Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity

20-Hydroxyvitamin D-3 (20(OH)D-3), the major product of CYP11A1 action on vitamin D-3, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3), to metabolize 20(OH)D-3. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)(2)D-3 with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D-3 (25(OH)D-3). CYP24A1 hydroxylated 20(OH)D-3 to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D-3 (20,24(OH)(2)D-3) and 20,25-dihydroxyvitamin D-3 (20,25(OH)(2)D-3). The catalytic efficiency of CYP24A1 for 20(OH)D-3 metabolism was more than 10-fold lower than for either 25(OH)D-3 or 1,25(OH)(2)D-3 and no secondary metabolites were produced. The two major products, 20,24(OH)(2)D-3 and 20,25(OH)(2)D-3, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)(2)D-3 or the parent 20(OH)D-3, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D-3. (C) 2012 Elsevier Inc. All rights reserved.