Article pubs.acs.org/JAFC
Se-Methyl‑L‑selenocysteine Induces Apoptosis via Endoplasmic Reticulum Stress and the Death Receptor Pathway in Human Colon Adenocarcinoma COLO 205 Cells Yen-Chen Tung,† Mei-Ling Tsai,‡ Fang-Ling Kuo,‡ Ching-Shu Lai,§ Vladimir Badmaev,∥ Chi-Tang Ho,† and Min-Hsiung Pan*,§,⊥,# †
Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, United States Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan § Institute of Food Sciences and Technology, National Taiwan University, Taipei 106, Taiwan ∥ American Medical Holdings Incorporated, 1440 Forest Hill Road, New York, New York 10314, United States ⊥ Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan # Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan ‡
ABSTRACT: Selenomethionine (SeMet) and Se-methyl-L-selenocysteine (MSeC) are natural organoselenium compounds found in garlic, onion, and broccoli. In addition, these compounds have lower toxicity and better anticancer activities than inorganic Se. This study investigated the effects of MSeC treatment on the growth of COLO 205 human colorectal adenocarcinoma cells and evaluated the mechanisms related to the MSeC-induced effects. When COLO 205 cells were treated with 200 μM MSeC for 24 h, MSeC caused 80% apoptosis in cells. MSeC increased the expression of Fas and FasL, followed by the cleavage of caspase-3, caspase-8, DNA fragmentation factor (DFF45), and poly(ADP-ribose) polymerase (PARP). MSeC also increased the levels of Bax protein and decreased the levels of Bid and Bcl-2 proteins. However, MSeC did not cause apoptosis through reactive oxygen species (ROS) stress but instead through endoplasmic reticulum (ER) stress. The cleavage of caspase-12 and caspase-9 was shown to increase the growth arrest and protein levels of DNA-damage inducible genes (GADD) 153 and 45. MSeC also downregulated the ERK1/2 and PI3K/AKT protein levels and upregulated the p38 and JNK protein levels in COLO 205 cells. These results showed that the mechanism by which MSeC induced apoptosis in COLO 205 cells involved caspase activation, the extrinsic apoptotic pathway, and the regulation of ER-stress-induced apoptosis. KEYWORDS: Se-methyl-L-selenocysteine, apoptosis, endoplasmic reticulum stress, COLO 205 cells
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INTRODUCTION Colorectal cancer (CRC) is the third most common cancer worldwide and the second most frequent cause of cancer death in men and women.1 Most cases of CRC are thought to arise from adenomatous polyps, and a routine colonoscopy may be a good tool for detecting adenomatous polyps early, thereby decreasing the incidence and mortality of CRC.2 However, CRC may arise between two colonoscopy examinations.3 The occurrence of CRC is highly related to diet. Red meat and alcohol consumption are recognized dietary risk factors for the development of CRC.4 Selenium (Se) is an essential micronutrient required by the human body and is an important element of glutathione peroxidase, which protects the body.5,6 Se can be obtained from inorganic compounds, such as selenites and selenates, or in organic forms, such as selenoamino acids, selenopeptides, and selenoproteins.5,7,8 Although the inorganic forms of Se are common sources of Se and have been shown to effectively induce apoptosis in different cancer cells, they may also cause toxicity in normal cells.9 An increasing number of studies have shown that organic Se, such as selenomethionine (SeM) and Se-methyl-L-selenocysteine (MSeC), have better anticancer activities and lower toxicities than inorganic Se.10 In 1971, Shamberger and Willis indicated that people living in areas with © 2015 American Chemical Society
high dietary Se consumption have lower mortality rates because of gastrointestinal tract, lung, and breast cancers.11,12 Moreover, epidemiological surveys have also demonstrated that selenium deficiency in the body can increase the risk of colonic, gastric, lung, and prostate carcinomas.7 Se can be obtained from food and is abundant in natural foods, such as nuts, eggs, fish, liver, chicken, and garlic.5 It seems that Se may be a supplement for CRC prevention. Thus, in the present study, we investigated the organoselenium compounds SeM, MSeC, N-γ-(L-glutamyl)L-selenomethioine (GSeM), and N-γ-(L-glutamyl)-Se-methyl-Lselenocysteine (GMSeC) for their inhibitory activities on the cell growth of human COLO 205 colorectal adenocarcinoma cells. We identified the most potential anticancer compound and explored the mechanism underlying the promotion of apoptosis in cancer cells. MSeC was shown to inhibit COLO 205 cancer cell growth through multiple underlying mechanisms to promote apoptosis. Altogether, these results indicate a potential application for MSeC in the prevention of colon cancer. Received: Revised: Accepted: Published: 5008
