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Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611
3 To whom correspondence should be addressed. E-mail: percival{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • antineoplastic transformation • antioxidant • cancer • mango • phytochemicals
Epidemiological evidence supports the health benefits of fruit and vegetable consumption beyond that which can be accounted for by nutrient content. Fruit and vegetable consumption is associated with a reduced risk of heart disease and some cancers. There is no agreement about whether this reduction is due to a combination of nutrients, replacement of fat in the diet, or the presence of nonnutritive phytochemicals in these plant foods. Although the specific roles of nonnutritive phytochemicals are not yet understood, studies have demonstrated mechanisms relating to antioxidant potential, as well as the ability to interact with vital cell processes. The broad, overall goal of our laboratory is to understand how phytochemicals derived from whole foods interact in cancer cell culture models. The development of in vitro screening procedures to identify chemopreventative foods or their chemopreventative fractions is important in identifying potentially beneficial foods or diets best suited for cancer prevention.
The mango (Mangifera indica L.) is a phytochemically dense food with high levels of carotenoids and phenolic compounds (1–3). Gorenstein et al. (4) examined 8 tropical fruits and showed that ripe mango had the highest gallic acid content and total polyphenolics compared with other fruits. Mercandante and Rodriquez-Amaya (5) examined the wide variety of carotenoids in the mango, finding that all-trans-violaxanthin was the predominant carotenoid, followed by all-trans-ß-carotene. Botting et al. (6) examined the antimutagens in 25 plant foods, using the Salmonella typhimurium mutagenicity assay against heterocyclic amine 2-amino-3-methylimidazo [4,5-f] quinoline. Data obtained from that study indicated strong antimutagenic properties in several plants, including mango. The presence of polyphenolics, carotenoids, and antimutagens in the mango suggests significant anticancer activity. Our specific objective here was to extend these observations to determine the anticancer activity of mango in mammalian cell culture systems.
Two cell culture assays were developed to examine in vitro anticancer activity. Cell cycle kinetics of HL-60 cells were evaluated in the presence and absence of different mango fractions. Isolated single compounds such as quercetin are known to inhibit the cell cycle of these cells (7), but whole foods have not been widely assayed in this model system. Since the compounds found in mango may interact synergistically, additively, or antagonistically (8), we wanted to examine the impact of the whole food, as well as fractions of the food, on cells. Another model system employed in this study was cell transformation assay. Neoplastic transformation of BALB/c 3T3 cells was chemically induced with a polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP)4, and the inhibition of foci formation by mango fractions was determined.
While testing mango, we were also interested in developing laboratory screening methods to determine the anticancer potential of foods. Although it is evident that anticancer activity determined in cell culture models cannot be extrapolated to food consumption, our goal was to find a biologically relevant methodology to study and compare foods that might have anticancer potential. We hypothesized that the mango would show evidence of substantial anticancer activity due to its rich composition of phytochemicals.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Seed and peels were removed from the edible fruit of mangos (M. indica L., cv. Palmer) that had been harvested at the height of the season (May–June) in Homestead, Florida. Mangos were examined and only sound, fully ripened fruits were used. The fruits were pureed and stored in aliquots at –20°C until needed. The puree was thawed, mixed 1:1 (w:v) with MilliQ water and sonicated. After centrifugation at 9000 x g for 20 min at 5°C, the supernatant was set aside. The pellet was resuspended in MilliQ water, sonicated, and centrifuged and the procedure was repeated a third time before supernatants were pooled and sterile filtered through a 0.22-µm pore filter. Sterile supernatant samples were stored at –85°C until utilized in cell culture. This preparation is referred to as whole mango juice and is devoid of pectin and other large polysaccharides.
Additional mango preparations were obtained from the whole mango juice by methanol extraction. The whole mango juice was bound to a SepPak C18 cartridge (Waters Association). The fraction containing compounds that did not bind to the cartridge was collected (Unbound), as well as fractions that were sequentially eluted with 25% methanol, 50% methanol, and 100% methanol, respectively. The solvent was removed from each of these fractions under reduced pressure and all fractions were adjusted to a 4x concentration with water. Data expression is based on single-strength, not concentrated, mango juice. Results of the HPLC-PDA analysis of these sequential fractions are shown in Supplemental Figure 1.
Antioxidant activity
Three methods were used to compare the antioxidant activity of mango juice and fractions obtained from the SepPak C18 cartridge.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) was prepared in methanol at 2.5 mmol/L. Ten to 50 µL of each sample were brought to 1.0 mL with the DPPH solution. The decrease in absorbance at 517 nm was monitored and the final reading taken when the reaction reached a plateau between 15 and 30 min. The absorbance of samples with DPPH at steady state was subtracted from the absorbance of control DPPH. The change in absorbance was compared with a standard curve of ascorbic acid and the DPPH antioxidant activity was expressed in ascorbic acid equivalents.
