KR20190137397A - A COMPOSITION FOR ANTICANCER COMPRISING LIPOTEICHOIC ACID, TNF-α AND IFN-γ - Google Patents

Abstract

The present invention provides an anticancer composition comprising lipoteichoic acid, TNF-α, and IFN-γ as active components. The present invention not only enhances efficiency of the anticancer effect by synthesizing a derivative for an active site of lipoteichoic acid, but also has an excellent anticancer effect because cancer cells die within 24 hours.

Description

Anti-cancer composition containing lipoteicoic acid, TNF-α and IFN-γ as active ingredients {A COMPOSITION FOR ANTICANCER COMPRISING LIPOTEICHOIC ACID, TNF-α AND IFN-γ}

The present invention relates to a composition for anticancer comprising lipoteichoic acid, TNF-α and IFN-γ as an active ingredient.

Colorectal cancer is a malignant tumor of the colon and rectum, with the world's 2000 incidence (945,000 new cases, 9.4% of cancers worldwide) and mortality (492,000 deaths, 7.9% of all cancers) being the third of all cancers. Higher, gender-specific proportions occur in males and females (male: female 1.1: 1).

Colorectal cancer is closely related to environmental factors. In particular, the incidence rate is increasing due to westernized eating habits, and related factors are known to be due to excessive animal fat, sugar, alcohol intake and fiber, antioxidant vitamins, lack of intake of vegetables or fruits.

In particular, eating a lot of animal fats and meat, unlike intake of fiber foods such as vegetables and grains, the amount of stool is small and the contents take a long time to pass through the large intestine.

In addition, the excretion of bile acids and sterols increases, and the composition of the bacterial species present in the large intestine causes changes in the types of bacteria that chemically change these substances. Therefore, a lot of carcinogens are generated, and the time for the carcinogen stays in contact with the colon becomes long, so that colorectal cancer frequently occurs.

In the case of colorectal cancer, a typical solid cancer cell in which a large number of cells grow in the form of agglomerates around blood vessels is one of the cancers in which treatment methods are extremely difficult to cure.

That is, it is not easy to completely remove colon cancer cells because the drug does not penetrate properly to the center of the agglomerated colon cancer cells.

Currently, there are few methods for treating colorectal cancer with drugs. Surgical treatment such as surgery or radiation therapy is the only limited method for treating colorectal cancer, and it is difficult to cure colorectal cancer.

Therefore, many researchers have conducted a lot of research to effectively inhibit the growth of colon cancer cells as well as most cancer cells by developing a method that can effectively inhibit the growth of colon cancer cells.

Apoptosis is an important mechanism by which most anticancer drugs exert the anti-proliferative effect of cancer cells. Active apoptosis is an active death caused by regulation of the expression and activity of several genes and proteins in response to a programmed signal inside the cell.

Self-killing is easily observed in many normal physiological phenomena of life. Self-killing, for example, plays an important role in the various morphological changes observed in the early developmental stages of life and in the functional self-organization of the immune or nervous system. In addition, even after becoming an adult, it functions essentially as a regulation of tissue homeostasis, removal of damaged cells, and defense against infection.

Self-killing is also deeply involved in the pathogenesis of various diseases. The occurrence of abnormal self-killing can be a cause of degenerative neurological diseases, immune system abnormalities, and cardiovascular disease, and abnormal suppression of self-killing can cause cancer. .

Looking more closely at diseases in which apoptosis is abnormally issued or suppressed outside of normal control processes, cancers induced by abnormal expression of genes such as p53, p16 and Bcl-2, HIV, Herpes and flu viruses can And autoimmune diseases such as diabetes, rheumatoid arthritis, multiple sclerosis and work experience.

As such, self-killing not only plays an important role in maintaining various physiological functions of living organisms, but is also closely related to the pathogenesis of various diseases.

Self-killing of each cell constituting the individual is a means of removing these abnormal cells from the individual, i.e. an irreversible genetic wound, to prevent the development of tumors by genetically damaged cells or induction of inappropriate differentiation by differentiation stimulants. It is a common means to remove cells with.

This concept is supported by the fact that commonly used anticancer drugs induce cancer cell death through a process of autokilling associated with cancer cell proliferation inhibition.

