Article pubs.acs.org/JAFC
Evaluation of Cytotoxic and Apoptotic Effects of Individual and Mixed 7‑Ketophytosterol Oxides on Human Intestinal Carcinoma Cells Junlan Gao,† Shaopeng Chen,‡ Lele Zhang,† Beijiu Cheng,† An Xu,‡ Lijun Wu,*,‡ and Xin Zhang*,† †
School of Life Sciences, Anhui Agricultural University, 130 Changjiang West Road, Hefei 230036, People’s Republic of China Institute of Technical Biology & Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China
‡
ABSTRACT: Phytosterol oxidation products (POPs) are constituents of the human diet. Definitive information on the toxic or biological effects of POPs is limited and in some cases contradictory. This study evaluates the cytotoxicity of four individual 7ketophytosterol oxides, including 7-ketositosterol (7K-SI), 7-ketocampesterol (7K-CA), 7-ketobrassicasterol (7K-BR), 7ketostigmasterol (7K-ST), and a mixture of 7-ketophytosterols (7K-MIX) toward a human intestinal carcinoma (HIC) cell line. Results showed that all tested compounds reduced cell proliferation in a dose-dependent manner; especially 7K-SI and 7K-CA exhibited higher activities. Both compounds increased early apoptotic cells and caused cell cycle arrest in the G1 phase with cell accumulation in the S phase. No evidence of cell death was observed induced by 7K-ST and 7K-MIX. Furthermore, 7K-SI, 7KCA, and 7K-BR induced apoptosis by enhancing caspase-3 activity and the modulatory effects of Bcl-2, while 7K-ST and 7K-MIX did not involve caspase-3 activation and Bcl-2 down-regulation. KEYWORDS: 7-ketophytosterol oxides, HIC cells, cytotoxicity, apoptosis, cell cycle
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INTRODUCTION
Previous studies demonstrated quantitatively the cytotoxic effects of POPs, mainly focused on β-sitosterol oxides and stigmasterol oxides, on different mammalian cell types, involving mouse macrophage-derived cell line (C57BL/6),18 human monocytic blood line (U937),19,20 colonic adenocarcinoma cell line (Caco-2),20−23 human hepatoma cell line (HepG2),20,24 Hela cells,25 and human breast adenocarcinoma (MCF-7) cells.26 These cells underwent apoptosis when treated with certain POPs. In relation to the cytotoxicity of 7-keto oxide derivatives of phytosterols, analogous compounds arising from different phytosterols displayed a different toxicity level, and not all 7-ketosterols induced apoptosis in these tested cell lines. 7-Ketositosterol was found to be cytotoxic and apoptotic in U937 cells and HepG2 cells.19 In contrast, there was no any evidence of apoptosis for the 7-ketostigmasterol in the U937 cells and Caco-2 cells.27 Given these novel findings, it is interest to examine all individual 7-keto oxides derived from different phytosterols to obtain a more comprehensive insight into the cytotoxicity of POPs. The objective of the present study was to systematically compare and evaluate the effects of four individual 7ketophytosterol oxides (7K-SI, 7K-CA, 7K-BR, and 7K-ST) and a blend of 7-ketophytosterols (7K-MIX) toward human intestinal carcinoma cells (HIC cells). The HIC cell line was chosen because it originated from human small bowel cancerous tissue with the features of cancer cells and epithelial cells. The small intestine is part of the digestive system and
Sterols, also known as steroid alcohols, are a subgroup of the steroids and an important class of organic molecules. Cholesterol is vital to animal cell membrane structure and function. Sterols of plants, called phytosterols, may inhibit the uptake of both dietary and endogenously produced cholesterol from the intestine, resulting in a decrease of cholesterol and LDL levels in serum.1,2 Moreover, phytosterols may possess anticarcinogenic, antiatherosclerotic, and anti-inflammatory effects, so they have become important health supplements in a range of function foods over the past 20 years.3−5 Phytosterols include β-sitosterol, campesterol, and stigmasterol, among others. Because of their chemical structure similar to cholesterol with a double bond at the C5−C6 position, phytosterols are also susceptible to oxidation forming phytosterol oxidation products (POPs).6,7 In general, 7-keto oxide derivatives are the most abundant POPs followed by 7βhydroxy oxides and epoxide derivatives. POPs have been detected in plant sterol-enriched and nonenriched