1 alpha ,25-Dihydroxyvitamin D sub(3) inhibits the human H295R cell proliferation by cell cycle arrest: A model for a protective role of vitamin D receptor against adrenocortical cancer

• Published in: The Journal of steroid biochemistry and molecular biology 2014-03, Vol.140, p.26-33
• Main Authors: Pilon, Catia, Urbanet, Riccardo, Williams, Tracy A, Maekawa, Takashi, Vettore, Silvia, Sirianni, Rosa, Pezzi, Vincenzo, Mulatero, Paolo, Fassina, Ambrogio, Sasano, Hironobu, Fallo, Francesco
• Format: Article
• Language: English
• ISSN: 0960-0760
• DOI: 10.1016/j.jsbmb.2013.11.008

Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1 alpha ,25-dihydroxyvitamin D sub(3) [1 alpha ,25(OH) sub(2)D sub(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 [ super(3)H]thymidine cell incorporation after treatment with 1 alpha ,25(OH) sub(2)D sub(3) at increasing doses. The effect of 1 alpha ,25(OH) sub(2)D sub(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 transition phase, was evaluated in cells treated with 1 alpha ,25(OH) sub(2)D sub(3) before and after VDR gene silencing. 1 alpha ,25(OH) sub(2)D sub(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 alpha ,25(OH) sub(2)D sub(3) cell exposure. CDK4 activation was reduced by 10 nM 1 alpha ,25(OH) sub(2)D sub(3) but was not affected by 1 alpha ,25(OH) sub(2)D sub(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 alpha ,25(OH) sub(2)D sub(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 immunohistochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth.