CN111358935A - Application of polypeptide in preparing anti-tumor and/or tumor metastasis inhibiting medicine and medicine - Google Patents

Abstract

The invention discloses an application of MEPE derived polypeptides in preparing anti-tumor and/or tumor metastasis inhibition drugs and drugs, wherein the MEPE derived polypeptides are: cyclic peptides obtained by adding one Cys to the N-terminal and the C-terminal of the polypeptide shown in SEQ ID NO. 1 or the polypeptide composed of the 5 th to 19 th amino acids in the amino acid sequence of the polypeptide shown in SEQ ID NO. 1 and cyclizing the added Cys. The polypeptide with the functions of resisting tumor and inhibiting tumor metastasis has the advantages of small molecular weight, easy synthesis, low immunogenicity and the like, and has good treatment effect on tumor and tumor metastasis.

Description

Application of polypeptide in preparing anti-tumor and/or tumor metastasis inhibiting medicine and medicine

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of MEPE derived polypeptides in preparation of anti-tumor and/or tumor metastasis inhibition medicines and medicines.

Background

TDL23 is a peptide containing 23 amino acids derived from extracellular matrix phosphoglycoprotein (MEPE), and has been reported to show that TDL23 can promote the production of bone, cartilage and tooth bone, and can be used for treating bone and cartilage related diseases (see patent documents 1 and 2; non-patent documents 1, 2 and 3), but there is no report that TDL23 is used for anti-tumor and/or inhibiting tumor metastasis. ERG15 is polypeptide derived from TDL23, which is composed of 5 th to 19 th amino acids of TDL23, while cERG15 is formed by adding one Cys to the N-terminal and C-terminal of ERG15 respectively for cyclization, and ERG15 and cERG15 are not reported to be used for resisting tumors and/or inhibiting tumor metastasis.

Documents of the prior art

Patent document

Patent document 1: WO2008091632A2

Patent document 2: CN1458935A

Non-patent document

Non-patent document 1: hayashibara et al, A synthetic peptide fragment of humanMEPE stimulants new Bone formation in vitro and in vivo.J. Bone MinerRes.2004 Mar; 19(3):455-62.

Non-patent document 2: liu H et al, Dentonin, a fragment of MEPE, enhanced dense step cell promotion.J Dent Res.2004 Jun; 83(6):496-9.

Non-patent document 3: sprowson AP et al, ASARM-truncated MEPE and TDL23 enhanced osteogenesis by promoting osteoprogenitor addition.J Orthop Res.2008Sep; 26(9):1256-62.

Disclosure of Invention

Based on this, the present inventors have unexpectedly found that the polypeptide SEQ ID NO: the polypeptides shown in 1-3 (named as TDL23, ERG15 and cERG15 respectively) can not only resist tumors, but also inhibit the metastasis of the tumors.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that: the MEPE derived polypeptide is used in the drugs for resisting tumors and/or inhibiting tumor metastasis, and the MEPE derived polypeptide is:

a) the polypeptide shown as SEQ ID NO. 1 is named TDL 23; or

b) 1, and the polypeptide consisting of amino acids 5 to 19 in the amino acid sequence of the polypeptide shown in SEQ ID NO, is named as ERG 15; or

c) The polypeptide obtained by end modification of the polypeptide consisting of the amino acids at the 5 th to 19 th positions in the amino acid sequence of the polypeptide shown in SEQ ID NO. 1 is named as cERG 15.

Further, the polypeptide ERG15 has the amino acid sequence as shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Further, the polypeptide cERG15 has an amino acid sequence shown in SEQ ID NO. 3.

Further, the medicament is in a liquid dosage form or a freeze-dried powder.

Further, the tumor is one of liver cancer, lung cancer, esophageal cancer, breast cancer, pancreatic cancer, skin cancer, uterine cancer and rectal cancer.

Further, the tumor is liver cancer.

The invention also provides a medicament for resisting tumors and/or inhibiting tumor metastasis, which contains the MEPE-derived polypeptide.

Further, the MEPE-derived polypeptide is the only or main active ingredient in the drug. In order to achieve better therapeutic effect, the MEPE-derived polypeptide of the present invention can be used in combination with commonly used antitumor drugs in the prior art, including but not limited to cisplatin, paclitaxel, gemcitabine, etc.

Further, the medicament is in a liquid dosage form or a freeze-dried powder.

Further, the medicine also comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be routinely selected by those skilled in the art according to the dosage form of the drug and the like.

Further, the effective amount of the MEPE-derived polypeptide in the drug is 0.01 wt% to 99.99 wt%, and when the drug is formulated into a liquid dosage form, the solvent is preferably physiological saline.

