RADICAL INDUCED LIPID ISOMERIZATION

POSTIGO, AL

BOLOGNA, ITALIA

Workshop; COST ACTION CM0603 FREE RADICALS IN CHEMICAL BIOLOGY; 2008

ISOF-CNR. ISTITUTO PER LA SINTESI ORGANICA E LA FOTOREATTIVITA-CONSIGLIO NAZIONALE DELLE RICERCHE

The double bond geometry of most of the naturally occurring unsaturated fatty acid residues is cis. Due to the relevance of fatty acids as structural components of cell membranes and as biologically active molecules, the change of the cis geometry means a change of the associated functions and activities. The finding that the cis to trans isomerization is effective in phospholipids by the intervention of radical species led to the discovery that there can indeed occur an endogenous formation of trans fatty acids, whose significance in biological systems started to be addressed with in vitro and in vivo studies. Studies of liposome models simulating the formation of isomerizing species and evaluating their ability to interact with the hydrophobic part of the membrane bilayer has contributed to the gain in knowledge of the fundamental features of the lipid isomerization in membranes. Further work is in progress for the identification of the real culprits of the in vivo lipid isomerization, and recent results are shown on oleic acid micelles, where *NO2 radicals are not able to induce double bond isomerization in comparison with amphiphilic thiol, such as 2-mercaptoethanol. H2S and sulfur-containing amino acid residues are two of the possible species involved in this process at a biological level. An update of the scenario of the geometrical isomerization in membranes by free radicals is provided, together with applications and perspectives in life sciences. <!--[if !supportEmptyParas]--> <!--[endif]--> Offspring of long-lived individuals are a useful model to discover biomarkers of longevity. The lipid composition of erythrocyte membranes from 41 nonagenarian offspring was compared with 30 matched controls. Genetic loci were also tested in 280 centenarians and 280 controls to verify a potential genetic predisposition in determining unique lipid profile. Gas chromatography was employed to determine fatty acid composition, and genotyping was performed using Taqman assays. Outcomes were measured for erythrocyte membrane percentage content of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids (omega-6 and omega-3), geometrical isomers of arachidonic and oleic acids, and total trans-fatty acids. Also, allele and genotyping frequencies at endothelial-nitric oxide synthase and delta-5/delta-6 and delta-9 desaturase loci were considered. Erythrocyte membranes from nonagenarian offspring had significantly higher content of C16:1 n-7, trans C18:1 n-9, and total trans-fatty acids, and reduced content of C18:2 n-6 and C20:4 n-6. No association was detected at endothelial-nitric oxide synthase and delta-5/delta-6 and delta-9 desaturase loci that could justify genetic predisposition for the increased trans C18:1 n-9, monounsaturated fatty acids and decreased omega-6 synthesis. We concluded that erythrocyte membranes derived from nonagenarian offspring have a different lipid composition (reduced lipid peroxidation and increased membrane integrity) to that of the general population. <!--[if !supportEmptyParas]--> <!--[endif]--> Nowadays, no data are available concerning the potential use of dual COX/5-LOX inhibitors as anticancer agents in colon cancer treatment. Here we report, for the first time, that the dual COX/5-LOX inhibitor licofelone triggers apoptosis in a dose- and time-dependent manner in HCA-7 colon cancer cells. Induction of apoptosis was related to the recruitment of the intrinsic mitochondrial apoptotic pathway, as shown by loss in mitochondrial membrane potential, cytochrome c release, caspase-9 and 3 activation and poly-(ADP-ribose)polymerase-1 cleavage. Moreover, licofelone induced the cleavage of the full length p21(Bax) into p18(Bax), a more potent inducer of the apoptotic process than the uncleaved form. Pre-treatment of HCA-7 cells with the pan-caspase inhibitor z-VAD-fmk significantly blocked licofelone-induced apoptosis, confirming that this process occurred primarily in a caspase-dependent pathway. We also present evidences that licofelone was able to affect the arachidonic acid cascade, as it blocked the activity of 5-LOX and COXs enzymes, and it induced, through the phosphorylation of cytoplasmic phospholipase A(2), the release of unesterified arachidonic acid from HCA-7 membrane phospholipids. However, apoptosis induction was not related to the ability of licofelone to affect the arachidonic acid cascade, since neither exogenous PGE(2) and LTB(4) addition, nor pharmacological inhibition of cytoplasmic phospholipase A(2), were able to rescue HCA-7 cells from apoptosis. Even if further studies are needed to clarify the mechanism of licofelone-induced apoptosis, this study suggest that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer treatment.