LACTOBACILLUS PLANTARUM MICROORGANISMS HAVING ANTI-CANCER ACTIVITY, COMPOSITION INCLUDING SAME, AND METHOD FOR PREVENTING OR TREATING CANCER USING SAME

Abstract

Provided are a microorganism as Lactobacillus plantarum having anti-cancer activity, a composition including the same, and a method of preventing or treating a cancer by using the same.

Claims

1. A microorganism as Lactobacillus plantarum LMT17-31 (Accession No.: KCTC-14283BP) having anti-tumor activity.

2. A pharmaceutical composition for preventing or treating a cancer comprising, as an active ingredient, the microorganism of claim 1 or a culture or extract thereof.

3. The composition of claim 2, wherein the cancer is a solid cancer.

4. The composition of claim 2, wherein the composition is administered in combination with an immune checkpoint inhibitor.

5. The composition of claim 4, wherein the immune checkpoint inhibitor is a CTLA4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor.

6. A food composition for preventing or ameliorating a cancer comprising, as an active ingredient, the microorganism of claim 1 or a culture or extract thereof.

Description

DESCRIPTION OF DRAWINGS

[0043] FIG. 1 shows representative optical microphotographs of a selected Lactobacillus plantarum LMT17-31 strain and a KCTC3108 strain as a type strain.

[0044] FIGS. 2A and 2B are diagrams showing results of measuring size of a tumor after the L. plantarum LMT17-31 strain is administered to mice having a tumor.

[0045] FIGS. 3A, 3B, 3C, 3D, 3E, and 3F are diagrams showing results of analyzing CD8 T cells infiltrating into a tumor and secretion of cytokines therefrom after the L. plantarum LMT17-31 strain is administered to mice having a tumor.

BEST MODE

[0046] Hereinafter, the present invention will be described in detail with reference to Examples below. However, these Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited by these Examples.

Example 1: Isolation and Identification of Lactobacillus plantarum LMT17-31 Strain

[0047] 1. Isolation of L. plantarum Strain

[0048] An L. plantarum strain was isolated from a rice wine sample. First, the sample was spread on an MRS medium (Difco, USA) and cultured anaerobically at 30° C. As a pretreatment method of the sample, the sample was obtained aseptically and diluted with 180 ml of a 0.85% NaCl solution, and then, a stock solution of the rice wine was homogenized with a stomacher for 5 minutes. The homogenized sample was diluted by stages in a tube containing 9 ml of a sterilized 0.85% NaCl solution to prepare a rice wine sample. The rice wine sample was spread on an MRS plate medium (Difco, USA) and cultured at 37° C. for 2 days to 3 days. Then, colonies that appeared thereon were sorted according to shape and color, and were purely isolated again.

[0049] 2. Identification of L. plantarum Strain

[0050] (1) Analysis of Morphological Characteristics

[0051] The selected L. plantarum strain was cultured in an MRS plate medium (Difco, USA), and morphological characteristics of the colonies thereof were observed. The morphological characteristics of the colonies appeared in the MRS plate medium for the selected L. plantarum strain and the KCTC3108 type strain of the L. plantarum are shown in Table 1 below.

TABLE-US-00001 TABLE 1 LMT17-31 KCTC3108 Shape Circular Circular Size 1 mm 1 mm Color Cream Cream Transparency Opaque Opaque Protuberance Convex Convex Surface Smooth Smooth Aerobic growth and + + development Anaerobic growth + + and development

[0052] FIG. 1 shows representative optical microphotographs of the selected L. plantarum LMT17-31 strain and the KCTC3108 strain. As shown in FIG. 1, the LMT17-31 strain was a rod-shaped bacillus and was similar in appearance to the typical genus Lactobacillus.

[0053] (2) 16S rDNA Analysis

[0054] The 16S rRNA gene of the isolated LMT17-31 strain was amplified, and the nucleotide sequence of the amplified 16S rRNA gene was analyzed. The amplification was performed by PCR using the genomic DNA of the LMT17-31 strain as a template and an oligonucleotide of SEQ ID NO: 1 and an oligonucleotide of SEQ ID NO: 2 (Macrogen Inc.) as primer sets. The nucleotide sequence of the 16S rDNA of the isolated LMT17-31 strain is shown as a sequence of SEQ ID NO: 3. The nucleotide sequence of the identified 16S rDNA was compared with the nucleotide sequence of the known 16S rDNA by using NCBI blast (http://www.ncbi.nlm.nih.gov/). As a result, the 16S rDNA of LMT17-31 was found to have 100% sequence identity to the L. plantarum species. In addition, as a result of phylogenetic tree analysis, LMT17-31 was found to be the same as the L. plantarum species. As a result, the LMT17-31 strain was identified as a new strain belonging to a newly found L. plantarum species.

