Selective cytotoxic activity of new lipophilic hydroxytyrosol alkyl ether derivatives

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ "This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/jf400796p ." Selective cytotoxic activity of new lipophilic 1 hydroxytyrosol alkyl ether derivatives 2 3 4 José Manuel Calderón-Montaño † , Andrés Madrona ‡ , Estefanía Burgos-Morón † , Manuel Luis 5 Orta § , Santiago Mateos § , José Luis Espartero ‡ and Miguel López-Lázaro †* 6 7 † Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain 8 ‡ Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of 9 Seville, Spain 10 § Department of Cell Biology, Faculty of Biology, University of Seville, Spain 11 12 13 14 *Corresponding Author (Tel: + 34 954 55 63 48; Fax: + 34 954 55 60 74; E-mail: 15 [email protected]) 16 2 Abstract 17 18 Recent data suggest that hydroxytyrosol, a phenolic compound of virgin olive oils, has 19 anticancer activity. This communication reports the synthesis of decyl and hexadecyl 20 hydroxytyrosyl ethers, as well as the cytotoxic activity of hydroxytyrosol and a series of 21 seven hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 non-22 malignant lung fibroblasts. Hydroxytyrosyl dodecyl ether (HTDE) showed the highest 23 selective cytotoxicity and possible mechanisms of action were investigated; results suggest 24 that HTDE can moderately inhibit glycolysis, induce oxidative stress and cause DNA damage 25 in A549 cells. The combination of HTDE with the anticancer drug 5-flurouracil induced a 26 synergistic cytotoxicity in A549 cancer cells but not in non-malignant MRC5 cells. HTDE 27 also displayed selective cytotoxicity against MCF7 breast cancer cells vs MCF10 normal 28 breast epithelial cells in the 1-30 micromolar range. These results suggest that the cytotoxicity 29 of HTDE is more potent and selective than that of parent compound hydroxytyrosol. 30 31 Keywords: olive oil, lipophilic derivatives, phenolic compounds, cancer, anticancer, 32 cytotoxic 33 3 INTRODUCTION 34 Epidemiological studies suggest that a Mediterranean diet may reduce the risk of 35 developing degenerative pathologies such as cardiovascular diseases and cancer. 1-4 Since 36 olive oil is the main source of fat in the Mediterranean diet, the number of reports describing 37 the beneficial properties of olive oil has notably increased in recent years. This olive oil 38 popularity has mainly been attributed to its high content on monounsaturated fatty acids and 39 to its richness in phenolic compounds. 3-9 40 The presence of phenolic compounds in olive oil has attracted much attention due to 41 their known biological activities and health effects. Particular attention has been placed on 3,4 42 dihydroxyphenylethanol (hydroxytyrosol), an o-diphenolic compound that is present in virgin 43 olive oil as secoiridoid derivatives or acetate esters and has shown a variety of 44 pharmacological activities. 10-15 Accumulating preclinical evidence suggests that 45 hydroxytyrosol has cancer chemopreventive and chemotherapeutic potential. In vitro 46 experiments have shown that hydroxytyrosol inhibits proliferation and induces apoptosis in a 47 variety of cancer cell lines from different origin, including breast, colon and leukemia cell 48 lines. 