Use of a kind of oligopeptide from Phascolosoma esculenta and 5-FU in the preparation of drug for resisting colorectal cancer

Abstract

This invention discloses the application of an oligopeptide derived from *Sipunculus nudus* and 5-FU in the preparation of drugs for treating colorectal cancer, belonging to the field of biomedical technology. This invention isolates an oligopeptide from *Sipunculus nudus*. Experimental verification shows that this small-molecule oligopeptide, when combined with 5-FU at high concentrations, significantly inhibits the proliferation of colon cancer cells, exhibiting a dose-dependent synergistic effect. Oligopeptide 659 and 5-FU synergistically activate the mitochondrial apoptosis pathway; combined administration enhances γ-H2AX focal point formation and exacerbates DNA double-strand breaks, thereby synergistically inhibiting CRC cell proliferation. In vivo analysis of the antitumor effect of peptide 659 using NOD-SCID female mice inoculated with DLD-1 cells further demonstrates the anti-colorectal tumor effect of oligopeptide 659. Analysis of mouse autopsies showed that, compared to the control group, oligopeptide 659 inhibited tumor growth in mice.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an oligopeptide and 5-FU derived from Sipunculus nudus in the preparation of a drug for treating colorectal cancer. Background Technology

[0002] 5-FU monotherapy resistance is a major challenge in clinical chemotherapy, especially in solid tumors such as colorectal cancer, leading to treatment failure and poor prognosis. Its mechanisms involve multiple levels, and current research suggests that these mechanisms collectively cause decreased sensitivity of cancer cells to 5-FU, necessitating the combination of targeted therapies or adjustments to treatment strategies to reverse resistance.

[0003] *Phascolosoma esculenta*, a species of sipuncula, possesses unique bioactive components (such as polysaccharides, peptides, terpenoids, and antimicrobial proteins) that exhibit potential pharmacological effects including anti-inflammatory, antioxidant, antitumor, and immunomodulatory properties. Extracts from the body wall of *Phascolosoma esculenta* are rich in sulfated polysaccharides, which can alleviate inflammatory responses by inhibiting the NF-κB signaling pathway. Antimicrobial peptides isolated from its coelomic fluid show broad-spectrum inhibitory activity against multidrug-resistant bacteria (such as *Staphylococcus aureus* and *Escherichia coli*). Furthermore, crude extracts of this species significantly scavenge free radicals and induce tumor cell apoptosis in in vitro experiments, suggesting its potential in the development of antioxidants and anticancer drugs. However, research on the mechanism of action, structural optimization, and in vivo pharmacodynamics of its active components is still in its early stages, and few studies on the medicinal use of *Phascolosoma esculenta* have mentioned purified products; most studies focus on crude extracts.

[0004] Studies have shown that crude extracts from Sipunculus nudus contain a mixture of polysaccharides and protein peptides, which have certain effects on delaying aging, improving memory, fighting fatigue, enhancing immunity, and protecting the liver. However, there are few research reports on its anti-tumor effects. Summary of the Invention

[0005] The purpose of this invention is to provide the application of oligopeptides derived from Sipunculus nudus and 5-fluorouracil (5-FU) in the preparation of drugs for treating colorectal cancer, thereby solving the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of the present invention is an oligopeptide derived from Sipunculus nudus, the amino acid sequence of which is shown in SEQ ID NO.6.

[0008] The second technical solution of the present invention is the application of the oligopeptide and 5-fluorouracil in the preparation of drugs for colorectal cancer.

[0009] The third technical solution of the present invention is a drug for treating colorectal cancer, comprising the oligopeptide and 5-fluorouracil.

[0010] The fourth technical solution of the present invention is the application of the oligopeptide in the preparation of drugs for colorectal cancer.

[0011] The fifth technical solution of the present invention is a drug for treating colorectal cancer, comprising the oligopeptide.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] This invention isolates an oligopeptide from *Sipunculus nudus*. Experimental results demonstrate that this small-molecule oligopeptide, when combined with 5-FU at high concentrations, significantly inhibits the proliferation of colon cancer cells, exhibiting a dose-dependent synergistic effect. This result provides experimental evidence for developing a chemosensitizing strategy based on peptide drugs. In vivo analysis of the antitumor effect of peptide 659 using NOD-SCID female mice inoculated with DLD-1 cells further confirms the anti-colorectal tumor activity of oligopeptide 659. Analysis of the results after mouse dissection showed that, compared to the control group, oligopeptide 659 inhibited tumor growth in mice (P<0.05). Attached Figure Description

[0014] Figure 1 The content of oligopeptides hydrolyzed by seven proteolytic enzymes and the free radical scavenging rate were determined.

[0015] Figure 2 The effects of different peptides on the viability of Hcoepic, DLD-1, and HCT-15 cells (200 μM, 48 h). Where: A: Hcoepic cell viability after treatment with different peptides; B: DLD-1 cell viability after treatment with different peptides; C: HCT-15 cell viability after treatment with different peptides. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

[0016] Figure 3The cell viability of CRC cells at different concentrations of 659 and 5-FU was measured. A, B, and C represent the cell viability of DLD-1 cells, with 5-FU concentrations of A: 5 μM; B: 10 μM; and C: 20 μM, respectively, for single and combined treatment groups. D, E, and F represent the cell viability of HCT-15 cells, with 5-FU concentrations of D: 5 μM; E: 10 μM; and F: 20 μM, respectively, for single and combined treatment groups. The treatment concentrations of 659 for both cell types were 50, 100, 150, and 200 μM. Compared with the control group: *: P < 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001. Compared with the 5-FU monotherapy group: s: P < 0.05. ss: P < 0.01. sss: P < 0.001. ssss: P < 0.0001. Compared with the corresponding concentration of 659 monotherapy group: #: P < 0.05. ##: P < 0.01. ###: P < 0.001. ####: P < 0.0001.

[0017] Figure 4 Cell viability of CRC cells under different time periods after drug administration. A, B, and C represent the cell viability of DLD-1 cells under single-drug and combined-drug administration, with detection time periods of A: 24 h; B: 48 h; and C: 72 h, respectively. D, E, and F represent the cell viability of HCT-15 cells under single-drug and combined-drug administration, with detection time periods of D: 24 h; E: 48 h; and F: 72 h, respectively. Compared with the control group: *: P < 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001. Compared with the 5-FU single-drug group: s: P < 0.05. ss: P < 0.01. sss: P < 0.001. ssss: P < 0.0001. Compared with the corresponding concentration of 659 alone: ​​#: P<0.05. ##: P<0.01. ###: P<0.001. ####: P<0.0001.

[0018] Figure 5 EdU staining results of DLD-1 cells under different drug administration groups. A: Staining results of DLD-1; B: Staining results of HCT-15; C: Statistical analysis of EdU staining results. Compared with the control group: ***P<0.001. ****P<0.0001.

