APRIL 2004 VOLUME 17, NUMBER 4 © Copyright 2004 by the American Chemical Society
Articles Induction of Apoptosis in Colorectal Carcinoma Cells Treated with 4-Hydroxy-2-nonenal and Structurally Related Aldehydic Products of Lipid Peroxidation James D. West,† Chuan Ji,† Stephen T. Duncan,‡ Venkataraman Amarnath,§ Claus Schneider,| Carmelo J. Rizzo,‡ Alan R. Brash,| and Lawrence J. Marnett*,†,‡ Departments of Biochemistry, Chemistry, Pathology, and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, and Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-0146 Received November 28, 2003
The oxidation of polyunsaturated fatty acids during oxidative stress gives rise to a series of toxic R,β-unsaturated aldehydes, including the electrophile 4-hydroxy-2-nonenal (4-HNE) and the related aldehydes, 4-hydroperoxy-2-nonenal (4-HPNE) and 4-oxo-2-nonenal (4-ONE). We synthesized these compounds, as well as the resolved enantiomers of 4-HNE, and compared their toxicities and apoptotic responses in the human colorectal cancer cell line RKO. All of these molecules execute similar death responses at comparable doses over almost identical time frames in RKO cells. The apoptotic response induced by 4-HPNE, 4-ONE, and 4-HNE enantiomers involves activation of caspases, proteolysis of downstream caspase targets, and nucleosomal DNA fragmentation. The results presented herein suggest that these molecules commonly activate certain signaling pathways that control cell death irrespective of their reactive properties.
Introduction Numerous diseases, including cancer, cardiovascular disease, and neurodegenerative disease, are associated with oxidative damage of biological macromolecules. Free radicals produced during oxidative stress damage proteins, DNA, carbohydrates, and lipids (1-3). Polyunsatu-
rated fatty acids, such as linoleic acid and arachidonic acid, are particularly susceptible to oxidative damage during oxidative stress. Following oxidation, the damaged lipids can decompose to form a series of reactive R,βunsaturated aldehydes, including malondialdehyde, acrolein, and 4-HNE1 (4, 5). Such aldehydes function as secondary cellular toxins by reacting with proteins and
* To whom correspondence should be addressed. Tel: (615)343-7329. Fax: (615)343-7534. E-mail:
[email protected]. † Department of Biochemistry. ‡ Department of Chemistry. § Department of Pathology. | Department of Pharmacology.
1 Abbreviations: 4-HNE, 4-hydroxy-2-nonenal; 4-HPNE, 4-hydroperoxy-2-nonenal; 4-ONE, 4-oxo-2-nonenal; PARP, poly(ADP-ribose) polymerase-1; DFF, DNA fragmentation factor; zVAD-fmk, carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone; DEVD-fmk, carbobenzoxy-AspGlu-Val-Asp-fluoromethyl ketone; WST-1, 4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate.
10.1021/tx034248o CCC: $27.50 © 2004 American Chemical Society Published on Web 02/24/2004
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are comparable in their ability to alter cell signaling and induce apoptosis.
Experimental Procedures
Figure 1. Decomposition of 13-HPODE to 4-HPNE, 4-ONE, and 4-HNE.
