JP2013253024A - Inhibitor of vascularization and cell growth - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein and peptide having vascularization inhibition effects, and a peptide inhibiting proliferation of cancer cells.SOLUTION: There are provided an inhibitor of vascularization and cell growth comprising S100A12 or a partial peptide of S100A12, and an inhibitor of vascularization and cell growth comprising an expression vector incorporated with a nucleic acid encoding S100A12 or a partial peptide of S100A12.

Description

  The present invention relates to an angiogenesis and cell growth inhibitor.

Cancer accounts for about 30% of Japanese causes of death, and the development of new cancer therapies is socially desired. So far, many anticancer drugs have been researched and developed and contributed to the progress of cancer treatment. However, it is no exaggeration to say that cancers or tumors are of various types, and the effective drugs differ for each tumor. Therefore, it is indispensable to develop therapeutic methods and therapeutic agents effective for cancer treatment in the future.
In recent years, the S100 protein family has attracted attention in connection with the treatment of cancer or tumors. S100 protein is a calcium ion binding protein having an EF hand motif, and is composed of 20 or more families. S100 protein is known to function in the cell in relation to the maintenance of calcium ion homeostasis, cell proliferation, differentiation and regulation of apoptosis. In addition, its expression has been confirmed in acute and chronic allergic inflammation sites, and it has been reported to attract mast cells and monocytes (Non-patent Document 1).
S100 family proteins are thought to play an important role in physiological activity by binding to divalent cations (calcium, zinc, copper ions) and dimerizing. Initially, S100 protein was identified as binding to RAGE (Receptor for advanced glycation end products), but in recent years, some S100 family proteins have been suggested not to interact with RAGE. Is also present.

S100A12 (calgranulin C, EN-RAGE), a member of the S100 protein family, has an EF hand motif for the calcium ion binding of the helix loop helix (Non-Patent Document 2), and is known as a RAGE ligand that interacts with the C domain of RAGE. (Non-patent Document 3). The RAGE binding domain of S100A12 has an amino acid sequence similar to that of amphoterin (residues 153-180) (Non-patent Document 4). S100A12 exists as a homodimer and is known to be converted into a tetramer or hexamer in the presence of calcium ions or zinc ions. In vivo, S100A12 is abundant in non-dividing neutrophils and accounts for about 5% of the total cytoplasmic protein. In particular, expression is observed in acute and chronic allergic inflammation sites.
So far, the inventors have clarified that S100A12 suppresses the growth and migration ability of LK-2 human lung cancer cells (Non-Patent Document 5), and in developing anticancer agents, the cancer cells of S100A12 Expectations are gathered for its usefulness as a factor having a growth inhibitory action.
However, many details remain unclear regarding the details of the effects of S100A12 on cells or living organisms.

Yan et al. (2008) J Biol Chem. 283, 13035-13043 Delangelica et al. (1994) J Biol Chem. 269, 28929-28936 Xie et al. (2007) J Biol Chem. 282, 4218-4231 Heizmann et al., (1998) Biometals 11, 383-397. Abstracts of the 19th Annual Meeting of the Japanese Society for Cancer Metastasis (p.42) Kanazawa June 16-17, 2010

The inventors found for the first time that S100A12 also has an anti-angiogenic effect. Furthermore, it was clarified that the peptide derived from S100A12 has both a cancer cell growth inhibitory effect and an angiogenesis inhibitory effect.
Then, this invention aims at provision of the peptide which suppresses the proliferation of a cancer cell.
Furthermore, this invention aims at provision of the protein and peptide which have an angiogenesis inhibitory effect.

The inventors of the present application have found that peptides derived from S100A12 (SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10) significantly suppress the growth of lung cancer cells, malignant mesothelioma cells, and gastric cancer cells.
Furthermore, the inventors have S100A12 and peptides derived from S100A12 suppress the proliferation of human vascular endothelial cells, and significantly suppress the proliferation promoting effect of vascular endothelial cells by VEGF, an angiogenesis promoting factor, It has been found that it inhibits the migration ability and lumen formation ability of vascular endothelial cells.
Based on the above findings, the present invention has been completed.

That is, the present invention relates to the following (1) to (9).
(1) An angiogenesis inhibitor and a cell growth inhibitor comprising the S100A12 protein and a pharmaceutically acceptable carrier.
(2) An angiogenesis inhibitor and a cell growth inhibitor comprising a recombinant vector containing a nucleic acid encoding S100A12 protein and a pharmaceutically acceptable carrier.
(3) An angiogenesis inhibitor and a cell growth inhibitor comprising a peptide shown in the following (a) or (b) and a pharmaceutically acceptable carrier.
(A) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 (b) In the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10, one or several A peptide consisting of an amino acid sequence having amino acid substitution, deletion or insertion, and having angiogenesis inhibitory activity and cell growth inhibitory activity (4) a nucleic acid encoding a peptide shown in (a) or (b) below An angiogenesis inhibitor and a cell follicle growth inhibitor comprising a recombinant vector and a pharmaceutically acceptable carrier.
(A) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 (b) In the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10, one or several A peptide comprising an amino acid sequence having an amino acid substitution, deletion or insertion, and having angiogenesis-inhibiting activity and cell-growth-inhibiting activity (5) Angiogenesis inhibition according to any one of (1) to (4) above and A therapeutic agent for a disease caused by angiogenesis, comprising a cell vesicle growth inhibitor.
(6) The therapeutic agent according to (5) above, wherein the disease caused by angiogenesis is diabetic vascular disorder, age-related macular degeneration, psoriasis vulgaris, rheumatoid arthritis, osteoarthritis, or corneal neovascularization.
(7) An anticancer agent comprising the angiogenesis inhibitor and cell follicle growth inhibitor described in (3) or (4) above.
(8) The anticancer agent according to (7), wherein the cancer is lung cancer, malignant mesothelioma, gastric cancer or pancreatic cancer.
(9) The peptide shown in the following (a) or (b).
(A) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10 (b) Substitution, deletion or insertion of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10 A peptide comprising an amino acid sequence having an angiogenesis inhibitory activity and a cell growth inhibitory activity

  By using a drug comprising a peptide derived from S100A12 disclosed in the present invention, treatment of cancer, particularly treatment of lung cancer, malignant mesothelioma, and stomach cancer can be effectively performed.

