JP2019131475A - Cancer metastasis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cancer metastasis inhibitor. SOLUTION: A cancer metastasis inhibitor containing at least one selected from the group consisting of cells of lactic acid bacteria belonging to the genus Enterococcus and components of the cells. [Selection diagram] None

Description

  The present invention relates to a cancer metastasis inhibitor and the like.

  According to the 2015 demographic statistics released by the Ministry of Health, Labor and Welfare, the top cause of death is malignant neoplasms for both men and women. A malignant neoplasm is a cell population that has abnormally grown due to DNA damage, infiltrates or metastasizes to surrounding tissues, and causes normal organs to malfunction, and is generally called cancer. According to the latest cancer statistics of the National Cancer Center, there were 370,346 people who died of cancer in 2015 (219,508 men, 150,838 women), and the number of sites with the highest number of deaths was the lung The second place is the large intestine and the third is the stomach.

  Cancer treatment methods include surgical removal of cancer cell mass, chemotherapy that suppresses or kills cancer cells with anticancer drugs, radiation therapy that kills cancer cells with radiation, activation There are immunotherapy in which the immune cells are returned to the body to suppress or kill the growth of cancer cells, antibody therapy in which a target molecule necessary for the growth of cancer is inhibited by a specific antibody, and a therapeutic method using them in combination.

  Surgery to remove the cancer is an effective treatment. If the cancer tissue is completely removed by surgery, the cancer is completely cured. However, if cancer cells deeply infiltrate important tissues and the excision significantly reduces the patient's life and quality of life (QOL), they cannot be completely removed and remain with chemotherapy and radiation therapy. It is done to kill cells.

  Drugs used in chemotherapy include alkylating drugs, antimetabolites, antitumor antibiotics, microtubule inhibitors, hormone analogs, platinum preparations, topoisomerase inhibitors, biologics (cytokines), molecular targeted drugs, non- There are specific immunostimulants and the like. Alkylating agents, antimetabolites, antitumor antibiotics, platinum preparations, and topoisomerase inhibitors are agents that inhibit DNA synthesis and replication and kill cancer cells. Hormone analogs are drugs that have an effect on hormone-dependent tumors. Molecular targeted drugs are drugs corresponding to cancer-specific molecular biological characteristics, including those that inhibit cancer with antibodies and small molecules that act on enzymes to inhibit cancer metabolism. Non-specific immunostimulators suppress cancer cell growth by activating immune cells (Non-patent Document 1). All of them suppress cancer cell growth, but other than molecular targeted drugs, they have a strong influence on normal cells and have a problem of strong side effects.

  There are also reports of using lactic acid bacteria preparations that increase immunity as a treatment with mild side effects. For example, Patent Document 1 discloses a method of continuously ingesting live bacteria of lactic acid bacteria for 10 years or more before the onset in order to reduce the risk of developing breast cancer. Patent Document 2 describes a food containing lactic acid bacteria that suppresses the β-catenin pathway and suppresses the growth of cancer cells. Patent Document 3 describes that an enzyme-treated product of lactic acid bacteria suppresses tumor growth, but does not describe immunostimulation or metastasis suppression.

  Survival of cancer patients has increased due to the combination of surgical resection and chemotherapy, but cancer is still the number one cause of death in Japan. It can be seen that it is not enough for eradication. In particular, the cancer characteristic of metastasis is troublesome. If the cancer affected area expands to multiple places, it will be necessary to extend the operation time and increase the number of times, and the burden on the patient will increase, making it impossible to apply surgery to those with weak physical strength or the elderly. In addition, it is necessary to increase the dosage in chemotherapy, but there are some cases where treatment is dropped from side effects. However, until now, effective drugs and methods that suppress cancer metastasis and facilitate cancer treatment have not been developed.

Japanese Patent Laying-Open No. 2015-178530 JP2013-203669A JP 09-227392 A

Today's treatment (2016 edition)

  An object of the present invention is to provide a cancer metastasis inhibitor.

