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. 2015 Nov 23:15:413.
doi: 10.1186/s12906-015-0940-9.

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

Sihui Ma  1   2 , Di Huang  3   4 , Mengxin Zhai  5   6 , Lubing Yang  7 , Sen Peng  8 , Changxu Chen  9 , Xiaoru Feng  10 , Qiang Weng  11 , Bolin Zhang  12   13 , Meiyu Xu  14   15
Affiliations

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

Sihui Ma et al. BMC Complement Altern Med. .

Abstract

Background: Walnut is unique because they have a perfect balance of n-6 and n-3 polyunsaturated fatty acids. The increasing market demand of walnut lipids results in the large amount of the oil extraction residue. The walnut residue is rich in nutritional proteins, and the uneconomic use of the by-product discouraged the development of walnut industry. Anticancer peptides have recently received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. The aim of this study was to investigate whether anticancer bioactive peptide is contained in walnut.

Methods: Walnut residual protein was hydrolyzed separately by five different proteases. The sequential purification of the hydrolysates was carried out by ultra-filtration, gel filtration chromatography and RP-HPLC to obtain a cancer cell growth inhibitory peptide. Cell cycle distribution, Annexin V-FITC/PI double staining, TUNEL assay, western blot and immunofluorescence for LC3-II assay were used to detect apoptosis and autophagy on cells. Cytokine production was measured by ELISA kits, macrophage phagocytosis was measured by neutral red uptake assay, nitric oxide production was measured by Griess reagent.

Results: The hydrolysates of walnut residual protein produced by papain under the optimal conditions (5 % substrate concentration and an enzyme-substrate ratio of 10 % at temperature 60 C for 3 h), showed significant growth inhibitory activity on MCF-7. The amino acid sequence of the purified peptide was identified as CTLEW with a molecular weight of 651.2795 Da. It is a novel bio-peptide with an amphiphilic structure. CTLEW induced both apoptosis and autophagy on MCF-7 cells, inhibited the cancer cells growth of Caco-2 and HeLa significantly, but did not show any cytotoxic activity against non-cancerous IEC-6 cells. Moreover, the bio-peptide enhanced proliferation and IL-2 secretion of spleen lymphocytes, promoted phagocytosis and NO production of macrophages.

Conclusion: These results suggested that a novel bio-peptide, CTLEW inducing apoptosis and autophagy on MCF-7 cells can be released from walnut residual protein through papain hydrolyzing under the certain condition. The bio-peptide shows selective inhibition towards cancer cells growth and immunomodulatory activity.

