JP5190849B2 - Antitumor agent for digestive organ cancer - Google Patents

Description

  The present invention relates to an antitumor agent for digestive organ cancer, more specifically, CSF (Competence and Sporulation Factor or Competence and sporulation stimulating factor) produced by probiotics or plantaricin A (plantaricin A) as an active ingredient. It relates to anti-tumor agents and the like.

  Enterobacteria are symbiotic organisms that live in the intestinal tract throughout the life of a host such as a human, and play many roles such as maintaining intestinal homeostasis, building the immune system, and protecting against pathogenic microorganisms. Over 800 types of intestinal bacteria coexist in the human intestinal tract and secrete various physiologically active substances to maintain a close relationship between the host and the intestinal bacteria (see Non-Patent Documents 1 to 3). . Among intestinal bacteria, bacteria that show useful effects on the living body, such as Bacillus subtilis and Lactobacillus, are called probiotics, and include Clostridium difficile and Helicobacter pylori. (Helicobacter pylori) is known to have an antibacterial action against pathogenic bacteria and an intestinal epithelial protective action, and is widely applied to the treatment of inflammatory diseases of the digestive tract (see Non-Patent Documents 4 and 5). In addition, it has been pointed out that abnormalities of the intestinal bacterial flora are involved in the pathogenesis of gastrointestinal tumors, and prophylactic effects are expected (see Non-Patent Document 6). Regarding cancer suppression of probiotics, bacteria themselves and their culture supernatants suppress the growth of human-derived cancer cell lines (see Non-Patent Document 7), and reduce the incidence of tumors in animal models of chemical carcinogenesis ( Non-patent document 8), anti-tumor effect and metastasis-suppressing effect in tumor-transplanted mice (see non-patent document 9) are known. In addition, regarding the cancer suppression mechanism, cytokines such as IFN-γ, IL-1β, and TNF-α are induced to modify the immune function of the host (see Non-Patent Document 9), and genotoxicity is suppressed ( Non-patent document 10), the probiotic cell itself adsorbs chemical carcinogens and mutagens (see non-patent document 11), and probiotics produce carcinogenic promoters and carcinogens by intestinal spoilage bacteria. (See Non-Patent Document 12) has been clarified.

  On the other hand, in recent years, various bacterial phenomena have been explained based on the concept of quorum sensing. Quorum sensing, sometimes translated as a cell density-dependent control mechanism, is defined as a phenomenon in which bacteria senses the density and regulates specific gene expression, and controls communication between bacteria and collective behavior (For example, refer nonpatent literature 13-15 etc.). Such inter-bacterial communication and collective behavior include bioluminescence, collective transfer, conjugative transmission, bacteriocin production for other bacteria, expression of virulence factors for the host, sporulation, biofilm formation, competence, etc. It is considered that a change in gene expression occurs when the signal molecule reaches a minimum threshold (see, for example, Non-Patent Documents 15 and 16).

  For example, in Lactobacillus plantarum C11 strain, when planted at a low bacterial density of 0.1% or less, plantaricin, which is one of bacteriocin, cannot be produced in lactic acid bacteria. Since it was observed that plantaricin production was recovered by adding about 1% of the culture solution during lysine production to the medium, plantaricin A was identified as a plantaricin production inducer contained in the culture solution ( Non-Patent Document 17 and Non-Patent Document 19). For plantaricin A, a bacteriocin-containing preparation as an additive to feed for raising farm animals (see Patent Document 1), and for plantaricin, a method for preparing a flavor component that is stable against the growth of spoilage pathogenic microorganisms (see Patent Document 2) ) Has been proposed for use as a bacteriocin.

  In Bacillus subtilis, it is known that sporulation occurs when glucose in the medium is depleted, but sporulation and competence are controlled by extracellular signal molecules. One of them is called CSF. CSF is thought to stimulate the expression of competence genes by being transported into cells by SpoOK oligopeptide permease and suppressing RapC phosphatase (see Non-Patent Document 18).

