Isolation of a novel bio-peptide from walnut residual protein inducing apoptosis and autophagy on cancer cells

Materials and chemicals

Walnuts were obtained from Hebei Jingpin Fruits Co., Ltd (Hebei, P.R. China). Proteases
were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell lines of MCF-7 (human
breast cancer cell line), Caco-2 (human colon cancer cells), Hela (human cervical
cancer cells) and IEC-6 (rat small intestinal crypt epithelial cells) were obtained
from the Cell Culture Centre of the Institute of Basic Medical Sciences, Chinese Academy
of Medical Sciences (Beijing, P.R. China).

Preparation of different walnut residual protein hydrolysates

Walnuts were ground and defatted with petroleum ether. The defatted flour was dried
in the drying oven overnight at 50 °C as walnuts oil extraction residue. Walnut residual
protein (WRP) was extracted from walnuts oil extraction residue by NaOH solution (pH 9.0)
at ratio 1:15 (w/v) for 1 h (45 °C). After pH adjustment to 4.5, the precipitate obtained
by centrifugation at 4000?×?g for 15 min and the supernatants were lyophilized. The
protein hydrolysates were prepared with alkaline protease, papain, pepsin, trypsin
and neutral protease, respectively. WRP was dissolved in distilled water at a concentration
of 30 mg/ml and hydrolyzed for 3 h separately using alkaline protease at pH 9.0, 50 °C,
papain at pH 7.0, 60 °C, pepsin at pH 2.5, 37 °C, trypsin at pH 8.0, 40 °C and neutral
protease at pH 7.5, 55 °C. Samples were taken out after 3 h and heated in a boiling
water bath at 95 °C for 10 min to inactive enzyme’activity. The samples were then
centrifuged at 4000?×?g for 15 min and the supernatants were lyophilized.

Ultrafiltration

The protein hydrolysates were sequentially filtered through cellulose membranes (Ultrafiltration
system Millipore, Bedford, Mass., USA) with molecular weight cutoffs (MWCO) of 10,
5, and 3 kDa (Millipore). Peptide fractions derived from walnut protein hydrolysates
were: 10–5 kDa, 5–3 kDa and??3 kDa fraction, respectively. All permeates were lyophilized
and stored at ?20 °C until further analysis.

Cell culture

MCF-7 and Hela cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco,
Rockville, MD, USA) containing 10 % heat-inactivated fetal calf serum (FCS) (Hyclone)
and 0.5 % penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5 %
CO
₂
. Caco-2 and IEC-6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco, Rockville, MD, USA)) supplemented with 10 % FCS (Hyclone), 0.1 mM nonessential
amino acid, 0.5 % penicillin/streptomycin at 37 °C in a humidified atmosphere containing
5 % CO
₂
.

Cell Viability Assay

Cell viability was assessed by MTT assay 19]. Briefly, cells were seeded into 96-well microplates at a density of 3?×?10
⁴
cells per well. After overnight incubation, cells were treated with various concentrations
of samples and cultured for 48 h. After incubation, 20 ?l of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide, 5 mg/mL) reagent was added to each well and incubated for 4 h at 37 °C in
the dark. The culture medium containing the MTT solution was replaced by 200 ?L DMSO
(dimethyl sulfoxide) and shaken in the dark for 15 min at room temperature for complete
dissolution of the MTT formazan productions. The optical density of each well was
measured by absorbance at 570 nm using a Benchmark Plus microplate reader (Bio-Rad,
Hercules, USA). The cell viability and inhibitory rate was calculated as follows:
Cell Viability (%)?=?b/a?×?100; Inhibitory Rate (%)?=?[(a???b)/a]?×?100, Where a is the optical density without sample, and b is the optical density in the presence of sample. The IC
₅₀
value is the concentration that inhibits 50 % of the cell viability.

Gel filtration chromatography

The fraction with the highest antioxidant activity after ultrafiltration separation
was re-dissolved in phosphate buffer(pH 7.0), then separated using a HiPrep 16/60
Sephacryl S-100HR column (16?×?600 mm, Amersham Pharmacia Biotech, Piscataway, NJ,
USA) which was eluted with phosphate buffer at a flow of 1.0 ml/min, and monitored
at 280 nm. The fractions with the desired peaks were pooled, desalted using a HiPrep™
26/10 desalting column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and lyophilized
for MTT assay.

