calcitriol and Leukemia--Myeloid

1,25-Dihydroxyvitamin D(3) (1,25D) used to treat human acute myeloid leukemia (AML) cells induces features of normal monocytes, but the mechanisms underlying this response are not fully understood. We hypothesized that one or more microRNAs (miRNA) known to control mouse hematopoiesis and lineage commitment might contribute to the ability of 1,25D to control the malignant phenotype. Here we report that 1,25D markedly induces expression of miR-32 in human myeloid leukemia cells, in which it targets the 3'-untranslated region of the mRNA encoding the proapoptotic factor Bim to reduce its expression. RNAi-mediated suppression of the miRNA-processing enzymes Drosha and Dicer increased Bim levels, in support of the concept that Bim is under miRNA control in AML cells. Antisense-mediated suppression of miR-32 was sufficient to upregulate Bim expression in AML cells. Conversely, ectopic expression of miR-32 downregulated Bim expression and increased the differentiation response to 1,25D treatment in a manner that was associated with increased cell survival. The positive effects of miR-32 on cell survival were confirmed by evidence of increased cell death in AML cells preexposed to antisense miR-32 before treatment with arabinocytosine, a chemotherapeutic drug used to treat human AML. Together, our findings indicate that miR-32 blockade is sufficient to elevate Bim expression and sensitize AML cells to chemotherapy-induced apoptosis. Thus, agents which can inhibit miR-32 expression may offer clinical utility by enhancing therapeutic efficacy in human AML.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Differentiation; Cytarabine; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid; Membrane Proteins; MicroRNAs; Monocytes; Oligoribonucleotides, Antisense; Proto-Oncogene Proteins; U937 Cells; Up-Regulation; Vitamin D

1Alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of steroid sulphatase (STS) in myeloid cells [Hughes et al., 2001, 2005]. This was attenuated by inhibitors of phospholipase D (PLD) (n-butanol, 2,3-diphosphoglyceric acid, C(2)-ceramide) and phosphatidate phosphohydrolase (PAP) (propranolol and chlorpromazine), but was unaffected by inhibitors of phospholipase C. The 1alpha,25(OH)(2)D(3)-induced STS activity was also attenuated by inhibitors of protein kinase Calpha and protein kinase Cdelta (Go 6976, HBDDE and rottlerin), but not by an inhibitor of protein kinase Cbeta (LY379196). Additionally, 1alpha,25(OH)(2)D(3)-induced STS activity was attenuated by inhibitors of RAS (manumycin A), RAF (GW5074), MEK (PD098059 and U1026) and JNK (SP600125), but not p38 (PD169316). 1alpha,25(OH)(2)D(3) produced a rapid and long lasting stimulation of the ERK-MAP kinase signalling cascade in HL60 myeloid leukaemic cells. This 'non-genomic' effect of 1alpha,25(OH)(2)D(3) blocked by pharmacological antagonists of nuclear vitamin D receptors (VDR(nuc)) and does not appear to require hetero-dimerisation with the retinoid-X receptor (RXR). Inhibitors of the Src tyrosine kinase (PP1), RAS (manumycin A), RAS-RAF interactions (sulindac sulphide and RAS inhibitory peptide), RAF (GW5074 or chloroquine), and protein kinase Calpha (HBDDE) abrogated the 1alpha,25(OH)(2)D(3)-stimulated increase in ERK-MAP kinase activity. Taken together, these results show that 1alpha,25(OH)(2)D(3)/VDR(nuc) activation of the RAS/RAF/ERK-MAP kinase signalling pathway plays an important role in augmenting STS activity in human myeloid leukaemic cell lines.