  Angiogenesis can be suppressed by using an agent comprising S100A12 and a peptide derived from S100A12 of the present invention. Furthermore, treatment of diseases caused by increased angiogenesis, such as diabetic angiopathy (retinopathy), age-related macular degeneration, cancer, psoriasis vulgaris, rheumatoid arthritis, osteoarthritis, corneal neovascularization is effective Can be done automatically.

It is the result of having examined the antitumor effect to a human lung cancer cell (LK2) and a human malignant mesothelioma cell (MSTO-211H). The graph on the left shows the change in tumor volume, and the graph on the right shows the tumor weight of the control and S100A12 administration groups. It is the result of investigating the influence which the peptide of sequence number 3 has on the proliferation of human lung cancer cell line LK2. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of having investigated the influence which the peptide of sequence number 9 (peptide B) and sequence number 11 (peptide C) exerts on the proliferation of human lung cancer cell line LK2. The vertical axis represents the degree of cell proliferation as a relative value. S100A12 was used at a concentration of 100 μg / ml, and the peptide was used at a concentration of 450 μg / ml. P <0.01 It is the result of having investigated the influence which the peptide of sequence number 3 has on the proliferation of human malignant mesothelioma cell line E10. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of having investigated the influence which the peptide of sequence number 3 has on the proliferation of human malignant mesothelioma cell line E1. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of having investigated the influence which the peptide of sequence number 3 has on the proliferation of human malignant mesothelioma cell line A-MESO. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of having investigated the influence which the peptide of sequence number 3 has on the proliferation of human malignant mesothelioma cell line Y-MESO. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of investigating the influence which the peptide of sequence number 3 has on the proliferation of human pancreatic cancer cell line KP2. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of investigating the influence which the peptide of sequence number 3 has on the proliferation of human gastric cancer cell line MKN45. The vertical axis represents the degree of cell proliferation as a relative value. P <0.01 It is the result of investigating the influence of S100A12 on vascular endothelial cells in tumors. It is the data which analyzed the number of blood vessels in a tumor after administration in the experiment of intratumoral administration shown in FIG. This is the result of staining blood vessels with CD31 antibody specific for vascular endothelial cells. The left graph summarizes the number of blood vessels per field of view of the microscope, and the right microscopic image shows an immunostained image of a typical blood vessel. It is the result of investigating the influence of S100A12 on vascular endothelial cells in vivo. Matrigel's paraffin block was subjected to HE staining, DAPI staining, and immunostaining with CD31. It is the result of investigating the influence of S100A12 on vascular endothelial cells in vivo. It is the result of counting the number of cells shown in FIG. It is the result of investigating the influence of S100A12 peptide (KELANTIKNIKDKAVI (SEQ ID NO: 7) on vascular endothelial cells in vivo. The paraffin block of Matrigel was subjected to HE staining, DAPI staining and immunostaining with CD31. It is the result of investigating the influence on the vascular endothelial cell in vivo of S100A12 peptide (KELANTIKNIKDKAVI (sequence number 7)). It is the result of counting the number of cells shown in FIG. It is the result of having investigated about the influence on the proliferation activity of a HUVEC cell (use HuMedia). The upper row shows the growth promoting effect of HUVEC cells by VEGF, the middle row shows the growth promoting effect of HUVEC cells by FGF, and the lower row shows that the growth of HUVEC cells itself is suppressed by S100A12 in a concentration-dependent manner. The vertical axis represents the relative value with respect to the number of cells in the control cell group in which PBS was added instead of S100A12. It is the result of investigating the influence on the migration ability of HUVEC cells (using HuMedia). The left graph shows the effect of S100A12 on the migration ability in the presence of VEGF. The right figure is a photomicrograph showing the state of cell migration to the scratched area. It is the result of having analyzed the lumen formation ability of a HUVEC cell. The graph on the left is obtained by quantifying the area of the lumen posting part from the image obtained by photographing the lumen forming part. The figure on the right is a photomicrograph of the lumen forming part. The examination result regarding the mechanism of the angiogenesis inhibitory effect by S100A12. A shows the outline of the experiment. B shows the results of Western blot analysis of the gene product phosphate shown in the figure.

  The present invention relates to an angiogenesis inhibitor and a cell growth inhibitor comprising S100A12 or a partial peptide of S100A12. Furthermore, the present invention also relates to an angiogenesis inhibitor and a cell growth inhibitor comprising an expression vector incorporating a nucleic acid encoding S100A12 or a partial peptide of S100A12. And an angiogenesis inhibitor and cell growth inhibitor comprising a partial peptide of S100A12 or S100A12, and an angiogenesis inhibitor and cell growth inhibitor comprising an expression vector incorporating a nucleic acid encoding the partial peptide of S100A12 or S100A12 In addition, S100A12 or a partial peptide of S100A12 or an expression vector for expressing them may be reached in vivo (in vivo tissue where cell growth should be suppressed, or intracellular in the tissue) It may contain a carrier or the like necessary to reach (1).

Here, “S100A12” is a protein consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. “Protein consisting of substantially the same amino acid sequence” means about 60% or more, preferably about 70% or more, more preferably about 80% of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, most preferably about 99% amino acid sequence, and has angiogenesis inhibitory activity and cytostatic activity or interacts with RAGE (Receptor for advanced glycation end products) It is a protein.
Alternatively, the protein consisting of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 is 1 in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. Or consisting of an amino acid sequence in which several (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to 5) amino acids have been deleted, substituted or added, and angiogenesis It may be a protein having inhibitory activity and cytostatic activity or interacting with RAGE.

The “partial peptide of S100A12” is a peptide consisting of a part of the amino acid sequence constituting S100A12. For example, it is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10. Are peptides consisting of identical amino acid sequences. “Peptide consisting of substantially the same amino acid sequence” means about 60% or more, preferably about 70% or more, more preferably about 80%, of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, most preferably about 99% amino acid sequence, and a peptide that has angiogenesis inhibitory activity and cytostatic activity or interacts with RAGE.
Alternatively, the peptide consisting of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 is 1 in the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10. Or consisting of an amino acid sequence in which several (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to 5) amino acids have been deleted, substituted or added, and angiogenesis A peptide having inhibitory activity and cytostatic activity or interacting with RAGE may be used.
The peptide consisting of the amino acid sequence of SEQ ID NO: 7 is known as an attractant for mast cells and monocytes (Non-patent Document 1), but this peptide has angiogenesis inhibitory activity and cell growth inhibitory activity. This is the first discovery disclosed by the present invention.

