Novel analogs of 1,25-dihydroxyvitamin D₂ combined with a plant polyphenol as highly efficient inducers of differentiation in human acute myeloid leukemia cells

1α,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] is known to act as a powerful differentiation inducer in various types of cancer cells, including acute myeloid leukemia (AML) cells. However, supraphysiological concentrations of 1,25(OH)₂D₃ required to induce terminal maturation of AML cells can cause lethal hypercalcemia in vivo. Here we characterized the differentiation-inducing effects of novel double-point modified analogs of 1,25-dihydroxyvitamin D₂ [1,25(OH)₂D₂], PRI-5201 and PRI-5202 [Pietraszek et al. (2013) Steroids, 78:1003–1014], on HL60, U937 and MOLM-13 human AML cells in comparison with their direct precursors (PRI-1906 and PRI-1907, respectively) and 1,25(OH)₂D₃. The results demonstrated the following order of potency for the tested compounds: PRI-5202 > PRI-1907 > PRI-5201 > PRI-1906 ≥ 1,25(OH)₂D₃, as determined by measuring the expression of cell surface markers of myeloid differentiation. Particularly, the sensitivity of different AML cell lines to PRI-5201 and PRI-5202 was 3-15-fold and 13-50 fold higher, respectively, compared to that of 1,25(OH)₂D₃. Importantly, all the analogs tested at 0.25–1 nM concentrations retained the ability of 1,25(OH)₂D₃ to cooperate with the rosemary polyphenol carnosic acid, which strongly potentiated their prodifferentiation activity in a cell type-dependent manner. These synergistic effects were associated with increased induction of the vitamin D receptor (VDR) protein expression. However, surprisingly, carnosic acid was able to significantly enhance only 1,25(OH)₂D₃-induced transactivation of the direct repeat 3 (DR3)-type vitamin D response element (VDRE), whereas no such cooperation was seen with 1,25(OH)₂D₂ analogs. Furthermore, dose-response analysis revealed that 1,25(OH)₂D₃ was more efficacious than the analogs in inducing VDRE activation. This suggests that the superior prodifferentiation activity of the analogs, as compared to 1,25(OH)₂D₃, may be due to their potential for enhanced activation of the differentiation-related VDRE(s) that differ from the DR3-type element tested in this study. Collectively, the results demonstrate that the new double-point modified 1,25(OH)₂D₂ analogs are much stronger inducers of myeloid differentiation than 1,25(OH)₂D₃ and that their efficacy can be further enhanced by combination with plant polyphenols. These combinations warrant their further mechanistic and translational exploration in AML and other types of cancer.