Article pubs.acs.org/JAFC
Purification and Characterization of a Novel Hemagglutinin with Inhibitory Activity toward Osteocarcinoma Cells from Northeast China Black Beans Xiuli Dan,† Jack Ho Wong,† Evandro Fei Fang,‡ Francis Chun Wai Chan,§ and Tzi Bun Ng*,† †
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, United States § School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China ‡
ABSTRACT: In the present study, we isolated a novel hemagglutinin from an edible legume and explored its growth-inhibitory effect on osteocarcinoma and liver cancer cells. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono Q, and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion dependent and stable over a wide range of temperature and pH values. Antiproliferative activity was observed in the tumor cell lines MG-63 and HepG2 but not in the normal cell line WRL 68. Osteocarcinoma cells treated with the hemagglutinin underwent obvious cell shrinkage, chromatin condensation, mitochondrial membrane depolarization, and apoptosis. The mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were found to be up-regulated to different extents after treatment of this hemagglutinin. KEYWORDS: anticancer drug, apoptosis, cytokine, hemagglutinin, lectin, protein purification
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INTRODUCTION Osteocarcinoma is listed as one of the top ten most common cancers in children and teenagers; among 800 new cases diagnosed annually in America alone, half of the patients are children or teens.1 The most common sites for osteosarcoma are long bones in the legs and arm bones. Metastasis was found in more than 80 percent of patients who received only surgical treatment.2 Lung is usually the most popular second host place for osteocarcinoma cells, which may represent the metastasis site in more than 85% cases.3 According to Gill et al., the cure rate for nonmetastatic cancer is around 66%.1 However, once metastases have occurred, the cure rate will drop to half as much, i.e., 33%. Ayan followed 94 patients who had been diagnosed with osteosarcoma and received combined chemotherapy of epirubicin, cisplantin, and ifosfamide for 3 years, and he found that the survival rate of these patients reached 72.3%.4 This result displayed great value and the potential of chemotherapy. However, the survival rate is far from satisfactory, and side effects of chemotherapy are by no means negligible. Research aiming at ascertaining safer and more effective therapies is warranted. Lectins/hemagglutinins belong to a group of proteins which initiate sugar binding by using water bridges, oligomerization, or post-translational modification.5 Lectin was originally found in castor beans at the end of the 19th century and subsequently in a variety of organisms, like legumes, mushrooms, animals, bacteria, and viruses, etc.6 Lectins play protective roles in plants, participate in growth in mushrooms, and are involved in the migration of lymphocytes in mammals.7−9 Animal lectins are present in extremely low quantities, which contribute to low yields and poor flexibility in production. On the contrary, lectins occur in plants in abundance, especially in legumes from © 2015 American Chemical Society
which a variety of lectins have been isolated and characterized. Lam et al. purified a hemagglutinin from Phaseolus vulgaris cv. French bean and demonstrated its inhibitory function toward HIV-1 reverse transcriptase and antiproliferative effect on hepatoma HepG2 cells and breast cancer MCF-7 cells.10 Lei et al. explored the potential of concanavalin A in antihepatoma therapy and revealed its working mechanism: Con A could specifically bind to mannose on the cell membrane, leading to the preference of glycoprotein internalization to mitochondria and finally triggering lethal autophagy.11 However, the lectin purified from French bean does not show any antiproliferative activity on tumor cells and fails to promote the production of nitric oxide; besides, it does not induce a mitogenic response from splenocytes and exhibits no antifungal activity.12 Lectins or hemagglutinins which exhibit carbohydrate specificity by binding reversibly to specific glycoproteins have multiple potential applications in agriculture, medicine, and biotechnological research. It has been well documented that lectins play important roles in cellular and molecular recognition and have been exploited as carriers of chemotherapeutic agents, biomarkers of specific microorganisms, potential diagnostic reagents, and anticancer drugs. Lectins or hemagglutinins purified from different species or even the same species may show differences in structure, molecular size, and carbohydrate specificity, resulting in different biological properties.13−16 Received: Revised: Accepted: Published: 3903
