KR20130048946A - Anti-cancer composition comprising lactobacillus reuteri cs132 or culture fluid of thereof - Google Patents
Anti-cancer composition comprising lactobacillus reuteri cs132 or culture fluid of thereof Download PDFInfo
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- KR20130048946A KR20130048946A KR1020110113885A KR20110113885A KR20130048946A KR 20130048946 A KR20130048946 A KR 20130048946A KR 1020110113885 A KR1020110113885 A KR 1020110113885A KR 20110113885 A KR20110113885 A KR 20110113885A KR 20130048946 A KR20130048946 A KR 20130048946A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- 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
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- 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
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- 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
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
Abstract
Description
The present invention relates to an anticancer composition comprising Lactobacillus luteri CS132 or a culture thereof, and more particularly, to Lactobacillus luteri CS132 or a culture thereof having anticancer activity that inhibits cell proliferation of cancer cells and promotes cell death. It relates to a pharmaceutical composition for anticancer.
In recent years, cancer deaths have increased in number of causes of death. In particular, in recent years, the incidence of cancer has increased due to a change in diet, that is, a higher proportion of meat and a lower proportion of vegetarian diet.
High-fat, high-protein diets are known not only to alter bile and cholesterol metabolism, but also to enhance the metabolic activity of bacteria that produce carcinogenic factors from their metabolites. Increasing the intake of meat is also known to increase the growth of harmful bacteria due to changes in intestinal pH and induce enzymes involved in carcinogenesis. Relatively reduced intake of dietary fiber leads to poor discharge of these harmful bacteria, leading to intestinal diseases and even serious conditions such as cancer.
On the other hand, lactic acid bacteria (lactic acid bacteria) is a bacterium that decomposes carbohydrates and uses the same to make lactic acid. Lactic acid bacteria can be classified into five genera, which are divided into Streptococcus, Lactobacillus, Leukonostoc, Bifidobacteria and Pediococcus. The streptococcus microorganism, a streptococcus strain, is a homozygous fermenting microorganism. It is known to inhibit spoilage bacteria and pathogens by fermenting milk to produce lactic acid. Lactobacillus microorganisms are homozygous or heterozygous fermenting lactic acid bacterium, which are common bacteria in the fermentation process of dairy products and vegetables. The microorganisms of the genus Lucococcus are heterozygous and mainly involved in the fermentation of vegetables. Bifidobacterial microorganisms are organisms that do not grow in the presence of oxygen, and fermentation of sugars produces L (+) -like lactic acid, which can be metabolized to infants. Finally, the microorganism of Pediococcus is a homozygous fermenting microorganism in the form of sycamore, which is present in kimchi or pickled food and is involved in the fermentation of meat such as sausage.
These lactic acid bacteria maintain the acidic intestinal pH to inhibit the reproduction of harmful bacteria such as Escherichia coli and Crosstridium sp. And improve diarrhea and constipation, as well as play a role in vitamin synthesis, anticancer action, and serum cholesterol reduction. do. In particular, lactic acid bacteria have a specific protein that can strongly bind to the mucous membrane and epithelial cells of the intestine, thus helping to prevent the growth of harmful bacteria. In addition, lactic acid bacteria are known to promote the proliferation of macrophages to enhance the cognitive and bactericidal ability of macrophage intestinal harmful bacteria, and to promote the secretion of immune-related substances (Gabriela perdigon et al. , J. of food Protection 53: 404-410, 1990; Katsumasa sato et al., Microbiol. Immunol., 32 (7): 689-698, 1988). Among them, lactobacillus microorganisms produce acidophilin to inhibit the growth of harmful bacteria such as foreign bacteria, Salmonella and Staphylococcus, and to inhibit the growth of diarrheal bacteria to normalize the intestinal microflora, thereby stopping diarrhea .
Therefore, the discovery of lactic acid bacteria having excellent anticancer activity among the beneficial lactic acid bacteria in the body and it is expected that it can be used safely in the manufacture of food as well as pharmaceutical products with excellent anticancer activity.
