Abstract
Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.
Highlights
Neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, are characterized by progressive synaptic and axonal degeneration that ends in neuronal death in specific regions of the brain
When applied together with 0.5 mM copper, myricetin at 5 and 10 μg/mL exacerbated the toxic effects of copper and further reduced viability of SH-SY5Y cells
Similar to the control group, SH-SY5Y cells treated with 10 μg/mL myricetin showed an epithelial-like flat phenotype
Summary
Neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, are characterized by progressive synaptic and axonal degeneration that ends in neuronal death in specific regions of the brain. Oxidative stress (OS) is one of the major pathological mechanisms contributing to neuronal damage and loss of function in these devastating diseases. Free radicals, which include a broad-spectrum of reactive oxygen and nitrogen species (ROS and RNS, respectively), are highly reactive moieties with an unpaired electron that are capable of inducing oxidative damage of cellular macromolecules and other components, resulting in their functional impairment [1,2]. Copper ions initiate redox cycling reactions and ROS formation, creating an oxidative environment that results in neuronal injury [7,8]. When the intracellular mechanisms involved in copper coordination are saturated, free copper ions may exert deleterious effects through direct and nonspecific binding to sulfhydryl groups (thiols) and amino groups of various cellular proteins, disturbing their normal cellular functions. It is known that increased copper may compromise the proteolytic activity of the ubiquitin-proteasome system, further promoting neuronal dysfunction in neurodegeneration [13]
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