A polypeptide and its application in the preparation of antitumor drugs

Abstract

This invention relates to a polypeptide and its application in the preparation of antitumor drugs, belonging to the field of antitumor drug technology. The amino acid sequence of the polypeptide of this invention is shown in SEQ ID NO: 1. It can selectively inhibit CRC cells (including CT26, MC38, and HCT116) without toxicity to other cells (including human intestinal epithelial cells CCD841 and HIEC6), exhibiting high application safety. Furthermore, the oncolytic mechanism of the polypeptide of this invention reduces the possibility of tumor cells developing drug resistance. It can be used to inhibit CRC tumor cells, thereby achieving the control and treatment of CRC.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, and in particular to a polypeptide and its application in the preparation of antitumor drugs. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide, with a high mortality rate. More than 900,000 people die from CRC each year, while over 1.8 million are diagnosed. Treatment for CRC includes surgery, chemotherapy, radiotherapy, and immunotherapy, with surgery being the preferred treatment. Due to the high invasiveness and metastatic nature of CRC, the 5-year survival rate is only about 50%. 5-Fluorouracil (5-FU) is the first-line therapy for CRC; however, its systemic toxicity and the development of drug resistance severely limit its efficacy. Similarly, while preoperative radiotherapy combined with surgery is the recommended treatment for locally advanced CRC, the low sensitivity of CRC patients to preoperative radiotherapy also limits treatment success rates.

[0003] Therefore, there is an urgent need for novel anti-tumor drugs that can inhibit tumor growth, are less likely to induce drug resistance, and have low toxicity, in order to achieve the treatment of colorectal tumors. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polypeptide that effectively inhibits tumor growth and its application in the preparation of anti-tumor drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a polypeptide, wherein the polypeptide is a polypeptide and / or its derivatives having an amino acid sequence as shown in SEQ ID NO: 1.

[0007] The polypeptide of this invention can effectively inhibit the growth of colorectal cancer tumor cells, reduce their survival rate, and is non-toxic to normal intestinal cells.

[0008] Furthermore, the derivative of the polypeptide is a phosphorylated amino acid of the N-terminal amino acid of the polypeptide. This is beneficial for improving the therapeutic effect on the intestines.

[0009] In a specific embodiment of the present invention, the chemical structural formula of the polypeptide derivative is shown in Formula I:

[0010]

[0011] Secondly, the present invention also provides a method for preparing the polypeptide, comprising the following steps:

[0012] S1: Following the amino acid sequence of the polypeptide, the amino acids are inoculated into Wang resin using a polypeptide synthesizer to obtain peptide resin;

[0013] S2: The peptide resin from step S1 is cleaved with trifluoroacetic acid to obtain crude peptides;

[0014] S3: Purify the crude polypeptide from step S2 to obtain the polypeptide.

[0015] Further, in step S3, the crude polypeptide from step S2 is purified by reversed-phase high-performance liquid chromatography.

[0016] Further, in step S1, amino acids are added to Wang resin one by one from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide.

[0017] Furthermore, the preparation method in step S1 includes the following steps:

[0018] S11: Protect the first amino acid at the C-terminus of the polypeptide with a 9-fluorenylmethoxycarboxyl group to obtain Fmoc-X, incorporate it into Wang resin, and then remove the 9-fluorenylmethoxycarboxyl group to obtain X-Wang resin.

[0019] S12: The second amino acid at the C-terminus of the polypeptide is protected with a 9-fluorenylmethoxycarboxyl group and a triphenylmethyl group to obtain Fmoc-Y-Trt-OH, which is then combined with X-Wang resin to form a peptide bond, resulting in a dipeptide with a protecting group.

[0020] S13: Following steps S11 and S12, replace the corresponding amino acids according to the amino acid sequence of the following polypeptide sequence, synthesizing sequentially from the C-terminus to the N-terminus of the polypeptide until the preparation is complete, and obtain peptide resin.

