CN110498850B - Polypeptide, derivative thereof and application thereof in preparing medicine for preventing and treating tumors - Google Patents

Abstract

The invention discloses a polypeptide that specifically binds to β-catenin, which is a TRIB3 mutant whose amino acid sequence is substituted, deleted or added with one or several amino acids and has TRIB3 activity. Also disclosed is a derivative of the polypeptide, which is a chimeric peptide formed by linking the polypeptide with a transmembrane peptide. The invention discloses an anti-tumor pharmaceutical composition containing the polypeptide or polypeptide derivative as an active ingredient. Also disclosed is the application of the polypeptides, polypeptide derivatives and pharmaceutical compositions in the preparation of medicines for preventing and/or treating TRIB3 and β-catenin-related diseases, especially tumors. The pharmaceutical composition has the ability to specifically bind to β-catenin, and can block the binding ability of TRIB3 and β-catenin protein, and at the same time, can reduce the protein level of TRIB3. The prepared medicine has the advantages of obvious curative effect, less toxic and side effects and safe use.

Description

Polypeptide, derivative thereof and application thereof in preparing medicine for preventing and treating tumors

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide, a derivative thereof and application thereof in preparing a medicament for preventing and treating tumors.

Background

The Wnt/beta-catenin signal channel has abnormal activation in various tumors (colon cancer, liver cancer, lung cancer, pancreatic cancer, glioma and breast cancer), and plays a promoting role in tumorigenesis, tumor proliferation, tumor metastasis, tumor dormancy, tumor immunity, tumor resistance, tumor dryness maintenance and the like. Beta-catenin, as an effector molecule of the signal pathway, is usually phosphorylated by its degradation complex members Axin-2, APC and GSK-3 beta, and then enters the ubiquitination pathway for degradation. When the Wnt signal is abnormally activated, the degradation complex member is disintegrated, and the beta-catenin accumulated in the cytoplasm is translocated into the nucleus to form a transcription complex with the LEF/TCF family, so that the transcription of various carcinogenic factors is promoted, and the development process of the tumor is promoted.

At present, targeted inhibitors for the pathway mainly comprise small molecule drugs, antibody drugs, RNA interference drugs and natural products. In the research for more than thirty years, except that a few drugs enter clinical trials, most drugs are still in preclinical research, and no targeted drugs are seen on the market. The types of action of the drugs in the research are mainly divided into four types: inhibiting the binding of ligand Wnt-3a to cell membrane surface receptors LRP5/6 and Frizzled; reducing the protein expression level of beta-catenin; inhibiting nuclear translocation of beta-catenin; inhibiting the activity of beta-catenin/TCF transcription complex in nucleus. The first class of drugs has poor universality because the process of binding Wnt-3a to a receptor is not the only factor for increasing the beta-catenin, such as the mutation of the beta-catenin per se or APC (active carbon dioxide) which is a component of a degradation complex of the beta-catenin in various colon cancers is a key step for inducing the abnormal activation of the pathway. The second class of drugs mainly comprises agonists of beta-catenin degradation complex members such as GSK-3 beta, Axin-2 and PKA, and the application of the drugs is limited due to mutation of upstream signal proteins such as beta-catenin; in addition, since β -catenin maintains the normal structure of cell membrane and cytoskeleton under physiological conditions, there may be a great risk of down-regulating the protein level of β -catenin. Therefore, the regulation center of gravity of the beta-catenin should be put into two aspects of inhibiting the beta-catenin from entering the nucleus or inhibiting the activity of the beta-catenin in a transcription complex in the nucleus. At present, the development difficulty of the medicine is greatly increased due to the weak targeting specificity of the nuclear-entering inhibitor. And some compounds or polypeptide medicines for interrupting the interaction of the beta-catenin and the transcription cofactor TCF4 directly act on downstream molecules of the signal channel, have strong targeting property and small side effect, and have good prospect of drug development and development inhibition of tumors.