  As a result of diligent research in view of the above problems, the present inventors have found that at least one selected from the group consisting of bacterial bodies of lactic acid bacteria belonging to the genus Enterococcus and its bacterial cell components has a cancer metastasis-inhibiting action. I found it. Moreover, it discovered that the dead cell obtained by the method including the process by a lytic enzyme and its cell component have a cancer metastasis suppression effect, although the immunostimulatory effect is very low. As a result of further research based on these findings, the present inventors have completed the present invention.

That is, the present invention includes the following embodiments:
Item 1. A cancer metastasis inhibitor comprising at least one selected from the group consisting of lactic acid bacteria belonging to the genus Enterococcus and its bacterial components.
Item 2. Item 2. The cancer metastasis inhibitor according to Item 1, wherein the lactic acid bacterium belonging to the genus Enterococcus is Enterococcus faecalis.
Item 3. Item 3. The cancer metastasis inhibitor according to Item 1 or 2, wherein the lactic acid bacterium belonging to the genus Enterococcus is Enterococcus faecalis NF-1011 strain (patent biological deposit center accession number FERM BP-10902).
Item 4. Item 4. The cancer metastasis inhibitor according to any one of Items 1 to 3, wherein the cell is a dead cell.
Item 5. Item 5. The cancer metastasis inhibitor according to Item 4, wherein the dead cell is a dead cell prepared by a method comprising treatment of live cells with a lytic enzyme.
Item 6. Item 6. The cancer metastasis inhibitor according to any one of Items 1 to 5, wherein the bacterial cell component is a water-soluble component.
Item 7. Item 7. The cancer metastasis inhibitor according to any one of Items 1 to 6, which is an oral preparation form.
Item 8. Item 8. The cancer metastasis inhibitor according to any one of Items 1 to 7, which is a food composition.
Item 9. Item 8. The cancer metastasis inhibitor according to any one of Items 1 to 7, which is a food additive.
Item 10. Item 8. The cancer metastasis inhibitor according to any one of Items 1 to 7, which is a pharmaceutical.

  According to the present invention, a cancer metastasis inhibitor can be provided. Lactic acid bacteria belonging to the genus Enterococcus, which are active ingredients, and their bacterial components are substances that have been ingested as foods. Therefore, according to the agent of the present invention, cancer metastasis can be suppressed while further reducing side effects.

The result of the macrophage activation test of Example 1 is shown. The vertical axis represents the TNF-α concentration in the culture supernatant, that is, the degree of macrophage activation, and the horizontal axis represents the sample added to the medium. The result of the breast cancer cell metastasis suppression test of Example 2 is shown. The vertical axis indicates the number of tumor colonies formed by metastasis, and the horizontal axis indicates the administered sample. The colony count count result of the melanoma cell metastasis suppression test of Example 3 is shown. The vertical axis indicates the number of tumor colonies formed by metastasis, and the horizontal axis indicates the administered sample. The lung photograph of the melanoma cell metastasis suppression test of Example 3 is shown. Black spots indicate colonies that have spread to the lungs.

  In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

  In one embodiment of the present invention, at least one selected from the group consisting of bacterial cells of lactic acid bacteria belonging to the genus Enterococcus and components of the bacterial cells (sometimes referred to as “essential components” in this specification). The present invention relates to a cancer metastasis inhibitor (also referred to as “the agent of the present invention” in the present specification). This will be described below.

  Lactic acid bacteria belonging to the genus Enterococcus are not particularly limited. (Enterococcus gallinarum), Enterococcus flavescens and the like. Among these, Preferably enterococcus faecalis, Enterococcus faecium etc. are mentioned, More preferably, enterococcus faecalis is mentioned. Moreover, among Enterococcus faecalis, preferably Enterococcus faecalis NF-1011 strain (patent biological deposit center accession number FERM BP-10902) which is a strain isolated from feces of healthy individuals.