DNA and inducing alterations in cell proliferation, cell cycle progression, and apoptosis (3, 4, 6). 4-HNE is an abundant and highly reactive electrophile produced during lipid peroxidation that is considered to be the principal cytotoxic product of lipid peroxidation. The chemical reactions that lead to 4-HNE production have been the subject of considerable investigation. A likely pathway involves the breakdown of ω-6-derived polyunsaturated fatty acid hydroperoxides [e.g., 13hydroperoxy-9,11-octadecadienoic acid (13-HPODE)] to 4-HPNE, which can dehydrate to 4-ONE or be reduced to 4-HNE (Figure 1) (7-9). Both 4-HPNE and 4-ONE have been isolated as products of lipid peroxidation, and 4-ONE has been demonstrated to be a powerful electrophile capable of reacting with protein and DNA at rates significantly greater than HNE (10, 11). 4-HPNE and 4-HNE are produced as mixtures of enantiomers that may exhibit differential reactivity with protein and DNA. In fact, reaction of racemic 4-HNE with calf thymus DNA produces an unequal mixture of diastereomeric propanodeoxyguanosine adducts (12). The major product from the reaction was subsequently shown to be derived from 4-(S)-HNE (13). Little is known about the biological responses elicited by either 4-HPNE or 4-ONE. In addition, it is not known if the stereochemistry of the 4-hydroxyl group is an important determinant of biological activity. To address these issues, we have synthesized 4-HPNE, 4-ONE, and the resolved enantiomers of 4-HNE and evaluated their ability to induce apoptosis in a human colon carcinoma cell line, RKO. This cell line was chosen because it has previously been used to characterize the effects of HNE, including the detailed analysis of its effects on the biochemical pathways leading to apoptosis (14). The results of our experiments indicate that all members of the “4-HNE pathway” exhibit toxicity in RKO cells and
Reagents. All chemicals, unless otherwise noted, were from Sigma-Aldrich (Madison, WI). Racemic 4-HNE was obtained from Cayman Chemical (Ann Arbor, MI). zVAD-fmk was obtained from Promega (Madison, WI). DEVD-fmk was obtained from Calbiochem (La Jolla, CA). zVAD-fmk and DEVD-fmk were stored as a 20 mM stock solution in Me2SO. Synthesis of 4-HPNE. 4-HPNE. 4-HPNE was prepared by autoxidation of 3(Z)-nonenal, which was synthesized as previously described (15). In the autoxidation reaction, 3(Z)-nonenal (50 mg, 0.36 mmol) was dissolved in a 1:1 mixture of water/ acetonitrile and bubbled with oxygen for 15 h at room temperature. For purification of 4-HPNE, aliquots of the autoxidation reaction were injected on an Alltech Econosil C18 HPLC column (10 mm × 250 mm) eluted with a solvent of acetonitrile/water/ acetic acid (60/40/0.01 by volume) at a flow rate of 3 mL/min and monitored by UV detection at 220 nm. The retention time for 4-HPNE was 9.5 min. To the collected fractions, an equal volume of water was added, and the solution was extracted twice with dichloromethane. The combined organic phases were washed with water, and the organic layer was evaporated to yield 4-HPNE (10%). 1H NMR spectra of 4-HPNE were recorded in CD3CN using residual CH3CN as an internal reference (δ ) 1.92 ppm): 9.58 ppm, d, J ) 7.8 Hz, 1 H; 6.9 ppm, dd, J ) 15.9 Hz, 6.2 Hz, 1 H; 6.25 ppm, ddd, J ) 15.9 Hz, 7.8 Hz, 1.2 Hz, 1 H; 4.6 ppm, q, J ≈ 6.5 Hz, 1 H. Synthesis of 4-ONE. 1. 