Topics: Calcium Signaling; Cell Line, Tumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Leukemia, Myeloid; MAP Kinase Signaling System; Phospholipase D; Protein Kinase C-alpha; Protein Kinase C-delta; raf Kinases; ras Proteins; Receptors, Calcitriol; Signal Transduction; src-Family Kinases; Steryl-Sulfatase; Tumor Cells, Cultured; Type C Phospholipases; Vitamin D

The goal of differentiation therapy is to induce cancer cells to stop proliferating and to express characteristics of normal cells. Vitamin D analogues, such as the deltanoids, are being evaluated as differentiation agents in the treatment of several human cancers (e.g., myeloid leukemias); however, these compounds have a tendency to produce hypercalcemia in patients receiving therapy. A combination of a differentiation-inducing deltanoid with a compound that blocks entry of calcium into cells (e.g., ketoconazole) may offer a new approach to differentiation therapy and address the problem of hypercalcemia. We investigated whether various ketoconazole-deltanoid combinations would alter cellular differentiation or intracellular calcium homeostasis in comparison with deltanoids used alone.. Cultured human leukemia HL60 cells were treated with ketoconazole-deltanoid combinations. Markers of differentiation (expression of CD11b and CD14 antigens and of non-specific esterase) were measured by flow cytometry and cytochemistry; cell cycle distribution was measured by flow cytometry of propidium iodide-stained cells. Expression of differentiation-related genes was assessed by northern blotting and immunoblotting, and changes in intracellular calcium homeostasis were monitored by fluorescence analysis of fura-2-containing cells.. Ketoconazole strongly potentiated the differentiating activity of the deltanoids, which exhibited low potency when used alone. Ketoconazole-deltanoid combinations had little effect on HL60 cell-cycle distribution, although the cells did stop proliferating and they differentiated. Ketoconazole-deltanoid combinations produced only minor changes in intracellular calcium homeostasis compared with changes produced by 1,25-dihydroxyvitamin D3, either alone or in combination with ketoconazole.. These results suggest that ketoconazole may be useful in combination with vitamin D analogues in the differentiation therapy for myeloid leukemias.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Calcium; Calcium Channels; CD11 Antigens; Cell Differentiation; Cell Division; Drug Synergism; HL-60 Cells; Humans; Ketoconazole; Leukemia, Myeloid; Lipopolysaccharide Receptors; Monocytes; Vitamin D

Singlet oxygen adducts of various vitamin D derivatives, 6,19-dihydro-6,19-epidioxyvitamin D (vitamin D endoperoxides, 2 and 2'), were chemically synthesized, and their biological activity in inducing differentiation of a human myeloid leukemia cell line (HL-60 cells) was examined. The potency of the endoperoxides derived from vitamin D derivatives possessing the 1 alpha-hydroxyl group such as 1 alpha, 25-dihydroxyvitamin D3 endoperoxides (2b and 2b') was markedly (10(-2)) diminished relative to the respective parent vitamin D compounds. In contrast, 25-hydroxyvitamin D3 endoperoxides [25-(OH)D3 endoperoxides, 2a and 2a'] and their analogues fluorinated at the 24- or 26- and 27-positions were 2.5-10 times more potent than 25-hydroxyvitamin D3 (1a) in spite of the absence of the conjugated triene structure typical of vitamin D compounds. The potency of these vitamin D endoperoxides (2 and 2'), especially those lacking the 1 alpha-hydroxyl group, in inducing differentiation of HL-60 cells was not correlated with their activity in binding to the cytosol receptor for 1 alpha, 25-dihydroxyvitamin D3 (1b). The binding efficiency to the receptor was relatively lower than the differentiating activity. To examine the action of vitamin D endoperoxides, carbon analogues of 25-(OH)D3 endoperoxides, two C-6 epimers of 25-hydroxy-6,19-dihydro-6,19-ethanovitamin D3 (6 and 6'), were synthesized. The carbon analogues (6 and 6') had no potential to induce differentiation of HL-60 cells. These results suggest that vitamin D endoperoxides (2 and 2') express their biological activity probably after being converted to some other compounds.

Topics: Binding, Competitive; Calcifediol; Cell Differentiation; Cell Line; Chemical Phenomena; Chemistry; Cholecalciferol; Cytosol; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid; Oxidation-Reduction; Oxygen; Phagocytosis; Receptors, Calcitriol; Receptors, Steroid; Singlet Oxygen; Structure-Activity Relationship