A polypeptide for enhancing the sensitivity of gastric cancer to cisplatin chemotherapy and its application

Abstract

The present invention provides a polypeptide for enhancing the sensitivity of gastric cancer to cisplatin chemotherapy and its application, belonging to the technical field of biomedicine. The polypeptide of the present invention is derived from the 2nd - 17th positions of the precursor protein thymosin β4 (TYB4). Through experiments, it is found that the polypeptide is closely related to drug resistance in gastric cancer cells. After being treated with the polypeptide of the present invention, the cisplatin-treated drug-resistant gastric cancer cells show significant induction of apoptosis in drug-resistant gastric cancer cells, inhibit the activity and migration of drug-resistant gastric cancer cells, and have no obvious toxic and side effects on normal gastric mucosal cells. The polypeptide of the present invention has the advantages of low immunogenicity, strong tissue permeability, low production cost, and being easy to modify to enhance in vivo stability and biological activity. Therefore, its application in the anti-tumor field has broad clinical application prospects and provides a new direction for the development of gastric cancer treatment drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a polypeptide for enhancing the sensitivity of gastric cancer to cisplatin chemotherapy and its application. Background Art

[0002] A tumor refers to a new growth formed by the hyperplasia of local tissue cells under the action of various tumorigenic factors. According to the cell characteristics of the new growth and the degree of harm to the body, tumors are divided into two major categories: benign tumors and malignant tumors. Malignant tumors can be divided into carcinomas and sarcomas. A carcinoma refers to a malignant tumor derived from epithelial tissue, and a sarcoma refers to a malignant tumor that occurs in mesenchymal tissues, including fibrous connective tissue, fat, muscle, blood vessels, bone, and cartilage tissues. For example, a malignant tumor formed by the epithelial cells of the large intestine mucosa is called colorectal mucosa epithelial carcinoma, abbreviated as colorectal cancer, and a malignant tumor formed by the skin epithelium is called skin epithelial carcinoma, abbreviated as skin cancer, and so on. During the occurrence and progression of tumors, special biological functions are formed, including unlimited proliferation, evasion of growth inhibitory genes, inhibition of apoptosis, induction of angiogenesis, and activation of invasion and metastasis.

[0003] Gastric cancer is a malignant tumor with a relatively high incidence rate, ranking fourth among malignant tumors and second among the causes of tumor-related deaths. The chemotherapy regimen based on platinum compounds represented by the combination of surgery and cisplatin (Cisplatin, DDP) is the main means for the treatment of gastric cancer. However, the 5-year survival rate of patients with advanced gastric cancer is still less than 30%. The phenomena of primary resistance and acquired resistance of tumors to cisplatin limit its clinical application, and cisplatin-resistant tumor cells will also develop cross-resistance to other chemotherapy drugs. Therefore, developing factors that can enhance the sensitivity of cisplatin chemotherapy to improve the chemotherapy response rate and minimize side effects can benefit gastric cancer patients during chemotherapy treatment, which has great clinical value.

