Meccanismi coinvolti nell'effetto prp-apoptotico dell'acido docosaesaenoico (DHA) nelle linea cellulare di adenocarcinoma pancreatico umano PaCa-44
Author(s)
Loppi, Barbara
Date Issued
January 23, 2006
Type
Doctoral Thesis
Abstract
Polyunsaturated fatty acids have been indicated to induce anti-proliferative and/or apoptotic effects in various tumor cells, although the mechanisms is unknown. In order to investigate the ability of fatty acids to induce growth inhibition and apoptosis in the human PaCa-44 pancreatic cancer cell line, alpha-linoleic acid (18:2, n-6; LA), butyric acid, oleic acid and docosahexaenoic acid (DHA) were supplemented at 200 µM concentration in the medium of cell cultures. Ours results showed that all fatty acids inhibited cell growth, while only DHA induced apoptosis in PaCa-44 cell line.
Investigating the mechanism underlying DHA induced apoptosis, an oxidative process
was implicated, considering that butylated hydroxytoluene (BHT), ascorbic acid (Vitamin C) and Vitamin E prevent lipid peroxidation and reversed apoptosis.
Furthermore we showed that DHA induced intracellular depletion of reduced glutathione (GSH), without affecting oxidized glutathione (GSSG). Intracellular GSH depletion is reversed by metionine and cystathionine, two specificic inhibitors of carrier-mediated GSH extrusion, inhibition of GSH efflux completely reversed apoptosis and lipid peroxidation.
Depletion of intracellular GSH level sensitizes cells to oxidative damage, decreasing activity of selenium-dependent glutathione peroxidase (GSH-Px) enzyme. Pretreatment with sodium selenate was accompanied by an increased level of GSH-Px activity, decreased of lipid peroxidation, GSH depletion and completely reversed apoptosis. These results provide the first evidence for a possible causative role of
oxidative stress in DHA-induced apoptosis.
Furthermore we investigated the implication of genetic mechanisms in apoptosis DHA-induced through activation of nuclear receptors Peroxisome Proliferator –Activated Receptors (PPARs). Treatments with Vitamin A (all-trans Retinoic Acid, at-RA) and Badge (Bhisphenol A Diglycidyl Ether), inhibitor of PPAR-γ, indicate that PPARs may be involved in DHA-induced apoptosis. The regulation of apoptosis
through PPARs may be connected to modulation of lipoxygenases enzymes (LOXs).
Indeed, PCR analysis demonstrated that treatment with DHA induced increased and/or decreased level of LOXs mRNA. These results document the antiproliferative and pro-apoptotic activities of DHA and suggest the interplay of two mechanisms, oxidative and genetic regulation, in DHA-induced apoptosis.
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