commercial foods8,9 and also found in the human plasma.10−12 The potential health risks of cholesterol oxidation products (COPs), including cytotoxicity,13 atherosclerosis,14 mutagenesis,15 and carcinogenesis,16 have been well documented. Compared with the relatively comprehensive studies of COPs, definitive information on the potential toxic or biological effects POPs is limit and in some cases contradictory.2 The limited evidence demonstrated that POPs produced atherogenicity and inflammation and distinct levels of cytotoxicity.6,7,17 In most cases, a blend instead of single POPs has been used for toxicity studies because of the lack of commercial availability of most individual POP standards. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1035
October 20, 2014 December 24, 2014 December 26, 2014 December 26, 2014 DOI: 10.1021/jf505079v J. Agric. Food Chem. 2015, 63, 1035−1041
Article
Journal of Agricultural and Food Chemistry
For cell cycle analysis, cells were centrifuged and fixed in 2 mL of methanol/PBS (9:1, v/v) at 0 °C overnight, washed twice in PBS, and resuspended in 200 μL of PBS containing 0.25 mg/mL RNase A for 30 min and then stained in 50 μL of PI (0.2 mg/mL in PBS) with incubation in the dark at 4 °C for 30 min. The fluorescence of 1 × 104 cells was then analyzed by BD FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Caspase Activity Assay. The measurement of caspase-3 activity was performed using a Caspase-3 Assay Colorimetric Kits (SigmaAldrich, Hamburg, Germany) according to the manufacturer’s protocol with some modifications. Briefly, HIC cells (1 × 106) were seeded in 60 mm culture dishes and exposed to 7-ketosterol oxides for 24 h and then collected and lysed at 0 °C. Lysates were clarified by centrifugation at 10000g (4 °C) for 10 min, and the supernatants were carefully transferred to a new microcentrifuge tube without disturbing the cell-debris pellet in the collection tube. Protein (50−200 ng) was incubated with 50 μL of reaction buffer and 5 μL of caspase-3 (AcDEVD-pNA) colorimetric substrate at 37 °C for a further 4 h. The cleavage of the peptide substrate was monitored at 405 nm using a 96well microplate reader (Bio-Rad, Hercules, CA, USA). The samples were analyzed in triplicate. Bcl-2 Content. The Bcl-2 level in HIC cells exposed to 7ketosterol oxides for 24 h was quantified using a Human Bcl-2 Platinum ELISA Kit (BMS244/3, eBioscince, San Diego, CA, USA). The supernatant from cells was removed; cells (5 × 106) were washed once with PBS and harvested by scraping and gentle centrifugation. PBS was aspirated, and an intact cell pellet was frozen at −80 °C for lysis at a later date. The cultured cells were resuspended in lysis buffer and incubated 60 min at room temperature with gentle shaking, and extracts were transferred to microcentrifuge tubes and centrifuged at 1000g for 15 min. According to the supplier’s instructions, the supernatant was added to the wells of the ELISA plate. The absorbance was measured at 450 nm using a Fluostar Optima Microplate Reader (BMG Labtech, Ortenberg, Germany), and the data were expressed as a percentage of the untreated control sample. Statistics. All data points represented the mean value (SE) of at least three independent experiments. One-way analysis of variance (ANOVA) and the LSD test were applied to determine differences in cytotoxic effects, apoptosis effects, changes of cell-cycle arrest, and changes in expression of caspase-3 and Bcl-2 between treated and control cultures. Statistical differences were considered significant at a value of p < 0.05 and reported as p < 0.05 and p < 0.01.
functions to break down food and nutrients to be absorbed into the body, and the intestinal epithelium is the first physiological barrier after oral intake of the compounds. In order to obtain a more comprehensive insight into the cytotoxicity and apoptotic mechanisms of 7-ketophytosterols, cell viability, cell cycle analysis, and cell apoptosis induced by these compounds were studied. For comparison, the cytotoxic effects of 7-ketocholesterol (7K-CH) were also assessed in the HIC cell line. In addition, several modulators of apoptosis (caspase-3 and Bcl-2) were investigated to determine whether these compounds were responsible for the intrinsic pathway of apoptosis in HIC cancer cells.