[0004] Although cisplatin has toxic side effects on the body, due to its broad spectrum and effectiveness in killing tumors, it is still used in many tumors, especially in the treatment of some solid tumors such as lung cancer, gastric cancer, head and neck tumors, ovarian cancer, etc. Cisplatin is a cell cycle non-specific drug. Its mechanism of action is that after entering the cell, the Cl- in the cisplatin molecule is replaced by water molecules to form a hydrate, which reacts with nucleophilic substances in the cell such as nucleic acids and sulfhydryl-containing proteins. The platinum atom binds to the N7 of DNA purine in the form of a covalent bond for intrastrand cross-linking or interstrand cross-linking, inducing double-strand DNA damage and triggering apoptosis. The molecular mechanism of cisplatin-induced drug resistance in gastric cancer is complex, including: ① ATP-binding cassette transporters (ABC) actively transport chemotherapeutic drugs across the membrane. When multi-drug resistance-related protein modulators or chemosensitizers bind to ABC proteins (such as P-glycoprotein, multi-drug resistance-related proteins), it will reduce their efflux of chemotherapeutic drugs and increase the intracellular concentration of chemotherapeutic drugs, thereby enhancing the effect of drugs on tumor cells. Such multi-drug resistance-related protein modulators or chemosensitizers include: calcium channel blockers (verapamil and its derivatives), cyclosporins (cyclosporin A and its derivatives), calmodulin inhibitors (phenothiazine compounds such as triflupromazine), antimalarial drugs (such as quinine), etc.; Currently, the use of such drugs to reverse the multi-drug resistance phenotype of tumor cells and increase the effect of chemotherapeutic drugs on tumor cells has begun to enter clinical trials, but their severe kidney toxicity, liver toxicity, gastrointestinal reactions and nervous system reactions limit their clinical application; ② The detoxification effect of glutathione S-transferase makes chemotherapeutic drugs ineffective, such as acidic GST; ③ Abnormal activity of DNA topoisomerase, such as drugs targeting ToPⅡ, form covalent complexes by binding and cross-linking with DNA, leading to the death of tumor cells. Such drugs include etoposide, teniposide and doxorubicin, etc.; ④ Mutation of the oncogene p53 and apoptosis-related pathways, etc. However, the above chemosensitizing drugs have not achieved satisfactory clinical efficacy and have toxic side effects on normal tissues, especially liver and kidney functions, and thus cannot be widely used clinically.

[0005] At present, the FDA and EMA have approved more than 20 anti-tumor peptides, among which Kyprolis, SomaKitTOC, Lutathera and Gallium Dotatoc Ga 68 have all been marketed. Polypeptides play an important role in anti-tumor effects due to their low toxicity and side effects, high activity, small molecular weight, and easy penetration into tumor cells. For example, Bryostatin 1 is one of the most abundant and well-studied peptides in the Bryostatin family, and it has shown significant anti-tumor activity in phase I trials of patients with malignant melanoma, lymphoma and ovarian cancer. Patent CN03158296.6 discloses a polypeptide ND100 with anti-tumor effects, which is composed of 1Ala, 1Glu, 1Gly, 1Leu, 2Pro, 1Thr, 1Tyr. Patent CN202110866068.8 discloses an anti-tumor polypeptide, which includes MDX1VDQSAVGFEYQGX2TEX3HASQX4GX5TX6X7VQX8EPAPGAPMGX9VTAT or an amino acid sequence having at least 90% sequence identity therewith; wherein, X1, X6, X9 are selected from R or Q; X2, X3, X4, X5, X7, X8 are selected from K or Q.

[0006] Therefore, there is an urgent need to provide a polypeptide with better anti-tumor effects to provide new ideas for the treatment or prevention of tumors. Summary of the Invention

[0007] In view of the above deficiencies, the present invention provides a polypeptide for improving the sensitivity of gastric cancer to cisplatin chemotherapy and its application. The polypeptide of the present invention is derived from positions 2-17 of the precursor protein thymosin β4 (TYB4). Through experiments, it is found that the polypeptide is closely related to the drug resistance of gastric cancer cells and can improve the sensitivity of drug-resistant gastric cancer cells to cisplatin. The drug-resistant gastric cancer cells treated with the polypeptide of the present invention show significant induction of apoptosis of drug-resistant gastric cancer cells after the action of cisplatin, inhibit the activity and migration of drug-resistant gastric cancer cells, and have no obvious toxic and side effects on normal gastric mucosal cells. The polypeptide of the present invention has the advantages of small immunogenicity, strong tissue permeability, low production cost, and easy modification to enhance in vivo stability and biological activity. Therefore, its application in the anti-tumor field has broad clinical application prospects, and there is no report on the use of this polypeptide to improve the sensitivity of gastric cancer to cisplatin chemotherapy at present.

[0008] In order to achieve the above invention purpose, the technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides an anti-tumor polypeptide, and the sequence of the polypeptide is as shown in SEQ ID NO.1.

[0010] Specifically, the polypeptide of the present invention is derived from the amino acid sequence at positions 2 - 17 of its precursor protein Thymosin Beta 4 (TYB4, precursor protein ID: P62328).