Therefore, the present inventors isolated and identified a new strain of Lactobacillus luteri belonging to the lactic acid bacterium, Lactobacillus ruteri CS132 (KCTC 11452BP), and this strain inhibits cancer cell proliferation and promotes apoptosis of cancer cells. The present invention has been completed by confirming the fact that it has anticancer activity.
Accordingly, it is an object of the present invention to provide a pharmaceutical composition for anticancer comprising Lactobacillus luteri CS132 (KCTC 11452BP) or a culture thereof as an active ingredient.
In addition, another object of the present invention to provide a health functional food for the prevention or improvement of cancer, including Lactobacillus Luteri CS132 (KCTC 11452BP) or a culture thereof as an active ingredient.
In order to achieve the above object, the present invention provides a pharmaceutical composition for anticancer comprising Lactobacillus luteri CS132 (KCTC 11452BP) or a culture thereof as an active ingredient.
In one embodiment of the present invention, the Lactobacillus luteri CS132 (KCTC 11452BP) or its culture may be one having anticancer activity through the activity of promoting apoptosis of cancer cells.
In one embodiment of the present invention, the cancer may be cervical cancer, stomach cancer or skin cancer.
In one embodiment of the present invention, the composition contains the Lactobacillus Luteri CS132 (KCTC 11452BP) strain in the number of 1.5 x 10 4 cells ~ 1.2x10 7 cells, or the culture solution of the Lactobacillus Lutheri CS132 (KCTC 11452BP) strain It may be contained in a volume ratio of 10 to 30% relative to the total volume.
In addition, the present invention provides a health functional food for the prevention or improvement of cancer, including Lactobacillus Luteri CS132 (KCTC 11452BP) or a culture thereof as an active ingredient.
In one embodiment of the present invention, the cancer may be cervical cancer, stomach cancer or skin cancer.
Lactobacillus luteri CS132 (KCTC 11452BP) according to the present invention or its cultures have excellent anti-proliferative effect of cancer cells and at the same time have anti-cancer activity through activity of apoptosis through promoting expression of apoptosis factor, There is an effect that can be usefully used in the manufacture of medicines for the treatment and the production of health functional food.
Figure 1 shows the results of analyzing the anticancer activity of Lactobacillus luteri CS132 according to the present invention, 1a shows the results of CCK-8 analysis, 1b shows the results of TUNEL analysis, in Figure 1b a is adipose progenitor cells B represents cervical cancer cells, c represents gastric cancer cells, and d represents skin cancer cells.
Figure 2 shows the results of analyzing the anticancer activity of Lactobacillus luteri CS132 according to the present invention, 2a shows the staining results using the Hoechst 33258 reagent, 2b shows the DNA fragmentation through the UV detector after agarose gel electrophoresis It shows the result confirmed.
Figure 3 shows the results of Western blot performed to confirm whether the Lactobacillus Luteri CS132 strain of the present invention in each cancer cell line has the activity of regulating the expression of autologous apoptosis-related factors.
The present invention is characterized by providing a new strain Lactobacillus luteri CS132 (KCTC 11452BP) having anticancer activity through inhibiting cell proliferation of cancer cells and promoting cancer cell apoptosis and an anticancer composition using the same.
In general, Lactobacillus is a gram-positive organ anaerobic bacterium, which is known to act as an intestinal synthesis of vitamins, amino acids, and proteins, as well as to enhance immunity and promote digestion, and to stabilize intestinal bacteria. In addition, by producing organic acids such as acetic acid and lactic acid in the intestine of normal people, it not only inhibits the growth of intestinal rot bacteria, but also reduces the growth inhibition of pathogenic bacteria in the colon, and has many beneficial effects on intestinal diseases such as diarrhea and constipation. It has been reported to have antimutagenic activity by adsorption of mutagenic substances.
Therefore, the inventors of the present invention are expected to be able to use a variety of high-functional foods, pharmaceuticals if you find a strain having anti-cancer activity in Lactobacillus sp. .