TRIB3 (Tribbels Homologue 3) is one of the human pseudokinase Tribbels homologous protein family members, has a joint protein-like function, and is involved in the assembly of various protein complexes. It was first identified in Drosophila and found that this protein inhibits mitosis, regulates cell proliferation, migration and morphogenesis during development. TRIB3 plays an important role in regulation and control in physiological and pathological processes such as glycolipid metabolism, adipocyte differentiation, apoptosis, stress, collagen expression and the like through interaction with various proteins. TRIB3 exhibited high expression in various tumor cell lines and human tumor tissues. In our previous studies, TRIB3 was found to interact with Smad3, enhance TGF-beta/Smad 3 signaling pathway activity, and promote tumor invasion. In addition, we have also found that hyperinsulinemia (insulin) and insulin-like growth factor 1(IGF-1) in the tumor microenvironment can up-regulate TRB3 expression. TRIB3 interacts with autophagy receptor P62, inhibiting the binding of P62 to LC3 and ubiquitinated substrates, causing the accumulation of P62 and ubiquitinated proteins with secondary inhibition of proteasome function. Inhibition of autophagy and ubiquitin proteasome systems by TRIB3 links malignant progression in diabetes and tumors.

The research finds that TRIB3 and beta-catenin have direct interaction in the cell nucleus and jointly promote the generation and development of tumors. However, the interaction area of the beta-catenin and the TCF4 protein is too large, so that the intervention of small molecular drugs cannot play a good interrupting role. In addition, TRIB3 is an important protein in the tumor development process, and a drug targeting the protein is not reported at present. Therefore, how to provide a medicament, which can weaken the transcriptional activity of beta-catenin/TCF 4 by breaking the interaction of beta-catenin/TRIB 3, and further effectively treat tumors related to Wnt/beta-catenin signaling pathways is a difficult problem to be solved in the field.

Disclosure of Invention

In order to overcome the defects that the Wnt/beta-catenin signal channel regulation mechanism is complex, the drug development process is slow, and a drug for effectively inhibiting the beta-catenin transcriptional activation activity is lacked in the prior art, the invention provides a polypeptide, a derivative thereof and application thereof in preparing a drug for preventing and treating tumors.

One of the technical schemes provided by the invention is as follows: a polypeptide specifically binding to beta-catenin is a TRIB3 mutant with the activity of TRIB3, wherein the amino acid sequence of the polypeptide is subjected to substitution, deletion or addition of one or more amino acids.

Preferably, it is a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1. The amino acid sequence is shown as SEQ ID NO: the polypeptide T3A6 shown in 1 can be specifically combined with beta-catenin, not only can inhibit a Wnt/beta-catenin signal channel, but also can reduce the protein level expression of TRIB 3.

One of the technical schemes provided by the invention is as follows: the derivative of the polypeptide specifically binding with the beta-catenin is a chimeric peptide formed by connecting the polypeptide with a cell-penetrating peptide.

Preferably, the cell-penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide.

More preferably, the cell-penetrating peptide is Pep2, and the amino acid sequence thereof is shown in SEQ ID NO: 2, respectively.

Most preferably, the amino acid sequence of the chimeric peptide is as set forth in SEQ ID NO: 3, respectively.

One of the technical schemes provided by the invention is as follows: an anti-tumor pharmaceutical composition, the active component of which contains the polypeptide or the derivative of the polypeptide.

Preferably, the active ingredient is a single active ingredient.

More preferably, the pharmaceutical composition further comprises other compounds having anti-tumor activity.

The active component is a compound with the function of preventing or treating tumors. In the pharmaceutical composition, the polypeptide for targeted inhibition of beta-catenin transcription activity and TRIB3 protein level can be used as an active ingredient alone or together with other compounds with antitumor activity.

Preferably, a pharmaceutically acceptable carrier and/or excipient is also included. The carrier or excipient is a conventional pharmaceutical carrier or excipient in the art, and the carrier or excipient can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the polypeptide and 0.01-99.99% of a pharmaceutical carrier or excipient, wherein the percentage is the mass percentage of the pharmaceutical composition.

More preferably, the route of administration is by injection or orally.