  The cells of lactic acid bacteria belonging to the genus Enterococcus are not particularly limited as long as they are the entire components of the lactic acid bacteria belonging to the genus Enterococcus, and may be live cells or dead cells. The microbial cell may be a dried product such as a lyophilized product.

  Viable cells of lactic acid bacteria belonging to the genus Enterococcus can be ordered from domestic distribution agencies such as ATCC, IFO and JCM, international distribution agencies, and can also be isolated from organisms. Since these microorganisms are bacteria generally present in foods, bacteria used for food production, or bacteria isolated from the stool of healthy persons, there is no risk of side effects.

  In addition, since it can be easily obtained in a large amount by culturing, the use of viable bacterial cells obtained by culturing is inexpensive and economical. Viable cells of lactic acid bacteria belonging to the genus Enterococcus can also be proliferated by culturing according to a known method. For example, the lactic acid bacteria are seeded in an appropriate amount of a sterilized Rogoza liquid medium, and statically cultured at 35 to 37 ° C. for 10 to 16 hours to obtain a preculture solution. A large amount of viable cells can be obtained by static culture in the same manner as the medium. When using viable cells, for example, the culture solution itself may be used, or the solid content of the culture solution (for example, a precipitate obtained by precipitating viable cells from the culture solution by centrifugation, etc., and then necessary) Depending on the condition, a precipitate obtained by washing with physiological saline or the like may be used, or a suspension obtained by suspending in a suspension of the solid content (for example, an isotonic solution such as physiological saline). A turbid liquid or the like may be used.

  The dead cell of lactic acid bacteria belonging to the genus Enterococcus is not particularly limited, and can be, for example, a cell wall disrupted product of live cells. This cell wall destruction may be the whole cell wall or a part of the living cell body. Examples of the cell wall destruction treatment method include heat treatment, treatment with physical force, treatment with a lytic enzyme, or a combination of these. Among these, a method including treatment with a lytic enzyme is preferable, and at least one selected from the group consisting of (a) treatment with a lytic enzyme, and (b) treatment with heat treatment and physical force is more preferable. And a method including treatment (preferably heat treatment), more preferably (a) at least one treatment (preferably selected from the group consisting of heat treatment and treatment by physical force) after treatment with a lytic enzyme (preferably And a method including performing a heat treatment.

  Although the temperature of heat processing will not be specifically limited if it is 100 degreeC or more, Preferably it can be the temperature (for example, 110-125 degreeC) which can be autoclaved. The heat treatment time is not particularly limited as long as part or all of the cell wall can be destroyed, and can be appropriately set according to the temperature of the heat treatment. The heat treatment time can be, for example, about 1 minute or more, preferably 5 to 20 minutes, more preferably about 5 to 15 minutes.

  The method of treatment by physical force is not particularly limited as long as part or all of the cell wall can be destroyed. For example, an ultrasonic treatment, a French press, etc. are mentioned.

  The enzyme used for the treatment with the lytic enzyme is not particularly limited as long as part or all of the cell wall can be destroyed, and an enzyme that is universally used to lyse bacteria can be widely used. Examples of the lytic enzyme include lysozyme, actinase, zymolyce, kitalase, mutanocillin, achromopeptidase and the like. Among these, lysozyme is preferable. A lytic enzyme may be used individually by 1 type, and may be used in combination of 2 or more type.

  The treatment conditions with the lytic enzyme can be appropriately set according to the type of lytic enzyme, the amount of lysing target (viable cells), and the like. For example, the lytic enzyme may be added to the live cell suspension so as to have a final concentration of 0.01 to 1 mg / mL and treated at 30 to 40 ° C. for 1 to 10 hours.

  The lactic acid bacteria belonging to the genus Enterococcus may be used singly or in combination of two or more.

  The bacterial cell component of the lactic acid bacterium belonging to the genus Enterococcus is not particularly limited as long as it is a component constituting the bacterial cell of the lactic acid bacterium. The bacterial cell component is preferably a water-soluble component. The water-soluble component is obtained, for example, by removing solid content from a water-containing solvent suspension of dead cells by centrifugation or the like.