[0021] In a specific embodiment of the present invention, when preparing the derivative of the polypeptide, the Fmoc-Y-Trt-OH used when binding the amino acid to be phosphorylated with X-Wang resin is N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphotyrosine (Fmoc-Tyr(HPO3OBzl)-OH).

[0022] Further, in step S2, the peptide resin from step S1 is cut with a cutting reagent composed of trifluoroacetic acid, water and triisopropylchlorosilane.

[0023] In a specific embodiment of the present invention, the mass ratio of trifluoroacetic acid, water and triisopropylchlorosilane is 85-95:5-10:1-5, preferably 95:2.5:2.5.

[0024] Further, in step S2, the peptide resin is mixed with the cleavage reagent and reacted at 20-25°C in the dark for 1-3 hours. After filtration, a filtrate and a precipitate are obtained. The precipitate is washed with trifluoroacetic acid to obtain a washing solution. The filtrate and washing solution are mixed and concentrated using a rotary evaporator. The precipitate is then precipitated with anhydrous diethyl ether at -18--24°C for 1-3 hours. After centrifugation, the precipitate is collected, washed, and dried to obtain crude peptide.

[0025] In a specific embodiment of the present invention, the reaction is carried out at 20°C in the dark for 2 hours.

[0026] In a specific embodiment of the present invention, anhydrous diethyl ether is used to precipitate the sample for 3 hours at -20°C.

[0027] Furthermore, the volume of anhydrous diethyl ether is 5 to 20 times that of the liquid after concentration by rotary evaporator, preferably 10 times.

[0028] Furthermore, the centrifugation conditions are 2000–3000g for 5–20 minutes.

[0029] In a specific embodiment of the present invention, the centrifugation conditions are 2500g centrifugation for 10min.

[0030] Further, the crude polypeptide from step S2 was dissolved in a 70-90% (v / v) acetonitrile aqueous solution, filtered, and eluted using a C18 reversed-phase atmospheric pressure column. The eluent was a mixture of methanol and sodium sulfate aqueous solution at a volume ratio of 30:70 to 70:30, the flow rate was 0.5-1 mL / min, the detection wavelength was 200-230 nm, the main peak was collected, and the product was lyophilized to obtain the eluted product.

[0031] The elution product was further purified using a reverse-phase C18 column. Elution buffer A was 0.1–0.12% (v / v) trifluoroacetic acid aqueous solution; elution buffer B was 0.1–0.12% (v / v) trifluoroacetic acid aqueous acetonitrile solution. The elution concentration was 25% (v / v) B–40% (v / v) B, the elution time was 10–12 min, and the flow rate was 0.5–1 mL / min. The main peak was then collected as above and lyophilized to obtain the polypeptide.

[0032] In a specific embodiment of the present invention, the crude polypeptide product of step S2 is dissolved in 90% (v / v) acetonitrile aqueous solution, filtered, and eluted using a C18 reversed-phase atmospheric pressure column. The eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30, the flow rate is 1 mL / min, the detection wavelength is 220 nm, the main peak is collected, and the product is lyophilized to obtain the eluted product.

[0033] The elution product was further purified using a reverse-phase C18 column. Elution buffer A was a 0.1% (v / v) trifluoroacetic acid aqueous solution; elution buffer B was a 0.1% (v / v) trifluoroacetic acid aqueous acetonitrile solution. The elution concentration was 25% (v / v)B to 40% (v / v)B, the elution time was 12 min, and the flow rate was 1 mL / min. The main peak was then collected as above and lyophilized to obtain the polypeptide.

[0034] Thirdly, the present invention also claims protection for the use of the polypeptide in the preparation of antitumor drugs.

[0035] Furthermore, the antitumor drug includes drugs that inhibit tumor growth.

[0036] In a specific embodiment of the present invention, the antitumor drug includes an anti-colorectal cancer drug.

[0037] The polypeptide described in this invention exhibits oncolytic activity, reducing the survival rate of CT26, MC38, and HCT116 colon cancer cells. Its oncolytic activity is further enhanced after dephosphorylation treatment with alkaline phosphatase (ALP). Furthermore, the concentration of ALP is higher in tumor cells and the intestinal environment, demonstrating further selectivity for the application environment. No cytotoxic effects were observed on human intestinal epithelial cells (CCD841 and HIEC6).