1,1-Dimethoxynon-2(E)-en-4-one. A solution of Dess-Martin periodinane (2.6 g, 6 mmol) in dry dichloromethane (20 mL) was cooled in a cold water bath, and a solution of 1,1-dimethoxynon-2(E)-en-4-ol (1 g, 4.9 mmol) in the same solvent (5 mL) was added dropwise (16, 17). The resulting suspension was stirred at room temperature for 40 min, and ether (40 mL) was added followed by saturated NaHCO3 solution (30 mL) containing Na2S2O3 (5 g). When the solid had dissolved, the phases were separated, and the ether layer was washed with NaHCO3 solution, brine, and water (20 mL each). The organic phase was dried and evaporated. The residue was purified by column chromatography (4:1 hexanesethyl acetate) to yield 0.8 g of 1,1-dimethoxynon-2(E)-en-4-one (80%). The NMR spectrum was identical to previous reports (18). GC-MS m/z 199 (1, M - H)+, 169 (30, M - CH3O)+, 101 (100). 2. 4-ONE. A solution of 1,1-dimethoxynon-2(E)-en-4-one (0.2 g, 1 mmol) in dichloromethane (25 mL) was stirred with Montmorillonite K-10 (400 mg) for 1 h and filtered (19). The solid was washed with the same solvent (2 × 5 mL). The filtrate was concentrated and purified by flash chromatography (5:1 hexanes-ethyl acetate) to yield 4-ONE (140 mg, 90%) as a colorless oil. 1H and 13C NMR spectra were identical to those reported earlier (18). GC-MS m/z 153 (15, M - H)+, 128 (100, M - CO)+. It was stored in dichloromethane under argon at -80 °C for 4-6 weeks without decomposition. Synthesis of 4-HNE Enantiomers. 1. Oct-2(E)-en-1-ol. In a flame-dried, round bottom flask was placed LiAlH4 (6.45 g, 170 mmol) in anhydrous ether (100 mL). The stirred suspension was cooled to -40 °C under argon. A solution of oct-2(E)-enal (9.95 g, 79 mmol) in ether (100 mL) was added dropwise over 30 min. The reaction mixture was stirred at room temperature for 3 h. The reaction was quenched by the slow addition of water (50 mL), which resulted in the formation of a precipitate. The precipitate was dissolved in 1 N H2SO4 (50 mL). The separated aqueous phase was extracted with ether (3 × 15 mL). The organic extracts were dried over sodium sulfate, filtered, and concentrated to give trans-oct-2-en-1-ol (9.94 g, 99% yield) as a yellow oil. The product was used without further purification. 1H NMR (300 MHz, CDCl ): δ 5.60 (m, 2H), 4.04 (d, J ) 5.4 3 Hz, 1 H), 2.02 (q, J ) 7.26 Hz, 2 H), 1.27-1.43 (m, 6 H), 0.89 (t, J ) 6.9 Hz, 3 H). 2. (-)-(2S,3S)-Epoxy-1-octanol. Epoxidation reactions were carried out under conditions described by Gao et al. (20). In a
Induction of Apoptosis by 4-HNE and Related Compounds flame-dried, round bottom flask was placed activated 4 Å powdered molecular sieves (0.78 g) and anhydrous CH2Cl2 (100 mL) under argon; the flask was cooled to -40 °C. In a separate flask, L-(+)-diethyl tartate (1.10 g, 5.64 mmol) was stirred in anhydrous CH2Cl2 (10 mL) over molecular sieves for 15 min and transferred to the reaction flask. In a separate flask, Ti(O-iPr)4 (1.33 g, 4.67 mmol) was stirred in CH2Cl2 (10 mL) over molecular sieves for 15 min and added to the reaction mixture. In a separate flamed-dried flask, cumene hydroperoxide (9.13 g, 60 mmol) was stirred in anhydrous CH2Cl2 for 15 min and added to the reaction mixture. The reaction mixture was stirred for 40 min at -40 °C, after which time a solution of 2(E)-octen-1-ol (3.0 g, 23.44 mmol) in anhydrous CH2Cl2 (10 mL) was added to the reaction mixture dropwise. The reaction mixture was stirred for 5 h at -20 °C and warmed to 0 °C. The