KR20200115381A - Faecalibacterium sp. and anti-cancer composition comprising the same - Google Patents

Abstract

The present invention provides a Faecalibacterium sp. microorganism. In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or a food composition for preventing or alleviating cancer, all of which comprise one or more selected from the group consisting of a fungus body of a Faecalibacterium cancerinhibens microorganism having anticancer activity, a culture of the microorganism, a pulverized material of the microorganism, and an extract of the microorganism.

Description

Microorganisms of the genus Faecalibacterium and an anticancer composition comprising the same {Faecalibacterium sp. and anti-cancer composition comprising the same}

The present invention relates to a microorganism of the genus Pacalibacterium having anticancer activity, and an anticancer composition comprising the microorganism, a pharmaceutical composition for preventing or treating cancer, or a food composition for preventing or improving cancer.

Microbiome refers to microorganisms existing in a specific environment and the entire genetic information. In addition to the human body, microbiome information is being used in various fields such as animals, agriculture, the ocean, and the environment. In particular, with the development of human microbiome research based on advances in genetic information analysis and data analysis technology, The diagnostic industry and healthcare industry are expected to grow.

Human intestinal microbes not only decompose various substances that human enzymes cannot decompose and convert them into nutrients that can be absorbed by human cells, as well as inhibit the growth of externally-derived harmful bacteria to prevent pathogen infection. These intestinal bacteria themselves or various metabolites secreted by the bacteria stimulate numerous immune cells present in the intestinal cells to activate or regulate the human immune response. In addition, the human microbiome is regarded as the second genome of humans due to the number and diversity of symbiotic microbial genes that are more than 100 times that of humans, and its importance is recognized.

In particular, due to the development of human microbiome research, research results showing that intestinal microbes are directly or indirectly related to a number of human diseases are increasing rapidly, and research showing that intestinal microbes are also related to various cancers of the human body is also increasing. Conventional cancer treatment involves a combination of chemotherapy, surgery, hormone therapy, and/or radiation therapy to remove neoplastic cells in a patient. Cancer chemotherapy is preferably based on the use of drugs that kill replicating cells faster than the agent kills normal cells in the patient. Surgery is used to remove the tumor mass, but once the cancer has metastasized, it has little effect. Radiation is only effective in localized areas. All of these approaches present significant drawbacks and additional risks, such as increased susceptibility to infection.

Chemotherapy drugs often cause two complications that require treatment with antibiotics, which can eventually lead to dysbiosis: mucositis (a wound at the weakened mucosal gate associated with bacterial translocation) and neutropenia. do. Thus, there is a strong need for the development of improved cancer treatments that support constructive interactions between immunity and treatments such as chemotherapy and/or radiation, even if not synergistic effects.

An example of the present invention is to provide a microorganism of the genus Faecalibacterium having anticancer activity.

An example of the present invention is a microorganism of the genus Faecalibacterium having anticancer activity; A culture of the microorganism; Lysate of the microorganism; And one or more extracts selected from the group consisting of the microorganisms, cultures and lysates; to provide an anticancer composition comprising at least one selected from the group consisting of, for example, a pharmaceutical composition for preventing or treating cancer.

Another example of the present invention is the microbial cells of the genus Faecalibacterium having anticancer activity; A culture of the microorganism; Lysate of the microorganism; And one or more extracts selected from the group consisting of the microorganisms, cultures and lysates; to provide an anticancer food composition comprising at least one selected from the group consisting of, for example, a food composition for preventing or improving cancer.

In order to achieve the object of the present invention, the present inventors studied the genus Faecalibacterium , which is a genus constituting the major bacteria in the intestine, to isolate and identify new microorganisms, and the isolated microorganisms of the genus Paecalibacterium are cancerous tumors in a mouse model. The present invention was completed by confirming that it has a production and proliferation inhibitory activity.

Hereinafter, the present invention will be described in more detail.

An example of the present invention is a microorganism of the genus Faecalibacterium having anticancer activity, specifically Faecalibacterium cancerinhibens, and more specifically Faecalibacterium cancerinhibens , CLCC1. About. More specifically, Faecalibacterium according to the present invention Cancerinhibens are characterized by having anti-cancer activity, for example anti-cancer activity against colon cancer and/or liver cancer.

In detail, the microorganisms of the genus Pacalibacterium of the present invention may be isolated from the intestinal flora. The microorganism of the genus Faecalibacterium having anticancer activity according to the present invention, more specifically the microorganism of Faecalibacterium cancerinhibens , has at least one or more of the following characteristics:

(1) Anti-cancer activity, specifically preventing the occurrence of cancer, delaying or inhibiting the growth rate of a tumor, reducing the size or volume of a tumor, or inhibiting or delaying metastasis of cancer, one example is Faecalibacterium cancerinhibens is an activity of reducing tumor volume to 10 to 90% when administered to a mouse subcutaneously transplanted with a cancer cell line, compared to a control group without administration of the microorganism,

(2) 16S rRNA having a nucleotide sequence of SEQ ID NO: 1 or a 16S rRNA comprising a nucleotide sequence of 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more of nucleotide sequence identity thereto. Having,

이거나, 혐기 조건 하에서 배양되는 것인,(3) Culture temperature of microorganisms 20 to 40

Or that is cultured under anaerobic conditions,

(4) Lipid production pattern, specifically 17:0 iso 3OH fatty acids are not included, and more specifically, 15:1 w8c fatty acids, 19:0 cyclo w10c/19w6 fatty acids, 20:1 w9c fatty acids, 14:0 3OH/ 16:1 iso I fatty acids, 15:0 3OH fatty acids, 16:0 iso fatty acids and 16:0 iso 3OH fatty acids, including one or more fatty acids selected from the group consisting of, in particular Faecalibacterium contains PL3, which is not in prausnitzii , and

(5) Production of butyric acid, specifically Faecalibacterium Has 2 to 50 times higher butyric acid content than prausnitzii .

Faecalibacterium according to the invention Genus microorganisms, for example It has anticancer activity of Faecalibacterium cancerinhibens , for example, it can prevent the occurrence of cancer, delay or inhibit the growth rate of the tumor, reduce the size or volume of the tumor, or have the activity of inhibiting or delaying metastasis of cancer. , Is not limited thereto. Examples of specific anticancer activity may include an activity of inhibiting and/or delaying metastasis of cancer due to inhibition of tumor growth rate. The tumor or cancer includes both gastrointestinal cancer and non-gastrointestinal cancer, and non-gastrointestinal cancer includes all other cancers except gastrointestinal cancer, and includes, for example, gastrointestinal cancer.

Specifically, at least about 10%, 20%, 30 compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same subject prior to treatment, or compared to the corresponding activity in another subject not receiving treatment. %, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% can have the activity of reducing the tumor size, the number of cancer cells, or the growth rate of the tumor. In one embodiment of the present invention, the colon cancer cell line-transplanted mouse administered with a Faecalibacterium cancer inhibis microorganism has a tumor volume of about 30 to 40% reduced compared to a mouse not administered with the microorganism. It was confirmed that the microbial effect of reducing the size of the tumor and inhibiting the growth rate of the tumor on colon cancer.

In the present invention, “cancer” refers to the physiological condition of an animal, typically characterized by abnormal or uncontrolled cell growth. For example, cancer and cancer pathologies include metastasis, interference with normally functioning surrounding cells, release of cytokines or other secreted products at abnormal levels, suppression or augmentation of inflammatory or immunological responses, neoplasia, precancer ( premalignancy, malignancy, surrounding or distant tissues or organs, such as lymph node invasion. In the present invention, the cancer may be gastrointestinal cancer and non-gastrointestinal cancer, and non-gastrointestinal cancer is cancer excluding gastrointestinal cancer and includes gastrointestinal cancer.

The gastrointestinal cancer is a malignant tumor that occurs in the gastrointestinal tract or the gastrointestinal tract, such as the esophagus, stomach, small intestine, or large intestine, and is found to be advanced enough to make complete resection of the cancer impossible in many cases because most of it has no subjective symptoms. Belongs to poor cancer. The gastrointestinal cancer or gastrointestinal cancer may be, for example, one or more cancers selected from the group consisting of esophageal cancer, gallbladder cancer, liver cancer, biliary tract cancer, pancreatic cancer, gastric cancer, small intestine cancer, colon cancer, colon cancer, anal cancer, and rectal cancer, but are limited thereto. Not, and in one example, it may be colon cancer and/or liver cancer.

The non-gastrointestinal cancer includes malignant tumors arising from tissues other than the gastrointestinal tract by excluding the gastrointestinal tract, for example, leukemia, prostate cancer, breast cancer, bladder cancer, kidney cancer, multiple myeloma, cervical cancer, thyroid cancer, ovarian cancer. , Urethral cancer, osteosarcoma, glioblastoma, brain tumor or lymphoma, but is not limited thereto.

In more detail, the anticancer activity according to the present invention may be an anticancer activity against colon cancer and/or liver cancer, for example, may be an activity that inhibits the occurrence or progression of liver cancer and/or colon cancer. Specifically, it may be to delay the occurrence of the tumor or inhibit the growth rate of the tumor, and may further have an activity to prevent metastasis of cancer due to the inhibition of the growth rate of the tumor.

