CN107474115B

CN107474115B - Polypeptide and application thereof in preparing medicine for treating and/or preventing tumors

Info

Publication number: CN107474115B
Application number: CN201610402144.9A
Authority: CN
Inventors: 胡卓伟; 李珂; 王凤
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-06-08
Filing date: 2016-06-08
Publication date: 2020-09-15
Anticipated expiration: 2036-06-08
Also published as: WO2017211317A1; CN107474115A

Abstract

The invention provides a polypeptide and application thereof in preparing a medicament for treating and/or preventing tumors. The amino acid sequence of the polypeptide is shown as a sequence table SEQ ID No.1, or two or more amino acids in the amino acid sequence shown as the sequence table SEQ ID No.1 are replaced by non-natural amino acids with side chains capable of being connected, and the derivatives comprise chimeric peptides formed by connecting the polypeptide and cell-penetrating peptides, fusion peptides formed by the polypeptide and viruses, methylated polypeptides, glycosylated polypeptides and pegylated polypeptides. The polypeptide or the polypeptide derivative can increase the number of PML nucleosomes in a targeted mode, and therefore the polypeptide or the polypeptide derivative can be applied to preparation of medicines for treating and/or preventing tumors.

Description

Polypeptide and application thereof in preparing medicine for treating and/or preventing tumors

Technical Field

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

Background

The tumor suppressor PML (PML) has been a research hotspot of the scientific community due to its association with the pathogenesis and disease progression of Acute Promyelocytic Leukemia (APL). The PML gene located on chromosome 15 and RAR alpha gene located on chromosome 17 are fused together, so as to encode and produce fusion protein PML-RAR alpha, which is the main carcinogenic factor for APL pathogenesis. The PML gene contains 9 exons and spans 53kb throughout the genome. The PML gene forms 6 nuclear and 1 cytoplasmic isoforms of PML due to differences in breakpoints. Wherein PML-I is the longest isomer and comprises 882 amino acids; PML VII is the shortest isomer, consisting of 435 amino acids. The N-terminal 418 amino acids of the PML protein amino acid sequence are common to all isoforms and comprise a number of conserved domains, such as the ring finger domain (R), two cysteine/histidine rich B-Box domains (B) and an alpha helical coiled coil domain (CC), which are also called RBCC domains or TRIM domains. The RBCC domain of PML plays a key role in its biological regulatory function.

PML protein is mainly enriched in nucleus and distributed in chromatin in a dotted structure. Such a punctate structure is named PML nucleosome. PML nucleosomes are heterogeneous in structure and function, and are also dynamic structures whose RBCC domain is critical for the formation of PML nucleosomes. Depending on the cell type, stress status and nutritional status, the size of PML nucleosomes ranges from 0.1 μm to 1 μm, with approximately 5-30 PML nucleosomes per nucleus. In addition, PML nucleosomes are thought to be the site for the aggregation or recruitment of related proteins (e.g., P53, DAXX, etc.). Although many of the proteins present on the PML nucleosome are constitutive, most of the proteins are dynamically altered within the PML nucleosome. PML exerts its regulatory effect by inhibiting or upregulating the function of related proteins (e.g., P53, DAXX, etc.) after their recruitment to the nucleosome by interacting with these proteins. PML nucleosomes can also serve as core sites for the regulation of post-translational modifications of related proteins. For example, PML proteins regulate the expression of related genes by modulating certain transcription factor activities; PML proteins and PML nucleosomes also regulate repair of DNA damage and mediate the growth of telomeres to maintain genomic stability. Thus, PML nucleosomes are capable of modulating various cellular functions, such as participation in apoptosis and cellular senescence, inhibiting cell proliferation, maintaining genomic stability, and antiviral function.