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MATERIALS AND METHODS
Materials. 7K-CH was purchased from Sigma-Aldrich Company (St. Louis, USA). 7K-ST and 7K-MIX were separately chemically synthesized from stigmasterol (Sigma-Aldrich Company, St. Louis, MO, USA) and phytosterols (Cognis, Saint-Fargeau-Ponthierry, Seineet-Marne, France) as described by Gao et al.28 The other three individual POPs (7K-SI, 7K-CA, and 7K-BR) were not commercially available and therefore were separated by semipreparative HPLC and characterized as described in the literature.28 Except 7K-BR and 7KMIX, the purity of each individual 7-ketosterol compound was ∼95%. 7K-BR contained 52% 7-ketobrassicasterol and 48% 7-ketoavenasterol, whereas the blend of 7K-MIX contained 45% 7K-SI, 32% 7K-CA, 10% 7K-BR, and 9% 7K-AV. The profile of this mixture reflected the composition of related phytosterol oxides in the plant oil.28 The chemical structures of these compounds are available in the literature.28 Cell Culture and Treatment. The HIC cell line was obtained from Chinese Academy of Medical Sciences Tumor Cell Culture (Beijing, China). HIC cells were cultured in DMEM/high glucose medium (Hyclone, Beijing, China) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate at 37 °C and 5% CO2 in a humidified incubator. Cells were routinely harvested by treatment with a 0.25% trypsinEDTA solution. Cultures were allowed to reach 80% confluence before experiments were performed. For treatment, all above-mentioned 7-ketosterols were separately dissolved in 100% ethanol and added to the cells to a final concentration of 30, 60, or 120 μM. The concentrations used here were physiologically significant and shown in some published studies.21,22,29 Equivalent quantities of ethanol were added to the control cells, and the final concentration did not exceed 0.5% (v/v) in the culture medium. Cells were then harvested after 24 h treatment for determination of cell viability, apoptotic effect, cell cycle, caspase-3 activity, and Bcl-2 content analysis. Cell Viability. Cell viability was determined by using the Cell Counting Kit-8 (CCK8) assay (Dojindo, Kumamoto, Japan), which is based on a reaction catalyzed by dehydrogenase of the mitochondria. Within this assay, cells were adjusted to a density of 2 × 104 cells/well in 96-well plates. After incubation with different concentrations of 7ketosterol oxides, the culture medium was discarded, cells were washed with PBS, and then 200 μL of 10% CCK8 in culture medium was added and incubated at 37 °C for 2 h. Absorbance was read at 450 nm with a Fluostar Optima microplate reader (BMG Labtech, Ortenberg, Germany) and expressed as the viable cell number as a percentage (%) of control cells. Measurements were made in triplicates. Cell Apoptosis and Cell Cycle Assay. HIC cells (5 × 105) were seeded in the wells of a 6-well plate and harvested by trypsinization (0.25% trypsin with EDTA) after exposure to different doses of 7ketosterol oxides for 24 h. With the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA), cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) according to the manufacturer’s protocol. Apoptotic cells were then analyzed by BD FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
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RESULTS Cell Viability Effects of 7-Ketosterols on HIC Cells. CCK8 cell viability assay was used to evaluate the cytotoxicity of the tested compounds after 24 h of exposure. Viability of the control sample was set at 100%. As shown in Figure 1, as the positive control, 30 and 60 μM 7K-CH caused 50% and 38% HIC cell growth inhibition (p < 0.01), respectively, whereas 60 μM of five 7-ketophytosterol oxides only caused 10−20% growth inhibition. This indicated that HIC cells were more sensitive to 7-ketocholesterol than 7-ketophytosterols. After treatment with 120 μM 7-ketophytosterol oxides for 24 h, all individual test compounds induced a significant reduction (p < 0.01) of cell viability relative to the negative control, and the cell survival was 37%, 39%, 56%, and 79% for 7K-SI, 7K-CA, 7K-BR, and 7K-ST treated cells, respectively. In contrast, 7KMIX caused no significant change in cell viability at all test concentrations. 7-Ketophytosterols Induce Apoptosis on HIC Cells. We then elucidated whether the cytotoxic effects of 7ketosterols were mediated through apoptosis. At low dose of 30 μM, all 7-ketophytosterol oxides caused no obvious change in the percentage of apoptotic cells. At 60 μM, there was a significant increase (p < 0.05) in apoptotic cells by all tested compounds treatment compared with the control group. At 1036
DOI: 10.1021/jf505079v J. Agric. Food Chem. 2015, 63, 1035−1041
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(Figure 3C), or 7K-ST (Figure 3E), the proportion of cells in G1 phase decreased from 40.5% to 26.8%, from 40.2% to 34.5%, and from 39.9% to 35.3%, respectively, whereas 7K-SI and 7K-CA caused the accumulation of cells in S phase from 42.0% to 52.9% and from 41.0% to 51.5% (p < 0.05), respectively. At all concentrations tested, 7K-BR (Figure 3D) and 7K-MIX (Figure 3F) did not show a significant difference (p > 0.05) in G1 and S phases. In addition, all the tested compounds did not lead to increase in the sub-G1 phase population. Effect of 7-Ketophytosterols on Caspase-3 Activity in HIC Cells. To assess whether 7-ketophytosterols induce caspase activation, which plays an important role in the execution phase of cell apoptosis, caspase-3 activity was evaluated. As shown in Figure 4, relative to the control group, significantly increased caspase-3 activity was observed in HIC cells treated with 60 and 120 μM 7K-SI, 7K-CA, and 7KBR for 24 h in a dose-dependent manner. Caspase-3 activity was enhanced 3-, 2-, and 1.3-fold relative to control group in response to the incubation with 120 μM 7K-SI, 7K-CA, and 7K-BR, respectively (p < 0.01). In contrast, caspase-3 activity remained at a basal level when HIC cells were treated with 7KST and 7K-MIX at all of the concentrations tested. Effect of 7-Ketosterols on Bcl-2 Protein in HIC Cells. To further verify the apoptotic effect, we investigated the changes in activity of the apoptosis-related proteins triggered by the tested 7-ketosterols. Results showed that, compared with control cells, the HIC cell protein levels of Bcl-2 declined after treatment with 60 μM 7K-CH, 120 μM 7K-SI, and 120 μM 7KCA, which favors the induction of apoptosis (Table 1). The Bcl-2 protein level was increased after treatment with 7K-BR at 30 μM, while it was decreased after treatment at 120 μM (p < 0.01). Bcl-2 remained at a basal level in cells treated with 7K-ST and 7K-MIX (60 μM and 120 μM) relative to the untreated control.
