CN113354711B - Anticancer bioactive peptide and synthesis method thereof - Google Patents

Abstract

The invention discloses an anticancer bioactive peptide, which comprises an amino acid sequence shown as SEQ ID NO. 1. The anticancer bioactive peptide is safe and effective, and has an obvious effect.

Description

Anticancer bioactive peptide and synthesis method thereof

Technical Field

The invention relates to the technical field of anticancer, in particular to an anticancer bioactive peptide and a synthesis method thereof.

Background

Cancer is still one of the leading causes of death in the world to date, and the pain and medical expense of cancer treatment pose a serious threat to the physical and mental health and quality of life of cancer patients and their families. At present, clinical surgical treatment is mainly suitable for patients with early cancer, and patients with late cancer are usually treated by chemotherapy and radiotherapy, but most of chemotherapeutic drugs have poor curative effects and great damage to organisms, so that a more effective method for treating cancer is urgently needed to be found.

Polypeptide anticancer drugs are hot spots in recent years of research and development of anticancer drugs, and bioactive peptides are peptide compounds with special physiological effects on organisms. Generally, the amino acid sequence is composed of 20 natural amino acids in different compositions and arrangements, and the function of the amino acid sequence is determined by the composition sequence of the amino acids. The bioactive peptide has small molecular weight, high targeting property and low toxicity, and has important research value and prospect in the clinical treatment of cancer. However, the active peptide anticancer drugs applied to clinic to date are few, and the ever-increasing demands of the anticancer drug market are far from being met.

Disclosure of Invention

The invention aims to provide an anticancer bioactive peptide and a synthesis method thereof, which are safe and effective and have obvious effect.

In order to achieve the aim, the invention provides an anticancer bioactive peptide, which comprises an amino acid sequence shown as SEQ ID NO. 1.

A method for synthesizing anticancer bioactive peptide comprises the following steps:

s1, removing a fluorenylmethoxycarbonyl (Fmoc) protecting group: selecting proper modified resin to synthesize target peptide, firstly adding 20% piperidine/dimethylformamide (pip/DMF) solution into a reactor to fully soak the resin, and then placing the reactor on a rocker to shake;

s2, cleaning: removing the solution in the reactor, adding N, N-Dimethylformamide (DMF) into the reactor to immerse the resin, then placing the reactor on a rocker and shaking, removing the filtrate, and repeating the cleaning process for three times;

s3, detection resin: placing a detection reagent A, B and a resin point into the same test tube, placing the test tube in an environment at 100 ℃, checking whether the color of the resin is changed, and if the color of the resin is changed, indicating that the Fmoc group is successfully removed;

s4, coupling: adding the prepared amino acid solution into resin, then adding N, N' -diisopropylcarbodiimide/dimethylformamide (DIC/DMF) solution into a reactor, and placing the reactor on a rocker to shake;

s5, detection resin: placing the detection reagents A and B and a resin point into a test tube, placing the test tube in an environment at 100 ℃, checking whether the color of the resin is changed, and if the color of the resin is not changed, indicating that the coupling of the amino acid is successful;

s6, cleaning: washing the coupled resin in the same manner as step S2;

s7, coupling: selecting proper amino acid solution to continue coupling, and repeating the steps S4-S7;

s8, detection: the purity and molecular weight of active peptides were determined using High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS).

Therefore, the anticancer bioactive peptide and the synthesis method are safe and effective, and have obvious effects.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is an HPLC chromatogram and corresponding peak values of biologically active peptides against cancer;

FIG. 2 is a MS spectrum and corresponding peaks of bioactive peptides against cancer;

FIG. 3 is a diagram showing the effect of CCK-8 in detecting PEP01 on the proliferation of human immortalized keratinocyte HaCaT cells;

FIG. 4 is a CCK-8 assay for the effect of PEP01 on melanoma SK-MEL-103 cell proliferation;

FIG. 5 is a CCK-8 assay for the effect of PEP01 on cervical cancer HeLa-S3 cell proliferation;

FIG. 6 is a CCK-8 assay for the effect of PEP01 on lung cancer A549 cell proliferation;

FIG. 7 is a graph showing the effect of PEP01 on the ability of cells to migrate;

FIG. 8 is a graph of the effect of PEP01 on wound healing of cell scarification;

FIG. 9 is an apoptosis assay.

Detailed Description

The technical solution of the present invention is further illustrated by the accompanying drawings and examples.