Several studies have demonstrated that the PML protein is a multifaceted protein that, in addition to APL, also functions as tumor suppressor in nonhematopoietic tumors. The tumor-inhibiting effect of PML has been demonstrated in several types of cancer, such as breast, lung, colon, prostate, and bladder cancer. Research shows that the expression level of PML protein in various tumor cells is obviously reduced, and the number of PML nucleosomes is reduced. The over-expression of PML protein can inhibit the proliferation of tumor cell, block cell cycle and promote the aging and apoptosis of tumor cell. In contrast, cells in which the PML gene is knocked out exhibit strong proliferative activity and resistance to apoptosis induced by ultraviolet rays or cytokines. Mice with the PML gene knocked out have also demonstrated a significant upregulation in the incidence of chemocarcinogen-induced tumors. The above studies all indicate that PML is a tumor suppressor and targeting increasing the number of PML nucleosomes is a potential target for tumor therapy. Therefore, the research and development of the matter for increasing the quantity of PML nucleosomes in a targeted manner have good prospect of drug development for inhibiting tumor occurrence and development.

In recent years, the chemical synthesis of highly active and highly selective synthetic polypeptide drugs has become a new research focus. Among them, alpha-helical peptides with stable structure are favored by researchers due to their higher drugability. However, the common alpha helical peptide has poor cell permeability and is easily hydrolyzed by proteases. Therefore, the polypeptide target drugs have a plurality of defects at present. From the above, it is highly desirable to obtain highly active, highly selective synthetic polypeptide drugs that target an increase in the number of PML nucleosomes.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a polypeptide and application thereof in preparing a medicament for treating and/or preventing tumors, aiming at the current situation that a synthetic polypeptide medicament which can increase the number of PML nucleosomes in a targeted manner, has high activity and high selectivity is lacked at present.

Through intensive research and repeated experiments, the inventor of the invention finds that the polypeptide S160 (the amino acid sequence of which is shown in the sequence table SEQ ID No. 1) capable of targeting PML nucleosome formation is obtained, but the biological stability of the polypeptide S160 is lower. This defect of low biostability is directly related to the inability of the polypeptide S160 to stably form the alpha-helical conformation required for activity in solution. Therefore, the inventors have conducted targeted studies and experiments, and found that if an amino acid residue at a specific position in the polypeptide S160 is replaced with an unnatural amino acid to which a side chain can be linked, such as S-pentenylalanine (S5), the modified polypeptide has a stable secondary structure of α -helix, and the modified polypeptide has extremely high affinity, stability against enzymatic hydrolysis, and cell-penetrating property, thereby having extremely high α -helix stability and metabolic stability, and being capable of inhibiting proliferation and metastasis of various tumor cells, and thus being applied to the preparation of a drug for treating and/or preventing tumors. Based on the research work of the inventor, the invention provides the following technical scheme.

One of the technical schemes provided by the invention is as follows: a polypeptide for increasing the number of PML nucleosomes in a targeted mode or a derivative of the polypeptide, wherein the amino acid sequence of the polypeptide is shown as a sequence table SEQ ID No.1, or two or more amino acids in the amino acid sequence shown as the sequence table SEQ ID No.1 are replaced by unnatural amino acids with connectable side chains, and the derivative is a chimeric peptide formed by connecting the polypeptide and a cell-penetrating peptide, a fusion peptide formed by the polypeptide and a virus, a methylated polypeptide, a glycosylated polypeptide or a pegylated polypeptide.

In the invention, the polypeptide with the amino acid sequence shown in the sequence table SEQ ID No.1 is called polypeptide S160.

In the present invention, the unnatural amino acid to which the side chain can be attached is an unnatural amino acid which is conventional in the art, and preferably S-pentenylalanine (S5). In the polypeptide, the number of the substituted amino acids is two, and the positions of the substituted amino acids are the ith position and the (i + 3) th position respectively, or the ith position and the (i + 4) th position, wherein i is more than or equal to 1 and less than or equal to 7, and i is a positive integer.

