WO2019199048A1 - Lactococcus lactis gen3013 strain and a composition for preventing or treating cancer comprising the same - Google Patents

Lactococcus lactis gen3013 strain and a composition for preventing or treating cancer comprising the same Download PDF

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WO2019199048A1
WO2019199048A1 PCT/KR2019/004262 KR2019004262W WO2019199048A1 WO 2019199048 A1 WO2019199048 A1 WO 2019199048A1 KR 2019004262 W KR2019004262 W KR 2019004262W WO 2019199048 A1 WO2019199048 A1 WO 2019199048A1
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cancer
gen3013
strain
lactococcus lactis
preventing
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French (fr)
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Han Soo Park
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Genome And Company
Gwangju Institute Of Science And Technology
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/231—Lactis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to a novel Lactococcus lactis GEN3013 strain and a composition for preventing or treating cancer the same.
  • the Lactococcus lactis GEN3013 strain of the present invention exerts not only the effect of suppressing the proliferation of cancer cells per se , but also the effect of reducing the motility of cancer cells and inhibiting angiogenesis, which is remarkably superior to the known Lactococcus lactis strains.
  • Lactococcus lactis GEN3013 strain of the present invention exhibits activities of anti-inflammation, anti-oxidant, and immune enhancement.
  • the high cost for the treatment of cancer comprises direct medical expenses, as well as indirect expenses such as reduced economic activities, rehabilitation costs, and patient care costs, which may put economic burden on a family of a cancer patient and also all societies.
  • a new anti-cancer agent which is especially low-cost and safe, is now being required.
  • the tumor microenvironment is preserved by unlimited the proliferation of cells, angiogenesis derived from hypoxia, controlling functions of immune cells infiltrating cancer tissues, wherein the cells move or metastasize to other issues through blood or lymphatic vessels, finally leading to death.
  • a synthetic anti-cancer agent developed for treating cancers inhibits metabolism of cancer cells to suppress the proliferation of cells or induce apoptosis. Otherwise, DNAs of double stranded helix are unfolded during cell division to inhibit the DNA replication procedures, based on which cell division is blocked and apoptosis is induced, finally suppressing the proliferation of cancer cells or eliminating cancer cells.
  • the synthetic anti-cancer agent attacks even normal cells. As such, a drug-induced side effect such as hair loss, diarrhea, or fever is caused.
  • a targeted anti-cancer agent for reducing the side effect and enhancing anti-cancer activities has been developed, where it targets a mutated gene involving cancer generation, not normal cells, to enhance a synthetic anti-cancer agent. However, a resistance mechanism to most of the targeted anti-cancer agents is expressed within 2 to 3 years. In this case, the targeted anti-cancer agent is not effective for the treatment any longer.
  • One of cancer characteristics is to generate hypoxia in tissues resulting from the unlimited the proliferation, followed by producing abnormal microvessels for the survival under the hypoxia circumstance, receiving nutrients, and maintaining the proliferation.
  • an anti-cancer agent targeting angiogenesis-related factors has been developed and used as a treating agent for patients.
  • the angiogenesis-related anti-cancer agent antagonizes angiogenesis, it does not sufficiently suppress the fast the proliferation of cancer cells. As such, it is administered to clinical patients in combination with other anti-cancer agents.
  • an immuno-oncology agent such as immune checkpoint inhibitor for controlling immunity of a patient, which is now used as a treating agent.
  • the treatment with PD-1/PD-L1 is highly responsive for skin or lung cancer patients, with improved quality of life.
  • the immuno-oncology agent controls the functions of immune cells in the tumor microenvironment to suppress the proliferation of cancer cells and increase activity of immune cells.
  • the immuno-oncology agent does not exhibit the same anti-cancer effect for all patients.
  • any biomarker specific to the immuno-oncology agent has not clearly been established.
  • Cancers are caused by exposure to a toxin-induced chemical, DNA damage due to stress, virus or harmful bacteria infection, chronic inflammation, etc.
  • Such cancer-inducing mechanism is not limited to a specific cancer and is observed in almost all carcinoma. It is thus known that the alleviation of information caused by infection and anti-oxidant activities to reduce abnormally high concentration of reactive oxygen species (ROS) not only lowers the stimulation to cancer cells, but also alleviate the proliferation of cancer cells.
  • ROS reactive oxygen species
  • the maintenance of immunity and homeostasis is most important in health care. When immunity is lowered, the exposure to hazardous environment or infection easily cause a disease.
  • the consistent treatment with anti-cancer agents and antibiotics against a disease weakens immunity in body and slow the drug effect for alleviating or treating a disease, which may lead to a secondary disease.
  • Lactic acid bacteria dwelling in the digestive tract of human plays a role to provide useful nutrients by degrading fibroid materials and composite proteins.
  • live microorganisms that benefits the host in health by improving the intestinal microbial environment in the GI tract of animals including human are collectively designated as probiotics.
  • Lactococcus lactis GEN3013 strain exerts not only the effect of suppressing the proliferation of cancer cells, reducing the motility of cancer cells, inhibiting angiogenesis, but also anti-inflammation and anti-oxidant activities, which arrived at the present invention.
  • the present invention is to identify a strain exhibiting anti-cancer and anti-inflammation effects, to study phylogenetic characteristics of the strain, and to utilize the findings for the development of a novel anti-cancer agent or therapeutic agent for inflammatory diseases or immunological diseases.
  • the present invention aims to provide a novel Lactococcus lactis strain with excellent effect of preventing or treating cancer.
  • the present invention aims to provide a pharmaceutical composition for preventing or treating cancer comprising the Lactococcus lactis strain.
  • the present invention aims to provide a food composition or an animal feed composition for preventing or improving cancer comprising the Lactococcus lactis strain.
  • the present invention in order to achieve the aims above, provides Lactococcus lactis GEN3013 strain.
  • the strain was deposited in Biological Resource Center affiliated in Korea Research Institute of Bioscience and Biotechnology under Accession Number KCTC13426BP in the name of Lactococcus lactis GNC3013 on December 13, 2017, which was changed into Lactococcus lactis GEN3013 on April 3, 2018.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising Lactococcus lactis GEN3013 strain.
  • the Lactococcus lactis GEN3013 strain may refer to the strain itself or include cytoplasmic fractions obtained by lysis of the strain.
  • the present invention provides a method for preventing or treating cancer, which comprises administering Lactococcus lactis GEN3013 strain to a subject in need thereof in a therapeutically effective amount.
  • subject as used herein may be a mammal, and preferably, human.
  • Lactococcus lactis GEN3013 strain of the present invention is characterized in exhibiting all effects of anti-cancer, anti-inflammation, anti-oxidant, and immunity enhancement.
  • the cancer may be, but is not limited to, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft-tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mastocytoma, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, gastric cancer, renal cancer, colon cancer, haematopoietic tumor, or metastatic cancers thereof.
  • the cancer may be lung cancer, colon cancer, gastric cancer, breast cancer, or liver cancer.
  • the inflammatory disease may be, but is not limited to, osteoarthritis, rheumarthritis, gout, spondylitis ankylopoietica, tendinitis, aponeurositis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, asthma, atopy, Crohn’s disease, or ulcerative colitis.
  • Lactococcus lactis GEN3013 strain of the present invention may exhibit anti-cancer effect by suppressing the proliferation of cancer cells and reducing the motility of cancer cells. Also, Lactococcus lactis GEN3013 strain of the present invention may exhibit anti-cancer effect by suppressing the expressions of vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), and angiopoietin 2 (Ang2).
  • VEGF vascular endothelial growth factor
  • Ang1 angiopoietin 1
  • Ang2 angiopoietin 2
  • Lactococcus lactis GEN3013 strain of the present invention may exhibit anti-inflammation and anti-cancer effect by suppressing the expression of TNF- ⁇ .
  • TNF- ⁇ is a cytokine secreted by immunocyte during chronic or acute inflammation response, such as infection in human body, external injury, septicemia, or rheumarthritis. If the concentration of TNF- ⁇ increases, it causes damage in intracellular lipid and glucose metabolism. While TNF- ⁇ is known as a cytokine inducing cell necrosis, studies were reported that when continuous stimulation of TNF- ⁇ is transmitted to the cells, it affects intracellular metabolism to generate oncogene. As such, the abnormal proliferation of cells occurs to finally promote the development of cancer.
  • the Lactococcus lactis GEN3013 strain of the present invention increases the production of IFN-gamma and reduces the production of IL-10, which may exhibit anti-cancer or immunity enhancement effects.
  • IFN-gamma is a protein secreted by NK cells and T lymphocytes when immunostimulation occurs. It is known that the reaction with a plasma membrane receptor activates immunocytes through JAK-STAT signal to suppress the proliferation of cancer cells or kill cancer cells.
  • IL-10 is one of the immune factors produced during inflammation response in macrophage, T cell, NK cell, and dendritic cell.
  • the expression of PD1 is increased by IL-10, which induces the expression of PD-L1 of tumor.
  • the signal transmission between the immune cells and the tumor is performed to promote the mechanism that reduces the effect of anti-cancer agent.
