The effects of hydrogen sulphide on proliferation of SH-SY5Y human neuroblastoma cells

Q Aldossari, RE Roberts, SPH Alexander. University of Nottingham, Nottingham, UK

Hydrogen sulphide (H2S) is generated by three enzymatic routes (1). Two enzymes are able to use pyridoxal phosphate-dependent metabolism of L-cysteine, CBS (cystathionine β-synthase) and CSE (cystathionine γ-lyase). The third pathway involves cysteine aminotransferase to generate 3-mercaptopyruvate, which is utilised by MPST (3-mercaptopyruvate sulphurtransferase), a pyridoxal phosphate-independent enzyme. Hydrogen sulphide has complex effects on neuronal survival, with both neurotoxic and neuroprotective effects reported. We have used the SH-SY5Y human neuroblastoma cell line to explore the effects of hydrogen sulphide on neuronal viability in vitro.

Differentiation of SH-SY5Y cells was conducted over six days in the presence of 10 µM retinoic acid, using the MTT tetrazolium assay as an index of cell numbers. Sodium sulphide at concentrations of 10-9-10-5 M was used as a source of exogenous hydrogen sulphide, while endogenous production of H2S was assessed using the methylene blue method (2). Immunoblotting was used to detect expression of endogenous H2S-producing enzymes. In some experiments, medium deficient in L-cysteine, L-glutamine and pyruvate (DMEM/CGP-) was used to minimise production of endogenous H2S in culture. Three or more passages of cells were assessed for each condition, assessing statistical significance using ANOVA with post-hoc Bonferroni analysis.

Na2S increased numbers of differentiated SH-SY5Y cells at 24 h exposure in serum-free conditions (10-9 M 155 ± 8; 10-8 M 154 ± 8; 10-7 M 158 ± 9; 10-6 M 166 ± 9; 10-5 M 171 ± 9 % control; all P<0.001) or in the presence of 5 % serum (10-9 M 129 ± 6; 10-8 M 134 ± 6; 10-7 M 130 ± 6; 10-6 M 138 ± 5; 10-5 M 147 ± 7 % control; all P<0.01). At 48 h exposure in serum-free conditions, only higher concentrations of Na2S evoked significant effects on cell numbers (10-6 M 139 ± 12, P<0.05; 10-5 M 159 ± 15 % control, P<0.001). Similarly, only the highest concentration of Na2S evoked a significant effect in 5 % serum at 48 h (10-5 M 139 ± 11 % control, P<0.05).

Cell numbers were reduced at both 24 and 48 h using DMEM/CGP- compared to complete DMEM in either the absence (74 ± 1 and 67 ± 2 %, P<0.001) or presence (86 ± 1 and 82 ± 2 % control, P<0.001) of 5 % serum. Replacing L-cysteine in the medium had no effect at 24 h in serum-free conditions (74 ± 1 %), but increased cell numbers under all other conditions (serum-free 48 h 77 ± 2 %; 5 % serum 24 h 97 ± 1; 48 h 100 ± 2 % control, all P<0.001).

Although a band was identified at 45 kDa with an anti-CSE antibody in rat liver, the antibody failed to identify immunoreactivity in SH-SY5Y cells. CBS immunoreactivity in SH-SY5Y cells was detected at 60 kDa, but levels decreased following differentiation. A doublet of immunoreactivity at 33 & 36 kDa was identified using an antibody directed against MPST, which was also reduced following differentiation. Using L-cysteine as substrate, H2S production was inhibited in the absence of pyridoxal phosphate or in the presence of 100 µM AOAA or PPG. Using 3-mercaptopyruvate as substrate allowed detection of H2S production, but at much lower activities than rat liver.

We conclude that serum appears to reduce the stimulatory effects of Na2S on cell numbers, which are particularly evident at 24 h compared to 48 h exposure. These data indicate the utility of SH-SY5Y cells as models for the investigation of the effects of exogenous and endogenous H2S.

1. Papapetropoulos A et al. (2015). Br J Pharmacol in press PM:24909294

2. Rashid S et al. (2013). Br J Pharmacol 168: 1902-1910. PM:23215842