Among them, the cell-penetrating peptide of the present invention is a cell-penetrating peptide that is conventional in the art as long as it can assist in delivering the polypeptide into a cell to function. Generally, the cell-penetrating peptide is a short peptide molecule consisting of 10-30 amino acids. Preferably, the cell-penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide, more preferably to the N-terminus of the polypeptide. The chimeric peptide of the present invention is a chimeric peptide conventional in the art, and is preferably formed from the polypeptide of the present invention.

The fusion peptide is conventional in the field, as long as the N-terminal hydrophobic conserved region of the polypeptide is an active peptide segment for mediating the virus fusion process, namely the fusion peptide region is contained. Preferably, the fusion peptide undergoes a conformational transition during insertion into the host cell membrane.

The methylation described herein is a methylation routine in the art, and preferably is a process in which a methyl group is catalytically transferred from an active methyl compound (e.g., S-adenosylmethionine) to another compound. The glycosylation described herein is conventional in the art and is preferably a process in which a sugar is transferred to a protein by a glycosyltransferase and forms a glycosidic bond with an amino acid residue on the protein. The pegylation according to the present invention is a pegylation that is conventional in the art, and preferably refers to a process of attaching polyethylene glycol to an active protein molecule by biotechnology, thereby changing the activity thereof.

In the present invention, preferably, the amino acid sequence of the polypeptide is shown in any one of sequence tables SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No. 12.

In the present invention, amino acid substitutions, deletions or additions may be appropriately made in the amino acid sequences shown in the above-mentioned SEQ ID Nos. 1 to 12, as long as the modified amino acid sequence is still capable of increasing the number of PML nucleosomes and maintaining the activity before modification.

The second technical scheme provided by the invention is as follows: the application of a polypeptide for increasing the number of PML nucleosomes in a targeted mode or a derivative of the polypeptide in preparing a medicine for treating and/or preventing tumors.

The tumor of the present invention is a tumor that is conventional in the art. Preferably liver cancer, lung cancer, breast cancer, intestinal cancer or leukemia. Wherein the liver cancer is the conventional liver cancer in the field, and preferably primary liver cancer or secondary liver cancer. The lung cancer is conventional lung cancer in the field, preferably small cell lung cancer or non-small cell lung cancer. The breast cancer is conventional in the art, preferably non-invasive breast cancer, early invasive breast cancer, invasive specific type of breast cancer or invasive non-specific type of breast cancer. The intestinal cancer is a cancer of the intestine as is conventional in the art, preferably colon or rectal cancer. The leukemia is a leukemia conventional in the art, preferably a lymphocytic leukemia or a non-lymphocytic leukemia.

In the present invention, the prevention is a conventional prevention in the art, preferably meaning prevention or reduction of 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.

The third technical scheme provided by the invention is as follows: an anti-tumor pharmaceutical composition comprising the above polypeptide or a derivative of the polypeptide targeted to increase the number of PML nucleosomes as an active ingredient.

In the invention, the active ingredient refers to a compound with the function of preventing or treating tumors. In the pharmaceutical composition, the polypeptide or the derivative of the polypeptide, which is targeted to increase the number of PML nucleosomes, can be used as an active ingredient alone or together with other compounds having antitumor activity.

The route of administration of the pharmaceutical composition of the present invention is preferably injection administration or oral administration. 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.

In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The medicinal carrier is a conventional medicinal carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable medicinal auxiliary material. 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 or the derivative of the polypeptide for increasing the number of PML nucleosomes in a targeted manner, and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

In the present invention, 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.

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 or the polypeptide derivative can increase the number of PML nucleosomes in a targeted mode, and therefore the polypeptide or the polypeptide derivative can be applied to preparation of medicines for treating and/or preventing tumors. The prepared medicine has the advantages of obvious curative effect, less toxic and side effect and safe use in treating and/or preventing tumor diseases.

Drawings

FIG. 1 shows the effect of immunofluorescence to verify the effect of polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 on PML nucleosome number. The ordinate is the average of the number of PML nucleosomes contained in a single cell, in units of one.

FIG. 2 is a graph showing the results of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the growth of hepatoma cell HepG 2. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control is no drug administration group.