[0011] In certain embodiments, through ProtParam analysis, it was found that the half-life of the polypeptide of the present invention in eukaryotic cells is 1.9 h; the aliphatic index is 30.63, and the grand average of hydropathicity (GRAVY) is -1.581, indicating that the polypeptide of the present invention has poor hydrophilicity and strong lipophilicity, and is easily transported into tumor cells by diffusion or endocytosis.

[0012] On the other hand, the present invention provides the use of the above polypeptide in the preparation of anti-tumor drugs.

[0013] Specifically, the drug further comprises a pharmaceutically acceptable carrier.

[0014] More specifically, the carrier is any one or more of a sustained release agent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.

[0015] Specifically, the tumors include, but are not limited to, bladder cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer or testicular cancer.

[0016] More specifically, the tumor is gastric cancer.

[0017] On the other hand, the present invention provides an anti-tumor drug, and the drug comprises the above polypeptide.

[0018] Specifically, the drug further comprises a pharmaceutically acceptable carrier.

[0019] More specifically, the carrier is any one or more of a sustained release agent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.

[0020] Specifically, the tumors include, but are not limited to, bladder cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer or testicular cancer.

[0021] More specifically, the tumor is gastric cancer.

[0022] On the other hand, the present invention provides the use of the above polypeptide in the preparation of a drug for enhancing the chemosensitivity of gastric cancer to cisplatin.

[0023] Specifically, the drug further comprises a pharmaceutically acceptable carrier.

[0024] More specifically, the carrier is any one or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorbent carrier, a surfactant or a lubricant.

[0025] Specifically, the tumors include but are not limited to bladder cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer or testicular cancer.

[0026] More specifically, the tumor is gastric cancer.

[0027] In another aspect, the present invention provides a drug for enhancing the sensitivity of gastric cancer to cisplatin chemotherapy, and the drug comprises the above polypeptide.

[0028] Specifically, the drug further comprises a pharmaceutically acceptable carrier.

[0029] More specifically, the carrier is any one or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorbent carrier, a surfactant or a lubricant.

[0030] Specifically, the tumors include but are not limited to bladder cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer or testicular cancer.

[0031] More specifically, the tumor is gastric cancer.

[0032] In another aspect, the present invention provides the use of the above polypeptide in combination with cisplatin in the preparation of an anti-tumor drug.

[0033] Specifically, the drug further comprises a pharmaceutically acceptable carrier.

[0034] More specifically, the carrier is any one or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorbent carrier, a surfactant or a lubricant.

[0035] Specifically, the tumors include but are not limited to bladder cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer or testicular cancer.

[0036] More specifically, the tumor is gastric cancer.

[0037] In another aspect, the present invention provides an anti-tumor drug, and the drug comprises the above polypeptide and cisplatin.

[0038] Specifically, the drug further comprises a pharmaceutically acceptable carrier.

[0039] More specifically, the carrier is any one or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.

[0040] Specifically, the tumors include but are not limited to bladder cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer or testicular cancer.

[0041] More specifically, the tumor is gastric cancer.

[0042] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0043] (1) The present invention provides an anti-tumor polypeptide, which is derived from positions 2-17 of the precursor protein thymosin β4 (TYB4). It has poor hydrophilicity and strong lipophilicity, and is easy to enter tumor cells through diffusion or endocytosis.

[0044] (2) The present invention firstly discovers that the polypeptide is closely related to drug resistance in gastric cancer cells and can improve the sensitivity of drug-resistant gastric cancer cells to cisplatin. The drug-resistant gastric cancer cells treated with the polypeptide of the present invention show significant induction of apoptosis of drug-resistant gastric cancer cells after the action of cisplatin, inhibit the activity and migration functions of drug-resistant gastric cancer cells, and have no obvious toxic side effects on normal gastric mucosal cells.

[0045] (3) The polypeptide of the present invention has the advantages of low immunogenicity, strong tissue permeability, low production cost, and easy modification to enhance in vivo stability and biological activity. Therefore, its application in the anti-tumor field has broad clinical application prospects and provides new ideas and directions for the development of gastric cancer treatment drugs. Description of the Drawings

[0046] Figure 1 It is a standard curve graph.