In one embodiment of the present invention, the liver cancer means all types of malignant tumors occurring in the liver, preferably hepatocellular carcinoma (HCC) or intrahepatic bile duct cancer (Cholangiocarcinoma, CC), and more preferably liver It may be one or more cancers selected from the group consisting of blastoma, cholangiocarcinoma, cholangiocystic adenocarcinoma, or liver cancer resulting from viral infection. Hepatocellular carcinoma is the most major histological subtype, accounting for 70-85% of primary liver cancer. In one embodiment of the present invention, the liver cancer may be liver cell carcinoma (hepatoma).

In one embodiment of the present invention, the colon cancer includes malignant tumors occurring at one or more sites selected from the group consisting of ascending colon, transverse colon, descending colon, sigmoid colon and rectal mucosa, for example, colorectal cancer carcinoma). The colon cancer may be one or more types selected from the group consisting of adenocarcinoma, lymphoma, malignant carcinoma, leiomyosarcoma, Kaposi's sarcoma, and squamous cell carcinoma, but is not limited thereto.

Faecalibacterium according to the invention The genus microbes, In a human cancer cell line, a mouse model in which a cancer cell line was subcutaneously transplanted, or a mouse model in which a cancer cell line was transplanted into the tissue, isolated from the feces of a healthy adult male, the effect of inhibiting cancer development and the growth of carcinoma was confirmed. In one embodiment of the present invention, Faecalibacterium from feces of a healthy adult male The cancerinhibens CLCC1 microorganism was isolated to confirm the effect of inhibiting the growth of tumors in mice induced or transplanted with colon cancer or liver cancer.

Faecalibacterium provided by the present invention Genus microorganisms, for example Faecalibacterium cancerinhibens has an activity of inhibiting tumor growth in a cancer cell line or a cancer cell line transplanted animal model, and the tumor growth inhibition may delay or inhibit the growth rate of a tumor, or reduce the size or volume of a tumor. In addition, the microorganism may have an activity of inhibiting the occurrence of tumors or inhibiting or delaying metastasis of cancer. The tumor growth inhibitory activity can be measured, for example, using the volume/size of the tumor or the weight of the tumor.

Faecalibacterium provided by the present invention When cancerinhibens was administered to an animal model in which a cancer cell line was subcutaneously transplanted, the tumor growth inhibition rate according to the tumor weight was 5% or more, 10% or more, 15% or more, 20% or more, 25% compared to the control group to which the microorganism was not administered. It may be greater than or equal to 30%, and the tumor growth inhibition rate according to the weight of the tumor may be calculated by Equation 1 below.

In Equation 1, IR is the tumor growth inhibition rate (IR) according to the tumor weight, T1 is the average tumor weight in each experimental group, and C1 is the average tumor weight in the negative control group.

Faecalibacterium provided by the present invention When the cancerinhibens CLCC1 microorganism was administered to an animal model in which a colon cancer cell line was subcutaneously transplanted, the tumor growth inhibition rate according to Equation 1 was 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30 % Or more, for example, 5 to 90%, 5 to 70%, 5 to 50%, 10 to 90%, 10 to 70%, 10 to 50%, 15 to 90%, 15 to 70%, 15 To 50%, 20 to 90%, 20 to 70%, 20 to 50%, 25 to 90%, 25 to 70%, 25 to 50%, 30 to 90%, 30 to 70%, or 30 to 50% I can.

Specifically, in one embodiment of the present invention, Faecalibacterium Using Equation 1 above based on the results of tumor weight measurements in three experimental groups of an animal model transplanted with a colon cancer cell line administered with a cancerinhibens CLCC1 microorganism at a low, medium or high concentration, and a negative control without administration of the microorganism. Tumor growth inhibition rate was measured. As a result of measuring the weight of tumors according to the administration of Faecalibacterium cancerinhibens CLCC1 in mice subcutaneously transplanted with a human colorectal cancer cell line, it was significantly lower in all three experimental groups administered with the microorganism compared to the negative control group. Specifically, in the experimental group administered the microorganism at a low concentration (2x10 ⁶ cells/head), the tumor growth inhibition rate was 39.6%, in the experimental group administered at a medium concentration (2x10 ⁷ cells/head), the tumor growth inhibition rate was 32.4%, and at a high concentration (2x10 ⁶ cells/head). ⁸ cells/head) showed a tumor growth inhibition rate of 39.3% in the experimental group, and it was confirmed that tumor growth of colon cancer was inhibited by more than 30% in all experimental groups.

Faecalibacterium provided by the present invention When cancerinhibens was administered to an animal model in which a cancer cell line was subcutaneously transplanted, the relative reduction in tumor volume according to the tumor volume was 5% or more, 10% or more, 15% or more, 20% or more, compared to the control group to which the microorganism was not administered. It may be 25% or more, or 30% or more, and the tumor growth inhibition rate according to the tumor volume may be calculated by Equation 2 below.

In Equation 2, T2 is the average tumor volume in each experimental group, and C2 is the average tumor volume in the negative control group.

When Faecalibacterium cancerinhibens CLCC1 was administered to a tumor animal model transplanted with a cancer cell line according to an example of the present invention, the tumor volume was 90% or less, 85% based on 100% of the tumor volume of the negative control group without administration of the microorganism. Or less, 80% or less, 77% or less, 70% or less, 67% or less, and the tumor volume is a value selected from the lower limit of 10% or more, 15% or more, 18% or more, and 20% or more, and the upper limit of 90% or less, It may have a numerical range combined with a numerical value selected from 85% or less, 80% or less, 77% or less, 70% or less, and 67% or less.

If the cancer is colon cancer, Faecalibacterium When the cancerinhibens CLCC1 microorganism is administered, the tumor volume is 10 to 90%, 10 to 85%, 10 to 80%, 10 to 77%, based on 100% of the tumor volume of the negative control group to which the microorganism is not administered. , 10 to 70%, 10 to 67%, 20 to 90%, 20 to 80%, 20 to 70%, 20 to 67%, 30 to 90%, 30 to 80%, 30 to 70%, 30 to 67% , 40-90%, 40-80%, 40-70%, 40-67%, 50-90%, 50-80%, 50-70%, 50-67% or 60-67% level reduction activity It may be to have.

Specifically, in a mouse subcutaneous tumor model using a human colon cancer cell line The average tumor size of starting the microorganism administered the same day (one day) is, but like in 105mm ³ all four groups, after 36 days is in the average tumor volume in the negative control group, while shown to 4330mm ^3, CLCC1 microorganism is administered group All showed a volume of 3000 mm ³ or less, and it was confirmed that they had a tumor volume of about 60 to 67% compared to the control group, and the effect of Pacalybacterium cancer inhibis on inhibiting the growth of colon cancer was confirmed.

When the cancer is liver cancer, the Faecalibacterium cancerinhibens CLCC1 microorganism provided by the present invention has a tumor volume of 90% or less, 85% based on 100% of the liver cancer tumor volume of the control group without administration of the microorganism when the microorganism is administered. Or less, 80% or less, 77% or less, 10 to 90%, 10 to 85%, 10 to 80%. Activity that decreases to a level of 10 to 77%, 15 to 90%, 15 to 85%, 15 to 80%, 15 to 77%, 18 to 90%, 18 to 85%, 18 to 80%, or 18 to 77% It may be to have.

The reduction in the tumor volume of liver cancer according to the present invention showed that the volume of liver cancer tumors was 19.5% to 75.3%, and an average of 32.9% was lower in the experimental group to which CLCC1 was administered orally compared to the control group. Therefore, it can be seen that the orally administered CLCC1 microorganism has the activity of inhibiting the development and progression of tumors in liver cancer.

In one embodiment of the present invention, Faecalibacterium As a result of transplanting subcutaneous tumor cells to mice administered with cancerinhibens CLCC1 strain and control mice not administered with the strain, and confirming the incidence and growth rate of liver cancer using MR imaging, when Faecalibacterium cancerinhibens CLCC1 strain was administered, liver cancer compared to the control group. The tumor volume was an average of 32.9% lower, indicating a delay in cancer development and a growth rate of tumors.

In one embodiment of the present invention, after transplanting the liver cancer cell line Hep55.1c to the left lobe of the liver of an 8-week-old mouse to cause liver cancer, Faecalibacterium As a result of comparing the tumor volume of the experimental group to which cancerinhibens microbe was administered and the control group without administration, Faecalibacterium when the tumor size on the day of transplantation was set to 1, Faecalibacterium Tumor growth in the experimental group administered with the cancerinhibens microorganism was significantly suppressed compared to the control group.

In an embodiment of the present invention, liver cancer is caused through a subcutaneous tumor transplantation experiment of a liver cancer cell line, and Faecalibacterium in the process of developing liver cancer for 55 days. If cancerinhibens CLCC1 strain was administered orally, Faecalibacterium It was confirmed that tumor incidence and progression were statistically significantly inhibited in the experimental group to which the cancerinhibens CLCC1 strain was administered orally compared to the control group (FIGS. 4A to 4C ). On average, compared to the control group, in the experimental group administered with CLCC1 orally, the tumor volume was 19.5% to 75.3%, and the average value was 32.9% lower. Therefore, it can be seen that the orally administered CLCC1 strain has an activity of inhibiting tumor development and progression.