FIG. 3 is a graph showing the results of polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the growth of lung cancer cells A549. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control is no drug administration group.

FIG. 4 is a graph showing the results of polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the growth of breast cancer cells MDA-MB-231. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control is no drug administration group.

FIG. 5 is a graph showing the results of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the growth of HCT-7 cells in intestinal cancer. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control is no drug administration group.

FIG. 6 shows the results of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the growth of leukemia cell K562. The abscissa is the time of administration in days. The ordinate is the number of cells in ten thousand. The control is no drug administration group.

FIG. 7 is a graph showing the results of inhibition of migration of hepatoma cell HepG2 by polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11. The ordinate is the ratio of the repaired area after cell scratching in percent. The control is no drug administration group.

FIG. 8 is a graph showing the results of polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting migration of lung cancer cells A549. The ordinate is the ratio of the repaired area after cell scratching in percent. The control is no drug administration group.

FIG. 9 is a graph showing the results of polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the migration of breast cancer cell MDA-MB-231. The ordinate is the ratio of the repaired area after cell scratching in percent. The control is no drug administration group.

FIG. 10 is a graph showing the results of inhibiting migration of HCT-8 cells of intestinal cancer by the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11. The ordinate is the ratio of the repaired area after cell scratching in percent. The control is no drug administration group.

FIG. 11 is a graph showing the results of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibiting the formation of leukemia cell K562 clone. The ordinate is the number of colony formations in units of counts. The control is no drug administration group.

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 Synthesis of the polypeptide

The amino acid sequence of the polypeptide S160 is shown in a sequence table SEQ ID No. 1. The polypeptide S160 was synthesized and purified by Beijing Saibaosheng Gene technology, Inc.

Two unnatural amino acids S-pentenylalanine (S5) were introduced for solid phase polypeptide chain synthesis. And after the synthesis of the solid-phase polypeptide chain is finished, performing olefin metathesis (RCM) cyclization by using ruthenium as a catalyst to obtain the target polypeptide. Finally, the target polypeptide is cleaved from the resin and purified. The above-mentioned steps for solid phase peptide chain synthesis and purification are carried out by Zhongji peptide Biochemical Co., Ltd. Wherein, two S-pentenyl alanines are inserted into the i th and i +4 th positions in the amino acid sequence of the polypeptide A2, so as to obtain the modified polypeptides with different sequences (the amino acid sequences are shown in the sequence table SEQ ID No. 2-SEQ ID No.12), and the specific insertion sites are as follows:

S160：Ala-Lys-Cys-Phe-Glu-Ala-His-Gln-Trp-Phe-Leu-Lys-His-Glu-Ala；

S160-S1：S5-Lys-Cys-Phe-S5-Ala-His-Gln-Trp-Phe-Leu-Lys-His-Glu-Ala；

S160-S2：Ala-S5-Cys-Phe-Glu-S5-His-Gln-Trp-Phe-Leu-Lys-His-Glu-Ala；

S160-S3：Ala-Lys-S5-Phe-Glu-Ala-S5-Gln-Trp-Phe-Leu-Lys-His-Glu-Ala；

S160-S4：Ala-Lys-Cys-S5-Glu-Ala-His-S5-Trp-Phe-Leu-Lys-His-Glu-Ala；

S160-S5：Ala-Lys-Cys-Phe-S5-Ala-His-Gln-S5-Phe-Leu-Lys-His-Glu-Ala；

S160-S6：Ala-Lys-Cys-Phe-Glu-S5-His-Gln-Trp-S5-Leu-Lys-His-Glu-Ala；

S160-S7：Ala-Lys-Cys-Phe-Glu-Ala-S5-Gln-Trp-Phe-S5-Lys-His-Glu-Ala；

S160-S8：Ala-Lys-Cys-Phe-Glu-Ala-His-S5-Trp-Phe-Leu-S5-His-Glu-Ala；

S160-S9：Ala-Lys-Cys-Phe-Glu-Ala-His-Gln-S5-Phe-Leu-Lys-S5-Glu-Ala；

S160-S10：Ala-Lys-Cys-Phe-Glu-Ala-His-Gln-S5-Phe-Leu-Lys-His-S5-Ala；

S160-S11：Ala-Lys-Cys-Phe-Glu-Ala-His-Gln-Trp-S5-Leu-Lys-His-Glu-S5。

example 2 circular dichroism method for detecting alpha helix rate of polypeptide