Meanwhile, only lactic acid bacteria were isolated from human feces using lactic acid bacteria selection medium, and among the isolated strains, strains having excellent acid resistance, bile resistance, and antibiotic resistance were selected, and the selected strains were molecules based on the 16S rDNA sequence. A phylogenetic analysis was performed to identify Lactobacillus ruteri strains. The identified strains were named “Lactobacillus ruteri CS132,” and were deposited with the Korea Biotechnology Research Institute Gene Bank on Jan. 6, 2009, and deposited with the accession number KCTC 11452BP. Deposited.
On the other hand, the inventors of the present invention, while studying the new in vivo function of the isolated and identified Lactobacillus Luteri CS132 strain, it was first identified that the strain has anti-cancer activity that inhibits the proliferation of cancer cells and promotes cancer cell death .
That is, according to one embodiment of the present invention, a culture supernatant obtained by culturing Lactobacillus luteri CS132 according to the present invention is obtained, and the supernatant is treated to cervical cancer cell lines, gastric cancer cell lines and skin cancer cell lines, respectively, and then cancer cells. The degree of proliferation of was analyzed. First, as a result of investigation through CCK-8 analysis widely used in the art, in the case of the group treated with the cancer cell line, the culture supernatant of Lactobacillus luteri CS132 according to the present invention was not a cancer cell as a control. Cell proliferation of cancer cells was suppressed compared to the group treated with fat precursor cells, and the culture of Lactobacillus ruteri CS132 was also examined in the results of TUNEL analysis, DNA fragmentation analysis and chromatin condensation to analyze the degree of cell death. When supernatants were treated with cancer cell lines, apoptosis of cancer cells was promoted, DNA fragmentation and chromatin were observed. It appeared to be even generated axial development (see Figs. 1 and 2).
Therefore, the present inventors have found that the Lactobacillus luteri CS132 or its culture according to the present invention effectively inhibits the proliferation of cancer cells and at the same time has an excellent activity of promoting apoptosis of cancer cells.
On the other hand, cancer refers to the rapid increase and growth of the number of cells exponentially due to the loss of self-killing function due to the damage of DNA of the cell and the lack of control of cell proliferation at the check point of the cell cycle. Is a protein that inhibits rapid cleavage is known as tumor protein 53 (tumor protein 53). In addition, p53 is a transcription factor that is activated when DNA is damaged, regulates cell cycle and apoptosis, inhibits cell proliferation of cells with damaged DNA, or induces apoptosis in severe cell damage. Representative tumor suppression protein.
Therefore, cancer occurs when the p53 gene is damaged or mutated. Recently, researches for treating cancer through the development of p53 expression or activity promoter have been conducted.
In addition, apoptosis of cells is regulated by apoptosis inducing factors and apoptosis inhibitors, and recently, the regulation of autologous apoptosis has attracted attention as a new target of anticancer therapy. Autologous apoptosis is an important phenomenon that causes active cell death, which is essential for the maintenance and development of tissue homeostasis. As described above, p53 acts as an important factor controlling cell survival and death. When p53 becomes active due to DNA damage, p53 induces cell cycle delay and suppression of cell proliferation.
In addition, important proteins that induce apoptosis are proteins belonging to the Bcl-2 family (family) and these are important regulators of the autoapoptotic pathway. Proteins belonging to the Bcl-2 family include Bcl-x (L), which inhibits apoptosis, and Bax and Bak, which induces apoptosis.
Therefore, a method of inducing autologous apoptosis or inactivating a factor that inhibits autologous apoptosis in cancer cells may also be a method of treating cancer.
The present inventors performed an experiment to determine whether the Lactobacillus luteri CS132 of the present invention can regulate the expression of p53, Bcl-2 and Bax, which are autologous apoptosis factors in cancer cells, that is, another embodiment of the present invention. In Example, the culture supernatant of Lactobacillus luteri CS132 was treated to a cancer cell line, and then the degree of expression of p53, Bax and Bcl-2 expressed in the cancer cell line was performed by Western blotting, and the culture supernatant was treated. The expression of p53 and Bax, which are apoptosis promoting factors, was increased in the cancer cells, compared to the control group, which was not.
