Discovering the activity of dietary phytochemicals at the level of intracellular signal transduction pathways is now considered the basis to suggest their eventual health effects. The bioactivity of these molecules is generally ascribed to redox and antioxidant properties, with a growing body of evidence indicating that most compounds are to be considered for their roles as modulators of redox-mediated signalling cascades, including those relevant to either survival or cell death(Reference Leonarduzzi, Sottero and Poli1). Moreover, whatever their properties, bioavailability has to be ascertained so that the impact on human health and nutritional importance of dietary phytochemicals can be assessed. Betalains are nitrogen-containing pigments occurring in the Cariophyllales order of plants, including beetroot and cactus pear, and in some fungal genera(Reference Strack, Vogt and Schliemann2, Reference Stintzing and Carle3). Indicaxanthin (Ind, Fig. 1), the yellow betalain characterising the edible fruit of the cactus Opuntia ficus indica, has recently emerged as a radical scavenger and antioxidant(Reference Butera, Tesoriere and Di Gaudio4, Reference Tesoriere, Allegra and Butera5) with peculiar physico-chemical characteristics, allowing the molecule to interact with and locate in membranes(Reference Turco Liveri, Sciascia and Allegra6, Reference Turco-Liveri, Sciascia and Lombardo7) and the potential to act at the level of body cells and tissues. Anti-inflammatory and protective effects of Ind have been shown in vitro, in endothelial cell cultures, where it can inhibit the cytokine-induced redox state alteration and modulate the expression of adhesion molecules(Reference Gentile, Tesoriere and Allegra8), and in either healthy or pathological erythrocytes(Reference Tesoriere, Butera and Allegra9, Reference Tesoriere, Allegra and Butera10). Other studies showed modulatory activity of Ind on the contractility of isolated mouse ileal muscle(Reference Baldassano, Tesoriere and Rotondo11, Reference Baldassano, Rotondo and Serio12). Different from the majority of dietary phytochemicals, Ind has appeared to be quite stable in absorptive gastrointestinal conditions(Reference Tesoriere, Fazzari and Angileri13), is not metabolised by human enterocytes(Reference Tesoriere, Gentile and Angileri14) or hepatocytes(Reference Reynoso, Giner and Gonzalez de Mejia15) and is bioavailable, reaching plasma micromolar concentrations after a dietary supplementation with cactus pear fruits(Reference Tesoriere, Allegra and Butera16). In the present study, we investigated the activity of Ind in THP-1 cells, a human monocyte–macrophage cell line, against the cytotoxicity of 7-ketocholesterol (7-KC). Oxysterols are a family of bioactive lipids generated in the body through either enzymatic or non-enzymatic oxidation of cholesterol or absorbed from the diet(Reference Brown and Jessup17). These compounds are involved in physiological processes(Reference Kandutsch and Chen18–Reference Bjorkhem, Andersson and Diczfalusy21); however, there is evidence that some of them have deleterious effects(Reference Brown and Jessup22, Reference Colles, Maxson and Carlson23). The potential of oxysterols to trigger pro-oxidative, pro-inflammatory and cytotoxic reactions, widely documented in a number of cells of the vascular wall, including human artery smooth muscle(Reference Pedruzzi, Guichard and Ollivier24, Reference Nishio and Watanabe25) and endothelial cells(Reference Luthra, Dong and Gramajo26–Reference Lemaire, Lizard and Monier28) as well as in immunocompetent cells such as monocyte/macrophages(Reference O'Callaghan, Woods and O'Brien29, Reference Lemaire-Ewing, Prunet and Montange30), led to the consideration that these compounds and 7-KC, in particular(Reference Brown and Jessup22), are important contributors to the progression of vascular dysfunction(Reference Miguet-Alfonsi, Prunet and Monier31, Reference Hulten, Lindmark and Diczfalusy32) and development of atheromas. The level of 7-KC in plasma of healthy subjects is from 0·001 to 4·7 μm(Reference Brown and Jessup22), whereas the amount measured in atherosclerotic plaque may be more than 40-fold higher(Reference Garcia-Cruset, Carpenter and Guardiola33, Reference Vaya, Aviram and Mahmood34). In the present study we simulated a pathophysiological condition by challenging human monocyte/macrophage THP-1 cells with 16 μm-7-KC, a concentration that can be reached in atherosclerotic plaque of hypercholesterolaemic subjects. Here, we report that Ind, when co-incubated at a concentration comparable with that measured in human plasma after a dietary supplementation with cactus pear fruits, totally prevents the 7-KC-induced apoptosis of THP-1 cells by interfering with molecular mechanisms known to be involved in the cytotoxicity of this oxysterol.
Molecular structure of indicaxanthin.
Experimental methods
Unless stated otherwise, all reagents and materials were from Sigma Chemical Company and solvents were of the highest purity or HPLC grade.
