Small peptides for preventing and treating colon cancer and use thereof
By designing short peptides that target and inhibit G3BP1 lactation, mimicking its key sites and competitively binding to lactase, the problem of colon cancer cell proliferation and migration was solved, achieving a significant tumor inhibition effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA AGRI UNIV
- Filing Date
- 2025-10-27
- Publication Date
- 2026-07-28
AI Technical Summary
Current technologies lack effective interventions for G3BP1 lactation in colon cancer cells, making it difficult to effectively inhibit tumor cell proliferation and migration.
A short peptide was designed and synthesized that targets and inhibits G3BP1 lactation. By mimicking the key lactation sites of G3BP1, it competitively binds to lactases and inhibits the lactation modification of G3BP1, thereby blocking the autophagy process and inhibiting the proliferation and migration of colon cancer cells.
This short peptide can significantly inhibit the growth of colon cancer cells and tumor formation, providing a new molecular targeted therapy strategy. Its effectiveness has been verified through in vitro cell experiments and in vivo animal experiments with xenografts.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and in particular relates to a small peptide for the prevention and treatment of colon cancer and its application. Background Technology
[0002] Colorectal cancer (CRC) is one of the leading causes of death and morbidity among digestive system malignancies worldwide. With the Westernization of dietary patterns, changes in lifestyle, and population aging, the incidence of colorectal cancer is showing a significant upward trend. The development of colorectal cancer is closely related to multifactorial and multi-stage molecular pathological processes, including metabolic reprogramming, inflammatory responses, abnormal activation of signaling pathways, and imbalances in autophagy regulation. Current clinical treatments mainly include surgical resection, chemotherapy, radiotherapy, and targeted therapy; however, these methods suffer from high recurrence rates, strong drug resistance, and poor prognosis, necessitating the exploration of new molecular targets and intervention strategies.
[0003] In the tumor microenvironment, the high glycolysis of tumor cells leads to the production of large amounts of lactic acid. Lactic acid is not only a metabolic waste product, but also a signaling molecule involved in the lactation of proteins. Existing research has shown that protein lactation plays a key role in regulating gene transcription, autophagy, and immune escape, making it a cutting-edge area of tumor metabolism research in recent years.
[0004] G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein 1) is a stress granule-associated protein with multiple functions, including regulating mRNA stability, signal transduction, and cellular stress responses. Multiple studies have shown that G3BP1 is highly expressed in various tumors and promotes tumor proliferation and migration. Our previous research found that G3BP1 in colon cancer cells exhibits significant lactation modification. Lactic acidified G3BP1 can activate autophagy, thereby providing tumor cells with energy and a survival advantage, promoting sustained tumor growth.
[0005] However, there is currently a lack of effective intervention methods to target the lactation regulation of G3BP1. Summary of the Invention
[0006] In view of this, the purpose of the present invention is to provide a small peptide for the prevention and treatment of colon cancer and its application.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a short peptide that targets and inhibits G3BP1 lactation, the amino acid sequence of which is shown in SEQ ID NO:8.
[0008] Preferably, the concentration of the short peptide is 5~20 μM.
[0009] This invention also provides the application of the aforementioned short peptide in the preparation of drugs that target and inhibit G3BP1 lactation.
[0010] The present invention also provides the use of the aforementioned short peptide in the preparation of drugs for the prevention and treatment of colon cancer.
[0011] The present invention also provides the use of the aforementioned short peptide in the preparation of a drug for inhibiting the growth of colon cancer cells and tumor formation.
[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention, through structural simulation and functional verification, screened a polypeptide that can specifically target G3BP1 in colon cancer cells. This polypeptide can mimic the key lactation site of G3BP1, competitively bind to lactase, inhibit the level of G3BP1 lactation modification, thereby blocking the G3BP1-mediated autophagy process and ultimately inhibiting the proliferation and migration of colon cancer cells.
