Anti-cancer peptide and application thereof

Abstract

The invention provides an anticancer peptide and application thereof, and relates to the technical field of biological anticancer. According to the application, the anti-cancer peptide is obtained through specific modification based on an amino acid sequence of a grass carp interleukin 21 gene. The anti-cancer peptide has an effect of inhibiting proliferation of intestinal cancer cells, can induce apoptosis of the intestinal cancer cells, shows lower toxicity compared with traditional anti-cancer drugs, and can selectively act on the cancer cells, so that damage to normal tissues is reduced. As a bioactive peptide, the anti-cancer peptide has the advantages of low molecular weight, strong tissue penetrating power, low toxicity to normal cells and the like, is expected to become a safer and more effective treatment choice, and has important value for developing an intestinal cancer treatment method with small side effect and high anti-cancer activity.

Description

Technical Field

[0001] The present invention relates to the field of biological anticancer technology, and in particular to an anticancer peptide and application thereof. Background Art

[0002] Cancer is a leading cause of death worldwide, with high morbidity and mortality. It is estimated that in 2022, there will be nearly 20 million new cancer cases and 9.7 million cancer deaths worldwide. Cancer can cause great harm to the body, including compression of tissues and nerves, infection and bleeding, and is prone to recurrence and metastasis. Therefore, cancer is extremely harmful to humans.

[0003] Anticancer peptides are a class of bioactive peptides with antitumor activity, which are widely present in a variety of organisms. Anticancer peptides have many advantages in treating tumors, such as low molecular weight, strong tissue penetration, good biocompatibility, and not easy to cause multidrug resistance. They can inhibit the proliferation of cancer cells and induce apoptosis of cancer cells to a certain extent. They are of great significance for developing new anticancer drugs and improving the effect of cancer treatment. In addition, anticancer peptides are usually less toxic to normal cells and can selectively act on cancer cells, thereby reducing damage to normal tissues and improving treatment safety. However, natural anticancer peptides also have some disadvantages. Although anticancer peptides have higher anticancer activity than traditional drugs, the activity of natural anticancer peptides is still low. In addition, natural anticancer peptides have a short half-life. In order to overcome these shortcomings, chemical modification can be used to improve stability, or new delivery systems can be developed to enhance their bioavailability, in order to further expand the application prospects of anticancer peptides. Summary of the invention

[0004] The present invention aims to provide a drug with small side effects and high anticancer activity for the treatment of colorectal cancer. Based on the above purpose, the present invention provides an anticancer peptide and its application, and the technical solution adopted is:

[0005] Provided is an anticancer peptide, which is obtained by modifying the amino acid sequence of a grass carp interleukin 21 gene, and the amino acid sequence is SEQ ID No.1.

[0006] The beneficial effects of the present invention are:

[0007] In the first aspect, the anticancer peptide provided in the present invention can inhibit the proliferation of intestinal cancer cells HCT116, while having little effect on the vitality of normal intestinal epithelial cells. Therefore, the present invention has anticancer effects and low toxicity.

[0008] Secondly, the anticancer peptides provided in the present invention can induce apoptosis of colorectal cancer cells and have certain potential in the treatment of colorectal cancer.

[0009] Thirdly, based on the above effects, the anti-cancer peptides provided by the present invention can effectively treat colorectal cancer, providing a new possibility for colorectal cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0011] Figure 1 Statistical graph showing the results of anticancer peptides inhibiting the proliferation of colorectal cancer cells

[0012] Figure 2 IC50 data statistics of anticancer peptides

[0013] Figure 3 Figure 2 shows the colony growth in the soft agar colony formation experiment.

[0014] Figure 4 Statistical graph of colony formation ability results of soft agar colony formation experiment

[0015] Figure 5 Statistical diagram of flow cytometry detection of M4-induced apoptosis of HCT116 cells

[0016] Figure 6 Statistical graph of the experimental results of WB detection of the effect of M4 on the relative content of apoptosis-related proteins

[0017] Figure 7 This is a statistical chart showing the effect of apoptosis inhibitors on cell activity detected by CCK8 method DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings herein can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative efforts are within the scope of protection of the present invention.