[0047] Figure 2 It is a graph of the bioinformatics analysis results of the endogenous differential polypeptide precursor protein.

[0048] Figure 3 It is a graph of the determination results of the endogenous differential polypeptide (*, P < 0.05; **, P < 0.01; #, P > 0.05 vs 1 μM).

[0049] Figure 4 It is a graph of the activity detection results of normal gastric mucosal cells GES-1 (#, P > 0.05 vs Scramble).

[0050] Figure 5 It is for PD-TYB4 2-17 It is a graph of the conservation analysis results of PD-TYB4 in humans and mice.

[0051] Figure 6 Figure showing the migration of SGC7901 / DDP cells (×200).

[0052] Figure 7 Figure showing the apoptosis of SGC7901 / DDP cells. Detailed implementation manners

[0053] The present invention will be further elaborated in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions indicated in the embodiments are generally in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0054] For those not indicating specific techniques or conditions in the embodiments, they are all in accordance with the techniques or conditions described in the literature in the field (such as referring to "Molecular Cloning: A Laboratory Manual", Third Edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press) or in accordance with the product instructions.

[0055] As used herein, the words "a", "an", and "the" mean "at least one" unless otherwise specifically stated.

[0056] When the terms "isolated" and "purified" are used in connection with a substance (such as a polypeptide, antibody, polynucleotide, etc.), it means that the substance is substantially free of at least one substance that may be included in its natural source. For example, an isolated or purified antibody means an antibody that is substantially free of cellular material (such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue source of the protein) or substantially free of chemical precursors or other chemicals during chemical synthesis. The term "substantially free of cellular material" includes preparations of polypeptides in which the polypeptide is separated from the cellular components of the cell (from which the polypeptide is isolated or recombinantly prepared).

[0057] The terms "protein" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. This term applies not only to naturally occurring amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues (e.g., artificial chemical mimics of the corresponding naturally occurring amino acids).

[0058] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are post-translationally modified in cells (e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The phrase "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid (an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group), but has a modified R group or a modified backbone (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to a chemical compound that has a different structure from the common amino acids but a similar function.

[0059] Amino acids can be referred to herein by their well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise expressly specified, the terms "polynucleotide", "oligonucleotide", "nucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably and are analogous to the amino acids referred to by their commonly accepted single-letter codes. Similar to amino acids, they encompass both naturally occurring and non-naturally occurring nucleic acid polymers. A polynucleotide, oligonucleotide, nucleotide, nucleic acid, or nucleic acid molecule can be composed of DNA, RNA, or a combination thereof.

[0060] As used herein, the term "biological sample" refers to a whole organism or a subgroup of its tissues, cells, or components (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, cord blood, urine, vaginal fluid, and semen). "Biological sample" also refers to a homogenate, lysate, extract, cell culture, or tissue culture prepared from a whole organism or a subgroup of its cells, tissues, or components, or a fraction or part thereof. Finally, "biological sample" refers to a culture medium, such as a nutrient broth or gel that has been used to culture an organism, which contains cellular components, such as proteins or polynucleotides.

[0061] The polypeptides or fragments used in this method can be obtained from nature as naturally occurring proteins by conventional purification methods or can be obtained by chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be used for synthesis include:

[0062] (1) Peptide Synthesis, Interscience, New York, 1966;

[0063] (2) "The Proteins", Volume 2, Academic Press, New York, 1976;

[0064] (3) "Peptide Synthesis" (in Japanese), Maruzen Co., 1975;

[0065] (4) "Basics and Experiment of Peptide Synthesis" (in Japanese), Maruzen Co., 1985;

[0066] (5) "Development of Pharmaceuticals" (Volume 2) (in Japanese), Volume 14 (peptide synthesis), Hirokawa, 1991;

[0067] (6) WO99 / 67288; and

[0068] (7) Barany G. and Merrifield R.B., Peptides Volume 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100 - 118.

[0069] Alternatively, any known genetic engineering method for generating polypeptides can be employed to obtain the protein (e.g., Morrison D.A. et al., J Bacteriol. October 1977; 132(1): 349 - 51; Clark - Curtiss J.E. and Curtiss R 3rd. Methods Enzymol. 1983; 101: 347 - 62). For example, a suitable vector is prepared that contains a polynucleotide encoding the protein of interest in an expressible form (e.g., downstream of regulatory sequences including a promoter), transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, by inserting the gene into a vector that expresses foreign genes.