JP 2002-262780 A JP 2007-267636 A Eckburg PB, et al. Nature, 1635-1638,2005 Sonnenburg JL, et al. Nature Immunol, 569-573, 2004 Hooper LV, et al. Nature Immunol, 269-273, 2003 Steidler L, Hans W, Schotte L, et al. Science 289; 1352-1355, 2000 Abbott A. Nature 427; 284-286, 2004. Chu FF, EsworthvRS, ChuPG, et al. Cancer Res. 64 (3); 962-8, 2004. L. Baricault et al. Carcinogenesis, 16; 245-252, 1995 B.R.Goldin and S.L.Gorbach, J. Natl.Cancer Inst., 64; 263-265, 198 T.Matsuzaki, Int.J.Immunother., 9; 23-28, 1993 H.Horie et al. Eur.J. Cancer Prev., 12; 101-107, 2003 X.B.Zhang and T.Ohta, Can.J.Microbiol., 39; 841-845, 1993 I.R.Rowland et al. Carcinogenesis, 19; 281-285,1998 Hardman et al, Antonievan Leeuvenhoek, 74,199-210 (1998) Gray, K.M.Trends Microbiol., 5 184-188 (1997) Miller et al., Annual Review of Microbiology Vol.55 165-199 (2001) Bassler et al., Cell, 125, 237-246, (2006) Diep et al., Mol. Microbiol., 18, 631-639, (1995) Solomon et al., Genes and Development 10: 2014-2024, (1996) J. Nakayama, Japanese Journal of Lactic Acid Bacteria, 2-13, 2001

  The specific tumor suppressor produced by probiotics has not been identified, and details of the mechanism of action are unknown. In addition, as a premise that the probiotics are effective, it is necessary that the bacteria themselves engraft in the intestinal epithelium, and that the engrafted bacteria must be in an environment where they can fully act. Due to variations in the intestinal flora, the presence of pathogenic bacteria, the influence of other drugs, etc., the intestinal environment of each individual varies widely. The effect of therapy is not stable (MacFarlan LV. Am J Gastroenterology, 2006). In addition, the current probiotic treatment sometimes requires a large amount of money and special facilities for purification and storage of viable bacteria.

  The subject of the present invention is a safe, inexpensive and effective novel anti-tumor effect comprising a short peptide derived from an intestinal bacterium symbiotic in the human intestinal tract and capable of being easily synthesized chemically. The purpose is to provide new drugs and health foods.

  The present inventors have absorbed a specific active substance such as a peptide secreted by intestinal bacteria called probiotics into the intestinal epithelial cells by the cell membrane transporter of human and mouse intestinal epithelial cells, and thus has an intestinal protective action. Has been identified and various active substances have been identified. Then, in the process of examining various functions in the intestine of the identified active substance, the present inventors happened to have plantaricin A secreted by Lactobacillus plantarum and CSF secreted by Bacillus subtilis. It has been found that the substance has an antitumor effect against, and that the activation of insulin-like growth factor binding protein, which is an antitumor factor, is involved in these substances, and the present invention has been completed.

  That is, the present invention relates to (A) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; (B) one or several amino acids deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing Or a peptide consisting of an added amino acid sequence and having antitumor activity against digestive organ cancer; (C) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing; (D) SEQ ID NO: 2 of the sequence listing A peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having antitumor activity against digestive organ cancer; The present invention relates to an antitumor agent for digestive organ cancer such as colon cancer. Hereinafter, the peptide described in any of (A) to (D) above may be referred to as “the present peptide”. The “peptide having antitumor activity against digestive organ cancer” may be “a peptide having an inducing activity of an insulin-like growth factor binding protein”.

  The present invention also relates to a method of using the peptide to prepare an antitumor agent against digestive organ cancer such as colorectal cancer, a method of administering the peptide to a patient with gastrointestinal cancer such as colorectal cancer, Administering anti-tumor agents against gastrointestinal cancers such as colorectal cancer and the recombinant vectors containing DNA encoding the peptide to patients with gastrointestinal cancers such as colorectal cancer, which contains a recombinant vector containing the encoded DNA as an active ingredient A method, an activator of insulin-like growth factor binding protein containing the peptide as an active ingredient, and the addition of the peptide, which are used for the prevention and treatment of digestive organ cancer such as colorectal cancer. The present invention relates to foods or food materials with a certain indication.