The inventor of the invention proves that the SEQ ID NO: 1-3 in a Transwell migration test and an anti-tumor and tumor metastasis test, the polypeptide has the functions of obviously inhibiting the migration of hepatoma cell HCCLM3, reducing the number of lesions of hepatoma cell HCCLM3 metastasized to lung tissues and reducing the tumor volume, and therefore, can be used for treating tumors and resisting the metastasis of tumors.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides polypeptides TDL23, ERG15 and cERG15 with the functions of resisting tumors and inhibiting tumor metastasis, which have the advantages of small molecular weight, easy synthesis, low immunogenicity and the like, and have good treatment effect on tumors and tumor metastasis.

Drawings

FIG. 1 is an HPLC map of a polypeptide of the present invention, wherein FIG. 1A is TDL23, FIG. 1B is ERG15, and FIG. 1C is cERG 15;

FIG. 2 is a graph showing the results of the reduction of the number of liver cancer cell HCCLM3 cells migrated by the polypeptide of the present invention;

FIG. 3 is a graph showing the results of the reduction of the cell mobility of HCCLM3 in hepatocarcinoma cells by the polypeptide of the present invention;

FIG. 4 is a graph showing the results of reduction of tumor volume by the polypeptide of the present invention;

FIG. 5 is a graph showing the results of reducing the number of lung metastasis.

Detailed Description

The present invention will be described in further detail below with reference to specific embodiments of examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

In the examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.

EXAMPLE I Synthesis of polypeptide

The polypeptides TDL23, ERG15 and cERG15 are synthesized by a solid phase synthesis process, the purity is more than 95%, HPLC (high performance liquid chromatography) maps of TDL23, ERG15 and cERG15 are respectively shown in a figure 1A, a figure 1B and a figure 1C, and 500mg is synthesized.

EXAMPLE II Transwell migration test

Human highly metastatic hepatoma carcinoma cells HCCLM3 were purchased from ATCC.

The test method comprises the following steps: resuscitating frozen HCCLM3 hepatocarcinoma cells, adding fresh MEM culture medium containing appropriate amount of serum, and culturing at 37 deg.C in CO₂The incubator is used for culture and passage for standby.

HCCLM3 liver cancer cells in logarithmic growth phase are collected and treated with drugs: culturing the control group in serum-free RMPI-1640 culture medium; the experimental groups were treated with serum-free RMPI-1640 medium containing different concentrations (1, 10, 100. mu.M) of TDL23, ERG15, cERG15, respectively, for 12 hours each.

The cells of each treatment group were collected and counted at 1 × 10⁵The individual cells were resuspended in 100. mu.l serum-free medium and added to the upper chamber of a Transwell cell culture plate, and 600. mu.l complete medium was added to the lower chamber.

37℃、5％CO₂After 24 hours of incubation, the chamber was removed, the cells in the upper chamber were wiped off with a cotton swab, fixed with 4% paraformaldehyde for 20min, washed 1 time with PBS, stained with crystal violet for 10min, washed 1 time with PBS, and the cells were examined microscopically for passage through the wells, if any, other experimental groups were terminated, and photographed for counting the number of passed cells.

And (3) test results: TDL23, ERG15 and cERG15 all inhibited the migration of HCCLM3, which is a liver cancer cell, and showed a decrease in the number of migrated cells and a marked dose dependence, compared to the control group (see FIG. 2). Among them, the inhibitory effects of the medium and high dose groups of TDL23, crerg 15 and the high dose group of ERG15 had very significant statistical significance (P < 0.01), and the inhibitory effect of the medium dose group of ERG15 had significant statistical significance (P < 0.05).

Scratch test

A solution of Fibronectin (FN) 50. mu.l in a concentration of 10. mu.g/ml was added to a 6-well plate, and the mixture was placed in a refrigerator at 4 ℃ overnight, and then RPMI-1640 medium containing 10% FBS was added the next day and sterilized with UV light for 2 hours.

The concentration to be in logarithmic growth phase is about 1 × 10⁶The suspension of HCCLM3 is inoculated into 6-well culture plate, and placed at 37 deg.C and 5% CO₂Culturing in an incubator by a conventional method.

After the cells were observed to grow into a monolayer under an inverted microscope, they were treated with mitomycin for 1 hour to inhibit cell division. The cell plates were scored with a 10. mu.l sterile micropipette tip as straight as possible. The scraped cells were removed by washing 2-3 times with PBS and serum-free medium was added.

Culturing the control group in serum-free RMPI-1640 culture medium; the experimental groups were treated with serum-free RMPI-1640 medium containing different concentrations (1, 10, 100. mu.M) of TDL23, ERG15, cERG15, respectively. The cells were further cultured in a 37 ℃ 5% CO2 incubator, and then sampled 24 hours after the administration treatment, and photographed. The healing of the scratch was observed under an inverted microscope.

Image Pro-Plus 6.0 software measures scratch width of any 3 parts of each group of cell scratches, calculates the cell migration rate (0h scratch width-24 h scratch width)/0 h scratch width to reflect the movement and migration capacity of each group of tumor cells, and performs statistical analysis on the data.

