Effect of 20-epi-1α,25-dihydroxyvitamin D3 on the proliferation of human neuroblastoma:: role of cell cycle regulators and the Myc-Id2 pathway

The antiproliferative effects of 1alpha,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] and its epimer, 20-epi-1alpha,25-dihydroxyvitamin D-3 [20-epi-1,25(OH)(2)D-3], in six human neuroblastoma (NB) cell lines (SH-SY5Y, NB69, SK-N-AS, IMR5, CHP134, and NGP) were investigated. We determined the ability of 1,25(OH)(2)D-3 and 20-epi-1,25(OH)(2)D-3 to influence cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell proliferation by bromodeoxyuridine (BrdU) incorporation, and their antineoplastic effect on colony formation in a soft agar assay. A concentration-dependent decrease in cell viability, inhibition of DNA synthesis, and suppression of clonal proliferation was observed with both compounds. 20-epi-1,25(OH)(2)D-3 was more potent in suppressing the proliferation of all six NB cell lines. To understand the mechanisms of action, we examined the effect of 20-epi-1,25(OH)(2)D-3 on the Myc-Id2 cell proliferative network and also on key regulators of the cell cycle. For the first time, we show that 20-epi-1,25(OH)(2)D-3 down-regulated Myc and Id2 expression by western blot analysis. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that 20-epi-1,25(OH)(2)D-3 induced the expression of retinoic acid receptor-beta and p21(Cip1), and down-regulated the expression of cyclin D1 resulting in decreased phosphorylation of retinoblastoma protein (pRB). In sum, we show that 20-epi-1,25(OH)(2)D-3 exerts strong antiproliferative effects by regulating key growth control networks (Myc-Id2-pRB) in NB cells. (C) 2003 Elsevier Science Inc. All rights reserved.