Figure 1. Cell viability measured by the CCK8-test. HIC cells were exposed to 0.5% ethanol (control), 30, 60, and 120 μM of 7ketophytosterol oxides (7K-SI, 7K-CA, 7K-BR, 7K-ST, and 7K-MIX) or 30 and 60 μM of 7K-CH for 24 h. Values are mean ± SE of three separate experiments and are marked when significantly different from control levels (*p < 0.05, **p < 0.01).
high dose of 120 μM, 7K-SI, 7K-CA, 7K-BR, 7K-ST, and 7KMIX caused early apoptotic cells to 64.0%, 53.6%, 17.5%, 15.9%, and 14.8%, respectively (Figure 2).
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DISCUSSION The present study was focused on the biological implications of individual and mixed 7-ketophytosterol oxides on HIC cell line. A further objective was to assess the death pathways related to Bcl-2 and caspase-dependence induced by these phytosterol oxides. We found that 7-ketophytosterol oxides displayed different cytotoxic and apoptotic effects on HIC cells. Except 7K-ST and 7K-MIX, other 7-ketophytosterol oxides (7K-SI, 7K-CA, and 7K-BR) were capable of inducing growth inhibition and apoptosis on HIC cells in a dose-dependent manner after 24 h treatment. At 120 μM, the order of 7-ketophytosterol oxide toxicity was 7K-SI > 7K-CA > 7K-BR > 7K-ST > 7K-MIX. 7KSI was the most cytotoxic and apoptotic compound, exerting the strongest reduction in cell viability and inducing cell apoptosis, whereas 7K-ST (120 μM) resulted in decreased cell viability with low level apoptosis in HIC cells. The cytotoxicity of 7K-CA was demonstrated for the first time, in the present study: cell apoptosis was induced when HIC cells were exposed to 7K-CA (60 and 120 μM), but its cytotoxic and apoptotic effects were less than those of 7K-SI. In addition, 60 μM 7KCH induced even higher cytotoxicity than 120 μM 7K-SI. This finding was in agreement with the results of Maguire et al.19 and Ryan et al.20 that β-sitosterol oxides had general toxic effects similar to COPs but higher concentrations of POPs were required to obtain comparable results.
Figure 2. Percentage of apoptosis in HIC cancer cells following exposure for 24 h to 30, 60, and 120 μM of 7-ketophytosterol oxides (7K-SI, 7K-CA, 7K-BR, 7K-ST, and 7K-MIX) or 30 and 60 μM of 7KCH. All compounds were dissolved in ethanol and equivalent quantities of ethanol were added to control cells. Apoptotic cells were assessed with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). Values are mean ± SE of three separate experiments and are marked when significantly different from control levels (*p < 0.05, **p < 0.01).
Effect of 7-Ketophytosterols on Cell Cycle. The effect of 7-ketosterols on cell cycle progression was determined by flow cytometry with PI-based staining. Results showed that after 24 h of 7-ketosterol treatment, the normal cell cycle progression was disrupted in HIC cells (Figure 3). At 60 μM, 7K-CH increased the percentage of cells in G1 phase from 40.5% to 79.4% and decreased the percentage of cells in S phase from 43.5% to 25.8% (p < 0.05) (Figure 3A). In contrast, when the cells were exposed to 120 μM of 7K-SI (Figure 3B), 7K-CA 1037
DOI: 10.1021/jf505079v J. Agric. Food Chem. 2015, 63, 1035−1041
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Figure 3. Changes in cell cycle phase populations of HIC cells exposed to 30, 60, and 120 μM of 7-ketophytosterol oxides (7K-SI (B), 7K-CA (C), 7K-BR (D), 7K-ST (E), and 7K-MIX (F)) or 30 and 60 μM of 7K-CH (A) for 24 h. Values are mean ± SE of three separate experiments and are marked when significantly different from control levels (*p < 0.05, **p < 0.01).