Therefore, suppression of self-killing plays an important role in the cancer process because the disturbance of the self-killing process induces the survival and growth of the damaged or injured cells. In addition, it has been reported that cancer preventive substances induce self-killing of these abnormal cells, and the induction of self-killing by them is associated with at least their cancer prevention activity.

Conventional treatments for colorectal cancer are known as capecitabine, leucoperin, and irinotecan, but these may cause side effects such as liver failure, gastric emptying, and skin damage.

In addition, as a method of treating colorectal cancer, genes and gene therapy for genes that play an important role in cell death, such as Bcl-2 family, p53, Inhibitory apoptosis proteins (IAPs), caspases, are performed. This method has a problem that affects normal cells as well as cancer cells.

In addition, the substances currently being studied are used as a combination therapy with anticancer drugs, which have been shown to enhance the therapeutic effect, but at the same time, problems with the development of resistance to the anticancer drugs and the death of normal cells have been pointed out.

Therefore, it is urgent to develop a new method of treatment that can identify cancer cell specific killing induction and immediate effect.

On the other hand, as recent studies have revealed that the invention of colorectal cancer can be reduced by controlling the composition of intestinal bacteria, probiotics and prebiotics have attracted attention as a new method for treating colorectal cancer. Probiotics and prebiotics increase the intestinal flora of useful microorganisms such as lactobacillus and Bifidobacteria and, conversely, reduce the number of harmful microorganisms.

These methods can prevent tumor formation by inhibiting intestinal inflammatory responses and enhancing immune function and anticancer activity. In addition, probiotics can bind to carcinogens in meat, inhibit their activity, and reduce the production of bacterial enzymes that hydrolyze carcinogen precursors such as β.

Therefore, while the development of anti-cancer agents that specifically act on cancer cells, the present inventors have found that the composition of lipoeichoic acid derived from lactic acid bacteria and a mixture of TNF-α and IFN-γ affects normal cells. Without paying attention to the induction of self-killing of cancer cells, it was intended to use it as a material for anticancer drugs.

The present invention aims to provide an anticancer composition that specifically kills only cancer cells in order to solve the problem that the existing anticancer agent induces normal cell death as well as cancer cells.

According to an aspect of the present invention, there is provided an anticancer composition comprising lipoteichoic acid, TNF-α and IFN-γ as active ingredients.

In one embodiment, the composition may induce self-killing of cancer cells within 24 hours.

In one embodiment, the cancer may be at least one selected from the group consisting of liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, rectal cancer, pancreatic cancer and blood cancer.

In one embodiment, the lipoteichoic acid may be derived from the cell wall of lactic acid bacteria.

In one embodiment, the concentration of TNF-α and IFN-γ may be 1 ng / mL or more, respectively.

In one embodiment, the composition may include at least 100 μg / mL of the lipoteicoic acid, at least 5 ng / mL of the TNF-α, and at least 5 ng / mL of IFN-γ.

In one embodiment, the lipoteichoic acid, TNF-α and IFN-γ can be administered simultaneously, separately or sequentially.

In one embodiment, the lipoteichoic acid, TNF-α and IFN-γ may exhibit anticancer activity through synergism in the body.

According to another aspect of the present invention, there is provided a pharmaceutical composition for cancer prevention and treatment comprising the composition.

According to another aspect of the present invention, there is provided a dietary supplement for cancer prevention and improvement comprising the composition.

According to another aspect of the present invention, there is provided an anticancer adjuvant composition comprising the composition.

According to another aspect of the present invention, a method of administering lipoteichoic acid, TNF-α and IFN-γ simultaneously, separately or sequentially in an animal Cancer treatment methods are provided.

According to the present invention, the anticancer composition has fewer side effects than conventional anticancer agents because lipoteichoic acid, TNF-α and IFN-γ act on a target cell, thereby activating a signaling system associated with cancer cell specific death. .