It is the figure which shows the effect of CSF and PBS (control) with respect to the nude mouse which transplanted the human colon cancer origin SKCO-1 cell, and the figure which shows the tumor diameter and the mass of the tumor part in case1 and case2. The tumor diameter and tumor mass in the group to which CSF was administered showed values lower than those in the control, respectively, and it was confirmed that CSF had a colon cancer growth inhibitory effect. It is a figure which shows the tumor diameter and the mass of the tumor part in the photograph which shows the effect of plantaricin A and PBS (control) with respect to the nude mouse which transplanted the human colon cancer origin SKCO-1 cell, and case1 and case2. The tumor diameter and tumor mass in the group to which CSF was administered showed values lower than those in the control, and it was confirmed that plantaricin A had an effect of inhibiting the growth of colorectal cancer. CSF and plantaricin A are added to human colon cancer-derived Caco-2 / bbe cells, the respective culture supernatants are collected, and protein array analysis is performed. The results are shown in FIG. As a result, CSF (see FIG. 3A) and plantaricin A (see FIG. 3B) each specifically induced insulin-like growth factor binding proteins (IGFBPs), which are antitumor factors. I confirmed.

  The antitumor agent for digestive organ cancer of the present invention is not particularly limited as long as it contains the peptide of the present invention as an active ingredient or a recombinant vector containing a DNA encoding the peptide of the present invention as an active ingredient. The method of the present invention is not particularly limited as long as the peptide is used for preparing an antitumor agent for digestive organ cancer such as colorectal cancer, and as an activator of the insulin-like growth factor binding protein of the present invention, There is no particular limitation as long as the peptide is an active ingredient, and the food or food material of the present invention is characterized by the addition of the peptide and is used for the prevention and treatment of digestive organ cancer. There is no particular limitation as long as it is a digestive organ cancer, and examples of the digestive organ cancer include esophageal cancer, stomach cancer, biliary tract cancer, pancreatic cancer, and colon cancer. Among them, colon cancer can be preferably exemplified. That.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is plantaricin A encoded by Lactobacillus plantarum C11plnA consisting of 26 amino acid residues (see Non-Patent Document 17), and SEQ ID NO: 2 in the sequence listing. The peptide consisting of the amino acid sequence shown in is a peptide consisting of the C-terminal 5 amino acid residues of a 40 amino acid long peptide encoded by Bacillus subtilis phrC (see Non-patent Document 18). In the present invention, the “amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing” is, for example, 1 to 5, preferably 1 to 3. More preferably, it means an amino acid sequence in which any number of one amino acid is deleted, substituted or added. Specifically, the N-terminus of plantaricin A consisting of 26 amino acid residues is 3 or 4 The deleted 23-mer and 22-mer peptides can be exemplified (for example, see Anderssen et al. Applied and Environmental Microbiology Vol 64, No 6 2269-2272). In the present invention, the “amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing” is, for example, 1 to 2, preferably 1 An amino acid sequence in which an arbitrary number of amino acids are deleted, substituted or added, particularly an amino acid sequence in which 1 to 2, preferably 1 arbitrary number of amino acids are substituted or added. Furthermore, in the present invention, the “peptide having antitumor activity against digestive organ cancer” refers to a peptide that activates an insulin-like growth factor binding protein and reduces digestive organ cancer such as colon cancer when locally administered. .

  The method for obtaining and preparing the present peptide is not particularly limited, and it can be prepared as a naturally derived peptide, as a chemically synthesized peptide, or as a recombinant peptide produced by a gene recombination technique. When the peptide is obtained as a naturally derived peptide, the present peptide can be obtained by appropriately combining peptide isolation / purification methods from microbial cells expressing the peptide. When the peptide is prepared by chemical synthesis, the peptide can be synthesized according to a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method). Moreover, this peptide can also be synthesize | combined using various commercially available peptide synthesizers. When the present peptide is prepared by a gene recombination technique, the present peptide can be prepared by introducing DNA encoding the peptide into a suitable expression system. Among these, it is preferable to synthesize the peptide according to a chemical synthesis method capable of easily preparing a relatively short peptide according to the present invention in a large amount, and particularly suitable for preparation using a commercially available peptide synthesizer Can be listed.

