title: 1α,25-Dihydroxyvitamin D₃ inhibits the human H295R cell proliferation by cell cycle arrest: a model for a protective role of vitamin D receptor against adrenocortical cancer.
abstract: Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃)]--vitamin D receptor (VDR) axis in the growth of adrenocortical cancer (ACC). The presence of VDR in various adrenocortical tissues, including ACC, was also investigated. DNA synthesis was evaluated by [³H]thymidine cell incorporation after treatment with 1α,25(OH)₂D₃ at increasing doses. The effect of 1α,25(OH)₂D₃ on cell cycle and apoptosis was analyzed with a flow cytometer. Cyclin-dependent kinase 4 (CDK4) expression, a molecular marker of G1-S cell cycle transition phase, was evaluated in cells treated with 1α,25(OH)₂D₃ before and after VDR gene silencing. 1α,25(OH)₂D₃ treatment inhibited cell proliferation by 20% at a dose of 1 nM, in parallel with steroid secretion decrease. A cell cycle arrest in G1, with no change in apoptotic cell proportion, was observed after 10 nM 1α,25(OH)₂D₃ cell exposure. CDK4 activation was reduced by 10 nM 1α,25(OH)₂D₃ but was not affected by 1α,25(OH)₂D₃ after VDR gene silencing. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors. VDR immunostaining was evident in benign tumors but it was weak in ACC tissues.
  
  
  CONCLUSIONS
  Slightly supra-physiological concentrations of 1α,25(OH)₂D₃ have a moderate anti-proliferative effect on H295R cells. Anti-proliferative effect was due to cell cycle arrest in G1 phase, without inducing apoptosis. The low mRNA expression levels at qRT-PCR as well as the weak immunohistochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth.
authors.affiliation: Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy.
authors.forename: Catia
authors.initials: C
authors.lastname: Pilon
authors.affiliation: Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Urbanet
authors.affiliation: Department of Medical Sciences, University of Torino, Torino, Italy.
authors.forename: Tracy A
authors.initials: TA
authors.lastname: Williams
authors.affiliation: Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Maekawa
authors.affiliation: Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Vettore
authors.affiliation: Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, Arcavacata di Rende (CS), Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Sirianni
authors.affiliation: Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, Arcavacata di Rende (CS), Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Pezzi
authors.affiliation: Department of Medical Sciences, University of Torino, Torino, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Mulatero
authors.affiliation: Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy.
authors.forename: Ambrogio
authors.initials: A
authors.lastname: Fassina
authors.affiliation: Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Sasano
authors.affiliation: Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy. Electronic address: francesco.fallo@unipd.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Fallo
mesh_terms: D000302:Adrenal Cortex / Q000378:metabolism; D000306:Adrenal Cortex Neoplasms / Q000188:drug therapy*; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D006801:Humans; D018167:Receptors, Calcitriol / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4
keywords: Adrenocortical cancer; H295R cells; Vitamin D