  • IL-10 constantly induces the activity of regulatory T cell wherein the regulatory T cell inhibits normal T cell activity of killing tumor cells.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer, characterized in comprising Lactococcus lactis GEN3013 strain; and an anti-cancer agent or an immune-oncology agent.
  • the present invention provides a method for preventing or treating cancer, which comprises administering Lactococcus lactis GEN3013 strain; and an anti-cancer agent or an immune-oncology agent to a subject in need thereof.
  • the anti-cancer agent may be, but is not limited to, oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, doxorubicin, and the like.
  • the immune-oncology agent may be, but is not limited to, anti-PD1, anti-PDL1, anti-PDL2, anti-A2AR, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-KIR, anti-CTLA, anti-Tim3, or anti-LAG3.
  • Lactococcus lactis GEN3013 strain and an anti-cancer agent or an immune-oncology agent may be administered sequentially or simultaneously to a patient in need thereof.
  • the present invention relates to a food composition or an animal feed composition for preventing or improving cancer comprising Lactococcus lactis GEN3013 strain.
  • the food composition may be, but is not limited to, a health functional food, a dairy product, a fermented product, or a food additive.
  • the novel Lactococcus lactis GEN3013 strain of the present invention has the effect of suppressing the proliferation of different cancer cell lines. Further, the Lactococcus lactis GEN3013 strain of the present invention exerts the effect of reducing the motility of cancer cells and inhibiting angiogenesis, which is remarkably superior to the known Lactococcus lactis strains.
  • Lactococcus lactis GEN3013 strain of the present invention may be used for inflammatory diseases or immunological diseases, as it has anti-inflammation, anti-oxidant, or immunity enhancement effect.
  • Lactococcus lactis GEN3013 strain of the present invention not only exhibits excellent anti-cancer effect when administered alone, but also has more remarkable anti-cancer effect when administered in combination with other anti-cancer agents or immune-oncology agents in comparison to each administration thereof.
  • Fig. 1 is the results showing cell adhesion of Lactobacillus rhamnosus LGG and GEN3013 strain to Caco-2 cells.
  • Fig. 2 is the results showing the motility of cancer cells when cancer cell lines are not treated or treated with GEN3013 strain.
  • Fig. 3 shows the expression of VEGF in HCT116 cell line non-treated or treated with GEN3013 strain as confirmed through PCR.
  • Fig. 4 shows the expression of Ang1 and Ang2 in HCT116 cell lines non-treated or treated with GEN3013 strain as confirmed through real-time PCR.
  • Fig. 5 shows the increased the expression of TNF- ⁇ by LPS, in RAW264.7 cells non-treated or treated with GEN3013 strain.
  • Fig. 6 is the results showing the effect of GEN3013 strain for increasing the production of IFN-gamma as confirmed by ELISA kit.
  • Fig. 7 is the results showing the effect of GEN3013 strain for inhibiting the production of IL-10 as confirmed by ELISA kit.
  • Fig. 8 is the results showing the effect of GEN3013 strain for inhibiting reactive oxygen species.
  • Fig. 9 is the results showing the proliferation of cells when cancer cell lines were treated with either one of GEN3013 strain or an anti-cancer agent (oxaliplatin or pemetrexed), or the combination.
  • Fig. 10 is the graph showing the effect of inhibiting tumor when GEN3013 strain or an anti-cancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
  • Fig. 11 is the graph showing the effect of inhibiting tumor when GEN3013 strain or an antibody for treating cancer (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
  • Fig. 12 is the results obtained in immune cells separated from spleen of mice when GEN3013 strain or an anti-cancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
  • Fig. 13 is the results obtained in immune cells separated from tumor of mice when GEN3013 strain or an anti-cancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
  • Fig. 14 is the results obtained in immune cells separated from spleen of mice when GEN3013 strain or an antibody for treating cancer (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
  • Fig. 15 is the results obtained in immune cells separated from tumor of mice when GEN3013 strain or an antibody for treating cancer (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
  • the present inventors have constantly studied to find probiotics having remarkable anti-cancer effect and finally found that a novel Lactococcus lactis GEN3013 strain exhibits superior anti-cancer effect, whereby arriving at the present invention.
  • the GEN3013 strain exerts the remarkable effect of suppressing the proliferation on various cell lines from, for example, lung cancer, colon cancer, gastric cancer, breast cancer, and liver cancer.
  • the motility of cancer cells is remarkably reduced. Unlike normal cells, cancer cells feature in being able to move with the proliferation even in case where damage occurs on the cells.
  • the reduced motility of cancer cells means a lowered possibility of metastasis of cancer.
  • the GEN3013 strain has the effect of inhibiting metastasis of cancer.
  • the GEN3013 strain may inhibit the expression of all of VEGF (vascular endothelial growth factor), Ang1 (Angiopoietin1), and Ang2 (Angiopoietin2), as a main factor involving angiogenesis.
  • VEGF vascular endothelial growth factor
  • Ang1 Angiopoietin1
  • Ang2 Angiopoietin2
  • Angiogenesis is one of the characteristics of cancer cells. Once angiogenesis is inhibited, the delivery of nutrients to cancer cells through blood vessels can be blocked to suppress the proliferation of cancer cells.
  • Immunity is the basic factor in human health.
  • the depression of immunity causes the proliferation of harmful bacteria, occurrence of diseases, and progress to chronic diseases.
  • a secondary disease may be developed.
  • the GEN3013 strain of the present invention increases immunity to enhance anti-cancer activities in a patient under anti-cancer treatment.
  • ROS reactive oxygen species
  • ROS ROS-induced retinopathy
  • GEN3013 strain of the present invention reduces ROS and suppress the proliferation of cancer cells, so that the development of different diseases is prevented.
  • the GEN3013 strain of the present invention increases the production of IFN-gamma, which is an immune factor secreted by cytotoxic T cells having the suppression activity of the proliferation of tumor cells, and reduces the production of IL-10, which is an immune factor secreted by T regulatory cells suppressing the activities of cytotoxic T cells, which results in effective immune oncology.
  • the cancer cell lines treated with GEN3013 have more remarkable effect of suppressing the cell proliferation than those treated with an anti-cancer agent.
  • the cancer cell lines treated with the combination of GEN3013 and an anti-cancer agent have more excellent effect of suppressing the cell proliferation than those treated with either one of GEN3013 or an anti-cancer agent.
  • GEN3013 exhibits increased anti-cancer activities in combination with a known anti-cancer agent.
  • the Lactococcus lactis GEN3013 strain of the present invention exerts the effect of suppressing the proliferation of cancer cells, reducing the motility of cancer cells, and inhibiting angiogenesis. Therefore, the Lactococcus lactis GEN3013 strain of the present invention may be used as a remarkable anti-cancer agent and also can be administered in combination with a conventional synthetic anti-cancer agent or immuno-oncology agent.
  • the GEN3013 strain may significantly reduce the expression of TNF- ⁇ derived by LPS as an inflammation inducer to prevent or treat both of inflammatory disease and cancer.
  • the present invention provides a method for preventing or treating cancer, inflammatory disease, immune disease, etc., by administering the Lactococcus lactis GEN3013 strain to the body.
  • composition of the present invention may be used as a medicinal drug, a health functional food, a dairy product, a fermented product, a food additive, or an animal feed.
  • Feces of a 20-month old infant were obtained in order to isolate lactic acid bacteria in human intestine.
  • a strain was isolated from the infant feces by utilizing selective media for Lactobacillus spp. and Bifidobacterium spp .
  • the collected feces samples were diluted in a phosphate buffered saline (PBS) solution including 0.85% NaCl to a 10 -5 , 10 -6 fold dilution, followed by spreading the same in media of De Man Rogosa, Sharpe agar (MRS broth; Difco, USA) with the addition of 0.5% cysteine, and being subjected to anaerobic culture at 37°C for 48 hours, to obtain colonies.
  • PBS phosphate buffered saline
  • the selected strain was subcultured in MRS broth and BL broth, and then stored in MRS broth and BL broth containing 20% glycerol in freezer at 80 °C.
  • Lactococcus lactis When the sequence of rRNA of the strain was analyzed, it was identified as Lactococcus lactis .
  • the strain was deposited in Biological Resource Center affiliated in Korea Research Institute of Bioscience and Biotechnology under Accession Number KCTC13426BP in the name of Lactococcus lactis GNC3013 on December 13, 2017, which was changed into Lactococcus lactis GEN3013 on April 3, 2018.
  • the indicator strains were Staphylococcus aureus KCTC3881, P. Aeruginosa KCTC 2004, E. Coli KCTC 1682, and L. Innocua KCTC 3586, which were cultured, spread on agar plate, and placed on a sterilized disc.
  • Lactococcus lactis GEN3013 as a test strain was placed on a sterilized disc, cultured at 37 °C for 24 hours, followed by measuring the size of inhibition zone. The results were shown in Table 3 below.
  • GEN3013 strain was subcultured in MRS broth containing 0.5 % glucose and 0.05% cysteine.
  • MRS broth containing 0.5 % glucose and 0.05% cysteine which was adjusted to have pH 2, 2.5, 3 and 3.5.