[0019] It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings. At the same time, in the description of the present invention, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0020] The invention provides an anticancer peptide, which is an anticancer peptide obtained by modifying the amino acid sequence of a grass carp interleukin 21 gene, and the amino acid sequence of the anticancer peptide is SEQ ID No.1.

[0021] The following is an experiment to verify the anti-cancer effect of M4.

[0022] Step S1, experimentally verifying that M4 inhibits the proliferation of colorectal cancer cell HCT116.

[0023] Step S11: Detect cell viability using CCK8 method.

[0024] Step S111, experimental principle of detecting cell viability using CCK8 method.

[0025] CCK8 reagent contains water-soluble tetrazolium salt (WST-8), which is reduced to a highly water-soluble yellow formazan product by mitochondrial dehydrogenase in living cells under the action of electron carriers. The amount of formazan generated is proportional to the number of living cells, and cell viability can be analyzed by measuring absorbance.

[0026] Step S112: Design of experiment for detecting cell viability using CCK8 method.

[0027] Step S1121, dilute HCT116 cells to 1×10 4 After 100 μL / well of the culture medium was inoculated into a 96-well plate, the plate was cultured at 37°C with 5% CO2 overnight.

[0028] Step S1122, discard the supernatant, add 100 μL of culture medium containing different concentrations of M4 (0, 1.25, 2.5, 5, 10, 20, 40 μM) to each well, incubate at 37°C 5% CO2 for different time periods (6 h, 12 h, 24 h), and set up four groups of replicates for each concentration and each time period.

[0029] Step S1123: After the incubation time is up, add 10 μL of CCK8 reagent to each well and incubate at 37° C. with 5% CO 2 for 4 hours.

[0030] Step S1124, using an ELISA reader to measure the absorbance at a wavelength of 450 nm. A "cell number-OD value" curve is drawn according to the number of cells in each well and the measured OD value to obtain the activity of the corresponding cells.

[0031] Step S113, analyzing the results of cell viability detection using the CCK8 method.

[0032] The results of cell viability detection by CCK8 method are shown in Figure 1 . As the concentration of M4 increased, the viability of HCT116 cells decreased. After 6 hours of incubation, when the concentration of M4 reached 2.5μM, the viability of HCT116 cells decreased significantly. After 12 hours of incubation, the cell viability was lower than that at 6 hours of incubation, but after that, the cell viability reached a plateau. This result shows that M4 inhibits the proliferation of intestinal cancer cells HCT116.

[0033] In the control group, after adding different concentrations of M4 to CCD 841CoN cells and incubating them for different times, when the M4 concentration reached above 20 μM, the viability of CCD 841CoN cells decreased significantly, indicating that low concentrations of M4 had little effect on the viability of normal intestinal epithelial cells.

[0034] Step S12: Calculate IC50.

[0035] Step S121, IC50 calculation principle.

[0036] IC50 refers to the half-inhibitory concentration of the drug being measured. The calculation principle of IC50 is to measure the inhibitory effect of the drug at different concentrations, draw a curve of the relationship between drug concentration and inhibition rate, and then calculate the drug concentration when the inhibitory effect reaches 50%.

[0037] Step S122: IC50 calculation process.

[0038] Step S1221, calculating the average OD value of the parallel control groups at different time points according to the results of detecting cell viability using the CCK8 method.

[0039] Step S1222: Divide the OD value of each dosing well by the OD value of the control group to obtain the cell survival rate and calculate the cell inhibition rate, inhibition rate = (1-survival rate) × 100%.

[0040] Step S1223, taking the logarithm of the concentration of M4 in each group, and performing a linear regression with the inhibition rate of the cells to obtain an equation.

[0041] Step S1224: According to the equation, calculate the drug concentration when the inhibition rate is 50%.

[0042] Step S123: analysis of IC50 calculation results.

[0043] IC50 calculation results are shown in Figure 2. After 6 hours of incubation, the IC50 of M4 for HCT116 cells was 14.89μM, and after 12 hours of incubation, the IC50 of M4 for HCT116 cells decreased to 10.62μM. The IC50 of M4 for CCD 841CoN cells was much higher than that for HCT116 cells. This result further shows that M4 has an inhibitory effect on the proliferation of colorectal cancer cells, but has little effect on the proliferation of normal intestinal epithelial cells. This result not only reflects the specificity of M4 as an anticancer peptide, but also suggests that the optimal incubation time after adding M4 may be 12 hours.

