24-oxo metabolites of vitamin D-3 analogues: Disassociation of their prominent antileukemic effects from their lack of calcium modulation

The seco-steroid hormone, 1 alpha ,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] inhibits proliferation and induces differentiation of malignant cells incl uding those of the hematopoietic system. The 24-oxo metabolite of 1,25(OH), D, also has prominent antiproliferative activities against various cancer c ells. We chemically synthesized five novel 24-oxo vitamin D, analogues and evaluated their abilities both to inhibit clonal growth and induce differen tiation of myeloid leukemia cells and to cause hypercalcemia. The 1 alpha , 25-dihydroxy-16-ene-D-3 [1,25(OH)(2)-16-ene-D-3] and 1 alpha ,25-dihydroxy- 16-ene-19-nor-D-3 [1,25(OH)(2)-16-ene-19-nor-D-2] and their 24-oxo metaboli tes showed greater potency than 1,25(OH)(2)D-3 in their abilities to inhibi t clonal proliferation of HL-60, NB4, and U937 leukemic cell lines as measu red by methylcellulose soft-gel assay. Their inhibition of clonal growth wa s irreversible as analyzed by pulse exposure studies. The synthetic analogu es also had greater potency than 1,25(OH),D, to induce differentiation of H L-60 and NB4 cells as measured by generation of superoxide, nonspecific est erase production, and induction of CD11b and CD14 cell surface antigens and to increase the proportion of these cells in the G(0)-G(1) phase of the ce ll cycle. For most assays, the 24-oxo metabolite was slightly more potent t han the unmodified analogue, and 50% activity was usually found in the nano molar range. These analogues and their 24-oxo metabolites also inhibited fr esh leukemic cell clonal proliferation. Expression of p27(KIPI), a cyclin-d ependent kinase inhibitor that plays an important role in blocking the cell cycle, was found by Western blot analysis to be induced by the analogues a nd their 24-oxo metabolites in both HL-60 and U937 cells, suggesting a poss ible mechanism by which these analogues inhibit leukemic growth. Notably, t he calcemic activity tested by injections of 1 alpha ,25-dihydroxy-16-ene-2 4-oxo-19-nor-D-3 in mice was at least 12-fold less than 1 alpha ,25(OH)(2)- 16-ene-19-nor-D-3. Taken together, chemically synthesized 24-oxo metabolite s of 1 alpha ,25(OH)(2)-16ene-D-3 and 1 alpha ,25(OH)(2)-16-ene-19-nor-D-3 irreversibly inhibited proliferation and induced differentiation of acute m yeloid leukemia cells with minimal toxicity; these compounds may have a rol e in the maintenance phase of therapy for acute myeloid leukemia.