The oxygen radical absorbance capacity (ORAC) method (9) was modifed (10) to adapt to a 96-well Molecular Devices fmax fluorescent microplate reader (485 nm excitation and 538 nm emission). Whole mango juice and methanol fractions were monitored for their ability to inhibit oxidation of fluorescein in the presence of the peroxyl radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride. The rate of oxidation was measured every 2 min and the area under the decay curve was compared with a blank (ORAC = 0) and a standard (0–50 µmol/L) curve of Trolox, a water-soluble analogue of vitamin E. Data were expressed in µmol/L Trolox equivalents.
Total soluble phenolics were analyzed using the Folin-Ciocalteu assay as previously performed (8), with data expressed as gallic acid equivalents.
Linear regression was performed with SigmaStat 2.03 (SPSS, Inc.) to examine relations among the analyses.
Transformation inhibition assay
Cell culture. BALB/c 3T3 cells, clone A-31, were acquired from the American Type Culture Collection and are 1 of 2 cell choices for this assay (11). Cells were grown in DMEM/F12, 10% FBS, 2 mmol/L L-glutamine and antibiotics in a humidified 5% CO2 atmosphere at 37°C and passed twice weekly to maintain a subconfluent state.
Colony-forming efficiency assay. This assay was used to determine the toxicity level of whole mango juice concentrations prior to selecting the level to be used in the transformation inhibition assay. BALB/c 3T3 cells were seeded at 500 cells per 60 mm dish. The cells were allowed to attach for 24 h and were then treated with 4 mg/L BaP or mango for 24 h. The cells were washed with PBS and the medium replaced. Uninoculated control cells were treated in the same manner. After 7–10 d, cells were fixed with methanol and stained with Giemsa. Colonies with at least 50 cells were enumerated microscopically. Concentrations were considered toxic if the mean colony-forming efficiency was <80% of the control. Only noncytotoxic concentrations were used in the transformation inhibition assay.
Transformation inhibition assay.
The transformation inhibition assay was based on the transformation assay with level II amplification, as described by Perocco et al. (12–14). Cells were maintained in DMEM/F12 at 1 x 104 cells/25 cm2 flask in 4 replicate flasks per treatment. After 24 h, cells were treated with 4 mg/L of BaP in culture medium for 72 h. Culture medium was removed, the monolayer washed twice with PBS, and fresh medium added. Whole mango juice was added to the flasks on day 7, at concentrations from 0.1 to 0.00001% (v:v). Negative control cells were treated with the DMSO vehicle and positive control cells were treated only with BaP. At confluency (12–14 d), the cells from flasks of the same treatment were trypsinized, pooled, counted, and divided into 8–9 replicates per treatment at 1 x 105 cells/25 cm2 flask for amplification. All flasks were incubated and fed twice weekly for 
6 wk, at which time they were fixed with methanol and stained with Giemsa. As the stability of the phytochemicals had not been determined, the whole mango juice was added fresh from frozen stock at each medium change. The transformation assay was performed twice. In one experiment, a linear regression analysis of the number of foci versus the concentration of mango was performed with SigmaStat software. In the second experiment, a 1-way ANOVA, followed by Student-Neuman-Keuls post hoc analysis, was used to detect differences in the mean number of foci per plate among the different levels tested. Differences were considered significant at P < 0.05.
Scoring of foci
The criteria set by Reznikoff et al. (15,16) and adopted by the IARC/NCI/CPA working group (11) were used to score foci in BALB/c 3T3 cells. Scorable foci are a mass of roughly 50–100 transformed cells with 3 phenotypic qualities: marked cellular disorientation, loss of contact inhibition as reflected in cellular piling, and invasiveness into the contiguous monolayer of contact-inhibited cells. Type III foci are dense, multilayered, and basophilic, with a random orientation of cells at the focus edge and invasion into the monolayer. The transformed cells are predominantly spindle-shaped (11). Type II foci have a more ordered and defined edge than Type III foci (11). The dense, highly stained areas in each flask were examined microscopically to determine foci type. Only Type II and Type III foci >1.0 mm in size were counted.
Cell cycle kinetics
HL-60 cells, a human promyelocytic leukemic cell line, were obtained from the American Type Culture Collection. This cell was chosen because of its short doubling time and usefulness in rapidly screening fractions for cell cycle inhibitory activity. In addition, these studies provide a background from which to examine the mechanism of cell death by bioactive compounds in future studies. Cells were maintained in HEPES-buffered RPMI 1640 medium, 10% fetal bovine serum, antibiotics, and 2 mmol/L glutamine at cell concentrations from 2 to 10 x 108 cells/L in a humidified 5% CO2 atmosphere at 37°C.