The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

Preferably, the pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the result sought.

One of the technical schemes provided by the invention is as follows: the polypeptide, the polypeptide derivative or the pharmaceutical composition and the application of TRIB3 in preparing the medicines for preventing and/or treating the diseases related to the beta-catenin, in particular to the tumor.

One of the technical schemes provided by the invention is as follows: application of TRIB3 inhibitor or antagonist in preparing medicine for preventing and treating diseases related to beta-catenin, especially tumor.

Preferably, the tumors in the two technical schemes are selected from intestinal cancer, liver cancer, lung cancer, pancreatic cancer or breast cancer.

More preferably, the intestinal cancer is colon cancer or rectal cancer; the liver cancer is primary liver cancer or secondary liver cancer; the lung cancer is non-small cell lung cancer or small cell lung cancer; the pancreatic cancer is pancreatic ductal adenocarcinoma and pancreatic acinar cell carcinoma; the breast cancer is non-invasive breast cancer, early invasive breast cancer, invasive special type breast cancer or invasive non-special type breast cancer.

Such prevention is conventional in the art and preferably refers to preventing or reducing tumor development after use in the presence of possible tumor factors. The treatment is conventional in the art and preferably means reducing the extent of the tumor, or curing the tumor to normalize it, or slowing the progression of the tumor.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the polypeptide, the polypeptide derivative and the pharmaceutical composition can inhibit beta-catenin transcription activity, can reduce Wnt/beta-catenin pathway activity, and can reduce protein level expression of TRIB3, so that the polypeptide, the polypeptide derivative and the pharmaceutical composition are applied to preparation of double-target drugs for treating and preventing tumors. The prepared medicine has the advantages of obvious curative effect, less toxic and side effect and safe use in treating tumor diseases.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The PBS described in the examples refers to phosphate buffer at a concentration of 0.1M and a pH of 7.2.

The room temperature described in the examples is a room temperature which is conventional in the art, and is preferably 15 to 30 ℃.

The experimental results are expressed by mean value plus or minus standard error, and the significant difference is considered to be existed by comparing that p is less than 0.05 and p is less than 0.01 through parameter or nonparametric variance test.

Example 1 peptide fragments binding to beta-catenin protein were screened by surface plasmon resonance.

Firstly, TRIB3 is segmented and cut into different polypeptide segments, and peptide segment synthesis is carried out by a polypeptide solid phase synthesizer, and the process is carried out by Beijing Saibaosheng Gene Co. The whole screening process was performed in a surface plasmon resonance instrument Biacore T200. The screening method comprises the following steps:

1) purified protein beta-catenin (available from Beijing Yiqiao Shenzhou Co.) was coupled to a CM5 chip (available from GE Co.) via an amino group, unbound protein was washed off at a flow rate of 10. mu.L/min, and the chip surface was equilibrated for 2 hours, washed and equilibrated with glycine buffer pH 2.5.

2) 200 μ L of polypeptide fragments (200, 100, 50, 25, 12.5nM) at different concentrations were injected automatically, the whole process being carried out at 25 ℃. The buffer used was HBS-EP buffer (0.01MHEPES, 0.15M NaCl, 3mM EDTA, 0.005% Surfactant P20.

3) Simulating a binding curve of the polypeptide with different concentrations and the beta-catenin by using Biacore T200 random analysis software to obtain a polypeptide with stronger binding capacity with the beta-catenin:

T3A 6: Leu-Ser-Ala-Pro-Ala-Arg-Cys-Leu-Val-Arg-Cys-Leu-Arg (i.e., a polypeptide with the amino acid sequence shown in SEQ ID No: 1).

KD＝2.88E-7，Rmax(RU)＝160。

Example 2 ELISA method for verifying the binding of peptide fragment T3A6 and protein beta-catenin

The specific operation steps are as follows:

1. human β -catenin and Bovine Serum Albumin (BSA) were diluted to 10. mu.g/ml with PBS, 100. mu.l of each well was added, and 96-well ELISA plates were coated overnight at 4 ℃.