In one embodiment of the present invention, when generating the liver in mice by transplantation experiments between the hepatoma cell line and, oral administration of Faecalibacterium spp CLCC1 in carcinogenesis process for 39 days, CLCC1 in the experimental group orally administered CLCC1 strain It was confirmed that cancer incidence and progression were statistically significantly suppressed compared to the control group not administered (FIGS. 5A to 5C ). Therefore, it can be confirmed that the CLCC1 strain has excellent tumor incidence and growth inhibitory activity.

Faecalibacterium according to the invention The genus microorganism, for example, Faecalibacterium cancerinhibens, is a 16S rRNA having a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more of nucleotide sequence identity thereto. It may be a microorganism having a 16S rRNA containing.

Faecalibacterium according to the invention Cancerinhibens microorganisms may be cultured at a temperature of 20 to 40°C, 25 to 40°C, 30 to 40°C, 35 to 40°C or 37°C, and may be characterized by being cultured under anaerobic conditions.

Faecalibacterium according to the invention cancerinhibens is Faecalibacterium prausnichii ( Faecalibacterium) , which is a microorganism in the genus Faecalibacterium. prausnitzii ) has different characteristics of lipid production patterns.

Faecalibacterium according to the invention cancerinhibens do not contain 17:0 iso 3OH fatty acids. In one embodiment of the present invention, Faecalibacterium cancerinhibens CLCC1 microorganism and Faecalibacterium As a result of analyzing the polar lipid content of prausnitzii species, CLCC1 was found to be Faecalibacterium. It was confirmed that it has additional PL3, which is not present in prausnitzii (Fig. 3b). Faecalibacterium according to the invention cancerinhibens are 15:1 w8c fatty acids, 19:0 cyclo w10c/19w6 fatty acids, 20:1 w9c fatty acids, 14:0 3OH/16:1 iso I fatty acids, 15:0 3OH fatty acids, 16:0 iso fatty acids and 16:0 It may include one or more fatty acids selected from the group consisting of iso 3OH fatty acids.

In one embodiment of the present invention, Faecalibacterium As a result of comparing the fatty acid composition of cancerinhibens CLCC1 microorganism and Faecalibacterium prausnitzii species by gas chromatography analysis, it was found that there was a difference in the composition of major fatty acids between the two microorganisms. Specifically, 15:1 w8c fatty acid, 19:0 cyclo w10c/19w6 fatty acid, 20:1 w9c fatty acid, 14:0 3OH/16:1 iso I fatty acid, 15:0 3OH fatty acid, 16:0 iso fatty acid and 16: 0 iso 3OH fatty acid was detected only in CLCC1 microorganism, and 17:0 iso 3OH fatty acid was detected only in Faecalibacterium prausnitzii ATCC 27768.

Faecalibacterium according to the invention Cancerinhibens is a short chain fatty acid (SCFA) with a high production of butyric acid. Specifically, Faecalibacterium of the present invention The amount of butyric acid produced by the cancerinhibens CLCC1 strain was 2 to 50 times, 2 to 40 times, 2 to 30 times, 2 to 20 times, 2 to 15 times, 2 to 10 times, 5 to 50 times, 5 to 40 times compared to Faecalibacterium prausnitzii species. It may be times, 5 to 30 times, 5 to 20 times, 5 to 15 times, or 5 to 10 times. Specifically, Faecalibacterium cancerinhibens CLCC1 strain and Faecalibacterium SCFA analysis of culture supernatant from prausnitzii species, Faecalibacterium Cancerinhibens CLCC1 strain showed a 7.8-fold higher butyric acid content than Faecalibacterium prausnitzii species (FIGS. 3C to 3D ).

Preferred microorganisms in the genus Faecalibacterium according to the present invention were deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on January 3, 2019, and received accession number KCTC 13783BP, and the scientific name is Faecalibacterium cancer inhibins. cancerinhibens ).

Faecalibacterium according to the invention When the cancerinhibens strain is plated on agar medium and cultured, colonies within 3 mm or less than 2 mm are formed, and have a long rod-shaped cell shape when observed with an optical microscope. In one embodiment of the present invention, Faecalibacterium cancerinhibens has a genome size of 2.9Mbp, the number of protein coding regions (CDS) is 2695, the GC ratio is 56.1%, the number of rRNA genes is 21, the total number of tRNA genes. Were found to be 69. Faecalibacterium cancerinhibens CLCC1 according to the present invention, Faecalibacterium Compared to prausnitzii , 7.8 times higher content of butanoic acid was detected (FIGS. 3C to 3D ).

An example of the present invention is an example of the present invention is a microorganism of the genus Faecalibacterium having anticancer activity; A culture of the microorganism; Lysate of the microorganism; And one or more extracts selected from the group consisting of the microorganisms, cultures and lysates; to provide an anticancer composition comprising at least one selected from the group consisting of, for example, a pharmaceutical composition for preventing or treating cancer. Specifically, the cancer includes gastrointestinal cancer and non-gastrointestinal cancer, and may be, for example, colon cancer and/or liver cancer.

The composition of the present invention is for use in the prevention, treatment or amelioration of cancer. The composition of the present invention can be administered to a patient diagnosed with cancer or confirmed to be at risk of developing cancer. The examples demonstrate that administration of the compositions of the present invention can reduce tumor growth in a number of tumor models. The treatment of cancer using the composition of the present invention may further have an activity of reducing or reducing metastasis of cancer cells in addition to the tumor formation and/or tumor growth delaying activity of cancer cells.

Microorganisms of the genus Faecalibacterium according to the present invention used in the anticancer composition, for example, Faecalibacterium cancer inhibins ( Faecalibacterium cancerinhibens ) is as described above.

The anticancer agent according to the present invention alone or in other therapies such as surgery, radiation therapy, gene therapy, immunotherapy (eg, targeted antibody immunotherapy, CAR-T cell therapy), oncolytic virus, bone marrow transplantation, stem cell transplantation, Hormone therapy, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, chemotherapy, and the like. Additionally, persons at high risk of developing a proliferative disease may receive treatment to inhibit and/or delay the onset of the disease. The anticancer activity of the composition according to the present invention may be effective when combined with a more direct anticancer agent. Thus, in certain embodiments, the present invention provides a composition comprising Faecalibacterium cancerinhibens and an anticancer agent.

Microbial cells of the genus Faecalibacterium, which are active ingredients of the anticancer composition according to the present invention; A culture of the microorganism; Lysate of the microorganism; And one or more extracts selected from the group consisting of the microorganisms, cultures and lysates; at least one selected from the group consisting of, 0.00001% to 100% by weight, 0.001% to 99.9% by weight, 0.1% by weight of the total anticancer agent composition % To 99% by weight, more preferably 1% to 50% by weight.

The microorganisms of the genus Pacalibacterium may be in a cell-free form that includes the cells themselves, or does not contain cells. The lysate refers to a lysate obtained by crushing the microbial cells of the Pacalibacterium or a supernatant obtained by centrifuging the lysate. In the present specification, unless otherwise noted, the microorganisms of the genus Pacalibacterium having anticancer activity include the microorganisms of the microorganism; A culture of the microorganism; Lysate of the microorganism; And one or more extracts selected from the group consisting of the microorganisms, cultures, and lysates; is used to mean at least one selected from the group consisting of.

The anticancer composition according to the present invention may include freeze-dried cells. Freeze-drying of microbial cells can be performed by a person skilled in the art by a method known in the art. Alternatively, the composition of the present invention may comprise a culture of living living microorganisms. In one embodiment, the microorganism contained in the anticancer composition according to the present invention is not inactivated, for example, heat-inactivated. In some embodiments, the microorganisms included in the anti-cancer composition according to the present invention are not killed, eg, heat-killed. In some embodiments, the microorganisms included in the anticancer composition according to the present invention are not weakened, eg, heat-damped. In some embodiments, the microorganisms included in the anticancer composition according to the present invention may partially or wholly colonize the intestine.

The anticancer composition according to the present invention contains a therapeutically effective amount of the microorganism of the present invention. A therapeutically effective amount of the microorganism is sufficient to exert a beneficial effect on the patient. A therapeutically effective amount of the bacterial strain may be sufficient to cause intestinal transport and/or partial or full colonization of the patient. For example, a suitable daily dose of bacteria for adult humans may be from about 1 x 10 ³ to about 1 x 10 ¹¹ colony forming units (CFU); For example, from about 1 x 10 ⁷ to about 1 x 10 ¹⁰ CFU; In another example, it is about 1 x 10 ⁶ to about 1 x 10 ¹⁰ CFU.

The pharmaceutical composition according to the present invention can be administered to mammals including humans by various routes. The mode of administration may be any method commonly used, for example, may be administered by oral, dermal, intravenous, intramuscular or subcutaneous routes, and preferably orally.