Indicaxanthin preparation
Ind was isolated from cactus pear (O. ficus indica) fruits (yellow cultivar). The phytochemical was separated from a methanol extract of the pulp by liquid chromatography on Sephadex G-25(Reference Butera, Tesoriere and Di Gaudio4). Fractions containing the pigment were submitted to cryodesiccation and purified according to Stintzing et al. (Reference Stintzing, Schieber and Carle35). Briefly, the desiccated material was re-suspended in 1 % acetic acid in water and submitted to semi-preparative HPLC using a Varian Pursuit C18 column (250 × 10 mm inner diameter; 5 mm; Varian), eluted with a 20 min linear gradient elution from solvent A (1 % acetic acid in water) to 20 % solvent B (1 % acetic acid in acetonitrile) with a flow of 3 ml/min. Spectrophotometric revelation was at 482 nm. The elution volumes relevant to Ind were collected. Samples after cryodesiccation were re-suspended in 5 mm-PBS (pH 7·4) at a suitable concentration and used immediately or stored at − 80°C. Concentration of the samples was evaluated spectrophotometrically in a DU-640 Beckman spectrophotometer by using a molar coefficient at 482 nm of 42 800(Reference Piattelli, Minale and Prota36). Ind was filtered through a Millex HV 0·2 μm filter (Millipore) immediately before use.
Cell culture
THP-1 cells (American Type Culture Collection) were grown in Roswell Park Memorial Institute medium supplemented with 2 mm-l-glutamine, 10 % fetal bovine serum, 100 U (60 μg)/ml penicillin, 100 μg/ml streptomycin and 5 μg/ml gentamicin. Cells were maintained in log phase by seeding twice a week at a density of 3 × 108 cells/l in humidified 5 % CO2 atmosphere at 37°C. In all experiments, THP-1 cells were seeded in triplicate in twenty-four-well culture plates at a density of 1·25 × 105 cells/cm2 and made quiescent through overnight incubation. Then, cells were treated with 7-KC at a final concentration of 16 μm, alone or with Ind, and incubation times were as indicated in the text. 7-KC was delivered to the cells using tetrahydrofuran as a solvent, at a final concentration of 0·1 % (v/v). Untreated cells incubated with 0·1 % tetrahydrofuran were used as control. No differences were found between cells treated with tetrahydrofuran and untreated cells in terms of cell number, viability and reactive oxygen species (ROS) production. At the times indicated, cells were harvested and quadruplicate haemocytometer counts were performed. The trypan blue dye exclusion method was used to evaluate the percentage of viable cells.
Measurement of 7-ketocholesterol
7-KC was extracted with four volumes of a methanol–hexane mixture (1:3, v/v) from 3 × 106 cells treated with 16 μm-oxysterol for 12 h. 7-KC was analysed by HPLC using a cyano-bonded column (Luna 5 μm, 250 × 4·6 mm; Phenomenex) equipped with a CN-guard cartridge (2·0 × 4·0 mm; Phenomenex) and hexane at a flow rate of 1 ml/min. 7-KC was detected spectrophotometrically at 234 nm. Quantification was by reference to standard curves constructed with 5–100 ng of the purified compound, and by relating the amount of the compound under analysis to the peak area.
Cell cycle analysis
Cell cycle stage was analysed by flow cytometry. Aliquots of 1 × 106 cells were harvested by centrifugation, washed with PBS and incubated in the dark in a PBS solution containing 20 μg/ml propidium iodide and 200 μg/ml RNase, for 30 min, at room temperature. Then, samples were immediately subjected to fluorescence-activated cell sorting analysis by Epics XL™ flow cytometer using Expo32 software (Beckman Coulter). At least 1 × 104 cells were analysed for each sample.
Acridine orange and ethidium bromide morphological fluorescence dye staining
Acridine orange stains DNA bright green, allowing visualisation of the nuclear chromatin pattern. Apoptotic cells have condensed chromatin that is uniformly stained. Ethidium bromide stains DNA orange, but is excluded by viable cells. Dual staining allows separate enumeration of populations of viable non-apoptotic, viable (early) apoptotic, non-viable (late) apoptotic and necrotic cells. After treatment, the medium was discarded. Cells were washed with PBS first and then incubated with 100 μl of PBS containing 100 μg/ml of ethidium bromide plus 100 μg/ml of acridine orange. After 20 s, ethidium bromide/acridine orange solution was discarded and cells were immediately visualised by means of a fluorescent microscope equipped with an automatic photomicrograph system (Leica). Multiple photographs were taken at randomly selected areas of the well to ensure that the data obtained are representative.
Measurement of phosphatidylserine exposure
Flow cytometry by double staining with Annexin V/propidium iodide was used to detect externalisation of phosphatidylserine to the cell surface. Cells were adjusted at 1 × 106 cells/ml with combining buffer. Cell suspension (100 μl) was added to a new tube, and incubated with 5 μl Annexin V and 10 μl of 20 μg/ml propidium iodide solution at room temperature, in the dark or 15 min. Then, samples of at least 1 × 104 cells were subjected to fluorescence-activated cell sorting analysis by appropriate two-dimensional gating method.