2. Washed three times with PBS containing 0.1% Tween-20, plated with 200. mu.l of blocking solution (10% bovine serum PBS), and coated for 2h at 37 ℃.

3. Pouring off the sealing liquid for coating, and correspondingly adding 200 mul of 1 mug/ml polypeptide T3A6 solution; meanwhile, a positive control was set, 200. mu.l of 1. mu.g/ml TRIB3 protein (obtained from RD) solution was added, and the mixture was incubated at 37 ℃ for 1 hour.

4. The plate was washed five times with PBS containing 0.1% Tween-20, and 100. mu.l of anti-beta-catenin monoclonal antibody diluted with blocking solution 1:4000(v/v) was added to each well and incubated for 1h at room temperature.

5. Six washes were performed with PBS containing 0.1% Tween-20. A substrate developing solution (100mM sodium acetate pH6.0, 10. mu.l of 30% hydrogen peroxide and 100. mu.g/ml TMB per 50ml of the buffer) was prepared, 100. mu.l was added to each well, and after incubation at room temperature for 5 minutes, 50. mu.l of 0.1M dilute sulfuric acid was added to each well to terminate the reaction.

6. The results were reflected as the OD450 of the sample well, see Table 1.

TABLE 1 affinity of the polypeptides for beta-catenin

Table 1 shows that the T3A6 peptide segment is similar to positive control TRIB3 and has higher affinity with beta-catenin protein.

Example 3 method of Competition ELISA it was verified that peptide fragment T3A6 can compete for the binding of TRIB3 to beta-catenin protein

The specific operation steps are as follows:

1. human β -catenin and Bovine Serum Albumin (BSA) were diluted to 10. mu.l/ml with PBS and 100. mu.l of each well was added and 96-well ELISA plates were coated overnight at 4 ℃.

2. Washed three times with PBS containing 0.1% Tween-20, plated with 200. mu.l of blocking solution (10% bovine serum PBS), and coated for 2h at 37 ℃.

3. The coated blocking solution was decanted off and 200. mu.l of TRIB3 protein solution was added at 1. mu.g/ml and incubated at 37 ℃ for 1 h.

4. Washed five times with PBS containing 0.1% Tween-20; mu.l of blocking solution was added to each well as a negative control and 100. mu.l of horseradish catalase-labeled polypeptide T3A6 diluted with blocking solution and incubated for 1h at room temperature.

5. Washing six times with PBS containing 0.1% Tween-20; a substrate developing solution (100mM sodium acetate, pH6.0, 10. mu.l of 30% hydrogen peroxide per 50ml of the buffer, 100. mu.g/ml TMB) was prepared, 100. mu.l was added to each well, and after incubation at room temperature for 5 minutes, 50. mu.l of 0.1M dilute sulfuric acid was added to each well to terminate the reaction.

6. The results were reflected as the OD450 of the sample well, see Table 2.

TABLE 2 Competition of TRIB3 binding to beta-catenin protein by the polypeptide

Table 2 shows three replicates of polypeptide competition for binding of TRIB3 to β -catenin protein, and the results indicate that: T3A6 can compete for the binding of TRIB3 to the beta-catenin protein, while the negative control does not compete for binding.

Example 4 Dual-luciferase reporter gene experiments verify that the polypeptide Pep2-T3A6 inhibits the transcriptional activation activity of beta-catenin.

Peptide fragment T3A6 is connected with cell-penetrating peptide Pep2 (the amino acid sequence is shown as SEQ ID NO: 2), and derivative Pep2-T3A6 (the amino acid sequence is shown as SEQ ID NO: 3, and the nucleotide sequence is shown as SEQ ID NO: 4) of the peptide is formed, and the peptide fragment is synthesized by Sebain Gene technology Limited company, and has purity>98 percent. Dual-luciferase reporter gene experiment kit (Dual-

Reporter Assay System, cat # E1910) was purchased from Promega. The TOPFLASH, FOPFLASH and PRL-TK plasmids were purchased from addrene.