January 9, 2015 March 27, 2015 March 27, 2015 March 27, 2015 DOI: 10.1021/acs.jafc.5b00106 J. Agric. Food Chem. 2015, 63, 3903−3914
Article
Journal of Agricultural and Food Chemistry
Ethylenediaminetetraacetic acid (EDTA) at a final concentration of 10 mM was added to NCBBH dissolved in PBS. The mixture was incubated at 4 °C for 5 h with shaking to make sure the hemagglutinin had been fully demetalized. The residual hemagglutinating activity was determined after EDTA had been removed. If the activity decreased or was completely lost, one of the following cations (KCl, AgCl, CuSO4, MgSO4, MnSO4, CaCl2, ZnSO4, FeSO4, and FeCl3 at a final concentration of 40 mM) was added, and after incubation for 3 h, hemagglutinating activity was assayed again to see whether it had been restored. The inhibition of hemagglutinating activity by carbohydrates was determined. NCBBH was dissolved in PBS buffer which contained different concentrations of sugar (the highest concentration at 100 mM) and serially 2-fold diluted with the same buffer in microtiter Uplates. Wells with only PBS or PBS-containing sugars were taken as the control. The mixture was subsequently mixed with an equal volume of rabbit blood (2%). The changes of HU in the presence and absence of different sugars were recorded. Labeling NCBBH with FITC. FITC labeled NCBBH was prepared by following the procedure provided by the manufacturer. Briefly, purified NCBBH was dissolved in 50 mM borate buffer at a concentration of 2 mg/mL. FITC dye (Thermo Scientific, Waltham, MA) dissolved in DMF at 10 mg/mL was slowly added to NCBBH solution with its final concentration 15-fold molar higher than that of NCBBH. The mixture was incubated in the dark at room temperature for 1 h. The excess FITC was removed by dialysis in ddH2O overnight at 4 °C. The FITC-labeled NCBBH was lyophilized and stored at −20 °C. Anticancer Activity of NCBBH. Cell Viability Assay. Osteosarcoma MG-63 cells, hepatoma HepG2 cells, and human embryonic liver WRL 68 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin (PS) at 37 °C. Cell viability after treatment with NCBBH was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay. Briefly, cells (5000 cells/ 100 μL/well) were seeded in a 96-well microtiter plate. Medium was discarded after 24 h, and cells were treated with 2-fold serially diluted NCBBH with a concentration varying from 15 to 0 μM for 24 or 48 h. MTT solution (25 μL 5 mg/mL) was added to each well after removal of the old medium and the solution incubated for another 4 h. Dimethyl sulfoxide (150 μL) was then added to each well to dissolve the crystal formazan. The absorbance at 570 nm was measured by using a BIO-RAD microplate reader. Cells (5 × 103) were seeded on a 6-well culture plate for 24 h, followed by the treatment of different concentrations of NCBBH for 6 days. Medium was changed every 2 days. After being rinsed with PBS, cells were fixed in 4% formaldehyde and subsequently stained with crystal violet (0.4 g/L). The images were captured by using the Nikon Live Cell imaging System Ti-E. The number of cells was counted by using Image-Pro Plus 6.0. Cell density was compared between the wells treated with and without NCBBH. Binding of NCBBH on Cells. 1 × 105 cells were seeded on 6-well culture plates overnight, followed by treatment of 20 μg/mL of FTIClabeled NCBBH for 24 h. The cells were harvested and washed twice with PBS. The FITC signal was then measured by a flow cytometer. Confocal Imaging Microscopy. 3 × 104 cells were seeded on the cover glass in 24-well culture plates overnight. Cells were then cultured with 0.2 mg/mL of FITC-labeled NCBBH for 3 or 24 h. After fixation in 4% formaldehyde for 15 min and washing three times with PBS, cells were then stained by 1 μM Hoechst 33342 for 20 min. The cover glass with cells were taken out from the culture plates and mounted in 50% glycerol on glass slide. The image was taken on the Olympus FV1000-ZCD laser scanning confocal system with a 60× oil immersion objective lens (Tokyo, Japan) and analyzed by Olympus Fluoview Version 2.0. Assay of Changes in Nuclear Morphology of MG-63 Cells. MG-63 cells were incubated in the presence of 4.52 μM NCBBH for 24 h and stained with 1 μM Hoechst 33342 for 20 min after removal of the medium and rinsing with PBS. Nuclear morphology was observed under a Nikon Live Cell Imaging System Ti-E.
