Effect of 20- epi-1α,25-dihydroxyvitamin D 3 on the proliferation of human neuroblastoma: role of cell cycle regulators and the Myc–Id2 pathway

Abstract

The antiproliferative effects of 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] and its epimer, 20- epi-1α,25-dihydroxyvitamin D 3 [20- epi-1,25(OH) 2D 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) 2D 3 and 20- epi-1,25(OH) 2D 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) 2D 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) 2D 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) 2D 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) 2D 3 induced the expression of retinoic acid receptor-β 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) 2D 3 exerts strong antiproliferative effects by regulating key growth control networks (Myc–Id2–pRB) in NB cells.