Measurement of mitochondrial transmembrane potential
Mitochondrial transmembrane potential (ΔΨm) was assayed by flow cytofluorometry, using the cationic lipophilic dye 3,3′-dihexyloxacarbocyanine iodide(Reference Stintzing and Carle3) (Molecular Probes, Inc.) which accumulates in the mitochondrial matrix. Changes in mitochondrial membrane potential are indicated by a reduction in the 3,3′-dihexyloxacarbocyanine iodide-induced fluorescence intensity. Cells were incubated with 3,3′-dihexyloxacarbocyanine iodide(Reference Stintzing and Carle3) at a 40 nmol/l final concentration, for 15 min at 37°C. After centrifugation, cells were washed with PBS and suspended in 500 μl PBS. Fluorescent intensities were analysed in at least 1 × 104 cells for each sample.
Measurement of intracellular reactive oxygen species
ROS level was monitored by measuring fluorescence changes that resulted from intracellular oxidation of dichlorodihydrofluorescein diacetate. Dichlorodihydrofluorescein diacetate, at 10 μm final concentration, was added to the cell medium 30 min before the end of the treatment. The cells were collected by centrifugation for 5 min at 2000 rpm at 4°C, washed, suspended in PBS and immediately subjected to fluorescence-activated cell sorting analysis. At least 1 × 104 cells were analysed for each sample.
Measurement of cellular thiols
After treatment, cells were collected by centrifugation, washed twice with cold PBS containing 0·025 % butylated hydroxytoluene and lysed by sonication. Cell lysates were mixed with 10 % SDS and 30 μm of 5,5′-dithiobis-(2-nitrobenzoic acid) and incubated with shaking at room temperature for 30 min. The total amount of reduced thiols, including both protein thiols and glutathione, was measured spectrophotometrically at 412 nm.
Measurement of cytosolic calcium
Intracellular Ca2+ concentration in a single cell was measured using fluo-3/AM as a fluorescent Ca2+ probe, whose intensity is directly representative of cellular concentration of the ion. Fluo-3/AM, at 2 μm final concentration, was added into the cell medium 40 min before the end of the treatment. After centrifugation, cells were washed with PBS and suspended in 500 μl PBS. The fluorescent intensities were analysed by fluorescence-activated cell sorting analysis in at least 1 × 104 cells for each sample.
Western blot analysis
After treatment, cells were collected by centrifugation, washed twice with cold PBS and gently lysed for 60 min in ice-cold lysis buffer (10 mm-HEPES, 1·5 mm-MgCl2, 10 mm-KCl, 0·5 mm-phenylmethylsulphonyl fluoride (PMSF), 1·5 μg/ml soyabean trypsin inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0·1 mm-benzamidine and 0·5 mm-dithiothreitol (DTT)). The lysates were centrifuged at 13 000 g for 5 min and supernatants (cytosolic fraction) were immediately portioned and stored at − 80°C up to 2 weeks. The nuclear pellet was resuspended in 60 μl of high-salt extraction buffer (20 mm-HEPES (pH 7·9), 420 mm-NaCl, 1·5 mm-MgCl2, 0·2 mm-EDTA, 25 % (v/v) glycerol, 0·5 mm-PMSF, 1·5 μg/ml soyabean trypsin inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0·1 mm-benzamidine and 0·5 mm-DTT) and incubated with shaking at 4°C for 30 min. The nuclear extract was then centrifuged for 15 min at 13 000 g and the supernatant was portioned and stored at − 80°C. The protein concentration of each sample was determined by using the Bradford protein assay reagent (Bio-Rad). Protein samples (80 μg/line) were separated on 12 % SDS-PAGE and transferred to nitrocellulose membrane. The immunoblot was incubated overnight at 4°C with blocking solution (5 % skimmed milk), followed by incubation with anti-poly(ADP-ribose) polymerase (PARP) monoclonal antibody (clone D-1, catalogue no. SC-365315; Santa Cruz Biotechnology), anti-phospho-Bad polyclonal affinity-purified antibody (clone Ser 136, catalogue no. SC-7999; Santa Cruz Biotechnology), anti-NF-κB with its inhibitor κ Bα (Iκ-Bα) monoclonal antibody (clone 417208, catalogue no. MAB4299; R&D Systems), anti-phospho-Iκ-Bα polyclonal antibody (S32/S36, catalogue no. AF4809; R&D Systems), anti-NF-κB p65 monoclonal antibody (clone 532 301 catalogue No. MAB 5078; R&D Systems) or anti-NADPH oxidase-4 (NOX-4) (clone N-15, catalogue no. SC -21 860; Santa Cruz Biotechnology) for 1 h at room temperature. Blots were washed two times with Tween 20/Tris-buffered saline and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-IgG antibody (Dako Denmark) for 1 h at room temperature. Blots were again washed five times with Tween 20/Tris-buffered saline and then developed by enhanced chemiluminescence (Amersham Life Science). Immunoreactions were also performed using β-actin antibody as loading controls.