April 9, 2015 May 5, 2015 May 5, 2015 May 5, 2015 DOI: 10.1021/acs.jafc.5b01779 J. Agric. Food Chem. 2015, 63, 5008−5016
Article
Journal of Agricultural and Food Chemistry
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Trypan Blue Exclusion Assay. COLO 205 cells were cultured in the 24 well plate with the 2 × 105 cells/mL density. After overnight growth, cells were treated with 0, 50, 100, 200, and 250 μM MSeC for 24 h. After a 24 h co-culture with different concentrations of MSeC, the cell suspension was collected and dyed with trypan blue by a 1:1 ratio. Cells were then counted in a hemacytometer by a microscope (Olympus America, Inc., Lake Success, NY). Flow Cytometry. Incubated COLO 205 cells were seeded with the 2 × 105 cells/mL density in the 24 well plate overnight and treated with different concentrations of MSeC for 24 h. After 24 h, cells were harvested with phosphate-buffer saline (PBS), fixed with iced 100% ethanol, and then stored in −20 °C. Cells were resuspended with hypotonic buffer (0.5% Triton X-100 in PBS and 0.5 μg/mL RNase) and then stained with PI at 37 °C for 15 min, individually. Fluorescence intensity was quantified by flow cytometry (BectonDickinson, San Jose, CA). AO Staining Assay. COLO 205 cells were cultured in the 24 well plate with 2 × 105 cells/mL density overnight and treated with 200 μM MSeC for another 24 h. After 24 h, cells were harvested and suspended in the PBS. Equal volumes of cell suspension and AO were mixed, and the fluorescence intensity was observed by a microscope (Olympus America, Inc., Lake Success, NY). DNA Fragmentation. COLO 205 cells were seeded with the 2 × 105 cells/mL density in a 100 mm Petri dish overnight and then treated with different concentrations of MSeC for 24 h. Cells were also treated with 200 μM MSeC for 0, 3, 6, 9, 12, 18, and 24 h. Cells were harvested, centrifuged, and suspended in PBS. The cell solution was centrifuged, and then the cell lysate was collected and mixed with DNA lysis-PK buffer at 56 °C overnight. RNase A (0.5 μg/mL) was added to the cell lysate solution at 50 °C for 3 h, and an equal volume of phenol/chloroform/isoamyl (25:24:1) was added in the cell lysate solution to obtain DNA. DNA was loaded in the 2% agarose gel electrophoresis and run at 50 V for 120 min in Tris−borate/ ethylenediaminetetraacetic acid (EDTA) electrophoresis buffer (TBE). The gel was stained by ethidium bromide, visualized under ultraviolet (UV) light, and photographed. Caspase Activity. COLO 205 cells were seeded in 100 mm with the 2 × 105 cells/mL density Petri dish overnight and then treated with 200 μM MSeC for 0, 3, 6, 9, 12, 18, and 24 h. Harvest cells were washed by PBS and centrifuged, and then the supernatant was removed. Protein was extracted by mixing cell pellet with gold lysis buffer [50 mM tris(hydroxymethyl)aminomethane (Tris)−HCl at pH 7.4, 1 mM NaF, 150 mM NaCl, 1 mM ethylene glycol bis(2aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM phenylmethanesulfonyl fluoride, 1% NP-40, and 10 g/mL leupeptin] and then centrifuged at 12 000 rpm for 30 min at 4 °C. A total of 50 μg of each sample was mixed with the caspase substrate (acetyl-Asp-Glu-ValAsp-7-amino-4-methylcoumarin) at 30 °C for 1 h, and the fluorescence intensity was quantified by the ELISA reader at 360 nm excitation wavelength and 460 nm emission wavelength. Determination of Superoxide, Hydroperoxide, Glutathione, and Mitochondrial Transmembrane Potential. COLO 205 cells were incubated with the 2 × 105 cells/mL density in the 24 well plate overnight and treated with 200 μM MSC for 0, 15, 30, 60, and 120 min. Cells were harvested and washed by PBS twice and mixed separately with dihydroethidium (DHE), 5-cholomethylfluoroescein diacetate (CMFDA), 2′,7′-dicholofluoroescein diacetate (DCFHDA), and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) for 30 min, and the reactions were stopped by trypsin−EDTA at 37 °C and then analyzed by flow cytometry. Analysis of Antioxidant and Apoptosis. COLO 205 cells (2 × 105 cells/mL) were seeded in the 24 well plate overnight and 50 and 100 μM allopurinol (ALL), 200 and 400 units/mL catalase (CAT), 10 and 20 μM diphenylene iodonium (DPI), 2.5 and 5 μM Nacetylcysteine (NAC), and 100 and 200 μg/mL superoxide dismutase (SOD) were added for 1 h, individually. After the treatment with different antioxidants, the cells were treated with 200 μM MSeC for 24 h and the reaction was stopped by trypsin−EDTA and analyzed by flow cytometry.