  S100A12 or a salt thereof can be extracted / separated from a cultured cell or tissue derived from an animal (eg, human, rat, mouse, etc.) expressing S100A12 by a conventional technique in the art. Alternatively, it can also be prepared by culturing a transformant containing DNA encoding S100A12 in a state capable of being expressed, and extracting and separating from the culture. When preparing from animal tissues or cells, homogenize the tissues or cells, extract with acid, etc., and combine the resulting extract with various chromatographies such as hydrophobic chromatography, reverse phase chromatography, ion exchange chromatography, etc. Can be isolated and purified.

Moreover, the partial peptide of S100A12 or a salt thereof can be produced by a known peptide synthesis method. The peptide synthesis method may be any method known to those skilled in the art, such as a solid phase synthesis method and a liquid phase synthesis method. That is, the target peptide can be produced by condensing the partial peptide or amino acid that can constitute the polypeptide of the present invention and the remaining portion, and removing the protective group when the product has a protective group. After the synthesis reaction, the partial peptide of the present invention can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, high performance liquid chromatography, recrystallization and the like. Alternatively, the synthesis may be requested from a trader that performs peptide synthesis.
Alternatively, similar to S100A12, it can also be prepared by culturing a transformant containing DNA encoding the S100A12 partial peptide in an expressible state and extracting / separating it from the culture.

In addition, the C-terminus of S100A12 or a partial peptide thereof in the present invention is usually a carboxyl group (—COOH) or a carboxylate (—COO—), and the carboxyl group is an amide (—CONH ₂ ) or ester (—COOR). ) And the like may be chemically modified. Here, as R in the ester, a C _1-6 alkyl group (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl), a C _3-8 cycloalkyl group (for example, cyclopentyl, cyclohexyl), C _1-6 aryl group (for example, phenyl, α-naphthyl), phenyl-C _1-2 alkyl group (for example, benzyl, phenethyl), α-naphthyl-C _1-2 alkyl group (for example, α-naphthylmethyl) and the like Is mentioned. In addition, pivaloyloxymethyl ester, which is widely used as an oral ester, can be used. When S100A12 or a partial peptide thereof has a carboxyl group in the peptide chain other than the C-terminal, those in which the carboxyl group is amidated or esterified are also included in S100A12 or the partial peptide of the present invention. Examples of the ester in this case include the above-mentioned esters. Similarly, the N-terminus of S100A12 of the present invention or a partial peptide thereof is usually an amino group (—NH ₂ ), but the amino group is chemically modified with a C _1-6 acyl group such as a formyl group or an acetyl group. May be. In addition, the glutamyl group produced by cleavage of the N-terminal side in vivo is pyroglutamine oxidized, or a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group) , A guanidino group, etc.) that are chemically modified with an appropriate functional group (for example, formyl group, acetyl, etc.) and those having a sugar chain attached are also included in S100A12 of the present invention or a partial peptide thereof.

In the present invention, the “nucleic acid encoding S100A12” is a protein comprising the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or the amino acid represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. A nucleic acid encoding a protein consisting of an amino acid sequence substantially identical to the sequence. Examples of such a nucleic acid include a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, and a base represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. A nucleic acid that encodes a protein that hybridizes with a complementary strand of a nucleic acid comprising a sequence under highly stringent conditions and has angiogenesis-inhibiting activity, cell growth-inhibiting activity, or interacts with RAGE can also be mentioned.
Furthermore, in the present invention, “nucleic acid encoding a partial peptide of S100A12” means a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10, or SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: The nucleic acid which codes the peptide which consists of an amino acid substantially the same as the amino acid sequence represented by 10. Examples of such a nucleic acid include a nucleic acid having a base sequence represented by SEQ ID NO: 8 encoding SEQ ID NO: 7, and a complementary strand of a nucleic acid having a base sequence represented by SEQ ID NO: 8. And a nucleic acid that encodes a peptide that hybridizes under high stringency conditions with angiogenesis-inhibiting activity and cytostatic activity or interacts with RAGE.

In the present invention, the nucleic acid that can hybridize under stringent conditions is preferably about 70% or more of the base sequence of the complementary strand of the nucleic acid to be hybridized (for example, the nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2), More preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, and most preferably DNA containing a nucleotide sequence having a nucleic acid sequence homology of about 99%.
Here, stringent conditions are hybridization conditions that can be easily determined by those skilled in the art. In general, washing temperature, salt concentration, etc., depending on the length of the nucleic acid to be hybridized. It is a condition of. In general, the longer the nucleic acid to be hybridized, the higher the temperature for proper annealing, and the shorter, the lower the temperature.
Specifically, for example, low stringency conditions include washing in a 0.1 × SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. in the filter washing step after hybridization. To do. Examples of highly stringent conditions include washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step. By making the stringent conditions higher, nucleic acids with high homology can be obtained.

  The nucleic acid encoding the S100A12 protein or a partial peptide thereof is preferably cDNA or the like. However, when the nucleic acid is incorporated into an expression vector designed to receive appropriate splicing in vivo, the nucleic acid includes , Introns and the like may be included. That is, any nucleic acid having a structure capable of expressing S100A12 protein or a partial peptide thereof at a desired site in a living body can be used by using an appropriate expression vector. is there.

  As for the nucleic acid encoding S100A12, it is also possible to obtain known sequence information from a database or the like and obtain an S100A12-encoding nucleic acid derived from a desired tissue or animal species based on the sequence information. Cloning of a nucleic acid encoding S100A12 can be performed based on ordinary knowledge in the art. The nucleic acid encoding S100A12 can be obtained, for example, by preparing a cDNA library or the like from a cell containing the nucleic acid, or using a commercially available cDNA library or the like by a conventional screening method. Alternatively, RNA is prepared from cells expressing the nucleic acid, cDNA is synthesized by reverse transcriptase, and then PCR primers are prepared based on the nucleic acid sequence to amplify the cDNA. May be.

The “cell” in the present invention may be a cancer cell or a normal cell. For example, in the case of a normal cell, it may be a vascular endothelial cell. When the “cell” of the present invention is a vascular endothelial cell, S100A12 or a partial peptide thereof directly suppresses the proliferation of the vascular endothelial cell, or VEGF, which is an angiogenesis promoting factor, or other growth factor (for example, FGF, etc.) ), The proliferation of vascular endothelial cells can be suppressed.
The animal species of the “cell” is not particularly limited, and is, for example, an animal cell, preferably a human, rat, mouse, cow, horse, sheep, chicken, dog, cat or the like, particularly preferably a human cell. It is a cell.