In the present study, a novel hemagglutinin was purified from the seeds of Northeast China black beans, and its biological properties and antitumor and immumoregulatory effects were characterized.
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MATERIALS AND METHODS
Materials and Reagents. Northeast China black beans, a cultivar of Phaseolus vulgaris, were purchased from a local vender and authenticated by Professor Shiuying Hu, Honorary Professor of Chinese Medicine, The Chinese University of Hong Kong. Affi-Gel Blue gel, Mono Q 5/50 GL, Superdex 75 10/300 GL columns, and purifier were bought from GE Healthcare, UK. Human hepatocellular carcinoma HepG2 cell line, osteocarcinoma MG-63 cell line, and normal embryonic liver cell WRL 68 cell line were purchased from the American Type Culture Collection. All primary and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). TRIZOL and the reverse transcriptase PCR kit were acquired from Invitrogen (Waltham, MA). Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Warrington, Cheshire, UK). FITC fluorescent dye was purchased from Thermo Scientific (Waltham, MA). Purification of Hemagglutinin. Dried beans (60 g) were soaked in water overnight and then ground into a slurry. After centrifugation at 14000 rpm for 30 min at 4 °C and filtration through filter paper, the supernatant was adjusted with 2 M Tris−HCl buffer (pH 7.6) until the concentration of the buffer reached 20 mM, and it was then loaded on an Affi-Gel Blue gel column which was equilibrated with 20 mM Tris− HCl buffer (pH 7.6). Unadsorbed fraction and adsorbed fraction were, respectively, eluted with the starting buffer and 1 M NaCl in Tris−HCl buffer (20 mM, pH 7.6). Afterward, the bound fraction was dialyzed against water, lyophilized, dissolved in 20 mM NH4HCO3 buffer (pH 9.6), and chromatographed on a Mono Q column equilibrated with the same buffer. The bound fraction was eluted with three successive linear gradients of NaCl (0−0.2, 0.2−0.5, and 0.5−1 M, respectively, collected in 5, 20, and 5 mL of buffer) in the same buffer using fast protein liquid chromatography (FPLC). The active fraction was collected and processed as described in previous steps before loading on a Superdex 75 HR10/300 column attached to the FPLC system. The system was run with 0.1 M NaCl in 20 mM Tris−HCl buffer. Purified hemagglutinin from Northeast China black beans (NCBBH) was dialyzed and lyophilized before storage. Characterization of NCBBH. To determine the molecular weight of NCBBH, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 15% acrylamide gel) was performed in accordance with the procedure previously reported.17 Besides, a standard curve was constructed by running three marker proteins on the same FPLC-gel filtration system to help further determine the molecular weight. Amino acid sequence was analyzed by Edman degradation followed by HPLC system analysis or the protein was cleaved into small peptides and analyzed by mass spectrometry. Hemagglutinating activity assay was conducted by incubating a serially 2-fold diluted hemagglutinin (50 μL) with 50 μL of rabbit blood (2% in phosphate-buffered saline) in microtiter U-plates. Agglutination status of the red blood cells was recorded when the blank had fully precipitated. The hemagglutination unit (HU) was defined as the reciprocal of the highest dilution of the sample which exhibited hemagglutination, and the number of hemaglutination units per milligram of protein was recorded as specific activity.18 The stability of hemagglutinin at different temperatures and pH values was determined by the following methods. Purified hemagglutinin was dissolved in phosphate-buffered saline (PBS) and incubated at different temperatures for 30 min. After the mixture was fully cooled to room temperature, the remaining hemagglutinating activity of the mixture was assayed. pH stability was tested by employing the following steps: NCBBH was first dissolved in water and then adjusted with NaOH and HCl to different pH values (0−14). After incubation at room temperature for 30 min, the mixture was neutralized to pH 7. Hemagglutinating activity was then determined. 3904