[0019] Figure 6The results of DLD-1 and HCT-15 cell colony formation under different drug administration groups are shown. A represents the cell proliferation of each group after DLD-1 and HCT-15 cell colony formation on agar plates. B represents the effect rate of each drug on the colony formation of the two CRC cell lines, calculated as colony formation rate = (number of colonies formed / number of cells inoculated) × 100%. Compared with the control group: **: P < 0.01. ***: P < 0.001. ****: P < 0.0001. Compared with the 5-FU monotherapy group: s: P < 0.05. ss: P < 0.01. sss: P < 0.001. ssss: P < 0.0001. Compared with the 659 monotherapy group: ###: P < 0.001. ####: P < 0.0001.

[0020] Figure 7 The results of γ-H2AX staining of two types of colorectal cancer cells under different drug administration groups are shown. A: γ-H2AX staining results of DLD-1; B: γ-H2AX staining results of HCT-15; C: The relative fluorescence intensity of each group of colorectal cancer cells was calculated and compared based on the ratio of the microscopic fluorescence area to the total field of view. Compared with the control group: *: P < 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001.

[0021] Figure 8 Cell cycle results for different drug administration groups. A: DLD-1 cell cycle results; B: HCT-15 cell cycle results; C: Statistical analysis of DLD-1 cell cycle results; D: Statistical analysis of HCT-15 cell cycle results. Compared with the control group: ***P<0.001. ****P<0.0001.

[0022] Figure 9 The results of cell apoptosis under different drug administration groups are shown. A: Apoptosis of DLD-1 and HCT-15 cells; B: Apoptosis rate of each group, expressed as the percentage of apoptotic cells to total cells. Compared with the control group: *: P < 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001.

[0023] Figure 10 The results of 33342 staining of cells under different drug administration groups.

[0024] Figure 11 This represents the expression of apoptosis-related proteins DLD-1 and HCT-15.

[0025] Figure 12Analysis of the expression levels of apoptosis-related proteins DLD-1 and HCT-15. Where A: BAX expression; B: BCL-2 expression; C: Cyclin D1 expression; D: PCNA expression. Compared with the control group: **: P < 0.01. ***: P < 0.001. ****: P < 0.0001. Compared with the 5-FU monotherapy group: ss: P < 0.01. sss: P < 0.001. ssss: P < 0.0001. Compared with the 659 monotherapy group: ###: P < 0.001. ####: P < 0.0001.

[0026] Figure 13 This is an anatomical diagram of a NOD-SCID tumor-bearing mouse. A: Tumor tissue image; B: Mouse image.

[0027] Figure 14 This is a curve showing the tumor volume in NOD-SCID tumor-bearing mice after drug treatment.

[0028] Figure 15 Thermogram of tumor volume in NOD-SCID tumor-bearing mice after drug treatment. (Mean±SD, n = 5, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs control) Detailed Implementation

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0030] This invention provides an oligopeptide derived from Sipunculus nudus, the amino acid sequence of which is shown in SEQ ID NO.6.

[0031] This invention also provides the use of the oligopeptide and 5-fluorouracil in the preparation of drugs for treating colorectal cancer.

[0032] In some specific embodiments, the molar ratio of the oligopeptide to 5-fluorouracil is (10~50):1.

[0033] In some specific implementations, the colorectal cancer includes human colorectal adenocarcinoma and human colon cancer.

[0034] This invention also provides a drug for treating colorectal cancer, comprising the oligopeptide and 5-fluorouracil.

[0035] In some specific embodiments, the molar ratio of the oligopeptide to 5-fluorouracil is (10~50):1.

[0036] This invention also provides the use of the oligopeptide in the preparation of drugs for colorectal cancer.

[0037] This invention also provides a drug for treating colorectal cancer, comprising the oligopeptide.

[0038] Example 1

[0039] This embodiment uses Sipunculus nudus as the research target. The first experiment studied the optimal enzymatic hydrolysis conditions of Sipunculus nudus. The second experiment used LC-MS / MS (liquid chromatography-tandem mass spectrometry) technology to analyze the enzymatic hydrolysate of Sipunculus nudus obtained under the first experimental conditions to determine the polypeptide components contained therein.

[0040] 1. Experimental Materials

[0041] Experiment 1 used fresh, palatable Sipunculus nudus, purchased from the Zhanjiang Seafood Market in Guangdong Province. After removing sand and other impurities and cleaning thoroughly, the worms were turned inside out to remove internal organs and body fluid. They were then rinsed thoroughly again, dried completely until their weight no longer decreased, and then packaged and stored at -20℃ for later use. Experiment 2 used the protein hydrolysate obtained from the optimal enzymatic hydrolysis method in Experiment 1 for LC-MS / MS analysis, ensuring that the hydrolysate was sent for analysis within 48 hours.

[0042] 2 Experimental Methods

[0043] 2.1 Screening of hydrolytic enzymes and hydrolysis conditions derived from Sipunculus nudus oligopeptides.

[0044] The prepared *Sipunculus nudus* (PE) bodies were ground into powder, then mixed thoroughly with double-distilled water to obtain a suspension with a material-to-liquid ratio of 1:4. The pH was adjusted (optimal pH values ​​were selected from 6.5, 7, 7.5, 8, and 9.5). The temperature was adjusted (optimal temperatures were selected from 37℃, 45℃, 50℃, and 55℃). Different enzymes (optimal hydrolytic enzymes were selected from 7 types of hydrolytic enzymes: neutral protease, alkaline protease, trypsin, bromelain, papain, animal protein hydrolase, and flavor protease) were added at 0.5% (mass fraction) and subjected to isothermal enzymatic hydrolysis at different temperatures for 4 hours. The enzymes were inactivated at 100℃. After cooling to room temperature, the mixture was centrifuged at 10000 r / min and 4℃ for 20 minutes. The pH was adjusted to neutral with NaOH (1M) and HCl (1M), and then adjusted to neutral every 1 hour for 3 consecutive times. The final enzymatic hydrolysate was stored at -20℃ for later use.

[0045] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging rate is an important indicator for evaluating the ability of substances (such as antioxidants) to scavenge free radicals, and is commonly used in the determination of antioxidant activity in food, pharmaceuticals, and cosmetics. DPPH is a stable organic free radical, appearing deep purple in solution. When antioxidants react with DPPH free radicals, they neutralize the free radicals by donating hydrogen atoms or electrons, causing the solution color to lighten (from purple to yellow). The scavenging rate, calculated from changes in absorbance, reflects the proportion of free radicals neutralized, indicating the sample's antioxidant capacity. In this study, the optimal oligopeptide extraction method was determined based on the optimal oligopeptide content and DPPH free radical scavenging rate. The optimal proteolytic enzyme and hydrolysis method were screened to determine the optimal oligopeptide preparation scheme.

[0046] 2.2 Determination of water-soluble protein content from oligopeptide solution derived from Sipunculus nudus var. chinensis.