The first embodiment of the present invention is an angiogenesis inhibitor and a cell growth inhibitor comprising S100A12 protein and a pharmaceutically acceptable carrier. For example, in the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, one or several amino acids A protein comprising an amino acid sequence having substitution, deletion or insertion, and having angiogenesis inhibitory activity and / or cell growth inhibitory activity, or an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 An angiogenesis inhibitor and cell growth inhibitor comprising an amino acid sequence having one or several amino acid substitutions, deletions or insertions, and comprising a protein that interacts with RAGE and a pharmaceutically acceptable carrier It is.
The second embodiment of the present invention is a peptide comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10, or the amino acid represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10. A peptide consisting of an amino acid sequence having a substitution, deletion or insertion of one or several amino acids, and having an angiogenesis inhibitory activity and a cell growth inhibitory activity, or SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 is a peptide that consists of an amino acid sequence having substitution, deletion or insertion of one or several amino acids in the amino acid sequence represented by 10 and interacts with RAGE. Furthermore, it is an angiogenesis inhibitor and a cell growth inhibitor comprising the peptide and a pharmaceutically acceptable carrier.

  The amino acid substitution, deletion, insertion or addition may be present in the isolated naturally occurring amino acid sequence of S100A12 or a partial peptide thereof, or alternatively a gene (or a gene encoding S100A12 or a partial peptide thereof) cDNA) may be newly introduced by modification by a technique known in the art. Substitution of a specific amino acid residue can be achieved by using a commercially available kit or the like, for example, by substituting a base by the Guppedduplex method, the Kunkel method, or other known methods or a method analogous thereto.

The third embodiment of the present invention is an angiogenesis inhibitor and a cell growth inhibitor comprising a recombinant vector containing a nucleic acid encoding S100A12 protein and a pharmaceutically acceptable carrier.
The fourth embodiment of the present invention is an angiogenesis inhibitor and a cell growth inhibitor comprising a recombinant vector containing a nucleic acid encoding a partial peptide of S100A12 and a pharmaceutically acceptable carrier.

Since S100A12 and its partial peptide can effectively suppress cell proliferation, they can be effectively used for cancer treatment, particularly treatment for lung cancer, malignant mesothelioma, gastric cancer, pancreatic cancer and the like.
Further, S100A12 and its partial peptide suppress the proliferation of vascular endothelial cells, inhibit the effect of promoting angiogenesis by VEGF, suppress the migration ability of vascular endothelial cells, or form lumens by vascular endothelial cells. By suppressing, angiogenesis can be suppressed. Therefore, S100A12 and its partial peptides are diseases caused by enhanced angiogenesis, such as diabetic angiopathy (retinopathy), age-related macular degeneration, psoriasis vulgaris, rheumatoid arthritis, osteoarthritis, corneal neovascularization It can be used effectively for the treatment.
Accordingly, the present invention includes, as another embodiment, an anticancer agent comprising S100A12 and a partial peptide thereof, or an expression vector containing a nucleic acid encoding S100A12 or a partial peptide thereof, and treatment of a disease caused by angiogenesis. Agent or preventive agent is included.

  Encodes a partial peptide of S100A12, S100A12, or a partial peptide of S100A12 or S100A12 contained in the pharmaceutical as an active ingredient of a pharmaceutical such as a cytostatic of the present invention, an anticancer agent, and a therapeutic agent for diseases caused by angiogenesis An expression vector comprising a nucleic acid may be a physiologically acceptable salt thereof. Examples of the salt include alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, and calcium; ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris when there are many acidic groups. Salts of amines such as (hydroxymethyl) aminomethane, N, N-bis (hydroxyethyl) piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, L-glucamine; or lysine , Δ-hydroxylysine, arginine and other basic amino acids can be formed. When many basic groups are contained, salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, Organic acids such as tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid Or salts with acidic amino acids such as aspartic acid and glutamic acid.

  The medicament of the present invention is an active ingredient S100A12, a partial peptide of S100A12, or a nucleic acid encoding a partial peptide of S100A12 or S100A12, a pharmacologically acceptable salt thereof, a solvate thereof or a solvate thereof. Although the hydrate itself may be administered, it is generally desirable to administer it in the form of a pharmaceutical composition containing the above-mentioned substance as an active ingredient and one or more pharmaceutical additives. As the active ingredient of the medicament of the present invention, two or more of the above substances can be used in combination, and the above pharmaceutical composition contains various cancers or treatment and / or prevention of diseases caused by angiogenesis. It is also possible to mix other active pharmaceutical ingredients.

The type of the pharmaceutical composition is not particularly limited, and the dosage form is an agent suitable for parenteral administration such as injection or infusion such as intravenous injection or intramuscular injection, suppository, transdermal administration, transmucosal administration, etc. In addition to the mold, depending on the case, it may be a dosage form suitable for oral administration such as a tablet, pill, capsule, granule, fine granule, powder, or oral liquid. However, when a dosage form suitable for oral administration is used, it is desirable to particularly consider a composition that suppresses degradation by protease or peptidase.
For liquid preparations such as injections and infusions, it may be prepared as a powdered dosage form such as a lyophilized form and dissolved or suspended in water or other suitable solvent at the time of use. If necessary, it may be dissolved in physiological saline or a glucose solution, and a buffering agent or a preservative may be added.

  A person skilled in the art can appropriately select the type of pharmaceutical additive used for the production of the pharmaceutical composition, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical composition depending on the form of the composition. It is. As an additive for preparation, an inorganic or organic substance, or a solid or liquid substance can be used, and generally it can be blended between 1% by weight and 90% by weight based on the weight of the active ingredient. . Specifically, examples of such substances are lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium. , Ion exchange resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polyso Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.

  In order to produce injections, active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc. Dissolve in distilled water for injection together with an isotonic agent, filter aseptically and fill into ampoules, or add mannitol, dextrin, cyclodextrin, gelatin, etc. . In addition, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like may be added to the active ingredient and emulsified in water to give an emulsion for injection.

  To produce a rectal dosage form, the active ingredient is moistened with a suppository base material such as cacao butter, fatty acid tri, di- and monoglycerides, polyethylene glycol, etc., dissolved, poured into a mold and cooled, or the active ingredient is made of polyethylene. What is necessary is just to coat | cover with a gelatin film | membrane after melt | dissolving in glycol, soybean oil, etc.