  • the washed bacteria were inoculated such that the concentration is 2 ⁇ 10 6 CFU/ml, cultured in a non-growing state at 37°C for 2 hours, and the viable bacteria were counted.
  • the survival rate was calculated by comparing the number of the viable bacteria before the inoculation with that 2 hours after the inoculation as follows. The results were shown in Table 4 below.
  • Lactococcus lactis GEN 3013 strain has a good acid resistance, the survival in an acidic condition is advantageous.
  • the cells used for the experiment were cultured at 37°C in 5% CO 2 at 90% humidity condition, where the culture media was changed once every two or three days.
  • the cells were washed with phosphate buffered saline (PBS), separating the adhered cells by using a trypsin-EDTA solution, and placing in a 12-well plate such that it had 1 ⁇ 10 5 cell/well.
  • PBS phosphate buffered saline
  • the isolated strains as subcultured were inoculated in the cells cultured for 20 days such that the amount is 1 ⁇ 10 7 CFU/ml, and cultured at 37°C in 5% CO 2 for 2 hours. After washing twice with PBS, the viable bacteria were counted in MRS broth containing 0.05% cysteine. The adherence rate was calculated by comparing the initial number with the final number where the data were indicated in a fold unit based on the value of Lactobacillus rhamnosus LGG as a control.
  • Cell lines of lung cancer (A549, H1975, HCC827, H1299, SW900), colon cancer (HCT116, LoVo, SNU-C2A), gastric cancer (SNU216, AGS, MKN-28, MKN-1, SNU-601, SNU-1), breast cancer (Hs578T, BT20, MDA-MB-231), and liver cancer (HepG2, Hep3B) were used as human cancer cell lines for the assay, while cell lines of colon cancer (CT26, MC38), lung cancer (LLC1), and breast cancer (4T1) were used as mouse cancer cell lines.
  • Cancer cell lines were placed in a 96-well plate in an amount of 1 ⁇ 5 ⁇ 10 3 cells/well. After 24 hours, a lactic acid bacteria sample was added in 0.5% and cultured for 72 hours, followed by treating the reagent, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue, in each well to react for 2 hours, so that viable cells were reacted with mitochondria to change the yellow MTT into violet color. The culture media containing MTT were then all removed, adding 100 ⁇ l of DMSO in each well, measuring the concentration of the violet color as absorbance at 540 nm by using Multiplate Reader. The test results on human-derived cancer cell lines and mouse-derived cancer cell lines were shown in Tables 6 and 7 below.
  • Cancer type Cell line Suppression rate of the proliferation (%) ⁇ SD colon cancer CT26 76.73 ⁇ 1.01 MC38 55.04 ⁇ 4.71 lung cancer LLC1 68.22 ⁇ 10.83 breast cancer 4T1 78.82 ⁇ 1.51
  • the GEN3013 strain exerted remarkable effect for suppressing the cell proliferation on both human- and mouse-derived cancer cell lines.
  • Korean Patent Application No. 10-2001-0012973 discloses that lactis ssp. cremoris has IC 50 values of 54 ⁇ g/ml on SNU-1 and 75 ⁇ g/ml on SNU-C2A, while Lactococcus lactis ssp. lactis has the values of 11 ⁇ g/ml on SNU-1 and 23 ⁇ g/ml on SNU-C2A.
  • GEN3013 had IC 50 values of 6.43 ⁇ g/ml on SNU-1 and 8.82 ⁇ g/ml on SNU-C2A, which was much lower than IC 50 values of other strains that were already known in prior studies. As such, it was confirmed that GEN3013 exhibits remarkable effect for suppressing growth of different cancer cells.
  • cancer cells are featured in moving along with the proliferation even when damage occurs on the cells.
  • wound healing assay was performed.
  • A549 (5X10 5 cells) and HCT116 (6X10 5 cells) strains were fixed on a 6-well plate. When 90 to 95% of cells were proliferated, we damaged the cells with 1000p tip in a certain interval. The cells were treated with PBS or GEN3013 strains for 24 hours to observe the motility of the cells by microscope. The results were shown in Fig. 2.
  • A549 and HCT116 had 65.31 ⁇ 1.69 % and 33.82 ⁇ 5.86 % of the active motility of cancer cells, respectively.
  • the motility of cells was remarkably reduced to 17.60 ⁇ 5.19 % and 13.50 ⁇ 8.55 % in A549 and HCT116, respectively.
  • HCT116 cell lines were treated with GEN3013 for 24 hours, from which RNA was obtained to synthesize cDNA. With the same, the expression of vascular endothelial growth factor (VEGF) was analyzed in normal PCR, while the expression of Ang1 and Ang2 was analyzed in real-time PCR. The results were shown in Figs. 3 and 4.
  • VEGF vascular endothelial growth factor
  • a group treated with GEN3013 had the remarkably reduced expression of 121 isoform and 165 isoform of VEGF in comparison with a control. Further, the expression of 189 isoform was insignificant in a control, while no expression of 189 isoform was found in a group treated with GEN3013.
  • Ang1 and Ang2 which are a factor involving angiogenesis other than VEGF
  • the expression of Ang1 was rarely observed while no expression of Ang2 was observed.
  • GEN3013 inhibits angiogenesis and thus block the delivery of nutrients into cancer cells through blood vessels, which results in remarkable effect for suppressing the proliferation of cancer cells.
  • RNA In order to evaluate anti-inflammation effect of GEN3013 strain, RAW264.7 cells as mouse macrophage were treated with GEN3013 strain for 24 hours and then with an inflammation inducer, LPS 100 ng/ml, for 6 hours to obtain RNA.
  • cDNA was synthesized from 1 ⁇ g of RNA and used to evaluate the expression of TNF- ⁇ , which is known as an inflammation factor, in real-time PCR. The results were shown in Fig. 5.
  • PBMC peripheral blood mononuclear cells
  • GEN3013 was placed in a 96-well plate, adding isolated monocytes in an amount of 5 x 10 3 per well, slightly mixing so that the reaction with the strains occurred, and culturing for 2 hours. Through a reaction for 2 hours, GEN3013 directed monocytes to be differentiated into macrophages.
  • T cells expressing CD4 and CD8 were separated from the remaining PBMC cells in MACS buffer and LS column.
  • the separated T cells were diluted in 100 ⁇ l of RPMI media such that the amount was 5 x 10 4 cells, placing in the wells containing monocytes and GEN3013, and culturing for 48 hours to activate immune.
  • a group treated with GEN3013 increased the production of IFN-gamma, which is an immune factor secreted by cytotoxic T cells having the suppression activity of the proliferation of tumor cells, more than two times in comparison with a control treated with E.coli.
  • the GEN3013 strain exerts the effect of inhibiting the cell damage resulting from ROS in cell and of reducing the proliferation of cancer cells.
  • Lactococcus lactis GEN3013 strain was cultured in MRS liquid media (Difco) containing 0.5% glucose and 0.05% cysteine at 37°C for 24 hours, lysing bacteria to be used for the test.
  • A549 human lung cancer cells
  • LLC1 mouse lung cancer cells
  • HCT116 human colon cancer cells
  • LoVo human colon cancer cells
  • MC38 mouse colon cancer cells
  • Human cancer cell lines were cultured in RPMI media, while mouse cancer cell lines in DMEM media.
  • Culture media of all cancer cell lines contained 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco Life Technologies, Grand Island, NY, U.S.A.). The test using cell lines were proceeded while culturing at 37°C and 5% CO 2 .
  • Oxaliplatin or pemetrexed was used as an anti-cancer agent.
  • an experiment was performed based on the following method.
  • the different cancer cell lines were diluted and placed in each well of a 6-well plate in an amount of 1 to 2 X 10 3 , fixed for 24 hours, followed by treating each well with lactic acid bacteria and/or an anti-cancer agent and changing the media every two or three days.
  • the plate treated with lactic acid bacteria and/or the anti-cancer agent for 144 hours was treated with 4% formalin for 30 minutes to fix the cells, washed with PBS twice, dyed with a crystal violet solution for 5 minutes, and washed again with distilled water, to observe the cell proliferation.
  • the results were shown in Fig. 9.
  • the cancer cell lines treated with GEN3013 exhibited the superior suppression of the proliferation of cancer cells, in comparison to the cancer cell lines treated with an anti-cancer agent.
  • MC38 mouse colon cancer cells and LLC1 mouse lung cancer cells which are treated with GEN3013 alone, showed almost complete suppression of the cancer cell proliferation, which resulted from GEN3013.
  • cancer cell lines treated with the GEN3013 strain in combination with the anti-cancer agent exerted more remarkable effect for suppressing the cell proliferation than the cancer cell lines treated with either one of GEN3013 or the anti-cancer agent.
  • the lactic acid bacteria samples were administered to mice for 2 weeks to increase intestinal adherence and immunity before preparing a tumor model.
  • 2 X 10 5 MC38 cancer cells were then subcutaneously injected around a right hip of 8 C57BL/6 mice per group to prepare a tumor model.
  • the lactic acid bacteria samples were orally administered to the animal models for three weeks (from Monday to Saturday).