Statistics
Results are given as means and standard deviations. Three independent observations were carried out for each experiment, replicated three times. Statistical comparisons were made using a one-way ANOVA, followed by Fisher's test. P< 0·05 was considered statistically significant.
Results
Protective effect of indicaxanthin on 7-ketocholesterol-induced cell growth arrest and apoptosis
In comparison with control cells (vehicle alone), a 24 h incubation of human monocyte/macrophage THP-1 cells with 16 μm-7-KC caused a significant decrease in cell growth, whereas co-incubation with 7-KC and 0·1–2·5 μm-Ind resulted in a dose-dependent cell protection (Fig. 2(A)). Ind at a concentration of 2·5 μm completely restored the growth of cells exposed to 7-KC for at least 72 h (Fig. 2(B)). Ind alone did not modify THP-1 cell growth at 2·5 μm, nor at 25 μm (not shown). The distribution of THP-1 cells in the cell cycle phases after 24 h treatment with 7-KC, either alone or in association with Ind, is shown in Fig. 2(C). A significant percentage increase of cells in the G0/G1 phase, accompanied by a concomitant decrease in the percentage of cells in the S and G2/M phases and induction of a subG1-cell population, were observed in the presence of 7-KC. Co-incubation with 2·5 μm-Ind completely prevented the arrest of cell cycle progression caused by oxysterol (Fig. 2(C)).
Effect of indicaxanthin (Ind) on 7-ketocholesterol (7-KC)-induced growth and cell cycle arrest in THP-1 cells. (A) Dose-dependent effect of Ind in the presence of 7-KC (16 μm) after 24 h incubation; (B) time-dependent effects of 2·5 μm-Ind in the presence of 7-KC (16 μm); (C) percentage of propidium iodide-stained THP-1 cells in different phases of the cell cycle, as determined by flow cytometry: control (C, ■), cells treated with vehicle; 7-KC (□), cells treated for 24 h with 7-KC (16 μm); 7-KC/Ind (
), cells treated with 7-KC in the presence of Ind (2·5 μm). Values are means and standard deviation: of three experiments carried out in triplicate represented by vertical bars. a,b,c,dMean values with unlike letters were significantly different (P< 0·05; Fisher's test). (C) Representative of three experiments with comparable results.
The incorporation of 7-KC in THP-1 cells was measured either in the absence or in the presence of Ind. HPLC analysis of cell extracts after a 12 h incubation with 7-KC revealed 1·46 (sd0·6) μg of 7-KC/106 cells (n 6), which non-significantly varied upon addition of 2·5 μm-Ind (1·53 (sd 0·5) μg/106 cells, n 6).
It has widely been reported that oxysterol-induced cell toxicity is associated with pro-apoptotic events. Externalisation of plasma membrane phosphatidylserine, changes of mitochondrial transmembrane potential, variation of nuclear morphology and PARP-1 cleavage were evaluated in THP-1 cells treated with 7-KC. Flow cytometry analysis of Annexin V-fluorescein isothiocyanate and propidium iodide-stained cells after 12 h treatment with 7-KC indicated a high percentage of cells in early apoptosis, with externalised phosphatidylserine (Fig. 3(A)). Loss of mitochondrial transmembrane potential was indicated by decreased mitochondrial 3,3′-dihexyloxacarbocyanine iodide-red fluorescence (Fig. 3(B)). Treatment with 7-KC for 24 h caused changes to cell morphology, with an increase in the number of cells permeable to ethidium bromide, with fragmented and/or condensed nuclei and appearance of membrane blebbing (Fig. 3(C)). One of the key executioners of apoptotic cell death is caspase 3, responsible for proteolytic cleavage of many key proteins, including PARP-1. In comparison with control cells, high levels of the 89 kDa cleaved product from PARP-1, with a decrease of the 116 kDa native protein, were observed in 7-KC-treated THP-1 cells (Fig. 3(D)). Co-incubation with 7-KC and Ind resulted in a total prevention of 7-KC-dependent cell death induction, as shown by the absence of any apoptotic feature (Fig. 3(A)–(D)).
Effect of indicaxanthin (Ind) on cell apoptosis induced by 7-ketocholesterol (7-KC) in THP-1 cells. (A) Percentage of Annexin V/propidium iodide (PI) double-stained THP-1 cells, as determined by flow cytometry. (B) Fluorescence intensity of 3,3′-dihexyloxacarbocyanine iodide-treated cells, as determined by flow cytometry. (C) Fluorescence micrographs of ethidium bromide/acridine orange double-stained THP-1 cells in 200 × magnification. Inset shows 400 × magnification. (D) Poly(ADP-ribose) polymerase cleavage by immunoblotting with densitometric analysis of the immunoblots. Control, cells treated with vehicle; 7-KC, cells treated for 12 h (A, B) or 24 h (C, D) with 7-KC (16 μm); 7-KC/Ind, cells treated with 7-KC in the presence of Ind (2·5 μm). Representative images of three experiments with comparable results. Data of the densitometric analysis are means and standard deviations. a,bMean values with unlike letters were significantly different (P< 0·05; Fisher's test). (A colour version of this figure can be found online at http://www.journals.cambridge.org/bjn).
