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National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
2To whom correspondence should be addressed. E-mail: nagao{at}nfri.affrc.go.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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20 µmol/L. Although neoxanthin induced evident apoptotic cell death, (R/S)-neochrome inhibited the cell proliferation without obvious apoptosis induction. These results indicate that dietary neoxanthin is partially converted into (R/S)-neochrome by intragastric acidity before intestinal absorption and that (R/S)-neochrome exhibits an antiproliferative effect on PC-3 cells by the induction of cytostasis.
KEY WORDS: • carotenoid • neoxanthin • neochrome • in vitro digestion • cell cycle
Epidemiologic studies demonstrated that the consumption of diets rich in fruits and vegetables is associated with a decreased risk of certain forms of cancer (1). As a representative of carotenoids in fruits and vegetables, ß-carotene was hypothesized to be a chemopreventive phytochemical (2). However, several human intervention trials did not prove the hypothesis (3). Hence, other phytochemicals, including carotenoids other than ß-carotene, may contribute to the lowered cancer risk associated with the intake of fruits and vegetables.
Among the carotenoids, in addition to ß-carotene and lutein, 5,6-epoxyxanthophylls such as neoxanthin and violaxanthin (Fig. 1) are ubiquitously distributed in the photosynthetic organs of higher plants (4,5). The 5,6-epoxyxanthophylls are essential components of the light-harvesting pigment-protein complex II of higher plants (6), and are precursors of abscisic acid, a plant hormone (7). Neoxanthin is therefore one of the major carotenoids in green leafy vegetables, and constitutes a considerable portion of dietary carotenoids. Neoxanthin was reported to inhibit chemically induced carcinogenesis in hamster buccal pouch (8). Recently, we demonstrated that neoxanthin reduced the viability of prostate cancer cells to a greater extent than other carotenoids present in foodstuffs such as ß-carotene, ß-cryptoxanthin, lutein, and lycopene (9). Among men in the United States, prostate cancer is the most frequent form of cancer and the second leading cause of cancer death (10). Because the high consumption of vegetables is epidemiologically associated with a reduced risk of prostate cancer (11), various dietary phytochemicals, including carotenoids, may contribute to prostate cancer chemoprevention (12).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Preparation of carotenoids from spinach. All procedures for carotenoid preparation and extraction were carried out under dim yellow light to minimize the degradation and isomerization of carotenoids by light irradiation. Lutein, neoxanthin, and violaxanthin were isolated from spinach by alumina column chromatography (14,15). Each carotenoid fraction was then further purified by preparative HPLC on a TSK-gel ODS 80Ts column (10 x 250 mm; Tosoh) with acetonitrile:methanol:water (75:15:10 for lutein, or 65:15:20 for violaxanthin and neoxanthin, by vol) containing 0.1% (wt/v) ammonium acetate as a mobile phase.
The purity of each carotenoid prepared was >99%, based on the peak area of all components absorbed at each specific wavelength in HPLC analysis. The extinction coefficient of each carotenoid was used for quantification (16). The geometrical conformation of the purified neoxanthin was confirmed as 9'-cis (9'-cis-neoxanthin) by the nuclear Overhauser effect spectroscopy (NOESY) spectrum, especially the NOE correlation between H-8' and H-11', recorded on an AVANCE 800 NMR instrument (Bruker BioSpin). The 9'-cis conformation of neoxanthin was reported to be the sole molecular form in plant photosynthetic organs, such as spinach leaves (5).
Preparation of neochrome stereoisomers.
Stereoisomers of neochrome (Fig. 1A) were prepared from the purified neoxanthin by acid-catalyzed epoxide–furanoid rearrangement (13). In brief, 50 µL of 0.1 mol/L HCl was added to the ethanolic solution (5 mL) of neoxanthin (
2 µmol), and the UV-visible absorption spectrum was monitored. After the rearrangement was completed (30 min), neochrome was extracted with diethyl ether. Each stereoisomer was then separately purified by preparative HPLC, and quantified using the extinction coefficient as described above. The stereochemical configuration at the 8' position was determined by the 1H NMR spectra (17). The all-trans geometrical conformation of each stereoisomer confirmed by the NOESY spectrum, especially the NOE correlation between H-8' and Me-19', indicated that the 9'-cis conformation of neoxanthin was converted to the all-trans conformation of (R/S)-neochrome during the rearrangement.