16-20 Several mechanisms of action have been shown to participate in its cytotoxic 49 activity, such as cell cycle arrest, cytochrome c release, activation of caspase 3, c-jun or bcl-2, 50 and inhibition of CDK6, HER2, fatty acid synthase, or PI3K/Akt/NF-kappa B pathway 51 (reviewed in reference 11). Recent animal experiments have revealed that hydroxytyrosol can 52 also inhibit cancer cell growth in vivo. 21 53 Our previous work reported the syntheses of lipophilic hydroxytyrosol derivatives in 54 the form of esters 22 and ethers 23 , and the evaluation of their antioxidant activity in relation to 55 that of hydroxytyrosol. 24-26 These studies revealed that the introduction of an acyl or alkyl 56 side chain in the hydroxytyrosol structure did not reduce, or even improved, the antioxidant 57 4 capacity of hydroxytyrosol. We have studied the absorption, metabolism and digestive 58 stability of these compounds 27-29 and have also observed that some of these hydroxytyrosol 59 derivatives exhibit antiplatelet, anti-inflammatory and neuroprotective effects. 30,31 In this 60 article we report the synthesis of two new alkyl derivatives of hydroxtyrosol, as well as the 61 evaluation of the cytotoxic activity on cancer and non-malignant cells of hydroxytyrosol and a 62 series of its ether derivatives (ethyl, butyl, hexyl, octyl, decyl, dodecyl and hexadecyl ethers). 63 Interestingly, hydroxytyrosol isolated from olive oil waste waters (OOWW) has been used for 64 the preparation of these compounds to give an added value to this type of residue. 65 66 MATERIAL AND METHODS 67 68 Chemicals 69 Hydroxytyrosol was obtained from olive oil waste waters as described elsewhere. 23 70 Hydroxytyrosol alkyl ether derivatives were prepared from hydroxytyrosol as described 71 below. Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) was 72 purchased from Biomol International. All other compounds used in this work were obtained 73 from Sigma. All compounds were dissolved in DMSO and added to the cells after appropriate 74 dilutions in cell culture medium. Final DMSO concentrations were never higher than 0.3%. 75 76 Cell Lines 77 The human A549 lung cancer cell line, the human embryo lung fibroblastic MRC5 cell 78 line and the human MCF7 breast adenocarcinoma cell line were maintained in DMEM 79 5 supplemented with 2 mM glutamine, 50 µg/mL penicillin, 50 µg/mL streptomycin and 10% 80 fetal bovine serum. The human MCF10 breast epithelial cell line (kindly provided by Dr. D. 81 Ruano and Dr. P. Daza) was maintained in a 1:1 mixture of Dulbecco's modified Eagle's 82 medium and Ham's F12 medium supplemented with 20 ng/mL epidermal growth factor, 100 83 ng/mL cholera toxin, 10 µg/mL insulin and 500 ng/mL of hydrocortisone (95%) and horse 84 serum (5%). To study the possible DNA damage response induced by the tested compounds, 85 the following parental and DNA repair-deficient cell lines were used: AA8: parental Chinese 86 hamster ovary cells; V3-3: AA8 cells mutated in XRCC7 (DNA-PK), non-homologous end 87 joining (NHEJ) deficient; KO40: AA8 cells mutated in FANCG, Fanconi anemia (FA) 88 deficient; VC8: V79 Chinese hamster lung cells mutated in BRCA2, homologous 89 recombination (HR) deficient; VC8B2: VC8 cells complemented with human BRCA2 (HR 90 proficient); EM9-V: AA8 cells mutated in XRCC1 (DNA ligase III), base excision repair 91 (BER) deficient; EM9-XH: EM9 cells complemented with XRCC1 (BER proficient), HCT-92 116: human colon cancer cells mutated in MLH1, mismatch repair (MMR) deficient; and 93 HCT-116 + c3: HCT-116 cells complemented with chromosome 3 (with MLH1 gene; MMR 94 proficient). 32,33 These DNA repair-deficient cell lines, kindly provided by Dr. Thomas 95 Helleday, were cultured in DMEM. All cell lines were cultured at 37 °C in a humidified 96 atmosphere containing 5% CO 2 . Cell culture reagents were obtained from Life Technologies. 