[0047] The soluble protein content was determined using the BCA (Bicinchoninic Acid Assay) protein concentration assay kit from Beyotime Biotechnology Co., Ltd. After incubating the reaction solution with the reagents in a 60°C water bath for 30 minutes, the absorbance was measured at a wavelength of 562 nm. Note that absorbance measurement should be completed within 10 minutes after color development to avoid precipitation or discoloration of the complex. A standard curve should be prepared using standard proteins of known concentrations for each experiment, and parallel experiments should be performed to reduce error.

[0048] 2.3 Determination of DPPH free radical scavenging rate of oligopeptides derived from Sipunculus nudus.

[0049] Dissolve DPPH powder in anhydrous ethanol or methanol (final concentration 0.2 mM) and store in the dark. Add 500 μl of the enzymatic hydrolysate to 500 μl of 0.2 mM DPPH solution, maintain room temperature at 25°C, and mix thoroughly by shaking under complete darkness. After mixing, let stand for 30 min. Take the supernatant and measure the absorbance (detection wavelength 517 nm), record as A0. Next, take 500 μl of the above DPPH solution and 500 μl of distilled water, mix thoroughly, and take 500 μl of the standard solution and 500 μl of anhydrous ethanol, mix thoroughly, and measure the absorbance at a wavelength of 517 nm to create a standard curve. Finally, plot the curves by the clearance rate of samples with different concentrations and calculate the half-maximal inhibitory concentration (IC50). 50 IC 50 The smaller the size, the stronger the antioxidant capacity.

[0050] 2.4 Sample Pretreatment of Oligopeptide Enzymatic Hydrolysate Derived from Sipunculus nudus

[0051] 2.4.1 Pretreatment of peptide samples <1K (1000 Daltons)

[0052] Dissolve the sample in dd H2O, transfer the dissolved sample to a 1 kD ultrafiltration tube, and centrifuge at 12000 rcf for 10 min at 4℃. Collect oligopeptide samples smaller than 1 kD in a 1.5 mL EP tube. Add DTT solution to dilute the sample to a target final concentration of 10 mmol / L. Place the tube in a 56℃ water bath, ensuring the liquid level is completely below the water bath level, and reduce in the dark for 1 h. After complete reduction, add IAA solution to a target final concentration of 50 mmol / L, and continue the reaction in the dark for 40-50 min. Finally, desalt the sample using a self-packed desalting column. After complete desalting, place the tube in a 45℃ vacuum centrifuge to evaporate the solvent until the weight no longer decreases.

[0053] 2.4.2 Sample pretreatment of 1K-3K peptides

[0054] Dissolve the sample in ddH2O, transfer the dissolved sample to a 1 kD ultrafiltration tube, and centrifuge at 12000 rcf for 10 min at 4℃. Collect oligopeptides larger than 1 kD and transfer them to a 3 kD ultrafiltration tube, centrifuge at 12000 rcf for 10 min at 4℃. Collect oligopeptide samples smaller than 3 kD in a 1.5 mL EP tube. Add DTT solution to dilute to a target final concentration of 10 mmol / L. Place the tube in a 56℃ water bath, ensuring the liquid in the tube is completely below the liquid level in the water bath, and reduce at a constant temperature in the dark for 1 h. After complete reduction, add IAA solution to a target final concentration of 50 mmol / L, and continue the reaction in the dark for 40-50 min. Finally, desalt using a self-packed desalting column. After complete desalting, place the tube in a 45℃ vacuum centrifuge to evaporate the solvent completely until the weight no longer decreases.

[0055] 2.4.3 Pretreatment of 3K-10K peptide samples

[0056] Dissolve the sample in ddH2O, transfer the dissolved sample to a 3kD ultrafiltration tube, and centrifuge at 2000 rcf for 10 min at 4℃. Collect oligopeptides larger than 3 kD and transfer them to a 10 kD ultrafiltration tube, centrifuge at 12000 rcf for 10 min at 4℃. Collect oligopeptide samples smaller than 10 kD in a 1.5 mL EP tube. Add DTT solution to dilute to a target final concentration of 10 mmol / L. Place the tube in a 56℃ water bath, ensuring the liquid in the tube is completely below the liquid level in the water bath, and reduce in the dark for 1 h. After complete reduction, add IAA solution to a target final concentration of 50 mmol / L, and continue the reaction in the dark for 40-50 min. Finally, desalt using a self-packed desalting column. After complete desalting, place the tube in a 45℃ vacuum centrifuge to evaporate the solvent until the weight no longer decreases.

[0057] 2.5 LC-MS / MS Detection of Samples Treated with Oligopeptide Enzymatic Hydrolysate Derived from Sipunculus nudus

[0058] 2.5.1 Capillary Liquid Chromatography Conditions

[0059] (1) Pre-column: The condition parameters are 300 μm id × 5 mm, RPLC C18, 5 μm, 100 Å;

[0060] (2) Analytical column: The conditions are 150 μm id×150 mm, RPLC C18, 1.9 μm, 100 Å;

[0061] (3) Mobile phase A: 0.1% analytical grade formic acid was used;

[0062] (4) Mobile phase B: 0.1% analytical grade formic acid, 80% ACN;

[0063] (5) Flow rate: 600 nL / min;

[0064] (6) Analysis time for each component: 66 min.

[0065] Table 1. Gradient elution separation effect in chromatographic analysis

[0066]

[0067] 2.5.2 Mass spectrometry conditions: divided into primary mass spectrometry parameters and secondary mass spectrometry parameters.

[0068] Table 2. Primary and Secondary Mass Spectrometry Parameters

[0069]

[0070] 2.6 Database Retrieval

[0071] The raw mass spectrometry files were retrieved from the target protein database using MaxQuant (1.6.2.10), and the retrieval parameters are shown in Table 3.

[0072] Table 3 Database Retrieval Parameters

[0073]

[0074] 2.7 Data Processing and Analysis

[0075] The experiment was repeated three times. The experimental data were statistically analyzed using SPSS 21.0. Depending on the data type, ANOVA, t-test, or linear regression were used. P < 0.05 was considered statistically significant.

[0076] 3. Experimental Results

[0077] 3.1 Screening results of oligopeptide enzymatic digestion experiments derived from Sipunculus nudus.

[0078] The results showed that various proteases could be used to hydrolyze Sipunculus nudus to obtain a certain amount of enzymatic polypeptides, and the protein content of each hydrolysate met the testing standards, indicating satisfactory levels. Further analysis of the free radical scavenging ability of the hydrolysates revealed that all hydrolysates possessed a certain capacity to scavenge free radicals. Figure 1 It was found that the neutral protease hydrolysis yielded the highest polypeptide content (12.40 mg / mL), and its DPPH free radical scavenging rate was also significantly increased, reaching 68.02%. Furthermore, the flavor protease hydrolysis also showed outstanding polypeptide content and free radical scavenging ability, at 11.26 mg / mL and 60.56%, respectively. Compared with the other five groups, the hydrolysates of *Sipunculus nudus* obtained by neutral and flavor protease hydrolysis showed the highest polypeptide content and free radical scavenging rate, with the neutral protease exhibiting the best effect; all differences were statistically significant (P<0.05). Subsequent experiments will use neutral protease as the tool enzyme for the hydrolysis of PE, and further experiments will be conducted on the hydrolysates.

