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

Abstract 1α,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 non-malignant 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 (S1P) 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 [ 3 H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P 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.