KR102259799B1 - 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 ingredients.

Description

Anticancer composition containing lipoteichoic 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 an anticancer composition comprising lipoteichoic acid, TNF-α and IFN-γ as active ingredients.

Colorectal cancer refers to a malignant tumor that occurs in the colon and rectum, and has the third highest worldwide incidence (945,000 new cases, 9.4% of all cancers) and mortality (492,000 deaths, 7.9% of all cancers) worldwide in 2000. high, and when compared by gender, it occurs in males and females at a similar rate (male: female 1.1:1).

The incidence of 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 the main causes of excessive animal fat, sugar, alcohol intake, and insufficient intake of fiber, antioxidant vitamins, and vegetables or fruits.

In particular, if you eat a lot of animal fat and meat, unlike the intake of fibrous foods such as vegetables or grains, the amount of stool is small and it takes a lot of time for the contents to pass through the large intestine and be excreted.

In addition, the excretion of bile acids and sterols increases, and the composition of bacterial species present in the large intestine changes, and the types of bacteria that chemically change these substances increase. Therefore, a lot of carcinogens are produced, and the time the carcinogens stay in the colon and come into contact with them becomes longer, which leads to frequent occurrence of colorectal cancer.

In the case of colorectal cancer, it is a representative solid cancer cell in which a large number of cells around blood vessels grow in a lump with each other.

In other words, it is not easy to completely remove colorectal cancer cells because the drug does not penetrate properly to the center of the lumped colorectal cancer cells.

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

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

Apoptosis is an important mechanism of action that most anticancer drugs exert an inhibitory effect on cancer cell proliferation. It is an active death that occurs by regulating the expression and activity of various genes and proteins according to a signal programmed inside the cell.

Apoptosis is easily observed in many normal physiological phenomena of living things. For example, apoptosis plays an important role in various morphological changes observed in the early developmental stage of an organism and in the functional self-organization of the immune system or nervous system. In addition, even after adulthood, it is essential to control the number of tissue homeostatic cells, to remove damaged cells, and as a defense mechanism against infection.

Self-death is also deeply involved in the pathogenesis of various diseases, and abnormal apoptosis can cause degenerative brain disease, immune system abnormalities, and cardiovascular diseases, and abnormal inhibition of auto-death can cause cancer. .

If we look in more detail at diseases in which apoptosis is abnormally issued or suppressed outside the normal regulatory process, cancers induced by abnormal expression of genes such as p53, p16 and Bcl-2, HIV, Herpes, and influenza viruses are caused by several infectious diseases. , and autoimmune diseases such as diabetes, rheumatoid arthritis, multiple sclerosis and myasthenia gravis.

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

The apoptosis of each cell constituting an individual is a means of removing these abnormal cells from an individual in order to prevent the development of a tumor caused by induction of inappropriate differentiation by genetically damaged cells or differentiation stimulants, that is, an irreversible genetic wound. It is a common means for removing cells with

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

Therefore, inhibition of apoptosis plays an important role in the cancer process because disturbance of the apoptosis process induces the survival and growth of damaged or damaged cells. In addition, it has been reported that substances having a cancer-preventing effect induce apoptosis of these abnormal cells, and the induction of apoptosis by them is at least related to their cancer-preventing activity.

Capecitabine, Leucovorin, and Irinotecan are known as conventional colorectal cancer treatment agents, but these may cause side effects such as liver failure, gastrostomy, and skin damage.

In addition, as a treatment method for colorectal cancer, therapeutic drugs and gene therapy for genes that play an important role in cell apoptosis, i.e., Bcl-2 family, p53, inhibitory apoptosis proteins (IAPs), and caspases are performed. This method has a problem in that it affects not only cancer cells but also normal cells.

In addition, the substances currently being studied are used as combination therapy with anticancer drugs, which showed results of enhancing the therapeutic effect, but at the same time, problems such as generation of resistance to anticancer drugs and normal cell death are pointed out.

Therefore, it is urgent to develop a new treatment method that can confirm cancer cell-specific death induction and immediate effect.