[0038] Fourthly, the present invention provides an anti-colorectal cancer drug, the drug containing the polypeptide.

[0039] Furthermore, the anti-colorectal cancer drug also contains pharmaceutically acceptable additives and / or excipients and / or carriers.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] This invention provides a polypeptide targeting colorectal cancer (CRC), which can selectively inhibit CRC cells (including CT26, MC38, and HCT116) without toxicity to other cells (including human intestinal epithelial cells CCD841 and HIEC6), exhibiting high application safety. Furthermore, the oncolytic mechanism of this polypeptide reduces the possibility of tumor cells developing drug resistance. It can be used to inhibit CRC tumor cells, thereby achieving the control and treatment of CRC. Attached Figure Description

[0042] Figure 1 The results of electrospray mass spectrometry for the polypeptide KY of this invention are shown.

[0043] Figure 2 The results show the oncolytic activity of the polypeptide KY of this invention.

[0044] Figure 3 The results show the in vitro safety of the polypeptide KY of this invention.

[0045] Figure 4 The results show the in vivo safety of the polypeptide KY of this invention. Wherein, a represents mouse body weight; b represents the weight of the mouse heart, liver, kidney, lung, and spleen; c represents blood biochemical indicators of the mouse; and d represents the H&E staining results of the mouse liver and kidney.

[0046] Figure 5 Fluorescence electron microscopy results for detecting the oncolytic mechanism of peptide KY. a) Image of CT26 cells stained with propidium iodide (PI) / Hoechst (scale bar, 100 μm); b) Quantitative analysis results of cells successfully stained with PI; c) TEM image (scale bar, 5 μm). ***P<0.001.

[0047] Figure 6 Statistical results of tumor size in mice to detect the in vivo activity of peptide KY. In the figures, a is a schematic diagram of the experimental protocol; b is tumor volume; c is tumor volume relative to day 0; d is a photograph of the tumor obtained from dissection; and e is the weight of the tumor obtained from dissection. Scale bar = 1 cm. *P<0.05, **P<0.01, ***P<0.001, no statistically significant difference in ns. Detailed Implementation

[0048] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0049] Example 1: Design and preparation of polypeptide KY

[0050] I. Experimental Methods

[0051] 1. This invention designs polypeptide KY using machine learning.

[0052] First, a random peptide library of 50 million sequences was constructed;

[0053] Subsequently, the random peptide library was filtered using a multi-filter approach:

[0054] The first-stage filtration retains only peptides with a hydrophobic moment range of 0.5–0.7 and a hydrophobicity ≥0.5. This is because, among all physicochemical properties, the hydrophobic moment is one of the most relevant to the antibacterial activity of antimicrobial peptides. Although the hydrophobic moment increases antibacterial and anticancer activity, it also increases toxicity to non-tumor cells; therefore, a moderate threshold was chosen.

[0055] The second stage of filtration includes: (1) setting the number of W (tryptophan) to 2, since multiple Ws are a favorable structure for promoting peptide self-assembly and disrupting bacterial membranes; (2) setting the number of R (arginine) to ≥1, given that the sequence generator contains two cationic amino acids, as R plays a more important role than K (lysine) in disrupting bacterial cell membranes; and (3) setting the number of non-cationic hydrophilic S (serine) to ≥1, in order to moderately reduce the percentage of hydrophobic amino acids.