[0055] The present inventors named LMT17-31 lactic acid bacteria as “L. plantarum LMT17-31” (Accession number: KCTC 14283BP), which was deposited in the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on Aug. 26, 2020.

Example 2: Analysis of Physiological Activity Characteristics of L. plantarum LMT17-31 Strain

[0056] 1. Sugar Fermentation Characteristics of L. plantarum LMT17-31 Strain

[0057] The sugar metabolism characteristics of the selected LMT17-31 strain were verified with the API 50 CHL kit (BioMetrieux, France) according to the experimental method provided by the supplying company. Table 2 shows the sugar fermentation characteristics of the identified LMT17-31 strain.

TABLE-US-00002 TABLE 2 LMT17-31 No. Carbohydrate 24 hours 48 hours 1 Glycerol − − 2 Erythritol − − 3 D-arabinose − − 4 L-arabinose + + 5 D-ribose + + 6 D-xylose − − 7 L-xylose − − 8 D-adonitol − − 9 Methyl-βD-xylopyranoside − − 10 D-galactose + + 11 D-glucose + + 12 D-fructose + + 13 D-mannose + + 14 L-sorbose − − 15 L-rhamnose − − 16 Dulcitol − − 17 Inositol − − 18 Mannitol + + 19 D-sorbitol + + 20 Methyl αD-mannopyranoside + + 21 Methyl αD-glucopyranoside + + 22 N-acetylglucosamine + + 23 Amygdalin + + 24 Arbutin + + 25 Esculin + + 26 Salicin + + 27 D-cellobiose + + 28 D-maltose + + 29 D-lactose + + 30 D-melibiose + + 31 D-sucrose + + 32 D-trehalose + + 33 Inulin − − 34 D-melezitose + + 35 D-raffinose + + 36 Amidon − − 37 Glycogen − − 38 Xylitol − − 39 Gentibiose + + 40 D-turanose + + 41 D-lyxose − − 42 D-tagatose − − 43 D-fucose − − 44 L-fucose − − 45 D-arabitol − − 46 L-arabitol − − 47 Potassium gluconate + + 48 Potassium 2-cetogluconate − − 49 Potassium 5-ketogluconate − −

[0058] 2. Evaluation of Stability of L. plantarum LMT17-31 Strain

[0059] (1) Investigation of Acid Resistance

[0060] To evaluate the acid resistance of the L. plantarum LMT17-31 strain, an experiment was conducted as follows. The LMT17-31 strain was inoculated into a sterilized MRS broth and cultured at 37° C. for 16 hours. Then, 1% of the strain was inoculated into a different sterilized MRS broth having a pH of 2.5 adjusted by HCl, and cultured at 37° C. for 2 hours. The samples were collected immediately after the strain inoculation and after 2 hours of the incubation, diluted in a fresh MRS broth, spread on an MRS plate medium, and cultured at 37° C. for 24 hours. Then, the number of colonies on the MRS plate medium was counted to measure the number of bacteria.

[0061] As shown in Table 3, the L. plantarum LMT17-31 strain showed 44.2% acid resistance to the acidity of pH 2.5, and the KCTC3108 type strain had 67.1% acid resistance to the same acidity.

TABLE-US-00003 TABLE 3 LMT17-31 KCTC3108 MRS (pH 6.8) (number of 2.2 × 10.sup.9 7.6 × 10.sup.8 cells/plate) MRS (pH 2.5) (number of 1.0 × 10.sup.9 5.1 × 10.sup.8 cells/plate) Survival rate (%) 44.2 67.1

[0062] (2) Investication of Bile Resistance

[0063] To confirm the influence of bile acid on the growth of the L. plantarum LMT17-31 strain, an experiment was conducted as follows. The selected strain was inoculated into a sterilized MRS broth and cultured at 37° C. for 24 hours. Considering that the concentration of bile salts in the intestinal tract is about 0.1%, an MRS broth containing 0.3% of bile salts (Sigma, USA) was prepared and 1% of the strain was inoculated thereinto to be cultured at 37° C. for 2 hours. The samples were collected immediately after the strain inoculation and after 2 hours of the incubation, diluted in a fresh MRS broth, spread on an MRS plate medium, and cultured at 37° C. for 24 hours. Then, the number of colonies on the MRS plate medium was counted to measure the number viable strain cells. As a control group, the incubation was performed in the same manner in an MRS broth without 0.3% bile acid, and the number of viable strain cells was measured. Table 4 shows the results of measuring the bile salt resistance.

