Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1α,25-dihydroxyvitamin D 3 in human promyelocytic leukemia (HL-60) cells

Abstract

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1α,25-dihydroxyvitamin D 3 [1α,25-(OH) 2D 3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B 2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H 2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1α,25-( OH) 2 D 3≧1α,25-( OH) 2-26,27-( CH 3) 2 D 3>1α,25-( OH) 2-26,27-( C 2 H 5) 2 D 3⪢1α,25-( OH) 2-26,27-( C 3 H 7) 2 D 3 . These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1α,25-(OH) 2D 3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1α-OH group from 1α,25-(OH) 2D 3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1α-OH groups in the 1α,25-(OH) 2D 3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1α,25-(OH) 2D 3 receptor of HL-60 cells, suggesting a 1α,25-(OH) 2D 3 receptor-mediated induction mechanism. Thromboxane synthesis capacity, a feature of the phenotype of HL-60 cells differentiated along the monocytic lineage, was used as a differentiation marker for the development of more potent 1α,25-(OH) 2D 3 compounds as potential differentiation-inducing drugs.