  • the lactic acid bacteria samples to be orally administered were diluted in 200 ⁇ l of PBS in an amount of CFU 1*10 9 per mouse.
  • Oxaliplatin (3 mg/kg, Sellekchem) or anti-PD1 (2 mg/kg, BioXCell) as an anti-cancer agent was intraperitoneally injected every Monday and Thursday after the cancer is induced.
  • the effect of inhibiting tumor was observed in a group treated with any one of lactic acid bacteria; and oxaliplatin or anti-PD1, and with the combination. The results were shown in Figs. 10 and 11.
  • the combination administration exhibited more remarkable effect of inhibiting tumor than the single administration of GEN3013 or the anti-cancer agent.
  • Example 11 Based on the results of Example 11, an animal test was performed in order to analyze the invasion of immune cells into tumor with GEN3013. Upon completing the test, tumor and spleen were separated from the mice, lysed, and isolated the immune cells. The isolated immune cells were reacted with fluorescence antibodies, which corresponds to a marker of the immune cells depending on the function, followed by analyzing by FACs. The results were shown in Figs. 12 to 15.
  • a group treated with GEN3013 had increased distribution of CD4 T cell, CD8 T cell, CD8 effector T cell, and NK cell, which play an important roll on anti-cancer immune response, in comparison with a control.
  • the number of regulatory T cells controlling activities of T-cells were significantly reduced.
  • the increased immune response was more remarkable in a group treated with the combination of GEN3013 and the anti-cancer agent.

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Abstract

The present invention relates to Lactococcus lactis GEN3013 strain and a composition for preventing or treating cancer and inflammatory diseases, comprising the same. Specifically, the Lactococcus lactis GEN3013 strain of the present invention exerts not only the effect of inhibiting the proliferation of cancer cells, reducing the motility of cancer cells, inhibiting angiogenesis, and increasing anti-cancer immune response, but also the effect of reducing the expression of inflammation factor, to prevent or treat cancers or inflammatory diseases. Further, Lactococcus lactis GEN3013 strain of the present invention exhibits more excellent anti-cancer effect when combined with other anti-cancer agents or antibodies for treating cancer.

Description

LACTOCOCCUS LACTIS GEN3013 STRAIN AND A COMPOSITION FOR PREVENTING OR TREATING CANCER COMPRISING THE SAME
The present invention relates to a novel Lactococcus lactis GEN3013 strain and a composition for preventing or treating cancer the same. Specifically, the Lactococcus lactis GEN3013 strain of the present invention exerts not only the effect of suppressing the proliferation of cancer cells per se, but also the effect of reducing the motility of cancer cells and inhibiting angiogenesis, which is remarkably superior to the known Lactococcus lactis strains.
Further, the Lactococcus lactis GEN3013 strain of the present invention exhibits activities of anti-inflammation, anti-oxidant, and immune enhancement.
The cancer incidence is increasing every year due to different social and environmental factors. Cancer is a representative disease under very serious consideration, while anti-cancer treatment has very widely been studied globally including basic research in cellular level. However, mechanism leading to cancer has not yet been established in a completely clear manner. Further, for the prevention of recurrence and with consideration of difficulties in achieving a full recovery, increasing demands on a new anti-cancer agent still exist.
Meanwhile, the high cost for the treatment of cancer comprises direct medical expenses, as well as indirect expenses such as reduced economic activities, rehabilitation costs, and patient care costs, which may put economic burden on a family of a cancer patient and also all societies. As such, a new anti-cancer agent, which is especially low-cost and safe, is now being required.
In cancer, the tumor microenvironment is preserved by unlimited the proliferation of cells, angiogenesis derived from hypoxia, controlling functions of immune cells infiltrating cancer tissues, wherein the cells move or metastasize to other issues through blood or lymphatic vessels, finally leading to death.
A synthetic anti-cancer agent developed for treating cancers inhibits metabolism of cancer cells to suppress the proliferation of cells or induce apoptosis. Otherwise, DNAs of double stranded helix are unfolded during cell division to inhibit the DNA replication procedures, based on which cell division is blocked and apoptosis is induced, finally suppressing the proliferation of cancer cells or eliminating cancer cells. However, the synthetic anti-cancer agent attacks even normal cells. As such, a drug-induced side effect such as hair loss, diarrhea, or fever is caused. A targeted anti-cancer agent for reducing the side effect and enhancing anti-cancer activities has been developed, where it targets a mutated gene involving cancer generation, not normal cells, to enhance a synthetic anti-cancer agent. However, a resistance mechanism to most of the targeted anti-cancer agents is expressed within 2 to 3 years. In this case, the targeted anti-cancer agent is not effective for the treatment any longer.
One of cancer characteristics is to generate hypoxia in tissues resulting from the unlimited the proliferation, followed by producing abnormal microvessels for the survival under the hypoxia circumstance, receiving nutrients, and maintaining the proliferation. As such, an anti-cancer agent targeting angiogenesis-related factors has been developed and used as a treating agent for patients. However, while the angiogenesis-related anti-cancer agent antagonizes angiogenesis, it does not sufficiently suppress the fast the proliferation of cancer cells. As such, it is administered to clinical patients in combination with other anti-cancer agents.
Recently, the active and vibrant research on an anti-cancer agent and the tumor microenvironment has led to develop an immuno-oncology agent such as immune checkpoint inhibitor for controlling immunity of a patient, which is now used as a treating agent. In particular, it is known that the treatment with PD-1/PD-L1 is highly responsive for skin or lung cancer patients, with improved quality of life. The immuno-oncology agent controls the functions of immune cells in the tumor microenvironment to suppress the proliferation of cancer cells and increase activity of immune cells. However, the immuno-oncology agent does not exhibit the same anti-cancer effect for all patients. Further, any biomarker specific to the immuno-oncology agent has not clearly been established. Several problems exist, for example, no drug effect in the variation of JAK-STAT gene, risks of autoimmune disease due to a characteristic of a produced antibody, high cost for the treatment, and the like.
Cancers are caused by exposure to a toxin-induced chemical, DNA damage due to stress, virus or harmful bacteria infection, chronic inflammation, etc. Such cancer-inducing mechanism is not limited to a specific cancer and is observed in almost all carcinoma. It is thus known that the alleviation of information caused by infection and anti-oxidant activities to reduce abnormally high concentration of reactive oxygen species (ROS) not only lowers the stimulation to cancer cells, but also alleviate the proliferation of cancer cells.
The maintenance of immunity and homeostasis is most important in health care. When immunity is lowered, the exposure to hazardous environment or infection easily cause a disease. The consistent treatment with anti-cancer agents and antibiotics against a disease weakens immunity in body and slow the drug effect for alleviating or treating a disease, which may lead to a secondary disease.
Lactic acid bacteria dwelling in the digestive tract of human plays a role to provide useful nutrients by degrading fibroid materials and composite proteins. As such, live microorganisms that benefits the host in health by improving the intestinal microbial environment in the GI tract of animals including human are collectively designated as probiotics.
The research on anti-cancer activities of lactic acid bacteria has started since the publication of Metchnikoff in the 1990s regarding extended life span by lactic acid bacteria. Specifically, Bogdanov et al. published that Lactobacillus bulgaricus remarkably inhibited the growth of Sarcoma 180 as an ascites tumor cell line that was transplanted into an ascites of a test animal. Further, Matsuzaki et al. published that when Lactobacillus casei was administered to 3LL and Line-10 hapatoma as a cancer model similar to human cancer metastasis, metastasis to lung and lymph node was significantly inhibited. They also disclosed that when Lactobacillus casei was consecutively administered via an intravenous injection of a mouse (100 mg/kg × 4) into B16-F10 melanoma where metastatis frequently occurs, superior cancer treatment effect was achieved. While many researchers then tried to develop a strain having anti-cancer activities, there has not yet been any case where a strain having remarkable anti-cancer activities was developed as a medicinal drug.
Based on the above, while the present inventors developed and studied probiotics having remarkable anti-cancer activity, it was confirmed that Lactococcus lactis GEN3013 strain exerts not only the effect of suppressing the proliferation of cancer cells, reducing the motility of cancer cells, inhibiting angiogenesis, but also anti-inflammation and anti-oxidant activities, which arrived at the present invention.
The present invention is to identify a strain exhibiting anti-cancer and anti-inflammation effects, to study phylogenetic characteristics of the strain, and to utilize the findings for the development of a novel anti-cancer agent or therapeutic agent for inflammatory diseases or immunological diseases.
Accordingly, the present invention aims to provide a novel Lactococcus lactis strain with excellent effect of preventing or treating cancer.
In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer comprising the Lactococcus lactis strain.
Further, the present invention aims to provide a food composition or an animal feed composition for preventing or improving cancer comprising the Lactococcus lactis strain.
The present invention, in order to achieve the aims above, provides Lactococcus lactis GEN3013 strain. The strain was deposited in Biological Resource Center affiliated in Korea Research Institute of Bioscience and Biotechnology under Accession Number KCTC13426BP in the name of Lactococcus lactis GNC3013 on December 13, 2017, which was changed into Lactococcus lactis GEN3013 on April 3, 2018.