[0070] The medicament of the present invention may comprise conventional pharmaceutical excipients and / or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmotic pressure regulators, buffers and pH regulators. Suitable additives include physiological biocompatible buffers (such as tromethamine hydrochloride), additional chelating agents (such as DTPA or DTPA-bisamide) or calcium chelate complexes (such as calcium DTPA, CaNaDTPA-bisamide), or, optionally, calcium or sodium salts are added (such as calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). The pharmaceutical composition of the present invention can be packaged for use as a liquid, or it can also be freeze-dried.

[0071] For solid compositions, conventional non-toxic solid carriers can be used; for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. of pharmaceutical grade.

[0072] In addition to the above, the medicament may also contain other pharmaceutically active ingredients, as long as they do not inhibit the in vivo function of the polypeptide. For example, the composition may contain chemotherapeutic agents commonly used in the treatment of cancer.

[0073] Example 1: Screening and preparation of anti-tumor polypeptide

[0074] I. Screening of endogenous differential polypeptides in cisplatin-resistant and cisplatin-sensitive gastric cancer tissues

[0075] The Label Free quantitative proteomics method was used to screen for differential endogenous polypeptides in cisplatin-resistant and sensitive gastric cancer tissues.

[0076] The basic process is as follows:

[0077] (1) Polypeptide extraction and sample treatment in cisplatin-resistant and cisplatin-sensitive gastric cancer tissues: Appropriate amounts of cisplatin-resistant and cisplatin-sensitive gastric cancer tissues were ground in liquid nitrogen and then protein lysate was added, and after pipetting and mixing evenly, PMSF with a final concentration of 1 mM, EDTA of 2 mM, and DTT of 10 mM were added. After mixing evenly, ultrasonic treatment was carried out on ice for 10 min. Centrifuge at 4°C and 12,000 r / min for 30 min, and take the supernatant into a new centrifuge tube. An equal amount of protein was ultrafiltered with a 10 kD ultrafiltration tube (centrifuged at 4°C and 12,000 r / min for 30 min), and the permeate was collected, which is the polypeptide sample.

[0078] (2) Polypeptide concentration determination was performed using the bicinchoninic acid (BCA) method: ① Preparation of BCA working solution: Prepare an appropriate amount of BCA working solution by mixing BCA reagent A, BCA reagent B, and BCA reagent C at a volume ratio of 50:48:2, and mix well; ② Preparation of standard solutions: Use the standard polypeptide solution provided by the Pierce Quantitative Colorimetric Peptide Assay kit to prepare different concentrations according to its instructions, which are 0, 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, 1 mg / mL respectively; ③ Take 4 μL of each sample and mix it with 16 μL of water, then add 180 μL of BCA working solution. Mix well by shaking and react at 37 °C for 15 min; ④ Use a SPECTRAMAX microplate reader to read the absorbance at 480 nm. Calculate the protein concentration of the sample based on the standard curve and the volume of the sample used. The standard curve is as shown in Figure 1 shown, and the sample concentrations are shown in Table 1 below.

[0079] Table 1. List of sample concentrations

[0080] Serial number Sample name Sample type Polypeptide concentration (μg / μL) Total amount of polypeptide (μg) 1 MG1 0.196 18.6 2 MG2 0.468 44.5 3 MG3 Polypeptide serum 0.349 33.2 4 NY1 0.228 21.7 5 NY2 0.315 29.9 6 NY3 0.518 49.2

[0081] (3) Take equal amounts of samples for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis: According to the peptide quantification results, dissolve the peptides with a concentration of 0.25 μg / μL in a mass spectrometry loading buffer (2% acetonitrile 0.1% formic acid) for mass spectrometry analysis.