[0079] 3.2 Separation effect of total ion chromatogram (TIC)

[0080] After molecular weight fractionation (<1kDa, 1–3kDa, 3–10kDa), the total ion chromatogram showed that the peaks of each component were sharp and the baseline was stable, indicating that molecular sieve and gradient elution program (4%→95% B phase, 66 min) can effectively separate oligopeptides of different molecular weights. Among them, the fraction with FW<1kDa was detected with high abundance signal in the peak group with retention time of 15 min-25 min.

[0081] 3.3 Database search results and analysis of high-scoring peptides

[0082] The raw mass spectrometry results obtained from LC-MS / MS analysis were analyzed using MaxQuant software (1.6.2.10) to obtain the resolution results of potential peptides. Among them, there were 172 peptides with a molecular weight <1 kDa, 196 peptides with a molecular weight of 1 kDa-3 kDa, and 16 peptides with a molecular weight of 3 kDa-10 kDa. Based on the peptide scores, 17 peptides with a molecular weight <1 kDa were selected and listed in Table 4. Considering that peptides with a molecular weight <1 kDa are more likely to enter cells and exert their effects, this embodiment selected the peptides listed in Table 4 for synthesis and subsequent research on their anti-colorectal cancer effects and mechanisms.

[0083] Table 4. High-molecular-weight peptides with a molecular weight <1kDa

[0084]

[0085] Example 2

[0086] 1. Experimental Materials

[0087] HcoEpiC cells (normal human colonic epithelial cells), DLD-1 cells (human colorectal adenocarcinoma cells), and HCT-15 cells (human colon cancer cells) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). Following the amino acid sequences of oligopeptides derived from *Sipunculus nudus* obtained in Part I, a batch of 17 oligopeptides with a purity >99.5% were synthesized by Jier Biochemical (Shanghai) Co., Ltd., batch number 1014659. 5-Fluorouracil abcam, catalog number: AB142387, purity >98%, CAS number 51-21-8.

[0088] 2. Preparation methods of main reagents in the experimental section

[0089] 2.1 High-glucose complete medium (Dulbecco's Modified Eagle Medium, DMEM)

[0090] Remove high-glucose DMEM basal medium, inactivated FBS, and antibiotics from a 4°C freezer. Ensure all items and procedures are sterile before preparation. Add 445 mL of high-glucose DMEM basal medium to a sterile container. Using a pipette, add 50 mL of 10% (v / v) inactivated FBS (pre-sterilized and inactivated) fetal bovine serum to the DMEM high-glucose medium. Add 5 mL of 1% antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL), seal tightly, shake well, and check the pH and osmotic pressure to obtain the final high-glucose DMEM complete medium. Detailed records and management of the medium should be maintained, including preparation date, composition, volume, and usage history. Simultaneously, transfer an appropriate amount of medium to a 48-well plate and incubate in a constant temperature incubator at 37°C (5% CO2). After 48 hours, examine the 48-well plate under a microscope for colony growth. If no colonies, other microorganisms, or impurities are present, the plate can be used for subsequent cell culture.

[0091] 2.2 Non-sterile PBS buffer

[0092] Prepare a water bath and sterilized measuring cylinder, magnetic stirrer, rotor, 1000 mL glass bottle and 2000 mL beaker, glass rod, etc. Pour one packet of PBS powder into the beaker, add RO water to a final volume of 2000 mL, stir with a glass rod in a 60℃ water bath, then place on a magnetic stirrer and continue stirring thoroughly for 30 minutes. Let stand for 10 minutes, then stir again until the PBS powder is completely dissolved. Bring the volume to a final volume of 2000 mL. Use a pH meter to check and adjust the pH of the PBS to between 7.2 and 7.4. Pour the solution into two 1000 mL glass bottles, label them, and store at room temperature until needed.

[0093] 2.3 Oligopeptide 659 stock solution (100 mM)

[0094] Accurately weigh 20 mg of 659 powder into a sterile EP tube, add DMSO in a clean bench and shake until the 659 powder is completely dissolved, prepare an oligopeptide solution with a final concentration of 100 mM, dispense 100 μL into sterile EP tubes, and store at -20°C for later use.

[0095] 2.4 Preparation of 5-FU stock solution (10 mM)

[0096] Accurately weigh 13.01 mg of 5-FU powder using sterile weighing paper and an analytical balance (accuracy 0.01 mg) and place it into a sterile centrifuge tube. Add the weighed 5-FU powder to 10 mL of sterile DMSO and vortex for approximately 2-5 minutes until completely dissolved. Filter the solution through a 0.22 μm sterile syringe filter into a sterile centrifuge tube. Finally, aliquot the filtered mother liquor into sterile 1 mL EP tubes and store at -20°C for later use.

[0097] 3 Experimental Methods

[0098] 3.1 In vitro cell experiments

[0099] The experimental cells (DLD-1, HCT-15 and HcoEpiC) were revived and passaged, and the effects of 17 oligopeptides (200 μM) on the proliferation of CRC cells were screened using the MTT assay.

[0100] Seventeen peptides were treated with 200 µM solutions of 17 colorectal cancer DLD-1 cells, HCT-15 cells, and HcoEpiC cells, respectively. After 48 h, the effects of the peptides on the proliferation of DLD-1, HCT-15, and HcoEpiC cells were detected using the MTT assay. The results showed (see...). Figure 2 (A) Most peptides, at a concentration of 200 µM, promoted the proliferation of normal colorectal HcoEpiC cells, except for peptides 661, 662, 663, 828, 830, 835, and 836. Peptides exhibited different opposing effects on colorectal DLD-1 and HCT-15 cells; some significantly inhibited proliferation, while others significantly promoted it. Figure 2 As shown in Figure B, peptides 690, 830, 831, 834, 835, and 836 significantly promoted the proliferation of DLD-1 cells, while peptides 659 and 655 inhibited their proliferation. Among these, peptide 659 showed the most significant inhibitory effect (P < 0.05). Peptides 628, 633, 655, 656, 657, 658, 660, 661, and 663 showed no significant difference compared to the control group (P > 0.05). Figure 2 As shown in the results, only peptides 659 and 662 exhibited significant inhibitory effects on HCT-15 cells, with peptide 659 showing the most pronounced inhibitory effect (P < 0.05). Other peptides at this concentration and treatment time did not show significant inhibitory effects on HCT-15 cells, with no statistically significant differences (P > 0.05). This indicates that after 48 hours of drug treatment, peptide 659 at the 200 μM concentration group significantly inhibited the growth of both types of CRC tumor cells compared to the 0 μM group, a statistically significant effect (P < 0.05), and its effect was the most prominent among the 17 peptides.