Therefore, through these results, the present inventors have found that the Lactobacillus luteri CS132 strain of the present invention has anticancer activity through the action of promoting the expression of genes that induce apoptosis in cancer cells while simultaneously reducing the expression of apoptosis inhibitory factors. I could tell the truth.
Therefore, the present invention may provide an anticancer pharmaceutical composition comprising Lactobacillus luteri CS132 (KCTC 11452BP) or a culture thereof as an active ingredient.
The Lactobacillus luteri CS132 (KCTC 11452BP) strain according to the present invention can be cultured in large quantities by a conventional lactic acid culture method. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. For example, a Man-Rogosa-Sharp (MRS) liquid medium and a liquid medium to which milk is added can be used.
The culture can be carried out under conventional lactic acid culture conditions, for example, can be incubated for about 10-72 hours at about 15-45 ℃. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, and this step may be performed according to the needs of those skilled in the art. The concentrated cells can be preserved so as not to lose their activity by freezing or freeze drying according to conventional methods.
In addition, the pharmaceutical composition for anticancer according to the present invention may include the crushed cell wall fraction of Lactobacillus luteri CS132 (KCTC 11452BP) strain, live bacteria, dead bacteria, dry bacteria or culture medium of the strain as an active ingredient, Lactobacillus Luteri CS132 (KCTC 11452BP) strain may be obtained by removing and concentrating the culture medium as described above, or a suspension in the form of mixed strains in the culture medium may be used.
In addition, the culture medium of the strain includes a culture solution itself cultured in a liquid medium, filtrate (centrifuged supernatant) and the like by removing the strain by filtration or centrifugation of the culture solution.
In the composition according to the present invention, the Lactobacillus lusteri CS132 (KCTC 11452BP) strain is contained in the composition in the number of 1.5x10 4 cells ~ 1.2x10 7 cells, or the culture solution of the Lactobacillus lusteri CS132 (KCTC 11452BP) strain is based on the total volume of the composition It may be included in a volume ratio of 10 to 30%.
The anticancer pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of Lactobacillus luteri CS132 or a culture thereof alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.
As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat or prevent cancer. The pharmaceutically effective amount of Lactobacillus luteri CS132 or the culture medium of the strain according to the present invention is 0.1 to 100 mg / day / kg body weight, preferably 1 ~ 10 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the type of cancer and its severity, the age, weight, health condition, sex, route of administration and duration of treatment.
As used herein, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human. The pharmaceutical composition may further include a carrier, an excipient and a diluent, and examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber. , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives. In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.
The pharmaceutical compositions according to the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. The dosage of the active ingredient may be appropriately selected depending on several factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient.
In addition, the pharmaceutical composition of the present invention can be administered in parallel with known compounds having the effect of preventing or treating cancer.
Furthermore, Lactobacillus luteri CS132 or its dispensing liquid according to the present invention can also be used as an active ingredient of a food composition for the prevention or improvement of cancer.
Since the Lactobacillus lusteri CS132 strain used in the present invention can be used as a food grade and can be safely ingested without a separate purification process for removing toxicity, a culture solution of Lactobacillus luteri CS132 having cancer cell line inhibitory activity is used in food. When added and ingested can get anti-cancer effect.
The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. For example, as a health food, Lactobacillus luteri CS132 of the present invention or a culture thereof may be prepared in the form of tea, juice, and drink for drinking, granulated, encapsulated, and powdered. In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage corned beef) Breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese), edible vegetable oils, margarine, vegetable protein , Retort foods, frozen foods, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the Lactobacillus Luteri CS132 and the culture thereof according to the present invention.
In addition, in order to use Lactobacillus luteri CS132 or its culture according to the present invention in the form of food additives can be prepared in powder or concentrate form. The preferred content of Lactobacillus luteri CS132 or its culture in the food composition of the present invention may be about 0.01 to 90 g per 100 g of food.