reaction was quenched by pouring into a beaker containing an ice-cold solution of ferrous sulfate-tartaric acid solution (33 g of ferrous sulfate heptahydrate and 10 g of DL-tartaric acid in 100 mL of deionized water), and this mixture was stirred for 10 min. The aqueous layer was extracted with ether (2 × 30 mL). The combined organic layers were stirred for 1 h at 0 °C with icecold 30% NaOH in saturated brine (10 mL). The separated, aqueous phase was extracted with ether (2 × 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and evaporated. Purification by flash column chromatography was performed, eluting with 10% ethyl acetate in hexanes, to give (2S,3S)-epoxy-1-octanol (2.01 g, 67%) as a white, crystalline solid. [R]D25 -40.2 (c 0.4, CHCl3). 1H NMR (300 MHz, CDCl3): δ 3.65 (ddd, J ) 2.4, 7.9, 14.9 Hz, 1 H), 3.36 (ddd, J ) 5.1, 7.4, 12.5 Hz, 1 H), 2.67-2.70 (m, 2 H), 1.53 (br s, 1 H), 1.11-1.39 (m, 8 H), 0.72 (t, J ) 7.14 Hz, 3 H). 3. (-)-(2R,3S)-Epoxyoctanal. To a stirred solution of oxalyl chloride (1.91 mL, 3.82 mmol) in CH2Cl2 (25 mL) cooled to -50 to -60 °C was added dimethyl sulfoxide (0.542 mL, 7.64 mmol) in CH2Cl2 (5 mL) dropwise (21). The reaction mixture was stirred for 2 min, and then, (2S,3S)-epoxy-1-octanol (0.5 g, 3.45 mmol) in CH2Cl2 (5 mL) was added dropwise. The reaction was stirred for 15 min, after which triethylamine (2.42 mL, 17.36 mmol) was added. The reaction mixture was stirred for 5 min and warmed to room temperature. Water (50 mL) was added, and the aqueous phase was extracted with CH2Cl2 (1 × 50 mL). The combined organic layers were washed with saturated brine (100 mL), dried over magnesium sulfate, filtered, and evaporated. The residue was diluted with CH2Cl2 (5 mL) and successively washed with 1% aqueous HCl, water, 5% Na2CO3, and water. The CH2Cl2 was evaporated. Purification by flash column chromatography was performed, eluting with 10-40% ethyl acetate in hexanes, to give (2S,3S)-epoxyoctanal (0.35 g, 71%) as a yellow oil. [R]D21.9 - 42° (c 0.3, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.91 (d, J ) 4.6 Hz, 1 H), 3.13 (td, J ) 1.5, 4.2 Hz, 1 H), 3.01 (dd, J ) 11.5, 4.6 Hz, 1 H), 1.22-1.57 (m, 8 H), 0.82 (t, J ) 7.2 Hz, 3H). 4. 4-(S)-(+)-HNE. In a flame-dried, 100 mL round bottom flask was placed methoxymethylenetriphenylphosphorane (2.11 g, 6.15 mmol) in THF (30 mL). The suspension was cooled to -40 °C. Potassium tert-butoxide (5.5 mmol, 1.0 M in THF) was added dropwise. The solution was stirred for 5 min and then cooled to -78 °C. A solution of (2S,3S)-epoxyoctanal (0.35 g, 2.46 mmol) in THF (5 mL) was added dropwise to the Wittig reagent (22). The reaction mixture was stirred for 15 min and allowed to warm to room temperature over 40 min. The reaction was quenched by adding water (50 mL), and the aqueous phase was extracted with CH2Cl2 (3 × 15 mL), dried over magnesium sulfate, filtered, and concentrated. Purification by flash column chromatography gave 4-(S)-HNE (0.248 g, 65%) as a pale yellow oil. The enantiomeric excess was estimated to be 88% based on chiral stationary phase HPLC analysis of the 2,4-dinitrophenylhydrazone derivative on a Chiralcel OD-H column with UV detection. [R]D21.9 +12.47 (c 0.46, CHCl3). 1H NMR (400 MHz, CDCl3): δ 9.49 (d, J ) 7.9 Hz, 1 H), 6.77 (dd, J ) 4.7, 15.7 Hz, 1 H), 6.23 (ddd, J ) 1.5, 7.9, 15.7 Hz, 1 H), 4.33 (m, 1 H), 2.04 (br s, 1 H), 1.18-1.56 (m, 8 H), 0.83 (t, J ) 6.9 Hz, 3 H).