  In order to produce an external preparation for skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc., and if necessary, moistened and kneaded to make an ointment, or rosin, alkyl acrylate polymer After being kneaded with an adhesive such as polyalkyl, it is spread on a non-woven fabric such as polyalkyl to obtain a tape.

  In order to produce a solid preparation for oral administration, an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose and polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium, and wet or dry granulate to form granules. In order to produce tablets, these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer, etc., and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also. In order to produce capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.

  The dose and frequency of administration of the medicament of the present invention are not particularly limited, and depending on conditions such as prevention of worsening / development of the disease to be treated and / or purpose of treatment, type of disease, patient weight and age, etc. It is possible to select appropriately according to the judgment. In general, the dose per day for an adult in oral administration is about 0.01 to 1000 mg (active ingredient weight), and can be administered once or several times a day or every few days. it can. When used as an injection, daily doses of 0.001 to 100 mg (active ingredient weight) are preferably administered continuously or intermittently to adults.

  The medicament of the present invention can be prepared as a sustained-release preparation such as a delivery system encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such materials can be readily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE), through a filter of appropriate pore size so as to be of a size suitable for use. Purified by evaporation.

The medicament of the present invention can be included as a pharmaceutical composition in the form of a kit together with instructions for administration in a container or pack. When the pharmaceutical composition according to the present invention is supplied as a kit, different constituents of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. The reason why the components are packaged separately in this way is to enable long-term storage without losing the function of the active component.
Reagents contained in the kit are supplied in any type of container in which the components remain active for an extended period of time, are not adsorbed by the container material, and are not subject to alteration. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle having a stopper that can be penetrated by a hypodermic needle.

  The kit also includes instructions for use. Instructions for using the kit comprising the pharmaceutical composition may be printed on paper or other material and supplied as an electrically or electromagnetically readable medium such as a CD-ROM, DVD-ROM or the like. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

Furthermore, the present invention provides a method for treating a disease, comprising administering to a patient a medicament or pharmaceutical composition comprising S100A12 or a partial peptide thereof.
The treatment method of the present invention includes treatment methods for various cancers and diseases caused by angiogenesis.
Here, “treatment” means to prevent or alleviate the progression and worsening of the pathology of the disease in a mammal that is or may be affected by the disease. It is used to mean a therapeutic treatment aimed at preventing or alleviating the progression and worsening.
The “mammal” to be treated means any animal classified as a mammal, and is not particularly limited. For example, in addition to humans, pet animals such as dogs, cats, rabbits, cows, pigs, sheep , Livestock animals such as horses. Particularly preferred “mammals” are humans.

  Next, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples.

1. Intratumoral administration of S100A12 Intratumoral administration of S100A12 was found to significantly suppress cancer growth in vivo in nude mouse tumor-bearing systems (subcutaneously transplanted human lung cancer cells LK2 and human malignant mesothelioma MSTO-211H cells). It was.
1-1. Antitumor effect on human lung cancer cells LK2 Cultured human lung cancer cells LK2 were 2 × 10 ⁷ cells, 12 nude mice (BALBC NU / NU male 4-5 weeks old, purchased from Japan SLC) (6 control mice, S100A12) The mice were inoculated subcutaneously on the back of 6 mice. After confirming the tumor 7-14 days after inoculation, intratumoral administration of S100A12 (SEQ ID NO: 1, the same applies hereinafter) was started. Every other day, once / day (250 μg / 100 μl PBS) was administered intratumorally. Control mice received 100 μl PBS intratumorally.
Over time, the tumor volume ^{(mm 3) lw 2/2} (l: length, w: width, Unit mm) was calculated and weighed weight excised tumor on day 28.
1-2. Anti-tumor effect on human malignant mesothelioma cells Cultured human malignant mesothelioma MSTO-211H cells, 2 × 10 ⁷ cells, 14 nude mice (BalbC nu / nu male 4-5 weeks old, Japan SLC) (control) The mice were inoculated subcutaneously on the back of seven mice and seven mice with S100A12 administration. After confirming the tumor 7-14 days after inoculation, intratumoral administration of S100A12 was started. Daily, once per day (300 μg / 100 μl PBS) was administered intratumorally for 24 days. Control mice received 100 μl PBS intratumorally.
Over time, the tumor volume ^{(mm 3) lw 2/2} (l: length, w: width, Unit mm) was calculated and weighed weight by the tumors excised at 24 days.

FIG. 1 summarizes the antitumor effects on human lung cancer cells LK2 and human malignant mesothelioma cells. The upper panel shows the anti-tumor effect in human malignant mesothelioma MSTO-211H (N = 7), and the lower panel in human lung cancer cells LK2 (N = 6). The left panel shows the tumor-suppressing effect over time, and the right panel shows the excised tumor weight.
In all cases, S100A12 significantly suppressed tumors (P values are shown in the figure).

2. Identification of active site involved in S100A12 growth inhibition.
Partial peptides of S100A12, KELANTIKNIKDKAVI (SEQ ID NO: 7), KELANTIKNIKDKAV (SEQ ID NO: 9) and ELANTIKNIKDKAVI (SEQ ID NO: 10) are human lung cancer cells LK2, human malignant mesothelioma E10 cells, human malignant mesothelioma E1 cells, The proliferation of human malignant mesothelioma A-MESO cells, human malignant mesothelioma Y-MESO cells and human gastric cancer MKN45 cells was significantly suppressed in vitro.
First, in order to identify the active site involved in the growth inhibition of S100A12, several partial peptides were synthesized, and the growth inhibition effect was examined using cultured LK2 cells. Recombinant S100A12 protein was used as a positive control.