Therefore, the present invention can provide a health functional food for preventing or improving cancer comprising Lactobacillus luteri CS132 (KCTC 11452BP) or a culture thereof as an active ingredient.
Lactobacillus luteri CS132 or the culture thereof according to the present invention is characterized by excellent anticancer activity can be effectively used as an effective component of the anticancer composition, in particular, the culture medium is usually most of the utility value is discarded such anticancer By identifying the activity, the present invention is of great industrial value.
In addition, the cancer that can be used for the purpose of preventing or treating the Lactobacillus Luteri CS132 or the culture thereof according to the present invention is not limited thereto, and may be cervical cancer, stomach cancer or skin cancer, and anticancer activity against this kind of cancer. Since has been demonstrated through one embodiment of the present invention can be effectively used in the treatment of the cancer.
Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.
< Example 1>
Lactobacillus Lutheri CS132 Isolation and Identification of Strains
Healthy male volunteers were recruited to collect the feces and all catalase negative strains were selected for use in subsequent studies using MRS medium as the selection medium. Identification of the strains was performed based on the characteristics of Lactobacillus as presented in Bergey's Manual by performing bacterioscopy, gram staining, growth at 15 ° C. and 45 ° C. and fermentation tests at various carbon sources. Water was identified based on morphological necropsy and biochemical profile by API-50 CHL test kit (bioMerieux Vitek Inc., Hazel Wood, Mo., USA). In order to analyze the morphology, gram, spore and capsule staining were performed.
As a result, the isolated bacteria are Gram-positive bacteria that grow simultaneously under aerobic and anaerobic conditions, do not form spores, have no motility, and have a bacillus form, and perform molecular siRNA analysis based on 16S rDNA sequence. As a result, it was identified as Lactobacillus lutery, showing 95% 16s rDNA homology with Lactobacillus lutery standard strain.
Therefore, the present inventors named the strain Lactobacillus reuteri CS132 (Lactobacillus reuteri CS132), and deposited on January 6, 2009 to the Korea Biotechnology Research Institute Gene Bank (Accession No. KCTC 11452BP).
< Example 2>
Lactobacillus
Lutheri
CS132
Of anticancer activity
<2-1> Lactobacillus Lutheri CS132 Analysis of Cancer Cell Proliferation Inhibitory Activity of Strains
The present inventors performed the following experiment to determine the anticancer activity of Lactobacillus reuteri CS132 strain.
First, cervical, gastric and skin cancer cell lines as cancer cells were cultured in DMEM (Gibco-BRL) medium containing 10% FBS (Hyclone) and 1% A / A (Gibco-BRL) as anticancer activity assays. Silver was incubated in a humid incubator maintained at 37 ° C. of 5% carbon dioxide. Adipose progenitor cells were also isolated from adipocytes, i.e., adipose tissue cells were washed three times with PBS solution containing 10% antibiotic-antimycotic, followed by 0.06% collagenase type I and 1 extracellular matrix. After digestion with% BSA (bovine serum albumin), the mixture was stirred for 2 hours in a water bath at 37 ° C. After filtration using 40 uM nylon mesh (BD Falcon, MA, USA), red blood cells were removed using erythrocyte lysis buffer containing 0.15 M NaCl, 1.0 mM potassium bicarbonate, and 0.1 mM EDTA, and the remaining cells were removed. Cell culture flasks were aliquoted and cultured under the same conditions as above. The cells were then incubated in DMEM (Gibco-BRL) medium containing 10% FBS (Hyclone) and 1% A / A (Gibco-BRL). After 24 hours, the cells were washed with PBS and suspended. The medium was replaced twice a week and passaged three times to obtain adipose precursor cells.