The specific detection steps are as follows:

1. plasmid TOPFLASH and plasmid PRL-TK which respectively carry out cotransfection reaction on beta-catenin/TCF 4 transcriptional activity in HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-231 breast cancer cells with the confluence of 60% (all the cells are purchased from the institute of basic medicine of Chinese academy of medical sciences), and mutant plasmid FOPFLASH and plasmid PRL-TK of the TOPFLASH are used as negative controls of the whole experiment.

2. 12 hours after transfection, 10. mu.M of Pep2-T3A6 or control peptide was added to the cells. The control peptide, scrambled polypeptide, scrambled the sequence of T3A6 of Pep2-T3A6 (as shown in SEQ ID No: 5).

After 3.12 hours, 250. mu.l of each lysate was added to each well, and after 15min, 10. mu.l of the supernatant was collected and added to a white 96-well plate, 50. mu.l of luciferase substrate was added for wavelength measurement, and 50. mu.l of stopbuffer was added for wavelength measurement again.

4. The ratio of the first and second values is the luciferase activity, and the results are shown in tables 3-7.

The result shows that the Pep2-T3A6 can reduce the transcriptional activity of beta-catenin/TCF 4 in HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-231 breast cancer cells.

TABLE 3 polypeptide inhibits the transcriptional activity of beta-catenin/TCF 4 in HCT-8 cells

	Control peptides	Pep2-T3A6	P value
				topFLASH*	2235±234	1124±56	0.0001
FOPFLASH#	102±3	103±2	/

*: fluorescence value F (Top) of firefly collected from a sample transfected with TOP-Flash and Renilla luciferin vectors/fluorescence value R (Top) of Renilla collected; #: the fluorescence value F (Fop) of the firefly acquired from a sample transfected with FOP-Flash and renilla fluorescein carriers/the fluorescence value R (Fop) of the renilla acquired; /: no significant difference exists; the same applies below.

TABLE 4 polypeptide inhibits the transcriptional activity of beta-catenin/TCF 4 in A549 cells

	Control peptides	Pep2-T3A6	P value
				topFLASH	2545±145	1235±36	0.0002
FOPFLASH	63±5	68±2	/

TABLE 5 polypeptide inhibits the transcriptional activity of beta-catenin/TCF 4 in HepG2 cells

	Control peptides	Pep2-T3A6	P value
				topFLASH	2954±156	1426±14	0.0001
FOPFLASH	107±3	89±2	/

TABLE 6 polypeptide inhibits the transcriptional activity of beta-catenin/TCF 4 in SW1990 cells

	Control peptides	Pep2-T3A6	P value
				topFLASH	2925±127	1486±10	0.0001
FOPFLASH	123±3	112±1	/

TABLE 7 polypeptide inhibits the transcriptional activity of beta-catenin/TCF 4 in MDA-MB-231 cells

	Control peptides	Pep2-T3A6	P value
				topFLASH	2634±23	1322±27	0.0001
FOPFLASH	77±1	84±2	/

Example 5 Dexamidone to verify the Effect of the polypeptide on the half-life of TRIB3 protein

1. Collecting colon cancer cells HCT-8 in logarithmic growth phase, and adjusting cell concentration with DMEM medium to prepare 20 ten thousand/mL cell suspension.

2. 2mL of the cell suspension prepared in step 1 was added to a 6-well plate for culture, and after 12 hours, the culture medium was replaced with a new one, and 1. mu.g/mL of the polypeptide Pep-T3A6 prepared in example 1 was added, respectively. The scrambled control peptide groups were added with an equal volume of solvent PBS.

After 3.12 hours, adding protein synthesis inhibitor Cycloheximide (CHX) according to time points, so that the action time is respectively 8h, 4h, 2h, 1h and 0 h; the control group was added with an equal volume of solvent.

4. Collecting cells, adding RIPA lysate (purchased from Shanghai Binyan biotechnology, Inc., adding protease inhibitors PMSF and leupeptin, aprotinin and other inhibitors before use according to the instruction), and performing ice lysis for 30 min; centrifuging at 12000rpm at 4 deg.C for 30 min; the supernatant was aspirated, the protein was quantified by BCA method, the protein was adjusted to a uniform concentration according to the quantification result, 5 Xloading buffer was added, and denaturation was carried out at 98 ℃ for 10 min.