[0101] 3.2 Effects of MTT assay on the proliferation of oligopeptides alone and in combination with 5-FU on CRC cells

[0102] The above experimental results verified that oligopeptide 659 has a significant anti-CRC effect. To improve the clinical application value of oligopeptides, and considering previous literature reports that colorectal cancer cells are sensitive to 5-FU, as well as previous laboratory results verifying that 5-FU has a certain inhibitory effect on CRC at a concentration of 5 μM, DLD-1 cells and HCT-15 cells were treated with different concentrations of 659 (50, 100, 150, 200 μM) in combination with 5-FU (5, 10, 20 μM) for 48 h. The effects and interactions of different concentrations of 659 alone and different concentrations of 5-FU in combination were observed.

[0103] The results show that ( Figure 3 For DLD-1 cells, compared with the control group, both 5-FU (5, 10, 20 μM) and 659 (50, 100, 150, 200 μM) alone significantly inhibited DLD-1 cells (P < 0.05). The inhibitory effect of both drugs on tumor cells increased with increasing drug concentration, with only a decreasing trend between the same drugs (P > 0.05). However, 659 alone was less effective than 5-FU (P < 0.05). The combined use of both drugs showed a stronger anti-DLD-1 cell effect than either drug alone, and the difference was statistically significant (P < 0.05). The inhibitory effect of the combined use of both drugs also tended to increase with increasing drug concentration, but the difference between the combined use groups was not statistically significant (P > 0.05). For HCT-15 cells, compared with the control group, 659 (50, 100, 150, 200 μM) alone showed an inhibitory trend, but the difference was not statistically significant (P>0.05). When 5-FU was used alone, the inhibitory effect at a concentration of 5 μM was not statistically significant (P>0.05), but the differences at 10 μM and 20 μM were statistically significant and highly statistically significant (P<0.05, P<0.01). However, when the two drugs were used in combination, compared with the control group, the lowest combined dose (5 μM-5-FU+50 μM-659) showed a significant increase in inhibitory effect, and the effect was stronger than that of the single drugs (P<0.05). The inhibitory effect of the combination increased with increasing concentration. However, when 5-FU was used at doses of 10 μM and 20 μM, there was no difference in inhibitory effect between the combination of 5-FU and 659 (150, 200 μM). The results suggest that the combined use of the two drugs can significantly enhance the anti-tumor effect, but there was no significant difference in the inhibitory effect between the two groups when 659 (150, 200 μM) and 5-FU (5, 10, 20 μM) were used together (P>0.05).

[0104] To improve the clinical application value of 659, reduce the dosage of 5-FU, and decrease the adverse reactions of chemotherapy drugs, based on the above experimental results, 5 μM 5-FU and 659 (50, 100, 150, 200 μM) were used in combination to observe their effects on two types of CRC cells at different treatment times (24h, 48h, 72h).

[0105] The result is Figure 4 It was found that, for DLD-1 cells, after 24 hours of drug treatment, there was no significant difference in the inhibitory effect of 5-FU and 659 at various concentrations compared with the control group (P>0.05). However, all four groups treated with the two drugs together showed significant inhibitory effects on DLD-1 cells (P<0.05). The inhibitory effect of the two drugs on tumor cells increased with increasing drug concentration in the range of 659 (50, 100, 150 μM), but 659 did not show a better effect at 200 μM. However, there was no significant difference between the combined treatment groups (P>0.05). After 48 hours of drug treatment, compared with the control group, 5-FU alone significantly inhibited tumor cells, but 659 showed no inhibitory effect on tumor cells at concentrations (50, 100, 150 μM) (P>0.05). At a concentration of 200 μM, it significantly inhibited tumor cells. However, all four groups using the two drugs in combination showed a more significant inhibitory effect on DLD-1 cells, superior to the 5 μM dose group using 5-FU alone (P<0.05), but there was no significant difference between the combination groups (P>0.05). After 72 hours of treatment, compared with the control group, except for 659 alone at a concentration of 50 μM which did not show a significant inhibitory effect, all other groups significantly inhibited tumor cell growth. The inhibitory effects of the four combination groups were similar to those of the 5-FU alone group, showing no significant trend of superior inhibition compared to 5-FU alone. This suggests that the combination of the two drugs has the best effect within the 48-hour timeframe.

[0106] For HCT-15 cells, after 24 hours of drug treatment, compared with the control group, 5-FU and 659 alone at a concentration of 200 μM significantly inhibited tumor cells (P < 0.05), while other concentrations of 659 showed no significant inhibitory effect (P > 0.05). All four groups treated with the two drugs in combination showed significant inhibitory effects on HCT-15 cells (P < 0.05). The inhibitory effect of the two drugs in combination on tumor cells was significantly better than that of 5-FU alone in the 659 (100, 150 μM) range, but the effect was optimal at a concentration of 150 μM. There was no significant difference between the combination groups (P > 0.05). After 48 hours and 72 hours of drug treatment, compared with the control group, the trend was consistent with that at 24 hours, but overall, the drugs showed significant inhibitory effects on tumor cells at 48 hours, but 659 showed the best inhibitory effect at a concentration of 150 μM. The results suggest that the combined use of the two drugs has the best effect on HCT-15 cells within a 48-hour timeframe.

[0107] Depend on Figure 3 and Figure 4 The results showed that for both types of CRC cells, the combined use of the two drugs was significantly more effective than the single use of any one drug. Based on the above results and data on the effects of the drugs on tumor cells, the research group calculated the synergistic index (q) of the combined use of the two drugs using the rate method, and analyzed the results. A q value < 0.85 indicates that drugs A and B have antagonistic effects; a q value between 0.85 and 1.15 indicates that the effects of drugs A and B are additive; and a q value > 1.15 indicates that drugs A and B have a synergistic effect.

[0108] Table 5. q values ​​of different concentrations of 5-FU combined with 659 on DLD-1 and HCT-15 cells

[0109]

[0110] The results (Table 5) showed that when the two drugs were used together, the q-value for each concentration combination was greater than 0.85, indicating good additive or synergistic effects. Specifically, for DLD-1 cells, the q-values ​​of 659 at concentrations of 150 μM and 200 μM combined with 5 μM 5-FU were the highest, indicating the best synergistic effect. A similar trend was observed for HCT-15 cells, but the q-value was slightly lower than that for DLD-1 cells. This invention further validated the q-values ​​of different drug combinations at different treatment times using 5 μM 5-FU and different concentrations of oligopeptide 659. The results showed that the q-value was the highest and the effect was best when the duration of action of the drug and colorectal cancer cells was 48 h, with the effect on DLD-1 cells being superior to that on HCT-15 cells.

[0111] Based on the above analysis, it was found that compared with the use of single drugs (oligopeptide 659 or 5-FU alone), the combination therapy of 150 μM oligopeptide 659 and 5 μM 5-FU significantly inhibited the proliferation of colorectal cancer cells at the 48-hour time point, which has better research value and good clinical significance. This combination therapy will be used in subsequent experiments.