The composition of the present invention may contain a pharmaceutically acceptable excipient or carrier. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent may be chosen with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain any suitable binders, lubricants, suspending agents, coatings, solubilizing agents as or in addition to carriers, excipients or diluents.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and may be divided into several times a day at regular intervals according to the judgment of the doctor or pharmacist. It can also be administered. For example, the daily dosage may be 0.1 to 500 mg/kg, preferably 0.5 to 300 mg/kg, based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be high or low depending on individual differences.

The compositions of the present invention may be probiotics, and probiotics may be combined with at least one suitable prebiotic compound. The prebiotic compounds are generally oligo- or polysaccharides or non-digestible carbohydrates such as sugar alcohols, which are not degraded or absorbed in the upper digestive tract. Known prebiotics include commercially available products such as inulin and transgalacto-oligosaccharides.

The composition of the present invention may be encapsulated so as to transport microorganisms of the genus Pacalibacterium into the intestine. Encapsulation protects the composition from degradation until transported to the target site through rupture by chemical or physical stimuli, such as physical disruption that can be caused by, for example, changes in pressure, enzyme activity or pH. Any suitable encapsulation method can be used. Exemplary encapsulation techniques include entrapment in a porous matrix, adhesion or adsorption on the surface of a solid carrier, self-aggregation by agglomeration or crosslinking agents, and mechanical containment after a microporous membrane or microcapsule.

An example of the present invention is an example of the present invention is a microorganism of the genus Faecalibacterium having anticancer activity; A culture of the microorganism; Lysate of the microorganism; And at least one extract selected from the group consisting of the microorganisms, cultures and lysate; anticancer composition comprising at least one selected from the group consisting of, for example, a food composition for preventing or improving cancer or an anticancer functional food composition will be.

Microorganisms of the genus Faecalibacterium according to the present invention used in the anticancer composition, for example, Faecalibacterium cancer inhibins ( Faecalibacterium cancerinhibens ) is as described above.

In the present invention, food refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through a certain amount of processing process, and food, food in the usual sense It is intended to include all additives, health functional foods, beverages and beverage additives. In the present invention, a beverage refers to a generic term for drinking to quench thirst or to enjoy taste, and is intended to include a functional beverage. The beverage may be in liquid, syrup and/or gel form.

The content of microorganisms in Pacalibacterium as an active ingredient contained in the food composition is not particularly limited appropriately depending on the type of food and desired use, for example, of microorganisms in Pacalibacterium, which is an active ingredient of the total food weight. At least one selected from the group consisting of cells, the microorganism culture, the lysate of the microorganism, and the extract of the microorganism is 0.00001% to 100% by weight, 0.001% to 99.9% by weight, 0.1% to 99% by weight, further Preferably, it may be added in an amount of 1% to 50% by weight and 0.01 to 15% by weight. For example, the food composition may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The composition of the present invention can be formulated as a food product. For example, food can provide nutritional benefits in addition to the therapeutic benefits of the present invention, such as nutritional supplements. Similarly, a food composition can be formulated to be more attractive to consume the composition by enhancing the taste of the composition of the present invention or by more similar to a general food item than a pharmaceutical composition.

The microorganisms of the genus Pacalibacterium according to the present invention and a composition for preventing or treating cancer containing the same, have an activity of inhibiting the occurrence and growth of cancer, and are especially ingested by humans in the form of a pharmaceutical composition or health functional food. It can be used for the prevention, treatment and/or improvement of cancer.

Figure 1a is Faecalibacterium This is a photograph showing the colony morphology formed after culturing cancerinhibens CLCC1 in Reinforced clostridial media (RCM) medium for 3 days.
Figure 1b is Faecalibacterium This is a photograph of the appearance of cells observed with an optical microscope in the exponenetial growth phase during liquid culture of cancerinhibens CLCC1.
Figure 1c is Faecalibacterium This is a photograph of cells taken with a scanning electron microscope in the exponenetial growth phase during liquid culture of cancerinhibens CLCC1.
Figure 2a is Faecalibacterium This is a picture showing the phylogenetic position obtained through 16S rRNA analysis of cancerinhibens CLCC1.
Figure 2b is Faecalibacterium This is a picture showing the phylogenetic position using 92 key genes in the genome of cancerinhibens CLCC1.
2C is a diagram showing the relationship between the Faecalibacterium cancerinhibens CLCC1 strain and related species based on the mean nucleotide identity (ANI) value.
Figure 2d is Faecalibacterium This is a table comparing the ANI values of cancerinhibens CLCC1 and related species.
Figure 3a is Faecalibacterium This is a graph of the results of gas chromatography analysis comparing the fatty acid composition of cancerinhibens CLCC1 and Faecalibacterium prausnitzii strains.
Figure 3b is Faecalibacterium Photographs showing the results of two-dimensional chromatography analysis performed to compare polar lipids of cancerinhibens CLCC1 and Faecalibacterium prausnitzii strains.
Figure 3c is Faecalibacterium This is a GC-MS analysis graph of short chain fatty acid analysis of cancerinhibens CLCC1.
Figure 3d is Faecalibacterium This is a GC-MS analysis graph of prausnitzii short-chain fatty acid analysis.
4A to 4C are graphs showing changes in tumor volume over time after induction of liver cancer in order to measure the effect of Faecalibacterium cancerinhibens CLCC1 on inhibiting cancer incidence in mice induced liver cancer by the method of Example 4. 4A is a dot graph showing each individual with a sword, FIG. 4B is a graph comparing the average values of the experimental group and the control group, and FIG. 4C is a line graph showing the change in the volume of tumors for each individual.
5A to 5C are Faecalibacterium in a liver transplantation model. Cancerinhibens CLCC1 strain on the cancer incidence inhibitory effect test results and the dot graph and line graph of each result. FIG. 5A is a result of directly comparing the size of a tumor, FIG. 5B is a result of expressing the change in tumor size in fold, and FIG. 5C indicates a delta value.
6A is a graph showing changes in tumor volume according to the dose of Faecalibacterium cancerinhibens CLCC1 strain in mice subcutaneously transplanted with human colorectal cancer cell lines.
6B is a graph showing the results of measuring tumor weight according to the dose of Faecalibacterium cancerinhibens CLCC1 strain in mice subcutaneously transplanted with human colorectal cancer cell lines.
6C is a result of visual observation of a change in tumor size according to the dose of Faecalibacterium cancerinhibens CLCC1 strain in a mouse subcutaneously transplanted with a human colorectal cancer cell line.
6D is a result of visual observation by separating tumor tissues from the negative control group (G1) and each experimental group (G2 to G4) after the administration period of the Faecalibacterium cancerinhibens CLCC1 strain in mice subcutaneously transplanted with a human colon cancer cell line.
6E is a result of performing H&E staining in order to confirm cell necrosis according to administration of the Faecalibacterium cancerinhibens CLCC1 strain in the tumor tissue of a mouse subcutaneously transplanted with a human colon cancer cell line.
6F is a result of performing TUNEL staining in order to confirm apoptosis according to administration of Faecalibacterium cancerinhibens CLCC1 strain in tumor tissue of a mouse subcutaneously transplanted with a human colorectal cancer cell line.
7 is a result of observing the change in the size of the tumor according to the administration of the Faecalibacterium cancerinhibens CLCC1 strain in the tumor tissue of a mouse subcutaneously transplanted with a mouse colon cancer cell line.
8 shows an experimental design for administering various test agents in an animal model having cancer in order to test anticancer efficacy according to an example of the present invention.

Hereinafter, the present invention will be described in more detail by examples. However, the scope of the present invention is not limited by these examples by the following examples.

Example 1. Isolation of microorganisms

The bacteria of the genus Faecalibacterium are dominant bacteria in the intestines of healthy adults, accounting for about 10% of all intestinal bacteria, and are known to have direct or indirect effects on health.

Faecalibacterium Cancerinhibens CLCC1 strain was isolated by diluting and smearing the feces of a healthy adult male in their twenties in Reinforced clostridial media (RCM) medium and culturing in an anaerobic culture condition at room temperature at 37°C.

Faecalibacterium cancerinhibens CLCC1 has a characteristic of forming colonies of up to 2 to 3 mm when cultured on RCM agar medium for 3 days, and showing a long rod-shaped cell shape when observed with an optical microscope. 1A and 1B show the result of observation of the colony morphology and the morphology of cells using an optical microscope. Figure 1c shows a photograph of the shape of the cell using a scanning electron microscope.

Example 2. Phylogenetic analysis of microorganisms

2-1. Genetics Sequencing

Faecalibacterium isolated by the method of Example 1 The cancerinhibens CLCC1 strain was cultured in RCM liquid medium at room temperature under anaerobic culture conditions at 37° C. for 1 day, and then centrifuged to obtain microorganisms. The entire genome was isolated from the obtained microorganisms using FastDNA SPIN Kit for Soil (MP Biomedicals).

After making a sequencing library using the PacBio sequencing library kit, whole genome sequencing was performed using the PacBio RS II platform, and genome sequencing information was analyzed using BIOiPLUG's Whole Genome analysis pipeline, our genome data analysis platform.