On the other hand, as it has been found that the invention of colorectal cancer can be reduced by controlling the composition of intestinal bacteria by recent research, probiotics and prebiotics are attracting attention as new colorectal cancer treatment methods. Probiotics and prebiotics increase the intestinal flora of useful microorganisms such as lactobacillus and Bifidobacteria , and conversely reduce the number of harmful microorganisms.

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

Therefore, while the development of anticancer agents that specifically act on cancer cells is on the rise, the present inventors found that a composition containing lipoteichoic acid derived from lactic acid bacteria and TNF-α and IFN-γ affects normal cells. It was intended to be used as a material for anticancer drugs by focusing on inducing apoptosis of cancer cells without giving them.

An object of the present invention is to provide an anticancer composition that specifically kills only cancer cells in order to solve the problem that existing anticancer agents induce normal cell death as well as cancer cells.

According to one 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 can induce apoptosis of cancer cells within 24 hours.

In one embodiment, the cancer may be one or more selected from the group consisting of liver cancer, stomach cancer, colorectal 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 the TNF-α and IFN-γ may be 1 ng/mL or more, respectively.

In one embodiment, the composition may include 100 μg/mL or more of the lipoteichoic acid, 5 ng/mL or more of TNF-α, and 5 ng/mL or more of IFN-γ.

In one embodiment, the lipoteichoic acid, TNF-α and IFN-γ may be administered simultaneously (simultaneous), separately (separate), or sequentially (sequential).

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

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

According to another aspect of the present invention, there is provided a health functional food for preventing and improving cancer 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, lipoteichoic acid, TNF-α and IFN-γ simultaneously (simultaneous), separately (separate), or sequentially (sequential) of animals other than humans comprising the step of administering A method of treating cancer is provided.

According to the present invention, the anticancer composition has fewer side effects than existing anticancer drugs because lipoteichoic acid, TNF-α and IFN-γ act on target cells, thereby activating a signal transduction system related to apoptosis specifically for cancer cells. .

In particular, the anticancer effect is excellent by synthesizing a derivative for the active site of lipoteichoic acid, and the anticancer effect is excellent because cancer cells are killed within 24 hours.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be 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 a composition according to an embodiment of the present invention. Blood cancer cells (THP-1) and colorectal cancer cells (HT-29) are cancer cells, and cells isolated from the mouse pancreas (splenocytes), primary cell lines (fibroblasts), and primary colon cells (CCD18co) are normal cells.
2 is an image of observation of changes in colorectal cancer cells (HT-29) according to the composition of the sample according to an embodiment of the present invention for 5 days.
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.
4 is a graph measuring the change of colorectal cancer cells (HT-29) according to the ligand concentration according to an embodiment of the present invention for 3 days.
5 is a graph measuring apoptosis of colorectal cancer cells (HT-29) according to various ligands according to an embodiment of the present invention.
6 to 8 confirm that apoptosis of cancer cells was induced by the composition according to an embodiment of the present invention. Figure 6 is DNA laddering, Figure 7 is Nucleus shrinking, Figure 8 is a Tunnel assay.
9 to 12 are results of verifying the anticancer effect of the composition according to an embodiment of the present invention in mice.
9 is an image of an anticancer experiment on CT-26 mouse colon cancer cells, and FIG. 10 is a graph measuring the weight of cancer cells 3 weeks after the experiment.
11 is a graph of measuring the size of cancer cells after 3 weeks of experimentation.
12 is an image of measuring the size of cancer cells extracted 3 weeks after the experiment.

Terms used in the present specification have selected general terms that are currently widely used as possible while taking functions of the present invention into consideration, but this may vary according to the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Unless otherwise defined, all terms used herein including technical or scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. Any numerical limitation given throughout this specification shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation were expressly written.

When a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Various scientific dictionaries containing the terms contained herein are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, several methods and materials are described. The present invention is not limited to specific methods, protocols, and reagents, as it may be used in various ways depending on the context used by those skilled in the art.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and therefore is not limited to the embodiments described herein.