Each of the cells thus prepared (fat progenitor cell line, cervical cancer cell line, gastric cancer cell line and skin cancer cell line) was dispensed in a 96 well plate with a cell number of 1.5 x 10 4 cell / well and incubated for 24 hours. The culture supernatant of the Lactobacillus luteri CS132 of the present invention was treated in an amount of% (v / v), wherein the culture supernatant of the Lactobacillus luteri CS132 was cultured in MRS broth (Becton dickinson, cockeysville, MD). The supernatant obtained by centrifugation at 4000 rpm for 10 minutes was used.
As described above, each cell line treated with the culture supernatant of Lactobacillus luteri CS132 was analyzed for the anticancer activity of Lactobacillus luteri CS132 through the following experimental methods.
In the anticancer activity assay, the degree of cancer cell proliferation was first examined by CCK-8 analysis using the ELISA method. That is, CCK-8 (corresponding to the volume ratio of 1/10 based on the volume of the culture medium in each of the cultured cells) Dojondo molecular technologies, Rockville, USA) and 4 hours later, cell survival was examined using an ELISA meter at 450 nm.
In addition, TUNEL analysis was performed for other anti-cancer activity assays, each of the prepared cell lines (each cell line treated with the culture supernatant of Lactobacillus luteri CS132) Click-iTTUNEL Alexa Fluor Imaging Assay Kit (Invitrogen, USA) Was used to measure the degree of cell death according to the kit using the method.
In addition, DNA cleavage and chromatin condensation were analyzed for other anti-cancer activity assays. First, DNA cleavage was performed by dispensing each cell line in a number of 1.2 × 10 7 cells / well in a 100 mm dish, incubating for 24 hours, and then lactobacillus. The culture supernatant of Luteri CS132 was treated with 20% (v / v) of each culture medium, and after another 24 hours, the Qigen mini-prep kit (Qigen, Max-volmer-strabe, Germany) was used. DNA was extracted, and 5 ug of DNA extracted from each cell line was loaded on 1% agarose gel, followed by electrophoresis, and the degree of DNA cleavage was analyzed using a UV detector.
In addition, chromatin condensation analysis was performed using Hoescht 33258, which stains nuclei in cells. This method is based on the principle that when chromatin is condensed and fragmented, the staining reagent penetrates the cell membrane and is stained blue. As a method of measuring by using, each cell was treated with the culture supernatant of Lactobacillus luteri CS132, and then the cells were washed twice with cold PBS and fixed for 4 minutes with 4% formaldehyde. The fixed cells were then washed again with PBS and then treated with 5 ug / ml of Hoescht 33258 for 10 minutes in the absence of light. Cells were then washed three times with cold PBS and reagent was added dropwise onto the cells. Since the nuclei of the cells were observed by fluorescence microscope.
Based on these experiments, the culture supernatant of the Lactobacillus luteri CS132 of the present invention was treated with cervical cancer, gastric cancer and skin cancer cell lines and the fat precursor cells used as a control, and the proportion of the culture supernatant of the Lactobacillus luteri CS132 was proportional to the treatment time. The cell viability of each cancer cell line was reduced, especially for cervical cancer cells, and the cell proliferation was reduced to 10, 24, 31 and 40% with 16, 24, 48 and 75 hours of incubation. In gastric cancer, cell proliferation decreased by 22, 24, 41, and 49% with time, and skin cancer decreased by 17, 22, 28, and 35%. It was confirmed that the cell proliferation is inhibited in cancer cell lines, in particular, it was found that the gastric cancer cell proliferation inhibitory effect is superior to other cancers. In the TUNEL analysis, cancer cell lines also showed a significant increase in cell death compared to adipose progenitor cells (see FIGS. 1A and 1B).
As a result of DNA fragmentation and chromatin condensation analysis, nuclear cell condensation was observed in all cancer cell lines treated with the culture supernatant of the Lactobacillus luteri CS132 of the present invention, as shown in FIGS. 2A and 2B. It was confirmed that anger was also induced. On the other hand, cell deaths such as DNA fragmentation and chromatin condensation were not observed in non-cancer cell lines.
Therefore, the present inventors have found that the Lactobacillus ruteri CS132 or the culture thereof according to the present invention has a cancer cell proliferation inhibitory activity and apoptosis promoting activity.