5. Taking part of the sample, and performing SDS-polyacrylamide gel electrophoresis according to the method described in molecular cloning experimental guidelines; after electrophoresis, immunoblot detection was performed.

6. And (3) carrying out quantitative analysis on the immunoblotting result by using Gel-Pro Analyzer32Analyzer4.0, drawing a time-dependent TRIB3 content change curve, and determining the time required for reducing the TRIB3 protein content to 50% of the time when CHX acts for 0h, namely the half-life of the TRIB3 protein. The results are shown in Table 8.

TABLE 8 Effect of the Polypeptides on the half-life of cellular TRIB3 protein

The above table shows that the half-life of TRIB3 protein was 0.4 + -0.1 hr under the action of Pep2-T3A6, while the half-life of TRIB3 protein was 2.5 + -0.1 hr under the action of scarmbled control peptide. The Pep2-T3A6 can obviously reduce the half-life of the TRIB3 protein.

Example 6 cell counting experiments to verify that the polypeptide Pep2-T3A6 inhibits the growth of tumor cells

The specific operation steps are as follows:

1. HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-231 breast cancer cells in logarithmic growth phase are collected to adjust the cell concentration, and cell suspension with the concentration of 15 ten thousand/mL is prepared.

2. 1mL of the cell suspension prepared in step 1 was added to a 12-well plate and cultured (wherein the culture medium for HepG2, HCT-8 and MDA-MB-231 cells was DMEM medium, and the culture medium for A549 and SW1990 cells was RPMI1640 medium, both purchased from Invitrogen; the culture temperature was 37 ℃ and the volume of the culture medium was 1mL), and after 12 hours, new medium was replaced, and 1. mu.g/mL of the above-mentioned scrambled control peptide and Pep2-T3A6 were added, respectively. Passages were performed every other day and counted. Growth curves were plotted after 12 days of culture. The results are shown in Table 9.

TABLE 9 polypeptide inhibition of tumor cell growth

Cell name/number of cells (ten thousand)	Control peptides	Pep2-T3A6	P value
				HCT-8	634±12.436	325±11.359	0.0001
HepG2	743±13.335	314±12.234	0.0002
				A549	687±11.343	200±14.233	0.0001
SW1990	456±12.435	168±11.264	0.0001
				MDA-MB-231	835±12.234	426±11.234	0.0001

The results in the above table indicate that the polypeptide Pep2-T3A6 can inhibit the growth of HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-23 breast cancer cell 1.

Example 7 cell scratch experiment to verify healing of polypeptides after inhibition of tumor cell scratching

The specific operation steps are as follows:

1. firstly, a marking pen is used at the back of the 6-hole plate, a straight ruler is used for drawing a transverse line, and the transverse line penetrates through the through hole.

2. Adding 5X 10 of the solution into each hole respectively⁵After the tumor cells are cultured in an incubator at 37 ℃ overnight, the cells adhere to the wall. The tumor cells are colon cancer cells HCT-8, liver cancer cells HepG2, lung cancer cells A549, pancreatic cancer cells SW1990 and breast cancer cells MDA-MB-231 in logarithmic growth phase.

3. The tip is used for scratching the ruler on the next day, and is perpendicular to the transverse line at the back as much as possible.

4. The cells were washed 3 times with PBS, the scraped cells were removed, and fresh medium was added, along with 1. mu.g/mL of the above scrambled control peptide and Pep2-T3A 6.

5. Then put into 5% (v/v) CO at 37 DEG C₂The incubator was used for cultivation, and samples were taken after 24 hours and photographed. The results are shown in Table 10.

TABLE 10 polypeptide inhibition of tumor cell migration

The larger the area ratio of damage repair indicates the stronger the migration ability of tumor cells and the stronger the healing ability after cell scratching. The results in Table 10 demonstrate that the polypeptide Pep2-T3A6 can reduce the healing capacity of HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-23 breast cancer cells after scarification.