The cell cycle of HL-60 cells was analyzed after incubation with whole mango juice or the methanol fractions. Mango juice samples were added to 2.5 x 108 cells/L at 0.0, 0.5, 1.0, and 2.0% of the culture medium volume. These concentrations were empirically determined in preliminary studies to give a range of responses. Methanol extracts were added at 2% of the culture-medium volume. Cells were harvested at 24 and 48 h, washed with PBS, and prepared for propidium iodide DNA staining. Cells were fixed for 30 min in ice-cold absolute ethanol, centrifuged at 1000 x g for 6 min at 4°C, and then washed twice in PBS. RNase (125 µL of 500,000 units/L in 0.038 mol/L sodium citrate) was added to the pellet, vortexed, and incubated for 15 min at 37°C. Propidium iodide (125 µL of 5 g/L in 0.038 mol/L sodium citrate) was added, vortexed, and incubated at room temperature between 30 min and 2 h. Analysis was conducted by flow cytometry with a FACScan flow cytometer (BD Immunocytometry Systems), using 488 nm excitation and 620 nm emission. The histogram showed 2 peaks, G0/G1 and G2/M phase DNA, while the S phase DNA comprised the area between the 2 peaks. Histograms were analyzed using WinMDI Flow cytometry software (version 2.8, build 1301–19–2000) to determine the percentage of cells in the G0/G1 and G2/M peaks. The percentage of cells in S phase was determined by difference. A 1-way ANOVA was performed on results within a phase and was followed by a Student-Neuman-Keuls all pairwise multiple comparison test. Differences between the means were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Transformation inhibition assay.
Whole mango juice exhibited antineoplastic transformation activity in both experiments. Spontaneous formation of foci averaged 0–2 foci per flask in negative cultures. Flasks containing only BaP (positive cultures) ranged from 6 to 19 foci per flask. The numbers of foci in cell cultures treated with BaP and mango were inversely correlated with the dose (P = 0.043, R2 = 0.79) (Fig. 1). In the second experiment, the BaP positive cultures averaged 16 foci per flask (Table 2). The addition of 0.01% whole mango juice reduced the number of foci 50% relative to the positive controls, whereas the addition of 0.1% whole mango juice reduced the number of foci 70%.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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The antioxidant activity in mango was assessed by 3 in vitro methods. ORAC, which detects peroxyl radicals generated from 2,2'-azobis (2-amidinopropane); Folin's assay, which detects metal reduction by unpaired electrons; and DPPH, a stable free radical that attracts unpaired electrons. The results of these methods correlated strongly with one another, yet the distribution of antioxidant activity among the fractions in each assay was different. Elution with 25% methanol resulted in a fraction of mango that contained 13% of the total ORAC activity, but only 2.4 and 7.2% of the total activity in Folin's and DPPH assays, respectively. Thus, this fraction appears to contain phytochemicals able to sequester peroxyl radicals. The fraction eluted with 50% methanol had low peroxyl radical scavenging activity but had 30% of the total DPPH activity. The 100% methanol eluate showed very low antioxidant activity when assessed by any method, probably due to a low concentration of total phytochemicals (see Supplemental Figure 1). This suggests that the various fractions of mango juice have different health benefits and anticancer properties.
Whole mango juice and methanol extracts of mango juice demonstrated anticancer activity by inhibiting the growth cycle of an immortal cancer cell line. In a dose-dependent manner, the addition of mango resulted in a greater percentage of the cell population in the G0/G1 phase and significantly fewer cells in the S and G2/M phases when assessed at 24 h. Interestingly, the 50% methanol extract of mango was the only fraction that significantly arrested cells in the G0/G1 phase of the cell cycle. Neither the whole mango juice nor the unbound fraction resulted in significant growth arrest, even though these fractions had the greatest total antioxidant activity. High antioxidant activity suggests a high concentration of phytochemicals, and therefore, more anticancer activity. Further work is underway to identify the polyphenolic composition of these fractions. A diversity of hydrolyzable tannins are present in the mango (3) (see Supplemental Figure 1).
Mango also exhibited anticancer activity by inhibiting neoplastic transformation in a dose-dependent manner. Carcinogenesis has 3 stages: initiation, promotion, and progression. In this model, BaP initiates carcinogenesis by forming DNA adducts (17), oxidative stress (18), and other assaults on the cell. Mango was added on day 7, 3 d after BaP was removed. The ability of mango to inhibit foci formation was therefore not during initiation but rather at a later stage of carcinogenesis, presumably during the promotion stage of carcinogenesis, insofar as progression is considered irreversible. This model suggests that there are additional functions of phytochemicals as anticancer agents beyond their antioxidant capacity.
In summary, mango contains compounds that have antioxidant activity, growth-arresting activity, and antipromotion activity. Whether these anticancer properties are maintained after digestion, absorption, and metabolism is unknown. Although anticancer activity has been related to antioxidant activity in many studies, our data suggest that the anticancer activity is a result of other functions and not solely the antioxidant activity. Although neoplastic transformation is not a good screening tool due to the length of the assay, it has demonstrated here the functional aspects of the whole food in a complex in vitro system. The detection of alterations in cell cycle progression that likely contribute to the antineoplastic effect is a more rapid means by which to screen foods or fractions of foods for their anticancer activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supplemental Figure 1 and Table 1 are available with the online posting of this paper at www.nutrition.org.
4 Abbreviations used: BaP, benzo[a]pyrene; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ORAC, oxygen radical absorbance capacity.
Manuscript received 25 October 2005. Initial review completed 2 December 2005. Revision accepted 1 February 2006.
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LITERATURE CITED |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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