Inhibition of ALV-A-induced apoptosis in DF-1 cells via inactivation of nuclear transcription factor κB by anthocyanins from purple corn (Zea mays L.)

journal of functional foods 10 (2014) 274–282

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Inhibition of ALV-A-induced apoptosis in DF-1 cells via inactivation of nuclear transcription factor κB by anthocyanins from purple corn (Zea mays L.) Dan Wang a, Yongdong Lei a,b, Yue Ma a, Li Zhang a, Xiaoyan Zhao a,* a

Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Science, Beijing Key Laboratory of Agricultural Products of Fruits and Vegetables Preservation and Processing, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 100097, China b College of Food, Shihezi University, Shihezi 832003, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

Ten anthocyanins were identified from purple corn (Zea mays L.), Jingzi variety, by high-

Received 18 February 2014

performance liquid chromatography tandem mass spectrometry in order to assess their anti-

Received in revised form 12 June

avian leukosis virus subgroup A (ALV-A) induced cell apoptosis in vitro. The inhibitory activity

2014

and mechanism were evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

Accepted 17 June 2014

bromide assay, Trans AM™ and western blotting. Total anthocyanins from purple corn (TAPC)

Available online

exhibited a dose-dependent effect on decreasing apoptosis of chicken embryo fibroblasts, DF-1 cell, infected with ALV-A in the range of 4–12 µg/mL. Meanwhile, TAPC decreased trans-

Keywords:

fer levels of nuclear transcription factor kappa B (NF-κB) p50/p65 from cytoplasm to nucleus,

Anthocyanin

and downregulated the expression of NF-κB p50/p65 which promoted DF-1 cell cytopathic

Purple corn

effects evaluated by western blotting. TAPC inhibited ALV-A induced apoptosis in DF-1 cells

ALV-A

via inactivation of NF-κB p50/p65. © 2014 Elsevier Ltd. All rights reserved.

DF-1 NF-κB

1.

Introduction

Anthocyanins have potential application as natural food colorants and exhibit antioxidation, antiinflammation, and anticarcinogenic activities in vitro (Inagaki, Morimura, Shigematsu, Kida, & Akutagawa, 2005; Kim et al., 2006; Lee, Lim, & Choung, 2013; Lerma, Peinado, & Peinado, 2013; Matsui et al., 2001; Nizamutdinova et al., 2009). Moreover, it mediates human myeloid leukemic HL-60 cells apoptosis via the p38-FasLand

* Corresponding author. Tel.: +86-10-51503053; fax: +86-10-51503053. E-mail address: [email protected] (X. Zhao). http://dx.doi.org/10.1016/j.jff.2014.06.022 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

Bid pathway in a dose dependent manner of the anthocyanin content (Chang, Huang, Hsu, Yanga, & Wang, 2005). Purple corn (Zea mays L.) is an important source of anthocyanin with the antimicrobial activity, inhibiting proliferation of human colorectal adenocarcinoma HT-29 cells, and exhibits an additive interaction with other phenolic acids (Jing et al., 2008; Zhao et al., 2009). However, the antivirus activities of purple corns have not yet been investigated. Avian leukosis virus infected meat-type chickens can form plaques which can induce huge economic losses (Lewis,