In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising Lactococcus lactis GEN3013 strain. Specifically, the Lactococcus lactis GEN3013 strain may refer to the strain itself or include cytoplasmic fractions obtained by lysis of the strain.
Alternatively, the present invention provides a method for preventing or treating cancer, which comprises administering Lactococcus lactis GEN3013 strain to a subject in need thereof in a therapeutically effective amount. The term “subject” as used herein may be a mammal, and preferably, human.
In particular, the Lactococcus lactis GEN3013 strain of the present invention is characterized in exhibiting all effects of anti-cancer, anti-inflammation, anti-oxidant, and immunity enhancement.
In the present invention, the cancer may be, but is not limited to, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft-tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mastocytoma, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, gastric cancer, renal cancer, colon cancer, haematopoietic tumor, or metastatic cancers thereof. Preferably, in the present invention, the cancer may be lung cancer, colon cancer, gastric cancer, breast cancer, or liver cancer.
In the present invention, the inflammatory disease may be, but is not limited to, osteoarthritis, rheumarthritis, gout, spondylitis ankylopoietica, tendinitis, aponeurositis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, asthma, atopy, Crohn’s disease, or ulcerative colitis.
Lactococcus lactis GEN3013 strain of the present invention may exhibit anti-cancer effect by suppressing the proliferation of cancer cells and reducing the motility of cancer cells. Also, Lactococcus lactis GEN3013 strain of the present invention may exhibit anti-cancer effect by suppressing the expressions of vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), and angiopoietin 2 (Ang2).
Also, Lactococcus lactis GEN3013 strain of the present invention may exhibit anti-inflammation and anti-cancer effect by suppressing the expression of TNF-α. Specifically, TNF-α is a cytokine secreted by immunocyte during chronic or acute inflammation response, such as infection in human body, external injury, septicemia, or rheumarthritis. If the concentration of TNF-α increases, it causes damage in intracellular lipid and glucose metabolism. While TNF-α is known as a cytokine inducing cell necrosis, studies were reported that when continuous stimulation of TNF-α is transmitted to the cells, it affects intracellular metabolism to generate oncogene. As such, the abnormal proliferation of cells occurs to finally promote the development of cancer.
In addition, the Lactococcus lactis GEN3013 strain of the present invention increases the production of IFN-gamma and reduces the production of IL-10, which may exhibit anti-cancer or immunity enhancement effects. IFN-gamma is a protein secreted by NK cells and T lymphocytes when immunostimulation occurs. It is known that the reaction with a plasma membrane receptor activates immunocytes through JAK-STAT signal to suppress the proliferation of cancer cells or kill cancer cells.
IL-10 is one of the immune factors produced during inflammation response in macrophage, T cell, NK cell, and dendritic cell. The expression of PD1 is increased by IL-10, which induces the expression of PD-L1 of tumor. As such, the signal transmission between the immune cells and the tumor is performed to promote the mechanism that reduces the effect of anti-cancer agent. IL-10 constantly induces the activity of regulatory T cell wherein the regulatory T cell inhibits normal T cell activity of killing tumor cells. Thus, it is known that if the expression of IL-10 in the tumor microenvironment is high, prognosis for patients with malignant tumors is not good.
The present invention relates to a pharmaceutical composition for preventing or treating cancer, characterized in comprising Lactococcus lactis GEN3013 strain; and an anti-cancer agent or an immune-oncology agent. Alternatively, the present invention provides a method for preventing or treating cancer, which comprises administering Lactococcus lactis GEN3013 strain; and an anti-cancer agent or an immune-oncology agent to a subject in need thereof.
The anti-cancer agent may be, but is not limited to, oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, doxorubicin, and the like.
The immune-oncology agent may be, but is not limited to, anti-PD1, anti-PDL1, anti-PDL2, anti-A2AR, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-KIR, anti-CTLA, anti-Tim3, or anti-LAG3.
In the present invention, Lactococcus lactis GEN3013 strain; and an anti-cancer agent or an immune-oncology agent may be administered sequentially or simultaneously to a patient in need thereof.
The present invention relates to a food composition or an animal feed composition for preventing or improving cancer comprising Lactococcus lactis GEN3013 strain.
The food composition may be, but is not limited to, a health functional food, a dairy product, a fermented product, or a food additive.
The novel Lactococcus lactis GEN3013 strain of the present invention has the effect of suppressing the proliferation of different cancer cell lines. Further, the Lactococcus lactis GEN3013 strain of the present invention exerts the effect of reducing the motility of cancer cells and inhibiting angiogenesis, which is remarkably superior to the known Lactococcus lactis strains.
In addition, the Lactococcus lactis GEN3013 strain of the present invention may be used for inflammatory diseases or immunological diseases, as it has anti-inflammation, anti-oxidant, or immunity enhancement effect.
In particular, the Lactococcus lactis GEN3013 strain of the present invention not only exhibits excellent anti-cancer effect when administered alone, but also has more remarkable anti-cancer effect when administered in combination with other anti-cancer agents or immune-oncology agents in comparison to each administration thereof.
Fig. 1 is the results showing cell adhesion of Lactobacillus rhamnosus LGG and GEN3013 strain to Caco-2 cells.
Fig. 2 is the results showing the motility of cancer cells when cancer cell lines are not treated or treated with GEN3013 strain.
Fig. 3 shows the expression of VEGF in HCT116 cell line non-treated or treated with GEN3013 strain as confirmed through PCR.
Fig. 4 shows the expression of Ang1 and Ang2 in HCT116 cell lines non-treated or treated with GEN3013 strain as confirmed through real-time PCR.
Fig. 5 shows the increased the expression of TNF-α by LPS, in RAW264.7 cells non-treated or treated with GEN3013 strain.
Fig. 6 is the results showing the effect of GEN3013 strain for increasing the production of IFN-gamma as confirmed by ELISA kit.
Fig. 7 is the results showing the effect of GEN3013 strain for inhibiting the production of IL-10 as confirmed by ELISA kit.
Fig. 8 is the results showing the effect of GEN3013 strain for inhibiting reactive oxygen species.
Fig. 9 is the results showing the proliferation of cells when cancer cell lines were treated with either one of GEN3013 strain or an anti-cancer agent (oxaliplatin or pemetrexed), or the combination.
Fig. 10 is the graph showing the effect of inhibiting tumor when GEN3013 strain or an anti-cancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
Fig. 11 is the graph showing the effect of inhibiting tumor when GEN3013 strain or an antibody for treating cancer (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
Fig. 12 is the results obtained in immune cells separated from spleen of mice when GEN3013 strain or an anti-cancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
Fig. 13 is the results obtained in immune cells separated from tumor of mice when GEN3013 strain or an anti-cancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
Fig. 14 is the results obtained in immune cells separated from spleen of mice when GEN3013 strain or an antibody for treating cancer (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
Fig. 15 is the results obtained in immune cells separated from tumor of mice when GEN3013 strain or an antibody for treating cancer (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
The present inventors have constantly studied to find probiotics having remarkable anti-cancer effect and finally found that a novel Lactococcus lactis GEN3013 strain exhibits superior anti-cancer effect, whereby arriving at the present invention.
Surprisingly, the GEN3013 strain exerts the remarkable effect of suppressing the proliferation on various cell lines from, for example, lung cancer, colon cancer, gastric cancer, breast cancer, and liver cancer.
Further, when treating cancer cell lines with the GEN3013 strain, the motility of cancer cells is remarkably reduced. Unlike normal cells, cancer cells feature in being able to move with the proliferation even in case where damage occurs on the cells. The reduced motility of cancer cells means a lowered possibility of metastasis of cancer. As such, the GEN3013 strain has the effect of inhibiting metastasis of cancer.
Further, the GEN3013 strain may inhibit the expression of all of VEGF (vascular endothelial growth factor), Ang1 (Angiopoietin1), and Ang2 (Angiopoietin2), as a main factor involving angiogenesis.
Angiogenesis is one of the characteristics of cancer cells. Once angiogenesis is inhibited, the delivery of nutrients to cancer cells through blood vessels can be blocked to suppress the proliferation of cancer cells.
Immunity is the basic factor in human health. The depression of immunity causes the proliferation of harmful bacteria, occurrence of diseases, and progress to chronic diseases. Further, when immunity is depressed as a side effect of anti-biotics or a treatment agent of a disease, a secondary disease may be developed. As such, the GEN3013 strain of the present invention increases immunity to enhance anti-cancer activities in a patient under anti-cancer treatment.
One of the distinct features in inflammatory diseases is the increased reactive oxygen species (ROS). It has long been known that ROS destroy bacteria and kill human cells. Further, ROS in a moderate concentration act on signal transduction pathway of cells while the exposure to ROS in a high concentration for a longer time causes non-specific damage in protein, lipid, and nucleic acid. ROS play an important role on normal physiological process such as protein phosphorylation, oxidation/reduction regulation of transcription factor and ion channel. As another main function, ROS involves biosynthesis procedures including the production of thyroid hormone and crosslinked bond of extracellular matrix. Excessive ROS cause neurodegeneration diseases resulting from mitochondrial dysfunction such as neurogenic muscle weakness, ataxia, retinitis, MELAS syndrome, MERRF syndrome, Kearns-Sayre syndrome, blepharoplegia, pigmentary retinopathy, disturbances in cardiac conduction, increased cerebrospinal fluid, or cardiovascular disease. Further, it is well known that most of cancer cells have high activities of ROS to result in the abnormal proliferation of cells. Thus, the GEN3013 strain of the present invention reduces ROS and suppress the proliferation of cancer cells, so that the development of different diseases is prevented.