The alpha helix rate of the polypeptide was measured by circular dichroism spectroscopy (purchased from Jasco, Japan). The polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 prepared in example 1 were dissolved in an aqueous solution, and the on-machine concentration of the circular dichroism chromatograph was adjusted to 1mg/mL, as shown in Table 1. Wherein, the alpha helix ratio refers to the percentage of the number of peptide fragments of the polypeptide which maintain the alpha helix of the secondary structure to the number of peptide fragments of the total polypeptide.

Table 1 shows that the alpha helix rates of the polypeptides S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 are obviously higher than that of the polypeptide S160, and the improvement of the alpha helix rate of the polypeptides S160-S1-S160-S11 can enhance the stability of the polypeptides because the maintenance of the alpha helix rate of the polypeptides is an important index for increasing the stability of the polypeptides.

TABLE 1 circular dichroism method for determining alpha helix rate of polypeptide

Example 3 immunofluorescent staining validation of polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 targeting increases in the number of PML nucleosomes

The specific operation steps are as follows:

1. liver cancer cells in logarithmic growth phase HepG2 (purchased from basic medicine institute of Chinese academy of medical sciences) were collected, and the cell concentration was adjusted with DMEM medium (purchased from Invitrogen, USA) to prepare a 15 ten thousand/mL cell suspension.

2. 1mL of the cell suspension prepared in step 1 was added to a 12-well plate (a cell culture glass disc purchased from Biotechnology Ltd, Haidew, Beijing) and cultured in advance in the 12-well plate, and the cell culture glass disc was replaced with a new medium after 12 hours, and 1. mu.g/mL of each of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 prepared in example 1 was added thereto and cultured for 12 hours.

3. Cell culture medium supernatant was aspirated and washed 3 times with PBS. 4% (v/v) paraformaldehyde fixed for 10 minutes. PBS wash 2-3 times, each time for 5 minutes. Permeabilization with PBS containing 0.5% (v/v) TritonX-100 for 10 min. Further washed 3 times with PBS for 5 minutes each. Blocking solution [ PBS containing 3% (v/v) BSA ] for 15 min and standing at room temperature. PML primary antibody (from Novus) diluted 1: 1000 by volume (PBS dilution) was added and wet-boxed overnight at 4 ℃. The next day PBS was washed 3 times for 5 minutes each. FITC fluorescent-labeled secondary antibody diluted 1: 100 by volume was added to the flask at room temperature for 1 hour. PBS was washed 3 times for 5 minutes each. Nuclei were stained with DAPI (purchased from sequoia kummenseng biotechnology limited, beijing) containing the encapsulated tablet at room temperature for 4 minutes.

4. The number and size of PML nucleosomes in cells were observed using a Leica SP2 confocal fluorescence microscope, FITC excited with a 488nm wavelength laser, and DAPI excited with UV light. The results are shown in table 2 and fig. 1.

Table 2 shows that the polypeptides S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 significantly increase the number of intracellular PML nucleosomes compared to the polypeptide S160, and that the polypeptides S160-S1-S160-S11 enhance the antitumor activity of the PML protein since the formation of PML nucleosomes is a key process by which PML exerts its oncostatin.