[0056] Level 3 filtering maintains the protein secondary structure prediction (PSSpred) value of the peptide at ≥80% to support the possibility of forming α-helices and self-assembling structures;

[0057] After filtering, only 5,491 sequences were retained from 50 million sequences (a filtering efficiency of 99.989%) to proceed to the next stage of the pipeline;

[0058] Finally, using a data module containing two machine learning-based models, the steps include: (1) Acquiring datasets of anticancer properties and toxicity: Obtaining peptide datasets of anticancer properties and toxicity from the Antimicrobial Activity and Structure of Peptides (DBAASP) database according to custom screening conditions, and setting IC 50(1) Using 128 μM as the threshold, peptides in the peptide dataset are defined as having / not having anticancer peptide activity or having / not having toxicity; (2) Construction of automatic machine learning models: The peptide datasets in step (1) are loaded into the automatic machine learning framework AutoGluon, the peptide datasets are divided, the classifier is trained, and the model is evaluated through 5-fold cross-validation to obtain the trained model. The trained model is used to predict new peptide data and automatically optimize the parameters; (3) Selection of the best model: AutoGluon in step (2) automatically recommends 28 models (14 anticancer peptide and toxicity prediction models) for further evaluation. Based on the performance of the models, the best prediction models are obtained; (4) The best prediction model in step (3) is used to predict anticancer peptide sequences from the peptide dataset to obtain anticancer peptide candidates. A total of 97 anticancer peptides are obtained; (5) In order to improve the stability of anticancer peptides as much as possible, sequences with high self-assembly potential are selected. The sequences are selected from the public database AMP. Input the sequences of the 97 anticancer peptides in step (4) into Prediction(dbaasp.org), predict the self-assembly potential of the anticancer peptide sequences based on their in vitro agglutination potential, and select 8 peptide sequences with a self-assembly prediction value greater than 400 from the 97 anticancer peptides; (6) In the 8 peptide sequences in step (5), according to the different positions of tyrosine Y at the N-terminus or C-terminus of the peptide sequence, synthesize 2 peptide sequences with self-assembly potential prediction values ​​of 481.26 (amino acid sequence as shown in SEQ ID NO: 1: KSWWRKIFYFIRY) and 592.78 (amino acid sequence as shown in SEQ ID NO: 2: YKKVRSIWWYIFK), and phosphorylate the tail tyrosine. After the peptide with amino acid sequence as shown in SEQ ID NO: 1 is treated with peptide acid phosphorylation, the chemical structure is shown in Formula I, which is peptide KY. After the peptide with amino acid sequence as shown in SEQ ID NO: 2 is treated with acid phosphorylation, the chemical structure is shown in Formula II, which is peptide YK.

[0059]

[0060] The sequences and physicochemical parameters of peptides KY and YK are shown in Table 1.

[0061] Table 1. Sequences and physicochemical parameters of peptides KY and YK

[0062]

[0063]

[0064] 2. Preparation method of polypeptide KY

[0065] This application employs a solid-phase chemical synthesis method to sequentially synthesize polypeptide KY. The solid-phase chemical synthesis method includes the following steps:

[0066] (1) The peptide KY was synthesized one by one from the C-terminus to the N-terminus according to the amino acid sequence of the peptide KY, and the synthesis was completed by a peptide synthesizer.

[0067] First, Fmoc-X (where X is the first amino acid at the C-terminus of each peptide; Fmoc stands for 9-fluorenylmethoxycarbonyl, an amino protecting group) is inscribed into Wang resin. After removing the Fmoc group, X-Wang resin is obtained. Then, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-triphenylmethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide) is combined with X-Wang resin to form peptide bonds, resulting in a dipeptide with a protecting group. The above peptide bond formation steps are repeated, and the types of amino acids are replaced according to the amino acid sequence of the subsequent peptide sequence, synthesizing from the C-terminus to the N-terminus of the peptide sequentially until the synthesis is complete, resulting in peptide resin. When the amino acid to be phosphorylated is combined with X-Wang resin, the Fmoc-Y-Trt-OH used is N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphotyrosine (Fmoc-Tyr(HPO3OBzl)-OH).

[0068] (2) Add the cleavage reagent to the peptide resin in step (1), react at 20°C in the dark for 2 hours, filter to obtain filtrate; wash the precipitate with TFA (trifluoroacetic acid) to obtain washing solution; mix the washing solution with the above filtrate, concentrate with rotary evaporator, add 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, and a white powder will be precipitated. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain crude peptide product. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5.