In particular, by synthesizing a derivative for the active site of lipoteicoic acid to increase the efficiency of the anti-cancer effect, it is excellent because the cancer cells die within 24 hours.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a graph evaluating cell death according to cells of the composition according to an embodiment of the present invention. Hematological cancer cells (THP-1) and colon cancer cells (HT-29) are cancer cells, and splenocytes, primary fibroblasts, and primary colon cells (CCD18co) from the pancreas of mice are normal cells.
Figure 2 is an image observed for 5 days the change of colorectal cancer cells (HT-29) according to the composition of the sample according to an embodiment of the invention.
3 is a graph evaluating the death of colorectal cancer cells (HT-29) according to the concentration of TNF-α and IFN-γ according to the composition of the sample according to an embodiment of the present invention.
Figure 4 is a graph measuring the change in colorectal cancer cells (HT-29) for three days according to the ligand concentration in accordance with an embodiment of the invention.
5 is a graph measuring the death of colorectal cancer cells (HT-29) according to various ligands according to an embodiment of the present invention.
6 to 8 confirm that the self-killing of cancer cells is induced by the composition according to an embodiment of the present invention. 6 is DNA laddering, FIG. 7 is Nucleus shrinking, and FIG. 8 is a Tunnel assay.
9 to 12 are the results of verifying the anti-cancer effect of the composition according to an embodiment of the invention in the mouse.
9 is an anticancer experimental image of CT-26 mouse colorectal cancer cells, Figure 10 is a graph measuring the weight of cancer cells three weeks after the experiment.
11 is a graph measuring the size of cancer cells after 3 weeks of the experiment.
12 is an image measuring the size of the cancer cells extracted after three weeks of the experiment.

The terminology used herein is to select general terms that are currently widely used as possible in consideration of the functions in the present invention, but may vary according to the intention or precedent of the person skilled in the art, the emergence of new technologies and the like. In addition, in certain cases, there is also a term arbitrarily selected by the applicant, in which case the meaning will be described in detail in the description of the invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the contents throughout the present invention, rather than the names of the simple terms.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and are not construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

The numerical range includes the numerical values defined in the range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were clearly written. All minimum numerical limits given throughout this specification include all higher numerical limitations as if the higher numerical limit were clearly written. All numerical limitations given throughout this specification will include all better numerical ranges within the broader numerical range, as the narrower numerical limitations are clearly written.

When a part is said to "include" a certain component, this means that it may further include other components, without excluding other components, unless specifically stated otherwise.

Various scientific dictionaries that include the terms included herein are well known and available in the art. While any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, some methods and materials have been described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents, as it may be used in various ways.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

According to one aspect of the invention, there is provided an anticancer composition comprising lipoteicoic acid, TNF-α and IFN-γ as an active ingredient, according to another aspect of the present invention, lipoteicoic acid, TNF-α and Provided are methods of treating cancer comprising administering IFN- [gamma] simultaneously, separately or sequentially.

The "contained as an active ingredient" may have an anticancer effect, and may mean that it contains an effective amount to reduce the growth of cancer cells.

The anticancer effect is to induce self-killing of cancer cells by the composition of the present invention. The lipoteichoic acid and the cytokines TNF-α and IFN-γ can induce self-killing of cancer cells within 24 to 72 hours by binding to receptors on the cell surface and regulating signaling processes.

Since the anticancer effect does not require knock out for a specific gene, side effects are less than that of conventional anticancer agents, and the cancer treatment period can be shortened.

In addition, since the lipoteichoic acid and the cytokines TNF-α and IFN-γ do not induce self-killing in primary cells, they have fewer side effects than conventional anticancer drugs.

The cancer may be one or more from the group consisting of liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, rectal cancer, pancreatic cancer, and blood cancer, but is not limited thereto.

The lipoteichoic acid may be derived from the cell wall of the lactic acid bacteria, the lactic acid bacteria may be Lactobacillus sp.

The genus Lactobacillus Lactobacillus pentosus ( Lactobacillus pentosus ), Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ) or Lactobacillus acidocillus acidophilus ( ophilus ) It may be, but is not limited thereto.

The TNF-α and IFN-γ are cytokines secreted by immune cells. The IFN- [gamma] increases the expression of the caspase gene required for autophagy of cancer cells, and the TNF- [alpha] induces the continuous activity of caspase protein.

Previous studies did not show complete cell death despite the TNF-α and IFN-γ treatments, since anti-apoptotic factors such as Bcl2 inhibit cancer cell death.

However, in the present invention, the lipoteichoic acid may induce expression of anti-apoptotic factors and induce cell death by TNF-α and IFN-γ.

Inhibition of the expression of Bcl2 by the lipoteicoic acid does not occur in CCD18co, which is a primary cell line, and as a result, cell death by the combination of lipoteicoic acid, TNF-α and IFN-γ does not occur in normal cells.

