Effect of 20-epi-1alpha,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.
Gumireddy
Kiranmai
K
Department of Pediatrics, Brown University School of Medicine, Providence, RI 02905, USA.
Ikegaki
Naohiko
N
Phillips
Peter C
PC
Sutton
Leslie N
LN
Reddy
C Damodar
CD
D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019938:Cyclin D1 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D009447:Neuroblastoma / Q000473:pathology*; D016900:Neurofilament Proteins / Q000096:biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D018167:Receptors, Calcitriol / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis; D012097:Repressor Proteins; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D016900:Neurofilament Proteins; D016271:Proto-Oncogene Proteins c-myc; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C118756:dihydroxy-vitamin D3; C082925:retinoic acid receptor beta; C051774:neurofilament protein H; D019938:Cyclin D1; D014807:Vitamin D; D050760:Cyclin-Dependent Kinase Inhibitor p27