androgen induced genes in this subset, the expression of which decreased by ≥2 fold in the presence of enzalutamide or ralaniten. (B) Heatmap representing differentially regulated genes between LNCaP-RAL^R (group 1/blue) and LNCaP (group 2/black) cells treated with ralaniten (35 μM), enzalutamide (5 μM), or DMSO vehicle and stimulated with 1 nM R1881 (+) or EtOH vehicle (−). Four UGT2B isoforms (UGT2B11, UGT2B15, UGT2B17, and UGT2B28 were identified which were highly enriched in the resistant line (between 10- and 18-fold increase) compared to matched LNCaP samples (n = 2 per treatment group). (C) Transcript levels of UGT2B isoforms normalized to SDHA in additional samples validated the microarray data. Data are presented as mean ± SEM, n = 3 independent experiments. (D) Microsomes harvested from untreated parental LNCaP and LNCaP-RAL^R cells revealed increased basal expression of UGT2B isoforms in the resistant line. (E) The UGT-Glo assay quantified the amount of UGT enzymatic activity, by measuring the amount of a luciferin substrate which could be consumed through glucuronidation (unpaired Student’s t-test; n = 4; p = 0.0003). Data are presented as mean ± SEM: RAL, ralaniten; ENZ, enzalutamide.