Prevention by indicaxanthin of 7-ketocholesterol-induced oxidative stress and NF-κB activation
Because of their peculiar NOX-4 activity, macrophages generate basal levels of intracellular ROS(Reference Martyn, Frederick and von Loehneysen37). The pro-apoptotic activity of 7-KC has been associated with NOX-4 induction, ROS overproduction and variation of the redox status of cells(Reference Palozza, Simone and Catalano38–Reference Leonarduzzi, Vizio and Sottero40). The intracellular ROS level, as well as the level of total thiols, were monitored from 30 min to 24 h in THP-1 cells incubated with 7-KC, either in the absence or in the presence of Ind. In the absence of Ind, we observed a biphasic ROS production, with a significant peak after 1 h treatment and a sustained progressive increase between 3 and 24 h of observation (Fig. 4(A)). Cell total thiols progressively decreased along 24 h of exposure to 7-KC (Fig. 4(B)). Co-incubation of cells with 7-KC and 2·5 μm-Ind completely prevented the intracellular increase of ROS and thiol loss (Fig. 4(A) and (B)). The dose-dependent effect of Ind on ROS production, monitored after 24 h incubation in the presence of 7-KC, is shown in the inset. Notably, the level of ROS measured in the presence of 7-KC and Ind was significantly (P< 0·05) lower than control cells along the entire incubation time interval, which was not observed in cells incubated with Ind alone, neither at 2·5 μm (Fig. 4(A)) nor at 10 μm-Ind (data not shown).
Effect of indicaxanthin (Ind) on oxidative stress induced by 7-ketocholesterol (7-KC) in THP-1 cells. (A) Reactive oxygen species (ROS) production. The inset shows dose-dependent effects of Ind in the presence of 7-KC (16 μm) after 24 h incubation. (B) Thiol depletion. (C) NADPH oxidase-4 (NOX-4) expression in THP-1 cells treated with 7-KC (16 μm) alone or in combination with Ind (2·5 μm) at different time intervals. Control (C, ■), cells treated with vehicle; 7-KC (●), cells treated with 7-KC (16 μm); 7-KC/Ind (○), cells treated with 7-KC in the presence of Ind (2·5 μm). ▲, Ind. (A) and (B) Cellular ROS and total thiol were assayed using flow cytometry (dichlorodihydrofluorescein diacetate staining) and by 5,5′-dithiobis-2-nitrobenzoic acid reaction, respectively, as reported in the Experimental methods section. Data are means and standard deviations of three separate experiments carried out in triplicate. (C) NOX-4 by immunoblotting with densitometric analysis of the immunoblots. ■, Control; □, 7-KC; , 7-KC/Ind. Representative images of three experiments with similar results. Data of the densitometric analysis are means and standard deviations. a,b Mean values with unlike letters were significantly different (P< 0·05; Fisher's test). MFI, mean fluorescence intensity.
The expression of NOX-4 in THP-1 cells after 1 and 3 h of incubation with either 7-KC or 7-KC and Ind was measured by Western blotting analysis (Fig. 4(C)). With respect to control, densitometric analysis showed no variation of NOX-4 protein after a 1 h treatment of THP-1 cells with 7-KC, whereas a 2-fold enhanced level was evident after 3 h. Up-regulation of NOX-4 protein was prevented by co-incubation of cells with 7-KC and Ind.
As a redox-sensitive transcription factor, NF-κB has recently been found to be involved in the early response of monocytes–macrophages to 7-KC injury(Reference Palozza, Simone and Catalano38, Reference Huang, Liu and Li41). In quiescent cells, NF-κB combines with its inhibitor κ Bα (Iκ-Bα) in cytosol. Upon cell activation, Iκ-Bα undergoes phosphorylation and ubiquitination-dependent degradation, thus leading to nuclear translocation of NF-κB. A Western blotting analysis of THP-1 cells treated with 7-KC for 12 h showed a net decrease of the cytosolic Iκ-Bα with increase of its phosphorylated form (phospho-Iκ-Bα), accompanied by nuclear translocation of the NF-κB p65 subunit. NF-κB activation was completely prevented by co-incubating 7-KC and Ind (Fig. 5).