[0082] Data acquisition software: Thermo Xcalibur 4.0 (Thermo, USA)

[0083] Reverse-phase column information: C18 column (75 μm × 25 cm, Thermo, USA)

[0084] Chromatographic instrument: EASY-nLC 1200 (Thermo, USA)

[0085] Mass spectrometry instrument: Q_Exactive HF-X (Thermo, USA)

[0086] Chromatographic separation time: 120 min

[0087] A: 2% acetonitrile 0.1% formic acid

[0088] B: 80% acetonitrile 0.1% formic acid

[0089] Flow rate: 300 nL / min

[0090] Gradient:

[0091] Table 2. EASY-nLC liquid phase gradient

[0092] Time (min) B(％) 0 5 64 23 80 29 90 38 92 48 93 100 120 Stop

[0093] MS scan range (m / z): 300 - 1500, acquisition mode: DDA, Top 20;

[0094] Top 20 (select the 20 most intense precursor ions for MS / MS fragmentation);

[0095] MS1 resolution: 60000, AGC target: 3e6, maximum injection time: 20 ms, fragmentation method: HCD;

[0096] MS2 resolution: 15000, AGC target: 5e4, maximum injection time: 45 ms, fixed first mass: 100 m / z;

[0097] minimum AGC target: 8e3, Intensity threshold: 1.8e5, dynamic exclusion time: 30 s.

[0098] (4) Use the Sequest or Mascot module in ProteomeDiscoerer to search the database. ① Database selection: The databases currently used can be mainly divided into two categories. One is maintained by NCBI, and the other is maintained by EBI. The main methods for building databases are as follows: a) NCBInr full database; b) NCBInr classification databases, including animal full database, plant full database, microorganism full database, bacterial full database, etc.; c) SwissProt / UniProt classification databases, including animal full database, plant full database, microorganism full database, bacterial database, etc.; d) NCBInr corresponding species databases, including human, wheat, yeast, Escherichia coli, etc.; e) Other species databases, such as self - measured genome and transcriptome databases. When selecting a database, follow the following principle: If it is a sequenced organism, directly select the database of that species. If it is a non - sequenced organism, select the large - scale proteome database most relevant to the tested sample. ② Database search: The software version used for database searching is PEAKS Studio 8.5. When searching the database, submit the raw file to the PEAKS Studio 8.5 server, select the already established database, and then perform database searching. The relevant parameters are as shown in Table 3 below:

[0099] Table 3. Search parameters of PEAKS Studio 8.5

[0100] Item Value PEAKSStudio version 8.5 Protein Database uniprot-taxonomy_9606_unique.fasta Cys alkylation Iodoacetamide Dynamic Modification Oxidation (M), Acetyl (Protein N-Terminus) Static Modification Carbamidomethyl (C) Enzyme Name Trypsin (Full) Max. Missed Cleavage Sites 2 Precursor Mass Tolerance 10 ppm Fragment Mass Tolerance 0.05 Da

[0101] Note: The result filtering parameter is Peptide FDR ≤ 0.01.

[0102] (5) Perform data statistics and bioinformatics analysis on the obtained library search results.

[0103] Table 4. List of library search results

[0104] Total Spectrum Identified Spectrum Peptide number Protein number Protein group number 79273 3416 495 197 80

[0105] II. Determination of endogenous differential polypeptides in cisplatin-resistant and cisplatin-sensitive gastric cancer tissues