Chem. Res. Toxicol., Vol. 17, No. 4, 2004 455 5. 4-(R)-HNE. The R-enantiomer was synthesized by changing to the antipodal (-)-diethyl tartrate ligand for the Sharpless asymmetric epoxidation. Cell Culture. RKO human colorectal carcinoma cells were grown in McCoy’s 5A medium (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (U.S. Biotechnologies, Parker Ford, PA), 2 mM L-glutamine, and antibiotics at 37 °C and 5% CO2. Treatments were carried out with varying concentrations of 4-ONE, 4-HPNE, or 4-HNE enantiomers dissolved in methanol. The total concentration of methanol per culture was e0.1% of the total medium volume. Cells were approximately 40-60% confluent at the time of treatment. Cytotoxicity Assay. The WST-1 reagent (Roche, Indianapolis, IN) was used to assess toxicity of all compounds in RKO cells. Reactions were carried out as described in the manufacturer’s protocol. Briefly, approximately 3.5 × 104 cells were seeded on a 96 well plate, grown for 20 h, and treated with various concentrations of 4-HPNE, 4-ONE, or 4-HNE. The total volume of medium in each well was 100 µL. Following the incubation with compound for 24 or 48 h, 10 µL of WST-1 reagent was added to each well for 1 h at 37 °C and 5% CO2. Absorbance of each sample was measured using a 96 well plate reader at 450 nm with background subtraction at 690 nm. The difference in absorbances was calculated, and the percentage of WST-1 conversion was plotted against the concentration of the aldehyde used. IC50 values for each compound were determined from sigmoidal dose-response curves using Prism 4 (GraphPad Software, San Diego, CA). Preparation of Cell Lysates and Western Blotting. Cells were scraped off of flasks following treatment, centrifuged at 100g for 5 min, and washed two times with cold phosphatebuffered saline, pH 7.4. Cell pellets were lysed on ice in buffer containing 145 mM NaCl, 15 mM HEPES (pH 7.0), 10 mM EGTA, 0.1 mM MgCl2, 1.0% Triton X-100, and protease inhibitor cocktail (Roche) for 30 min. Debris from lysates was cleared by centrifugation at 16 000g for 5 min. The supernatant was recovered, and protein concentrations were quantified using bicinchoninic acid protein assay (Pierce, Rockford, IL) with bovine serum albumin as a standard. Proteins (30 µg) were separated by SDS-PAGE under reducing conditions and were transferred to a poly(vinylidene difluoride) membrane (Millipore, Bedford, MA) for 4 h at 0.2 A in a buffer containing 25 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol. Membranes were blocked for 1 h at room temperature in TTBS [100 mM Tris (pH 7.5), 150 mM NaCl, and 0.1%Tween-20] containing 5% nonfat dry milk. Rabbit primary antibodies against human caspase-3, caspase-9, PARP, and DFF45/DFF35 were obtained from Cell Signaling Technologies (Beverly, MA). Membranes were treated with the appropriate dilutions of antibodies for 1-2 h at room temperature and were washed three times for 10 min with TTBS. The appropriate secondary antibodies conjugated to horseradish peroxidase (Amersham, Piscataway, NJ) were applied at 1:5000 dilutions for 45 min at room temperature, and the membrane was washed four times with TTBS. Chemiluminescent reagents (Amersham) were added for 1-2 min, and blots were autoradiographed. DNA Fragmentation Analysis. The procedure for preparing DNA for analysis of fragmentation has been described elsewhere (14). Caspase-3 Activity Assay. A colorimetic CaspACE Assay system was purchased from Promega. The manufacturer’s protocol was followed using 35 µg of cell protein per reaction. Reactions were performed in triplicate and were monitored spectrophotometrically at 405 nm using a 96 well plate reader.
Results Synthesis of 4-HPNE, 4-ONE, and (+)- and (-)-4HNE. The procedures for synthesis of the target compounds are summarized in Figure 2. 4-HPNE was prepared via autoxidation of 3(Z)-nonenal by passing a
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Figure 2. Synthetic routes to 4-HPNE, 4-ONE, and (+)- and (-)-4-HNE.
Figure 3. Cytotoxicity assay for 4-HPNE, 4-ONE, and 4-HNE in RKO cells. RKO cells were treated with indicated concentrations of 4-HPNE (A), 4-ONE (A), or enantiomers of 4-HNE (B) for the indicated times. Cell viability was assessed colorimetrically using the tetrazolium salt WST-1. Results are plotted as a percentage of control (n ) 4 ( SD) and are representative of 2-3 independent experiments.
stream of O2 through a solution of the aldehyde in water/ acetonitrile. Extracts of the autoxidation mixture were injected onto a reversed phase HPLC column, and fractions containing 4-HPNE were collected, diluted with water, and extracted with CH2Cl2. 4-HPNE was isolated following evaporation of the CH2Cl2 at an overall yield of 10%.