<2-2> Lactobacillus Lutheri CS132 Analysis of the effect on the cancer-related factors of the strain
Through the above examples, the present inventors confirmed that the Lactobacillus luteri CS132 strain of the present invention has anticancer activity, and more specifically, examined whether the strain may also affect the expression of cancer-related factors. After treating the Lactobacillus luteri CS132 culture supernatant of the present invention to each cell line used in Example <2-1>, the expression levels of p53, Bcl-2 and Bax genes, which are cancer-related factors, were treated in the treated cell lines. Confirmation via Western blot. At this time, the Western blot was washed three times with each cell line treated with Lactobacillus luteri CS132 culture supernatant with PBS solution, and then RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris, pH 8.0, 50 mM NaCl, 50). mM NaF, 100 l Na3VO4, 1 mM DTT, and 50 lg / ml PMSF) and lysed each cell for 1 hour on ice. Subsequently, insoluble materials were removed by centrifugation at 12,000 rpm for 20 minutes. The supernatant was centrifuged on SDS-PAGE, followed by electrophoresis of the separated proteins onto nitrocellulose membrane. Antibodies against Bcl-2 and Bax) were used to examine the expression of these proteins.
As a result, as shown in Figure 3, the cervical cancer, gastric cancer and skin cancer cell lines treated with the Lactobacillus luteri CS132 culture supernatant of the present invention, the expression of p53, an anticancer factor, was increased compared to the control group not treated In addition, the expression of Bax, which promotes apoptosis, was also increased compared to the control group. On the other hand, expression of Bcl-2, a cancer promoting factor (ie, cancer cell death inhibiting factor), was shown to be decreased.
Therefore, the present inventors have found that the Lactobacillus luteri CS132 strain according to the present invention has anticancer activity through apoptosis promoting activity of cancer cells.
So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (6)
The Lactobacillus luteri CS132 (KCTC 11452BP) or its culture is anticancer pharmaceutical composition characterized in that it has an anticancer activity through the activity of promoting apoptosis of cancer cells.
The cancer is cervical cancer, stomach cancer or skin cancer, characterized in that the pharmaceutical composition for cancer.
The composition may contain Lactobacillus Luteri CS132 (KCTC 11452BP) strains in a number of 1.5 x 10 4 cells to 1.2x10 7 cells, or culture medium of Lactobacillus Luteri CS132 (KCTC 11452BP) strains in an amount of 10-30% based on the total volume of the composition. An anticancer pharmaceutical composition, characterized in that it is contained in a volume ratio.
The cancer is a functional food for the prevention or improvement of cancer, characterized in that the cervical cancer, stomach cancer or skin cancer.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015170158A1 (en) * | 2014-05-05 | 2015-11-12 | Giovanni Mogna | Therapy for use in the treatment of tumors, acquired immunodeficiency syndrome and leukemias by dual immune biostimulation |
| WO2015170159A1 (en) * | 2014-05-05 | 2015-11-12 | Giovanni Mogna | A composition for use in treating or preventing viral or bacterial infections in a subject undergoing anti-tumor chemotherapy, leukemia treatment or aids therapy comprising l. reuteri ler03 and/or l. salivarius ls06. |
| KR20160043809A (en) | 2014-10-14 | 2016-04-22 | 대한민국(농촌진흥청장) | Lactobacillus reuteri Black8, and Anti-tumor composition using the same |
| WO2019013401A1 (en) * | 2017-07-11 | 2019-01-17 | 이일규 | Composition for inhibiting oral pathogenic bacteria, containing lactobacillus reuteri cs 132 (kctc 11452bp) or culture product thereof |