[0013] In this study, both in vitro cell experiments (including analysis of cell proliferation, migration, and autophagy levels) and in vivo animal experiments with xenografts confirmed that this short peptide can significantly inhibit the growth of colon cancer cells and tumor formation. This discovery provides a new strategy and candidate drug molecule for molecular targeted therapy of colon cancer. Attached Figure Description
[0014] Figure 1 It is a polypeptide that inhibits G3BP1 lactation; Figure 2 The screening of peptides for G3BP1 lactation inhibition and autophagy inhibition (where A is lactation inhibition and B is autophagy inhibition). Figure 3 The lactation inhibition capacity and autophagy inhibition capacity of different concentrations of K393-Peptide-8 are shown (where A is the lactation inhibition capacity and B is the autophagy inhibition capacity). Figure 4 This is the design of a control peptide; Figure 5 These are the LCMS results for CtrL-peptide; Figure 6 These are the LCMS results for K393-peptide; Figure 7 These are the LCMS results for K393R-peptide; Figure 8 It refers to the effect of polypeptides on cell proliferation activity; Figure 9 It refers to the effect of polypeptides on cell proliferation capacity; Figure 10The in vivo xenograft experiment verifies the anti-tumor effect of the peptide (where A is a tumor image, B is tumor weight, and C is a tumor growth curve). Detailed Implementation
[0015] This invention provides a short peptide that targets and inhibits G3BP1 lactation. The amino acid sequence of the short peptide is shown in SEQ ID NO:8, specifically VQKVLSNRPIMFR. In this invention, the concentration of the short peptide is preferably 5-20 μM, more preferably 10-20 μM.
[0016] This invention also provides the application of the aforementioned short peptide in the preparation of drugs that target and inhibit G3BP1 lactation.
[0017] The present invention also provides the use of the aforementioned short peptide in the preparation of drugs for the prevention and treatment of colon cancer.
[0018] The present invention also provides the use of the aforementioned short peptide in the preparation of a drug for inhibiting the growth of colon cancer cells and tumor formation.
[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0020] Example 1
[0021] 1. Design and synthesis of peptides that inhibit G3BP1 lactation
[0022] (1) Peptide design
[0023] Based on the G3BP1 protein domain information and its lactation site prediction results (combined with PDB database structure analysis and literature reports), eight candidate short peptide sequences were designed and synthesized with the key lactation site K393 of G3BP1 as the core. These sequences are: K393-peptide-1 (DSEPVQKVLSNRP, SEQ ID NO:1), K393-peptide-2 (FDDSEPVQKVLSN, SEQ ID NO:2), K393-peptide-3 (EPVQKVLSNRPIM, SEQ ID NO:3), K393-peptide-4 (VQKVLSNRPIMFR, SEQ ID NO:4), K393-peptide-5 (DSEPVQKVLSNRP, SEQ ID NO:5), K393-peptide-6 (FDDSEPVQKVLSN, SEQ ID NO:6), and K393-peptide-7 (EPVQKVLSNRPIM, SEQ ID NO:4). NO:7) and K393-peptide-8 (VQKVLSNRPIMFR, SEQ ID NO:8) (as shown in NO:7) and K393-peptide-8 (VQKVLSNRPIMFR, SEQ ID NO:8) Figure 1 (As shown). Each peptide contains a conserved region similar to the amino acid environment surrounding the G3BP1 lactation site, aiming to mimic the G3BP1 lactation recognition structure, thereby competitively binding to lactases and inhibiting the lactation modification of G3BP1.
[0024] (2) Polypeptide synthesis
[0025] The polypeptide sequence was synthesized in vitro using the Fmoc solid-phase method, following the C-terminus to N-terminus sequence. A dried crude peptide was prepared through amino acid ligation, peptide chain ligation, and peptide chain shearing. The chemically synthesized crude peptide was purified and analyzed using high-performance liquid chromatography coupled with high-resolution mass spectrometry (purification conditions: mobile phase A was a 0.065% (v / v) trifluoroacetic acid aqueous solution dissolved in pure water; mobile phase B was a 0.05% (v / v) trifluoroacetic acid solution dissolved in acetonitrile; total flow rate: 1 ml / min; detection wavelength: 220 nm).