[0112] 3.3 Analysis of information related to oligopeptide 659 using the CAS database

[0113] The Chemical Abstracts Service (CAS) database (via the SciFinder platform) was used to detect and analyze information related to the selected preferred peptides (molecular weight <1 kDa). This was used to confirm their potential for further experimental development. If the selected preferred peptides are not recorded in the CAS system, they are considered innovative drugs with significant development potential. If the selected preferred peptides are recorded in CAS but have not yet been reported for pharmacodynamics or other biological effects, they are also considered innovative drugs worthy of further development. If the selected preferred peptides have already been widely reported in anti-tumor research, their development potential is limited. The specific detection steps are as follows: Log in to CAS SciFinder → Search by structure → Click “DrawStructure”, upload the 659 oligopeptide structure file → Select “Exact Search” (exact match) → Add restrictions → Molecular weight range: Set Molecular Weight <1000 Da → Type: Check “Peptides” → Click “Search” to obtain the results.

[0114] The amino acid sequence "GDEAQSKRG" was input and analyzed using the CAS SciFinder database. While a similar oligopeptide chain was detected in the CAS database, a short peptide based on 9 amino acids obtained using this experimental method has not yet been reported. Furthermore, a literature search in the CAS database revealed only four patents mentioning peptides similar to oligopeptide 659 in vaccine development based on bioinformatics predictions. All of these patents relied on bioinformatics methods to predict oligopeptide-ligand binding, aiming to predict the application of this peptide in cancer diagnosis, oligopeptide epitope identification, and vaccine development; none mentioned cell or animal experiments.

[0115] Example 3

[0116] Study on the in vitro synergistic inhibition of colorectal cancer cells by oligopeptide 659 and 5-FU

[0117] 1. Cell resuscitation and cell passage.

[0118] 2. EdU staining experiment to observe the effect of oligopeptide 659 alone and in combination with 5-FU on the proliferation of two types of CRC cells; crystal violet staining to observe the effect of oligopeptide 659 alone and in combination with 5-FU on the clonogenic assay of the two types of CRC cells; γ-H2AX fluorescence staining experiment to observe the effect of oligopeptide 659 alone and in combination with 5-FU on DNA damage in the nucleus of the two types of CRC cells; Western blotting to explore the effect of oligopeptide 659 alone and in combination with 5-FU on the expression of proliferation proteins in the two types of CRC cells; flow cytometry to detect the effect of oligopeptide 659 alone and in combination with 5-FU on the cell cycle of the two types of CRC cells; flow cytometry to detect the effect of 659 alone and in combination with 5-FU on CRC cell apoptosis; Hoechst 33342 staining to observe the effect of oligopeptide 659 alone and in combination with 5-FU on apoptosis of CRC colorectal cancer cells; Western blotting to explore the effect of oligopeptide 659 alone and in combination with 5-FU on the expression of apoptosis proteins in CRC cells.

[0119] 3. Experimental Results

[0120] 3.1 Results of Edu fluorescence staining of two types of CRC cells by oligopeptide 659 in synergistic effect with 5-FU

[0121] Depend on Figure 5 As can be seen, the fluorescence colors displayed in different groups of cells differed. The control group showed stronger fluorescence, mainly concentrated within the cell nucleus, exhibiting a large-scale sheet-like distribution, indicating significant and rapid cell proliferation. The oligopeptide 659 group showed scattered light spots, but the intensity, brightness, and density were significantly lower than the normal group, suggesting that oligopeptide 659 can significantly inhibit cell proliferation. Under the same conditions, the number of light spots in the 5-FU monotherapy group was further reduced, and the light spots in the oligopeptide 659 and 5-FU combined therapy group were almost invisible, suggesting that the combined use of the two drugs had a stronger inhibitory effect on cells. Next, within a fixed-size random field of view, the relative intensity of fluorescence was calculated by determining the ratio of positive cells to total cells. Compared with the blank control group, the cell proliferation ratio was significantly reduced in the 5-FU and oligopeptide 659 monotherapy groups and the 5 μM 5-FU + 150 μM oligopeptide 659 combined therapy group (P < 0.05), with the combined use of the two drugs showing the best effect. The results for both CRC cell types were consistent, suggesting that oligopeptide 659 combined with 5-FU significantly inhibited the proliferation of DLD-1 and HCT-15 cells (P<0.05). This also indicates that oligopeptide 659 may exert its anti-cancer effect by inhibiting cancer cell proliferation, and that oligopeptide 659, in synergy with 5-FU, can produce a good synergistic effect against colorectal cancer cells.

[0122] 3.2 Results of the clone formation experiment of oligopeptide 659 on two types of CRC cells

[0123] The results of the plate cloning method showed that ( Figure 6 In the control group of both CRC cell lines, the number of clones was dense, the morphology was irregular, the boundaries were blurred, and the cells were loosely arranged and connected. The number and quantity of clones in the oligopeptide 659 single-drug group were significantly reduced compared to the blank group, but increased compared to the 5-FU group. The group using oligopeptide 659 and 5-FU in combination had the fewest clones, were sparse, and had clear boundaries, indicating that the combined use of the two drugs had a significant killing effect on both groups of CRC cells. The colony formation rate was calculated as (number of clones formed / number of inoculated cells) × 100%. The results showed that compared to the blank control group, the 5 μM 5-FU group, and the 150 μM oligopeptide 659 group, the colony formation rate of the 5 μM 5-FU + 150 μM oligopeptide 659 combined-drug group was significantly lower. Compared to the 5 μM 5-FU group, the 150 μM oligopeptide 659 single-drug group was less effective than the 5 μM 5-FU single-drug group, and the difference was statistically significant (P < 0.05). However, the inhibitory effect of the 5 μM 5-FU + 150 μM oligopeptide 659 combination therapy on colorectal cells was significantly better than that of the 5 μM 5-FU alone group (P<0.05). The drugs had consistent effects on the two types of CRC cells, suggesting that the 5 μM 5-FU + 150 μM oligopeptide 659 combination therapy had a significant inhibitory effect on CRC cell proliferation (P<0.05).

[0124] 3.3 DNA damage effects of oligopeptide 659 combined with 5-FU on two types of CRC cells

[0125] In this embodiment, the experimental results of γ-H2AX fluorescence staining method are shown (see...). Figure 7 In the control group, almost no fluorescent focal points were observed in the cells, indicating that no significant DNA double-strand breaks occurred. However, in the other three groups treated with the corresponding drugs, obvious γ-H2AX fluorescent focal points (foci) were observed in the corresponding cells. The oligopeptide 659 monotherapy group had the fewest fluorescent focal points, followed by the 5-FU monotherapy group, while the combined drug group showed a dense distribution of green fluorescent dots. The relative fluorescence intensity of each group was calculated based on the ratio of the fluorescence range under the microscope to the total field of view. Figure 7 The results showed that, compared with the blank control group, the 5 μM 5-FU treatment group, the 150 μM oligopeptide 659 monotherapy group, and the 5 μM 5-FU + 150 μM oligopeptide 659 combination treatment group significantly increased the DNA damage rate of DLD-1 and HCT-15 cells (P<0.05), with the combination treatment group showing the most significant effect. This suggests that the combination of the two drugs has the strongest and most effective DNA damage effect on CRC cells.