  When using (administering) an antitumor composition against gastrointestinal cancer containing the peptide as an active ingredient as a pharmaceutical product, conventional pharmaceutically acceptable carriers, binders, stabilizers, excipients, solubilization Various compounding ingredients for preparation such as an agent and a solubilizing agent can be added. Examples of the dosage form include suspensions, powders, granules, capsules and the like. As the administration method, administration may be performed by directly inoculating tumor tissue, for example, intravenously In addition, it can be injected subcutaneously, intramuscularly, intraperitoneally, or the like, or may be administered orally and via the nasopharynx. The dose can be appropriately selected depending on various administration conditions such as prevention or treatment, administration method, severity, and patient age.

  In addition, the method for obtaining and preparing the DNA encoding the present peptide is not particularly limited, and an appropriate probe based on the amino acid sequence information shown in SEQ ID NO: 1 or SEQ ID NO: 2 disclosed in the present specification A target gene can be isolated by preparing primers and using them to screen a cDNA library in which the gene is predicted to exist, or can be prepared by chemical synthesis according to a conventional method. The recombinant vector containing the DNA encoding the peptide of the present invention is not particularly limited as long as it is a recombinant vector capable of expressing the peptide of the present invention. Such a recombinant vector is suitable for expressing DNA encoding the peptide as an expression vector. Can be built by integrating Such an expression vector is preferably one that can replicate autonomously in human cells, or one that can be integrated into the chromosome of human cells, and a promoter, enhancer, terminator at a position where DNA encoding the peptide can be expressed. Those containing a control sequence such as can be suitably used. For the DNA encoding the peptide, a drug delivery system based on normal gene therapy can be used.

  Examples of the food or food material of the present invention include yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, tea, sencha, oolong tea, sports drinks, and other beverages, pudding, cookies, bread, cake, jelly Baked confectionery such as rice crackers, Japanese confectionery such as sheep crab, bread confectionery such as frozen confectionery, chewing gum, noodles such as udon and soba, fish paste products such as kamaboko, ham and fish sausage, miso, soy sauce, dressing , And other seasonings such as sweeteners, dairy products such as cheese and butter, tofu, konjac, other boiled rice, dumplings, croquettes, salads, etc. This food or food material is intended for the food or food material containing the peptide. Therefore, the invention relating to the food or food material of the present invention includes a method of using the food or food material to which the peptide is added as a food or food material for antitumor against digestive cancer, or the peptide of the present invention. Of a method for producing a food or food material for anti-tumor against digestive organs characterized by adding the peptide to the food or food material Is also substantially included.

  The activator of insulin-like growth factor binding protein comprising the peptide of the present invention as an active ingredient is, for example, insulin-like growth factor binding when brought into contact with digestive organ cancer cells such as colon cancer cells in an aqueous medium. It refers to a substance that can induce protein into an aqueous medium, and a commercially available protein array can be used for detection of insulin-like growth factor binding protein. The activator of insulin-like growth factor binding protein of the present invention can be advantageously used as an antitumor agent for digestive organ cancer of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Anti-tumor activity of CSF against human colorectal cancer-derived SKCO-1 cells]
As a test CSF, a peptide consisting of 5 amino acid residues (ERGMT; SEQ ID NO: 2) synthesized by Hokkaido System Science Co., Ltd. was used. After anesthesia of 12-16 week old nude mice, 1 × 10 ⁶ cells / 100 μL colon cancer-derived SKCO-1 cells (ATCC, HTB-39) were transplanted subcutaneously at two sites on the back of nude mice. One of them was directly injected every 48 hours into the site where 100 μM of CSF 200 μL had been transplanted. As a control, 200 μL of PBS (manufactured by GIBCO, product number: 20012-027) was injected into the remaining one site every 48 hours. Four weeks later, each tumor part was excised, the tumor diameter and the mass of the tumor were measured, and a comparative examination of histological changes was performed. As shown in FIG. 1, in any experiment conducted twice (see FIG. 1, case 1 and case 2), the tumor diameter and the tumor mass in the group administered with CSF were lower than the values in the control, respectively. As shown, it was confirmed that CSF has an effect of inhibiting the growth of colorectal cancer.