The differences in susceptibility of cells to sterol oxide treatment might be related to the chemical structures of sterol oxides and the genetic background of the cell lines. 7-Ketosterol oxides are structurally similar compounds that possess the same sterol nucleus (one double bond at C5−C6 and a ketone group at C7 in the B-ring) but a small difference in the side chain (such as substitution at C24 or an additional
double bond between C22 and C23). We found that, in the present study, HIC cells were more sensitive to the 7-ketosterol oxides that had no double bond in the side chain (7K-CH, 7KSI, and 7K-CA) than other oxides with a double bond between C22 and C23 in the side chain (7K-ST and 7K-BR). The substitution of the double bond in the side chain (7K-ST) might influence the equilibrium between the ketone form and 1038
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and is sensitive to TNF and anti-Fas antibodies.30,31 Caco-2 cells express retinoic acid binding protein I and retinol binding protein II.32 HepG2 cells retain many functions of the normal liver cell, express 3-hydroxy-3-methylglutaryl-CoA reductase and hepatic triglyceride lipase activities, have decreased expression of apoA-I mRNA, and have increased expression of catalase mRNA in response to gramoxone (oxidative stress).33 Combined our experimental results that 7-ketosterol oxides displayed different cytotoxic and apoptotic effects on the HIC cell line, this suggested that the toxicity of sterol oxides on different cell lines depends on the physiological area in which they work. In addition, synergistic as well as inhibitory effects of individual sterol oxides in mixtures had been noted.34 Some studies indicated that oxysterols as a mixture did not elicit the same cytotoxic effects and defensive responses as the individual oxysterols.35−37 They suggested that major oxysterols were not universally cytotoxic and the simultaneous presence of several different oxysterol species might reduce the adverse effects of individual oxysterols.35 These finding were consistent with our results that 7K-MIX exerted a reduction of the toxic potential compared with the individual phytosterol oxides (7K-SI, 7KCA, and 7K-BR). The studies of cytotoxicity were also supplemented with cell cycle assay. Alemany et al. reported that exposure to neither 7K-ST nor 7K-CH led to increases in the sub-G1 phase population in Caco-2 cells.23 Similar results were found in HIC cells treated by all the tested compounds in the present study. In addition, 7K-CH (60 μM) caused cell cycle arrest in the G1 phase and decreased the percentage of cells in S phases, whereas 7K-SI and 7K-CA (120 μM) led to an accumulation in S phase, and a simultaneous decrease of cells engaged in the G1 phase in HIC cells. Therefore, there is not definite relationship between 7-ketosterol structures and their effect on the cell cycle. Apoptosis is a highly regulated physiologic cell death process that is critical for development, host defense, and the prevention of malignant transformation and inflammation throughout the body.38,39 There are two major pathways regulating apoptosis: the intrinsic pathway mediated by the mitochondria,40 and the extrinsic pathway induced by the death receptor.41,42 Recently, new apoptotic mechanisms including caspase-independent pathway and granzyme-initiated pathways have been shown to exist in lymphocytes.42,43 Regarding the death pathway of oxide derivatives of sterols, Prunet et al. demonstrated that caspase-3 was essential to trigger 7-ketocholesterol- and 7β-hydroxycholesterol-induced apoptosis, and these oxysterols simultaneously activate caspase3-dependent or -independent modes of cell death.44 Roussi et al. reported that 7β-hydroxysitosterol and 7β-hydroxycholesterol followed different death pathways despite their structural similarity.21 In the case of 7β-hydroxysitosterol, the intracellular caspase-9 and -3 was activated in Caco-2 cells. In contrast, 7βhydroxycholesterol did not activate caspase-3.21 They caused mitochondrial depolarization but not involve the Bcl-2 family proteins in Caco-2 cells.21,22 In addition, caspase-8 was not activated in cells treated with both hydroxysterols.21 Alemany et al. showed that 7K-CH regulate key enzymes HMG-CoA in cholesterol metabolism and cause alterations in mitochondrial membrane potential in Caco-2 cells.27 Considering the complexity of the sterol oxides involved in the death way, in our present study, Bcl-2 and caspase-3 were detected to preliminary confirm the intrinsic death pathway of
Figure 4. Caspase-3 activity (fold increase vs the control) in HIC cells following 24 h of incubation with 30, 60, and 120 μM of 7ketophytosterol oxides (7K-SI, 7K-CA, 7K-BR, 7K-ST, and 7K-MIX) or 30 and 60 μM of 7K-CH. Values are mean ± SE of three separate experiments and are marked when significantly different from control levels (*p < 0.05, **p < 0.01).