LK-2 cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, 250 ng / ml amphoterin B at 37 ° C., 5% CO2. _The culture was performed under _two conditions. Other human cancer cultured cells were cultured in the same manner. The cells used were human gastric cancer MKN45 and human pancreatic cancer KP2 purchased from Cell Bank. Human malignant mesothelioma A-MESO and human malignant mesothelioma Y-MESO were provided by Dr. Sekido of Aichi Cancer Center. got. In addition, human malignant mesothelioma E10 and human malignant mesothelioma E1 were provided by Dr. Hirota of Internal Medicine, Ehime University.
Cell proliferation was measured using Cell Counting Kit 8 (Dojindo, Kumamoto, Japan). This cell proliferation measurement method is a measurement method using the dehydrogenase activity in living cells as an index, and is a tetrazolium salt WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitro. Phenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt] is reduced to form formazan, and the number of viable cells is measured. 3.0 × 10 ³ LK-2 cells were seeded in a 96-well plate and cultured in 100 μl of RPMI medium per well. Recombinant S100A12 was added and cultured for 48 hours. Thereafter, 10 μl of WST-8 solution was added per well and cultured at 37 ° C. for 4 hours. Absorbance at 450 nm was measured using a spectrophotometer Multiscan JX (Thermo Labsystems, Waltham, Mass.), and the absorbance at 690 nm was used as a reference.

Recombinant S100A12 protein was expressed in E. coli, and the expression product was purified before use. E. coli (BL21) was transformed with the pET28a-S100A12 vector, and the resulting overexpressed E. coli was cultured in 50 ml of LB medium at 37 ° C. for 15 hours. The cultured 50 ml LB medium was put into 1 L of LB E. coli solution, and further cultured at 37 ° C. After culturing for 3 hours, IPTG (Isopropyl-β-D (−)-thiogalactopylanoside) was added to a final concentration of 1 mM and cultured for 5 hours. After 5 hours, the obtained LB medium was centrifuged at 8,000 rpm for 10 minutes to obtain a precipitate. The precipitate was resuspended in 12.5 ml PBS (−) and crushed with a sonicator UP-50H (dr. Heelscher GmbH, Teltow, Germany). The supernatant was obtained by centrifugation at 12,000 rpm for 30 minutes at 4 ° C. To the supernatant, 3 ml of 50% Ni-NTA slurry (equilibrated with PBS (−)) was added, rotated at 4 ° C. for 30 minutes, and centrifuged at 2,800 rpm for 10 minutes. Ni-NTA (protein bound) was washed 5 times with PBS (-) containing 10 mM imidazole and packed in a column. S100A12 protein was eluted stepwise (50 mM, 100 mM, 250 mM, 500 mM imidazole / PBS (−)). Each fraction was sterilized with a filter (Syringe Drive Filter Unit). Endotoxins derived from bacteria were removed from the recombinant protein using EndoTrap blue 1/1. Recombinant protein was dispensed in 300 μl aliquots and stored at −20 ° C. The protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).
The partial peptide of S100A12 was synthesized in the Institute of Medical Science, the University of Tokyo.
As a result of the experiment, a partial peptide consisting of the sequences of SEQ ID NO: 7 (referred to as peptide A), SEQ ID NO: 9 (referred to as peptide B) and SEQ ID NO: 10 (referred to as peptide C) is prominent against human lung cancer cell LK2. The growth inhibitory effect was shown (FIGS. 2 and 3). Furthermore, peptide A also showed a significant growth inhibitory effect on other cancer cells (FIGS. 4 to 9).

3. 3. Effect of S100A12 and S100A12 peptide on vascular endothelial cells 3-1. Inhibitory effect of S100A12 on angiogenesis in tumor First, the angiogenesis inhibitory effect of S100A12 was examined in vivo.
A paraffin block was prepared by fixing formalin-fixed LK2 tumor (refer to the description of 1-1 above for the tumor) and control tumor administered S100A12. After preparing a slice specimen, blood vessel immunohistochemical staining was performed using an antibody of CD31 antigen (anti-CD31 rabbit antibody) expressed in vascular endothelial cells. The number of blood vessels per microscope visual field was counted and statistically processed.

Immunostaining was performed as follows.
The paraffin-fixed sample was immersed in xylene for 3 minutes for 5 minutes, washed with ethanol (washed three times in the order of 100%, 90%, and 80%), and finally washed with running water. The deparaffinized sample was autoclaved (121 ° C., 10 minutes) with 10 mM citrate buffer (pH 6) to activate the antigen. When the temperature of the citrate buffer reached room temperature, it was washed 3 times with PBS and immersed in 3% hydrogen peroxide for 5 minutes for deperoxidase treatment. Hydrogen peroxide was washed with running water, then washed with PBS, and blocked with a nonspecific reaction blocking reagent (Dako) for 10 minutes. After blocking, the sample was reacted with a stock solution of rabbit polyclonal CD31 antibody (ab28365; Abcam) as a primary antibody (4 ° C., 16 hours). The primary antibody was washed 3 times with PBS, and then reacted with Envision-Rabbit (Dako), which is a secondary antibody, at room temperature for 1 hour. After the reaction, it was washed with PBS, reacted with DAB until vascular endothelial cells were stained brown, and washed with running water. After washing, counterstaining was performed with hematoxylin (Dako).

In each tumor section immunostained with the CD31 antibody, the stained blood vessels were photographed with a microscope (50 times) for each of five visual fields. The number of blood vessels in each field of view taken was measured, the average number of blood vessels in 5 fields of view was calculated, and the number of blood vessels per field of view of each tumor was taken. From the number of blood vessels per visual field of each tumor, the average number of blood vessels per visual field of three tumors, standard deviation, and P-Value between the control group and the S100A12 administration group were calculated and evaluated by T-TEST.
As shown in FIG. 10 (left panel), administration of S100A12 significantly reduced the number of new blood vessels as compared to control (p = 0.01). The right panel of FIG. 10 shows a typical blood vessel immunostaining image.