[Anti-tumor activity of plantaricin A against SKCO-1 cells derived from human colon cancer]
Plantaricin A was synthesized as a test plantaricin A by synthesizing a peptide consisting of 26 amino acid residues shown in SEQ ID NO: 1. Similar to the method of Example 1, human colon cancer-derived SKCO-1 cells were transplanted into two subcutaneous sites on the back of nude mice, and 200 μL of 100 nM of the above-mentioned plantaricin A was transplanted every 48 hours. The site was injected directly. Four weeks later, the tumor was removed and compared with the control. In any of the experiments conducted twice (see FIG. 2, case 1 and case 2), the tumor diameter and the mass of the tumor in the group administered with plantalysin A showed values lower than those in the control, respectively. It was confirmed that ricin A has an effect of inhibiting the growth of colorectal cancer.

[Examination of anti-tumor substance inducing ability of CSF using Caco-2 / bbe cells]
100 nM CSF (diluted with 1 mL of cell culture medium) was added to human colon cancer-derived Caco-2 / bbe cells (ATCC: CRL-2102). Similarly, as a control, 100 nM PBS (diluted with 1 mL of cell culture medium) was added to the Caco-2 / bbe cells. After reacting at room temperature for 24 hours, each culture supernatant was collected, and the peptides contained in the culture supernatant were described as a protein array kit (RayBio (registered trademark) Human Cytokine Antibody Array V, manufactured by RayBiotech). Analysis was carried out using the instructions in the manual, and substances specifically induced by CSF were identified. The result is shown in FIG. 3A. As a result, it was confirmed that CSF specifically induced insulin-like growth factor binding proteins (IGFBPs), which are antitumor factors.

[Study of antitumor substance-inducing ability of plantaricin A using Caco-2 / bbe cells]
As in Example 3, 100 nM plantaricin A (diluted with 1 mL of cell culture medium) was added to Caco-2 / bbe cells derived from human colorectal cancer, and after 24 hours, the culture supernatant was collected and contained in the culture supernatant The peptides were analyzed using the protein array kit. The result is shown in FIG. 3B. As a result, it was confirmed that plantaricin A specifically induced IGFBPs.

  The composition for activating insulin-like growth factor binding protein of the present invention comprising the present peptide such as plantaricin A secreted by Lactobacillus plantarum or CSF secreted by Bacillus subtilis as an active ingredient is an antitumor composition against digestive organ cancer Useful as a product.

Claims (8)

The antitumor agent with respect to digestive organ cancer which uses the peptide in any one of the following (A)-(B) as an active ingredient.
(A) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) a peptide comprising an amino acid sequence in which one amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having antitumor activity against digestive organ cancer; The antitumor agent according to claim 1, wherein the digestive organ cancer is colon cancer. A method of using any of the following peptides (A) to (B) for preparing an antitumor agent against digestive organ cancer.
(A) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) a peptide comprising an amino acid sequence in which one amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having antitumor activity against digestive organ cancer; The method according to claim 3, wherein the digestive organ cancer is colon cancer. The antitumor agent with respect to digestive organ cancer which uses the recombinant vector containing DNA which codes the peptide in any one of the following (A)-(B) as an active ingredient.
(A) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) a peptide comprising an amino acid sequence in which one amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having antitumor activity against digestive organ cancer; 6. The antitumor agent according to claim 5, wherein the digestive organ cancer is colon cancer. An activator for an insulin-like growth factor binding protein comprising any one of the following peptides (A) to (B) as an active ingredient.
(A) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) a peptide comprising an amino acid sequence in which one amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having antitumor activity against digestive organ cancer; The activator according to claim 7, wherein the digestive organ cancer is colon cancer.