  The agent of the present invention includes, for example, pharmaceuticals, food compositions [for example, health foods, nutritional supplements (balance nutritional foods, supplements, etc.), nutritional functional foods, and foods for specified health use in various fields. ], And can be used as a food additive.

  The formulation form of the agent of the present invention is not particularly limited, and can be a formulation form usually used in each application field depending on the application field of the agent of the present invention. By making an essential component into a concentrated state as necessary, even a large amount of cells or a processed product thereof can be prepared into an administrable dosage form. Examples of the dosage form include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules Preparations, dry syrups, liquids (including drinks, suspensions, syrups), jelly preparations suitable for oral intake (oral preparation forms), injections, patches, lotions, creams, etc. A formulation form suitable for parenteral intake (parenteral formulation form) is exemplified, and an oral formulation form is preferred. In particular, examples of the food composition include liquid, gel-like or solid foods such as balanced nutritional foods such as gums, confectionery such as tablet confectionery, and beverages such as nutritional beverages, powders, capsules and tablets.

  In addition to the essential components, the agent of the present invention may be appropriately mixed with other components according to the application field, formulation form, and the like. The ingredients that can be blended are not particularly limited, but include, for example, water, amino acids, alcohols, polyhydric alcohols, saccharides, gums, polysaccharides and other high molecular compounds, surfactants, antiseptic / antibacterial / bactericidal agents, In addition to pH adjusters, chelating agents, antioxidants, enzyme components, binders, disintegrants, lubricants, fluidizing agents, cooling agents, minerals, cell activators, nourishing tonics, excipients, increases Sticky agent, stabilizer, preservative, tonicity agent, dispersant, adsorbent, disintegration aid, wetting agent or wetting regulator, moisture-proofing agent, coloring agent, flavoring agent or fragrance, fragrance, reducing agent, Examples include solubilizers, solubilizers, foaming agents, thickeners or thickeners, solvents, bases, emulsifiers, plasticizers, buffers, and brighteners.

  The content of the essential components in the agent of the present invention is not particularly limited because it can be appropriately selected depending on the usage form. An essential amount of the essential component may be blended in the agent of the present invention, but the essential component is usually 0.001 to 60%, more preferably 0.01 to 40% by dry weight with respect to the weight of the agent of the present invention. %, Particularly preferably 0.1 to 30%.

  The amount of application (for example, administration, ingestion, inoculation, etc.) of the agent of the present invention can be appropriately increased or decreased depending on symptoms, patient age, body weight, formulation form and the like. As an essential ingredient per day for adults, 0.001 to 0.5 g / Kg body weight, preferably 0.002 to 0.1 g / Kg body weight can be normally applied, It can be applied in batches or in several batches.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Reference Example 1 Preparation of viable cell samples Enterococcus faecalis NF-1011 strain (patent biological deposit center accession number FERM BP-10902) was inoculated in 10 ml of Rogusa liquid medium and aerobically statically cultured at 37 ° C for 15 hours Pre-culture) to obtain a cell solution (seed) having a cell concentration of about 10 ⁹ cells / ml. This was seeded in 10 L of Rogosa liquid medium (bacterial cell concentration: 10 ⁶ cells / ml) and statically cultured at 37 ° C. for 16 hours to obtain a cell solution having about 10 ⁹ viable cells / ml. . The obtained bacterial liquid is collected by centrifugation (12,000 × g, 20 minutes), washed twice with physiological saline (0.85% aqueous sodium chloride solution), and suspended in 100 ml of distilled water. It became cloudy and a cell suspension was obtained.

Reference Example 2 Preparation of dead cell sample Lysozyme was added to the cell suspension as a live cell sample (Reference Example 1) to a final concentration of 0.1 mg / ml, treated at 37 ° C for 4 hours, and then at 110 ° C. Heat treatment was performed for 10 minutes to obtain a processed bacterial cell product.