Faecalibacterium in Table 1 below cancerinhibens The genome characteristics of the CLCC1 strain are shown. Faecalibacterium The total genome size of cancerinhibens CLCC1 strain was about 2.9Mbp, the number of protein coding regions (CDS) was 2695, and the GC ratio (GC ratio, %) was 56.1%. There were 21 total rRNA genes and 69 total tRNA genes.

Item value Genome size (bp) 2,941,967 Protein coding site number (Number of CDSs) 2,695 GC ratio (GC ratio, %) 56.1 Number of rRNA genes 21 Number of tRNA genes 69

2-2. 16S rRNA Phylogenetic analysis using analysis

Faecalibacterium sequence analyzed by the method of Example 2-1 Phylogenetic taxonomic analysis was performed by analyzing the rRNA of the cancerinhibens CLCC1 strain. The 16S rRNA sequence of the Faecalibacterium cancerinhibens CLCC1 strain is shown in SEQ ID NO: 1.

Specifically, similarity analysis of 16S rRNA was performed using the 16S-based identification for prokaryote pipeline of the EzBioCloud database, and phylogenetic analysis was performed using the MEGA program. Faecalibacterium identified by analyzing the 16S rRNA gene nucleotide sequence in FIG. 2A The phylogenetic location of the cancerinhibens CLCC1 strain was indicated.

16S rRNA similarity analysis result, Faecalibacterium Cancerinhibens CLCC1 strain showed 98% of 16S rRNA similarity to Faecalibacterium prausnitzii ATCC 27768(T). The phylogenetic position of CLCC1 is "Firmicutes phylum, Clostridia river, Clostridiales order, Ruminococcaceae family.

2-3. Phylogenetic taxonomic analysis using key genes

Using the genome sequence data obtained by the method of Example 2-1, a phylogenetic taxonomic analysis was performed through gene sequence analysis.

Specifically, phylogenetic analysis was performed using the similarity of 92 genes (up-to-date bacterial core gene (UBCG)) that can be used as taxonomic markers of bacteria. The 92 genes refer to genes provided by the UBCG (up-to-date bacterial core gene) pipeline (Na, SI, Kim, YO, Yoon, SH, Ha, SM, Baek, I. & Chun, J. (2018)).

Fig. 2b shows a picture showing the phylogenetic position of the relative species obtained by analyzing 92 key genes.

2-4. Average Nucleotide Analysis of the relationship using the average nucleotide identity (ANI)

Using the genome analysis data obtained by the method of Example 2-1, the relationship was analyzed by comparing ANI values between the Faecalibacterium cancerinhibens CLCC1 strain and the related species.

Specifically, Faecalibacterium cancerinhibens using the Genome-based identification for prokaryote (TrueBAC ID) pipeline, an analysis platform provided by ChunLab, Inc. The genome similarity value was analyzed by comparing the entire genome sequence between the CLCC1 strain and other related species.

Faecalibacterium based on ANI values in Figure 2c The relationship between cancerinhibens CLCC1 strain and related species is shown. Faecalibacterium in Figure 2d A comparison table of ANI values of cancerinhibens CLCC1 strain and related species is shown. Each row and column is 1 for Faecalibacterium cancerinhibens CLCC1, 2 for Faecalibacterium prausnitzii ATCC 27768(T), 3 is Fournierella massiliensis AT2(T), 4 is Intestinimonas butyriciproducens DSM 26588(T), 5 is Intestinimonas massiliensis GD2(T), 6 for Pseudoflavonifractor capillosus ATCC 29799(T), 7 for Ruthenibacterium lactatiformans 585-1(T), 8 for Subdoligranul um variabile DSM 15176(T), 9 for Gemmiger formicilis ATCC 27749(T) . (T) after each species name means type strain.

Faecalibacterium As a result of genome translation of cancerinhibens CLCC1 strain and comparison with related species, CLCC1 strain showed an average nucleotide identity (ANI) value of 85.99% at the genome level with Faecalibacterium prausnitzil , the closest relative at the whole genome level. It can be seen that this is a novel species that has not been isolated and reported. Faecalibacterium the Faecalibacterium spp identified by the above method It was named cancerinhibens CLCC1.

Example 3. Analysis of fatty acid content of microorganisms

Faecalibacterium cancerinhibens To analyze the molecular biological properties of the CLCC1 strain, Faecalibacterium Fatty acid composition of cancerinhibens CLCC1 strain and Faecalibacterium prausnitzii were compared.

Specifically, Faecalibacterium cancerinhibens CLCC1 strain and Faecalibacterium prausnitzii After culturing the strains in RCM medium at 37° C. and anaerobic conditions, fatty acids were obtained according to Miller's method (Miller, LT (1982) J. Clin. Microbiol. 18, 861-867) using about 40 mg of cells.

Agilent technologies 6890 Gas chromatography was used for the analysis of the extracted fatty acids, and the separation column was A30m X 0.320mm X 0.25μm Crosslinked Methyl siloxane column (HP-1). Figure 3a Faecalibacterium cancerinhibens CLCC1 strain and Faecalibacterium A graph of the results of gas chromatography analysis comparing the fatty acid composition of prausnitzii strain (ATCC 27768) is shown. In the graph of FIG. 3A, the names of fatty acids and their values (%, w/v) that each peak means It is shown in Table 2 below.

Type of fatty acid Content in CLCC1 (%) Content in ATCC 27768 (%) 16:00 32.42 42.28 18:1 w7 28.15 14.88 17:0 2OH 12.51 8.09 16:1 w7c/16:1 w6c 5.22 6.04 16:1 w9c 3.89 1.57 14:00 2.44 14.93 17:0 anteiso 2.42 0.99 16:1 w5c 2.17 0.83 18:1 w9c 1.88 0.86 18:00 1.59 2.17 16:1 2OH 1.41 0.51 20:1 w7c 0.84 0.43 18:1 w5c 0.81 0.32 15:0 2OH 0.75 0.5 18:0 10-methyl, TBSA 0.45 0.21 15:1 w8c 0.44 - 15:1 iso H/13:0 3OH 0.35 1.44 16:0 3OH 0.32 0.11 15:0 anteiso 0.21 0.2 12:00 0.2 1.59 15:0 iso 3OH 0.17 0.49 17:00 0.17 0.2 19:0 cyclo w10c/19w6 0.16 - 20:00 0.14 0.1 13:1 at 12-13 0.13 0.35 10:00 0.13 0.2 20:1 w9c 0.13 - 17:0 iso 0.12 0.17 14:0 3OH/16:1 iso I 0.12 - 15:0 3OH 0.11 - 16:0 iso 0.1 - 16:0 iso 3OH 0.05 - 17:0 iso 3OH - 0.54

As shown in Table 2 above, 15:1 w8c fatty acid, 19:0 cyclo w10c/19w6 fatty acid, 20:1 w9c fatty acid, 14:0 3OH/16:1 iso I fatty acid, 15:0 3OH fatty acid, 16:0 iso fatty acid and 16:0 iso 3OH fatty acid were detected only in CLCC1 strain, and 17:0 iso 3OH fatty acid was found in Faecalibacterium prausnitzii ATCC 27768 strain was detected only.

As a result of the comparative analysis of the molecular biological characteristics, Faecalibacterium cancerinhibens CLCC1 strain is Faecalibacterium It was confirmed that it is a different species from Faecalibacterium prausnitzii , a known species included in cancerinhibens . When comparing the fatty acid composition of the two microorganisms, it can be seen that there is a difference in the composition of the major fatty acids between the two microorganisms, and the results are shown in FIGS. 3A and 2.

Example 4. Polar lipid analysis of microorganisms

Thin layer chromatography (TLC) analysis was performed to compare the composition of polar lipids.

Specifically, polar lipids were extracted from a 50 mg microbial lyophilized sample using Minikin's method (Minikin, D.E., et al. (1984). J Microbial Meth 2, 233-241.). And the first development was performed in a solvent (chloroform:methanol:water = 65:25:3.8 (v/v)) in a ratio of chloroform, methanol and water 65:25:3.8 (v/v). Thereafter, the second in a solvent (chloroform:methanol:acetic acid:water = 40:7.5:6:1.8 (v/v)) in the ratio of chloroform, methanol, acetic acid and water 40:7.5:6:1.8 (v/v) Deployment was carried out. After the second development, it was stained using 5% (w/v) ethanolic molybdenum phosphate (ethanolic molybdatophosphoric acid).

In addition, when comparing the composition of polar lipids, Faecalibacterium cancerinhibens CLCC1 and Faecalibacterium prausnitzii was found to have one phosphatidylglycerol (PG), two unidentified phospholipids (PL1, PL2), and a number of unidentified lipids (L). In addition, it was confirmed that CLCC1 additionally has unidentified phospholipids (PL3), which are not found in Faecalibacterium prausnitzii , and the results are shown in FIG. 3B. As can be seen from the above results, Faecalibacterium according to the present invention cancerinhibens CLCC1 and Faecalibacterium It can be seen that the polar lipid production pattern of prausnitzii is different.