Example one

An anticancer bioactive peptide, which has an amino acid sequence shown in PEP01 in Table 1.

A method for synthesizing anticancer bioactive peptide comprises the following steps:

s1, removing Fmoc protecting groups: selecting proper modified resin to synthesize target peptide, firstly adding a pip/DMF solution with the volume of 20% into a reactor to fully soak the resin, and then placing the reactor on a rocker to shake; the modified resin is derived resin taking D301 resin as a carrier, and different modified resins have different effects.

S2, cleaning: removing the solution in the reactor, adding DMF (dimethyl formamide) into the reactor to immerse the resin, then placing the reactor on a rocker, shaking, removing the filtrate, and repeating the cleaning process for three times;

s3, detection resin: placing a detection reagent A, B and a resin point into the same test tube, placing the test tube in an environment at 100 ℃, checking whether the color of the resin is changed, and if the color of the resin is changed, indicating that the Fmoc group is successfully removed;

s4, coupling: adding the prepared amino acid solution into resin, then adding the DIC/DMF solution into a reactor, and placing the reactor on a rocker to shake;

s5, detection resin: placing the detection reagents A and B and a resin point into a test tube, placing the test tube in an environment at 100 ℃, checking whether the color of the resin is changed, and if the color of the resin is not changed, indicating that the coupling of the amino acid is successful;

s6, cleaning: washing the coupled resin in the same manner as step S2;

s7, coupling: selecting proper amino acid solution to continue coupling, and repeating the steps S4-S7;

s8, detection: the purity and molecular weight of the active peptides were determined using HPLC and MS methods.

As shown in FIG. 1-2, the purity of the active peptide (hereinafter referred to as PEP01) of the present invention was found to be 98.40% by HPLC and MS detection, and the molecular mass was 1088.

TABLE 1 Properties of active peptides

Test of

(I) cell culture

The melanoma SK-MEL-103 cell strain, the cervical cancer Hela-S3 cell strain and the lung cancer A549 cell strain used by the invention are mainly from other laboratories. The cell lines used contained 5% -10% fetal bovine serum (FBS, Gibco)^TM) 1 Xpenicillin streptomycin (Hyclone) in complete medium (DMEM) (Hyclone) and in 5% CO₂Cultured in an incubator at 37 ℃.

(II) toxicity test on Normal cells

In order to detect the toxicity of the active peptide PEP01 to normal cells, cell viability was detected using a cell proliferation and toxicity detection kit (CCK-8 kit). Human immortalized keratinocytes HaCaT were seeded into 96-well plates and cultured overnight. Then adding active peptide sequence (PEP01) shown in SEQ ID NO:1 at different concentrations to the test group for treatment for 24 hours, wherein the concentrations are 1, 10, 25, 50 and 100 mu g/mL respectively, and detecting the influence of the active peptide on the normal cell viability. The control group was added with the same volume of sterile water, and the total volume was 100. mu.L.

After a certain incubation time, 10. mu.L of CCK-8 detection reagent was added to each well and incubated at 37 ℃ for 2 h. And detecting the light absorption value under the condition of 450nm wavelength by using a multifunctional microplate reader, and repeating all experiments for 3 times.

As shown in fig. 3, PEP01 at concentrations of 1, 10, 25, 50, 100 μ g/mL had no significant effect on cell viability of HaCaT cells, indicating that the active peptide PEP01 is low or non-toxic to normal cells.

(III) in vitro anti-cancer Effect on different cancer cells

The melanoma SK-MEL-103 cell strain, the cervical cancer Hela-S3 cell strain and the lung cancer A549 cell strain are inoculated into a 96-well plate and cultured overnight. Then adding active peptide PEP01(1, 10, 25, 50, 100 mu g/mL) to treat for 48, 72 and 96 hours respectively, detecting the vitality of cells, and screening out the concentration and action time point with the best anticancer effect.

Meanwhile, the control group was added with the same volume of sterile water, and the total volume was 100. mu.L. After a certain incubation time, 10. mu.L of CCK-8 was added to each well and incubated at 37 ℃ for 2 h. The absorbance at a wavelength of 450nm was measured in a multifunctional microplate reader (Biotek, USA) and all experiments were repeated 3 times.

As shown in FIG. 4, cell viability was measured after 48h (A), 72h (B) and 96h (C) of treatment with 1, 10, 25, 50, 100 μ g/mL PEP 01. PEP01 has obvious inhibition effect on the proliferation of SK-MEL-103 cells, wherein PEP01 with the concentration of 10 mug/mL has the best inhibition effect on the SK-MEL-103 cells for 72 h. In addition, P <0.05, P <0.01, P < 0.0005.