Reference Example 3. Preparation of Dead Cell Body Water-Soluble Component Sample The treated cell product, which is a dead cell sample (Reference Example 2), was centrifuged (12000 × g, 15 minutes), and the supernatant was collected.

Example 1. Macrophage activation test Macrophages induced in the abdominal cavity of mice were collected with a 3% thioglycolate medium, and adjusted to 1 × 10 ⁶ cells / ml with RPMI medium (Nissui) containing 10% FCS. In a 96-well cell culture plate, the cells were cultured for 3 hours together with a live cell sample (Reference Example 1) or a dead cell sample (Reference Example 2), and the amount of TNF-α in the culture supernatant was determined by ELISA (RSD TNF- The measurement was performed using an α Mouse DuoSet.

  The results are shown in FIG. When a live cell sample (Reference Example 1) was added, it was confirmed that TNF-α was detected and macrophages were activated, but when a dead cell sample (Reference Example 2) was added, TNF was confirmed. -Α was hardly detected.

Example 2 Breast cancer cell metastasis inhibition test Male BALB / c mice 8 weeks old were divided into 2 groups, physiological saline was used for the control group, and dead cell samples (Reference Example 2) (60 mg / mouse) were used for the test group. It was orally administered every day during the period. On the third day from the start of administration, BJMC3879 mouse breast cancer cells (source: Osaka Medical University) (5 × 10 ⁵ cells / animal) were inoculated from the orbital venous plexus. On day 7 after breast cancer cell inoculation, the lungs were removed and fixed with paraformaldehyde, then embedded in paraffin, sliced, and stained with HE, and the number of tumor colonies on the sections was counted.

  The results are shown in FIG. It was found that the number of cancer cells engrafted in the lung was significantly less in the group administered with the dead cell sample (Reference Example 2).

Example 3 Melanoma Metastasis Inhibition Test Female C57BL / 6L-CAS mice are divided into 2 groups, the control group is physiological saline, the test group is dead cell water-soluble component sample (Reference Example 3) (dead cell sample (reference Example 2) 60 mg / animal) was orally administered daily during the study period. On the third day after administration, melanoma cells (1 × 10 ⁵ cells / animal) were inoculated from the orbital venous plexus. On the 14th day after melanoma cell inoculation, the lungs were removed and fixed with paraformaldehyde, and then clarified by immersion in glycerin. Under a stereomicroscope, photographs of melanoma engrafted in the lung were taken and the number of colonies was counted.

  The results are shown in FIG. It was found that the number of cancer cells engrafted in the lungs was significantly less in the group administered with the dead cell water-soluble component sample (Reference Example 3).

Claims (10)

A cancer metastasis inhibitor comprising at least one selected from the group consisting of bacterial cells of lactic acid bacteria belonging to the genus Enterococcus and bacterial cell components. The cancer metastasis inhibitor of Claim 1 whose lactic acid bacteria which belong to the said Enterococcus genus are Enterococcus faecalis. The cancer metastasis inhibitor according to claim 1 or 2, wherein the lactic acid bacterium belonging to the genus Enterococcus is Enterococcus faecalis NF-1011 strain (patent biological deposit center accession number FERM BP-10902). The cancer metastasis inhibitor according to any one of claims 1 to 3, wherein the cells are dead cells. The cancer metastasis inhibitor according to claim 4, wherein the dead cell is a dead cell prepared by a method including treatment of a living cell with a lytic enzyme. The cancer metastasis inhibitor according to any one of claims 1 to 5, wherein the bacterial cell component is a water-soluble component. The cancer metastasis inhibitor according to any one of claims 1 to 6, which is an oral preparation form. The cancer metastasis inhibitor according to any one of claims 1 to 7, which is a food composition. The cancer metastasis inhibitor according to any one of claims 1 to 7, which is a food additive. The cancer metastasis inhibitor according to any one of claims 1 to 7, which is a medicine.