Journal of Steroid Biochemistry & Molecular Biology 140 (2014) 26–33 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology jo ur nal home p age: www.elsevier.com/locate/jsbmb 1,25-Dihydroxyvitamin D 3 inhibits the human H295R cell proliferation by cell cycle arrest: A model for a protective role of vitamin D receptor against adrenocortical cancer Catia Pilon a , Riccardo Urbanet a , Tracy A. Williams b , Takashi Maekawa c , Silvia Vettore a , Rosa Sirianni d , Vincenzo Pezzi d , Paolo Mulatero b , Ambrogio Fassina a , Hironobu Sasano c , Francesco Fallo a,∗ a Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy b Department of Medical Sciences, University of Torino, Torino, Italy c Department of Pathology, Tohoku University School of Medicine, Sendai, Japan d Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, Arcavacata di Rende (CS), Italy a r t i c l e i n f o Article history: Received 22 July 2013 Received in revised form 28 October 2013 Accepted 7 November 2013 Keywords: Vitamin D H295R cells Adrenocortical cancer a b s t r a c t Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1,25- dihydroxyvitamin D 3 [1,25(OH) 2 D 3 )]–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 [ 3 H]thymidine cell incorporation after treatment with 1,25(OH) 2 D 3 at increasing doses. The effect of 1,25(OH) 2 D 3 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 tran- sition phase, was evaluated in cells treated with 1,25(OH) 2 D 3 before and after VDR gene silencing. 1,25(OH) 2 D 3 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 1,25(OH) 2 D 3 cell exposure. CDK4 activation was reduced by 10 nM 1,25(OH) 2 D 3 but was not affected by 1,25(OH) 2 D 3 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 1,25(OH) 2 D 3 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 immunohis- tochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Bio-activation of vitamin D 3 , the inert secosteroid precur- sor (cholecalciferol) obtained from dietary sources or de novo synthesis in the skin as a result of ultraviolet light-induced photolysis, involves two sequential hydroxylations in humans catalyzed by cytochrome P450 (CYP) 4 -containing enzymes. The first of these conversions is catalyzed in the liver by CYP2R1, ∗ Corresponding author at: Department of Medicine-DIMED, Clinica Medica 3, University of Padova, Via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 049 8212654; fax: +39 049 8218744. E-mail address: francesco.fallo@unipd.it (F. Fallo). a vitamin D 25-hydroxylase which metabolizes cholecalciferol to 25-hydroxycholecalciferol [25(OH)D 3 ]. The second step is the conversion in the kidney of 25(OH)D 3 to active metabolite 1,25-dihydroxycholecalciferol D3 [1,25(OH) 2 D 3 ] by CYP27B1 (25(OH)D 3 , 1-hydroxylase) [1]. In addition to the classical role in calcium and bone homeostasis, 1,25(OH) 2 D 3 (also known as cal- citriol) has been recognized to have several “non-calcemic” effects in a variety of cells after binding to vitamin D receptor (VDR; NR1I1), a member the nuclear receptor superfamily which includes recep- tors for steroids, thyroid hormones and retinoic acid. Depending on the cell type, the VDR forms homodimers or heterodimers with the retinoid X receptor (RXR; NR2B) to allow specific DNA binding. The binding of 1,25(OH) 2 D 3 with VDR-RXR complex is followed by the attachment of this complex to the vitamin D responsive ele- ments, which then initiate transcription in the promoter of target 0960-0760/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2013.11.008