[0064] As shown in Table 4, since the LMT17-31 strain maintained a survival rate of 66.4% at a concentration of 0.3%, which is a concentration higher than 0.1% that is similar to the actual concentration in the intestine, the LMT17-31 strain can be a basis sufficiently enough for the survival in the intestines of humans or animals.

TABLE-US-00004 TABLE 4 LMT17-31 KCTC3108 Control group (number of 2.2 × 10.sup.9 7.6 × 10.sup.8 cells/plate) 0.3% bile salts (number of 1.5 × 10.sup.9 5.5 × 10.sup.8 cells/plate) Survival rate (%) 66.4 72.4

[0065] (3) Investigation of Intestinal Adherence

[0066] To evaluate the degree of adherence to intestinal cells of the L. plantarum LMT17-31 strain, a Caco-2 cell line (KCLB 30037.1) of the human epithelial colorectal adenocarcinoma cells purchased from the Korea Cell Line Bank was used. The Caco-2 cells were inoculated into a Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) under conditions of 5% CO.sub.2 and 37° C. to reach 7×10.sup.4 cells/100 μl. The cells were cultured to form a cellular monolayer in a 96-well plate (Corning, USA).

[0067] Separately, the LMT17-31 strain cultured in the MRS broth was washed with PBS, suspended in an antibiotic-free DMEM medium, and then added at a concentration of 1×10.sup.7 CFU to the Caco-2 cells constituting the cellular monolayer. The cells were cultured for 2 hours under conditions of 5% CO.sub.2 and 37° C. To remove cells that failed to adhere to the Caco-2 cells, a washing process was performed thereon by using PBS 5 times, and 100 μl of 0.1% Triton X-100 was used to detach the adhered cells that were then spread on an MRS solid medium. After culturing the cells at 37° C. for 24 hours, the number of colonies on the plate medium was counted to investigate the intestinal adherence of the LMT17-31 strain.

[0068] Table 5 shows the number of L. plantarum LMT17-31 strain adhered to the intestinal epithelial cells. As shown in Table 5, the novel L. plantarum LMT17-31 strain of the present invention showed about 2% of adherence, and the comparative L. plantarum KCTC3108 strain showed about 1% of adherence, to the Caco-2 intestinal epithelial cells.

TABLE-US-00005 TABLE 5 LMT17-31 KCTC3108 Number of strains treated 1.2 0.8 (10.sup.7 CFU) Number of bacteria adhered 22.1 10.3 (10.sup.4 CFU) Adhesion rate (%) 1.87 1.32

Example 3: Evaluation of Anti-Tumor Efficacy of L. plantarum LMT17-31 Strain

[0069] 1. Induction of Mouse Model for Tumor and Administration of L. plantarum LMT17-31 Strain

[0070] C57BL/6 mice (male, weight: 20 g to 22 g) used for induction of a mouse model for tumor were purchased from Orient Bio INC., and were acclimated to the environment for 1 week prior to the start of an experiment. 2.5×10.sup.5 MC38 cells derived from C57BL/6 murine colon adenocarcinoma cells were injected into the subcutaneous tissue of the mouse back, and after 1 week of the tumor injection, the mice were separated into groups based on the tumor size (50 mm.sup.3 to 70 mm.sup.3). For 2 weeks after the group separation, the L. plantarum LMT17-31 strain was orally administered with PBS containing 1×10.sup.9 CFU strain per mouse by utilizing a zonde once every other day. As a positive control group, an anti-PD1 antibody (clone number: RMP1-14, Manufacturer: Bioxcell, product name: InvivoMAb anti-mouse PD-1) was intraperitoneally administered twice a week at 10 mg per kg of mouse weight. As a negative control group, PBS was orally administered. Regarding compositions of the experimental groups, a total of 4 groups with 8 animals in each group were prepared as shown in Table 6.

TABLE-US-00006 TABLE 6 Group 1 PBS Control group Group 2 Group treated with anti-PD1 antibody Group 3 Group treated with L. plantarum LMT17-31 Group 4 Group treated with anti-PD1 antibody + LMT17-31 in combination

[0071] The tumor size was measured until day 23 after the MC38 tumor cell line was injected into the mice, and the mice were sacrificed by using carbon dioxide to extract the tumor. Then, analysis on tumor-infiltrating immune cells was performed. The analysis results are shown in FIGS. 3A, 3B, 3C, 3D, 3E, and 3F. FIGS. 3A, 3B, 3C, 3D, 3E, and 3F are diagrams showing the results of analyzing CD8 T cells infiltrating into the tumor and secretion of cytokines therefrom after the L. plantarum LMT17-31 strain was administered to the mice having the tumor. The MC38 tumor cell line includes mouse colon adenocarcinoma cells induced by subcutaneous injection of dimethylhydrazine into C57BL/6 mice. The induced mouse model for tumor used in Examples herein was prepared by transplanting the MC38 tumor cells into the subcutaneous tissue of the mouse, indicating that the effect of orally administered bacteria can exhibit on inhibition of the cancer growth in the subcutaneous tissue and that the bacteria can affect not only a cancer related to the intestines but various solid carcinomas.