RP-HPLC

The fraction exhibiting the highest cytotoxic activity after gel filtration chromatography
was further purified on a Zorbax SB-C18 (250 mm?×?4.6 mm,5 um,Agilent,USA). The column
was eluted by a linear gradient of acetonitrile (0-40 %) containing 0.1 % Trifluoroacetic
acid (TFA) at a flow rate of 1.0 ml/min. The eluted peaks were detected at 214 nm
and the fraction was then lyophilized.

Molecular mass and amino acid sequence analysis

Accurate molecular mass and amino acid sequence of the active peptide was determined
by Hybrid Quadrupole TOF-LC/MS/MS mass spectrometer (AB Sciex Instruments, CA, USA)
coupled with ESI source. Generally, the peptide was infused into electrospray source
following dissolve in acetonitrile/water (1:1, v/v), and molecular mass was determined
by charged (M?+?H)
⁺
state in the mass spectrum. Following molecular mass determination and sequence information
was obtained by BioLynx analysis system.

Simulated gastrointestinal digestion of bioactive peptides

The pH of the sample was adjusted to pH 2.0 by 1 N HCl, and the mixture was incubated
with pepsin at 800 U/mL in a shaking bath for 2 h at 37 C with shaking at 100 rpm.
The sample was then neutralized with 1 M NaOH to pH 7.0 before addition of pancreatin
at 2 mg/mL and bile extract mixtureat 0.3 mg/mL for a further incubation of 2 h. Finally,
the digestion was terminated by boiling for 10min 20].

Cell cycle distribution analysis

MCF-7 cells were treated with or without test sample for 48 h. Cells were harvested
fixed with cold ethanol/destillata (3:1, v/v) at 4 °C overnight. Before analysis,
cells were washed with PBS and stained with a solution containing 100 ?L propidium
iodide, 100 ?g/mL RNase at 37 °C for 30 min in the dark. The fluorescence of stained
cells was analyzed by flow cytometry using a FACSCanto (Becton-Dickinson, CA, USA).

Annexin V-FITC/PI double staining assay

Apoptosis can be detected by translocation of phosphatidyl serine to the cell surface
using an annexin V-FITC antibody. MCF-7 cells were seeded in 6-well plates by density
of 3?×?10
⁴
cells/ml. Cells were treated with or without test sample for 48 h. The cells were
counted after trypsinization and washed twice with cold PBS. The cell pellet was resuspended
in 100 ?l of binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl
₂
) at a density of 1?×?10
⁵
cells per ml and incubated with 5 ?l of FITC-conjugated Annexin-V and 10 ?l of propidium
iodide (PI) for 15 min at room temperature in the dark. Annexin V-FITC and PI fluorescence
was monitored by flow cytometry (FACSAria, Becton–Dickinson, CA, USA). Cells that
stained positive for Annexin V were classified as early apoptotic cells; cells positive
for both PI and Annexin V staining were classified as late apoptotic cells; and PI
and Annexin V negative cells were classified as live cells 19].

TUNEL (TdT-mediated dUTP nick end labeling) assay

TUNEL technique has been extensively used for the detection and quantification of
apoptosis. MCF-7 cells were cultured on coverslips and then fixed with stationary
liquid (Anhydrous ethanol: chloroform: glacial acetic acid 6: 3:1), and digested in
proteinase K for 20 min at 37 °C, before endogenous peroxidase was blocked in 3 %
hydrogen peroxide. Terminal deoxynucleotidyl transferase (TdT) in reaction buffer
(containing a fixed concentration of digoxigenin-labelled nucleotides) was applied
to sections for 30 min at 37 °C, before the slides were placed in Stop/Wash buffer
for 10 min. Following washes, a prediluted peroxidase conjugated antibody was applied
for 30 min. Apoptotic cells were detected after incubation in the 3,3-diaminobenzidine
(DAB) chromogen for approximately 10 min and slides were counterstained with hematoxylin
21], 22].

