Selective Cytotoxic Activity of New Lipophilic Hydroxytyrosol Alkyl Ether Derivatives Jose ́ Manuel Calderó n-Montañ o, † Andre ́ s Madrona, ‡ Estefanía Burgos-Moró n, † Manuel Luis Orta, § Santiago Mateos, § Jose ́ Luis Espartero, ‡ and Miguel Ló pez-La ́ zaro* ,† † Department of Pharmacology, Faculty of Pharmacy, ‡ Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, and § Department of Cell Biology, Faculty of Biology, University of Seville, Seville 41012, Spain ABSTRACT: Recent data suggest that hydroxytyrosol, a phenolic compound of virgin olive oils, has anticancer activity. This communication reports the synthesis of decyl and hexadecyl hydroxytyrosyl ethers, as well as the cytotoxic activity of hydroxytyrosol and a series of seven hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 non- malignant lung fibroblasts. Hydroxytyrosyl dodecyl ether (HTDE) showed the highest selective cytotoxicity, and possible mechanisms of action were investigated; results suggest that HTDE can moderately inhibit glycolysis, induce oxidative stress, and cause DNA damage in A549 cells. The combination of HTDE with the anticancer drug 5-fluorouracil induced a synergistic cytotoxicity in A549 cancer cells but not in non-malignant MRC5 cells. HTDE also displayed selective cytotoxicity against MCF7 breast cancer cells versus MCF10 normal breast epithelial cells in the 1-30 μM range. These results suggest that the cytotoxicity of HTDE is more potent and selective than that of parent compound hydroxytyrosol. KEYWORDS: olive oil, lipophilic derivatives, phenolic compounds, cancer, anticancer, cytotoxic ■ INTRODUCTION Epidemiological studies suggest that a Mediterranean diet may reduce the risk of developing degenerative pathologies such as cardiovascular diseases and cancer. 1-4 Since olive oil is the main source of fat in the Mediterranean diet, the number of reports describing the beneficial properties of olive oil has notably increased in recent years. This olive oil popularity has mainly been attributed to its high content of monounsaturated fatty acids and to its richness in phenolic compounds. 3-9 The presence of phenolic compounds in olive oil has attracted much attention due to their known biological activities and health effects. Particular attention has been placed on 3,4-dihydroxy- phenylethanol (hydroxytyrosol), an o-diphenolic compound that is present in virgin olive oil as secoiridoid derivatives or acetate esters and has shown a variety of pharmacological activities. 10-15 Accumulating preclinical evidence suggests that hydroxytyrosol has cancer chemopreventive and chemother- apeutic potential. In vitro experiments have shown that hydroxytyrosol inhibits proliferation and induces apoptosis in a variety of cancer cell lines from different origin, including breast, colon, and leukemia cell lines. 16-20 Several mechanisms of action have been shown to participate in its cytotoxic activity, such as cell cycle arrest; cytochrome c release; activation of caspase 3, c-jun, or bcl-2; and inhibition of CDK6, HER2, fatty acid synthase, or PI3K/Akt/NF-kappa B pathway (reviewed in ref 11). Recent animal experiments have revealed that hydroxytyrosol can also inhibit cancer cell growth in vivo. 21 Our previous work reported the syntheses of lipophilic hydroxytyrosol derivatives in the form of esters 22 and ethers 23 and the evaluation of their antioxidant activity in relation to that of hydroxytyrosol. 24-26 These studies revealed that the introduction of an acyl or alkyl side chain in the hydroxytyrosol structure did not reduce, or even improved, the antioxidant capacity of hydroxytyrosol. We have studied the absorption, metabolism, and digestive stability of these compounds 27-29 and have also observed that some of these hydroxytyrosol derivatives exhibit antiplatelet, anti-inflammatory, and neuroprotective effects. 30,31 In this article we report the synthesis of two new alkyl deri- vatives of hydroxtyrosol, as well as the evaluation of the cyto- toxic activity on cancer and non-malignant cells of hydroxy- tyrosol and a series of its ether derivatives (ethyl, butyl, hexyl, octyl, decyl, dodecyl, and hexadecyl ethers). Interestingly, hydroxytyrosol isolated from olive oil waste waters (OOWW) has been used for the preparation of these compounds to give an added value to this type of residue. ■ MATERIALS AND METHODS Chemicals. Hydroxytyrosol was obtained from olive oil waste waters as described elsewhere. 23 Hydroxytyrosol alkyl ether derivatives were prepared from hydroxytyrosol as described below. Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) was purchased from Biomol International. All other compounds used in this work were obtained from Sigma. All compounds were dissolved in DMSO and added to the cells after appropriate dilutions in cell culture medium. Final DMSO concentrations were never higher than 0.3%. Cell Lines. The human A549 lung cancer cell line, the human embryo lung fibroblastic MRC5 cell line, and the human MCF7 breast adenocarcinoma cell line were maintained in DMEM supplemented with 2 mM glutamine, 50 μg/mL penicillin, 50 μg/mL streptomycin, and 10% fetal bovine serum. The human MCF10 breast epithelial cell line (kindly provided by Dr. D. Ruano and Dr. P. Daza) was maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium supplemented with 20 ng/mL epidermal growth factor, 100 ng/mL cholera toxin, 10 μg/mL insulin, and 500 ng/mL hydrocortisone (95%)/horse serum (5%). To study the Received: October 1, 2012 Revised: May 2, 2013 Accepted: May 2, 2013 Published: May 2, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 5046 dx.doi.org/10.1021/jf400796p | J. Agric. Food Chem. 2013, 61, 5046-5053