[0069] (3) Equilibrate the column with 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid) for 30 min, dissolve the polypeptide from step (2) in 90% (v / v) acetonitrile aqueous solution, filter, use a C18 reversed-phase atmospheric pressure column, use gradient elution (the eluent is a mixture of methanol and 0.2 mol / L sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), the flow rate is 1 mL / min, the detection wavelength is 220 nm, collect the main peak, freeze dry, and obtain the gradient elution product;

[0070] Further purification was performed using a reverse-phase C18 column. Elution buffer A was 0.1% (w / w) TFA aqueous solution; elution buffer B was 0.1% (w / w) TFA acetonitrile solution. The elution concentration was 25% (v / v) B to 40% (v / v) B, the elution time was 12 min, and the flow rate was 1 mL / min. The main peak was collected again and lyophilized to obtain the purified peptide.

[0071] (4) Identification of peptides: The purified peptides obtained in step (3) were analyzed by electrospray mass spectrometry.

[0072] II. Experimental Results

[0073] like Figure 1 As shown, the purity of the prepared polypeptide KY is greater than 95%.

[0074] Example 2: IC50 assay for the antitumor activity of peptides KY and YK 50 value

[0075] I. Experimental Methods

[0076] Peptides KY and YK were dissolved in PBS to obtain 256 μM solutions of peptide KY and YK. 0.32% (v / v) alkaline phosphatase (ALP) (1 unit / μL) was added to each of the 256 μM peptide KY and YK solutions, and the mixtures were incubated at 37 °C for at least 10 h and then stored overnight at -20 °C to obtain ALP-treated peptide KY solution (KY-ALP) and ALP-treated peptide YK solution (YK-ALP).

[0077] Cytotoxicity of different concentrations (8, 16, 32, 64, and 128 μM) of peptide KY, ALP-treated peptide KY, peptide YK, and ALP-treated peptide YK solutions diluted in PBS to each cell line (CT26, MC38, HCT116, CCD841, and HIEC6) was assessed using the Cell Counting Kit-8 (CCK-8, APExBIO Technology LLC, USA). Cells were cultured overnight in 96-well plates (4000–6000 cells / well) supplemented with 10% (v / v) FBS (fetal bovine serum). Cell viability was assessed using the CCK-8 assay, and the data were analyzed to plot a semi-logarithmic curve of viability versus peptide concentration, and the IC50 was calculated. 50 Values. Cells treated with culture medium served as a control group. Each experiment was repeated at least three times, and each experiment consisted of three technical parallel experiments.

[0078] II. Experimental Results

[0079] As shown in Table 2, the IC50 of peptide KY on colon cancer cell lines... 50 The value was lower than that of peptide YK, indicating that peptide KY had a better inhibitory effect on colon cancer than peptide YK. The IC50 value of peptide KY against normal intestinal cell lines... 50The concentration was slightly lower than that of peptide YK, but this concentration far exceeded the activity concentration range of peptide KY. The oncolytic activity of peptide KY was further enhanced after dephosphorylation with ALP treatment. Furthermore, alkaline phosphatase concentrations were higher in tumor cells and the intestinal environment, indicating that peptide KY exhibited further selectivity for its application environment.

[0080] Table 2. IC50 of the peptides against colon cancer and normal intestinal cell lines. 50 Value (μM)

[0081]

[0082] Example 3: Detection of the in vitro oncolytic activity of peptide KY

[0083] I. Experimental Methods

[0084] The peptide KY was dissolved in PBS to obtain a peptide KY solution with a concentration of 256 μM. 0.32% (v / v) alkaline phosphatase ALP (1 unit / μL) was added to the 256 μM peptide KY solution, mixed well, and incubated at 37 °C for at least 10 h. The solution was then stored overnight at -20 °C to obtain an ALP-treated peptide KY solution.