The concentration of the TNF-α and IFN-γ may be at least 1 ng / mL, respectively, preferably the anticancer composition is at least 100 μg / mL of the lipoteicoic acid, at least 5 ng / mL of the TNF-α, and IFN- [gamma] may comprise at least 5 ng / mL.

When the concentrations of the TNF-α and IFN-γ are less than 1 ng / mL, respectively, the effect of inhibiting self-killing of cancer cells may not be realized, and the upper limit of concentration of each component may be determined in consideration of the characteristics or the health state of the subject. have.

The lipoteichoic acid, TNF-α and IFN-γ can be administered simultaneously, separately or sequentially, and the lipoteicoic acid, TNF-α and IFN-γ May exhibit anticancer activity through synergistic action in the body.

Typically, the lipoteichoic acid, TNF-α, and IFN-γ are generally administered simultaneously as a composition, but even if each of the effective ingredients are administered to the human body at a time difference, the respective effective ingredients administered separately act simultaneously in the body. Levels of anticancer activity can be achieved.

According to another aspect of the present invention, there is provided a pharmaceutical composition for cancer prevention and treatment comprising the composition.

The cancer is a disease including colon cancer, liver cancer, breast cancer, pancreatic cancer and leukemia, malignant lymphoma, multiple myeloma, aplastic anemia and the like.

The term "prevention" means any action that reduces the frequency or extent of pathological phenomena. Prevention can be complete or partial. In this case, it may refer to a phenomenon in which the carcinogenic symptoms in the subject decrease compared with the case where the composition is not used.

The term "treatment" refers to all actions that are clinically involved in altering the natural process of the subject or cell to be treated, and may be performed during or to prevent a clinical pathology.

The desired therapeutic effect may prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, slow the progression of the disease, or Mitigating or temporarily alleviating the condition or improving the prognosis.

The composition may be administered in the form of oral delivery, parenteral delivery. The composition can be administered systemically or topically, and the administration can include oral and parenteral administration. The composition may be formulated with a suitable amount of pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

The composition may further comprise a carrier, excipient and diluent used in the manufacture of the pharmaceutical composition. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, but are not limited thereto.

In addition, the composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions.

Solid preparations for oral administration may be used in tablets, pills, powders, granules, capsules, and the like, wherein the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose in the compound and fractions thereof. Or gelatin can be mixed and prepared. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.

As the liquid preparation for oral administration, suspending agents, solvents, emulsions, syrups, and the like may be used, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like, in addition to water and liquid paraffin, which are simple diluents, may be used.

For parenteral administration, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories can be used. The non-aqueous solvent and suspension may be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The composition may be administered in a pharmaceutically effective amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level may be of the type and severity of the subject, age, sex, type of disease, drug Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts.

The composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

The object for the prevention or treatment of the cancer is not particularly limited, and any object can be applied. For example, it can be applied to any individual such as non-human animals such as monkeys, dogs, cats, rabbits, marmots, rats, mice, cows, sheep, pigs, goats, and humans, and the like, and the mode of administration is limited if it is a conventional method in the art. Includes without.

According to another aspect of the present invention, there is provided a dietary supplement for cancer prevention and improvement comprising the composition.

The health functional food means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the law on health functional foods, and the functional properties control nutrients or physiologically on the structure and function of the human body. It may mean ingestion for the purpose of obtaining a useful effect for health use such as action.

The health functional food may include a conventional food additive, and unless otherwise specified, the food additive may comply with the standards and standards for the corresponding item according to the General Regulations and General Test Law of the Food Additives Authorized by the Food and Drug Administration. Compliance can be determined by

The items described in the food additive orbital are, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extract, crystalline cellulose, high pigment, guar gum, sodium L-glutamate preparations, and noodles. Mixed preparations, such as an added alkali, a preservative, and a tar pigment, are mentioned.

The health functional food may be used in various ways, such as foods and beverages for the prevention or improvement of cancer, for example, various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives and the like.

The health functional food may be prepared and processed in any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and pills for the purpose of preventing or improving cancer.

Specifically, the health functional food in the form of tablets is a mixture of the compound, excipients, binders, disintegrants and other additives in a conventional manner, and then compression-molded with a lubricant, or directly compression-molded the mixture Can be prepared. In addition, the health functional food in the form of tablets may contain a mating agent and the like, if necessary, may be coated with a suitable coating agent.