[0126] 3.4 Oligopeptide 659 and its combination with 5-FU arrested the cell cycle of two types of CRC cells at the G0 / G1 phase.

[0127] Normal cell populations exhibit relatively stable proportions across different phases of the cell cycle (G0 / G1, S, and G2 / M). Most non-proliferating cell lines have a high proportion of cells in the G0 / G1 phase, typically reaching 50%-70%, with 10%-30% in the S phase and 10%-20% in the G2 / M phase. In tumor cells, due to their proliferative characteristics, the proportion of cells in the G0 / G1 phase is reduced. In this study, flow cytometry was used to detect the proportions of CRC cells treated with two different drugs for 48 hours in each treatment group. The results showed that compared to the control group (see...),... Figure 8 Both the oligopeptide 659 monotherapy group and the 5-FU monotherapy group could arrest both types of CRC cells in the G0 / G1 phase. The proportion of cells arrested in the G0 / G1 phase was even higher in the oligopeptide 659 and 5-FU combined therapy group, and the difference was statistically significant (P < 0.01). The results also showed that oligopeptide 659 and 5-FU had a more significant effect on DLD-1 in both types of CRC cells, arresting the vast majority of tumor cells in the G0 / G1 phase. Finally, the distribution of cells in different cell cycles was determined by calculating the percentage of cells in each cell cycle per unit quantity. The results showed that compared with the blank control group, the percentage of cells in the G0 / G1 phase was significantly increased in the 5 μM 5-FU group, the 150 μM oligopeptide 659 monotherapy group, and the 5 μM 5-FU + 150 μM oligopeptide 659 combined therapy group (P < 0.05). Compared with the blank control group, the number of G0 / G1 phase cells in the 5-FU monotherapy group and the oligopeptide 659 monotherapy group was significantly increased, but decreased compared with the combined treatment group, and the difference was statistically significant (P<0.05). The proportion of G0 / G1 phase cells in the treatment groups was significantly increased, while the proportion of S phase and G2 / M phase cells was decreased, indicating that CRC cell proliferation was significantly inhibited.

[0128] 3.5 Results of flow cytometry analysis of the apoptosis-promoting effects of oligopeptide 659 and its combination with 5-FU on two types of CRC cell lines.

[0129] In this experiment, flow cytometry was used to detect the effects of different drug treatments on two types of CRC cells 48 h later. Figure 9The results showed that after treatment with oligopeptide 659 and 5-FU on both types of CRC cells, the number of early apoptotic cells in the lower right quadrant and late apoptotic cells in the upper right quadrant significantly increased, especially in the group treated with oligopeptide 659 and 5-FU in combination (P < 0.01). The apoptosis rate was calculated by dividing the number of apoptotic cells into the percentage of total cells, and the apoptosis rate of each group was obtained. The results showed that, compared with the blank control group, the number of apoptotic cells in DLD-1 cells was significantly increased in both the 5-FU alone group and the 5 μM 5-FU + 150 μM oligopeptide 659 combination group (P < 0.05), with the combination group showing the most significant effect (P < 0.01). However, for HCT-15 cells, compared with the blank control group, the 5 μM 5-FU treatment group and the 5 μM 5-FU + 150 μM oligopeptide 659 combination treatment group significantly promoted HCT-15 apoptosis (P < 0.01), but the effect of the 150 μM oligopeptide 659 alone group was not significantly different from the control group (P > 0.05), suggesting that the drug is more effective against DLD-1. Combined with the experimental results in 3.4, it can be concluded that oligopeptide 659 and synergistic 5-FU have a dual effect of arresting the cell cycle and promoting apoptosis in colorectal cells, inducing both cell cycle arrest and apoptosis, indicating a stronger anti-tumor effect.

[0130] 3.6 Results of Hoechst 33342 assay on the apoptosis-promoting effects of oligopeptide 659 and its combination with 5-FU on two types of CRC cells

[0131] Depend on Figure 10 The Hoechst 33342 assay detected apoptosis in cellular cells of two CRC cells after 48 h of treatment with different drugs. Consistent results were observed in both CRC cell types compared to the control group. The number of apoptotic cells was significantly increased in the 5-FU-only group, the oligopeptide 659-only group, and the combined 5 μM 5-FU + 150 μM oligopeptide 659 group (P < 0.05), with significantly enhanced fluorescence intensity compared to the control group. The combined 5 μM 5-FU + 150 μM oligopeptide 659 group showed the most significant apoptotic effect on CRC cells, with the highest fluorescence intensity, and the difference was statistically significant (P < 0.05). This suggests that oligopeptide 659 and 5-FU can synergistically act on living tumor cells and accelerate their apoptosis.

[0132] 3.7 Oligopeptide 659 and its combination with 5-FU promote the apoptosis-inducing effects of two types of CRC cells.

[0133] BAX is a major pro-apoptotic member of the BCL-2 family; therefore, increased levels of BAX suggest that the drug may promote cell apoptosis. BCL-2, Cyclin D1, and PCNA are major inhibitors of apoptosis; decreased levels of these proteins suggest increased apoptosis. Figure 11-12 The results showed that, compared with the control group, the 5-FU monotherapy group, the oligopeptide 659 monotherapy group, and the 5 μM 5-FU + 150 μM oligopeptide 659 combination therapy group all promoted changes in the content of apoptosis-related proteins in CRC cells, significantly reduced the expression of proteins BCL-2, Cyclin D1, and PCNA, and significantly increased the expression of BAX protein, with the 5 μM 5-FU + 150 μM oligopeptide 659 combination therapy group showing the most significant effect (P < 0.001). Therefore, it can be concluded that the combination of 659 and 5-FU can significantly induce apoptosis in colorectal cancer cells, and the effect is significantly better than that of oligopeptide 659 monotherapy and 5-FU monotherapy (P < 0.05).

[0134] Example 4

[0135] Study on the synergistic effect of oligopeptide 659 with 5-FU against colorectal cancer in vivo

[0136] 1. Experimental Materials

[0137] 1.1 Experimental Subjects

[0138] Human colon cancer cell line, DLD-1 cells (human colorectal adenocarcinoma cells), was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). NOD-SCID female mice (4 weeks old) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Animal qualification certificate SCXK (Guangdong) 2023-0067, experimental environment qualification certificate SYXK (Guangdong) 2021-0262.

[0139] 2. Experimental Methods

[0140] 2.1 Establishing a mouse xenograft model using DLD-1

[0141] Five-week-old female NOD-SCID mice underwent shaving at the injection site. Using electric clippers, the hair was pushed against the direction of hair growth to expose the skin at the injection site in the right axilla of the mouse, with a hairless exposed area of ​​approximately 1.5cm × 1.5cm.