Further, the GEN3013 strain of the present invention increases the production of IFN-gamma, which is an immune factor secreted by cytotoxic T cells having the suppression activity of the proliferation of tumor cells, and reduces the production of IL-10, which is an immune factor secreted by T regulatory cells suppressing the activities of cytotoxic T cells, which results in effective immune oncology.
When different cancer cell lines are treated with any one of the GEN3013 strain; and a synthetic anti-cancer agent or immuno-oncology agent as known, or combination thereof, the cancer cell lines treated with GEN3013 have more remarkable effect of suppressing the cell proliferation than those treated with an anti-cancer agent. The cancer cell lines treated with the combination of GEN3013 and an anti-cancer agent have more excellent effect of suppressing the cell proliferation than those treated with either one of GEN3013 or an anti-cancer agent. As such, GEN3013 exhibits increased anti-cancer activities in combination with a known anti-cancer agent.
Thus, the Lactococcus lactis GEN3013 strain of the present invention exerts the effect of suppressing the proliferation of cancer cells, reducing the motility of cancer cells, and inhibiting angiogenesis. Therefore, the Lactococcus lactis GEN3013 strain of the present invention may be used as a remarkable anti-cancer agent and also can be administered in combination with a conventional synthetic anti-cancer agent or immuno-oncology agent.
Further, the GEN3013 strain may significantly reduce the expression of TNF-α derived by LPS as an inflammation inducer to prevent or treat both of inflammatory disease and cancer.
The present invention provides a method for preventing or treating cancer, inflammatory disease, immune disease, etc., by administering the Lactococcus lactis GEN3013 strain to the body.
The composition of the present invention may be used as a medicinal drug, a health functional food, a dairy product, a fermented product, a food additive, or an animal feed.
Hereinafter, a better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limitation of the present invention. The following examples are merely provided to explain in details to those skilled in the art.
[Example 1]
Isolation of Lactococcus lactis GEN3013 strain
Feces of a 20-month old infant were obtained in order to isolate lactic acid bacteria in human intestine. A strain was isolated from the infant feces by utilizing selective media for Lactobacillus spp. and Bifidobacterium spp.
The collected feces samples were diluted in a phosphate buffered saline (PBS) solution including 0.85％ NaCl to a 10-5, 10-6 fold dilution, followed by spreading the same in media of De Man Rogosa, Sharpe agar (MRS broth; Difco, USA) with the addition of 0.5% cysteine, and being subjected to anaerobic culture at 37℃ for 48 hours, to obtain colonies. Gene sequences of 16S rRNA of each strain were then identified by using colony PCR and sequencing technology. A strain having high similarity (100%) to Lactococcus lactis was named as GEN3013 (SEQ ID No. 1).
The selected strain was subcultured in MRS broth and BL broth, and then stored in MRS broth and BL broth containing 20% glycerol in freezer at 80 ℃.
[Example 2]
Identification of Lactococcus lactis GEN3013 strain
When the sequence of rRNA of the strain was analyzed, it was identified as Lactococcus lactis. The strain was deposited in Biological Resource Center affiliated in Korea Research Institute of Bioscience and Biotechnology under Accession Number KCTC13426BP in the name of Lactococcus lactis GNC3013 on December 13, 2017, which was changed into Lactococcus lactis GEN3013 on April 3, 2018.
1. Evaluation of sugar fermentation properties
The carbohydrate fermentation properties of the GEN3013 strain were evaluated by using API CHL kit (BioMetrieux Co. France). The results were shown in Table 1 below.
GEN3013
0 CONTROL -
1 GLY -
2 ERY -
3 DARA -
4 LARA +
5 RIB +
6 DXYL -
7 LXYL -
8 ADO -
9 MDX -
10 GAL +
11 GLU +
12 FRU -
13 MNE -
14 SBE -
15 RHA -
16 DUL -
17 INO -
18 MAN -
19 SOR -
20 MDM -
21 MDG -
22 NAG -
23 AMY -
24 ARB -
25 ESC -
26 SAL -
27 CEL -
28 MAL +
29 LAC +
30 MEL -
31 SAC +
32 TRE -
33 INU -
34 MLZ -
35 RAF +
36 AMD -
37 GLYG -
38 XLT -
39 GEN -
40 TUR -
41 LYX -
42 TAG -
43 DFUC -
44 LFUC -
45 DARL -
46 LARL -
47 GNT +
48 2KG -
49 5KG -
2. Evaluation of enzyme activities
In order to evaluate biochemical properties of the GEN3013 strain, the enzyme activities were analyzed by using API ZYM kit (BioMetrieux Co. France). The results were shown in Table 2 below.
No Type GEN3013
1 Control -
2 Alkaline phosphatase -
3 Esterase (C4) -
4 Esterase Lipase(C8) -
5 Lipase(C14) -
6 Leucine arylamidase +
7 Valine arylamidase -
8 Crystine arylamidase -
9 Trypsin -
10 a-chymotrypsin -
11 Acid phospatease -
12 Naphtol-AS-BI-phosphohydrolase +
13 a-galactosidase -
14 b-galactosidase +
15 b-glucuronidase -
16 a-glucosidase -
17 b-glucosidase -
18 N-acetyl-β-glucosaminidase -
19 a-mannosidase -
20 a-fucosidase -
[Example 3]
Evaluation of activities of Lactococcus lactis GEN3013 strain
1. Anti-biotic activities
In order to evaluate the effect of the GEN3013 strain for inhibiting the growth of harmful bacteria, a disc diffusion assay was performed. The indicator strains were Staphylococcus aureus KCTC3881, P. Aeruginosa KCTC 2004, E. Coli KCTC 1682, and L. Innocua KCTC 3586, which were cultured, spread on agar plate, and placed on a sterilized disc.
The Lactococcus lactis GEN3013 as a test strain was placed on a sterilized disc, cultured at 37 ℃ for 24 hours, followed by measuring the size of inhibition zone. The results were shown in Table 3 below.
Figure PCTKR2019004262-appb-T000001
2. Measurement of acid resistance
GEN3013 strain was subcultured in MRS broth containing 0.5 % glucose and 0.05% cysteine. In order to evaluate whether the strain is viable depending on pH change of the culture media, we prepared MRS broth containing 0.5 % glucose and 0.05% cysteine which was adjusted to have pH 2, 2.5, 3 and 3.5. The washed bacteria were inoculated such that the concentration is 2×106 CFU/ml, cultured in a non-growing state at 37℃ for 2 hours, and the viable bacteria were counted. The survival rate was calculated by comparing the number of the viable bacteria before the inoculation with that 2 hours after the inoculation as follows. The results were shown in Table 4 below.
Survival rate (%) = [(CFU/ml 120min) / (CFU/ml 0min)] × 100
Survival Rate (%)
Strain pH 2.0 pH 2.5 pH 3.0 pH 3.5
LGG 0.26 97.55 103.56 110.24
GEN3013 16.90 75.04 82.56 95.92
As shown in Table 4 above, at pH 2.0, the survival rate of Lactobacillus rhamnosus LGG as a control was 0.26% while that of Lactococcus lactis GEN3013 strain was 16.90%, which shows that the GEN3013 strain has remarkable viability in a high acidic environment.
As such, it was confirmed that since Lactococcus lactis GEN 3013 strain has a good acid resistance, the survival in an acidic condition is advantageous.
3. Evaluation of intestinal adherence
In order to evaluate intestinal adherence of GEN3013 strain, cell adhesion test was carried out on Caco-2 cell of human epithelial colorectal adenocarcinoma cell which was purchased from the Korean gene bank. For the cell culture, Minimum Essential Medium (MEM; Gibco, Carlsbad, CA, USA) was used by adding 20% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 ㎍/mL streptomycin (Gibco).
The cells used for the experiment were cultured at 37℃ in 5% CO2 at 90% humidity condition, where the culture media was changed once every two or three days. When cell differentiation reached at peak, the cells were washed with phosphate buffered saline (PBS), separating the adhered cells by using a trypsin-EDTA solution, and placing in a 12-well plate such that it had 1×105 cell/well.
The isolated strains as subcultured were inoculated in the cells cultured for 20 days such that the amount is 1×107 CFU/ml, and cultured at 37℃ in 5% CO2 for 2 hours. After washing twice with PBS, the viable bacteria were counted in MRS broth containing 0.05% cysteine. The adherence rate was calculated by comparing the initial number with the final number where the data were indicated in a fold unit based on the value of Lactobacillus rhamnosus LGG as a control.
The calculated results were shown in Fig. 1, from which it was confirmed that the GEN3013 strain has similar intestinal adherence to that of Lactobacillus rhamnosus LGG as a control. As such, it was found that the GEN3013 strain has superior intestinal adherence and thus high intestinal adaptability.
