Small peptide for preventing and treating colon cancer and application thereof

Abstract

The invention provides a small peptide for preventing and treating colon cancer and application thereof, and belongs to the technical field of biological medicine. Through structural simulation and functional verification, a polypeptide capable of specifically inhibiting G3BP1 lactylation in colon cancer cells in a targeted manner is screened out. The polypeptide inhibits the lactic acid modification level of G3BP1 by simulating a G3BP1 lactic acid recognition site and competitively combining with lactic acid enzyme, so that the G3BP1 mediated autophagy process is blocked, and finally proliferation and migration of colon cancer cells are inhibited. In-vitro cell experiments (including cell proliferation, migration and autophagy level analysis) and in-vivo transplantation tumor animal experiments prove that the oligopeptide can significantly inhibit growth and tumor formation of colon cancer cells. The discovery provides a new strategy and candidate drug molecules for molecular targeted therapy of colon cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a small peptide for preventing and treating colon cancer and application thereof. BACKGROUND

[0002] Colorectal cancer (CRC) is one of the malignant tumors of the digestive system with high morbidity and mortality worldwide. With the westernization of dietary structure, changes in lifestyle and population aging, the incidence of colon cancer is showing a significant upward trend. The occurrence of colon cancer is closely related to multiple factors and multiple stages of molecular pathological processes, including metabolic reprogramming, inflammatory response, abnormal activation of signal pathways and imbalance of autophagy regulation. The current main clinical treatment methods include surgical resection, chemotherapy, radiotherapy and targeted therapy, but the recurrence rate is high, the drug resistance is strong, and the prognosis is poor, so it is urgent to explore new molecular targets and intervention strategies.

[0003] In the tumor microenvironment, the high glycolysis of tumor cells leads to the generation of a large amount of lactic acid. Lactic acid is not only a metabolic waste, but also can act as a signal molecule to participate in the lactylation of proteins. Studies have shown that protein lactylation plays a key role in regulating gene transcription, autophagy, immune escape and other processes, and has become a frontier field 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 such as regulating mRNA stability, signal transduction and cellular stress response. Many studies have shown that G3BP1 is highly expressed in various tumors and promotes tumor proliferation and migration. Our previous studies have found that G3BP1 in colon cancer cells is significantly lactylated, and lactylated G3BP1 can activate the autophagy process, thereby providing energy and survival advantage for tumor cells and promoting the sustained growth of tumors.

[0005] However, there is currently a lack of effective intervention means for the lactylation regulation of G3BP1. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a small peptide for preventing and treating colon cancer and application thereof.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a short peptide for targeting inhibition of G3BP1 lactylation, and the amino acid sequence of the short peptide is shown in SEQ ID NO: 8.

[0008] Preferably, the concentration of the short peptide is 5-20 μM.

[0009] The application also provides the short peptide in the preparation of a drug for targeted inhibition of G3BP1 lactylation.

[0010] The application also provides the short peptide in the preparation of a drug for prevention and treatment of colon cancer.

[0011] The application also provides the short peptide in the preparation of a drug for inhibition of growth and tumor formation of colon cancer cells.

[0012] Compared with the prior art, the application has the following beneficial effects: The application screens a polypeptide capable of specifically targeting G3BP1 in colon cancer cells through structure simulation and function verification. The polypeptide can simulate a key site of G3BP1 lactylation, competitively bind to lactylation enzyme, inhibit the modification level of G3BP1 lactylation, block the autophagy process mediated by G3BP1, and finally inhibit the proliferation and migration of colon cancer cells.

[0013] In the research of the application, through in vitro cell experiments (including cell proliferation, migration and autophagy level analysis) and in vivo transplanted tumor animal experiments, it is verified that the short peptide can significantly inhibit the growth and tumor formation of colon cancer cells. This finding provides a new strategy and candidate drug molecule for molecular targeted therapy of colon cancer. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a polypeptide for inhibiting G3BP1 lactylation; Figure 2 is screening of G3BP1 lactylation inhibition ability and autophagy inhibition ability of the polypeptide (wherein A is the lactylation inhibition ability, and B is the autophagy inhibition ability); Figure 3 is the lactylation inhibition ability and autophagy inhibition ability of different concentrations of K393-Peptide-8 (wherein A is the lactylation inhibition ability, and B is the autophagy inhibition ability); Figure 4 is the design of the control peptide; Figure 5 is the LCMS result of CtrL-peptide; Figure 6 is the LCMS result of K393-peptide; Figure 7 is the LCMS result of K393R-peptide; Figure 8 is the influence of the polypeptide on cell proliferation activity; Figure 9 is the influence of the polypeptide on cell proliferation ability; Figure 10are tumor transplantation experiment in vivo to verify the anti-tumor effect of polypeptide (wherein A is tumor picture, B is tumor weight, C is tumor growth curve). DETAILED DESCRIPTION