Effect of indicaxanthin (Ind) on 7-ketocholesterol (7-KC)-induced Iκ-Bα (NF-κB with its inhibitor κ Bα) degradation and p65 nuclear translocation in THP-1 cells. Cellular (phospho Iκ-Bα and Iκ-Bα) and nuclear (p65) lysates were obtained as reported in the Experimental methods section. Representative image of three separate Western blotting analysis with comparable results and densitometric analyses of immunoblot. Data of the densitometric analysis are means and standard deviations. a,bMean values with unlike letters were significantly different (P< 0·05; Fisher's test). Control (■), cells treated with vehicle; 7-KC (□), cells treated for 12 h with 7-KC (16 μm); 7-KC/Ind (), cells treated with 7-KC in the presence of Ind (2·5 μm).
Inhibitory effect of indicaxanthin on 7-ketocholesterol-induced intracellular Ca2+ elevation
A rise in cytosolic-free Ca (Ca2+)i is considered an early step in the signalling cascade induced by oxysterols. A Fluo-3/AM staining, followed by flow cytometry analysis, was performed to measure the Ca2+ content in THP-1 cells after treatment with 7-KC, either in the absence or in the presence of Ind, during the time interval between 30 min and 6 h. In comparison with control cells, a 7-KC-dependent (Ca2+)i elevation was not evident at 30 min, whereas the Ca2+-associated fluorescence clearly increased at 1 h of treatment and high levels were maintained for the following 5 h (Fig. 6(A)). THP-1 cells co-incubated with 7-KC and Ind showed a staining intensity quite comparable with control, with total prevention of the oxysterol-induced increase of cytosolic Ca2+ along the entire incubation time (Fig. 6(A)). The dose-dependent effect of Ind on the intracellular Ca elevation, monitored after 6 h incubation in the presence of 7-KC, is shown in the inset.
7-Ketocholesterol (7-KC)-induced (A) increase of free intracellular Ca2+ (B) and decrease of phospho-Bcl-2 antagonist of cell death ser99 levels with densitometric analysis of the immunoblots in THP-1 and effect of indicaxanthin (Ind) cells. (A) Ca2+ levels were assayed after cell loading with fluo-3/AM followed by flow cytometry analysis. Data are means and standard deviations of three separate experiments carried out in duplicate. The inset shows dose-dependent effects of Ind in the presence of 7-KC (16 μm) after 6 h incubation. (B) Representative image of three separate Western blotting analyses with comparable results. a,bMean values with unlike letters were significantly different (P< 0·05; Fisher's test). Control, cells treated with vehicle; 7-KC, cells treated for 12 h with 7-KC (16 μm); 7-KC/Ind, cells treated with 7-KC in the presence of Ind (2·5 μm). MFI, mean fluorescence intensity.
7-KC-dependent (Ca2+)i elevation is associated with the activation of the phosphatase calcineurin, one target of which is the pro-apoptotic protein Bcl-2 antagonist of cell death (BAD)(Reference Berthier, Lemaire-Ewing and Prunet42). Phosphorylated BAD in its serine 75 and/or 99 is bound to the 14-3-3 protein in the cytoplasm(Reference Zha, Harada and Yang43). Once dephosphorylated, BAD translocates to the mitochondria, where it allows cytochrome c release following heterodimerisation(Reference Wang, Pathan and Ethell44) with the anti-apoptotic proteins Bcl-2 and Bcl-XL(Reference Yang, Zha and Jockel45). Western blot analyses performed with phospho-BAD ser 99-specific antibody showed that treatment of THP-1 cells with 7-KC decreased the phosphorylated BAD levels by more than 95 % compared with control cells, whereas co-treatment with Ind prevented this effect, thus inhibiting BAD activation (Fig. 6(B)).
Discussion
Cytotoxic oxysterols such as 7-KC have been implicated in the pathophysiology of atherosclerosis, where they induce apoptosis in cells of the vascular wall and in monocytes/macrophages(Reference Palozza, Serini and Verdecchia39, Reference Biasi, Leonarduzzi and Vizio46–Reference Lizard, Gueldry and Sordet48), thus contributing to the development of atheromatous plaque(Reference Tabas49). In the present study, evidence is provided that the phytochemical Ind, at a concentration achievable in human subjects after dietary supplementation with cactus pear fruit(Reference Tesoriere, Allegra and Butera16), prevents 7-KC-induced apoptosis of human monocyte/macrophage THP-1 cells by preventing alteration of the cell oxidative balance and dysregulation of intracellular Ca homeostasis. The 7-KC-induced arrest of cell cycle progression and other pro-apoptotic events, such as nuclear morphological changes, externalisation of phosphatidylserine in the plasma membrane, depolarisation of mitochondrial membrane and PARP cleavage, were completely inhibited by the co-incubated phytochemicals. As Ind did not affect the incorporation of 7-KC in the cells, its action was investigated taking into account oxysterol activities known to be associated with cell toxicity. Oxysterols affect cell processes by biochemical and membrane biophysical mechanisms(Reference Massey and Pownall50–Reference Vejux, Malvitte and Lizard52). As far as 7-KC is concerned, it has been suggested that it modulates cell processes possibly through effects within the plasma membrane(Reference Berthier, Lemaire-Ewing and Prunet42). 7-KC transfers spontaneously between membranes, partitions in the less densely packed Ld domains and has a tendency to destabilise membrane rafts(Reference Olkkonen and Hynynen53, Reference Massey and Pownall54), which may affect raft-associated functional proteins, including Ca channels(Reference Berthier, Lemaire-Ewing and Prunet42), and possibly perturb the activity of signalling enzymes including NADPH oxidase(Reference Han, Li and Villar55, Reference Shao, Segal and Dekker56). Indeed, two events have been reported as pivotal to stimulate an apoptotic process after challenging THP-1 cells with 7-KC, influx of free Ca into the cytosol and intracellular overproduction of ROS(Reference Leonarduzzi, Vizio and Sottero40, Reference Berthier, Lemaire-Ewing and Prunet42).