Oral administration of neoxanthin to mice. All procedures were conducted in accordance with the Guidelines for Experimental Animals of the National Food Research Institute, Japan. Male ICR mice (7 wk old; Clea Japan) were housed at 25°C with a 12-h light:dark cycle and acclimated with free access to an MF standard rodent diet (oriental Yeast) and tap water. After 7 d of feeding, the mice were deprived of food for 15 h before treatment. Neoxanthin (40 nmol) solubilized in 0.2 mL of mixed micelles was administered to each mouse by direct stomach intubation. The mixed micelles containing 12 mmol/L sodium taurocholate, 2.5 mmol/L monoolein, 7.5 mmol/L oleic acid, 1.25 mmol/L lysophosphatidylcholine, and 0.2 mmol/L neoxanthin were prepared as previously described (18). Control mice received mixed micelles prepared similarly without neoxanthin.
Two hours after the administration, each mouse was anesthetized with diethyl ether, and blood was collected from the caudal vena cava with a heparinized syringe. Plasma was prepared by centrifugation of blood at 1000 x g for 15 min at 4°C. Immediately after the blood sample was taken, the liver and small intestine were excised. The liver was rinsed with ice-cold saline, and the small intestinal contents were collected by washing out using a syringe with ice-cold saline. All samples were stored at –80°C and analyzed within 1 wk. The time point for sampling was selected because in previous studies, ß-carotene, lutein, and fucoxanthin administered orally to mice reached maximum concentrations in plasma at 2 h after the doses (18,19).
Carotenoids in the plasma and livers were extracted with dichloromethane as previously described (18). Carotenoids in the small intestinal contents were extracted with 4 volumes of dichloromethane:methanol (2:1, v/v). Analytical HPLC was carried out with an HP-1100 System (Agilent Technologies). A TSK-gel ODS 80Ts column (2 x 250 mm, Tosoh) attached to an ODS guard column (2 x 10 mm, Tosoh) was used with a mobile phase of acetonitrile:methanol:water (75:15:10, by vol) containing 0.1% (wt/v) ammonium acetate at a flow rate of 0.3 mL/min. The eluate was monitored with a photodiode array detector (250–550 nm), and carotenoids were quantified from their peak area at 450 nm by use of calibration curves of authentic standards.
In vitro digestion of spinach.
The in vitro digestion of spinach was carried out based on a method simulating the human digestion system (20,21). Washed spinach leaves were blanched for 2 min and cooled down quickly. The boiled leaves were then cut into small pieces and subsequently ground well in a mortar with a pestle. The ground sample (0.5 g) was then mixed in a 15-mL polypropylene tube with 3 mL of 0.5% pepsin solution (pH 2.0 or 3.0 with HCl) containing 3.6 mmol/L CaCl2, 1.5 mmol/L MgCl2, 49 mmol/L NaCl, 12 mmol/L KCl, and 6.4 mmol/L KH2PO4 (21). After the pH of the mixture was adjusted to 2.0 or 3.0 with 1 mol/L HCl, the mixture was incubated at 37°C in the dark with orbital shaking (Bio-Shaker BR-30 LF; TAITEC) at 210 rpm for 1 h (gastric phase digestion). The pH was then raised to 5 with 1 mol/L NaHCO3 followed by the addition of a mixture (3 mL) of pancreatin (4 g/L) and bile salt (25 g/L) dissolved in 0.1 mol/L NaHCO3. After the pH was further raised to 7.5 with 2 mol/L NaOH, the mixture was incubated again at 37°C with orbital shaking (210 rpm) for 2 h (intestinal phase digestion).