97 98 Preparation of ether derivatives of hydroxytyrosol 99 Hydroxytyrosyl alkyl ethers (10–16) were prepared from hydroxytyrosol (1) using a 100 method previously described by us. 23 The following compounds have been synthesized for the 101 first time: 102 6 1,2-bis(benzyloxy)-4-(2’-decyloxyethyl)benzene (7): white solid (86% yield). 1 H-NMR 103 (500 MHz, DMSO-d 6 ) δ ppm 7.37 (m, 10H, 2xPh), 6.96 (d, J=2.0 Hz, 1H, H 4 ), 6.92 (d, J=8.2 104 Hz, 1H, H 7 ), 6.71 (dd, 1H, H 8 ), 5.07 (s, 2H, CH 2 Ph in pos. 5), 5.06 (s, 2H, CH 2 Ph in pos. 6), 105 3.48 (t, J=6.9 Hz, 2H, H 1 ), 3.32 (t, J=6.5 Hz, 2H, H 1’ ), 2.68 (t, 2H, H 2 ), 1.43 (m, 2H, H 2’ ), 106 1.23 (m, 14H, H 3’ −H 9’ ), 0.83 (t, 3H, H 10’ ). 13 C-NMR (125 MHz, DMSO-d 6 ) δ ppm 148.2 (C 5 ), 107 146.6 (C 6 ), 137.4 y 137.3 (C ipso , Bn groups), 132.2 (C 3 ), 128.2−127.4 (C 3’’ , C 4’’ y C 5’’ , groups 108 Bn), 121.2 (C 8 ), 115.4 (C 4 ), 114.6 (C 7 ), 70.9 (C 1 ), 70.2 (CH 2 Ph in pos. 6), 70.1 (CH 2 Ph in pos. 109 5), 69.8 (C 1’ ), 35.0 (C 2 ), 29.1 (C 2’ ), 29.0−28.6 (C 4’ −C 7’ ), 25.6 (C 3’ ), 31.2 (C 8’ ), 22.0 (C 9’ ), 13.8 110 (C 10’ ). Elem. Anal. Calcd. for C 32 H 42 O 3 : C, 80.97; H, 8.92. Found: C, 88.85; H, 8.78. 111 1,2-bis(benzyloxy)-4-(2’-hexadecyloxyethyl)benzene (9): white solid (86% yield). 1 H-112 NMR (500 MHz, DMSO-d 6 ) δ ppm 7.37 (m, 10H, 2xPh), 6.96 (d, J=2.0 Hz, 1H, H 4 ), 6.93 (d, 113 J=8.2 Hz, 1H, H 7 ), 6.72 (dd, 1H, H 8 ), 5.08 (s, 2H, CH 2 Ph in pos. 5), 5.07 (s, 2H, CH 2 Ph in 114 pos. 6), 3.49 (t, J=7.0 Hz, 2H, H 1 ), 3.33 (t, J=6.5 Hz, 2H, H 1’ ), 2.69 (t, 2H, H 2 ), 1.45 (m, 2H, 115 H 2’ ), 1.23 (m, 10H, H 3’ −H 15’ ), 0.84 (t, 3H, H 16’ ). 13 C-NMR (125 MHz, DMSO-d 6 ) δ ppm 116 148.1 (C 5 ), 146.6 (C 6 ), 137.4 y 137.3 (C ipso , Bn groups), 132.2 (C 3 ), 128.2−127.4 (C 3’’ , C 4’’ y 117 C 5’’ , Bn groups), 121.3 (C 8 ), 115.5 (C 4 ), 114.7 (C 7 ), 70.9 (C 1 ), 70.2 (CH 2 Ph in pos. 6), 70.1 118 (CH 2 Ph in pos. 5), 69.8 (C 1’ ), 35.0 (C 2 ), 31.2 (C 14’ ), 29.1 (C 2’ ), 29.0−28.6 (C 4’ −C 13’ ), 25.6 119 (C 3’ ), 22.0 (C 15’ ), 13.8 (C 16’ ). Elem. Anal. Calcd. for C 38 H 54 O 3 : C, 81.67; H, 9.74. Found: C, 120 81.27; H, 9.35. 121 4-(2’-decyloxyethyl)benzene-1,2-diol (14): colourless oil (83% yield). 1 H-NMR (500 122 MHz, DMSO-d 6 ) δ ppm 8.63 (s, 1H, OH in pos. 6), 8.56 (s, 1H, OH in pos. 5), 6.59 (d, J=8.0 123 Hz, 1H, H 7 ), 6.57 (d, J=2.1 Hz, 1H, H 4 ), 6.43 (dd, 1H, H 8 ), 3.44 (t, J=7.2 Hz, 2H, H 1 ), 3.33 (t, 124 J=6.6 Hz, 2H, H 1’ ), 2.58 (t, 2H, H 2 ), 1.41 (m, 2H, H 2’ ), 1.24 (m, 10H, H 3’ −H 9’ ), 0.84 (t, 3H, 125 H 10’ ). 13 C-NMR (125 MHz, DMSO-d 6 ) δ ppm 144.8 (C 5 ), 143.3 (C 6 ), 129.7 (C 3 ), 119.3 (C 8 ), 126 116.1 (C 4 ), 115.3 (C 7 ), 71.4 (C 1 ), 69.9 (C 1’ ), 34.9 (C 2 ), 29.1 (C 2’ ), 29.0−28.6 (C 4’ −C 7’ ), 25.6 127 7 (C 3’ ), 31.2 (C 8’ ), 22.0 (C 9’ ), 13.8 (C 10’ ). Elem. Anal. Calcd. for C 18 H 30 O 3 : C, 73.43; H, 10.27. 128 Found: C, 73.41; H, 10.39. 129 4-(2’-hexadecyloxyethyl)benzene-1,2-diol (16): white solid (83% yield). 1 H-NMR (500 130 MHz, DMSO-d 6 ) δ ppm 8.59 (s, 2H, OH in pos. 5 and 6), 6.60 (d, J=8.0 Hz, 1H, H 7 ), 6.58 (d, 131 J=2.1 Hz, 1H, H 4 ), 6.43 (dd, 1H, H 8 ), 3.44 (t, J=7.2 Hz, 2H, H 1 ), 3.33 (t, J=6.6 Hz, 2H, H 1’ ), 132 2.59 (t, 2H, H 2 ), 1.45 (m, 2H, H 2’ ), 1.23 (m, 10H, H 3’ −H 15’ ), 0.85 (t, 3H, H 16’ ). 13 C-NMR (125 133 MHz, DMSO-d 6 ) δ ppm 144.8 (C 5 ), 143.3 (C 6 ), 129.7 (C 3 ), 119.3 (C 8 ), 116.1 (C 4 ), 115.3 (C 7 ), 134 71.4 (C 1 ), 69.9 (C 1’ ), 35.0 (C 2 ), 31.2 (C 14’ ), 29.1 (C 2’ ), 29.0−28.6 (C 4’ −C 13’ ), 25.6 (C 3’ ), 22.0 135 (C 15’ ), 13.8 (C 16’ ). Elem. Anal. Calcd. for C 24 H 42 O 3 : C, 76.14; H, 11.18. Found: C, 76.41; H, 136 10.71. 