As shown in FIG. 5, after treating with PEP01 of 0.1, 1, 10, 25, 50, 100 μ g/mL for 48h (A), 72h (B) and 96h (C), the cell viability was measured, and PEP01 had a significant inhibitory effect on proliferation of HeLa-S3 cells, wherein the inhibitory effect of PEP01 of 25 μ g/mL on HeLa-S3 cells for 72h was the best. In addition, P <0.05, P <0.01, P < 0.0005.

As shown in FIG. 6, cell viability was measured after 24h (A), 48h (B) and 72h (C) of treatment with 0.1, 1, 10, 25, 50, 100 μ g/mL PEP 01. PEP01 has obvious inhibition effect on the proliferation of A549 cells, wherein PEP01 with the concentration of 10 mu g/mL has the best inhibition effect on the treatment of A549 cells for 24 h. In addition, P <0.05, P <0.01, P < 0.0005.

(IV) cell migration assay

After placing the culture dish with a pen and the diameter of 60mm, drawing a horizontal line every 0.5-1cm by using a ruler for comparison. About 5X 10 addition to the well plate⁵And (4) cells. After the film is fully paved, the gun head is used for contrasting with the ruler, and the film is scratched perpendicular to the transverse line at the back. Cells were washed 3 times with Phosphate Buffered Saline (PBS) and serum free medium was added.

The optimal concentration of active peptide PEP01 was added and the control group was added with the same volume of sterile water. Photographs were taken under an inverted microscope (Nikon, JP) at 0, 6, 12 hours.

SK-MEL-103 cells were treated with 0. mu.g/mL and 10. mu.g/mL PEP01, respectively, for 12h and 24h to visualize wounds at 40-fold microscopy, and cell scratch widths were measured and calculated using Image J software. The cell mobility was calculated using the following formula: cell mobility (0h scratch width-post-incubation scratch width)/0 h scratch width × 100%. Wherein P <0.05, P <0.01, P < 0.0005.

As shown in FIGS. 7-8, PEP01 was able to significantly inhibit the migration of the melanoma SK-MEL 103 cells at 6h and 12 h.

(V) apoptosis test

The cell slide is taken and placed in a six-hole plate, and the cell is planted in the six-hole plate for overnight culture. Stimulating apoptosis of cells with the selected active peptide at optimal concentration and time, adding water with the same volume as control group, and using H₂O₂As a positive control. After culturing for a certain period of time, the culture medium was aspirated. Experiments were performed according to the Hoechst 33258 apoptosis kit. 0.5mL of the fixative was added and fixed for 15 minutes. Wash twice with PBS for 3 minutes each. 0.5mL of Hoechst 33258 staining solution was added thereto, and staining was performed for 5 minutes. Wash twice with PBS for 3 minutes each time, blotting off the liquid. Dropping a drop of the anti-fluorescence quenching mounting solution on the glass slide, and covering the cell slide with the cells. Photographs were observed under a fluorescence microscope (Nikon, JP).

As shown in fig. 9, (a) control group: SK-MEL-103 cells were treated with complete medium only. (B) Positive control group: 0.5% H before detection of apoptosis₂O₂SK-MEL-103 cells were treated for 15 min to induce apoptosis. (C) Experimental groups: SK treatment with 10. mu.g/mL PEP01MEL-103 cells for 72 hours and the degree of apoptosis was examined. (D) The number of cells in each group was counted using Image J. (E) Two hundred cells were randomly selected for each group and the apoptosis rate was calculated. The experiment was repeated three times. PEP01 treatment of cells at 10. mu.g/mL for 72h was found to induce significant apoptosis in the melanoma SK-MEL-103 cells.

Therefore, the anti-cancer bioactive peptide and the synthesis method are safe and effective, and have an obvious anti-cancer effect.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

<110> Wenzhou Kenn University (Wenzhou-Kean University)

<120> an anticancer bioactive peptide and its synthesis method

<141> 2021-06-04

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> bioactive peptide chain (peptide chain)

<400> 1

Glu Leu Gln Ser Thr Gly Arg Lys Val Ala

1 5 10

Claims (1)

1. An anticancer bioactive peptide, which is characterized in that: the amino acid sequence of the active peptide is ELQSTGRKVA.

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