[0106] Using the Label Free quantitative proteomics method, a total of 3,416 endogenous polypeptides were obtained, among which 495 polypeptides had significant differences (t-test, P < 0.05). Analysis found that these polypeptides were mainly composed of 8 - 20 amino acids, which was consistent with the properties of endogenous polypeptides produced by the proteasome degradation pathway; the molecular weight of the precursor proteins was concentrated in the range of 1 - 30 kDa, and the number of peptide segments contained was between 1 - 2 (see Figure 2 A - C). Given that endogenous polypeptides mainly originate from the degradation of protein precursors, functional analysis was performed on the precursor proteins of the differential polypeptides. The results showed that the functions of these precursor proteins were mainly involved in the PI3K - Akt signaling pathway, p53 signaling pathway, and apoptosis pathway (see Figure 2 D - E), and these signaling pathways were closely related to the apoptosis signal of cells. Further, according to the differential polypeptide screening principles of ① high mass spectrometry signal level, ② small intra-group difference, and ③ large inter-group difference, the selection range was further narrowed, and 4 polypeptides were selected from 45 qualified differential polypeptides (29 up-regulated and 16 down-regulated) for preliminary functional research. Among them, 2 endogenous polypeptides were derived from the precursor proteins of Zyxin and TYB4protein (Thymosin Beta 4) respectively, and both were differentially low-expressed in cisplatin-resistant gastric cancer tissues, with differential multiples of 0.21 and 0.23; 2 endogenous polypeptides were derived from the precursor proteins of Fibrinogen alpha chain and Hemoglobin subunit beta (Fragment) respectively, and both were differentially highly expressed in cisplatin-resistant gastric cancer tissues, with differential multiples of 5.02 and 5.04. After chemically synthesizing the polypeptides, gastric cancer cell lines resistant to cisplatin were intervened with different concentrations (1μm, 50μm, 100μm, 200μm, 300μm). The results showed that the polypeptide derived from TYB4 (PD-TYB4 2-17 ) had a significant effect on reducing the activity of cisplatin-resistant gastric cancer cells under the combined action with cisplatin (see Figure 3 A - C). Further, gastric mucosal epithelial cells were treated with 300μm of PD-TYB4 2-17 and 300μm of scramble control peptide. The results showed that PD-TYB4 2-17 had no obvious cytotoxicity to normal gastric mucosal cells (seeFigure 4 ) to prompt PD-TYB4 2-17 Combined with cisplatin, it inhibits the activity function of gastric cancer cells, which is closely related to the pathophysiology of gastric cancer cisplatin resistance.

[0107] III. Synthesize the linear polypeptide SDKPDMAEIEKFDKSK, and the polypeptide is synthesized from the C-terminus to the N-terminus. The specific steps are as follows:

[0108] ① Weigh 3g of Wang resin (substitution degree 0.3mmol / g) in a 150mL reactor and soak it with 50mL of dichloromethane (DCM). ② After 2h, wash the resin with 3 times the volume of the resin in nitrogen-dimethylformamide (DMF), then drain it, repeat this four times, drain the resin and set it aside. ③ Weigh an appropriate amount of the first amino acid lysine K at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) in a 50mL centrifuge tube, add 20mL of DMF to dissolve it, then add 3mL of N,N-diisopropylcarbodiimide (DIC) and shake it for 1min. After the solution is clarified, add it to the reactor, then add 3 times the molar amount of DMAP to the reactor, and then place the reactor in a shaker at 30℃ for reaction. ④ After 4h, use a certain amount of acetic anhydride to cap the head (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with 3 times the volume of the resin in DMF, drain it and set it aside. ⑤ Add a certain amount of 20% piperidine (piperidine / DMF=1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it. ⑥ Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1min). The resin has color, indicating that the deprotection is successful. ⑦ Weigh an appropriate amount of the second aminothreonine T and an appropriate amount of HOBT in a 50mL centrifuge tube, add 25mL of DMF to dissolve it, and then add 2.5mL of DIC and shake it for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction. ⑧After 1 hour, take a small amount of resin for testing, and use the ninhydrin method to test (two drops of test A and test B, 100℃ for 1min). If the resin is colorless, it means the reaction is complete; if the resin has color, it means the condensation is incomplete, and the reaction continues. ⑨After the reaction is complete, wash the resin four times with DMF, then drain it, add a certain amount of 20% piperidine (piperidine / DMF=1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain it to check whether the protection is removed. ⑩According to steps 7-9, connect the following amino acids SDKPDMAEIEKFDKSK in sequence. After removing the Fmoc of Lys, wash it four times with DMF, and then drain the resin with methanol. Then, the polypeptide was cleaved from the resin using 95% cleavage solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) (10 mL of cleavage solution was added per gram of resin), and centrifuged and precipitated four times with ice ether (cleavage solution: ether = 1:9). Finally, it was separated and purified by HPLC and then freeze-dried to obtain a polypeptide with a purity of 98%.