| KR102049700B1 (en) | 2019-07-31 | 2019-11-28 | (주) 에이투젠 | Composition for improving preventing or treating muscular disease including Lactobacillus reuteri ATG-F4 |
| KR20220152148A (en) | 2021-05-07 | 2022-11-15 | 주식회사 브이원바이오 | Limosillactobacillus reuteri VA102 and Anti-tumor composition using the same |
| CN117402768A (en) * | 2023-03-13 | 2024-01-16 | 广东悦创生物科技有限公司 | Lactobacillus reuteri KA1 and application thereof in preparation of anti-inflammatory and anti-tumor food and drug |
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2011
- 2011-11-03 KR KR1020110113885A patent/KR20130048946A/en not_active Application Discontinuation
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10413576B2 (en) | 2014-05-05 | 2019-09-17 | Giovanni Mogna | Therapy for use in the treatment of tumors, acquired immunodeficiency syndrome and leukemias by dual immune biostimulation |
| WO2015170158A1 (en) * | 2014-05-05 | 2015-11-12 | Giovanni Mogna | Therapy for use in the treatment of tumors, acquired immunodeficiency syndrome and leukemias by dual immune biostimulation |
| CN106573022A (en) * | 2014-05-05 | 2017-04-19 | G·莫格纳 | A composition for use in treating or preventing viral or bacterial infections in a subject undergoing anti-tumor chemotherapy, leukemia treatment or AIDS therapy comprising l. reuteri LER03 and/or l. salivarius LS06 |
| US11077153B2 (en) | 2014-05-05 | 2021-08-03 | Chiara Benassai et al. | Composition for use in treating or preventing viral or bacterial infections in a subject undergoing anti-tumor chemotherapy, leukemia treatment or aids therapy comprising L. reuteri LER03 and/or L. salivarius LS06 |
| RU2721565C2 (en) * | 2014-05-05 | 2020-05-20 | Джованни МОНЬЯ | Therapeutic agent for use in treating tumors, acquired immunodeficiency syndrome and leukemia by double immune biostimulation |
| WO2015170159A1 (en) * | 2014-05-05 | 2015-11-12 | Giovanni Mogna | A composition for use in treating or preventing viral or bacterial infections in a subject undergoing anti-tumor chemotherapy, leukemia treatment or aids therapy comprising l. reuteri ler03 and/or l. salivarius ls06. |
| KR20160043809A (en) | 2014-10-14 | 2016-04-22 | 대한민국(농촌진흥청장) | Lactobacillus reuteri Black8, and Anti-tumor composition using the same |
| CN110997897A (en) * | 2017-07-11 | 2020-04-10 | 李溢圭 | Oral pathogenic bacteria inhibiting composition comprising lactobacillus reuteri CS 132(KCTC11452BP) or culture thereof |
| KR20190006739A (en) * | 2017-07-11 | 2019-01-21 | 이일규 | Oral pathogen inhibiting composition comprising lactobacillus reuteri cs 132 (kctc 11452bp) or a culture thereof |
| WO2019013401A1 (en) * | 2017-07-11 | 2019-01-17 | 이일규 | Composition for inhibiting oral pathogenic bacteria, containing lactobacillus reuteri cs 132 (kctc 11452bp) or culture product thereof |
| KR102049700B1 (en) | 2019-07-31 | 2019-11-28 | (주) 에이투젠 | Composition for improving preventing or treating muscular disease including Lactobacillus reuteri ATG-F4 |
| WO2021020663A1 (en) | 2019-07-31 | 2021-02-04 | (주) 에이투젠 | Composition comprising lactobacillus reuteri atg-f4 for prevention or treatment of muscular disorder |
| KR20220152148A (en) | 2021-05-07 | 2022-11-15 | 주식회사 브이원바이오 | Limosillactobacillus reuteri VA102 and Anti-tumor composition using the same |
| CN117402768A (en) * | 2023-03-13 | 2024-01-16 | 广东悦创生物科技有限公司 | Lactobacillus reuteri KA1 and application thereof in preparation of anti-inflammatory and anti-tumor food and drug |
| CN117402768B (en) * | 2023-03-13 | 2024-03-22 | 广东悦创生物科技有限公司 | Lactobacillus reuteri KA1 and application thereof in preparation of anti-inflammatory and anti-tumor medicines |
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