Example 5. short chain fatty acid; SCFA ) analysis

Faecalibacterium prausnitzii and Faecalibacterium In order to examine the differences in the production patterns of short-chain fatty acids in cancerinhibens CLCC1 microorganisms, an analysis was requested to the Korea Institute of Medical Sciences and the distribution of short-chain fatty acids was performed.

Specifically, Faecalibacterium prausnitzii and Faecalibacterium cancerinhibens CLCC1 strain of the present invention were cultured in RCM medium for 16 hours, and then the cells were precipitated through centrifugation (4° C., 4,000 rpm, 10 min) to collect the supernatant. The collected supernatant was filtered using a 0.22um filter and then stored refrigerated. Samples for GC-MS for SCFA analysis from the supernatant were prepared using Takeshi Furuhashi's method (Takeshi Furuhashi, et al., Analytical Biochemistry, Volume 543, 2018, Pages 51-54). Agilent's 6890 series equipment was used for chain fatty acid analysis.

Faecalibacterium cancerinhibens CLCC1 and Faecalibacterium , another known species of the genus Faecalibacterium As a result of comparative analysis of short chain fatty acid (SCFA) present in the culture supernatant of prausnitzii , only butanoic acid was detected as SCFA in both samples. Also Faecalibacterium Compared to the sample prausnitzii Faecalibacterium A 7.8-fold higher content of butanoic acid was detected in cancerinhibens CLCC1 sample. 3C to 3D are graphs of the GC-MS analysis results.

In addition, when viewed as a percentage in each sample was acid, Faecalibacterium cancerinhibens CLCC1 culture supernatant in 91.64 percent showed the acid, in the culture supernatant of the acid Faecalibacterium prausnitzii was 76.40%. Faecalibacterium in Table 3 The results of short-chain fatty acid analysis of cancerinhibens CLCC1 strain are shown in Table 4, Faecalibacterium. The results of short-chain fatty acid analysis of prausnitzii are shown. Other substances indicated in Tables 3 to 4 below were classified as noise signals that could not accurately identify substances because they were detected in the substance library database, but their matching rate was very low. In Table 3 below, RT means retention time.

peak RT Library/ID Matching Rate (Quality) Area Percentage (% of total, %) One 2.6965 Oxirane 58 606,058 3.132 2 3.1915 Butanoic acid (CAS); n-Butyric acid 94 17,732,337 91.635 3 3.5174 Ethane, methoxy- (CAS); Methane 50 497,085 2.569 4 4.1933 2-Pentanone, 4-hydroxy-4-methyl- (CAS) 43 515,638 2.665

peak RT Library/ID Matching rate
(Quality) Area Rate (% of Total, %) One 2.6908 2-Decanone (CAS) 38 338,372 11.491 2 3.0167 Butanoic acid (CAS); n-Butyric acid 93 2,249,717 76.401 3 4.2057 2-Pentanone, 4-hydroxy-4-methyl- (CAS) 47 356,542 12.108

Butyric acid is known to have anticancer activity, and the Faecalibacterium cancerinhibens CLCC1 strain of the present invention is Faecalibacterium Compared to prausnitzii , the production of butyric acid is higher among short-chain fatty acids, which can be predicted to exhibit excellent anticancer activity.

Example 6. Cancer incidence inhibition efficacy test in subcutaneous tumor model using mouse liver cancer cell line

Faecalibacterium In order to confirm the ability of cancerinhibens CLCC1 strain to inhibit cancer incidence, the efficacy of inhibiting cancer incidence in a subcutaneous tumor model using a mouse liver cancer cell line was tested. In 10 6-week-old male C57BL/6 mice, liver cancer was developed through subcutaneous tumor transplantation of a liver cancer cell line (Hep55.1c), and oral administration of Faecalibacterium cancerinhibens CLCC1 strain during the development of liver cancer for 55 days was effective in the growth of liver cancer cells. We looked at the impact. The liver cancer cell line Hep55.1c is a mouse-derived hepatoma.

Specifically, Faecalibacterium cancerinhibens CLCC1 was orally administered from 4 days before the mouse liver cancer cell line was implanted into the subcutaneous tissue of the mouse. On the 4th day of oral administration, a mouse liver cancer cell line (Hep55.1c) was made to be 2.0 x 10 ⁶ cells and transplanted into the subcutaneous tissue of the right flank.

For the subcutaneous tumor model using the mouse liver cancer cell line, Faecalibacte rium cancerinhibens CLCC1 strain was orally administered once a day for 55 days and 5 times a week. Thereafter, the size (tumor volume) of the liver cancer tumor was measured using Magnetic Resonance (MR) imaging every 7 days from the day of transplantation (day 0) to the day 55.

Faecalibacterium The cancerinhibens CLCC1 strain was cultured in RCM medium, concentrated, and stored frozen to a final glycerol concentration of 15% (v/v) using PBS buffer and glycerol. And it was orally administered to mice at 200 ul (2x10 ⁸ cells) after thawing immediately before administration. As a control, 10 mice to which the CLCC1 strain was not administered were used. The control group was orally administered 200ul of PBS and glycerol (15%, v/v) solution containing no strain.

Tables 5 and 6 below show the tumor size results of the control and test bacteria in the liver cancer inhibition efficacy test of the isolated strain in the subcutaneous tumor model. Table 5 shows the results of changes in tumor volume for the control group, and Table 6 shows Faecalibacterium These are the results of changes in the volume of tumors in the experimental group administered with the cancerinhibens CLCC1 strain. In the table, SD means standard deviation.

In FIG. 4, the tumor volume according to the change of time after inducing cancer in the mouse is shown as a graph, and the changes in the size of the tumor are shown in numerical values in Tables 5 to 6 above.

As a result of the experiment, it was confirmed that the incidence and progression of liver cancer were significantly inhibited in the experimental group to which CLCC1 was administered orally compared to the control group. On average, compared to the control group, in the experimental group administered with CLCC1 orally, the tumor volume was 19.5% to 75.3%, and the average value was 32.9% lower, Faecalibacterium. It was confirmed that the incidence of liver cancer in the experimental group administered with the cancerinhibens CLCC1 strain was delayed and the rate of tumor growth progression was inhibited.

In addition, on the 4th day, the first tumor size measurement day after surgery, the size of the tumor in the experimental group administered with the Faecalibacterium canceerinhibens CLCC1 strain was significantly smaller than that of the control group, indicating that it had a cancer-inducing effect.

Therefore, it can be seen that the orally administered CLCC1 strain has an activity to inhibit the occurrence and progression of liver cancer.

Example 7. Inhibitory efficacy test in liver transplantation model using mouse liver cancer cell line

Faecalibacterium To examine the effect of cancerinhibens CLCC1 strain on the growth of liver cancer cells, 4 8-week-old male C57BL/6 mice developed liver cancer through a liver transplantation experiment with a liver cancer cell line, and Faecalibacterium cancerinhibens bacteria CLCC1 during the 39-day liver cancer development process. We investigated the effect of oral administration on the growth of liver cancer cells.

Specifically, the liver cancer cell line Hep55.1c was used, and the epigastric part of the 8-week-old male C57BL/6 mouse was opened and transplanted to the left lobe of the liver to become 5.0 x 10 ⁵ cells. After transplantation of a tumor cell line in the liver, after a recovery period for 4 days, the size of the tumor was confirmed through MRI, and 4 well-formed tumors were selected and used in subsequent experiments.

For 39 days from the day of the transplant operation (day 0), the CLCC1 strain was prepared in the same manner as in Example 4 and orally administered to 4 mice that completed the transplant operation, and the CLCC1 strain was not administered to the 4 control mice. The control group was orally administered 200ul of PBS and glycerol (15%, v/v) solution containing no strain. Thereafter, the size and relative size of the tumor and the relative value of growth were measured periodically.

The size of the tumor was measured by magnetic resonance (MR) imaging every 7 days, and the relative value of the tumor size was derived by calculating the relative size to the tumor size on the day of transplantation. 5A to 5C show the results and graphs of the cancer-inducing inhibitory effect test results of the CLCC1 strain in the liver transplantation model.

Figure 5a is a point graph and a line graph directly comparing the size of the tumor, Figure 5b is a point and line graph expressing the size change of the tumor in fold, and Figure 5c is the amount of change in the size of the tumor (tumor size and It is a point and line graph showing the difference in tumor size on the previous measurement day).

As a result, it was confirmed that the incidence and progression of liver cancer were significantly suppressed in the experimental group administered with CLCC1 orally compared to the control group not administered with the isolate. Results of the inhibition of liver cancer and tumor size change data are shown in FIGS. 5A to 5C.

Table 7 shows the relative growth (Fold) of tumor size, and Table 7 shows the relative growth (Delative) of tumor growth.

As can be seen in Tables 7 and 8 and FIGS. 5A to 5B, it was confirmed that tumor growth of the mice to which the CLCC1 strain was administered orally was significantly suppressed compared to the control group. Therefore, it can be confirmed that the Faecalibacterium kenserinhibins CLCC1 strain has excellent tumor growth inhibitory ability.