[0109] Example 2: Peptide PD-TYB4 2-17Verification of the impact on the biological functions of cisplatin-resistant gastric cancer cells

[0110] 1. Cell culture: The gastric cancer cisplatin-resistant cell line SGC7901 / DDP was purchased from Shanghai Bogu Biotechnology Co., Ltd. The SGC7901 / DDP cell line was inoculated in PRMI-1640 complete culture medium containing 10% fetal bovine serum (FBS) and cultured in an incubator at 37°C with 5% CO 2 . When the cell density reached 70 - 80% confluence, experiments were conducted. After incubating SGC7901 / DDP cells with 300 μM PD-TYB4 2-17 or 300 μM Scramble peptide (control group) for 6 h, 20 μM DDP at the final concentration was added and the cells were cultured for another 48 h. Experimental groups: DDP + Scramble group and DDP + PD-TYB4 2-17 group.

[0111] 2. Detection of SGC7901 / DDP cell viability: The CCK-8 assay was used to detect the viability of SGC7901 / DDP cells. The concentration of SGC7901 / DDP cells was adjusted to 1×10 9 / L with complete culture medium and inoculated into 96-well plates. After culturing the cells with different treatment factors for 48 h, the culture medium was discarded. 90 μL of PRMI-1640 complete culture medium and 10 μL of CCK-8 reagent were added to each well, and the cells were incubated for another 4 h. The absorbance values of each well were measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0112] 3. Detection of SGC7901 / DDP cell apoptosis: After culturing the cells with different treatment factors for 48 h, the cell density was adjusted to 1×10 8 / mL with pre-cooled PBS. 5 μL of Annexin V-FITC and 10 μL of propidium iodide were added respectively, and the cells were incubated at low temperature in the dark for 15 min. The apoptosis rate of SGC7901 / DDP cells was detected by flow cytometry.

[0113] 4. Detection of SGC7901 / DDP cell migration: The Transwell invasion assay was used to detect the migration of SGC7901 / DDP cells. Before the experiment, SGC7901 / DDP cells were starved for 24 h, collected, and the cell density was adjusted to 3×10 6 cells / mL with serum-free medium. 500 μL of the cell suspension was placed in the upper chamber of a Transwell insert (6.5 mm, 8.0 μm pore size; Corning), and 750 μL of PRMI-1640 complete culture medium containing 20% FBS was added to the lower chamber. The cells were cultured for 24 h. The invaded cells were fixed with formaldehyde, and the "adherent" cells were stained with 10% crystal violet. The number of cells was counted under a microscope to evaluate the migration ability of SGC7901 / DDP cells.

[0114] It was found by online analysis using UniProt that PD-TYB4 2-17 is derived from the 2nd to 17th amino acids (SDKPDMAEIEKFDKSK) of human TYB4 protein and is highly conserved in humans and mice ( Figure 5 ).

[0115] Further experimental data showed that: compared with the DDP+Sramble group, the proliferation activity and migration number of cells in the DDP+PD-TYB4 2-17 group decreased, and the apoptosis rate increased, with a statistically significant difference (P<0.05), as shown in Figure 6 and 7 , Table 5.

[0116] Table 5. Comparison of proliferation activity, migration and apoptosis rates of SGC7901 / DDP cells in each group

[0117]

[0118] Note: (*, P<0.05; **, P<0.01 vs DDP+Scramble group)

[0119] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims. Sequence Listing <110> Jiangsu Cancer Hospital <120> A polypeptide for improving the sensitivity of gastric cancer to cisplatin chemotherapy and its application <130> 20220628 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 16 <212> PRT <213> Artificial sequence <400> 1 Ser Asp Lys Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys Ser Lys 1 5 10 15

Claims

1. Use of a polypeptide in the preparation of a drug for reversing cisplatin resistance of gastric cancer cells in combination with cisplatin, characterized in that, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: the polypeptide is derived from the amino acids at positions 2-17 of its precursor protein thymosin β4.

3. The use according to claim 1, characterized in that: the drug further comprises a pharmaceutically acceptable carrier.

4. The use according to claim 1, characterized in that: the gastric cancer cells belong to the SGC7901 cell line.

5. An anti-tumor drug, characterized in that: the drug comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 and cisplatin.

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