journal of functional foods 10 (2014) 274–282

Chinnasamy, Morgan, & Harold, 2001; Young, Bates, & Varmus, 1993). The ubiquitous nuclear transcription factor kappa B (NFκB) has been implicated in the control of proliferation and apoptosis in many types of cancer cells (Cacho, Gallego, Bemad, Quílez, & Sánchez-Acedo, 2004). Many viruses, such as dengue virus, cytomegalovirus, Epstein–Barr virus, hepatitis B virus cause kinds of tumors by activation NF-κB (Cahir-McFarland, Davidson, Schauer, Duong, & Kieff, 2000; DeMeritt, Milford, & Yurochko, 2004; Jan et al., 2000; Pan, Duan, Sun, & Feitelson, 2001). The human T-cell leukemia virus type 1 (HTLV-1) transactivator can induce immortalization of primary human T-lymphocytes through a mechanism independent of NF-κB activation (Rosin et al., 1998). Also, previous researchers have suggested that the activation of NF-κB plays an important role in the apoptotic process of human hematopoietic cells (Bessho et al., 1994). ALV and the human T-cell leukemia virus type 1 (HTLV-1) are in the same genus, so whether the NF-κB routes also applies to ALV, and anthocyanin represses apoptosis through this pathway has not been reported. NF-κB, normally a latent protein residing in the cytoplasm as a complex with the inhibitory protein IκBa, can be activated by a variety of proinflammatory agents or through genotoxic stress or various viruses (Antony et al., 2003). This process is initiated by dissociation of the NF-κB complex, which exposes DNA binding domain, and transfers from cytoplasm into the nuclei, then bind with a variety of target genes involved in DF-1 cells survival. However, activation of NF-κB also transcriptionally upregulates its inhibitor protein IκBa, thereby tightly controlling NF-κB activity in normal cells. Which kind of NF-κB protein is activated after Avian leukosis virus subgroup A (ALV-A) infected DF-1, and whether total anthocyanins from purple corn (TAPC) can inactivate NF-κB activity have not previously been evaluated. Therefore, the aim of this study was to identify TAPC by highperformance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), and evaluated the effect of TAPC on NF-κB activity in DF-1 cells infected with Avian leukosis virus subgroup A (ALV-A) by methods of MTT, Trans AM™ and western blotting. Meanwhile, the role of the most abundant of anthocyanins in TAPC, cyanidin-3-O-glucoside (C3G) was studied compared with TAPC. Results would provide useful information for illustrating the mechanism of the inhibition of TAPC on cell apoptosis induced by ALV-A.

2.

Materials and methods

2.1.

Reagents and chemicals

Purple corn, DF-1 cell line and ALV-A strain were supplied by Beijing Academy of Agriculture and Forestry Sciences. Anhydrous ethanol, methanol, and acetonitrile were purchased from Dima Technologies (Beijing, China). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Chemie GmbH, Stuttgart, Germany). Standard of C3G was obtained from Polyphenols Laboratories AS (Sandnes, Norway). The solvents/chemicals used were chromatography-grade. Analytical-grade hydrochloric acid, glutamine, penicillin, streptomycin sulfate, sodium chloride, potassium chloride, monobasic potassium phosphate,

275

disodium hydrogen orthophosphate, dimethyl sulfoxide, ethylenediaminetetraacetic acid, nonfat milk, and polysorbate 20 (Tween 20) were obtained from Beijing Chemistry Co. (Beijing, China). Dulbecco’s modified Eagle’s medium (MEM), fetal bovine serum (FBS) were supplemented by Becton, Dickinson and Company (New York, NY, USA). Nuclear extract kit, mouse monoclonal antibodies, rabbit polyclonal antibodies, β-actin, GAPDH, IgG, NF-κB antibodies p52, p65, c-Rel, RelB, horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Sep-Pak C18 solid-phase extraction (SPE) cartridge (12 mL, 2000 mg) was obtained from Waters (Milford, MA, USA). Syringe filter units (0.22 µm) were supplied by Hercules (Beijing, China). Distilled water was used throughout.

2.2.

Preparation of total anthocyanins from purple corn

Purple corn was extracted with 60% (v/v) ethanol acidified with hydrochloric acid until pH = 3.0 at 50 °C for 120 min. The ratio of purple corn and extracted solution was 1:15 (v/v). The supernatants were evaporated to dryness at 40 °C with a rotary evaporator BÜchi R-3000 (BÜchi Labortechnic AG, Flawil, Switzerland), and filtered using a 0.22 µm syringe filter. The concentrated sample was then purified by using a SepPak C18 SPE cartridge which was conditioned consecutively with 8 mL of methanol and 8 mL of water. An aliquot of 1 mL of sample was loaded onto the SPE cartridge. Eluting with water and ethyl acetate removed sugars, organic acids, phenolic acids, and flavonoids, respectively. Then, methanol eluent was collected and concentrated with a rotary evaporator and dried through nitrogen. The product, TAPC, was further used for HPLCMS/MS analysis and antivirus experiments.

2.3.