[0085] 1. Detection of the oncolytic activity of peptide KY

[0086] (1) The cytotoxicity of different concentrations (8, 16, 32, 64, and 128 μM) of peptide KY diluted in PBS and peptide KY treated with ALP to each cell line (CT26, MC38, HCT116, CCD841, and HIEC6) was assessed using the Cell Counting Kit-8 (CCK-8, APExBIO Technology LLC, USA). Cells were cultured overnight in 96-well plates (4000–6000 cells / well) supplemented with 10% (v / v) FBS (fetal bovine serum). The final cell viability was assessed using the CCK-8 assay. Cultured cells served as a control group. Each experiment was repeated at least three times, and each experiment consisted of three technical parallel experiments.

[0087] (2) Hemolytic activity of peptide KY was detected using fresh red blood cells from C57BL / 6 mice. Red blood cells were harvested after centrifugation at 1000×g for 5 min, washed twice with 10 volumes of PBS, and resuspended in PBS to a concentration of 1% (v / v) to obtain a red blood cell suspension. 50 μL of the red blood cell suspension was mixed with 50 μL of peptide KY solution of different concentrations (8, 16, 32, 64, and 128 μM) dissolved in PBS or ALP-treated peptide KY solution, and incubated at 37℃ for 1 h. After 1 h, the mixture was removed, centrifuged at 1000×g for 5 min, and the supernatant was transferred to a new 96-well plate. The OD value was measured at 570 nm to determine the amount of hemoglobin released. Red blood cell suspensions prepared with PBS and 0.1% (v / v) Triton X-100 were used as negative and positive controls, respectively. Each experiment was repeated at least three times, and each experiment consisted of three technical parallel experiments.

[0088] (3) Twenty-five healthy female C57BL / 6 mice (6 weeks old) were randomly assigned to seven cages (5 mice per cage). The cages were individually placed in ventilated cages (IVCs) at a temperature of 20–24°C and humidity of 50–60%, with 60 air changes per hour and a 12 / 12-hour light / dark cycle. Mice were allowed to acclimatize to the facility for two days prior to the experiment. Mice were intravenously injected with 100 μL of PBS or different concentrations of polypeptide KY solution diluted with PBS (0, 5, and 10 mg / kg), and changes in body weight were monitored. At the end of the experiment (day 5), mice were sacrificed, and cervical dislocation and organ harvesting were performed. Heart, liver, kidney, lung, and spleen organs were weighed, fixed with 4% (w / v) paraformaldehyde, and stained with H&E. Blood samples were collected from the orbital veins of the mice. Automated biochemical analyzers are used to measure blood biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (ALP), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatine kinase (CREA), creatine kinase (CK), and low-density lipoprotein cholesterol (LDH), to reflect the extent of liver and kidney damage.

[0089] II. Experimental Results

[0090] like Figure 2 As shown, peptide KY exhibits oncolytic activity, reducing the survival rate of CT26, MC38, and HCT116 colon cancer cells, and its oncolytic activity is further enhanced after ALP treatment for dephosphorylation. Furthermore, alkaline phosphatase concentrations are higher in tumor cells and the intestinal environment, indicating that peptide KY demonstrates further selectivity for its application environment.

[0091] like Figure 3As shown, the peptide KY did not exhibit cytotoxicity against human intestinal epithelial cells (CCD841 and HIEC6) at the highest tested concentration. It did cause some damage to mouse blood cells at the highest concentration of 128 μM, but this concentration was much higher than its active concentration (5 mg / kg and 10 mg / kg).

[0092] like Figure 4 As shown, the safety results in mice indicated that no damage occurred even after 5 days of peptide KY injection, demonstrating its safety. Figure 4 As shown in figure a, the body weight of mice injected with peptide KY and those injected with PBS was not significantly different on day 5; Figure 4 As shown in b, mice injected with polypeptide KY and mice injected with PBS had essentially the same weight in heart, liver, kidney, lung, and spleen; Figure 4 As shown in c, there was no significant difference in blood biochemical parameters between mice injected with polypeptide KY and mice injected with PBS; Figure 4 As shown in d, after H&E staining, the liver and kidney sections of mice injected with polypeptide KY were normal.