NOX-4, the constitutively active homologue of the NOX family of membrane NADPH oxidase(Reference Katsuyama, Matsuno and Yabe-Nishimura57), is considered the source of ROS following stimulation of macrophages with oxidised lipids(Reference Palozza, Simone and Catalano38, Reference Palozza, Serini and Verdecchia39, Reference Larsson, Baird and Nyhalah47, Reference Lee, Qiao and Schröder58). The enzyme, which is known to continuously produce low ROS levels with a signalling function(Reference Martyn, Frederick and von Loehneysen37, Reference Leto, Morand and Hurt59, Reference Rhee, Kang and Jeong60), under physiological conditions does not affect macrophage viability. Increased levels of the enzyme activity are considered to depend on the protein expression(Reference Serrander, Cartier and Bedard61). In macrophages, NOX-4 can be over-expressed after certain stimuli such as oxLDL(Reference Lee, Qiao and Schröder58) and 7-KC(Reference Palozza, Simone and Catalano38, Reference Palozza, Serini and Verdecchia39), leading to a high and sustained ROS production. In THP-1 cells, in particular, up-regulation of NOX-4 has been shown to be required for 7-KC-induced apoptosis(Reference Palozza, Serini and Verdecchia39, Reference Lee, Qiao and Schröder58). Searching for the effect of Ind on the redox status of THP-1 cells after 7-KC treatment, the intracellular levels of ROS and thiols were monitored for 24 h from the addition of the oxysterol. A very early and slowly increasing ROS production was observed, followed by a sustained gradual rise due to over-expression of NOX-4, with a parallel depletion of total thiols, indicating a cell redox environment irreversibly compromised. Under these conditions, impairment of mitochondrial function is expected, with induction of an apoptotic sequence(Reference Mignotte and Vayssiere62, Reference Hall63). In accordance with our observations, a time- and peroxide-dependent decrease of reduced glutathione has been shown to be substantially implicated in the control of the 7-KC-induced apoptosis in U937 monocytes(Reference Lizard, Gueldry and Sordet48). The finding that co-incubated Ind completely prevented early oxidative events, as well as up-regulation of NOX-4, and maintained the cell redox balance suggested that downstream events sensitive to cell redox changes, including those contributing to apoptotic death, were affected by the phytochemicals. The observed early formation of ROS in a time preceding NOX-4 over-expression suggests some acute enzyme regulation by 7-KC. Molecular mechanisms of NOX-4 activation are still unknown; however, acute regulation of the NOX-4 activity has been reported, e.g. in adipose cells after stimulation with insulin(Reference Anilkumar, Weber and Zhang64, Reference Mahadev, Motoshima and Wu65), in human aortic endothelial cells by oxidised phospholipids through rac1 translocation(Reference Lee, Gharavi and Honda66) and in cardiac myocytes, where a local regulatory role of Ca2+ on the enzyme activity has been shown(Reference Zhang, Jin and Yi67). As far as immune-competent cells and oxysterols are concerned, a prompt overproduction of ROS has also been observed in murine macrophages after 7-KC stimulation(Reference Leonarduzzi, Vizio and Sottero40).
Specific molecular targets of oxidation mediating cellular responses to the NADPH oxidase may be different in different cells(Reference Chen, Craige and Keaney68); ROS, however, serve as a common intracellular messenger for NF-κB activation(Reference Schreck, Rieber and Baeuerle69, Reference Dröge70). In the present as well as other studies with human monocyte cell lines(Reference Palozza, Simone and Catalano38, Reference Huang, Liu and Li41), NF-κB activation has been found to be associated with 7-KC pro-inflammatory and pro-apoptotic effects. In line with the protection of the redox state of the cell environment, our data show that Ind inhibited the 7-KC-stimulated ROS-dependent activation of NF-κB.