HPLC-DAD/ESI-MS analysis of TAPC

TAPC after purifying by Sep-Pak C18 SPE cartridge were performed by Agilent 1200 series liquid chromatograph containing an autosampler coupled with a 6300 series ion trap mass spectrometer (Agilent, Santa Clara, CA, USA). An aliquot of 3 µL of the sample was injected onto an analytical scale Zorbax SBAO column (particle size, 1.8 µm; 100 × 3.0 mm; Agilent). The operating conditions were as follows: column oven at 25 °C; injection volume, 3 µL; eluent flow rate, 3 mL/min. The elution mode were A (0.5%, v/v, formic acid in water) and B (acetonitrile containing 0.5% formic acid) with the following gradient: 13–22% B from 0 to 8 min, 22–23% B from 8 to 15 min, 23–100% from 15 to 20 min, and isocratic 100% for 5 min, then 100–13% from 25 to 30 min, and isocratic at 13% from 30 to 35 min to equilibrate the column for the next injection. Diodearray detector (DAD) detection with 520 nm as detection wavelength was performed. TAPC were detected using an ion trap in the positive ion mode. The MS parameters were as follows: nebulizing pressure, 45 psi; source temperature, 110 °C; desolvation gas flow, 11 L/min nitrogen; desolvation temperature, 350 °C; 12 L/min nitrogen; smart parameter setting, compound stability, 20%; trap drive level, 100% scan ranger, m/z 100–1500.

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2.4.

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Cell culture and virus

DF-1 cell was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL of penicillin, and 100 mg/mL streptomycin sulfate. All cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. ALV-A was used at 103 infectious units per 7.5 × 105 DF-1 cell seeded into 25 mm2 culture flasks. The cells were allowed to attach for 24 h.

2.5.

Determination of cell viability

To evaluate the cytotoxicity of TAPC, a MTT colorimetric assay was performed to determine cell viability (Ferrari, Fornasiero, & Isetta, 1990). Cells were seeded in 96-well plates at a density of 1–2 × 105 cells/well at 37 °C for 48 h and treated with TAPC (4, 8, 12, 16 and 20 µg/mL) for 24, 48, 72 and 96 h. Then, media were removed and cells were incubated with MTT (0.5 mg/ mL) at 37 °C for an additional 4 hours. The viable cell number/ dish, which is proportional to the production of formazan after a solubilization with dimethyl sulfoxide, can be measured spectrophotometrically by multifunctional microplate reader (BIO RAD, Hercules, CA, USA) at 490 nm.

2.6. Reverse transcription-polymerase chain reaction (RT–PCR) Total RNA was isolated from DF-1 cells after the above treatments for 6 days according to the manufacturer’s instruction (Qiagen, NY, USA), and cDNA synthesis and PCR amplification were performed as previously described (Fenton, Maddula, & Trevor, 2005). For reverse transcription, 3 µL of total cellular RNA were used as templates in a 10 µL reaction containing 1 µL dNTPs (2.5 mM), 2 µL 5 × AMV Buffer, 1.5 µL Oligo primer dT20 (10 pmoL/mL), 0.25 µL RNase inhibiter, 0.5 µL AMV RTase (200 U/mL) and 1.75 µL DEPC water, then the reaction was performed at 42 °C for 1 h. Afterwards, 5 µL cDNA product were used as templates in PCR amplifications together with appropriate primers (for P27, upstream sequence: 5′GAATTCATGCCTGTAGTGATTAAGACAG-3′, downstream sequence: 5′-GTCGACCTAGGGCTGGATAGCAGACTACAT-3′; for ALV-A, upstream sequence: 5′-GTACCACCCTCACTTATCGG AAGG-3′, downstream sequence: 5′-ACCCACTGGCATTACCC AAACTAC-3′). The final products were analyzed on 1.0% agarose gel and detected by ethidium bromide staining.

2.7.

Anti-ALV-A invasion assay of TAPC

To evaluate the effect of TAPC for ALV-A-induced DF-1 cells apoptosis, the preventive and therapeutic experiments were carried out. Briefly, for the preventive assay, DF-1 cells were seeded onto 96-well plates at a concentration of 2–5 × 104 cells/ mL; media were removed while cells grew to 80% confluence, incubated with anthocyanin (4, 8, 12 and 16 µg/mL) at 37 °C for 2 h and then treated with ALV-A, then medium containing 2% FBS was added to the wells of the plate, finally measured cytostatic activity for 24, 48, 72 and 96 h by MTT assay. Following therapeutic assay, DF-1 cells grew to 80% confluence; media were removed, incubated with ALV-A at 37 °C for 2 h and then treated with anthocyanin (4, 8, 12 and 16 µg/mL), invading cells

were fixed with DMEM of containing 2% FBS to 0.2 mL, then cell viability was determined for 24, 48, 72 and 96 h by MTT assays.