After the intestinal phase digestion, the digest was immediately centrifuged at 4200 x g for 20 min at 4°C, and the supernatant was filtrated with a 0.2-µm filter to remove debris. Digested carotenoids were extracted from the filtrate with 4 volumes of dichloromethane:methanol (2:1, v/v). Total carotenoids in spinach leaves were also extracted from the ground spinach with dichloromethane:methanol (2:1, v/v) until the dichloromethane phase was apparently colorless. HPLC analysis was performed with the same apparatus and column as described above. Analytes were eluted at a flow rate of 0.2 mL/min with acetonitrile:methanol:water (75:15:10, by vol) containing 0.1% (wt/v) ammonium acetate for 0–45 min to elute xanthophylls, followed by methanol/ethyl acetate (70:30, v/v) containing 0.1% (wt/v) ammonium acetate for 45–70 min to elute ß-carotene. For MS analysis, the HPLC system was connected to an LCQ mass spectrometer (Thermo Finnigan) equipped with an interface of atmospheric pressure chemical ionization as previously described (18).
Antiproliferative activity on PC-3 Cells. PC-3 human prostate cancer cells were obtained from the American Type Culture Collection. The cells were cultured in DMEM supplemented with 10% FBS, 4 mmol/L L-glutamine, 40,000 IU/L penicillin, and 40 mg/L streptomycin (complete medium), in a 5% CO2 humidified atmosphere at 37°C.
For the cell proliferation assay, PC-3 cells were seeded in 96-well plates at a density of 5000 cells/well with 100 µL of complete medium and incubated for 24 h. The medium was then replaced with fresh complete medium supplemented with neoxanthin, (8'R)-neochrome, or (8'S)-neochrome. The carotenoids, dissolved in dimethyl sulfoxide (DMSO), were added to the culture medium at final concentrations of 1.25–20 µmol/L. The control cells received the vehicle (0.4% DMSO) alone. Just before (0 h) and 24, 48, and 72 h after the treatment, cell viability was evaluated by MTT assay (22) with a modified extraction buffer (23). The 50% inhibitory concentration (IC50) of the cell proliferation was calculated from a 4-parameter logistic regression equation in SigmaPlot software version 6.0 (SPSS).
For Western blot analysis, cells were seeded in 10-cm dishes (5 x 105 cells/dish with 10 mL of complete medium) and treated with the carotenoids as described above at a dose of 10 µmol/L for 48 h. After the treated cells were harvested and washed twice with ice-cold PBS, cells were sonicated twice for 10 s in a loading buffer (New England BioLabs). The cell lysates were then boiled for 5 min and chilled on ice. An aliquot of the lysates was taken and the protein was precipitated with 10% trichloroacetic acid to determine the protein content using a DC protein assay kit (Bio-Rad). An equal amount of protein (40 µg) in the loading buffer was subjected to 7.5% (for PARP) or 12% (for cleaved caspase-3 and ß-actin) SDS-PAGE and transferred to nitrocellulose membrane. After being blocked for 1 h at room temperature with 50 g/L bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20, membranes were incubated with specific antibodies overnight at 4°C, followed by alkaline phosphatase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive proteins were then visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate.
For cell cycle analysis and fluorescence microscopy, cells were seeded in 6-well plates (1 x 105 cells/well with 2 mL of complete medium) and treated with the carotenoids (10 µmol/L) for 48 h. Cell cycle analysis was carried out with a Beckton Dickinson FACScan flow cytometer using CellQuest software (BD Immunocytometry Systems). Briefly, cells were harvested with trypsin and fixed in 70% ethanol overnight at 4°C. The cells were then treated with RNase and stained with propidium iodide (25 mg/L in PBS). Ten thousand cells were examined for each sample. Cell cycle distribution was analyzed by ModFit LT software (Verity Software House). Fluorescence microscopy was performed with a Nikon IX70 inverted fluorescence microscope (Nikon) after Hoechst 33342 (Calbiochem) in PBS was added directly to the culture medium to a final concentration of 5 mg/L.