TABLE 2 cellular immunofluorescence assay to determine the effect of polypeptides on the number of cellular PML nucleosomes

Example 4 cell counting experiments demonstrated that the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 inhibit the growth of tumor cells

The specific operation steps are as follows:

1. liver cancer cells HepG2 (purchased from the institute of basic medicine of Chinese medical science institute), lung cancer cells A549 (purchased from the institute of basic medicine of Chinese medical science institute), colon cancer cells HCT-8 (purchased from the institute of basic medicine of Chinese medical science institute), breast cancer cells MDA-MB-231 (purchased from the institute of basic medicine of Chinese medical science institute) and leukemia cells K562 (purchased from the institute of basic medicine of Chinese medical science institute) in logarithmic growth phase are collected, the cell concentration is adjusted, 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 medium for HepG2, A549, HCT-8 and MDA-MB-231 cells was DMEM medium and the medium for K562 cells was 1640 medium, all purchased from Invitrogen; culture temperature was 37 ℃ and medium volume was 1mL), and after 12 hours, the medium was replaced with a new one, and 1. mu.g/mL of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 prepared in example 1 were added, respectively. Passages were performed every other day and counted. Growth curves were plotted after 12 days of culture. The experimental results are shown in FIGS. 2 to 6 and tables 3 to 7.

FIGS. 2-6 and tables 3-7 illustrate that the polypeptides S160-S1-S160-S11 are more capable of inhibiting the growth of tumor cells than S160.

TABLE 3 polypeptide inhibition of growth of hepatoma cell HepG2

TABLE 4 polypeptide inhibits growth of Lung cancer cell A549

TABLE 5 polypeptide inhibits growth of breast cancer cells MDA-MB-231

Polypeptide name	Number of cells (ten thousand)
		Control	539.000±39.219
S160	547.333±22.048
		S160-S1	294.000±38.527
S160-S2	327.333±41.176
		S160-S3	194.000±25.515
S160-S4	312.000±28.746
		S160-S5	255.333±20.515
S160-S6	267.000±27.465
		S160-S7	300.333±44.333
S160-S8	333.667±59.150
		S160-S9	207.000±4.583
S160-S10	282.333±42.990
		S160-S11	265.667±34.372

TABLE 6 polypeptide inhibits growth of HCT-7 cells of intestinal cancer

TABLE 7 polypeptide inhibition of growth of leukemia cells K562

Polypeptide name	Number of cells (ten thousand)
		Control	729.000±30.216
S160	714.000±43.301
		S160-S1	275.667±24.592
S160-S2	309.000±46.479
		S160-S3	267.333±26.766
S160-S4	330.333±56.628
		S160-S5	197.000±12.741
S160-S6	230.333±36.608
		S160-S7	297.000±45.044
S160-S8	297.000±45.044
		S160-S9	229.333±27.891
S160-S10	197.000±12.741
		S160-S11	236.555±60.628

Example 5 cell scratch experiment verification the specific operation steps for healing after inhibiting tumor cell scratches by the polypeptide 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. 5 × 10 was added to each well separately⁵The tumor cells are attached after being cultured in a DMEM medium at 37 ℃ in an incubator overnight. The tumor cells are liver cancer cells HepG2, lung cancer cells A549, colon cancer cells HCT-8 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 new medium was added, together with 1. mu.g/mL of the polypeptides S160, S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10, and S160-S11 prepared in example 1.

5. Then put into 5% (v/v) CO at 37 DEG C₂CulturingThe box was incubated, and samples were taken after 24 hours and photographed. The results are shown in FIGS. 7 to 10 and tables 8 to 11.

The results in FIGS. 7 to 10 and tables 8 to 11 show that the larger the area ratio of the lesion repair area, the stronger the migration ability of tumor cells and the stronger the healing ability after cell scratching. Therefore, the polypeptides S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 can reduce the healing capacity of the tumor cells after scratching.

