Effects of pomegranate chemical constituents/intestinal microbial metabolites on CYP1B1 in 22Rv1 prostate cancer cells.

The cytochrome P450 enzyme, CYP1B1, is an established target in prostate cancer chemoprevention. Compounds inhibiting CYP1B1 activity are contemplated to exert beneficial effects at three stages of prostate cancer development, that is, initiation, progression, and development of drug resistance. Pomegranate ellagitannins/microbial metabolites were examined for their CYP1B1 inhibitory activity in a recombinant CYP1B1-mediated ethoxyresorufin-O-deethylase (EROD) assay. Urolithin A, a microbial metabolite, was the most potent uncompetitive inhibitor of CYP1B1-mediated EROD activity, exhibiting 2-fold selectivity over CYP1A1, while urolithin B was a noncompetitive inhibitor with 3-fold selectivity. The punicalins and punicalagins exhibited potent CYP1A1 inhibition with 5-10-fold selectivity over CYP1B1. Urolithins, punicalins, and punicalagins were tested for their 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1 inhibitory activity in the 22Rv1 prostate cancer cell line. Urolithins A and B showed a decrease in their CYP1-mediated EROD inhibitory IC50 values upon increasing their treatment times from 30 min to 24 h. Urolithin C, 8-O-methylurolithin A, and 8,9-di-O-methylurolithin C caused a potent CYP1-mediated EROD inhibition in 22Rv1 cells upon 24 h of incubation. Neutral red uptake assay results indicated that urolithin C, 8-O-methylurolithin A, and 8,9-di-O-methylurolithin C induced profound cytotoxicity in the proximity of their CYP1 inhibitory IC50 values. Urolithins A and B were studied for their cellular uptake and inhibition of TCDD-induced CYP1B1 expression. Cellular uptake experiments demonstrated a 5-fold increase in urolithin uptake by 22Rv1 cells. Western blots of the CYP1B1 protein indicated that the urolithins interfered with the expression of CYP1B1 protein. Thus, urolithins were found to display a dual mode mechanism by decreasing CYP1B1 activity and expression.