4. Antibiotic resistance test
In order to evaluate antibiotic resistance of the GEN3013 strain, a minimum inhibitory concentration value was measured. Lactic acid bacteria with maximized activities through subculture was spread in media, placing in Etest (BioMetrieux Co. France) to culture for 24 hours, and measuring MIC values producing a clear zone. The results were shown in Table 5.
Amp* Tet Gen Kan Strep
MIC (μg/ml) 0.125 0.19 3 6 32
EFSA 2 4 32 64 32
*Amp (ampicillin), Tet (tetracycline), Gen (gentamicin), Kan (kanamycin), Strep (streptomycin)
As shown in Table 5 above, it was found that the strain has susceptibility to antibiotics of ampicillin, tetracycline, gentamicin, kanamycin, and streptomycin. When referring to the cutoff value indicated in EFSA (efsa journal 2012;10(6):2740), it was confirmed that the strain is safe for being used as lactic acid bacteria.
[Example 4]
Effect of Lactococcus lactis GEN3013 strain for suppressing the tumor proliferation
In order to evaluate whether Lactococcus lactis GEN3013 exhibits anti-cancer activities in different cancer cell lines, MTT assay was performed.
Cell lines of lung cancer (A549, H1975, HCC827, H1299, SW900), colon cancer (HCT116, LoVo, SNU-C2A), gastric cancer (SNU216, AGS, MKN-28, MKN-1, SNU-601, SNU-1), breast cancer (Hs578T, BT20, MDA-MB-231), and liver cancer (HepG2, Hep3B) were used as human cancer cell lines for the assay, while cell lines of colon cancer (CT26, MC38), lung cancer (LLC1), and breast cancer (4T1) were used as mouse cancer cell lines.
Cancer cell lines were placed in a 96-well plate in an amount of 1~5×103 cells/well. After 24 hours, a lactic acid bacteria sample was added in 0.5% and cultured for 72 hours, followed by treating the reagent, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue, in each well to react for 2 hours, so that viable cells were reacted with mitochondria to change the yellow MTT into violet color. The culture media containing MTT were then all removed, adding 100 ㎕ of DMSO in each well, measuring the concentration of the violet color as absorbance at 540 nm by using Multiplate Reader. The test results on human-derived cancer cell lines and mouse-derived cancer cell lines were shown in Tables 6 and 7 below.
Cancer type Cell line Suppression rate of proliferation (%) ± SD
lung cancer H1975 41.95 ± 2.97
HCC827 73.55 ± 1.80
A549 39.50 ± 1.59
SW900 56.59 ± 2.08
H1299 71.04 ± 2.10
colon cancer LoVo 63.40 ± 2.89
HCT116 80.61 ± 0.89
SNU-C2A 45.49 ± 1.22
gastric cancer SNU-216 76.11 ± 0.60
AGS 87.50 ± 1.62
MKN-28 64.43 ± 1.63
MKN-1 46.91 ± 3.29
SNU-601 70.13 ± 0.59
SNU-1 20.72 ±0.37
breast cancer Hs578T 58.44 ± 0.36
BT20 62.34 ± 1.20
MDA-MB-231 53.63 ± 3.24
liver cancer HepG2 49.05 ± 1.72
Hep3B 45.89 ± 1.15
Cancer type Cell line Suppression rate of the proliferation (%) ± SD
colon cancer CT26 76.73 ± 1.01
MC38 55.04 ± 4.71
lung cancer LLC1 68.22 ± 10.83
breast cancer 4T1 78.82 ± 1.51
As shown in Tables 6 and 7 above, the GEN3013 strain exerted remarkable effect for suppressing the cell proliferation on both human- and mouse-derived cancer cell lines.
Further, the effect of GEN3013 for suppressing growth of human-derived cancer cell lines was evaluated as an IC50 value as shown in Table 8 below.
SNU-1(μg/ml) SNU-C2A(μg/ml) HCT116(μg/ml) Hep3B(μg/ml)
GEN3013 6.43 8.82 5.68 10.47
Korean Patent Application No. 10-2001-0012973 discloses that lactis ssp. cremoris has IC50 values of 54 μg/ml on SNU-1 and 75 μg/ml on SNU-C2A, while Lactococcus lactis ssp. lactis has the values of 11 μg/ml on SNU-1 and 23 μg/ml on SNU-C2A.
As shown in Table 8 above, GEN3013 had IC50 values of 6.43 μg/ml on SNU-1 and 8.82 μg/ml on SNU-C2A, which was much lower than IC50 values of other strains that were already known in prior studies. As such, it was confirmed that GEN3013 exhibits remarkable effect for suppressing growth of different cancer cells.
[Example 5]
Effect of Lactococcus lactis GEN3013 strain for reducing the motility of cancer cells
Unlike normal cells, cancer cells are featured in moving along with the proliferation even when damage occurs on the cells. In order to evaluate whether the motility of cancer cells is reduced by GEN3013 strain, wound healing assay was performed. A549 (5X105 cells) and HCT116 (6X105 cells) strains were fixed on a 6-well plate. When 90 to 95% of cells were proliferated, we damaged the cells with 1000p tip in a certain interval. The cells were treated with PBS or GEN3013 strains for 24 hours to observe the motility of the cells by microscope. The results were shown in Fig. 2.
As shown in Fig. 2, based on the cell condition at 0 h of A549 and HCT116 cell lines as damaged, at 24 hours, A549 and HCT116 had 65.31± 1.69 % and 33.82 ± 5.86 % of the active motility of cancer cells, respectively. When treated with GEN3013, the motility of cells was remarkably reduced to 17.60 ± 5.19 % and 13.50 ± 8.55 % in A549 and HCT116, respectively.
As such, it was confirmed that GEN3013 strain inhibits cancer metastasis and thus exhibits excellent anti-cancer activities.
[Example 6]
Effect of Lactococcus lactis GEN3013 strain for inhibiting angiogenesis
In order to evaluate whether GEN3013 strain inhibits angiogenesis, which is one of main characteristics of cancer cells, the expression test of an angiogenesis-related factor was carried out.
HCT116 cell lines were treated with GEN3013 for 24 hours, from which RNA was obtained to synthesize cDNA. With the same, the expression of vascular endothelial growth factor (VEGF) was analyzed in normal PCR, while the expression of Ang1 and Ang2 was analyzed in real-time PCR. The results were shown in Figs. 3 and 4.
As shown in Fig. 3, a group treated with GEN3013 had the remarkably reduced expression of 121 isoform and 165 isoform of VEGF in comparison with a control. Further, the expression of 189 isoform was insignificant in a control, while no expression of 189 isoform was found in a group treated with GEN3013.
Further, as shown in Fig. 4, in terms of the expression of Ang1 and Ang2, which are a factor involving angiogenesis other than VEGF, in a group treated with GEN3013, the expression of Ang1 was rarely observed while no expression of Ang2 was observed.
As such, it was confirmed that GEN3013 inhibits angiogenesis and thus block the delivery of nutrients into cancer cells through blood vessels, which results in remarkable effect for suppressing the proliferation of cancer cells.
[Example 7]
Anti-inflammation effect of Lactococcus lactis GEN3013 strain
In order to evaluate anti-inflammation effect of GEN3013 strain, RAW264.7 cells as mouse macrophage were treated with GEN3013 strain for 24 hours and then with an inflammation inducer, LPS 100 ng/ml, for 6 hours to obtain RNA.
cDNA was synthesized from 1 ㎍ of RNA and used to evaluate the expression of TNF-α, which is known as an inflammation factor, in real-time PCR. The results were shown in Fig. 5.
As shown in Fig. 5, when the degree of the increased expression of TNF-α by LPS is defined as 1 relative index, a group treated with GEN3013 had no more than 0.5 of the degree of the increased expression of TNF-α. Since TNF-α is also known as a cancer inducer, the results of the remarkably reduced expression of TNF-α means that GEN3013 has both anti-inflammation and anti-cancer activities.
[Example 8]
T cell activity of Lactococcus lactis GEN3013 strain, and the effect of controlling the production of IFN-gamma and IL-10
PBMC was separated from human blood by using Ficoll, removing red blood cells with RBC lysis buffer, and isolating monocytes in LS column and MACS buffer.
GEN3013 was placed in a 96-well plate, adding isolated monocytes in an amount of 5 x 103 per well, slightly mixing so that the reaction with the strains occurred, and culturing for 2 hours. Through a reaction for 2 hours, GEN3013 directed monocytes to be differentiated into macrophages.
While reacting monocytes with GEN3013, T cells expressing CD4 and CD8 were separated from the remaining PBMC cells in MACS buffer and LS column. The separated T cells were diluted in 100 ㎕ of RPMI media such that the amount was 5 x 104 cells, placing in the wells containing monocytes and GEN3013, and culturing for 48 hours to activate immune.
After a certain time, cell culture in each well was collected in 1.5 ml tube, separating supernatants alone, and measuring the production of IFN-gamma and IL-10 in ELISA kit. The results were shown in Figs. 6 and 7.