4-ONE was synthesized in two steps from 1,1-dimethoxynon-2(E)-en-4-ol (18). The alcohol was oxidized to 1,1dimethoxynon-2(E)-en-4-one by Dess-Martin periodinane; then, the acetal was hydrolyzed to 4-ONE. The overall yield was 72%, and 4-ONE was isolated as a colorless oil following silica gel flash chromatography. The use of Montmorillonite K-10 for the final deprotection
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Table 1. Cytotoxic Potency of Molecules Studieda IC50 (µM) compound
24 h
48 h
racemic 4-HNE 4-HPNE 4-ONE 4-(R)-HNE 4-(S)-HNE
62 ( 2 41 ( 2 50 ( 3 57 ( 3 65 ( 2
59 ( 2 39 ( 2 59 ( 10 56 ( 2 62 ( 1
a IC 50 values (compound concentration at which cell viability was 50% of control; average ( SE) were determined as described in the Experimental Procedures for RKO cells treated with compounds for 24 or 48 h. Values were derived from the average of 8-20 replicates using the WST-1 cytotoxicity assay.
offered the advantage of easy removal of the catalyst after hydrolysis, thus minimizing side reactions. The enantiomers of 4-HNE were synthesized from 2(E)octenol (22). The alkene was subjected to Sharpless asymmetric epoxidation using cumene hydroperoxide and chiral Ti-tartrate catalysts. The enantiomeric epoxides were further oxidized to the enantiomeric epoxy-aldehydes, which were converted to the (+)- and (-)-enantiomers of 4-HNE by Wittig homologation. The overall yield was 31%, and the enantiomers of 4-HNE were isolated as pale yellow oils following silica gel flash chromatography. Toxicity of 4-HNE, 4-HPNE, and 4-ONE in RKO Cells. The apoptotic response initiated by racemic 4-HNE has been characterized in several cell types, including neuronal cells, endothelial cells, colorectal cancer cells, and cells of hematopoetic lineage (14, 23-26). Previously, we determined mechanisms of cytotoxicity for malondialdehyde and 4-HNE in RKO colorectal carcinoma cells (14, 27). To evaluate the cytotoxicity of 4-HNE enantiomers, 4-HPNE, and 4-ONE in RKO cells, the ability of treated cultures to reduce the tetrazolium salt, WST-1, to formazan was determined. All molecules exhibited comparable toxicity profiles following 12, 24, or 48 h treatments (Figure 3, 12 h treatment not shown), suggesting that they potentially work through similar mechanisms to execute their respective death responses. Quantitative comparison of each molecule’s cytotoxic potency revealed that 4-HPNE and, to a lesser extent, 4-ONE were slightly more toxic than 4-HNE and its enantiomers at 24 h (Table 1), with all molecules exhibiting an IC50 between approximately 40 and 65 µM. Because noticeable toxicity for all compounds was evident over this range, further experiments exploring the mechanism of the cell death response were carried out using these concentrations. Activation of Caspases in Apoptotic Responses Induced by 4-HNE, 4-HPNE, and 4-ONE. Racemic 4-HNE induces apoptosis in RKO cells via cytochrome c release from the mitochondria and activation of caspases-9 and -3 (14, 23, 26). To determine if 4-HPNE, 4-ONE, and stereoisomers of 4-HNE were capable of activating caspase-3, extracts from treated cells were incubated with the caspase-3 cleavage sequence linked to p-nitroaniline (DEVD-pNA) and assayed for the release of p-nitroaniline from the peptide. As is shown in Figure 4, dose-dependent activation of caspase-3 was observed in RKO cells treated for 24 h with all compounds. Only slight differences in caspase activities were observed among cells treated with enantiomers of 4-HNE (Figure 4). These differences, representing