According to one aspect of the present invention, there is provided an anticancer composition comprising lipoteichoic acid, TNF-α and IFN-γ as active ingredients, and according to another aspect of the present invention, lipoteichoic acid, TNF-α and Provided is a method for treating cancer comprising administering IFN-γ simultaneously, separately, or sequentially.

The "comprising as an active ingredient" is capable of exhibiting an anticancer effect, and may mean containing an effective amount to reduce the growth of cancer cells.

The anticancer effect is to induce apoptosis of cancer cells by the composition of the present invention. The lipoteichoic acid and the cytokines TNF-α and IFN-γ can induce apoptosis of cancer cells within 24 to 72 hours by regulating the signaling process after binding to a receptor on the cell surface.

Since the anticancer effect does not require a knock out of a specific gene, side effects are less compared to conventional anticancer drugs, 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 apoptosis in normal cells (primary cells), there are fewer side effects compared to conventional anticancer drugs.

The cancer may be one or more from the group consisting of liver cancer, stomach cancer, colorectal 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 lactic acid bacteria, and the lactic acid bacteria may be of the genus Lactobacillus sp .

The Lactobacillus genus Lactobacillus pento Saskatchewan (Lactobacillus pentosus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus Planta Room (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) or Lactobacillus know even Phil Ruth (Lactobacillus acidophilus) may be, but is not limited thereto.

The TNF-α and IFN-γ are cytokines secreted from immune cells. The IFN-γ increases the expression of the caspase gene required for apoptosis of cancer cells, and the TNF-α induces the continuous activity of the caspase protein.

In a previous study, despite the TNF-α and IFN-γ treatment, it did not show complete cell death, because anti-apoptotic factors such as Bcl2 inhibit apoptosis of cancer cells.

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

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

The concentration of the TNF-α and IFN-γ may be 1 ng/mL or more, respectively, and preferably, the anticancer composition is 100 μg/mL or more of the lipoteichoic acid, the TNF-α 5 ng/mL or more, and IFN-γ5 ng/mL or more.

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

The lipoteichoic acid, TNF-α and IFN-γ can be administered simultaneously (simultaneous), separately (separate), or sequentially (sequential), the lipoteichoic acid, TNF-α and IFN-γ can exhibit anticancer activity through synergistic action in the body.

In general, the lipoteichoic acid, TNF-α, and IFN-γ will generally be administered simultaneously as a composition, but even if each active ingredient is administered to the human body with a time lag, each individually administered active ingredient acts simultaneously in the body. It is possible to implement a level of anticancer activity.

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

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

The "prevention" means any action that reduces the frequency or degree of occurrence of a pathological phenomenon. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the cancer symptoms in the individual are reduced compared to the case where the composition is not used.

The "treatment" refers to any action that clinically intervenes to change the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it.

The desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or reduce the rate of disease progression. alleviating or temporarily ameliorating the condition, or improving the prognosis.

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

The composition may further include a carrier, excipient and diluent used in the preparation of the pharmaceutical composition. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injection solutions.

A solid preparation for oral administration may be a tablet, pill, powder, granule, capsule, etc., and the solid preparation may include at least one excipient to the compound and its fractions, for example, starch, calcium carbonate, sucrose, lactose. , or it can be prepared by mixing gelatin and the like. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

Liquid formulations for oral administration may include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be used in addition to simple diluents such as water and liquid paraffin.

Formulations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. The non-aqueous solvent and suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogelatin may be used.

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

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

It is not particularly limited as long as it is an individual for the purpose of preventing or treating the cancer, and any individual is applicable. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, guinea pigs, rats, mice, cattle, sheep, pigs, goats, etc. can be applied to any subject, and the method of administration is limited as long as it is a conventional method in the art. include without

According to another aspect of the present invention, there is provided a health functional food for preventing and improving cancer comprising the composition.

The health functional food means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and the functionality is to control nutrients or physiologically affect the structure and function of the human body. It may refer to ingestion for the purpose of obtaining useful effects for health purposes, such as action.