The hard capsules of the health functional food in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of additives such as the compound and excipients or granules or peeled granules thereof, and the soft capsules may contain the compounds and excipients. The mixture with additives, such as these, can be prepared by filling in capsule bases, such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The dietary supplement in the form of a cyclic form may be prepared by molding a mixture of the compound, excipient, binder, disintegrating agent, etc. in a suitable manner, and may be coated with sucrose or other suitable coating agent, or starch, talc, or a suitable substance. You can also give a favor.

The granular health functional food may be prepared by granulating a mixture of the compound, excipient, binder, disintegrating agent and the like in an appropriate manner, and may contain a flavoring agent, a copper, and the like as necessary.

In addition, the definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents and the like are described in documents known in the art and may include those having the same or similar functions.

According to another aspect of the present invention, there is provided an anticancer adjuvant composition comprising the composition.

The "anticancer adjuvant" means an agent that can improve, enhance or enhance the anticancer effect of the anticancer agent. For example, when the anticancer adjuvant is used in combination with an anticancer agent, the anticancer effect of the anticancer agent may be improved, improved or increased.

As another example, a concentration-dependent anticancer agent may be used as an anticancer adjuvant that can improve, enhance or enhance the anticancer effect of the anticancer agent when used in combination with an anticancer agent at a level that does not exhibit anticancer activity by itself. .

The anticancer adjuvant may be used as an anticancer agent or an anticancer adjuvant according to the treatment concentration, and may be used as an anticancer adjuvant in the treatment concentration range which does not exhibit anticancer activity by itself.

Those skilled in the art to which the present invention pertains will be able to apply by changing the type, introduction ratio, etc. of each configuration based on the description of the present invention, if the equivalent technical effect is implemented in spite of the above modification, the present invention It will be included in the technical idea of the.

Through the following examples, the present invention will be described in more detail, but the present invention is not limited by the following examples.

Example  1: induction of cancer cell specific death

To confirm the cancer cell specific killing induction of the composition of the present invention, various cells were treated with the composition and the degree of cell death was measured.

0 to 100 μg / mL of pLTA (lipotecoic acid isolated from the cell wall of Lactobacillus L. plantarum K8) was pretreated for 18 hours.

After treatment with 10ng / mL of TNF-α and IFN-γ for 24 hours, the degree of cell death was measured using the WST-1 Cell Proliferation Assay System (DoGenBio, Korea) (FIG. 1).

Referring to Figure 1, in the case of cancer cell lines HT-29 and THP-1 cells as the concentration of pLTA increases, the cell survival rate decreased. However, no cell death effects were observed in CCD18co (primary cells isolated from the large intestine), Fibroblasts (skin-derived fibroblasts) and Splenocytes, not cancer cells.

The results suggest that cell death by pLTA and TNF-α / IFN-γ treatment is specific for cancer cells, in particular colon cancer cells and hematologic cancer cells.

Example  2: induction of death of colorectal cancer cells over time

In order to observe the cancer cell specific killing process of Example 1 by time zone, HT-29 colorectal cancer cells were observed.

HT-29 colorectal cancer cells were seeded in 6 well plates and treated with 10 ng / mL TNF-α and IFN-γ, and treated with 100 μg / mL pLTA 18 hours before, 10 ng / mL TNF- The degree of cell death was observed for 5 days by dividing into the experimental group retreated with α and IFN-γ, and the control group without any treatment (FIG. 2).

Referring to FIG. 2, the control group treated with nothing rapidly increased the number of cells over time. In the TNF-α + IFN-γ experimental group, cell death was observed in Day1 and Day2, but the number of cancer cells increased from Day3.

However, in the pLTA and TNF-α / IFN-γ experimental groups, most cancer cells were killed at Day 1 and all cancer cells were killed at Day 2 and did not proliferate again.

The results indicate that pLTA and TNF-α / IFN-γ treatment can induce effective killing of colon cancer cells.

In the case of TNF-α + IFN-γ some cancer cell death was observed but proliferated again over time.

The results suggest that cancer treatments using only TNF-α and IFN-γ cannot be commercially available.