2.8.

Preparation of cytoplasmic and nuclear extracts

Cytosolic and nuclear protein fractions from DF-1 cells were prepared by the nuclear extract kit. Based on the therapeutic assay, 1 × 106 adherent DF-1 cells were seeded onto 75 mm2 culture flask, media were removed while cells grew to 80% confluence, incubated with ALV-A at 37 °C for 2 h and then treated with anthocyanin (4 and 12 µg/mL) and C3G (10 µg/mL), medium containing 2% FBS to 15 mL was added to culture flask, the protein fractions were prepared for 72 h according to the protocol of the manufacturer. The cytoplasmic and nuclear extracts were collected and stored at −80 °C. The protein contents of the cell lysate were determined by using the Bradford calorimetric assay method (Thermo, Waltham, MA, USA).

2.9.

Western blot analysis

To determine the protein expression of NF-κB in the cytoplasm and the nucleus, separate extracts were prepared. Ten micrograms of the cytoplasmic and the nuclear lysate from therapeutic experiments were subjected to 10% SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk in 0.1% polysorbate 20 (Tween 20) for 1 h and then probed with a diluted primary antibody. Primary antibodies, including mouse monoclonal antibodies against NF-κB p50 (1:1000 dilution) or NF-κB p65 (1:500) and rabbit polyclonal antibodies against β-actin (1:1000), GAPDH (1:1000) were used at the dilutions indicated. Blots were then washed with Tris-buffered saline, exposed for 1 hour to either horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies IgG (1:5000) as appropriate, and washed again. Bound antibodies were detected by chemiluminescence using a supersignal west pico chemiluminescent substrate kit (Pierce Biotechnology, Rockford, IL, USA, 500 mL) according to manufacturer’s instructions. Western blotting for β-actin and GAPDH served as two positive controls for total protein loaded.

2.10.

NF-κB DNA-binding activity measurement

Trans AM™ NF-κB family kit (Active Motif, Carlsbad, CA, USA) was used to detect NF-κB DNA-binding activity according to the protocol of the manufacturer. The nuclear and cytoplasmic extracts were obtained from DF-1 cells as described above. We recommended using 5 µg of the cytoplasmic and the nuclear lysate from therapeutic experiments in completely lysis buffer per well. Based on the instructions, binding of primary antibody, 100 µL of diluted NF-κB antibodies (1:1000; p50, p52, p65, c-Rel and RelB) were added, and binding of secondary antibody, 100 µL of diluted HRP-conjugated antibody (1:1000) were added. Then, colorimetric solution was used and incubated for 5 min at 25 °C in dark. The data were measured by a spectrophotometer within 5 min at 450 nm with an optional wavelength of 655 nm as reference.

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2.11.

Statistical analysis

The results were expressed as the mean value ± standard deviation. All statistical analyses were done with the Super ANOVA (version 1.11, Abacus Concepts Inc., Berkeley, CA, USA). Oneway ANOVA and multiple comparisons (Fisher’s least-significant difference test) were used to evaluate the significant differences of data at a criterion of P < 0.05.

3.

Results

3.1.

Identification of TAPC

Purple corn anthocyanin extract was separated by Sep-Pak C18 SPE cartridge. Sugars and acids can be washed away by water. The phenolic acids and flavonoids which were less polar compounds could be removed from the column with ethyl acetate. Anthocyanins were recovered with methanol. TAPC (a visible red fraction) can only be obtained through methanol eluting, confirming the existence of anthocyanin, which will be further separated by HPLC. The HPLC result showed that the TAPC consisted of 10 anthocyanins (Table 1). Furthermore, mass spectrometry was used to determine the molecular mass and the compounds. Compounds were detected in the positive ion mode and performed in a wider mass range scan (m/z 100–1500) with lower compound stability (20%). All components can be detected as their protonated adducts under current conditions (Pasch, Pizzi, & Rode, 2001). First, peak 1 was identified. Under the above condition, there was a single prominent protonated molecular ion peak showed at m/z 449 [M + H]+, indicating that only one compound existed in peak 1 (data not shown). MS/MS results are shown in Table 1. It produced one fragment ion at m/z 287 through losing one group with mass of 449 − 287 = 162 from parent ion, indicating sugar moiety as the lost fragment, furnishing the cyanidin aglycone with m/z 287. Finally, peak 1 was identified as C3G according to its uv/vis spectrum, mass spectra,