TABLE 8 polypeptide inhibition of hepatoma cell HepG2 migration

TABLE 9 polypeptide inhibits migration of lung cancer cells A549

Polypeptide name	Area ratio of damage repair
		Control	86.00±2.082
S160	45.67±6.064
		S160-S1	21.67±2.404
S160-S2	23.67±4.096
		S160-S3	22.33±1.856
S160-S4	28.33±0.67
		S160-S5	21.33±4.702
S160-S6	22.67±4.256
		S160-S7	18.00±0.5774
S160-S8	27.67±1.202
		S160-S9	22.00±1.732
S160-S10	26.00±1.000
		S160-S11	24.67±2.603

TABLE 10 polypeptide inhibits breast cancer cell MDA-MB-231 migration

TABLE 11 Polypeptides inhibiting migration of HCT-8 in colon cancer cells

Polypeptide name	Area ratio of damage repair
		Control	86.00±5.568
S160	49.00±3.512
		S160-S1	28.33±2.404
S160-S2	33.67±1.856
		S160-S3	22.33±1.856
S160-S4	28.33±0.6667
		S160-S5	28.00±2.082
S160-S6	29.67±6.489
		S160-S7	24.67±3.844
S160-S8	21.00±2.646
		S160-S9	22.00±1.732
S160-S10	26.00±4.933
		S160-S11	28.00±1.528

Example 6 clonogenic experiments validation of polypeptides inhibiting clonogenic of leukemia cells

The operation steps are as follows:

1. laying agar at the lower layer: 5% (w/w) agar was boiled in a water bath to completely melt, cooled to 50 ℃, and 9 times the volume of a 37 ℃ pre-warmed 1640 culture solution (purchased from Invitrogen corporation) was added thereto, mixed well, added to a 24-well plate (0.8 mL per well), and solidified at room temperature for use.

2. Laying upper agar: to 9.4mL of the cell suspension was added 0.6mL of 5% (w/w) agar at 50 ℃ and mixed well, and then added 0.8mL of the agar-plated 24-well plate. Solidifying at room temperature. The number of cells per well was 100. The preparation method of the cell suspension comprises the following steps: leukemia cells K562 were diluted with 1640 medium and adjusted to a concentration of 132 cells/mL.

3. The cells obtained in step 2 were incubated in 1640 medium at 37 ℃ for 3 weeks in an incubator, and the number of formed colonies was counted.

The results are shown in fig. 11 and table 12. The results in Table 12 demonstrate that the level of inhibition of leukemic cell clonogenic by the polypeptides S160-S1, S160-S2, S160-S3, S160-S4, S160-S5, S160-S6, S160-S7, S160-S8, S160-S9, S160-S10 and S160-S11 is significantly increased relative to the polypeptide S160.

TABLE 12 polypeptide inhibition of leukemia cell clonogenic formation

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.

Claims

1. A polypeptide for increasing the number of PML nucleosomes in a targeted manner is characterized in that the amino acid sequence of the polypeptide is shown as a sequence table SEQ ID No. 1; or, as shown in the sequence table SEQ ID No.1, two or more than two amino acids in the amino acid sequence are replaced by unnatural amino acids with connectable side chains, the unnatural amino acid with the connectable side chains is S-pentenoic alanine, and the amino acid sequence of the polypeptide is shown in any one of the sequence tables SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No. 12.

2. The use of the polypeptide for the targeted increase of the number of PML nucleosomes according to claim 1 in the preparation of a medicament for the treatment and/or prevention of a tumor, wherein the tumor is liver cancer, lung cancer, breast cancer, intestinal cancer or leukemia.

3. The use of claim 2, wherein the liver cancer is primary or secondary liver cancer; the lung cancer is small cell lung cancer or non-small cell lung cancer; the breast cancer is non-invasive breast cancer, early invasive breast cancer, invasive special type breast cancer or invasive non-special type breast cancer; the intestinal cancer is colon cancer or rectal cancer; the leukemia is lymphocytic leukemia or non-lymphocytic leukemia.

4. An anti-tumor pharmaceutical composition comprising the polypeptide of claim 1 targeted to increase the number of PML nucleosomes.

5. The pharmaceutical composition of claim 4, further comprising one or more pharmaceutically acceptable carriers.

6. The pharmaceutical composition of claim 4, wherein the polypeptide that targets an increased number of PML nucleosomes is used as an active ingredient.