Oleuropein shows copper complexing properties and noxious effect on cultured SH-SY5Y neuroblastoma cells depending on cell copper content.

The secoiridoid oleuropein is a non-flavonoid polyphenol, found in the fruit, leaves and food derivatives from Olea europea. Like other polyphenols it shows a very low toxicity towards healthy tissues and a protective action against cancer or neurodegeneration, but its mechanism of action is not yet understood. In the present report we have used optical and ESR spectroscopy as well as molecular modelling to demonstrate that oleuropein forms a complex with the transition metal copper; the dysmetabolism of this metal is suspected to be involved in both cancer and neurodegeneration. Experiments carried out with the aglycon derivative of oleuropein, produced by ß-glycosidase treatment of oleuropein glycoside, showed that also the aglycon forms copper-complexes, but with different spectroscopic features than the glycosidic form. Molecular modelling analysis confirmed that two oleuropein molecules (glycosidic or aglycon forms) can easily coordinate one copper ion. The relationship between oleuropein and copper was investigated in SH-SY5Y human neuroblastoma cells. When cells were depleted of copper by treatment with the copper chelator triethylenetetramine (Trien), that binds copper with higher affinity than oleuropein, oleuropein was less toxic than to copper-adequate cells. Conversely, incubation of SH-SY5Y cells with exogenous copper sulphate increased cell susceptibility to oleuropein. Furthermore SH-SY5Y cells differentiated by retinoic acid pre-treatment showed a lower level of copper, and were more resistant to oleuropein treatment. The oleuropein aglycon was not toxic towards SH-SY5Y cells. In conclusion, the copper-oleuropein complex may be involved in the toxicity of oleuropein towards tumour cells, depending on their copper level.

Features of ceruloplasmin in the cerebrospinal fluid of Alzheimer's disease patients.

The level of the apo-form of the copper enzyme ceruloplasmin (CP) is an established peripheral marker in diseases associated with copper imbalance. In view of the proposal that disturbances of copper homeostasis may contribute to neurodegeneration associated with Alzheimer's disease (AD), the present work investigates, by Western blot and non-reducing SDS-PAGE followed by activity staining, the features of CP protein, and the copper/CP relationship in cerebrospinal fluid (CSF) and serum of AD patients. Results show that only a fraction of total copper is associated with CP in the CSF, at variance with serum, both in affected and in healthy individuals. Furthermore, a conspicuous amount of apo-ceruloplasmin and a decrease of CP oxidase activity characterize the CSF of the affected individuals, and confirm that an impairment of copper metabolism occurs in their central nervous system. In the CSF of AD patients the decrease of active CP, associated with the increase in the pool of copper not sequestered by this protein, may play a role in the neurodegenerative process.

Ceruloplasmin (2-D PAGE) Pattern and Copper Content in Serum and Brain of Alzheimer Disease Patients.

A dysfunction in copper homeostasis seems to occur in Alzheimer's disease (AD). We previously evidenced that an excess of non-ceruloplasmin-copper (NCC) correlated with the main functional, anatomical as well as cerebrospinal markers of the disease. Aim of our study was to investigate ceruloplasmin isoforms as potential actors in this AD copper dysfunction. Our data show that AD patients have ceruloplasmin fragments of low molecular weight (<50 kDa) both in their serum and brain, contrary to healthy controls. Ceruloplasmin isoforms of higher molecular weight (115 and 135 kDa in serum and 135 kDa in brain), as well as copper levels in the brain, instead, do not seem to mark a difference between AD and healthy subjects. These data suggest a ceruloplasmin fragmentation in the serum of AD patients. Some clues in this direction have been found also in the AD brain.