Additive cell protective and oxidative stress reducing effects of combined treatment with cromolyn sodium and masitinib on MPTP-induced toxicity in SH-SY5Y neuroblastoma cells

Abstract

The suppression of oxidative-stress induced neurotoxicity by antioxidants serves as a potential preventive strategy for neurodegenerative diseases. In this study, we aimed to investigate the cell protective and antioxidant effects of masitinib and cromolyn sodium against toxin-induced neurodegeneration. First, human neuroblastoma SH-SY5Y cells were differentiated into neuron-like (d)-SH-SY5Y cells. The differentiated cells were confirmed by immuno-staining with anti-PGP9.5 antibody, a neuronal marker. Cell culture groups were formed, and a neurotoxin, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) was applied on cells followed by masitinib and/or cromolyn sodium treatments. Survival rate of cells were detected by MTT assay. Anti-inflammatory Transforming Growth Factor-β1 (TGF-β1) and nitric oxide (NO) levels and total oxidant and antioxidant capacities (TOC and TAC) in cell conditioned media (CM) were measured. Morphological analysis and apoptotic nuclear assessment of cells were also noted. When (d)-SH-SY5Y cells were exposed to neurotoxin, cell viability rates of these cells were found to be decreased in a concentration-dependent manner. CM of toxin applied group displayed higher levels of TOC/TAC ratios and NO levels compared to control (p < 0.01). Both masitinib and cromolyn sodium protected cells from toxin-induced cell death as revealed by ameliorated rates of viability, reversed toxin-induced elevation of TOC/ TAC ratios, and decreased NO levels in their CM (p < 0.01). Combined treatment significantly reduced TOC/TAC ratios and NO levels more effectively compared to mono-treatments. Both drugs also increased TGF-β1 levels significantly in cell CM. When these agents were tested for therapeutic effects against toxin-induced cell degeneration, better viability results were obtained by both masitinib and cromolyn sodium treatment, with significantly better amelioration provided by combined application of these drugs (p < 0.01). This study demonstrated new findings that combined treatment with cromolyn sodium, an FDA-approved drug of asthma, and masitinib, an orally administered drug with a low toxicity, exert neuroprotective and additive therapeutic effects. We propose that combination therapy of masitinib and cromolyn sodium may represent an innovative treatment in neurodegenerative diseases. Combination therapy may be more advantageous that it enables combined application of lower doses of both drugs, providing less side effects.