DOI: 10.1021/acs.jafc.5b00106 J. Agric. Food Chem. 2015, 63, 3903−3914
Article
Journal of Agricultural and Food Chemistry Flow Cytometry Assay for Apoptosis. Apoptosis-inducing activity of NCBBH was examined by using the Annexin-V (BD Pharmingen, San Jose, CA) and propidium iodide PI (Sigma-Aldrich, St. Louis, MO) staining methods. Briefly, MG-63 cells were treated with different concentrations of NCBBH for 48 h. Trypsinized cells were centrifuged (1500 rpm, 5 min), washed with PBS, and stained with 0.5 mg/mL Annexin V and PI (final concentration 5 μg/mL) in binding buffer (0.01 M HEPES, pH 7.4, containing 140 mM NaCl and 25 mM CaCl2) in darkness at room temperature for 15 min. Cells were then applied to a flow cytometer (after filtration). Assay of Mitochondrial Membrane Depolarization. The mitochondrial membrane potential of MG-63 cells was measured by JC-1 staining as previously reported, based on the principle that in healthy cells, JC-1 dye accumulates in the mitochondria and emits red fluorescence while JC-1 exists as a monomer in cytosol and emits green fluorescence when the mitochondrial membrane is depolarized.19 After treatment with NCBBH (1.25, 0.625, and 0 μM) for 24 h, MG-63 cells were collected and stained with 2.5 μg/mL JC-19 dye for 15 min in darkness before analysis using a flow cytometer. Western Blotting. After treatment of NCBBH for different durations, the MG-63 cells were lysed in ice-cold RIPA lysis buffer. The whole cell extracts were then added to the loading buffer and boiled for 5 min before loading on SDS-PAGE. After electrophoresis, the proteins on the gel were then transferred onto polyvinylidene fluoride membranes and blocked in 5% milk for 40 min. The membranes were subsequently incubated with the first antibody overnight at 4 °C and the second antibody for 2 h at room temperature. The bands were finally visualized by using ECL detection kits. Effect of N-Acetylgalactosamine (GalNAc) on the Antiproliferative Activity of NCBBH. To determine whether GalNAc could partially inhibit the antiproliferation effect of NCBBH, MG-63 cells and HepG2 cells were cultured with different concentration of NCBBH in the presence and absence of GalNAc for 48 h. The highest concentration of GalNAc was 5 mM which is close to the plasma sugar level of human body. The toxicity of GalNAc itself on the cancer cells was also determined by culturing cells in the medium containing different concentrations of GalNAc for 48h. Detection of Immunomodulatory Effect of NCBBH on Splenocytes. Real-Time PCR. The spleen was collected immediately after the mouse (C57BL/6J) was sacrificed and pressed gently through a sterile stainless steel sieve in PBS. The cell suspension was collected and centrifuged at 1500 rpm for 3 min, followed by the addition of lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 100 mM EDTA, pH 7.2) to lyse the blood cells. The splenocytes were collected by centrifugation at 1500 rpm and washed twice with PBS before culturing with different concentrations of NCBBH in DMEM for 4 h. The total RNA of splenocytes was extracted with TRIZOL and reverse-transcribed into cDNA with oligo-Dt primers using a Revert first strand cDNA synthesis kit. Real-time PCR was performed using SYBR Green. The target genes include IFN-γ, IL-2, IL-6-, IL-1β, TNFα, and glyceraldehyde-3-phosphate dehydrogenase known as GAPDH as the standard (shown in Table 1). Detection of Cytokine Secretion by Western Blotting. The splenocytes were prepared as described above and evenly separated into six cell culture dishes which contained different concentrations of NCBBH in the same final volume of FBS free DMEM. The cells were cultured for 24 h before the conditional medium was collected. The same volume of the conditional medium was taken for Western blotting by following the procedures described above. Statistics. Results were taken from three independent experiments (n = 3), and data were expressed as means ± standard deviation (SD). Scatter diagram and histograms were drawn using GraphPad Prim 5 (Graphpad Software, La Jolla, CA). For PCR experiments, one-way ANOVA followed by a Bonferronni posthoc test was used for the comparisons between multiple groups. A P value