3-2. In vivo angiogenesis inhibitory effect of S100A12 The effect of S100A12 on angiogenesis in vivo was examined by Matrigel plug assay.
The Matrigel plug assay is a method for evaluating angiogenesis by injecting Matrigel mixed with an angiogenesis-inducing factor (VEGF) and a test substance (S100A12 peptide) subcutaneously into a mouse and observing blood vessels formed in the Matrigel. is there.
First, on ice, 50 μl of S100A12 (1 mg / ml) and 50 μl of VEGF (1000 ng / ml) were mixed with 400 μl of Matrigel (BD Biosciences), and the mixture (500 μl) was subcutaneously applied to the back of C57 black mice (5 weeks old male). Injected. Matrigel was collected 21 days after the injection, and after formalin fixation, a paraffin block was prepared, and the section was stained with HE to analyze the state of angiogenesis. In angiogenesis, CD31 specifically expressed in vascular endothelial cells was analyzed by immunohistochemical staining.
Immunohistochemical staining was performed as follows.
The paraffin- fixed sample was immersed in xylene three times for 5 minutes, washed with ethanol (washed three times in the order of 100%, 90%, and 80%), and finally washed with distilled water.
Antigen activation The specimen subjected to deparaffinization treatment was autoclaved (121 ° C., 10 minutes) with 10 mM citrate buffer (pH 6) to activate the antigen. After the temperature of the citrate buffer reached room temperature, it was washed 3 times with PBS.
Blocking was performed with a blocking 5% BSA solution for 30 minutes.
Reaction of primary, secondary antibody, and DAPI Reaction was performed with a rabbit polyclonal CD31 antibody (ab28365; Abcam) stock solution (4 ° C., 16 hours). The primary antibody was washed 3 times with PBS, and then reacted with the secondary antibody, anti-rabbit IgG-FITC (1/200 dilution) (SC-2012; Santa Cruz) for 30 minutes at room temperature. The primary antibody was washed once with PBS, then reacted with DAPI solution (1/500) for 5 minutes, and after the reaction, washed with PBS three times.
When S100A12 was added, the vascular endothelial cell density increased by the addition of VEGF (middle of FIG. 11, FIG. 12) was significantly reduced (lower of FIG. 11, FIG. 12). From the above, it was revealed that S10012A significantly suppresses angiogenesis in vivo. In the DAPI staining in FIG. 11, the color is stained dark blue, but in the black and white drawing, it is stained white. Similarly, CD31 is dyed green in color, but is dyed white in black and white drawings. Merge is a diagram in which DAPI staining and CD31 staining are superimposed, but in a black and white drawing, it is stained white.

3-3. In vivo angiogenesis inhibitory effect of S100A12 peptide The effect of S100A12 peptide (SEQ ID NO: 7) on angiogenesis in vivo was examined by Matrigel plug assay.
First, 50 μl of S100A12 peptide (1 mg / ml) and 50 μl of VEGF (1000 ng / ml) are mixed with 400 μl of Matrigel (BD Biosciences) on ice, and the mixture (500 μl) is subcutaneously in the back of C57 black mice (5 weeks old male). Injected into. Matrigel was collected 21 days after the injection, and after formalin fixation, a paraffin block was prepared, and the section was stained with HE to analyze the state of angiogenesis. In angiogenesis, CD31 specifically expressed in vascular endothelial cells was analyzed by immunohistochemical staining.
The immunohistological staining was performed in the same manner as in S100A12.
When S100A12 peptide was added, the vascular endothelial cell density increased by the addition of VEGF (FIG. 13, middle, FIG. 14) was significantly reduced (FIG. 13, lower, FIG. 14). As in FIG. 11, in the DAPI staining in FIG. 13, the color is stained dark blue, but in the black and white drawing, it is stained white. Similarly, CD31 is dyed green in color, but is dyed white in black and white drawings. Merge is a diagram in which DAPI staining and CD31 staining are superimposed, but in black and white drawings, it is stained white.
From the above, it was revealed that the S10012A peptide significantly suppresses angiogenesis in vivo.

3-4. Effects of S100A12 on the proliferation ability, migration ability, and lumen formation of vascular endothelial cells Next, the effects of S100A12 on the proliferation ability, migration ability, and lumen formation of vascular endothelial cells were examined in vitro.
The cells used in this example are primary umbilical vein-derived cells (frozen HUVEC Strain No. 620760; Kurabo Industries). As the medium, HuMedia-EB2 (Kurabo) was mainly used. Regarding the influence on proliferation, an experiment using Medium 199 (M199; SIGMA) was also conducted. As additional factors, Kurabo's growth factor set (FBS, hEGF (10 μg / ml), hydrocortisone (1 mg / ml), antibacterial agent (gentamicin 50 mg / ml, amphoterin B 50 μg / ml), hFGF (5 μg / ml) , Heparin (10 mg / ml) was used. FBS was used in an amount of 1/50 of HuMedia, and other factors were added 1/1000 of HuMedia. Human VEGF recombinant was purchased from Wako Pure Chemical Industries.

First, the frozen cell solution was warmed in a 37 ° C. water bath, and thawed until a small amount of frozen cells remained, and the container was disinfected with 70% ethanol, and then gently thawed with a 1 ml pipette to completely thaw. 10 μl of the cell solution was taken, and the number of cells was counted with a hemocytometer. At the same time, the remaining cell solution was mixed with 4 times the growth factor set-added HuMedia-EB2. After counting the number of cells, it was adjusted with a growth factor set-added HuMedia-EB2 so as to be 2,500 cells / cm ² or more, and seeded on a plate. Thereafter, cell culture was performed in a 6 cm dish using HuMedia-EB2 to which a growth factor set was added, and subculture was performed using a subculture reagent set of Kurabo Industries.
Cell passage was performed as follows. After removing the culture solution, it was washed once with 1 ml of HEPES buffer, and 1 ml of trypsin / EDTA was added. The added trypsin / EDTA was removed after acclimating to the whole dish, and allowed to stand at room temperature for 1 to 3 minutes, and then observed with a microscope to confirm that the cells were detached. After confirming that the cells were detached, 1 ml of HEPES buffer was added and pipetted, mixed with 1 ml of neutralized trypsin that had been dispensed into a 15 ml tube, and then 1,500 rpm (desktop microcentrifuge 2420 KUBOTA ) For 3 minutes. After centrifugation, the supernatant was aspirated and 2 ml of fresh medium was added to the precipitated cells and pipetted. Cells dissociated by pipetting were counted with a hemocytometer, adjusted with a new medium so as to have a density of 2,500 cells / cm ² or more, and seeded.

3-4-1. Analysis of proliferation activity of HUVEC cells Using 6cm dishes, cells cultured with HuMedia-EB2 supplemented with growth factor set were collected, and the cell concentration was adjusted to 2,000 cells / well with fresh HuMedia-EB2 (containing growth factor set). After the preparation, 100 μl of the prepared cell suspension was seeded on a 96-well plate. After 24 hours, the medium was exchanged with HuMedia-EB2 supplemented with 1% FBS to which S100A12 was added so that the desired concentration (0, 10, 25, 50, 100 μg / ml) was obtained. One hour after the medium exchange, VEGF (final concentration 50 ng / ml), FGF (final concentration 50 ng / ml), or PBS was added. After 48 hours, the number of cells was measured with a cell counting kit.

