HIV-1 Protease Inhibitor Ritonavir Potentiates the Effect of 1,25-Dihydroxyvitamin D₃ to Induce Growth Arrest and Differentiation of Human Myeloid Leukemia Cells Via Inhibition of CYP24.

Ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explores the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] to its inactive form 1,24,25,(OH)₃. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)₂D₃ (10⁻⁷ M, 1h) induced expression of CYP24 by 80-fold, and pre-incubation of these cells with RTV (2X10⁻⁵ M, 3h) decreased 1,25(OH)₂D₃-stumulated expression of CYP24 transcripts by 30 %, resulting in increased levels of 1,25(OH)₂D₃ in culture media (50.7 vs 82.4 nmol/L). Importantly, pre-incubation of HL-60 cells with RTV (2X10⁻⁵ M, 3h) and then exposure to 1,25(OH)₂D₃ (10⁻⁷ M, 24 h) also increased intracellular levels of 1,25(OH)₂D₃ (1.20 vs 2.46 nmol/L) and potentiated the ability of 1,25(OH)₂D₃ to induce growth arrest of HL-60 cells. Clonogenic assay showed that either RTV (5X10⁻⁶ M) or 1,25(OH)₂D₃ (10⁻⁸ M) alone inhibited colonal growth of HL-60 cells by either a mean 30 ± 12 (±SD) % and 40 ± 12 %, respectively; when both were combined, colony formation was inhibited by a mean 80 ± 13 % (p<0.01). Measurement of CD14 expression, NBT reduction, and non-specific esterase staining showed that RTV augmented the prodifferentiative effects of 1,25(OH)₂D₃ in HL-60 cell. For example, 1,25(OH)₂D₃ (10⁻⁸ M, 2 days) induced approximately 48 % of HL-60 cells to become CD14 antigen positive. RTV (2X10⁻⁵ M, 48h) alone did not stimulate expression of CD14 antigen; when 1,25(OH)₂D₃ was combined with RTV, approximately 65 % of cells expressed CD14. Furthermore, androgen-independent DU145 human prostate cancer cells, that are resistant to growth inhibition by 1,25(OH)₂D₃, expressed 80-fold higher levels of CYP24 transcripts compared to HL-60 cells, and intracellular levels of 1,25(OH)₂D₃ were not detectable (<0.3 nmol/L) after exposure of these cells to 1,25(OH)₂D₃ (10⁻⁷ M, 24 h). Of note, pre-incubation of these cells with RTV (2X10⁻⁵ M, 3h) and then exposure to 1,25(OH)₂D₃ (10⁻⁷ M, 24 h) increased intracellular levels of 1,25(OH)₂D₃ to 0.94 nmol/L and sensitized DU145 cells to growth inhibition mediated by 1,25(OH)₂D₃ as measured by MTT assay. Taken together, inhibition of CYP24 might open a new paradigm for therapy using vitamin D compounds.