[0026] Experimental results: The purity of the synthesized peptides K393-peptide-1, K393-peptide-2, K393-peptide-3, K393-peptide-4, K393-peptide-5, K393-peptide-6, K393-peptide-7 and K393-peptide-8 was all greater than 95%.
[0027] 2. Screening of peptides that efficiently inhibit G3BP1 lactation
[0028] A stable human colorectal adenocarcinoma LS174T cell line expressing Flag-G3BP1 was constructed (purchased from the Cell Bank of the Chinese Academy of Sciences). Cells were cultured in 10cm dishes for 24 hours to achieve a cell density of 70%–80%. Eight designed and synthesized candidate peptides (final concentration set at 10 μM) were added, and cells were incubated at 37°C and 5% CO2 for 24 hours before collection. Total protein was extracted using RIPA lysis buffer (containing a mixture of 1× protease inhibitor and deacetylase inhibitor). After quantification, equal amounts of protein were used for immunoprecipitation (IP) experiments: 10 μl of Flag-agarose gel was added to each sample, and the mixture was incubated overnight at 4°C to enrich the Flag-G3BP1 complex. After washing, loading buffer was added, and the mixture was boiled for lysis and then separated by SDS-PAGE. The cells were then transferred to PVDF membranes, and the G3BP1 lactation level was detected using anti-lactylation antibody, with anti-G3BP1 used as an internal control. To further evaluate the regulatory effect of peptides on autophagy, Western blot analysis was performed on the expression changes of LC3-I / LC3-II and p62 in the same batch of cell lysates. By comparing the lactation inhibition rate of different peptides on G3BP1 and the expression trends of autophagy-related proteins, candidate short peptides that most significantly inhibit G3BP1 lactation and effectively suppress autophagy were identified for subsequent mechanism of action studies.
[0029] Experimental results: such as Figure 2 As shown. Compared to the control group (Ctrl) and seven other short peptides, K393-Peptide-8 significantly inhibited the lactation of G3BP1 (…). Figure 2 In addition to A), the levels of LC3-II / LC3-I and p62 in cells treated with K393-Peptide-8 were significantly increased. Figure 2 (B in the text). Therefore, among the eight designed peptides, K393-Peptide-8 showed the strongest ability to inhibit G3BP1 lactation and the fusion of autophagosomes and lysosomes in cancer cells, and was used for subsequent experiments.
[0030] 3. Dose-response relationship of peptide inhibition of G3BP1 lactation
[0031] A stable LS174T cell line expressing Flag-G3BP1 was constructed. The optimal short peptide K393-Peptide (K393-Peptide-8), selected through screening, was used to treat LS174T cells at different concentration gradients (0, 5, 10, 20 μM). After 24 h of treatment, changes in G3BP1 lactation and autophagy levels were detected (Western Blot analysis was used to detect G3BP1 lactation, LC3-II / LC3-I, and p62 expression). The optimal concentration range for the peptide to inhibit lactation and autophagy activity was determined, providing a reference for subsequent functional experiments.
[0032] Experimental results: such as Figure 3 As shown, 20 μM K393-Peptide showed the best inhibitory effect on G3BP1 lactation. Figure 3 (A) By measuring the LC3 and p62 levels of cells, it was found that the cell lines treated with 20 μM K393-Peptide had significantly increased LC3 and p62 levels, indicating that it significantly inhibited the level of autophagy.
[0033] 4. Effects of polypeptides on the function of colon cancer cells
[0034] (1) Design and synthesis of mutant peptides
[0035] like Figure 4 As shown, using the screened high-efficiency short peptide (K393-peptide) as a template, the key lactation site lysine (K) was mutated to arginine (R) to construct a K→R mutant peptide (denoted as K393R-peptide, VQRVLSNRPIMFR, SEQ ID NO:9). Simultaneously, the transmigration peptide portion (HLYVSPWGG, SEQ ID NO:10) of K393-peptide was synthesized separately as a control peptide (CtrL-peptide). K393-peptide, K393R-peptide, and CtrL-peptide were synthesized in vitro using the Fmoc solid-phase method. The crude peptides obtained from chemical synthesis were purified and analyzed using high-performance liquid chromatography coupled with high-resolution mass spectrometry.