previous research and the reference of standard (Jing et al., 2008; Pascual-Teresa, Santos-Buelga, & Rivas-Gonzalo, 2002). Other peaks were identified similarly as peak 1. The identified structures of 10 peaks were summarized in Table 1. The anthocyanins from purple corn belong to peonidin, cyanidin, pelargonidin or delphinidin aglycone, were bound with glucose or rhamonside, with glucose acylated with malonyl group. The relative amounts of anthocyanins calculated by their peak area are displayed in Table 1. The composition of the anthocyanins isolated from purple corn was as follows: C3G (1) : pelargonidin-3-O-glucoside (2) : cyanidin-3-(6″-malonylglucoside) (3) : peonidin-3-O-glucoside (4) : isocyanidin-3-(6″malonylglucoside) (5) : cyanidin-3-2malonlyglucoside (6) : pelargonidin-3-(6″-malonylglucoside) (7) : delphinidin-3-Oglucoside-5- rhamnoside (8) : peonidin-3-(6″-malonylglucoside) (9) : delphinidin-3-O-glucoside (10) = 37.13 : 6.56 : 2.21 : 8.24 : 27.15 : 2.69 : 5.85 : 7.95 : 0.65 : 1.57. It was clear that the major anthocyanins identified were cyanidin type (peaks 1 and 5), and C3G was the most prominent of the components.

3.2.

Proliferative response of ALV-A in DF-1 cells

Total cellular DNA was isolated from the DF-1 cells or total RNA was separated from suspension after the cell was inoculated with ALV-A and 4, 12 µg/mL anthocyanins for 5 days. The total DNA extract containing the common antigen p27 gene of ALVs was analyzed by PCR (Fig. 1A, lines 1–5), while the characterization of subgroup A gene from the total RNA extract was analyzed by RT-PCR (Fig. 1A, lines 7–11). A DNA fragment about 749 bp was amplified from the DNA of DF-1 cells treatment with ALV-A and 4, 12 µg/mL anthocyanin, or only treated with ALVA. Furthermore, the fragment of subgroup A gene (529 bp) was amplified from all ALV-A treatment samples. Experimental results agreed with expected amplified fragment. However, there were no DNA fragments amplified from normal DF-1 cells with the same primers. In addition, the morphology of ALV-A purified by centrifugation in millipore tube (Amicon® Ultra-15, 100 kD) were scanned by transmission electron microscopy (Fig. 1B). Outer membrane was removed during concentra-

Table 1 – HPLC-DAD/ESI-MS results, identification of anthocyanins and relative amounts of anthocyanins in TAPC. Peak label1

Rt (min)

UV/Vis λmax (nm)

m/z MH+

Fragment ions

1 2 3 4 5 6 7 8 9 10

17.8 20.8 21.4 22.0 22.9 24.1 24.7 24.9 28.4 29.4

517 508 516 516 516 516 506 510 518 514

449 433 535 463 535 621 519 611 549 465

287 271 287 301 287 287 271 465, 303 301 303

1

α

Compound identity

Percentage of anthocyanin calculated from the peak area (%)α

Cyanidin-3-O-glucoside Pelargonidin-3-O-glucoside Cyanidin-3-(6″-malonylglucoside) Peonidin-3-O-glucoside Isocyanidin-3-(6″-malonylglucoside) Cyanidin-3-2malonlyglucoside Pelargonidin-3-(6″-malonylglucoside) Delphinidin-3-O-glucoside-5-rhamnoside Peonidin-3-(6″-malonylglucoside) Delphinidin-3-O-glucoside

37.13 ± 0.35a 6.56 ± 0.31d 2.21 ± 0.30f 8.24 ± 0.60c 27.15 ± 0.70b 2.69 ± 0.24f 5.85 ± 0.15e 7.95 ± 0.10c 0.65 ± 0.07h 1.57 ± 0.10g

Refer to Fig. 1. Percentage of anthocyanin calculated from the peak area. Each value of percentage of anthocyanin is the mean of three replications ± standard deviation. Represent the values within each line. Values followed by the same letter are not significantly different (P < 0.05) by Duncan Multiple Range Test.