[0093] Example 4: Detection of the oncolytic mechanism of peptide KY

[0094] I. Experimental Methods

[0095] CT26 cells were administered at a rate of 4 × 10⁻⁶. 4 Cells were seeded at a rate of [number] cells / well in 96-well plates and cultured overnight. The ALP-treated KY solution from Example 3 was diluted to 16 μM with PBS to obtain a 16 μM ALP-treated KY solution. This 16 μM ALP-treated KY solution was added to 1640 medium and incubated with CT26 cells overnight, followed by gentle washing three times with PBS. Cells were stained with PI dye and then observed and photographed using an inverted fluorescence microscope (Nikon ECLIPSE Ti2-E).

[0096] II. Experimental Results

[0097] like Figure 5 a and Figure 5 As shown in b, after incubation with ALP-treated peptide KY solution, the non-membrane-permeable dye PI increased in CT-26 cells, as shown in Figure b. Figure 5 As shown in Figure c, cell membrane damage occurred after incubation with KY solution treated with ALP peptides.

[0098] Example 5: Detection of in vivo activity of peptide KY

[0099] I. Experimental Methods

[0100] After 4–5 weeks of acclimatization, C57BL / 6 mice were subcutaneously injected with CT26 cells (1.0 × 10⁻⁶) into the right lower abdomen. 6A colorectal tumor-bearing mouse model was established. When the tumor appeared and grew to a certain size, the mice were randomly divided into two groups (n=5 per group) and subjected to intervention for 7 consecutive days. The two groups of mice were intravenously injected with PBS and peptide KY solution (5 mg peptide / kg), respectively, while ALP (0.456 units / mouse) was injected intratumorally. Mouse weight and tumor volume were recorded daily. After the intervention, the mice were euthanized under isoflurane anesthesia. Mouse weight and tumor weight were collected.

[0101] II. Experimental Results

[0102] like Figure 6 Figure a shows a schematic diagram of the experimental scheme. Figure 6 b represents the tumor volume of mice after different treatment groups; Figure 6 c represents the volume of mouse tumors relative to day 0 after different treatment groups; Figure 6 d shows photographs of mouse tumors after different treatments obtained from dissection; Figure 6 e represents the weight of the mouse tumors obtained from dissection after different treatments.

[0103] CRC mice treated with peptide KY (intratumoral injection of ALP) had smaller and milder tumors compared to CRC mice injected with PBS. These results indicate that peptide KY enhances oncolytic activity and inhibits CRC tumor development under ALP induction.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A polypeptide derivative, characterized in that, The chemical structural formula of the polypeptide derivative is shown in Formula I: Ⅰ。 3. The method for preparing the polypeptide according to claim 1, characterized in that, Includes the following steps: S1: Following the amino acid sequence of the polypeptide, the amino acids are inoculated into Wang resin using a polypeptide synthesizer to obtain peptide resin; S2: The peptide resin from step S1 is cleaved with trifluoroacetic acid to obtain crude peptides; S3: Purify the crude polypeptide from step S2 to obtain the polypeptide.

4. The preparation method according to claim 3, characterized in that, In step S3, the crude peptide from step S2 is purified by reversed-phase high-performance liquid chromatography.

5. The use of the polypeptide of claim 1 or the polypeptide derivative of claim 2 in the preparation of an anti-colon cancer drug.

6. The application according to claim 5, characterized in that, The anti-colon cancer drugs include drugs that inhibit the growth of colon cancer tumors.

7. An anti-colon cancer drug, characterized in that, The drug contains the polypeptide of claim 1 and / or the polypeptide derivative of claim 2.

8. The anti-colon cancer drug according to claim 7, characterized in that, The anti-colon cancer drug also contains pharmaceutically acceptable additives.

9. The anti-colon cancer drug according to claim 7, characterized in that, The anti-colon cancer drug also contains pharmaceutically acceptable excipients.

10. The anti-colon cancer drug according to claim 7, characterized in that, The anti-colon cancer drug also contains a pharmaceutically acceptable carrier.

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