Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1α,25-dihydroxyvitamin D₃ in human promyelocytic leukemia (HL-60) cells

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], 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 B₂ synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H₂ (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1α,25-(OH)₂D₃≧1α,25-(OH)₂-26,27-(CH₃)₂D₃>1α,25-(OH)₂-26,27-(C₂H₅)₂D₃≫1α,25-(OH)₂-26,27-(C₃H₇)₂D₃. 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)₂D₃ molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1α-OH group from 1α,25-(OH)₂D₃ 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)₂D₃ 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)₂D₃ receptor of HL-60 cells, suggesting a 1α,25-(OH)₂D₃ 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)₂D₃ compounds as potential differentiation-inducing drugs.