Spermidine activates authophagy but does not rescue human neuroblastoma SH-SY5Y cells from effects of arginine starvation

Abstract

Background. Neuroblastoma is a malignant tumor of the sympathetic nervous system common in early childhood. Autophagy is manifested in neuroblastoma cells at basal levels, but is often upregulated in cells of the aggressive neuroblastoma forms. The aim of the study was to evaluate effects of polyamine spermidine and deficiency of arginine on cell viability and autophagy regulation in cells of human neuroblastoma. Materials and Methods. The human neuroblastoma SH-SY5Y cell line was an experimental model for the MTT assay of metabolic activity and cell viability upon exposure to different concentrations of spermidine in complete and arginine-free media. Assessing autophagy induction under arginine deficiency and spermidine treatment was carried out using fluorescence microscopy of neuroblastoma cells labeled with autophagic lysosomes-staining dye monodancylcadaverine. Results and Discussion. It was found that arginine withdrawal abrogates proliferation of SH-SY5Y cells. In the presence of arginine precursor, citrulline, in arginine-free medium, SH-SY5Y cells could not proliferate and, therefore, are auxotrophic for arginine. SH-SY5Y cells were more sensitive to arginine starvation than to starvation for indispensible amino acids lysine or leucine. It was also revealed that spermidine at low concentrations of 5-10 μM did not affect SH-SY5Y cells viability irrespective of arginine availability. However, at 50 and higher concentrations this polyamine was highly cytotoxic in arginine-sufficient or deficient media. Analysis of autophagy induction by spermidine and under arginine starvation revealed an increase in the number of autophagic lysosomes in SH-SY5Y cells and additive effect of the two stimuli. Conclusion. The described experiments revealed that arginine deprivation abrogated proliferation, led to a decrease in cell viability and induced autophagy in human neuroblastoma SH-SY5Y cells. Spermidine at the concentrations of 5-10 μM, while upinducing autophagy, did not improve survival of SH-SY5Y cells under arginine deprivation, whereas at a concentration above 50 μM spermidine had a strong cytotoxic effect. The main observation of this study is that autophagy can be readily manipulated in neuroblastoma cells by simultaneous deprivation for arginine and spermidine treatment. Keywords: cancer, neuroblastoma, SH-SY5Y, autophagy, spermidine