Statistical analysis. The results are expressed as means ± SD. Statistical analysis was carried out using ANOVA with Scheffé’s F-test in StatView ver. 4.5J statistical software (Abacus Concepts). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Antiproliferative effect of neoxanthin and neochrome on PC-3 cells. Both neoxanthin and (R/S)-neochrome inhibited the proliferation of PC-3 cells in a dose-dependent manner (Fig. 4). The IC50 values of the cell proliferation during the 72-h culture were 2.9, 1.2, and 5.3 µmol/L for neoxanthin, (8'R)-neochrome, and (8'S)-neochrome, respectively (Fig. 4B). Apoptotic chromatin condensation and nuclei fragmentation occurred only in neoxanthin-treated cells at a dose of 10 µmol/L, although the cell density was lower in both neoxanthin- and neochrome-treated cells than in vehicle-treated control cells (Fig. 5A). Cleavage of caspase-3 and PARP, biochemical hallmarks of apoptosis, was also observed only in the neoxanthin-treated cells (Fig. 5B). The treatment of cells with 10 µmol/L neoxanthin and (R/S)-neochrome significantly altered the cell cycle distribution (Fig. 5C and D). Treatment with both neoxanthin and (R/S)-neochrome led to a remarkable reduction of cells in the S phase (P < 0.01) and a significant accumulation in the G2/M phase [neoxanthin and (8'R)-neochrome, P < 0.01; (8'S)-neochrome, P = 0.02] compared with vehicle-treated cells. Cells in the G0/G1 phase also accumulated after treatment with (R/S)-neochrome [(8'R)-neochrome, P = 0.02; (8'S)-neochrome, P < 0.01].
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the present study, we demonstrated for the first time that a substantial portion of neoxanthin orally administered to mice was converted into neochrome stereoisomers in the gastrointestinal tract before absorption. Consequently, both neoxanthin and (R/S)-neochrome appeared in the plasma and liver. Because 5,6-epoxide in carotenoid is readily isomerized to 5,8-epoxide in an acidic condition (13), the epoxide–furanoid rearrangement of neoxanthin in mice was an expected result, one that would be mediated by gastric acid. However, the study in mice using purified neoxanthin did not consider the effect of the food matrix, which affects the bioavailability of dietary carotenoids (25–27). We therefore examined the digestion of spinach, one of the major dietary sources of neoxanthin, in vitro to evaluate the influence of intragastric acidity on the rearrangement of neoxanthin in the food matrix. As a result, the gastric phase acidity strongly affected the conversion of spinach neoxanthin into neochrome. Because the intragastric acidity of healthy humans, pH 1 at baseline, increases transiently to pH > 4 after meal ingestion and remains at pH > 2 for a few hours (28,29), the result suggests that the extent of the rearrangement of dietary neoxanthin would vary with the timing of neoxanthin-containing food intake. In addition, food processing and other food ingredients such as lipids may also modulate the rearrangement by affecting the interaction of dietary neoxanthin with gastric acid.
The present results strongly suggest the intestinal absorption of dietary neoxanthin in part as neochrome. Recently, we also demonstrated that orally administered fucoxanthin, a 5,6-epoxyxanthophyll in edible brown algae, was absorbed as fucoxanthinol in mice (18,30). However, until now, there was no direct evidence of the absorption of 5,6-epoxyxanthophylls in humans. Barua and Olson (31) reported that after a single oral dose (10 mg) of either violaxanthin or lutein 5,6-epoxide, no violaxanthin, lutein 5,6-epoxide, or any of their 5,8-epoxide metabolites in human plasma were detected, whereas ß-carotene 5,6-epoxide was well absorbed (32). Pérez-Gálvez et al. (33) reported that neither violaxanthin nor capsanthin 5,6-epoxide was detected in paprika oleoresin-ingested (violaxanthin, 2.38 mg; capsanthin 5,6-epoxide, 1.83 mg) human chylomicrons. In the present study, the digestion efficiency of neoxanthin during the in vitro digestion of spinach exceeded that of ß-carotene, and was comparable to or higher than that of lutein (Fig. 3E). However, our previous study (15) demonstrated that the uptake of carotenoids from mixed micelles by Caco-2 cells, a model of human intestinal epithelium, was simply dependent on the lipophilicity of the carotenoids; consequently, the uptake of relatively polar epoxyxanthophylls such as neoxanthin and violaxanthin was much lower than that of carotenoids commonly shown in human plasma such as ß-carotene, ß-cryptoxanthin, and lutein. Hence, the lack of evidence for the absorption of 5,6-epoxyxanthophylls in humans may be due in part to the lower uptake of polar carotenoids by intestinal epithelial cells. Furthermore, the generation of 5,8-epoxide stereoisomers with hypsochromic shift (13) may also make it difficult to detect 5,6-epoxyxanthophyll–derived carotenoids in plasma and other tissues. In the case of violaxanthin, a variety of 5,8-epoxide isomers (Fig. 1B) would be generated in the gastrointestinal tract as found in the present in vitro digestion study (Fig. 3B and C). In addition, a number of minor metabolites of carotenoids present in human serum (34) may interfere with the detection of a trace amount of 5,6-epoxyxanthophyll–derived carotenoids in HPLC analysis. To clarify the absorption and metabolism of dietary 5,6-epoxyxanthophylls in humans, further studies should be carefully designed to identify 5,6-epoxyxanthophyll–derived metabolites in biological samples.