Example 8 Transwell experiment verifies that the polypeptide Pep2-T3A6 can inhibit the invasion capacity of tumor cells

The specific operation steps are as follows:

1. the 100 Xfibronectin stock solution was taken out from a refrigerator at-20 deg.C, melted and mixed into 1 Xworking solution, and 30. mu.l was taken out and quickly coated on the outside of a transwell cell having a pore size of 8 μm to fill the bottom of the cell.

2. Taking out the Matrigel from a refrigerator at the temperature of 20 ℃ below zero, diluting the Matrigel by 8 times after melting, quickly adding 40 mu l of the Matrigel into the inner side of a transwell chamber with the aperture of 8 mu m, and quickly spreading the Matrigel on the bottom of the hole; the solidified chamber was placed in the well of a 24-well plate and 800. mu.l of complete medium was added to the lower chamber.

3. Collecting HCT-8, liver cancer cell HepG2, lung cancer cell A549, pancreatic cancer cell SW1990 and breast cancer cell MDA-MB-231 in logarithmic growth phase; then, the cell concentration was adjusted to prepare a cell suspension of 1000 ten thousand cells/ml.

4. Mu.l of cell suspension was added to the chamber, along with 1. mu.g/ml of the above scrambled control peptide and Pep2-T3A 6.

5. The plates were then removed from the CO2 incubator at 37 ℃ for culture.

6. After culturing for 12-24h, carrying out crystal violet staining on the cell colony, and taking a picture by using a microscope; the results are shown in Table 11 for the number of cells in the 40-fold mirror average single field.

TABLE 11 Polypeptides inhibiting tumor cell invasion

The result shows that the polypeptide Pep2-T3A6 obviously inhibits the invasion capacity of HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-23 breast cancer cells.

Example 9 nude mouse subcutaneous tumor experiment verifies that peptide fragment Pep2-T3A6 inhibits cancer cell growth

1. Experiment consumables and reagents: sterilized EP tube 1.5mL, sterile 10mL centrifuge tube, tip filter (100 mesh), absorbent cotton ball, forceps, alcohol cotton ball, sterile 1mL syringe, matrigel, PBS (filtration), pancreatin, serum.

2. Experimental animals and groups: 20 male nude mice (purchased from Beijing Wittingle laboratory animals Co., Ltd.) at 4-6 weeks were randomly divided into two groups: 10 control groups and 10 Pep2-T3A6 administration groups.

3. Cell preparation: digesting the HCT-8 cells labeled by luciferase which are cultured by adherence with pancreatin, and sucking off the pancreatin after reaching the pancreatin digestion time (at the moment, the cell state is a single cell and the adherence is just not fallen); the cells were stopped with 1% serum in PBS at 2 ml/dish, blown down, transferred to a 10ml centrifuge tube, and centrifuged at 1000 Xg for 5 min. Discarding the supernatant, resuspending PBS, and sieving with 100 mesh sieve once; cell count, according to cell: mixing matrigel 6:4, adjusting cell concentration to 5 × 10⁵/ml。

4. Tumor cell inoculation: 100 μ l of the cell suspension was inoculated subcutaneously under the right upper abdomen and near the underarm of nude mice.

5. And (3) observing the growth of the tumor: tumor cells were treated with the polypeptide one week after subcutaneous injection (4mg/kg body weight twice weekly) and tumor size was recorded with a vernier caliper. Tumor volume ═ (length × width)/2;

the results are expressed as mean + -SEM and the differences between the peptide groups and the scarmbled control peptide groups were examined using t test.

A larger tumor volume indicates a faster tumor growth. Subcutaneous tumor volumes in each group of mice 4 weeks after tumor inoculation are shown in tables 12-16.