Table 1. Bcl-2 Content in HIC Cells Following Exposure for 24 h to 30, 60, or 120 μM of Sterol Oxidesa Bcl-2 content (fold increase vs. the control) compound 7K-CH
7K-SI
7K-CA
7K-BR
7K-ST
7K-MIX
exposure (μM) cholesterol oxides 30 60 phytosterol oxides 30 60 120 30 60 120 30 60 120 30 60 120 30 60 120
mean
SE
0.80** 0.68**
0.02 0.06
1.12 0.91 0.66** 1.66 0.98 0.75** 1.34** 1.11 0.82** 1.15 0.98 1.03 1.07 0.97 0.93
0.09 0.07 0.08 0.03 0.06 0.03 0.08 0.08 0.01 0.01 0.01 0.12 0.04 0.04 0.10
Values are mean ± SE of three separate experiments and are marked with when significantly different from control levels (*p < 0.05, **p < 0.01). a
hydroxide adducts, which might be shifted by interaction with proteins at the intestinal epithelial level.23 Ryan et al. reported that β-sitosterol oxides produced different toxic effects in U937, Caco-2, and HepG2 cell lines, but without inducing apoptosis in either HepG2 or Caco-2 cells.24 Koschutnig et al. reported a decrease in HepG2 cell viability upon exposure to single β-sitosterol oxide but little effects induced by the mixture of polar oxides.21 Other studies indicated that 7-ketostigmasterol (30−120 μM) had no cytotoxic effects on U937 cells24 and Caco-2 cells.27,29 It is well-known that the U937 cell line can express the Fas antigen 1039
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(4) Shahidi, F. Functional foods: Their role in health promotion and disease prevention. J. Food Sci. 2004, 69, R146−R149. (5) Chen, Z. Y.; Ma, K. Y.; Liang, Y.; Peng, C.; Zuo, Y. Role and classification of cholesterol-lowering functional foods. J. Funct. Foods 2011, 3, 61−69. (6) Ryan, E.; McCarthy, F. O.; Maguire, A. R.; O’Brien, N. M. Phytosterol oxidation products: Their formation, occurrence, and biological effects. Food Rev. Int. 2009, 25, 157−174. (7) O’Callaghan, Y.; McCarthy, F. O.; O’Brien, N. M. Recent Advances in Phytosterol Oxidation Products. Biochem. Biophys. Res. Commun. 2014, 446, 786−791. (8) Grandgirard, A.; Martine, L.; Joffre, C.; Juaneda, P.; Berdeaux, O. Gas chromatographic separation and mass spectrometric identification of mixtures of oxyphytosterol and oxycholesterol derivatives: Application to a phytosterol-enriched food. J. Chromatogr. A 2004, 1040, 239−250. (9) Conchillo, A.; Cercaci, L.; Ansorena, D.; Rodriguez-Estrada, M. T.; Lercker, G.; Astiasarán, I. Levels of phytosterol oxides in enriched and nonenriched spreads: Application of a thin-layer chromatographygas chromatography methodology. J. Agric. Food Chem. 2005, 53, 7844−7850. (10) Grandgirard, A.; Martine, L.; Demaison, L.; Cordelet, C.; Joffre, C.; Berdeaux, O.; Semon, E. Oxyphytosterols are present in plasma of healthy human subjects. Br. J. Nutr. 2004, 91, 101−106. (11) Liang, Y. T.; Wong, W. T.; Guan, L.; Tian, X. Y.; Ma, K. Y.; Huang, Y.; Chen, Z. Y. Effect of phytosterols and their oxidation products on lipoprotein profiles and vascular function in hamster fed a high cholesterol diet. Atherosclerosis 2011, 219, 124−133. (12) Menéndez-Carreño, M.; Steenbergen, H.; Janssen, H. G. Development and validation of a comprehensive two-dimensional gas chromatography-mass spectrometry method for the analysis of phytosterol oxidation products in human plasma. Anal. Bioanal. Chem. 2012, 402, 2023−2032. (13) Wielkoszyński, T.; Gawron, K.; Strzelczyk, J.; Bodzek, P.; Zalewska-Ziob, M.; Trapp, G.; Srebniak, M.; Wiczkowski, A. Cellular toxicity of oxycholesterols. Bioessays 2006, 28, 387−398. (14) Poli, G.; Sottero, B.; Gargiulo, S.; Leonarduzzi, G. Cholesterol oxidation products in the vascular remodeling due to atherosclerosis. Mol. Aspects Med. 2009, 30, 180−189. (15) Kanhal, M. A.; Ahmad, F.; Othman, A. A.; Arif, Z.; Orf, S. A.; Murshed, K. A. Effect of pure and oxidized cholesterol-rich diets on some biochemical parameters in rats. Int. J. Food Sci. Nutr. 2002, 53, 381−388. (16) Homma, Y.; Kondo, Y.; Kaneko, M.; Kitamura, T.; Nyou, W. T.; Yanagisawa, M.; Yamamoto, Y.; Kakizoe, T. Promotion of carcinogenesis and oxidative stress by dietary cholesterol in rat prostate. Carcinogenesis 2004, 25, 1011−1014. (17) Hovenkamp, E.; Demonty, I.; Plat, J.; Lütjohann, D.; Mensink, R. P.; Trautwein, E. A. Biological effects of oxidized phytosterols: A review of the current knowledge. Prog. Lipid Res. 2008, 47, 37−49. (18) Adcox, C.; Boyd, L.; Oehrl, L.; Allen, J.; Fenner, G. Comparative effects of phytosterol oxides and cholesterol oxides in cultured macrophage-derived cell lines. J. Agric. Food Chem. 2001, 49, 2090− 2095. (19) Maguire, L.; Konoplyannikov, M.; Ford, A.; Maguire, A. R.; O’Brien, N. M. Comparison of the cytotoxic effects of β-sitosterol oxides and a cholesterol oxide, 7β-hydroxycholesterol, in cultured mammalian cells. Br. J. Nutr. 2003, 90, 767−776. (20) Ryan, E.; Chopra, J.; McCarthy, F.; Maguire, A. R.; O’Brien, N. M. Qualitative and quantitative comparison of the cytotoxic and apoptotic potential of phytosterol oxidation products with their corresponding cholesterol oxidation products. Br. J. Nutr. 2005, 94, 443−451. (21) Roussi, S.; Winter, A.; Gosse, F.; Werner, D.; Zhang, X.; Marchioni, E.; Geoffroy, P.; Miesch, M.; Raul, F. Different apoptotic mechanisms are involved in the antiproliferative effects of 7βhydroxysitosterol and 7β-hydroxycholesterol in human colon cancer cells. Cell Death Differ. 2005, 12, 128−135.