Example  3: TNF -α + IFN - γ treatment concentration  Caused by cell death

In order to observe the anticancer effect according to the treatment concentration of TNF-α + IFN-γ, experiments were conducted in HT-29 cells.

HT-29 cells treated with 100 μg / mL pLTA or not before 18 hours were treated with TNF-α and IFN-γ at concentrations of 0 to 10 ng / mL for 24 hours and then treated with WST-1 solution. The degree of death was measured (FIG. 3).

Referring to FIG. 3, HT-29 cell death begins to appear from 5 + 5 (5 ng / mL TNF-α + 5 ng / mL IFN-γ when only TNF-α + IFN-γ is treated without pLTA pretreatment. As cell concentration increased, cell death also increased.

On the other hand, when TNF-α + IFN-γ was treated with 100 μg / mL of pLTA for 18 hours, a slight cell death was observed from 0.125 + 0.125, and significant cell death was TNF-α + IFN-γ concentration. It appeared at 1 + 1. However, the most ideal colorectal cancer cell death was when 100 μg / mL pLTA and TNF-α / IFN-γ5 + 5 ng / mL were treated. No cancer cell regrowth was observed.

Example  4: cell death by ligand treatment time

Colorectal cancer cell line HT-29 cells were treated with 100 μg / mL pLTA, 0.5 μg / mL LPS, 10 ng / mL TNF-α, 10 ng / mL IFN-γ in combination or individually without pretreatment.

In order to verify the self-toxicity of the ligands used in the experiments, ligands of the indicated concentrations were treated alone.

In all experiments, the degree of cell death was measured using WST-1 solution after 24, 48 and 72 hours of ligand treatment (FIG. 4).

4, cell death by ligand alone was partially observed by pLTA and IFN-γ at day 3.

When treated with TNF-α + IFN-γ, it began to appear from the first day of treatment, and about 50% cell death was observed on the third day. Similar treatment of LPS and TNF-α + IFN-γ resulted in similar levels of cell death.

In addition, when co-treatment with pLTA and TNF-α (pLTA + T) or IFN-γ, about 50% cell death was observed on day 3.

On the other hand, treatment with pLTA and TNF-α + IFN-γ (pLTA + T + I) simultaneously resulted in cell death of about 45% on day 1, 75% on day 2 and about 95% on day 3.

The results suggest that effective killing of HT-29 cells, a colorectal cancer cell line, is caused by the combination of pLTA and TNF-α + IFN-γ.

Example  5: Measurement of Colorectal Cancer Cell Death by Various Ligands

To determine whether the combination of pLTA and TNF-α + IFN-γ acts specifically against cancer cell death, cell death by TNF-α + IFN-γ retreatment after pretreatment of various ligands was measured.

Ligand aLTA (lipotecoic acid isolated from S. aureus cell wall), pLTA (lipotecoic acid isolated from L. plantarum cell wall), LPS (lipopolysaccharide, the endotoxin cell wall component of Gram-negative bacteria), MPLA (monophosphoryl lipid A), PGLPS (LPS isolated from Porphyromonas gingivalis ), AALPS (LPS isolated from Aggregatibacter actinomycetemcomitans ), fPGN ( Shigella) Peptidoglycan isolated from flexneri ), P3C4 (synthetic TLR2 ligand) and pPGN (Peptidoglycan isolated from L. plantarum ) were pretreated for 18 hours and then 10 ng / mL of TNF-α + IFN-γ After treatment for 24 hours to determine the degree of cell death using a WST-1 solution (Fig. 5).

Referring to FIG. 5, colon cancer cell death by a combination of ligand and TNF-α + IFN-γ was observed when pre-treatment of gram-positive bacteria lipoteicoic acid.

LPS or peptidoglycan other than lipoteicoic acid did not induce such cell death.

Pretreatment with Pam3CSK4, a ligand of TLR2, resulted in some cancer cell death by TNF-α + IFN-γ, but was significantly weaker than lipoteichoic acid, the same TLR2 ligand.

The results suggest that only LTA among various bacterial derived ligands can specifically induce cancer cell death with TNF-α + IFN-γ.

Example  6: Apoptosis  Caused by cell death

Experiments were performed to determine whether apoptosis was caused by colon cancer cell death by a combination of pLTA and TNF-α + IFN-γ.