MATERIALS AND METHODS
Cell Culture. The cell line COLO 205 [CCL-222, American Type Culture Collection (ATCC), Rockville, MD] was cultured in the RPMI-1640 medium with 10% fetal bovine serum (Gibco, Grand Island, NY) and penicillin−streptomycin (Biowest, Nuaille, France) and maintained in a humidified 5% CO2 atmosphere at 37 °C. Chemicals and Reagents. SeM, GSeM, MSeC, and GMSeC are kindly provided by American Medical Holdings, Inc. (New York, NY) (Figure 1). The purity of those compounds is over 99.2% according to
Figure 1. Chemical structures of (A) SeM, (B) MSeC, (C) GSeM, and (D) GMSeC. high-performance liquid chromatography analysis. Trypan blue, acridine orange (AO), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-Akt and anti-PARP antibodies were purchased from Merck (Whitehouse Station, NJ); anti-Bcl-2 and anti-Bax were purchased from Santa Cruz Biotechnology (Dallas, TX); anti-Bid, anti-Fas, and anti-FasL were purchased from Becton-Dickinson (San Jose, CA); anti-DFF-45/ICAD and anticaspase 8 were purchased from MBL (Woburn, MA); anti-p44/42 (ERK1/2) was purchased from Cell Signaling Technology (Danvers, MA); anti-p53 was purchased from Oncogen (La Jolla, CA); and antiphospho-Akt and anti-caspase 3 were purchased from Novus (Littleton, CO). Cytotoxicity Assay. COLO 205 cells were seeded in the 24 well plate with the density of 2 × 104 cells/mL overnight. Cells were treated with 0, 50, 100, 200, and 250 μM SeM, GSeM, MSeC, and GMSeC for 24 h. Cell viability was assayed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and determined by scanning with an enzyme-linked immunosorbent assay (ELISA) reader at 570 nm. 5009
DOI: 10.1021/acs.jafc.5b01779 J. Agric. Food Chem. 2015, 63, 5008−5016
Article
Journal of Agricultural and Food Chemistry Western Blotting. COLO 205 cells were seeded in 100 mm with the 2 × 105 cells/mL density Petri dish overnight and then treated with 0, 50, 100, 200, and 250 μM for 24 h and 200 μM MSeC for 0, 3, 6, 9, 12, 18, 24 h, individually. Protein was extracted with gold lysis buffer (50 mM Tris−HCl at pH 7.4, 1 mM NaF, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethanesulfonyl fluoride, 1% NP-40, and 10 μg/mL leupeptin) by mixing the cells and gold lysis buffer on ice for 30 min, followed by centrifugation at 10000g for 30 min at 4 °C. The total protein (50 μg) was used in the 12% sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) at a constant current of 20 mA. Proteins were electrotransferred from gel to immobile membrane [polyvinylidene fluoride (PVDF), Millipore Corp., Bedford, MA] with transfer buffer (25 mM Tris−HCl at pH 8.9, 192 mM glycine, and 20% methanol). The membranes were blocked with blocking solution [20 mM Tris−base, 125 mM NaCl, 0.2% Tween 20, 1% bovine serum albumin (BSA), and 0.1% sodium azide] and then immunoblotted with primary antibodies [including Anti-Akt and antiPARP antibodies purchased from Merck (Whitehouse Station, NJ), anti-Bcl-2 and anti-Bax purchased from Santa Cruz Biotechnology (Dallas, TX), anti-Bid, anti-Fas, and anti-FasL purchased from BectonDickinson (San Jose, CA), anti-DFF-45/ICAD and anti-caspase 8 purchased from MBL (Woburn, MA), anti-p44/42 (ERK1/2) purchased from Cell Signaling Technology (Danvers, MA), anti-p53 purchased Oncogen (La Jolla, CA), and anti-phospho-Akt and anticaspase 3 purchased from Novus (Littleton, CO)] at 4 °C overnight. Detection was achieved by measuring the chemiluminescence of the blotting agent [electrochemiluminescence (ECL), Amersham Corp., Arlington Heights, IL], after exposure of the filters to Kodak X-Omat films. Statistical Analysis. All results were presented as means ± standard error of the mean (SE) in the independently performed experiments. The differences between the control group and different concentrations of Se compounds were analysis by Student’s t test. Data were considered statistically significant at a p value of