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Fig. 1 – PCR detection of the common ALV p27 gene and RT-PCR detection the particular ALV A gene in vivo (A), the morphology of ALV-A (B). Lanes 1–5 showed the common antigen p27 primers of ALVs analyzed by PCR assay, and lanes 7–11 were the subgroup A gene specific primers of ALVs by RT-PCR assay. Lane 6, DL 2000 bp marker; lanes 1 and 7, water control; lanes 2 and 8, DF-1 cell; lanes 3 and 9, DF-1 cell + ALV-A; lanes 4 and 10, DF-1 cell + ALV-A + 4 µg/mL TAPC; lanes 5 and 11, DF-1 cell + ALV-A + 12 µg/mL TAPC.

tion process, so only the inner membrane of ALV-A can been seen. Under the electron microscope, they were spherical with high electron density kernel and the diameter was 35–45 nm. The results were all according with the morphology of ALV, which showed that ALV-A proliferated after infecting DF-1 cells.

3.3.

The cytotoxicity of TAPC to DF-1 cells

In order to study the protection of TAPC on cell infected with ALV-A, the toxicology was determined by treating DF-1 cells with various concentrations of TAPC (4, 8, 12, 16 and 20 µg/ mL) for 24, 48, 72 and 96 h followed by a MTT assay. The results are shown in Fig. 2A. The remaining cell viability of sample treated with TAPC in four periods had the same trend, compared to the controls. During the concentration of 4–12 µg/ mL, TAPC promoted the growth of DF-1 cells with a dosedependent manner, while the treatment of 16 µg/mL TAPC was similar to the controls, which had no cytotoxicity to DF-1 cells. However, 20 µg/mL anthocyanins obviously decreased the remaining cell viability, which showed a strong cytotoxicity for cell growth. Concentrations of anthocyanins that had no cytotoxicity to DF-1 cells were used for the following experiments. Furthermore, trypan blue day exclusion assay was also performed and results were similar to those of MTT assay (data not shown), which confirmed the results of MTT assay.

3.4.

markedly protection activity of DF-1 cells infected with ALVA, and the preventive effect was better than therapeutic treatment in vitro.

3.5.

The effect of TAPC on NF-κB DNA-binding activity

Nuclear and cytoplasmic proteins from cultured DF-1 cells infected with ALV-A and treated with TAPC were subjected to analysis for NF-κB DNA-binding activity as measured by a Trans AM™ NF-κB Family Kit. Mostly NF-κB p50 and p65 were in the cytoplasm. When cell infected with ALV-A, p50 and p65 transferred from the cytoplasm to the nucleus (Fig. 3A and B). Four and 12 µg/mL anthocyanins treatments for 72 h reduced the nuclear NF-κB p50 and p65 DNA-binding activity in DF-1 cells, compared with the ALV-A positive control. The effect of treatment of 12 µg/mL was stronger than 4 µg/mL. However, in terms of the DNA-binding activity of NF-κB p52 or c-Rel, no significant inactivation was observed in all of the treatments (Fig. 3C and D). Moreover, NF-κB RelB also showed an unmarked feature (data not shown). These results suggested that anthocyanins only reduced the NF-κB DNA-binding activities through inhibiting the translocation and activation of p50 and p65 in DF-1 cells after infecting with ALV-A.

