1α,25(OH)
2-16-ene-D
3, a synthetic analog of the steroid hormone, 1α,25(OH)
2D
3, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal
in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)
2-16-ene-D
3 expresses its biological activities are still not completely understood. Our previous
in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)
2-16-ene-D
3 and 1α,25(OH)
2D
3 are metabolized differently. 1α,25(OH)
2-24-oxo-16-ene-D
3, an intermediary metabolite of 1α,25(OH)
2-16-ene-D
3 formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)
2-24-oxo-D
3, the corresponding intermediary metabolite of 1α,25(OH)
2D
3. In a subsequent
in vivo study, we also reported that 1α,25(OH)
2-24-oxo-16-ene-D
3 exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)
2-24-oxo-16-ene-D
3, in generating some of the key biological activities ascribed to its parent, we performed the present
in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)
2-16-ene-D
3 and 1α,25(OH)
2D
3 are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)
2-24-oxo-16-ene-D
3 in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)
2-16-ene-D
3 and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)
2D
3. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)
2-16-ene-D
3 and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)
2D
3 itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)
2-24-oxo-16-ene-D
3, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)
2-16-ene-D
3.
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