Example 8. Confirmation of anticancer effect in mouse subcutaneous tumor model using human colon cancer cell line

8-1. Experimental method to confirm anticancer effect

Faecalibacterium In order to confirm the ability of cancerinhibens CLCC1 strain to inhibit cancer incidence, the efficacy of inhibiting cancer incidence in a subcutaneous tumor model using a human colon cancer cell line was tested. HCT-116 cells were used as the colorectal cancer cell line, and nude mice (CAnN.Cg-Foxn1nu/CrlOri, SPF) were used as mice. The HCT-116 is a cancer cell line corresponding to human-derived colorectal carcinoma.

Specifically, the colon cancer cell line HCT-116 (2.5 Υ10 ⁷ cells/mL) was injected into mice that had undergone a one-week acclimatization period by administering 0.2 mL/head subcutaneously to the right side of the mouse using a disposable syringe.

When the size of the cancer cells grew to about 85-119 mm ³ after cancer cell transplantation, 10 mice per group were classified as a negative control group without CLCC1 administration and an experimental group 3 group receiving CLCC1 by concentration. Specifically, the test group were divided into three groups of high-low, medium, depending on the concentration of CLCC1, a low concentration is 2x10 ⁶ cells / head, jungnong degree of 2x10 ⁷ cells / head, a high concentration of F at a concentration of 2x10 ⁸ cells / head The cancerinhibens CLCC1 strain was administered. The strain was administered orally by syringe once daily for 36 days. The negative control group was administered only an excipient (a PBS solution containing 15% (v/v) glycerol).

Table 9 shows the information of the negative control group and each experimental group.

sign CLCC1 dosage Dosage Explanation Population (heads) G1 0 0.2mL/head Negative control 10 G2 2x10 ⁶ (cells/head) 0.2mL/head Low concentration experimental group 10 G3 2x10 ⁷ (cells/head) 0.2mL/head Medium concentration experimental group 10 G4 2x10 ⁸ (cells/head) 0.2mL/head High concentration experimental group 10

8-2. Tumor size change and growth inhibition rate following microbial administration

In order to confirm the anti-colorectal cancer effect of the F. cancerinhibens CLCC1 strain, the size change of the tumor was confirmed. Specifically, after the start of the strain administration test of Example 7-1, general symptoms such as appearance, behavior, and excretion were observed once a day, and the weight was measured once a week, the size of the tumor was measured twice a week, and the volume was calculated. I did. Figures 6c and 6d show the results of visual observation of the size of the tumor.

The tumor volume (Tv) was calculated by the method of Equation 3 below by measuring the perpendicular width (W) and the maximum length (L) of the tumor, respectively.

The results of confirming the change in tumor volume over 36 days are shown in Table 10 below and FIG. 6A. Compared to the negative control group (G1), it was confirmed that the tumor size decreased statistically significantly in all experimental groups administered with the CLCC1 strain. In Table 10 below, Mean means the average volume, and S.D. means the standard deviation.

* p<0.05, Significant difference from the negative control group (G1) by Dunnett's t-test.

** p<0.01, Significant difference from the negative control group (G1) by Dunnett's t-test.

Specifically, the average tumor size of starting the microorganism administered the same day (one day) is, but like in 105mm ³ all four groups, 36 days after, while the mean tumor volume from negative control appears to 4330mm ^3, CLCC1 strain is administered All of the experimental groups showed a volume of 3000 mm ³ or less, and it was confirmed that they had a tumor volume of about 60 to 67% compared to the control group, and the effect of inhibiting the growth of colon cancer of the Facalibacterium cancer inhibins was confirmed.

Also, based on the results of measuring the weight of the tumor, the tumor growth inhibition rate (IR) in each experimental group was calculated by the method of Equation 1 below.

[Equation 1]

In Equation 1, T is the average tumor weight in each experimental group, and C is the average tumor weight in the negative control group.

Tables 11 and 6b show the results of measuring tumor weights in each group after the end of the strain administration experiment, and Table 12 below shows the results of measuring average weights of mice in each group according to the experiment.

** p<0.01, Significant difference from the negative control group (G1) by Dunnett's t-test

In the negative control group (G1), the weight of the tumor extracted after necropsy was about 3.21 g, but in the experimental group, the weight of the tumor tissue was significantly lower as 1.94, 2.17 and 1.95 g at low, medium, and high concentrations, respectively. On the other hand, the total body weight in each group did not show a significant difference between the control group and the experimental group.

In the low concentration experimental group, the tumor growth inhibition rate was 39.6%, in the medium concentration experimental group, the tumor growth inhibition rate was 32.4%, and in the high concentration experimental group, the tumor growth inhibition rate was 39.3%, confirming that tumor growth was inhibited by more than 30% in all experimental groups. .

8-3. Necrosis of tumor cells and Apoptosis ( Apoptosis ) Confirm

After 36 days of CLCC1 strain administration, autopsies were performed, and H&E staining (Hematoxylin & Eosin staining) to see necrosis of tumor cells in tumor tissue and TUNEL assay to see apotosis were performed.

Figure 6e shows a picture of the result of H&E staining for necrosis analysis of tumor cells. There was no significant difference in the level of necrosis of tumor cells between the negative control group and the test group. Therefore, it can be seen that the cell line of Pacalibacterium cancer inhibins of the present application does not induce an inflammatory response due to tumor necrosis.

Figure 6f shows the results of the TUNEL staining analysis for the analysis of apoptosis of tumor cells. It was confirmed that the apoptosis of tumor cells was statistically significantly increased in the experimental group administered with Pacalibacterium compared to the negative control group. Therefore, it was confirmed that the decrease in tumor volume in each Pacalibacterium administration group was not due to necrosis of the tumor, but due to apoptosis of the tumor and did not induce an inflammatory response.

Table 13 shows the results of comparing the necrosis and apoptosis of tumor cells. In the table below, ± is minimal, + is mild, ++ is moderate, +++ is marked, and ++++ is severe, depending on the degree of cell death. Show. The number of dead cells corresponding to the corresponding stage is indicated under each symbol.

## p<0.01, Significant difference from the negative control group (G1) by Steel's t-test.

As can be seen in Table 13, the control group (Control, G1) showed a cell death rate of about 10.33%, and the low concentration experimental group (G2) showed a cell death rate similar to that of the control group, but the medium concentration (G3) And in the high concentration (G4) experimental group, more than 12.4% apoptosis was observed. Therefore, it was confirmed that the apoptosis of tumor cells increased due to the administration of the Pacalibacterium cancer inhibis microorganism.

Example 9: Using a colon cancer subcutaneous tumor model Faecalibacterium Evaluation of the efficacy of genus microorganisms

(1) animal model preparation

When the CLCC1 strain was administered, a mouse subcutaneously transplanted with colon cancer cells was used to confirm the anticancer efficacy against colon cancer, and a test was performed at Champion's Oncology, a non-clinical commissioned testing agency in the United States. Colon cancer cells were used as the mouse-derived colon cancer cell line MC38 cells, and the mouse strain was used as C57BL/6. The MC38 cells are cancer cell lines corresponding to mouse-derived colorectal carcinoma. The test group was as follows, and 10 mice were included per group.

Specifically, in 6-week-old male C57BL/6 mice, colon cancer was induced by subcutaneous tumor transplantation of the colon cancer cell line, MC38, and the effect of oral administration of Faecalibacterium cancerinhibens CLCC1 strain on the growth of colon cancer cells during colon cancer development. I looked at.

(2) Preparation of test formulation

In the same manner as the method for preparing the test formulations of the control and experimental groups of Example 8-1, the Faecalibacterium cancerinhibens CLCC1 strain of Example 1 was cultured in RCM medium, concentrated, and final glycerol using PBS buffer and glycerol. It was kept frozen at a concentration of 15% (v/v). And thawed just before the administration to the mouse maridang low concentration of viable cells in 200ul content (2x10 ⁷ cells / dose) (sample 1 corresponds to G3 of Example 8-1) and the high concentration of viable cells (2x10 ⁸ cells / dose) maridang 200ul (2x10 ⁸ cells ) (Sample 2 was orally administered to be equivalent to G4 in Example 8-1). As a control, 10 mice to which the CLCC1 strain was not administered were used.

Sample 3 was used as high-concentration dead cells (2×10 ⁸ cells/dose) by treating Sample 2 prepared above in an oven at 100° C. for 2 hours.

(3) Efficacy evaluation

As shown in the test design of FIG. 8, the test formulations of the control group and samples 1 to 3 were administered two weeks before inoculation of cancer cells, and administered once a day for a total of 40 days. Specifically, the test formulations of the control and samples 1 to 3 were orally administered once a day for 40 days, and after 14 days from the start of administration, colon cancer cells (5x10 ⁵ MC38 cells in 0.1ml PBS) were transferred to the subcutaneous tissue of the left flank. After inoculation and an engraftment period of 1 week, the size of the tumor was observed from the 22nd day to the 40th day for 18 days. Day 22, the start date of tumor observation, was regarded as day 0, and the size (tumor volume) of colon cancer tumors was measured using Magnetic Resonance (MR) imaging for 18 days. The change in the size of the tumor is measured by measuring the maximum length (L) and short axis (perpendicular width, W) of the tumor for 22 days after engraftment of cancer cells, and substituting it into Equation 3 below to determine the tumor volume (TV). ) Was calculated. The tumor volumes measured for the 18 days are shown in Table 13 and FIG. 7 below.