Colorectal cancer cell line HT-29 cells were pretreated with 100 μg / mL of pLTA for 18 hours and then retreated with 10 ng / mL of TNF-α + IFN-γ for 24 hours.

As a control group, a group treated with only 100 μg / mL of pLTA and a group treated with only 10 ng / mL of TNF-α + IFN-γ were used.

Apoptosis increased even when only TNF-α + IFN-γ (TI) was treated, but acute Apoptosis occurred when pLTA and TNF-α + IFN-γ were treated together.

Referring to FIG. 6, DNA laddering, which is evidence of representative apoptosis, was increased by pLTA and TNF-α + IFN-γ (pLTI).

Referring to FIG. 7, Nucleus shrinking was greatly increased by pLTA and TNF-α + IFN-γ.

Referring to FIG. 8, cells undergoing apoptosis were increased by pLTA and TNF-α + IFN-γ through a tunnel assay.

The results suggest that cell death by pLTA and TNF-α + IFN-γ occurs through Apoptosis.

Example  7: mouse anticancer experiment

In order to confirm the anticancer effect of the composition of the present invention, CT-26 (5x10 ⁵ cells / 200 μL), a mouse colon cancer cell line, was injected subcutaneously into the side of a Balb / c mouse (n = 3).

After 2 days, the experimental group was subcutaneously injected with 10 mg / kg of pLTA and 50 μg / kg of TNF-α and IFN-γ after 1 day (pLTA + TNF-α + IFN-γ).

Two days later, the experiment was repeated for three weeks by repeat injections of pLTA and TNF-α + IFN-γ. The control group was injected subcutaneously with PBS every three days.

The pLTA group was injected subcutaneously with only 100 mg / kg of pLTA at 3 day intervals and the TNF-α + IFN-γ group with 50 μg / kg of TNF-α + IFN-γ at 3 day intervals.

9, cancer cell growth of the TNF-α + IFN-γ wall pLTA + TNF-α + IFN-γ test group was inhibited compared to the control and pLTA alone treatment. On the last day of the experiment, cancer cells were separated from the mice and the weight and size of the cancer cells were measured (FIGS. 10 and 12).

Treatment with pLTA alone inhibited the growth of some cancer cells compared to the control.

On the other hand, in the experimental group treated with TNF- [alpha] + IFN- [gamma], the weight and size of cancer cells were greatly reduced, which may be due to the rather high concentration of TNF- [alpha] + IFN- [gamma] used in the experiment.

In addition, in the pLTA + TNF-α + IFN-γ treatment group, cancer cell growth was most suppressed.

Referring to Figure 12, pLTA and TNF-α + IFN-γ-treated cancer cells grew significantly after 3 weeks.

On the other hand, the growth of cancer cells injected subcutaneously with pLTA and TNF-α + IFN-γ was significantly inhibited compared to the control group. In particular, the growth of mouse cancer cells treated with pLTA + TNF-α + IFN-γ was most effectively inhibited.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

Claims (12)

Antipot cancer composition comprising lipoteikoic acid, TNF-α and IFN-γ as active ingredients. The method of claim 1,
A composition for inducing self-killing of cancer cells within 24 hours. The method of claim 1,
The cancer is at least one composition selected from the group consisting of liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, rectal cancer, pancreatic cancer and blood cancer. The method of claim 1,
The lipoteicoic acid is a composition derived from the cell wall of lactic acid bacteria. The method of claim 1,
Wherein the concentrations of TNF-α and IFN-γ are each 1 ng / mL or more. The method of claim 5,
At least 100 μg / mL of the lipoteicoic acid, at least 5 ng / mL of the TNF-α, and at least 5 ng / mL of IFN-γ. The method of claim 1,
Wherein the lipoteichoic acid, TNF-α and IFN-γ are administered simultaneously, separately, or sequentially. The method of claim 7, wherein
The lipoteicoic acid, TNF-α and IFN-γ are compositions exhibiting anticancer activity through synergism in the body. A pharmaceutical composition for preventing or treating cancer, comprising the composition of any one of claims 1 to 8. A health functional food for preventing or improving cancer comprising the composition of any one of claims 1 to 8. An anticancer adjuvant composition comprising the composition of any one of claims 1 to 8. A method for treating cancer in animals other than humans comprising administering lipoteicoic acid, TNF-α and IFN-γ simultaneously, separately, or sequentially.

Images