As shown in Fig. 6, a group treated with GEN3013 increased the production of IFN-gamma, which is an immune factor secreted by cytotoxic T cells having the suppression activity of the proliferation of tumor cells, more than two times in comparison with a control treated with E.coli.
Further, as shown in Fig. 7, a group treated with GEN3013 less produced IL-10, which is an immune factor secreted by T regulatory cells suppressing the activities of cytotoxic T cells, in comparison with a control treated with E.coli.
As such, it was confirmed that GEN3013 controls T cells involving anti-cancer and immune activities, and the production of immune factors secreted by the T cells.
[Example 9]
Anti-oxidant activity of Lactococcus lactis GEN3013 strain
In order to evaluate anti-oxidant activity of GEN3013, A549 cancer cell lines were treated with GEN3013 for 24 hours and then with 0.5 uM H2O2 for 5 hours, and measuring the amount of ROS in cells with DCFDA fluorescent dyes in FACs equipment. The results were shown in Table 9 below and Fig. 8.
PBS H2O2 H2O2 + GEN3013 (0.1%) H2O2 + GEN3013 (0.5%)
ROS (%) 4.7 64.8 54.6 35.6
As shown in Table 9 above, a group treated with PBS alone had 4.66% of ROS, while a group treated with H2O2 alone had 64.8% of the increased amount of ROS. A group treated with 0.1% or 0.5% GEN3013 had 54.6% and 35.6% of ROS, respectively. As such, it was found that GEN3013 exhibits the effect of reducing the amount of ROS and the increased concentration of GEN3013 leads to the increased inhibition of ROS.
Thus, it was confirmed that the GEN3013 strain exerts the effect of inhibiting the cell damage resulting from ROS in cell and of reducing the proliferation of cancer cells.
[Example 10]
Effect of Lactococcus lactis GEN3013 strain in combination with an anti-cancer agent for suppressing the proliferation of cancer cells (in vivo test)
Lactococcus lactis GEN3013 strain was cultured in MRS liquid media (Difco) containing 0.5% glucose and 0.05% cysteine at 37℃ for 24 hours, lysing bacteria to be used for the test.
A549 (human lung cancer cells), LLC1 (mouse lung cancer cells), HCT116, LoVo (human colon cancer cells), and MC38 (mouse colon cancer cells) were used in the test. Human cancer cell lines were cultured in RPMI media, while mouse cancer cell lines in DMEM media. Culture media of all cancer cell lines contained 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco Life Technologies, Grand Island, NY, U.S.A.). The test using cell lines were proceeded while culturing at 37℃ and 5% CO2.
Oxaliplatin or pemetrexed was used as an anti-cancer agent. In order to evaluate the suppression of the proliferation of cancer cells by a combination treatment of an anti-cancer agent and lactic acid bacteria, an experiment was performed based on the following method. The different cancer cell lines were diluted and placed in each well of a 6-well plate in an amount of 1 to 2 X 103, fixed for 24 hours, followed by treating each well with lactic acid bacteria and/or an anti-cancer agent and changing the media every two or three days. The plate treated with lactic acid bacteria and/or the anti-cancer agent for 144 hours was treated with 4% formalin for 30 minutes to fix the cells, washed with PBS twice, dyed with a crystal violet solution for 5 minutes, and washed again with distilled water, to observe the cell proliferation. The results were shown in Fig. 9.
As shown in Fig. 9, the cancer cell lines treated with GEN3013 exhibited the superior suppression of the proliferation of cancer cells, in comparison to the cancer cell lines treated with an anti-cancer agent. Especially, MC38 mouse colon cancer cells and LLC1 mouse lung cancer cells, which are treated with GEN3013 alone, showed almost complete suppression of the cancer cell proliferation, which resulted from GEN3013.
Further, the cancer cell lines treated with the GEN3013 strain in combination with the anti-cancer agent exerted more remarkable effect for suppressing the cell proliferation than the cancer cell lines treated with either one of GEN3013 or the anti-cancer agent.
[Example 11]
Effect of Lactococcus lactis GEN3013 strain in combination with an anti-cancer agent for suppressing the proliferation of tumor (in vivo test)
The lactic acid bacteria samples were administered to mice for 2 weeks to increase intestinal adherence and immunity before preparing a tumor model. 2 X 105 MC38 cancer cells were then subcutaneously injected around a right hip of 8 C57BL/6 mice per group to prepare a tumor model. Simultaneously with the injection of tumor cells, the lactic acid bacteria samples were orally administered to the animal models for three weeks (from Monday to Saturday). The lactic acid bacteria samples to be orally administered were diluted in 200μl of PBS in an amount of CFU 1*109 per mouse. Oxaliplatin (3 mg/kg, Sellekchem) or anti-PD1 (2 mg/kg, BioXCell) as an anti-cancer agent was intraperitoneally injected every Monday and Thursday after the cancer is induced. The effect of inhibiting tumor was observed in a group treated with any one of lactic acid bacteria; and oxaliplatin or anti-PD1, and with the combination. The results were shown in Figs. 10 and 11.
As shown in Figs. 10 and 11, the combination administration exhibited more remarkable effect of inhibiting tumor than the single administration of GEN3013 or the anti-cancer agent.
[Example 12]
Effect of Lactococcus lactis GEN3013 strain in combination with an anti-cancer agent for suppressing the proliferation of tumor (in vivo test)
Based on the results of Example 11, an animal test was performed in order to analyze the invasion of immune cells into tumor with GEN3013. Upon completing the test, tumor and spleen were separated from the mice, lysed, and isolated the immune cells. The isolated immune cells were reacted with fluorescence antibodies, which corresponds to a marker of the immune cells depending on the function, followed by analyzing by FACs. The results were shown in Figs. 12 to 15.
As shown in Figs. 12 to 15, a group treated with GEN3013 had increased distribution of CD4 T cell, CD8 T cell, CD8 effector T cell, and NK cell, which play an important roll on anti-cancer immune response, in comparison with a control. The number of regulatory T cells controlling activities of T-cells were significantly reduced.
The increased immune response was more remarkable in a group treated with the combination of GEN3013 and the anti-cancer agent.
It was confirmed from the results that controlling activities of immune cells, which results from GEN3013, involves the suppression of the tumor proliferation.
Deposit Information
● Depositary Authority: Korea Research Institute of Bioscience and Biotechnology
● Accession Number: KCTC13426BP
● Deposit Date: December 13, 2017
Figure PCTKR2019004262-appb-I000001
Figure PCTKR2019004262-appb-I000002

Claims (14)

  1. Lactococcus lactis GEN3013 strain (KCTC13426BP).
  2. A pharmaceutical composition for preventing or treating cancer, comprising Lactococcus lactis GEN3013 strain (KCTC13426BP).
  3. A pharmaceutical composition for preventing or treating cancer, comprising Lactococcus lactis GEN3013 strain (KCTC13426BP), characterized in that the Lactococcus lactis GEN3013 strain exhibits anti-cancer, anti-inflammation, anti-oxidant, and immunity enhancement effect.
  4. The pharmaceutical composition for preventing or treating cancer according to Claim 2, characterized in that the cancer is melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft-tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mastocytoma, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, gastric cancer, renal cancer, colon cancer, haematopoietic tumor, or metastatic cancers thereof.
  5. The pharmaceutical composition for preventing or treating cancer according to Claim 4, characterized in that the cancer is lung cancer, colon cancer, gastric cancer, breast cancer, or liver cancer.
  6. The pharmaceutical composition for preventing or treating cancer according to Claim 2, characterized in that the Lactococcus lactis GEN3013 strain exhibits anti-cancer effect by reducing the motility of cancer cells.
  7. The pharmaceutical composition for preventing or treating cancer according to Claim 2, characterized in that the Lactococcus lactis GEN3013 strain exhibits anti-cancer effect by suppressing the expression of vascular endothelial growth factor (VEGF).
  8. The pharmaceutical composition for preventing or treating cancer according to Claim 3, characterized in that the Lactococcus lactis GEN3013 strain suppresses the expression of TNF-α.
  9. The pharmaceutical composition for preventing or treating cancer according to Claim 3, characterized in that the Lactococcus lactis GEN3013 strain increases the production of IFN-gamma and reduces the production of IL-10.
  10. The pharmaceutical composition for preventing or treating cancer according to Claim 2, characterized in further comprising an anti-cancer agent.
  11. The pharmaceutical composition for preventing or treating cancer according to Claim 10, characterized in that the anti-cancer agent is at least one selected from the group consisting of oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, and doxorubicin.
  12. A food composition for preventing or improving cancer, comprising Lactococcus lactis GEN3013 strain (KCTC13426BP).
  13. The food composition for preventing or improving cancer according to Claim 12, characterized in that the food composition is a health functional food, a dairy product, a fermented product, or a food additive.
  14. An animal feed composition for preventing or improving cancer, comprising Lactococcus lactis GEN3013 strain (KCTC13426BP).
PCT/KR2019/004262 2018-04-12 2019-04-11 Lactococcus lactis gen3013 strain and a composition for preventing or treating cancer comprising the same WO2019199048A1 (en)

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