Although our previous study showed the potent antiproliferative effect of neoxanthin on prostate cancer cell lines (9), the present results indicate that dietary neoxanthin is converted in part into (R/S)-neochrome before the intestinal absorption. Therefore, we also examined the antiproliferative activity of (R/S)-neochrome on PC-3 human prostate cancer cells. At doses 2.5 µmol/L, (8'R)-neochrome suppressed the cell proliferation to a greater extent than neoxanthin (Fig. 4B). Thus, (8'R)-neochrome might act as a chemopreventive agent in vivo at lower concentrations; however, the tissue distribution of neochrome is not known at present. Both neoxanthin and neochrome dose dependently suppressed the proliferation of PC-3 cells. However, the antiproliferative effects of neoxanthin and neochrome likely occur by somewhat different mechanisms. In the present cell culture study, no neochrome stereoisomers were detected in neoxanthin-treated cells, whereas unchanged neoxanthin was shown predominantly (data not shown). Thus, the growth inhibition observed in neoxanthin-treated cells would be attributable to the action of neoxanthin itself or its metabolites, which were not generated via (R/S)-neochrome. Neoxanthin induced apoptosis in PC-3 cells and decreased cell viability at doses 
10 µmol/L during the 72-h culture. On the other hand, neither (8'R)- nor (8'S)-neochrome induced obvious apoptotic cell death; consequently, cell viability was retained at a level nearly identical to that of initial (0 h) cells even after the 72-h culture at a dose of 20 µmol/L (Fig. 4). This result suggests that the antiproliferative effect of (R/S)-neochrome on PC-3 cells is due to the induction of cytostasis rather than apoptotic cell death. The reduction of cells in the S phase (Fig. 5C and D) indicates restriction of the G1/S transition with the treatment of neoxanthin and (R/S)-neochrome. In addition, the accumulation of cells in the G2/M phase suggests that the treatment of neoxanthin and (R/S)-neochrome inhibited the G2/M transition and/or cell division. The cell cycle disturbance induced by neoxanthin and neochrome warrants further investigation of the cell cycle machinery and its upstream signaling pathways. The suppression of cell cycle propagation was proposed as a target for prostate cancer prevention by dietary phytochemicals (35). Recently, Obermüller-Jevic et al. (36) reported that lycopene inhibited the cell cycle progression of normal prostate epithelial cells. Hyperproliferation of prostate epithelium is a major cause of benign prostate hyperplasia.
In conclusion, the present results demonstrate that neoxanthin, a major carotenoid in green leafy vegetables, is converted into neochrome stereoisomers by gastric acid. The in vitro digestion study suggests that intragastric acidity strongly affects the extent of the epoxide–furanoid rearrangement of dietary neoxanthin and other 5,6-epoxyxanthophylls. The observed antiproliferative effect of neoxanthin and neochrome on PC-3 prostate cancer cells warrants further study, especially in regard to the metabolism, tissue distribution, and chemopreventive mechanism of neoxanthin.
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FOOTNOTES |
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3 Abbreviations used: DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; IC50, 50% inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NOE(SY), nuclear Overhauser effect (spectroscopy); PARP, poly (ADP-ribose) polymerase.
Manuscript received 17 May 2004. Initial review completed 4 June 2004. Revision accepted 20 June 2004.
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