The modulating effects of selected antidepressants and related drugs on markers of cellular resilience in cultured human neuroblastoma cells

Abstract

The neurodegenerative hypothesis of depression postulates that major depression is associated with impaired neuroplasticity, particularly in the hippocampus and prefrontal cortex, and that effectiveness of antidepressants may be partially attributed to the extent to which they exert neuroprotective properties. Therefore the current study investigated the effect of selected antidepressant drugs on the survival of cultured human neuroblastoma (SH-SY5Y) cells, before and after glutamate-induced excitotoxicity, as well as the effects of these drugs on pro- and anti-apoptotic pathways. The free fraction of plasma protein-bound drugs was determined in Ham's F12 culture medium + 10% foetal bovine serum by means of HPLC analysis. Cultured human SH-SY5Y cells were treated for 24 hours with and without 10 mM glutamate plus a low (pharmacological) or high free concentration of fluoxetine, mirtazapine, tianeptine, imipramine, myo-inositol, gabapentin or lithium. Thereafter mitochondrial activity was determined with the MTT cell viability assay. The effects of the drug treatments on genes encoding for pro-apoptotic factors Bcl2-associated X protein (Bax), caspase-3 and caspase-8, and anti-apoptotic factors brain derived neurotrophic factor (BDNF), nuclear factor kappa beta (NF-Kp), protein kinase B (Akt), cyclic adenosine monophosphate response element binding protein (CREB) and define B-cell CLL/lymphoma 2 (Bcl-2), was determined utilising quantitative real-time reverse transcriptase polymerase chain reaction (rt$^2$-PCR). Results from HPLC analyses indicate that fluoxetine, mirtazapine, tianeptine and imipramine are moderately bound to plasma albumin in culture medium and subsequently the free drug concentrations were determined. The drug treatments using pharmacological concentrations of lithium, myo-inositol, imipramine and gabapentin, but not of fluoxetine, mirtazapine or tianeptine, protected against glutamate induced excitotoxicity, as measured with the standard MTT assay, while no drug-induced changes in cell viability were observed in the absence of glutamate. The MTT assay results also indicated that the high concentration of lithium and gabapentin significantly increase the viability of the SH-SY5Y cells following glutamate cytotoxicity, while the high concentrations of other drugs failed to alter cell viability. The MTT data were supported by the results from rt 2-PCR, indicating that the low (pharmacological) concentrations of fluoxetine, imipramine, tianeptine and myo-inositol significantly increase anti-apoptotic gene expression in human neuroblastoma cells after glutamate induced excitotoxicity, while pro-apoptotic gene expression is significantly decreased with gabapentin, imipramine, lithium, mirtazapine, myo-inositol and tianeptine. In conclusion, at pharmacological concentrations all antidepressants displayed neuroprotective effects in the presence of excitotoxicity, but via different mechanisms.