  As a result, S100A12 (50, 100 μg / ml) suppressed the growth effect of VEGF (50 ng / ml) in a concentration-dependent manner (FIG. 15, upper panel). S100A12 (25, 50, 100 μg / ml) suppressed the proliferation effect of FGF (50 ng / ml) in a concentration-dependent manner (middle panel in FIG. 15). Furthermore, when the influence of S100A12 (10, 25, 50, 100 μg / ml) on the proliferation of HUVEC cells was analyzed, the proliferation of HUVEC cells was suppressed depending on S100A12 (lower panel in FIG. 15).

3-4-2. Evaluation of migration ability of HUVEC cells (scratch assay)
Using 6cm dish, collect cells cultured with growth media set-added HuMedia-EB2, and adjust the cell concentration to 1 × 10 ⁴ cells / well or higher with fresh HuMedia-EB2 (contains growth factor set) 1 ml of the prepared cell suspension was seeded on a 12-well plate. When the cells became confluent, the medium was exchanged with HuMedia-EB2 supplemented with 1% FBS to which S100A12 was added so as to obtain a desired concentration (25, 50, 100 μg / ml). 14 hours after the medium exchange, the cell layer of the plate in which the cells are layered was scratched with a yellow chip (BMbio), and then the medium was exchanged with fresh HuMedia-EG2 (1% FBS) containing each concentration of S100A12. In addition, VEGF (final concentration 50 ng / ml) was added. After 10 hours, formalin fixation was performed, and the cells were stained with CBB. Immediately after scratching and after fixing with formalin, the images were taken with a microscope, and the area of a region where no cells were present was measured with ImageJ from the image to determine the migration rate.
The results are shown in FIG. S100A12 (50, 100 μg / ml) significantly suppressed migration of HUVEC cells in a concentration-dependent manner.

3-4-3. Analysis of the ability of HUVEC cells to form lumens 300 μl of Matrigel (BD Biosciences) was added to a 24-well plate on ice and incubated at 37 ° C. for 30 minutes. Using a 6 cm dish, collect the cells cultured with HuMedia-EB2 supplemented with growth factor set, and after adjusting the cell concentration with fresh HuMedia-EB2 (containing 1% FBS) to 4 × 10 ⁴ cells / well, 500 μl of the prepared cell suspension was seeded. At the time of sowing, VEGF and S100A12 were added. After 14 hours, formalin fixation was carried out and observed under a microscope. In the photographed image, the lumen forming part was colored black with PowerPoint, and the area of the black part was measured with ImageJ (FIG. 17).
As a result, as shown in FIG. 17, S100A12 (100 μg / ml) significantly suppressed the lumen formation of HUVEC cells.

3-5. Next, in order to investigate the mechanism of the angiogenesis inhibitory action by S100A12, the phosphorylation state of the gene product involved in the signal transduction system by VEGF was examined by Western blot analysis.
After culturing the cells in HuMedia supplemented with the growth factor set, the medium was changed to 1% FBS-HuMedia EB2. S100A12 or PBS was added 5 hours after the medium change, and VEGF or PBS was added 1 hour later. Proteins were collected 5 and 15 minutes after addition of VEGF or PBS (see Figure 18A).

As a result of the experiment, S100A12 was found to be phosphorylated (p-VEGFR2, p-p38, p-Akt, p-Erk, p-JNK) (p- is the amino acid shown in FIG. 18B). It was revealed that phosphorylation was suppressed))). This experiment was performed by Western blotting using p-VEGFR2, p-p38, p-Akt, p-Erk, and p-JNK specific antibodies (purchased from CST Japan).
At 5 minutes after addition of VEGF, S100A12 caused the expression levels of p-VEGFR2, p-p38, p-Akt, p-Erk, and p-JNK to be 56%, 54%, 55%, 63%, and 61%, respectively. It was suppressed to. Furthermore, 15 minutes after the addition of VEGF, S100A12 suppressed the expression levels of p-VEGFR2, p-p38, and p-JNK to 42%, 52%, and 48%, respectively. The quantification of the expression level was corrected by the expression level of GAPDH.
The above results fully explain the anti-angiogenic effect of S100A12. In particular, it is known that phosphorylation of p-VEGFR2 is closely related to luminal formation of vascular endothelial cells. However, since S100A12 suppresses phosphorylation of p-VEGFR2, in vivo formation of lumen It is strongly suggested that S100A12 is involved in
In addition, S100A12 did not affect the expression level of GAPDH performed as a control.

  The present invention greatly contributes to the development of therapeutic agents and the establishment of therapeutic methods for cancer or diseases caused by increased angiogenesis.

Claims (9)

  An angiogenesis inhibitor and a cell growth inhibitor comprising the S100A12 protein and a pharmaceutically acceptable carrier.   An angiogenesis inhibitor and cell growth inhibitor comprising a recombinant vector comprising a nucleic acid encoding S100A12 protein and a pharmaceutically acceptable carrier. An angiogenesis inhibitor and cell growth inhibitor comprising a peptide shown in the following (a) or (b) and a pharmaceutically acceptable carrier.
(A) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 (b) In the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10, one or several A peptide comprising an amino acid sequence having amino acid substitution, deletion or insertion, and having angiogenesis inhibitory activity and cell growth inhibitory activity An angiogenesis inhibitor and a cell follicle growth inhibitor comprising a recombinant vector containing a nucleic acid encoding the peptide shown in the following (a) or (b) and a pharmaceutically acceptable carrier.
(A) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 (b) In the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10, one or several A peptide comprising an amino acid sequence having amino acid substitution, deletion or insertion, and having angiogenesis inhibitory activity and cell growth inhibitory activity   A therapeutic agent for a disease caused by angiogenesis, comprising the angiogenesis inhibitor and the cell follicle growth inhibitor according to any one of claims 1 to 4.   The therapeutic agent according to claim 5, wherein the disease caused by angiogenesis is diabetic vascular disorder, age-related macular degeneration, psoriasis vulgaris, rheumatoid arthritis, osteoarthritis, or corneal neovascularization.   An anticancer agent comprising the angiogenesis inhibitor and the cell vesicle growth inhibitor according to claim 3 or 4.   The anticancer agent according to claim 8, wherein the cancer is lung cancer, malignant mesothelioma, gastric cancer or pancreatic cancer. The peptide shown in the following (a) or (b).
(A) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10 (b) Substitution, deletion or insertion of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10 A peptide comprising an amino acid sequence having an angiogenesis inhibitory activity and a cell growth inhibitory activity