137 138 Cell proliferation assay 139 The MTT assay is a colorimetric technique for the quantitative determination of cell 140 viability. It is based on the capability of viable cells to transform the MTT salt (3-(4,5-141 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into a purple formazan dye. 142 Exponentially growing cells were seeded into 96-well plates and drugs were added 24 h later. 143 Cells were exposed to the drugs for 48 h (except in the experiments with the parental and 144 DNA repair-deficient cell lines, in which cells were exposure for 24 h and were then allowed 145 to grow for additional 24 h in drug-free medium to let them repair the possible DNA damage 146 induced by the drugs). After these 48 h, medium was removed and 125 µL MTT (1 mg/mL in 147 medium) was added to each well for 5 h. Then, 80 µL 20% SDS in 20 mM HCl was added to 148 dissolve the insoluble purple formazan product, plates were incubated for 10 hours at 37 ºC, 149 and optical densities were measured at 540 nm on a multiwell plate spectrophotometer reader. 150 Cell viability was expressed as percentage in relation to controls. All data were averaged from 151 8 at least three independent experiments and were expressed as means ± standard error of the 152 means (SEM). 153 154 Inhibition of glycolysis 155 Glycolysis inhibition was assessed by measuring concentrations of glucose (initial 156 product of glycolysis) and lactate (final product of glycolysis) in control and treated cells. 157 Briefly, 10 6 cells were exposed to the tested compounds for 8 h, and glucose and lactate 158 concentrations were determined in cell supernatants by using the Accutrend ® Plus analyzer 159 together with Accutrend glucose strips and BM-Lactate Strips (Roche Diagnostics). After 160 calibrating the instrument with glucose and lactate calibration strips, test strips were used to 161 determine glucose and lactate levels via colorimetric-oxidase mediator reactions according to 162 the manufacturer's instructions. 34 Results are expressed as percentage of lactate production 163 and percentage of glucose consumption in relation to untreated cells, and are shown as the 164 means ± standard error of the means (SEM) of three independent experiments. 165 166 Comet assay 167 The single-cell gel electrophoresis assay (comet assay) is a well established technique 168 for DNA damage detection. The images obtained with this assay resemble a "comet" with a 169 distinct head and tail; the head is composed of intact DNA, while the tail consists of damaged 170 (single-strand or double-strand breaks) DNA. This assay has been described in detail by Singh 171 et al. 35 We followed this protocol with minor modifications described previously. 36 Briefly, 172 standard slides were immersed in 1% normal melting agarose at 55 °C, left to allow the 173 agarose to solidify, and kept at 4 °C until use. After cell treatments, approximately 10000 174 15 Figure 5A shows that HTDE 3 µM can inhibit the growth of MCF7 breast cancer cells 321 without affecting the growth of MCF10 normal breast cells. It is unknown whether the oral 322 administration of HTDE can lead to such concentrations in plasma and tissues. Previous 323 experiments suggest that hydroxytyrosol alkyl ether derivatives are stable when digested in 324 vitro, are rapidly absorbed, and are metabolized only partially. 