[0015] The present application provides a short peptide for targeting inhibition of G3BP1 lactylation, and the amino acid sequence of the short peptide is shown in SEQ ID NO: 8, and specifically VQKVLSNRPIMFR. In the present application, the concentration of the short peptide is preferably 5-20 μM, and further preferably 10-20 μM.

[0016] The present application also provides the use of the short peptide in the preparation of a drug for targeting inhibition of G3BP1 lactylation.

[0017] The present application also provides the use of the short peptide in the preparation of a drug for preventing and treating colon cancer.

[0018] The present application also provides the use of the short peptide in the preparation of a drug for inhibiting the growth and tumor formation of colon cancer cells.

[0019] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0020] Example 1

[0021] 1. Design and synthesis of polypeptide for inhibiting G3BP1 lactylation

[0022] (1) Polypeptide design

[0023] Based on the G3BP1 protein domain information and the prediction results of its lactylation sites (combined with PDB database structure analysis and literature reports), taking the key lactylation site K393 of G3BP1 as the core, eight candidate short peptide sequences were designed and synthesized, which 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), K393-peptide-7 (EPVQKVLSNRPIM, SEQ ID NO: 7) and K393-peptide-8 (VQKVLSNRPIMFR, SEQ ID NO: 8) (as shown in Table 1).Figure 1 Each polypeptide comprises a conserved region similar to the amino acid environment around the G3BP1 lactylation site, aiming to mimic the G3BP1 lactylation recognition structure, thereby competitively binding to the lactylation enzyme and inhibiting the lactylation modification of G3BP1.

[0024] (2) Polypeptide synthesis

[0025] The polypeptide sequence was synthesized in the order of C-terminal to N-terminal by using the in vitro Fmoc solid-phase method. Through amino acid connection reaction, peptide chain connection reaction and peptide chain cleavage treatment, the dried crude peptide was prepared. The crude peptide obtained by chemical synthesis was purified and detected by using high performance liquid chromatograph and high resolution mass spectrometer (purification conditions: mobile phase A was 0.065% (V / V) trifluoroacetic acid aqueous solution dissolved in pure water, mobile phase B was trifluoroacetic acid solution with a volume fraction of 0.05% (v / v) dissolved in acetonitrile, total flow rate: 1 ml / min, detection wavelength: 220 nm).

[0026] Experimental results: the purity of the synthesized polypeptides 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 were all greater than 95%.

[0027] 2. Screening of polypeptides highly efficient in inhibiting G3BP1 lactylation

[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] The experimental results are shown in Figure 3 K393-peptide at 20 μM has the best inhibitory effect on G3BP1 lactylation Figure 3 A). By determining the LC3 and p62 levels of cells, it is found that the LC3 and p62 levels of cells treated with K393-peptide at 20 μM are significantly increased, indicating that the autophagy level of cells is significantly inhibited.

[0033] 4. Effect of polypeptides on the function of colon cancer cells

[0034] (1) Design and synthesis of mutant polypeptides

[0035] As shown in Figure 4 , the key lactylation site lysine (K) of the screened high-efficiency short peptide (K393-peptide) was mutated to arginine (R) to construct K→R mutant polypeptide (denoted as K393R-peptide, VQRVLSNRPIMFR, SEQ ID NO: 9). At the same time, the threading peptide part (HLYVSPWGG, SEQ ID NO: 10) in K393-peptide was synthesized alone as a control peptide (CtrL-peptide). K393-peptide, K393R-peptide and CtrL-peptide were synthesized by in vitro Fmoc solid phase method, and the crude peptides obtained by chemical synthesis were purified and detected by high performance liquid chromatograph and high resolution mass spectrometer.

[0036] The experimental results of LCMS of K393-peptide, K393R-peptide and CtrL-peptide are shown in Figures 5-7 , and their purities are 96.62%, 95.50% and 98.07%, respectively.