The health functional food may include normal food additives, and the food additives meet the standards and standards for the item in accordance with the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. suitability can be judged.

The items described in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, depigmentation, licorice extract, crystalline cellulose, high pigment, natural additives such as guar gum, sodium L-glutamate preparation, noodles Mixed formulations, such as an additive alkali agent, a preservative agent, and a tar dye agent, 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 manufactured and processed into 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 granulated with a mixture of the compound, excipient, binder, disintegrant, and other additives in a conventional manner, and then a lubricant is added thereto and compression-molded, or the mixture is directly compression-molded. can be manufactured. In addition, the health functional food in the form of tablets may contain a mating agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of the compound and additives such as excipients, or their granules or coated granules, and soft capsules include the above compounds and excipients. It can be prepared by filling a mixture with additives such as gelatin in a capsule base. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring can be prepared by molding a mixture of the compound, excipient, binder, disintegrant, etc. in an appropriate way, and if necessary, the skin is coated with sucrose or other suitable peeling agent, or starch, talc, or a suitable substance. You can also dress up with

The health functional food in the form of granules may be prepared in a granular form by an appropriate method of a mixture of the compound, excipients, binders, disintegrants, etc., and may contain flavoring agents, flavoring agents, etc. as necessary.

In addition, the term definitions for the excipients, binders, disintegrants, lubricants, 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" refers to an agent capable of improving, enhancing or enhancing the anticancer effect of an anticancer agent. For example, when the anti-cancer adjuvant is used together with an anti-cancer agent, the anti-cancer effect of the anti-cancer agent may be improved, enhanced or increased.

As another example, when an agent exhibiting concentration-dependent anti-cancer activity is used together with an anti-cancer agent at a level that does not show anti-cancer activity by itself, it can be used as an anti-cancer adjuvant that can improve, enhance or increase the anti-cancer effect of the anti-cancer agent. .

The anti-cancer adjuvant may be used as an anti-cancer agent or an anti-cancer adjuvant depending on the treatment concentration, and may be used as an anti-cancer adjuvant in a treatment concentration range that does not show anti-cancer 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, and if equivalent technical effects are realized despite the above modifications, the present invention It is said to be encompassed by the technical thought of

It is apparent that the present invention is further elaborated through the following examples, but the present invention is not limited by the following examples.

Example 1: Induction of cancer cell specific death

In order to confirm the cancer cell-specific apoptosis induction of the composition of the present invention, various cells were treated with the composition and the degree of cell death was measured.

For cancer cell lines isolated from humans, THP-1 (blood cancer cells), HT-29 (colon cancer cells), primary cell lines fibroblast, primary colorectal cells CCD18co, and splenocytes isolated from mouse pancreas, 0 to 100 μg/ mL of pLTA (lipoteichoic 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 FIG. 1 , in the case of cancer cell lines, HT-29 and THP-1 cells, the cell viability decreased as the concentration of pLTA increased. However, no apoptotic effect was observed in CCD18co (primary cells isolated from the colon), Fibroblasts (fibroblasts isolated from the skin) and Splenocytes, which were not cancer cells.

The above results suggest that apoptosis by treatment with pLTA and TNF-α/IFN-γ is specific to cancer cells, particularly colon cancer cells and blood cancer cells.

Example 2: Induction of apoptosis over time of colorectal cancer cells

In order to observe the cancer cell-specific apoptosis process of Example 1 for each time period, HT-29 colorectal cancer cells were observed.

After seeding HT-29 colorectal cancer cells into a 6 well plate, the experimental group treated with 10 ng/mL of TNF-α and IFN-γ, treated with 100 μg/mL pLTA 18 hours before, and then treated with 10 ng/mL of TNF- The degree of apoptosis was observed for 5 days by dividing the experimental group with α and IFN-γ re-treated, and the control group not treated with anything ( FIG. 2 ).