[0044] Step S13: soft agar colony formation test.

[0045] Step S131, soft agar colony formation test experimental principle.

[0046] The colony formation experiment is an effective method to determine the survival rate of cells. When a single cell is grown in vitro for more than 6 generations, the cell group composed of its offspring is called a clone or colony. At this time, each clone is 0.3-1.0mm in size and contains more than 50 cells. By counting the clone formation rate, the proliferation potential of a single cell can be quantitatively analyzed. The soft agar clone formation experiment is often used for cells growing in suspension. Some malignant tumor cells can proliferate not only in the adherent state, but also in the suspended state. Their ability to form clones in soft agar reflects their degree of malignancy. This method can be used for basic research on cell differentiation and efficacy testing of clinical tumor treatment.

[0047] Step S132: Soft agar colony formation test experimental design.

[0048] Step S1321, prepare cell suspension: digest the monolayer cells in the logarithmic growth phase conventionally and collect the cell pellet by centrifugation. Resuspend the cell pellet, count, and dilute to 1×10 2 Pieces / mL.

[0049] Step S1322, prepare agar: prepare two low melting point agarose solutions of 1.2% and 0.7% respectively with distilled water, sterilize by high pressure, and maintain at 40°C to prevent solidification. Mix 1.2% agarose and 2× complete medium in a ratio of 1:1, take 1.5 ml and add it to a 6-well plate. After cooling and solidification, it can be used as bottom agar and placed in an incubator for standby use.

[0050] Step S1323, inoculating cells: After mixing 0.7% agarose and 2× complete culture medium in a sterile tube at a ratio of 1:1, add 0.2 mL of cell suspension to the tube, mix thoroughly, and inject into a plate with 1.2% agarose as a bottom layer to form a double agar layer. Finally, spread a layer of culture medium containing M4 anticancer peptide.

[0051] Step S1324: culture for 2 to 3 weeks according to the growth rate of the cells.

[0052] Step S1325: take pictures with a microscope or camera, and calculate the clone formation rate.

[0053] Step S133: Analyze the results of the soft agar colony formation test.

[0054] Typical colony growth chart and colony count chart are shown in Figure 3 and Figure 4 , Figure 3 This is a picture of the colony growth in the soft agar colony formation test. Figure 4 This is a statistical chart of the colony count in the soft agar colony formation test. The colony numbers in the different M4 concentration groups are significantly less than that in the negative control group, which shows that M4 has an anti-cancer effect.

[0055] Step S2: experimentally verify the ability of M4 to induce apoptosis of HCT 116 cells.

[0056] Step S21, flow cytometry detection.

[0057] Step S211, flow cytometry detection experimental principle.

[0058] Flow cytometry can sort out different cell populations. The cells in this experiment were double-stained with PI and AnnexinV-FITC. Among them, PI is propidium iodide, which is a red fluorescent dye. It cannot penetrate the intact cell membrane of living cells, but can stain cells in the late stage of apoptosis, dead cells and damaged cell membranes; AnnexinV, that is, annexin V, can bind to phosphatidylserine (PS) that flips from the inside to the outside of the cell membrane in the early stage of apoptosis; FITC, that is, fluorescein isothiocyanate, is a green fluorescent dye; when AnnexinV combines with FITC, early apoptotic cells can be marked by green fluorescence. Therefore, using PI and AnnexinV-FITC double staining, it is possible to distinguish between living cells, early apoptotic cells, late apoptotic cells and dead cells. Figure 5 As shown: the Q5-1 / Q3-1 quadrant is PI-positive and AnnexinV-FITC-negative cells, i.e., dead cells; the Q5-2 / Q3-2 quadrant is PI-positive and AnnexinV-FITC-positive cells, i.e., late apoptotic cells; the Q5-3 / Q3-3 quadrant is PI-negative and AnnexinV-FITC-negative cells, i.e., living cells; the Q5-4 / Q3-4 quadrant is PI-negative and AnnexinV-FITC-positive cells, i.e., early apoptotic cells.

[0059] Step S212: flow cytometry detection experiment design.