Western blotting

MCF-7 cells were seeded in 6 cm dishes overnight and incubated with test sample for
the 48 h. Cells were harvested by trypsinization and washed twice with cold PBS. Total
cellular protein lysates were prepared by harvesting cells in protein extraction buffer
(50 mM Tris–HCl, pH 7.4, 1 mM NaF, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethanesulfonyl
fluoride; 1 % NP-40; and 10 mg/mL leupeptin) to the cell pellets on ice for 30 min,
followed by centrifugation at 10,000?×?g for 30 min at 4 °C. Equal amounts of protein
(50 mg) were separated by 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE). After electrophoresis, proteins were electroblotted to nitrocellulose
membranes, and subsequently incubated in blocking buffer (in PBS, 5 % skim milk, and
0.1 % Tween 20) at room temperature for 2 h. After blocking, the membranes were incubated
with the antibodies, rabbit anti-LC3 I/II (1:800), and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH, 1: 800). Subsequently, membranes were washed five times in a
wash buffer and incubated with appropriate donkey anti-rabbit horseradish peroxidase
conjugated secondary antibody (1:8000), and the bands were disposed with the Gel-Pro
Analyzer 4.0 (Media Cybernetics, USA) 19], 23].

Immunofluorescence for LC3-II

MCF-7 cells were seeded on coverslips overnight and then incubated with test sample
or rapamycin (RAPA) for the next 48 h. The cells fixed with 4 % (v/v) paraformaldehyde
for 30 min and then covered with 10 % (v/v) goat serum for 60 min at room temperature
followed by incubation with primary antibody of LC3-II in 1: 200 dilution at 4 °C
overnight. Cells were then probed with Alexa Fluor 488-goat anti-rabbit IgG secondary
antibody (1:200, Invitrogen Life Technologies, Carlsbad, CA, USA). Counterstaining
of cell nuclei was performed by Vectashield H-1200 mounting medium with DAPI. Fluorescent
staining was examined with a confocal fluorescence microscope (Nikon EZ-C1, Nikon,
Tokyo, Japan). 24]–26].

Measurement of cytokine production

For cytokine immunoassays, Mouse spleen lymphocytes were cultured for 24 h at a density
of 1?×?10
⁶
cells/ml in 96-well plates. Supernatants were removed at the indicated time, and IL-2
production was quantified by sandwich immunoassays using the protocol supplied by
ELISA kits (Beyotime Biotechnology, China).

Neutral red uptake assay for macrophage phagocytosis

Macrophages were prepared from Kunming mice as described Chen et al. 27]. The phagocytic ability of macrophage was measured by neutral red uptake. After cells
were cultured with test substances for 48 h, 100 ?l neutral red solutions (dissolved
in 10 mM PBS with the concentration of 0.075 %) was added and incubated for 1 h. The
supernatant was discarded and the cells in 96-well plates were washed with PBS twice
to remove the neutral red that was not phagocytized by RAW264.7 cells. Then cell lysate
(ethanol and 0.01 % acetic acid at the ratio of 1:1, 100 ?l/ well) was added to lyse
cells. After cells were incubated in room temperature overnight, the optical density
at 540 nm was measured by a micro-plate reader (Bio-Rad, Hercules, U.S.A.).

Nitric oxide (NO) production by macrophages

Measurement of nitrite in medium was used as an indicator of NO production. The macrophage
cells (5?×?10
⁵
cells/ml) were cultured in 96-well plates with test substances. After 24 h, culture
supernatants were collected and nitrite, the stable reaction product of NO with molecular
oxygen, was measured using Griess reagent. Equal volumes of Griess reagent and sample
were incubated together at room temperature for 10 min. Nitrite production was determined
by comparing the absorbance at 540 nm with a standard curve generated by NaNO
₂20], 28].

Statistical analysis

The data were expressed as means?±?S.D. The significance of difference was evaluated
with one-way ANOVA, followed by Student’s t- test to statistically identify differences
between the control and treated groups. Significant differences were set at *p??0.05, **p??0.01, and ***p??0.001.