[0142] Dilute the original cell suspension to 4 × 10⁻⁶. 7 The inoculation concentration was set at 1 cell / mL to complete the preparation of the inoculation cell suspension, which was then placed on ice for inoculation.

[0143] After stabilizing the mouse to expose its right axillary region and ensure the skin is taut, wipe the exposed area with a cotton ball containing 75% alcohol. Slowly insert a syringe 1 cm subcutaneously and gently and evenly inject 0.1 mL of the prepared DLD-1 cell suspension (4*10⁴ cells per 10⁻¹²) subcutaneously. 6 (One per needle). A small bump will appear at the injection site. After injection, pause for 3 seconds and then rotate the needle out.

[0144] 2.2 Experimental Procedure of the Anti-tumor Effect of Oligopeptide 659 Combined with 5-FU on Mouse Xenografts

[0145] A xenograft model was constructed using DLD-1. Tumors were then induced in mice, and when the tumors grew to 50-100 mm... 3 Subsequently, the tumor-forming mice were randomly divided into 8 groups, and each group was treated with the corresponding drug. 5-FU was treated with the commonly used antitumor dose of mice, 10 mg / kg. Oligopeptide 659 was treated with the mouse dose converted from cell dose, 100 mg / kg as the medium dose. The low dose was 50 mg / kg, which was twice the dose, and the high dose was 200 mg / kg, which was twice the dose, as shown in Table 6.

[0146] Table 6. Grouping of Oligopeptide 659 combined with 5-FU against colorectal cancer in mice

[0147]

[0148] Daily drug administration and tumor and weight tracking were performed according to grouping and body weight. Once the tumor reached a certain size (tumor weight not exceeding 10% of the mouse's body weight and average tumor diameter not exceeding 20 mm), blood was collected, the mice were euthanized, and the tumor tissue and other organ tissues were completely dissected for corresponding histopathological and serological analysis. The drug administration period for this experiment was 12 days. The recorded data and observations included the following: daily tracking and recording of mouse body weight and tumor size. After drug administration, NOD-SCID mice were photographed with tumors. Mice were euthanized by cervical dislocation with blood collected from the eyeballs. Tumor tissue was collected, weighed, and photographed. The tumor was divided in half, labeled, and one half was placed in a 10 mL centrifuge tube and fixed with paraformaldehyde; the other half was placed in a cryovial, immersed in liquid nitrogen for 5 min, and then stored at -80℃. The mouse heart, liver, spleen, lungs, and kidneys were dissected, weighed, and the liver and kidneys were transferred to 10 mL paraformaldehyde centrifuge tubes for later use.

[0149] HE staining, immunohistochemical detection, TUNEL staining, biochemical analysis of blood, and Western blot detection of mouse tumor tissue were performed.

[0150] 3. Experimental Results

[0151] 3.1 Successful establishment of a DLD-1 subcutaneous xenograft mouse model

[0152] To investigate the anti-colorectal tumor activity of the oral sipunculid peptide 659, NOD-SCID mice were first injected subcutaneously into the right axilla with DLD-1 colorectal tumor material. Approximately one week later, tumor formation was clearly visible in the right axilla of the mice, with a diameter of 5-8 mm and a volume of 100-150 mm². 3， Within the experimental requirements, the tumor formation rate was 100%, indicating that the mouse xenograft model was successfully established. Subsequently, the tumor-bearing mice were randomly divided into 8 groups and treated with the corresponding drugs for observation.

[0153] 3.2 Effects of Oligopeptide 659 Combined with 5-FU on the Growth of DLD-1 Tumors

[0154] Mice were administered the drug once daily at a fixed time, and tumor growth was recorded, with body weight measured every 3 days. The experiment was terminated after 12 days of administration when a difference in tumor diameter was observed between groups. Blood was collected from the mice's eyes via enucleation, followed by complete tumor removal and sampling of major organs. Tumor growth was observed in the mice. The positive control drug 5-FU group showed significant inhibition of tumor growth after 11 days (12 doses). The low-dose oligopeptide 659 group showed tumor inhibition, but not as strong as 5-FU alone. With increasing dose, the tumor-inhibiting effect of medium and high-dose 659 treatment groups increased, with high-dose 659 showing the strongest inhibitory effect on tumor growth. When 659 and 5-FU were used together, 659 showed good tumor-inhibiting effects even at low doses, superior to 5-FU alone. Furthermore, the synergistic anti-tumor effect between 659 and 5-FU increased with increasing 659 dose (see...). Figure 13 ).

[0155] After subcutaneous injection of tumor cells into mice, tumor growth was monitored daily, and the results were analyzed using a tumor volume curve. Figure 14 As can be seen, compared with the control group, 659 showed an inhibitory effect on tumor growth, and the effect increased with increasing dose. The inhibitory effect of high-dose 659 on tumors was similar to that of 5-FU alone. When 659 and 5-FU were used together, 659 showed better effect than 5-FU alone even at low doses, and the effect increased with increasing dose of 659. At high doses, a significant anti-tumor effect was observed (P<0.05).

[0156] Depend on Figure 15It can be seen that the control group had the darkest color, while the colors of all the drug-treated groups lightened to varying degrees. The group using a combination of high-dose 659 and 5-FU had the lightest color, followed by the group using a combination of medium-dose 659 and 5-FU, the group using a combination of low-dose 659 and 5-FU, the group using 5-FU alone, the group using a combination of high-dose 659 alone, the group using a combination of medium-dose 659 alone, and the group using a combination of low-dose 659 alone. Trends and... Figure 13 , 14 The results were consistent, indicating that 659 has an inhibitory effect on tumor growth, and the inhibitory effect is positively correlated with the drug concentration, showing a good dose-response relationship. The inhibitory effect on tumors is more obvious when used in combination with 5-FU, and the combination also reflects the dose-response relationship of 659.

[0157] from Figure 13-15 The three sets of figures show that the drugs have a consistent effect on tumors, and all of them demonstrate that the combination of oligopeptide 659 and 5-FU has a good synergistic effect. Furthermore, the anti-tumor effect increases with the increase of the dose of oligopeptide 659, and the dose-response relationship is obvious. High doses of oligopeptide 659 synergistically with 5-FU have a significant anti-tumor effect.

[0158] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of an oligopeptide derived from Sipunculus nudus and 5-fluorouracil in the preparation of a drug for treating colorectal cancer, characterized in that, The amino acid sequence of the oligopeptide is shown in SEQ ID NO.

6.

2. The application according to claim 1, characterized in that, The molar ratio of the oligopeptide to 5-fluorouracil is (10~50):

1.

3. A drug for treating colorectal cancer, characterized in that, Includes oligopeptides as shown in SEQ ID NO.6 and 5-fluorouracil.

4. The drug according to claim 3, characterized in that, The molar ratio of the oligopeptide to 5-fluorouracil is (10~50):

1.

5. The use of oligopeptides as shown in SEQ ID NO.6 in the preparation of drugs for treating colorectal cancer.

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