[0060] Step S2121: Adjust the HCT116 cell concentration to 1X10 by cell counting.5 / ml, and then 2ml of cells were inoculated into a 6-well plate, one well as a blank control group, and the remaining five wells as experimental groups, and cultured overnight at 37°C and 5% CO2.

[0061] Step S2122, discard the culture supernatant, add 2 mL of culture medium without M4 to the blank control group, add 2 mL of culture medium containing different concentrations of M4 (0 μM, 2.5 μM, 5 μM, 10 μM, 15 μM) to the experimental groups, and culture for 24 hours at a temperature of 37° C. and a CO2 concentration of 5%. The following steps are the same for the blank control group and the experimental group.

[0062] Step S2123, discard the culture supernatant, wash twice with PBS, then digest with 100 μL of trypsin, and then transfer to a centrifuge tube.

[0063] Step S2124: Adjust the HCT116 cell concentration to 1X10 by cell counting. 6 / ml, adjust the centrifuge speed to 1000rpm, centrifuge at 4℃ for 10 minutes, and discard the culture supernatant.

[0064] Step S2125: Add 1 mL of pre-cooled PBS to the centrifuge tube, gently shake to suspend the cells, adjust the centrifuge speed to 1000 rpm, centrifuge at 4° C. for 10 minutes, and discard the culture supernatant.

[0065] Step S2126. Repeat steps S2125 and S2126 twice.

[0066] Step S2127, resuspend the cells in the centrifuge tube in 200 μL binding buffer.

[0067] Step S2128, add 10 μL Annexin V-FITC to the centrifuge tube and mix gently, and react at room temperature for 15 minutes in the dark.

[0068] Step S2129, add 300 μL of binding buffer (total reaction volume 500 μL) and 5 μL of PI staining solution to the centrifuge tube, and use flow cytometry to detect within 1 hour. During the experiment, the blank control group is used to determine the gate, and then the cells in the experimental group are detected.

[0069] Step S213: analyzing the flow cytometry detection results.

[0070] The results of flow cytometry are shown in Figure 2. Figure 5. In the range of 0 to 15 μM (0 μM, 2.5 μM, 5 μM, 10 μM, 15 μM), the higher the concentration of M4 added, the lower the proportion of live cells, and the higher the total proportion of dead cells and apoptotic cells. Moreover, starting from 10 μM, the degree of decrease in the proportion of live cells and the degree of increase in the total proportion of dead cells and apoptotic cells become obvious. This shows that M4 can induce apoptosis of HCT116 cells and has an anti-cancer effect.

[0071] Step S22: immunoblotting test.

[0072] Step S221, immunoblotting experiment principle.

[0073] Western Blot is a method for separating and identifying proteins. Proteins in samples are separated by SDS-polyacrylamide gel electrophoresis, and antibodies are used to bind to proteins. Since antibodies specifically bind to target proteins, a clear band can be seen after development, and the width of the band can be used for protein quantitative analysis.

[0074] Step S222: Immunoblotting Experiment Design

[0075] Step S2221, sample preparation: HCT116 cells were treated with M4 at different concentrations (0 μM, 2.5 μM, 5 μM, 10 μM, 15 μM) for 24 hours, rinsed twice with pre-cooled PBS, added 100 μL RIPA lysis buffer to each well of a 6-well plate, lysed on ice for 30 min, transferred to a 1.5 mL centrifuge tube after mixing, and centrifuged at 4°C 12000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube, the concentration was measured by the BCA method, and the loading amount was calculated, the concentration was adjusted to 1X with 5X loading buffer, and the sample was placed in boiling water for 5 min until the protein was denatured.

[0076] Step S2222, electrophoresis: prepare polyacrylamide gel, wait for it to solidify, pour 1X Running Buffer into the tank, load sample into each well (load amount is 50 ng total protein), adjust the voltage to 60 V for electrophoresis, and terminate the electrophoresis when bromophenol blue runs out.

[0077] Step S2223, transfer: place the membrane in a dish containing methanol for 30 seconds, then pour it into 100 mL of 1X transfer buffer to terminate activation, cut off the separation gel part, assemble in the order of black plate, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and white plate, insert the transfer clip into the transfer rack, and transfer at 85V until bromophenol blue leaves the separation gel.

[0078] Step S2224, blocking: remove the membrane, transfer it to a plate containing 5% skim milk / TBST, and block it on a decolorizing shaker at room temperature for 2 hours.

