New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase

1 alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D-3), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and nonmalignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (SIP) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)(2)D-3 as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [H-3]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for Cl P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)(2)D-3 and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)(2)D-3 receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)(2)D-3 supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)(2)D-3 and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma. (C) 2012 Elsevier Ltd. All rights reserved.