Increase of cytoplasmic Ca2+ concentration is one of the most rapid cellular response upon exposure to various oxysterol congeners, including 7-KC, and precedes events of the apoptotic cascade(Reference Mackrill71, Reference Lordan, O'Brien and Mackrill72), i.e. activation of the Ca-dependent phosphatase calcineurin, dephosphorylation, activation and translocation to mitochondria of the pro-apoptotic protein BAD, release of cytochrome c in the cytosol and caspase activation(Reference Wang, Pathan and Ethell44, Reference Yang, Zha and Jockel45). Ind totally prevented the 7-KC-induced intracellular Ca elevation, as well as the downstream pro-apoptotic events such as dephosphorylation of BAD, mitochondrial depolarisation and caspase 3-dependent PARP cleavage, which provides a rationale for the cytoprotective effect through interference with the mitochondria-mediated apoptotic process(Reference Mignotte and Vayssiere62, Reference Panini and Sinensky73). Finally, under the conditions applied in the present study, the increase of intracellular ROS after treatment with 7-KC preceded Ca elevation. 7-KC has been reported to promote the influx of Ca2+ across the THP-1 plasma membrane through distinct mechanisms, including perturbation of the membrane lipid rafts followed by translocation of Ca2+ channels into the rafts(Reference Berthier, Lemaire-Ewing and Prunet42) and/or interaction with subunits of Ca channels, leading to modulation of their gating or electrophysiological properties(Reference Massey74). Ca ion channels may be redox regulated(Reference Ermak and Davies75, Reference Bogeski, Kappl and Kummerow76). The present results may suggest a role of ROS in promoting the 7-KC-stimulated Ca influx in our THP-1 cell system.
The protective activity of redox-active and antioxidant natural compounds against oxysterol-induced apoptosis of macrophages has been the object of some research(Reference Palozza, Simone and Catalano38, Reference Palozza, Serini and Verdecchia39, Reference Lizard, Gueldry and Sordet48). It has been shown that the ability of antioxidants to counteract 7-KC-induced apoptosis does not only depend, and cannot simply be explained, by their capability to inhibit production of ROS(Reference Lizard, Miguet and Besséde77). In this context, the present study shows that the anti-apoptotic effect of Ind is associated not only with maintaining of the cell redox balance, but also with the inhibition of the 7-KC-dependent elevation of Ca2+ in the cell cytosol. A mechanism explaining, at a molecular level, the activity of Ind cannot definitely be assessed at this time; however, previous studies and present findings allow some speculation to be made. In aqueous systems Ind can partition into either saturated or unsaturated phosphatidylcholine liposomes, where it locates at the so-called palisade domain, between the core hydrophobic region and the polar head groups(Reference Tesoriere, Allegra and Butera5–Reference Turco-Liveri, Sciascia and Lombardo7). Such a location would allow Ind to react with radicals generated and/or adsorbed onto the membrane surface(Reference Tesoriere, Allegra and Butera5), or possibly modulate membrane processes by biophysical mechanisms. We observed that Ind alone did not scavenge ROS formed by the basal activity of NOX-4; however, the enzyme appeared to be regulated negatively by the concurrent action of Ind and 7-KC. Biophysical variations of the membrane, in particular at the raft microdomain, are known to modulate NOX-4(Reference Han, Li and Villar55, Reference Shao, Segal and Dekker56). As destruction of membrane rafts is a consequence of 7-KC treatment(Reference Massey and Pownall50, Reference Olkkonen and Hynynen53), it is tempting to speculate that Ind and 7-KC interacting at the THP-1 cell membrane could bring about some stabilising effect, leading to modulate NOX-4 enzyme activity and possibly prevent opening of Ca channels. It is important, however, that no effect was elicited by Ind alone on the cell redox status, as in contrast with abnormal cells, e.g. the tumour ones, interference of redox-active compounds with the redox environment of normal cells might interfere with certain physiological cellular and organ functions, and would thus eventually be detrimental rather than beneficial.
Previous studies have shown that the regular consumption of cactus pear fruits positively affects the body's redox balance, decreases oxidative damage to lipids and improves antioxidant status in healthy human subjects(Reference Tesoriere, Butera and Pintaudi78). The present findings demonstrate that Ind, a highly bioavailable phytochemical(Reference Tesoriere, Allegra and Butera16), characteristic of these fruits, can prevent the 7-KC-induced apoptosis of human macrophages when assayed at nutritionally relevant concentrations. Though these seasonal fruits are not common, fruit derivatives, such as juices or concentrated extracts to be added to other food preparations (yogurt, other fruit juices, etc), can easily be prepared by industrial food manufacturers. The stability of Ind(Reference Tesoriere, Fazzari and Allegra79) may allow such treatments. Given the importance of oxidised cholesterol derivatives in the pathogenesis of atherosclerosis, the present findings providing new information on the bioactive potential of Ind further highlight the contribution of O. ficus indica fruits to human health.
Acknowledgements
The present study has been supported by grants from the Ministero dell'Istruzione, Università e Ricerca (University of Palermo, Fondi di Ateneo ex 60 %). L. T. planned the study and discussed the results; M. A., C. G., A. A. provided methodological assistance; M. A. L. coordinated the work and discussed the results. None of authors has a conflict of interest.