[0079] Step S2225, immunodetection: The membrane was transferred to the primary antibody ((5% skim milk: TBST = 1: 1000), and incubated overnight on a 4°C decolorizing shaker. Subsequently, the membrane was transferred to a TBST plate and washed three times on a room temperature decolorizing shaker, each for 10 minutes. The membrane was then transferred to the secondary antibody (5% skim milk: TBST = 1: 10000), incubated at room temperature for 2 hours, and the membrane was transferred to a TBST plate and washed three times on a room temperature decolorizing shaker, each for 10 minutes. Equal amounts of ECLA solution and B solution were mixed, and an appropriate amount was taken to cover the membrane, and the membrane was exposed with a Tianneng chemiluminescence instrument to develop the color and analyze the bands.

[0080] Step S223: analysis of immunoblotting results.

[0081] The results of the immunoblotting assay to detect the effect of M4 on the relative levels of apoptosis-related proteins are shown in Figure 6 The left picture is the experimental result, and the internal reference protein is β-actin protein. The right picture is the statistical chart of the experimental results. With the increase of M4 concentration, the relative contents of Bcl-2 protein and Pro caspase-3 protein decreased, and the relative contents of Bax protein and Cleaved caspase-3 protein increased. When the M4 concentration reached 2.5μM, the relative contents of Bax protein and Cleaved caspase-3 protein increased significantly. When the M4 concentration reached 10μM, the relative content of Bcl-2 protein decreased significantly.

[0082] Among them, Bcl-2 protein is an anti-apoptotic protein, Bax protein is a pro-apoptotic protein, and Cleaved caspase-3 protein is formed by cleavage of inactive Pro caspase-3 protein during cell apoptosis. Therefore, it can be shown that M4 does induce cell apoptosis, and in the process of M4-induced cell apoptosis, the Bcl-2 signaling pathway is inhibited, and the Bax and Cleaved caspase-3 signaling pathways are activated.

[0083] Step S23: using CCK8 method to detect the effect of apoptosis inhibitors on cell viability.

[0084] Step S231, experimental principle of detecting cell viability using CCK8 method.

[0085] The experimental principle of detecting cell viability by CCK8 method is shown in step S111.

[0086] Z-VAD-FMK is an irreversible caspase inhibitor that can penetrate the cell membrane and inhibit cell apoptosis caused by caspase activation.

[0087] Step S232: Design an experiment to detect the effect of apoptosis inhibitors on cell viability using the CCK8 method.

[0088] Step S2321: dilute HCT116 cells to 1×10 4 After 100 μL / well of the culture medium was inoculated into a 96-well plate, the plate was cultured at 37°C with 5% CO2 overnight.

[0089] Step S2322, discard the supernatant, set up four treatment groups (group 1 without drug, group 2 with M4, group 3 with Z-VAD-FMK (100 μM), group 4 with M4 and Z-VAD-FMK), and incubate at 37° C. 5% CO2 for 24 hours. Set up 4 replicates for each group.

[0090] Step S2323: After the test time is up, add 10 μL of CCK8 reagent to each well and incubate at 37° C. 5% CO 2 for 4 hours.

[0091] Step S2324: Measure the absorbance at a wavelength of 450 nm using an ELISA instrument. Make a "cell number-OD value" curve graph based on the number of cells in each well and the measured OD value to obtain the activity of the corresponding cells.

[0092] Step S233: Use CCK8 method to detect the effect of apoptosis inhibitors on cell viability. The results of using CCK8 method to detect the effect of apoptosis inhibitors on cell viability are shown in Figure 7 Compared with the control group, M4 significantly inhibited the viability of HCT116 cells, but after the addition of apoptosis inhibitor Z-VAD-FMK, the viability of HCT116 cells was significantly improved. This result indicates that apoptosis inhibitors can partially inhibit the death of HCT116 cells caused by M4.

Claims

1. An anticancer peptide, characterized in that The antimicrobial peptide is obtained by modifying the amino acid sequence of the grass carp interleukin 21 gene, and the amino acid sequence of the antimicrobial peptide is SEQ ID No.

1.

2. A drug for treating colorectal cancer, characterized in that: The medicine contains the anticancer peptide described in claim 1.

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