TABLE 12 Polypeptides inhibiting growth of colon cancer cell HCT-8 in mice

TABLE 13 polypeptide inhibits growth of lung cancer cell A549 in mice

TABLE 14 polypeptide inhibition of growth of hepatoma cells HepG2 in mice

TABLE 15 polypeptide inhibition of pancreatic cancer cell SW1990 growth in mice

TABLE 16 polypeptide inhibits growth of breast cancer cells MDA-MB-231 in mice

The result shows that the polypeptide Pep2-T3A6 can inhibit the growth of HCT-8 colon cancer cells, HepG-2 liver cancer cells, A549 lung cancer cells, SW1990 pancreatic cancer cells and MDA-MB-23 breast cancer cells in mice.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Huzhuowei

<120> polypeptide, derivative thereof and application thereof in preparing medicine for preventing and treating tumors

<130> P1711728C

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> T3A6

<400> 1

Leu Ser Ala Pro Ala Arg Cys Leu Val Arg Cys Leu Leu Arg

1 5 10

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Pep2

<400> 2

His Leu Tyr Val Ser Pro Trp

1 5

<210> 3

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Pep2-T3A6

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Leu Ser Ala Pro Ala Arg Cys Leu Val Arg Cys Leu Leu Arg His Leu

1 5 10 15

Tyr Val Ser Pro Trp

20

<210> 4

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> Pep2-T3A6 DNA

<400> 4

ctgtccgccc ccgcccggtg cctggtgcgg tgcctgctgc ggcacctgta cgtgtccccc 60

tgg 63

<210> 5

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> scarmbled polypeptide

<400> 5

His Leu Tyr Val Ser Pro Trp Gly Gly Leu Arg Val Arg Leu Cys Leu

1 5 10 15

Ala Leu Ser Pro Arg Ala Cys

20

Claims (12)

1. A polypeptide specifically binding to beta-catenin is characterized in that the polypeptide has an amino acid sequence shown as SEQ ID NO: 1.

2. The derivative of the polypeptide specifically binding to beta-catenin according to claim 1, wherein the derivative of the polypeptide is a chimeric peptide formed by connecting the polypeptide specifically binding to beta-catenin according to claim 1 with a membrane-penetrating peptide.

3. The derivative of the polypeptide that specifically binds to β -catenin according to claim 2, wherein the membrane-penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide.

4. The derivative of the polypeptide specifically binding to β -catenin according to claim 2 or 3, wherein the cell-penetrating peptide is Pep2, and the amino acid sequence thereof is shown as SEQ ID NO: 2, respectively.

5. The derivative of the polypeptide that specifically binds to β -catenin according to claim 4, wherein the amino acid sequence of the chimeric peptide is as shown in SEQ ID NO: 3, respectively.

6. An anti-tumor pharmaceutical composition, wherein the active ingredient comprises the polypeptide of claim 1 or a derivative of the polypeptide of any one of claims 2 to 5.

7. The anti-neoplastic pharmaceutical composition of claim 6, wherein the active ingredient is a single active ingredient.

8. The anti-neoplastic pharmaceutical composition of claim 7, wherein the active ingredient further comprises other compounds having anti-neoplastic activity.

9. The anti-tumor pharmaceutical composition according to claims 6 to 8, further comprising a pharmaceutically acceptable carrier, filler, diluent and/or excipient.

10. The anti-neoplastic pharmaceutical composition of claim 9, wherein the route of administration is injection or oral.

11. Use of the polypeptide specifically binding to β -catenin according to claim 1, the derivative of the polypeptide specifically binding to β -catenin according to any one of claims 2 to 5, and the anti-tumor pharmaceutical composition according to any one of claims 6 to 10 for preparing a medicament for treating intestinal cancer, liver cancer, lung cancer, pancreatic cancer, or breast cancer.

12. The use of claim 11, wherein the intestinal cancer is colon cancer or rectal cancer; the liver cancer is primary liver cancer or secondary liver cancer; the lung cancer is non-small cell lung cancer or small cell lung cancer; the pancreatic cancer is pancreatic ductal adenocarcinoma and pancreatic acinar cell carcinoma; the breast cancer is non-invasive breast cancer, early invasive breast cancer, invasive special type breast cancer or invasive non-special type breast cancer.