27-29 Indeed, HTDE and related 325 hydroxytyrosol alkyl ether derivatives were found to exert a neuroprotective effect in rats 326 when administered orally, therefore suggesting that HTDE is bioavailable after oral 327 administration. In any case, the possible low oral bioavailability and high metabolism of 328 HTDE could be overcome with a sustained intravenous infusion. This strategy could be used 329 to obtain and maintain cytotoxic concentrations of HTDE in plasma and tissues, which would 330 maximize its therapeutic potential. Future animal studies are needed to evaluate the anticancer 331 potential of HTDE in vivo. 332 The possible exploitation of the biological properties of hydroxytyrosol alkyl ether 333 derivatives described in this and previous reports 25,26,30,31 would require an adequate supply of 334 these derivatives. Several million tons per year of wastes are estimated to be produced 335 worldwide by the olive oil industry and, interestingly, hydroxytyrosol alkyl ether derivatives 336 can be synthesized easily and in high yield using hydroxytyrosol obtained from olive oil 337 waste waters. 23,48 338 In summary, since accumulating data suggest that the olive oil constituent 339 hydroxytyrosol has anticancer activity, we have evaluated the selective cytotoxic activity of a 340 series of hydroxytyrosol alkyl ether derivatives in relation to that of hydroxytyrosol, and have 341 investigated possible mechanisms involved in the cytotoxicity of the most selective 342 compound (HTDE, 15). 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Nutr. 2007, 47, 187-229. 495 496 497 23 We thank the Consejería de Innovación, Ciencia y Empresa of the Junta de Andalucía (Spain) 498 for financial support (grant no. P09-AGR-5098). 499 24 FIGURE CAPTIONS 500 Figure 1. Synthetic procedure of the alkyl hydroxytyrosyl ether derivatives (10–16). 501 502 Figure 2. Selective cytotoxicity of hydroxytyrosol and several alkyl hydroxytyrosyl ether 503 derivatives (10–16). The percentage of cell viability (± SEM) in A549 cancer cells and MRC5 504 normal cells exposed for 48 h to hydroxytyrosol (1), its alkyl ether derivatives (10-16) and 5-505 fluorouracil (5-FU) was determined with the MTT assay. 506 507 Figure 3. Possible mechanisms of action involved in the cytotoxicity of hydroxytyrosyl 508 dodecyl ether (HTDE, 15). A) Percentage of lactate produced by cells exposed for 8 h to 509 HTDE (15), dichloroacetate (DCA) and 5-fluorouracil (5-FU) in relation to untreated cells 510 (control). B) Percentage of glucose consumed by cells exposed for 8 h to HTDE, DCA and 5-511 FU in relation to untreated cells. C) Prevention of HTDE -induced cell death in A549 cells by 512 the superoxide dismutase mimetic MnTMPyP (MTT assay); A549 cells were exposed to 513 HTDE for 48 h in the presence and absence of MnTMPyP (added 1 h before HTDE). D) 514 Representative photographs of untreated cells, of cells treated with the DNA damaging agent 515 camptothecin (cpt), and of cells exposed for 2 h and 24 h to HTDE 32 µM and 100 µM (comet 516 assay). E) Quantification of DNA damage (comet assay) expressed as percent of DNA 517 damage in tail and as tail moment (tail length × percentage of DNA in the tail). F) Cells 518 mutated in XRCC1 (deficient in base excision repair) are more sensible than parental cells to 519 the cytotoxic activity of HTDE. Both cell lines were treated with several concentrations of 520 HTDE for 24 h and, after a recovery period of 24 h, cell viability was estimated with the MTT 521 assay. 522 31 TOC Graphic H O HO O Hydroxytyrosyl dodecyl ether (15)