[0072] 2. Evaluation of Antitumor Efficacy According to Administration of L. plantarum LMT17-31 Strain

[0073] FIGS. 2A and 2B are diagrams showing the results of measuring the tumor size after the L. plantarum LMT17-31 strain was administered to the mice having the tumor. The administration was performed according to Section 1. In detail, the rumor size was measured twice a week from day 7 to day 23 after the injection of the tumor cell line. To accurately measure the tumor size, the long axis and the short axis of the tumor were measured by using a vernier caliper, and the tumor size was calculated by the formula of ‘long axis×(short axis).sup.2/2’. In FIGS. 2A and 2B, PBS indicates a group administered with PBS as a negative control group, aPD1 indicates a group administered with anti-PD1 antibody as a positive control group, LMT17 indicates a group administered with the LMT17 strain as an experimental group, and LMT17-31+aPD1 indicates a group administered with the LMT17-31 strain and anti-aPD1 antibody in combination as an experimental group. FIG. 2A shows the tumor size according to the number of days after the cell injection, and FIG. 2B shows the tumor size on day 23 after the cell injection. In FIG. 2A, the numbers on the horizontal axis represent days on which the tumor size was measure, and that is, days elapsed after the administration of the tumor cells. In FIG. 2B, the bars indicate all tumor sizes for each subject on day 23 after the cell injection. As shown in FIGS. 2A and 2B, statistically significant tumor growth inhibition was observed in both the group administered with the LMT17-31 strain alone and the group administered with the LMT17-31 strain and anti-PD1 antibody in combination.

[0074] 3. Analysis of Tumor-Infiltrating Immune Cell and Evaluation of Functionality of Immune Cell

[0075] CD8 T cells that are tumor-infiltrating immune cells are important indicators of anticancer responses, and interferon gamma and granzyme B that are secreted by the CD8 T cells are functional cytokines showing the activation ability of immune cells. FIG. 3 shows the results of analyzing the CD8 T cells infiltrating into the tumor and the secretion of cytokines secreted by the CD8 T cells, after the L. plantarum LMT17-31 strain was administered to the mice having the tumor. The administration was performed according to Section 1. The tumor was excised on day 23 after the administration. The excised tumor was separated into single cells by using an RPMI1640 medium supplemented with 50 ug/ml of Liberase and 40 ug/ml of DNase I and utilizing a cell strainer. To observe the patterns of producing interferon gamma by T cells, the separated single cells were stimulated for 4 hours with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml of ionomycin in an RPMI1640 medium supplemented with 10% FBS. The PMA and ionomycin substances are substances that cause signal stimulation to activate T cells, and play the same role as the activation mechanism of T cells upon actual antigens, and thus can provide an environment in which an immune response seems to occur. Ionomycin as a Ca2+ ionophore increases a level of protein kinase C (PKC). In the case of PMA, by phosphorylating PKC, PMA synergizes for targeting CD4 T cells and CD8 T cells. After the stimulation, the cells were stained with the antibodies shown in Table 7 for the analysis on the immune cells and the identification of the production of interferon gamma, and then analyzed by using a CANTO II flow cytometry apparatus.

TABLE-US-00007 TABLE 7 Description Target Color Surface marker CD45 PE-Cy7 TCRβ APC-Cy7 CD4 PerCP-Cy5.5 CD8 Pacific blue Intracellular staining IFNγ FITC Granzyme B APC

[0076] FIGS. 3A, 3B, and 3C show the percentage of CD8 T cells in the tumor-infiltrating T cells, the percentage of IFNγ-expressing cells in the CD8 T cells, and the percentage of granzyme B-expressing cells in the CD8 T cells, respectively. FIGS. 3D, 3D, and 3F show the total number of tumor-infiltrating CD8 T cells, the number of IFNγ-expressing cells in the CD8 T cells, and the number of granzyme B-expressing cells in the CD8 T cells, respectively. As shown in FIG. 3, the percentage and number of the tumor-infiltrating CD8 T cells increased significantly in the group administered with the LMT17-31 strain, and the group administered with the anti-PD1 antibody and LMT17-31 strain in combination, compared to the PBS-treated group. Also, regarding the percentage and number of the CD8 T cells that produce activating factors, i.e., interferon gamma and granzyme B, a significant increase was shown in the group treated with the LMT17-31 strain, and the group treated with the anti-PD1 antibody and LMT17-31 strain in combination, compared to the PBS control group.