[0036] Experimental results: The LCMS results for K393-peptide, K393R-peptide, and CtrL-peptide are as follows: Figures 5-7 As shown, their purities are 96.62%, 95.50%, and 98.07%, respectively.
[0037] (2) Cell proliferation experiment (CCK-8 assay)
[0038] LS174T cells were seeded in 96-well plates (3 × 10⁶ cells per well). 3 Cells were divided into Control-peptide (CtrL-pep), K393R-peptide (K393R-pep), and K393-peptide (K393-pep) groups. After culturing at 20 μM for 24, 48, and 72 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance was measured at 450 nm. Cell viability was calculated, and concentration-time curves were plotted to evaluate the inhibitory effect of the peptides on cell proliferation.
[0039] Experimental results: such as Figure 8 As shown, compared with ctrl-peptide and K393R-Peptide, K393-Peptide can significantly inhibit cell proliferation activity.
[0040] (3) Transwell assay
[0041] LS174T cells in logarithmic growth phase were digested and collected, washed and resuspended in serum-free medium, and the cell concentration was adjusted to 5 × 10⁻⁶. 5 Cells / mL. Add 100 μL of cell suspension (5 × 10⁶ cells / mL). 4 Cells were seeded in the upper chamber of an 8 μm pore size Transwell chamber. 600 μL of culture medium containing a chemotactic agent (10% fetal bovine serum) was added to the lower chamber. The chamber was incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, unmigrated cells were gently wiped away from the upper layer of the chamber with a cotton swab. Cells that had migrated through the membrane were then fixed with 4% paraformaldehyde for 15 minutes and stained with crystal violet for 10 minutes. Finally, the chamber was rinsed and dried, and five fields of view were randomly selected under a microscope to count and statistically analyze the cells that had migrated through the membrane.
[0042] Experimental results: such as Figure 9 As shown, compared with ctrl-peptide and K393R-Peptide, K393-Peptide can significantly inhibit cell migration ability.
[0043] 6. In vivo antitumor effects of peptides
[0044] A subcutaneous xenograft tumor model of LS174T mice was constructed. Once the tumor volume reached approximately 100 mm³, the mice were randomly assigned to three groups (Ctrl-pep, K393R-Peptide, and K393-Peptide). The tumors were injected intratumorally at a predetermined dose (5 mg / kg) every two days for one week. Tumor volume (V = 0.5 × L × W²) and body weight changes were recorded periodically. At the end of the experiment, the tumors were weighed and photographed. The results were used to evaluate the efficacy of the short peptides in inhibiting tumor growth in vivo and to analyze the inhibitory effect of the peptides on tumor growth.
[0045] Experimental results: such as Figure 10 As shown, compared to Ctrl-peptide, K393R-peptide and K393-peptide, they can significantly inhibit the size of tumor cells ( Figure 10 A in the middle), weight ( Figure 10 (B) and tumor proliferation rate ( Figure 10 (C in the middle).
[0046] As demonstrated by the above embodiments, this invention, through structural simulation and functional verification, has screened out a polypeptide that can specifically target G3BP1 in colon cancer cells. This polypeptide can mimic the key lactation site of G3BP1, competitively bind to lactase, inhibit its lactation modification level, thereby blocking the G3BP1-mediated autophagy process, ultimately inhibiting the proliferation and migration of colon cancer cells, and also significantly inhibiting the growth of colon cancer cells and tumor formation.
[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A short peptide that targets and inhibits G3BP1 lactation, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO:
8.
2. The short peptide according to claim 1, characterized in that, The concentration of the short peptide is 5~20 μM.
3. The use of the short peptide according to claim 1 or 2 in the preparation of a drug for the prevention and treatment of colon cancer.
4. The use of the short peptide according to claim 1 or 2 in the preparation of a medicament for inhibiting the growth of colon cancer cells and the formation of colon cancer tumors.