7-ketosterols. It is becoming clear that the Bcl-2 family proteins play an important role in the regulation of apoptosis at the mitochondrial level. Dysregulation of the Bcl-2 proteins was proved to induce the destruction of mitochondrial membrane, which result in the release of cytochrome c and apoptosisinducing factors from the mitochondria and subsequently induces activation of the caspase cascade.45,46 Significant changes were found in expression of the apoptosis related Bcl-2 family proteins after treatment with phytosterol oxides. In the present study, 7K-CH (60 μM) and 7K-SI and 7K-CA (120 μM) caused a significant decrease in the level of antiapoptotic protein Bcl-2, which in turn resulted in the activation of caspase-3. Caspase-3 activity analysis showed that treatment of 7K-CH, 7K-SI, and 7K-CA did activate apoptosis proteins caspase-3 with dose-dependent manner. However, no significant changes in expression of Bcl-2 protein were found after 24 h treatment with 7K-ST and 7K-MIX in HIC cells. It might involve various apoptosis mechanisms or cell death pathway. In conclusion, our study demonstrated that HIC cells were significantly more sensitive to 7K-CH, 7K-SI, and 7K-CA treatment than other 7-ketophytosterol oxides. The cytotoxicity of 7K-CA was demonstrated at first time and analogue to 7KSI. 7K-SI and 7K-CA modulated cell viability, proliferation, and expression of apoptosis-related proteins. Since induction of cell cycle arrest was usually accompanied by growth inhibition, the research is required to identify which cell cycle regulators attribute to the alteration of specific phases of cells. Moreover, further study is required to clarify the precise pathway of POPsinduced apoptosis.
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AUTHOR INFORMATION
Corresponding Authors
*L.W. Phone/fax: +86 551 65591602. E-mail:
[email protected]. *X.Z. Phone/fax: +86 551 65786021. E-mail: xinzhang@ahau. edu.cn. Funding
This work was supported by the fund of the National Natural Science Foundation of China (Grant 21072002). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS POPs, phytosterol oxidation products; COPs, cholesterol oxidation products; 7K-CH, 7-ketocholesterol; 7K-SI, 7ketositosterol; 7K-CA, 7-ketocampesterol; 7K-ST, 7-ketostigmasterol; 7K-BR, 7-ketobrassicasterol; 7K-MIX, mixture of 7ketophytosterols; HIC, human intestinal cancer epithelial cells; HPLC, high performance liquid chromatography; CCK8, Cell Counting Kit-8
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REFERENCES
(1) Jones, P.; AbuMweis, S. Phytosterols as functional food ingredients: Linkages to cardiovascular disease and cancer. Curr. Opin. Clin. Nutr. 2009, 12, 147−151. (2) Ostlund, R. E.; Racette, S. B.; Stenson, W. F. Inhibition of cholesterol absorption by phytosterol-replete wheat germ compared with phytosterol-depleted wheat germ. Am. J. Clin. Nutr. 2003, 77, 1385−1389. (3) Weber, N.; Weitkamp, P.; Mukherjee, K. D. Cholesterol-lowering food additives: Lipase-catalysed preparation of phytosterol and phytostanol esters. Food Res. Int. 2002, 35, 177−181. 1040
DOI: 10.1021/jf505079v J. Agric. Food Chem. 2015, 63, 1035−1041
Article
Journal of Agricultural and Food Chemistry (22) Roussi, S.; Gosse, F.; Aoude-Werner, D.; Zhang, X.; Marchioni, E.; Geoffroy, P.; Miesch, M.; Raul, F. Mitochondrial perturbation, oxidative stress and lysosomal destabilization are involved in 7βhydroxysitosterol and 7β-hydroxycholesterol triggered apoptosis in human colon cancer cells. Apoptosis 2007, 12, 87−96. (23) Alemany, L.; Laparra, J.; Barberá, R.; Alegría, A. Relative expression of cholesterol transport-related proteins and inflammation markers through the induction of 7-ketosterol-mediated stress in Caco-2 cells. Food Chem. Toxicol. 2013, 56, 247−253. (24) Koschutnig, K.; Heikkinen, S.; Kemmo, S.; Lampi, A.-M.; Piironen, V.; Wagner, K.-H. Cytotoxic and apoptotic effects of single and mixed oxides of β-sitosterol on HepG2-cells. Toxicol. in Vitro 2009, 23, 755−762. (25) Sato, H.; Nishida, S.; Tomoyori, H.; Sato, M.; Ikeda, I.; Imaizumi, K. Oxysterol regulation of estrogen receptor α-mediated gene expression in a transcriptional activation assay system using Hela cells. Biosci., Biotechnol., Biochem. 