[Equation 3]

[Equation 2]

In Table 13 below, Mean means the mean tumor volume, and SD means the standard deviation. Table 13 shows the results of changes in tumor volume (volume, mm ³ ) in a subcutaneous tumor model in which the isolated strains according to the control group and samples 1 to 3 were transplanted with a colon cancer cell line.

Observation day Control Sample 1 Sample 2 Sample 3 Mean S.D. Mean S.D. Mean S.D. Mean S.D. One 28.63 19.64 18.3 13.12 29.1 18.21 19 14.64 4 47.63 27.39 44.8 33.41 60.7 89.62 42.3 42.41 7 109.38 60.98 92.6 59.67 100.6 111.84 85.5 89.91 8 174.63 33.93 171.7 124.72 231.4 319.62 124.2 97.38 11 367.63 83.97 295.2 201.81 303.8 360.13 277.2 265.91 13 633.75 218.75 437.4 307.75 446.1 457.75 396.8 388.22 15 934.13 329.64 686.6 495.19 785.5 694.4 707.4 679.62 18 1441.86 444.79 1296 979.09 1070.56 852.65 1094.89 897

As a result, changes in tumor volume obtained in the group to which the strains of Samples 1 to 3 were administered compared to the control group are shown in FIG. 7, respectively. Based on the last day of measurement, the tumor volume of the control group was set to 100%, and the tumor volume of Samples 1 to 3 was calculated as a percentage. 25.8% decrease), Sample 3 was 75.9% (24.1 decrease), and as a result of the test, it was confirmed that tumor growth was significantly inhibited in colon cancer model mice administered with CLCC1 compared to the control group.

It was confirmed that the tumor growth was statistically significantly inhibited even when the dead cells of CLCC1 were administered. In addition, looking at the decrease in tumor volume of Sample 1 and Sample 2 using live bacteria of the CLCC1 strain but with different concentrations, it was confirmed that the anticancer activity increased in a concentration-dependent manner under the same experimental conditions.

In addition, general symptoms such as appearance, weight, behavior, and excretion were observed at intervals of 1 to 3 days after the test formulation was administered. The test was performed for a total of 40 days after administration of the test formulation, and observed for 18 days after engraftment of cancer cells. The measured body weights (Kg) of the experimental animals are shown in Table 15 below.

Observation day Control Sample 1 Sample 2 Sample 3 One 20.60 20.79 20.60 21.17 4 20.03 20.62 21.31 21.64 7 20.24 20.47 21.42 21.55 8 20.26 20.41 21.22 21.52 11 20.05 20.44 21.06 21.22 13 20.42 20.44 21.17 21.28 15 20.54 20.77 21.13 21.38 18 20.50 20.93 21.27 21.09

As for the total body weight of the test animals, the weight of the mice corresponding to the control group after cancer cell engraftment tended to be lower than that of the test group, but there was no significant difference between the control group and the test group as a whole.

Korea Microorganism Conservation Center (overseas) KCCM13783BP 20190103

<110> ChunLab, Inc. <120> Faecalibacterium sp. and anti-cancer composition comprising the same <130> DPP20200280KR <150> KR 10-2019-0036156 <151> 2019-03-28 <150> KR 10-2019-0036155 <151> 2019-03-28 <150> KR 10-2019-0118200 <151> 2019-09-25 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1499 <212> DNA <213> Artificial Sequence <220> <223> Faecalibacterium cancerinhibens CLCC1 16S rRNA sequence <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgcgc ctaacacatg caagtcgaac 60 gagcgagaga gagcttgctt tctcgagcga gtggcgaacg ggtgagtaac gcgtgaggaa 120 cctgcctcaa agagggggac aacagttgga aacgactgct aataccgcat aagcccacgg 180 ctcggcatcg agcagaggga aaaggagcaa tccgctttga gatggcctcg cgtccgatta 240 gctagttggt gaggtaatgg cccaccaagg cgacgatcgg tagccggact gagaggttga 300 acggccacat tgggactgag acacggccca gactcctacg ggaggcagca gtggggaata 360 ttgcacaatg ggggaaaccc tgatgcagcg acgccgcgtg gaggaagaag gtcttcggat 420 tgtaaactcc tgttgttggg gaagataatg acggtaccca acaaggaagt gacggctaac 480 tacgtgccag cagccgcggt aaaacgtagg tcacaagcgt tgtccggaat tactgggtgt 540 aaagggagcg caggcgggaa gacaagttgg aagtgaaatc tatgggctca acccataaac 600 tgctttcaaa actgtttttc ttgagtagtg cagaggtagg cggaattccc ggtgtagcgg 660 tggaatgcgt agatatcggg aggaacacca gtggcgaagg cggcctactg ggcaccaact 720 gacgctgagg ctcgaaagtg tgggtagcaa acaggattag ataccctggt agtccacacc 780 gtaaacgatg attactaggt gttggaggat tgaccccttc agtgccgcag ttaacacaat 840 aagtaatcca cctggggagt acgaccgcaa ggttgaaact caaaggaatt gacgggggcc 900 cgcacaagca gtggagtatg tggtttaatt cgacgcaacg cgaagaacct taccaagtct 960 tgacatccct tgacagacat agaaatatgt tttctcttcg gagcaaggag acaggtggtg 1020 catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080 cttatggtca gttactacgc aagaggactc tggccagact gccgttgaca aaacggagga 1140 aggtggggat gacgtcaaat catcatgccc tttatgactt gggctacaca cgtactacaa 1200 tggcgttaaa caaagagaag caagaccgcg aggtggagca aaactcagaa acaacgtccc 1260 agttcggact gcaggctgca actcgcctgc acgaagtcgg aattgctagt aatcgtggat 1320 cagcatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatgaga 1380 gccgggggga cccgaagtcg gtagtctaac cgcaaggagg acgccgccga aggtaaaact 1440 ggtgattggg gtgaagtcgt aacaaggtag ccgtaggaga acctgcggct ggatcacct 1499

Claims (16)

Faecalibacterium genus microorganism (Faecalibacterium spp.) comprising a 16S rRNA gene comprising a nucleotide sequence having a sequence identity of 97% or more with the nucleotide sequence of SEQ ID NO: 1. The method of claim 1, wherein the microorganism is 15:1 w8c fatty acid, 19:0 cyclo w10c/19w6 fatty acid, 20:1 w9c fatty acid, 14:0 3OH/16:1 iso I fatty acid, 15:0 3OH fatty acid, 16: Microorganisms of the genus Pacalibacterium containing one or more fatty acids selected from the group consisting of 0 iso fatty acids and 16:0 iso 3OH fatty acids. The microorganism of the genus Pacalibacterium according to claim 1, wherein the microorganism does not contain 17:0 iso 3OH fatty acids. The microorganism of claim 1, wherein the microorganism has an anticancer activity that induces tumor growth inhibitory activity or apoptosis of cancer cells. The microorganism of the genus Pacalibacterium according to claim 1, wherein when the microorganism is administered to an animal model transplanted with a cancer cell line, a relative reduction in tumor volume is 5% or more compared to a control group to which the microorganism is not administered. The microorganism of the genus Pacalibacterium according to claim 1, wherein when the microorganism is administered to an animal model transplanted with a cancer cell line, the tumor growth inhibition rate according to the weight of the tumor is 5% or more compared to a control group to which the microorganism is not administered. The method of claim 1, wherein the microorganism is cultured under a temperature of 25 to 40 ℃ and anaerobic conditions, Pacalibacterium genus microorganism. The microorganism of claim 1, wherein the microorganism is Faecalibacterium cancer inhibins. The microorganism of claim 8, wherein the microorganism is Faecalibacterium cancer inhibins deposited with accession number KCTC 13783BP. Consisting of an extract of the microorganism of the genus Faecalibacterium (Faecalibacterium spp.) according to any one of claims 1 to 9, a culture of the microorganism, a lysate of the microorganism, and the lysate, a culture or a lysate An anti-cancer composition comprising at least one active ingredient selected from the group. The composition of claim 10, wherein the composition induces tumor growth inhibitory activity or apoptosis of cancer cells. The method of claim 10, wherein the cancer is esophageal cancer, gallbladder cancer, liver cancer, biliary tract cancer, pancreatic cancer, gastric cancer, small intestine cancer, colon cancer, colon cancer, anal cancer, rectal cancer, leukemia, prostate cancer, breast cancer, bladder cancer, kidney cancer, multiple myeloma , Cervical cancer, thyroid cancer, ovarian cancer, urethral cancer, osteosarcoma, glioblastoma, brain tumor or lymphoma composition. The composition of claim 10, wherein the cancer is at least one selected from the group consisting of colon cancer and liver cancer. The composition of claim 10, wherein the anticancer composition is a pharmaceutical composition or a food composition. 11. The composition of claim 10, wherein the composition is a probiotic. The composition of claim 15, wherein the composition comprises probiotics and prebiotics.

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