And (3) test results: TDL23, ERG15, creg 15 all reduced the width of the scratch of HCCLM3, which is a liver cancer cell, compared to the control group, thereby reducing the mobility of HCCLM3 cells and showing a significant dose dependence (see fig. 3). The medium and high dose groups of TDL23 and cERG15 and the high dose group of ERG15 have very significant statistical significance (P is less than 0.01) on the reduction effect of the migration rate of HCCLM3 of the liver cancer cells, and the medium dose group of ERG15 has significant statistical significance (P is less than 0.05) on the reduction effect of the migration rate of HCCLM3 of the liver cancer cells.

EXAMPLE III antitumor and anti-metastatic Effect

7-8 weeks old female Balb/c nude mice were inoculated subcutaneously on the right with HCCLM3 cells (5 × 10)⁶Only). 1 week after inoculation, mice with uniform tumor volume were randomly divided into 4 groups, vehicle control groups, and low, medium, and high dose TDL23 groups, each of which contained 15 mice.

Each group of mice was given the corresponding drug (saline, 1, 3, 10mg/kg TDL23) intravenously twice a week. The long and short diameters of the tumor were measured 1 time every other day with calipers and the tumor volume was calculated. Tumor volume was calculated using the following formula: tumor volume (mm)³) Long diameter × (short diameter)²/2。

Animals were sacrificed 10 weeks after treatment, lung tissue was harvested, immediately fixed in 4% paraformaldehyde overnight at 4 ℃, then paraffin embedded, sectioned, and stained with Hematoxylin and Eosin (HE). The lung metastases were counted under microscope.

And (3) test results: treatment with TDL23 at different doses reduced tumor volume compared to vehicle control. After 50 days of treatment, the TDL23 inhibited tumor volume by 35.3%, 55.8%, and 64.8% in the low, medium, and high dose groups, respectively (see FIG. 4), wherein the reduction of tumor volume in the medium and high dose groups was statistically significant (P < 0.01).

TDL23 treatment reduced the number of foci of hepatoma cell HCCLM3 metastasis to lung tissue compared to the control group and exhibited significant dose dependence (see fig. 5). Wherein, the effect of inhibiting the number of the lung metastasis in the medium and high dose groups has very significant statistical significance (P is less than 0.01).

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> Guangzhou Zhicheng medical science and technology Limited

Application of <120> polypeptide in preparation of anti-tumor and/or tumor metastasis inhibition drugs and drugs

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>23

<212>PRT

<213> Artificial sequence

<400>1

Thr Asp Leu Gln Glu Arg Gly Asp Asn Asp Ile Ser Pro Phe Ser Gly

1 5 10 15

Asp Gly Gln Pro Phe Lys Asp

20

<210>2

<211>15

<212>PRT

<213> Artificial sequence

<400>2

Glu Arg Gly Asp Asn Asp Ile Ser Pro Phe Ser Gly Asp Gly Gln

1 5 10 15

<210>3

<211>17

<212>PRT

<213> Artificial sequence

<220>

<221>DISULFID

<222>(1)..(17)

<400>3

Cys Glu Arg Gly Asp Asn Asp Ile Ser Pro Phe Ser Gly Asp Gly Gln

1 5 10 15

Cys

Claims (9)

1. The application of MEPE derived polypeptide in preparing anti-tumor and/or tumor metastasis inhibiting drugs is characterized in that the MEPE derived polypeptide is:

   a) 1, polypeptide shown as SEQ ID NO; or

   b) 1, and the polypeptide consisting of amino acids at positions 5-19 in the amino acid sequence of the polypeptide shown in SEQ ID NO; or

   c) 1, and cyclic peptides which are obtained by adding one Cys to the N-terminal and the C-terminal of the polypeptide consisting of the amino acids at the 5 th to 19 th positions in the amino acid sequence of the polypeptide shown in SEQ ID NO. 1 and cyclizing the Cys.
2. 2. The use of claim 1, wherein the polypeptide of b) has the amino acid sequence as set forth in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
3. 3. The use according to claim 1, wherein c) said polypeptide has the amino acid sequence shown as SEQ ID NO 3.
4. 4. Use according to any one of claims 1 to 3, wherein the medicament is in liquid form or in lyophilized powder form.
5. 5. The use according to any one of claims 1 to 3, wherein the tumour is one of liver cancer, lung cancer, oesophageal cancer, breast cancer, pancreatic cancer, skin cancer, uterine cancer and rectal cancer.
6. 6. The use of claim 5, wherein the tumor is liver cancer.
7. 7. An anti-tumor and/or tumor metastasis suppressing agent comprising the MEPE-derived polypeptide according to any one of claims 1 to 7.
8. 8. The medicament of claim 7, wherein the MEPE-derived polypeptide is the sole or primary active ingredient in the medicament.
9. 9. The medicament of claim 7 or 8, wherein the medicament is in a liquid dosage form or a lyophilized powder.

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