3.6. TAPC inhibit NF-κB p50 and p65 translocation from cytoplasm to nucleus

The protection of TAPC on cell infected with ALV-A

To investigate anti-ALV-A-induced cell apoptosis of anthocyanins, the preventive and therapeutic effects in vitro was applied by MTT assay (Fig. 2B). MTT assay revealed that 4–16 µg/mL of TAPC inhibited the death of cells induced by ALV-A in vitro, compared with ALV-A positive controls for different times. During the concentration of 4–12 µg/mL, the effectiveness was a dosedependent manner. For the same concentrations, the preventive treatment exhibited a higher OD value than therapeutic treatment in the same time. The results showed that TAPC had

To confirm the inhibitory effects, we performed western blot analysis on the expression of NF-κB p50 and p65 in the cytoplasm to the nucleus with β-actin and GAPDH as two internal controls, and results were shown in Fig. 3E. It was found that the expression level of NF-κB p50/p65 in the nucleus was low, and high in the cytoplasm in normal DF-1 cell. ALV-A significantly upregulated NF-κB p50 and p65 expression level in the nucleus of DF-1 cells. On the contrary, NF-κB p50 and p65 translocation were suppressed by anthocyanins treatment in a dose-dependent manner.

journal of functional foods 10 (2014) 274–282

A

1.2

OD Value

1.1 1.0

0 µg/mL 4 µg/mL 8 µg/mL 12 µg/mL 16 µg/mL 20 µg/mL

results showed that ALV-A enhancing the expression levels of NF-κB p50 and p65 target proteins in the nucleus of DF-1 cells. The NF-κB p50 expression were mitigated by the presence of TAPC and C3G (Fig. 4A), and C3G was significantly higher than TAPC under the same 10 µg/mL concentration. Meanwhile, NFκB p65 expression was also retarded by TAPC and C3G (Fig. 4B). Therefore, western blot analysis revealed that cyanidin-3glucoside exhibited more inhibitory effects. It presumed that C3G might be believed to be the major active component in anthocyanins that was most responsible for the anti-ALV-Ainduced cell apoptosis or different kind of anthocyanins contained antagonistic effect.

0.9 0.8 0.7 0.0

24

48

72

96

Time (h)

B 1.0

4.

0 ug/mL anthocyanins (A) 0 ug/mL A +ALV-A 4 ug/mL A +ALV-A 8 ug/mL A +ALV-A 12 ug/mL A +ALV-A 16 ug/mL A +ALV-A

Preventive effect

0.9

OD Value

0.8 0.7 0.6 0.2 0.0 1.0

24h

48h

72h 96h Therapeutic effect

24

48

72

0.9 0.8 0.7 0.6 0.2 0.1 0.0

279

96

Time (h) Fig. 2 – The cytotoxicity of TAPC to DF-1 cell (A); The preventive and therapeutic effects of TAPC for ALV-Ainduced DF-1 cells apoptosis (B). Bars in the figure represent standard deviations of four replications.

3.7. Comparison of the inhibitory effects of TAPC and C3G on NF-κB p50 and p65 activities C3G displayed strong growth inhibitory effect against human leukemia Molt 4B cells, and also it is one of most prominent of the anthocyanins in TAPC. The inhibitory effects on NF-κB p50 and p65 transcriptional level of C3G was evaluated by a quantitative western blot analysis to compare with TAPC. It was performed by using nuclear protein extracts following the protocol provided with the odyssey infrared imaging system. The