2004, 68, 1790−1793. (26) Newill, H.; Loske, R.; Wagner, J.; Johannes, C.; Lorenz, R. L.; Lehmann, L. Oxidation products of stigmasterol interfere with the action of the female sex hormone 17β-estradiol in cultured human breast and endometrium cell lines. Mol. Nutr. Food Res. 2007, 51, 888− 898. (27) Alemany, L.; Laparra, J.; Barberá, R.; Alegría, A. Evaluation of the cytotoxic effect of 7keto-stigmasterol and 7keto-cholesterol in human intestinal (Caco-2) cells. Food Chem. Toxicol. 2012, 50, 3106− 3113. (28) Gao, J.; Yue, Q.; Ji, Y.; Cheng, B.; Zhang, X. Novel synthesis strategy for the preparation of individual phytosterol oxides. J. Agric. Food Chem. 2013, 61, 982−988. (29) O’Callaghan, Y. C.; Foley, D. A.; O’Connell, N. M.; McCarthy, F. O.; Maguire, A. R.; O’Brien, N. M. Cytotoxic and apoptotic effects of the oxidized derivatives of stigmasterol in the U937 human monocytic cell line. J. Agric. Food Chem. 2010, 58, 10793−10798. (30) Sundstrom, C.; Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 1976, 17, 565−577. (31) Ralph, P.; Moore, M. A.; Nilsson, K. Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines. J. Exp. Med. 1976, 143, 1528−1533. (32) Trainer, D. L.; Kline, T.; McCabe, F. L.; Faucette, L. F.; Field, J.; Chaikin, M.; Anzano, M.; Rieman, D.; Hoffstein, S.; Li, D. J. Biological characterization and oncogene expression in human colorectal carcinoma cell lines. Int. J. Cancer 1988, 41, 287−296. (33) Busch, S. J.; Barnhart, R. L.; Martin, G. A.; Flanagan, M. A.; Jackson, R. L. Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3- methylglutaryl-CoA reductase gene expression in a human hepatoma cell line, HepG2. J. Biol. Chem. 1990, 265, 22474− 22479. (34) O’Sullivan, A. J.; O’Callaghan, Y. C.; O’Brien, N. M. Differential effects of mixtures of cholesterol oxidation products on bovine aortic endothelial cells and human monocytic U937 cells. Int. J. Toxicol. 2005, 24, 173−179. (35) Hall, M. C. The effect of oxysterols, individually and as a representative mixture from food, on in vitro cultured bovine ovarian granulosa cells. Mol. Cell. Biochem. 2006, 292, 1−11. (36) Aupeix, K.; Weltin, D.; Mejia, J. E. Oxysterol-induced apoptosis in human monocytic cell lines. Immunobiology 1995, 194, 4−5. (37) Leonarduzzi, G.; Sottero, B.; Poli, G. Oxidized products of cholesterol: dietary and metabolic origin, and proatherosclerotic effects. J. Nutr. Biochem. 2002, 13, 700−710. (38) Ferri, K. F.; Kroemer, G. Organelle-specific initiation of cell death pathways. Nat. Cell Biol. 2001, 3, E255−E263. (39) Bortner, C. D.; Cidlowski, J. A. Cellular mechanisms for repressing apoptosis. Annu. Rev. Pharmacol. Toxicol. 2002, 42, 259− 281. (40) Hockenbery, D.; Nunez, G.; Millirman, C.; Schreiber, R. D.; Korsmever, S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990, 348, 334−336.
(41) Zapata, J. M.; Pawlowski, K.; Hass, E.; Ware, C. F.; Godzik, A.; Reed, J. C. A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains. J. Biol. Chem. 2001, 276, 24242−24252. (42) Xu, G.; Shi, Y. Apoptosis signaling pathways and lymphocyte homeostasis. Cell Res. 2007, 17, 759−771. (43) Sarin, A.; Williams, M. S.; Alexander-Miller, M. A. Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 1997, 6, 209−215. (44) Prunet, C.; Lemaire-Ewing, S.; Menetrier, F.; Neel, D.; Lizard, G. Activation of caspase-3-dependent and -independent pathways during 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell death: A morphological and biochemical study. J. Biochem. Mol. Toxicol. 2005, 19, 311−326. (45) Hoetelmans, R. W. M.; van Slooten, H. J.; Keijzer, R.; Erkeland, S.; van de Velde, C. J. H.; van Dierendonck, J. H. Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells. Cell Death Differ. 2000, 7, 384−39232. (46) Youle, R. J.; Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 2008, 9, 47− 59.
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DOI: 10.1021/jf505079v J. Agric. Food Chem. 2015, 63, 1035−1041