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Figures

Fig. 1
Fig. 1
Effects of walnut residual protein and the enzyme protein hydrolysates on MCF-7 cell proliferation. After treated with test substances (0.5, 1, 2, 3, 4 mg/ml) for 48 h, cell viability was measured by MTT assay. WRP, walnut residual protein; WRPH-pa, walnut residual protein hydrolysate prepared with papain; WRPH-pe, walnut residual protein hydrolysate prepared with pepsin; WRPH-tr, walnut residual protein hydrolysate prepared with trypsin; WRPH-np, walnut residual protein hydrolysate prepared with neutral protease; WRPH-ap, walnut residual protein hydrolysate prepared with alkaline protease. Results were expressed as means ± S.D. of four separate experiments. Statistical significance test for comparison with control (untreated) group was done by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 2
Fig. 2
Effect of WRPH-pa in different hydrolytic conditions on MCF-7 cell proliferation. a WRPH-pa was hydrolyzed at temperature of 50, 55, 60, 65 and 70 °C, respectively with the conditions of time 3 h, substrate concentration 4 % and an enzyme-substrate ratio 10 %. b WRPH-pa was hydrolyzed at time of 1, 2, 3, 4 and 5 h, respectively with the conditions of temperature 60 °C, substrate concentration 4 % and an enzyme-substrate ratio 10 %. c WRPH-pa was hydrolyzed at substrate concentrations of 2, 3, 4, 5 and 6 %, respectively with the conditions of temperature 60 °C, time 3 h and an enzyme-substrate ratio 10 %. d WRPH-pa was hydrolyzed at enzyme-substrate ratio of 8, 9,10,11 and 12 %, respectively with the conditions of temperature 60 °C, time 3 h and substrate concentration 4 %. WRPH-pa, walnut residual protein hydrolysate prepared with papain
Fig. 3
Fig. 3
Isolation of bioactive peptides with cytotoxic activity on MCF-7 cells. a IC50 value of WRPH-pa with different molecular weights on MCF-7 cell proliferation. b Gel filtration chromatography of WRPH-pa with <3 kDa molecular weights on a HiPrep 16/60 Sephacryl S-100HR column. Insets show the IC50 value of fraction I to V on MCF-7 cell proliferation. c RP-HPLC chromatography of fraction V from gel chromatography. Insets show the IC50 value of fraction A to D on MCF-7 cell proliferation. d, e Identification of fraction C by TOF-LC/MS/MS coupled with ESI source. d Amino acid sequence analysis of the bioactive peptide. e Molecular mass analysis of the bioactive peptide. Results were expressed as means ± S.D. of four separate experiments
Fig. 4
Fig. 4
Effect of CTLEW on cell cycle distribution in MCF-7 cells. After treated with CTLEW (0, 0.5, 1.0 mg/ml) for 48 h, cells were stained by propidium iodide. The fluorescence of stained cells was analyzed by flow cytometry. a control (0 mg/ml of CTLEW); (b) 0.5 mg/ml of CTLEW; (c) 1.0 mg/ml of CTLEW. Data analysis was performed with ModFit LTTM cell cycle analysis software
Fig. 5
Fig. 5
Induction of apoptosis cell death by CTLEW in MCF-7 cells. a Annexin V-FITC/PI doubles staning assay. Cells were treated with (0.5 or 1.0 mg/ml) or without (control) CTLEW for 48 h, and were stained by annexinV-FITC/PI, respectively. Phosphotidylserine externalization in MCF-7 cells was determined by flow cytometry of annexinV-FITC/PI stained cells. b TUNEL apoptosis assay. Cells were treated with (0.5 or 1.0 mg/ml) or without (control) CTLEW for 48 h, cell tissue slice was labelled by digoxigenin and observed by immunocytochemical staining, the labelled yellow brown nucleus represents the apoptosis cells. Data were presented as mean ± S.D. of three separate experiments
Fig. 6
Fig. 6
Induction of autophagic cell death by CTLEW in MCF-7 cells. a Western blotting of LC3I/II. Cells were treated with (0.1, 0.5, and 1.0 mg/ml) or without (control) CTLEW for 48 h, and expression of LC3I/II in MCF-7 cells was detected by Western blotting. GAPDH was used as internal control and RAPA was used as a positive control. b Immunofluorescence for LC3-II. Cells were treated with (1.0 mg/ml) or without (control) CTLEW for 48 h, and formation of autophagic vacuoles was detected by immunofluorescent staining for LC3-II. RAPA was used as a positive control. Blue, DAPI-stained cell nuclei; Green, LC3-II-stained cytoplasm (100x magnification). Fluorescence intensity represents the expression of LC3-II. Data were presented as mean ± S.D. of three separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001, significantly different from the respective control group
Fig. 7
Fig. 7
Immunomodulatory activity of CTLEW. a Effects of CTLEW on mouse spleen lymphocytes proliferation. Cells were pretreated with (0.5, 1, 2, 3, 4 mg/ml) or without (control) CTLEW for 48 h. Con A (10 μg/ml) was used as a positive control. Cell viability was measured by MTT assay. b Effects of CTLEW on IL-2 cytokine production in mouse spleen lymphocytes. Cells were cultured for 24 h in the media with (0.5 mg/ml) or without (control) CTLEW. ConA (10 μg/ml) was used as a positive control. The amounts of IL-2 released into the culture media were measured by immunoassays. c Effect of CTLEW on phagocytosis of macrophages by neutral red uptake assay. After treated with (0.5 mg/ml) or without (control) CTLEW for 48 h, macrophage phagocytosis was determined by Neutral red uptake assay. LPS (1 μg/ml) was used as a positive control. (D), Effect of CTLEW on the NO production of macrophages. Cells were pretreated with (0.5 mg/ml) or without (control) CTLEW for 24 h. LPS (1 μg/ml) was used as a positive control. The supernatant nitrite levels were determined using Griess reagent. Results were expressed as means ± S.D. of four separate experiments. Statistical significance test for comparison with untreated group was done by Student’s t-test. *p < 0.05; **p < 0.01, ***p < 0.001
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