Discussion

Purple corn (Z. mays L.) is a natural source of anthocyanins. Anthocyanins from purple corn is stable over a wide range of temperatures and times, which is very important for maintaining their function (Zhao et al., 2008). This study found 10 disparate monomeric anthocyanins in Jingzi variety, with C3G and cyanidin-3-(6″-malonylglucoside) as its main components. C3G is largely present in the human diet, which is suggested to possess biological properties, such as suppressing 7,12dimethylbenz[a]anthracene-induced mammary carcinogenesis (Fukamachi, Imada, Ohshima, Xu, & Tsuda, 2008), and protecting skin cells against the adverse effects of UVB radiation. Moreover, it has been reported that the order of inhibitory efficacies of mesangial hyperplasia and matrix accumulation was C3G > Peonidin-3-O-glucoside > Cyanidin-3(6″-malonylglucoside) > Peonidin-3-(6″-malonylglucoside) > Pelargonidin-3-O-glucoside (Li et al., 2012). This study found that anthocyanins from purple corn in the range of 4–16 µg/ mL retarded ALV-A-induced apoptosis in vitro, and C3G exhibited mainly activities in anthocyanins extracts. NF-κB activation pathway is involved in the inflammation, cancer cell proliferation, invasion, and metastasis (Antony et al., 2003). Purple corn containing plentiful anthocyanins and dietary anthocyanidin delphinidin, C3G have been reported to involve NF-κB pathway (Cimino, Amvra, Canali, Saija, & Virgili, 2006; Han et al., 2009). Moreover, ALV and the human T-cell leukemia virus type 1 (HTLV-1) were in the same genus. Previous studies have shown that HTLV-1 transactivator (Tax) can activate major cellular signal transducing pathways including NF-kB and cAMP-responsive element binding protein (CREB) (Matsumoto, Shibata, & Fujisawa, 1997; Ross, Narayan, & Gree, 2000; Sun & Ballard, 1999). We wanted to discover whether the NF-κB routes also apply to ALV, and whether anthocyanins repressed apoptosis through this pathway. The results show that NF-κBs are expressed in the DF-1 cells and play an important role in the ALV-A infected process, while NF-κB p50/p65 is involved in inducing the breakdown of transcription pathway. It is presumed that ALV-A activated cellular signal transducing pathway (NF-κB) of DF-1 cells. Anthocyanins improved the ALV-A-triggered nuclear transcription factor system dysfunction by enhancing cytoplasmic NF-κB p50/p65 expression and repealing nuclear NF-κB p50/p65 expression, then, decreasing the cell apoptosis. Furthermore, anthocyanins with different chemical structures exhibit different extents of physiological

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Fig. 3 – The effect of TAPC on nuclear factor κB DNA-binding activity (A–D) and translocation of NF-κB p50 and p65 in DF-1 cells infected with ALV-A (E). β-Actin and GAPDH protein were used as an internal control. Bars in the figure represent standard deviations of three replications.

function (Cao, Sofic, & Prior, 1997). C3G exhibited stronger inhibitory effects than TAPC, indicating that C3G was most likely linked to ALV-A, which was mediated by a crosstalk between ALV-A and NF-κB signaling. In summary, this study determined the anthocyanin composition in purple corn (Z. mays L.) and investigated the potential for anthocyanins to prevent the DF-1 cells apoptosis induced by ALV-A. Furthermore, we revealed that anthocyanins protected the cell apoptosis via interruption of NF-κB signaling which was involved in the crosstalk between DF-1 cells and ALV-A (Fig. 5). NF-κB mostly consists of a heterodimer of p50 and p65, which combines with inhibitory κBα (IκBα) as an inactive complex in the cytoplasm in normal DF-1 cells. When ALV-A gets into the cells, it would bind to receptors RIPs, which

would activate the IKK kinase, resulting in the degradation of IκBα through phosphorylation by kinase. The heterodimer of p50 and p65 is transferred into the nucleus after separating from p-IκBa leading to activation, which mediated the transcription and translation of tumor gene. It was presumed that there were three kinds of inhibitory actions. First of all, anthocyanins probably improved the resistance of DF-1 cell to ALVA, preventing ALV-A from entering into the cells, thereby limiting the chances of infection. Furthermore, anthocyanins inactivated the NF-κB p50 and p65 through reducing the phosphorylation of kinase, leading to decreasing the progression of NFκB p50 and p65 from cytoplasm to nucleus. Lastly, anthocyanins downregulated the expression level of NF-κB p50 and p65 in the nucleus, which inhibited the transcription and transla-

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Fig. 5 – Proposed inhibitory mechanisms of TAPC on DF-1 cell apoptosis induced by ALV-A.

REFERENCES

Fig. 4 – Comparison of the inhibitory effects of TAPC and C3G on NF-κB p50 activities (A); Comparison of the inhibitory effects of TAPC and C3G on NF-κB p65 activities (B). GAPDH protein was used as an internal control. Respective blot data were obtained from three independent experiments. Bars in the figure represent standard deviations of three replications, and values not sharing a letter are significantly different at P < 0.5.

tion of target gene. TAPC inhibited the apoptosis in DF-1 cells induced by ALV-A via disturbing NF-κB signaling pathway, which would give helpful information for preventing the Avian leukosis.

Acknowledgments This project was supported by Beijing Nova program (Z131105000413023), China Agriculture Research System (CARS25) and National Natural Science Foundation of China (30972048).

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