Role of 1,25-dihydroxyvitamin D3 and extracellular calcium in the regulation of proliferation in cultured SH-SY5Y human neuroblastoma cells

Abstract

This study examines the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on SH-SYSY human neuroblastoma cells cultured in the presence of medium containing varying concentrations of calcium (0.1, 0.9, 1.4, 1.8 mM). Pyruvate kinase activity was assayed in SH-SY5Y cells incubated in variable calcium medium with or without 1, 10 or 100 nM 1,25(OH)2D3 for 48 h. The enzyme levels showed a significant increase in comparison with control, when the cells were incubated with 100 nM hormone in the presence of 0.1 mM calcium, while pyruvate kinase activity decreased, when the cells were treated with 100 nM 1,25(OH)2D3 in the presence of 1.8 mM calcium. The proliferative activity of SH-SY5Y was dependent on the extracellular concentration of calcium, being the highest at 1.8 mM calcium and completely absent at 0.1 mM calcium. In the presence of 1,25(OH)2D3, at the three concentrations used and after 48 h incubation, a significant decrease in cell number was always observed, without a direct correlation between 1,25(OH)2D3 effect and calcium concentration in the medium. [3H]Thymidine incorporation in SH-SY5Y cells significantly increased in comparison with control, when the 48 h incubation with 1, 10 or 100 nM 1,25(OH)2D3 was carried out in the presence of 0.1 mM calcium, while, at the other calcium concentrations, the hormone did not cause any significant change in this parameter. The treatment of SH-SYSY cells with 1 nM 1,25(OH)2D3 for 48 h did not affect cell morphology, when 0.1 mM calcium was present, while, in the medium containing 1.8 mM calcium, the treated cells showed a slight trend to differentiation. The differentiating effect of 10 microM all-trans retinoic acid, even if incomplete after 48 h treatment, was only observed in the cultures grown in 1.8 mM calcium, in comparison with those maintained in 0.1 mM calcium.