[0037] (2) Cell proliferation experiment (CCK-8 method)

[0038] LS174T cells were inoculated in 96-well plates (3×10 3 cells per well), and Control-peptide (CtrL-pep), K393R-peptide (K393R-pep) and K393-peptide (K393-pep) groups were set up. After 24, 48 and 72 h of culture at 20 μM, CCK-8 solution (10 μL per well) was added, and the absorbance was measured at 450 nm after 1 h of incubation at 37°C. The cell survival rate was calculated and the concentration-time curve was drawn to evaluate the inhibitory effect of polypeptides on cell proliferation.

[0039] The experimental results are shown inFigure 8 As shown in Figure 11, compared with Ctrl-peptide and K393R-Peptide, K393-Peptide can significantly inhibit the proliferation activity of cells.

[0040] (3) Transwell assay

[0041] Digest and collect the logarithmic growth phase LS174T cells, wash and resuspend with serum-free medium, adjust the cell concentration to 5 x 105cells / mL. 100 μL of cell suspension (5 x 105cells) is inoculated on the upper chamber of the Transwell chamber with a pore size of 8 μm. Add 600 μL of medium containing chemotactic agent (10% fetal bovine serum) to the lower chamber. Incubate the chamber in a 37°C, 5% CO2incubator for 24 hours. After incubation, gently wipe off the cells in the upper chamber that have not migrated with a cotton swab. Then, fix the cells that have crossed the membrane with 4% paraformaldehyde for 15 minutes, and stain with crystal violet staining solution for 10 minutes. Finally, rinse and dry the chamber, randomly select 5 fields of view under a microscope, and count and statistically analyze the cells that have crossed the membrane. 5 4 Digest and collect the logarithmic growth phase LS174T cells, wash and resuspend with serum-free medium, adjust the cell concentration to 5 x 105cells / mL. 100 μL of cell suspension (5 x 105cells) is inoculated on the upper chamber of the Transwell chamber with a pore size of 8 μm. Add 600 μL of medium containing chemotactic agent (10% fetal bovine serum) to the lower chamber. Incubate the chamber in a 37°C, 5% CO2incubator for 24 hours. After incubation, gently wipe off the cells in the upper chamber that have not migrated with a cotton swab. Then, fix the cells that have crossed the membrane with 4% paraformaldehyde for 15 minutes, and stain with crystal violet staining solution for 10 minutes. Finally, rinse and dry the chamber, randomly select 5 fields of view under a microscope, and count and statistically analyze the cells that have crossed the membrane.

[0042] Experimental results: As shown in Figure 11, compared with Ctrl-peptide and K393R-Peptide, K393-Peptide can significantly inhibit the proliferation activity of cells. Figure 9

[0043] 6. In vivo anti-tumor effect of polypeptide

[0044] Construct LS174T nude mouse subcutaneous tumor model, and randomly divide into groups (Ctrl-pep, K393R-Peptide and K393-Peptide) when the tumor volume reaches about 100 mm³. Inject intratumorally at a set dose (5 mg / kg) once every two days for 1 week of continuous administration. Measure the tumor volume (V = 0.5 x L x W²) regularly and record the body weight changes. After the experiment, weigh and take pictures of the tumors. The results are used to evaluate the effect of short peptides on the inhibition of tumor growth in vivo, and to analyze the inhibitory effect of polypeptides on tumor growth.

[0045] Experimental results: As shown in Figure 11, compared with Ctrl-peptide, K393R-Peptide and K393-Peptide can significantly inhibit the size (A in Figure 11), weight (B in Figure 11) and tumor proliferation rate (C in Figure 11) of tumor cells. Figure 10 Figure 10 Figure 10 Figure 10

[0046] ​​​​​​From the above examples, the application screens out a polypeptide capable of specifically targeting G3BP1 in colon cancer cells through structure simulation and function verification. The polypeptide can simulate the key site of G3BP1 lactation, competitively bind to lactation enzyme, inhibit the lactation modification level, block the autophagy process mediated by G3BP1, and finally inhibit the proliferation and migration of colon cancer cells. Moreover, it can also significantly inhibit the growth and tumor formation of colon cancer cells.

[0047] The above only describes the preferred embodiments of the application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered within the protection scope of the application.

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 that targets and inhibits G3BP1 lactation.

4. 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.

5. The use of the short peptide according to claim 1 or 2 in the preparation of a drug for inhibiting the growth of colon cancer cells and tumor formation.

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