Referring to FIG. 2 , in the control group that was not treated with anything, the number of cells increased rapidly over time. In the case of the TNF-α+IFN-γ experimental group, apoptosis was observed on Day 1 and Day 2, but the number of cancer cells increased from Day 3.

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

The above result means that effective killing of colorectal cancer cells can be induced by treatment with pLTA and TNF-α/IFN-γ.

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

The above results suggest that cancer therapeutics using only TNF-α and IFN-γ cannot be actually commercialized.

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

In order to observe the anticancer effect according to the treatment concentration of TNF-α+IFN-γ, it was tested in HT-29 cells.

HT-29 cells treated with or without 100 μg/mL pLTA for 18 hours were treated with TNF-α and IFN-γ at a concentration of 0 to 10 ng/mL for 24 hours, and then cells were treated with WST-1 solution. The degree of death was measured (FIG. 3).

Referring to Figure 3, when only TNF-α+IFN-γ was treated without pLTA pretreatment, HT-29 cell death started to appear from 5+5 (5 ng/mL TNF-α+5 ng/mL IFN-γ). Thus, as the treatment concentration increased, apoptosis also increased.

On the other hand, when 100 μg/mL of pLTA was pre-treated for 18 hours and then treated with TNF-α+IFN-γ, slight apoptosis was observed from 0.125+0.125, and significant apoptosis was at TNF-α+IFN-γ concentration appeared in 1+1. However, the most ideal colorectal cancer cell death is when 100 μg/mL pLTA and TNF-α/IFN-γ 5+5 ng/mL or more are treated, and no regrowth of cancer cells was observed at this time.

Example 4: Cell death according to ligand treatment time

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

In order to verify the toxicity of the ligand used in the experiment, the indicated concentration of the ligand was 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).

Referring to FIG. 4 , apoptosis by ligand-only treatment was partially observed by pLTA and IFN-γ on the 3rd day.

When treated with TNF-α+IFN-γ, it started to appear from the 1st day of treatment, and about 50% of cell death was observed on the 3rd day. A similar level of cell death was observed when LPS and TNF-α+IFN-γ were co-treated.

Also, when pLTA and TNF-α (pLTA+T) or IFN-γ were co-treated, about 50% of cell death was observed on the 3rd day.

On the other hand, when pLTA and TNF-α+IFN-γ were treated simultaneously, about 45% of cell death on day 1, 75% on day 2, and about 95% of cell death on day 3 were observed.

These 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 specifically acts on cancer cell death, cell death by TNF-α+IFN-γ retreatment after pretreatment with various ligands was measured.

Ligands aLTA (lipoteichoic acid isolated from the cell wall of S. aureus ), pLTA (lipoteichoic acid isolated from the cell wall of L. plantarum ), LPS (lipopolysaccharide, a component of the endotoxin cell wall of Gram-negative bacteria), MPLA (monophosphoryl lipid A), PGLPS ( LPS isolated from Porphyromonas gingivalis ), AALPS ( LPS isolated from Aggregatibacter actinomycetemcomitans ), fPGN ( peptidoglycan isolated from Shigella flexneri ), P3C4 (synthetic TLR2 ligand), pPGN ( L. peptidoglycan isolated from plantarum ) was pre-treated for 18 hours and then treated with 10 ng/mL of TNF-α+ IFN-γ for 24 hours, and then the degree of cell death was measured using WST-1 solution ( 5).

Referring to FIG. 5, colorectal cancer cell death by the ligand and TNF-α+IFN-γ combination was observed in the case of pretreatment with lipoteichoic acid of Gram-positive bacteria.

LPS or peptidoglycan other than lipoteichoic acid did not induce such apoptosis.

In the case of pretreatment with Pam3CSK4, a ligand of TLR2, cancer cell death by TNF-α+IFN-γ occurred slightly, but it was significantly weaker than that of lipoteichoic acid, which is the same TLR2 ligand.

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

Example 6: Cell death by apoptosis

An experiment was performed to determine whether colorectal cancer cell death by the combination of pLTA and TNF-α+IFN-γ was caused by apoptosis.

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

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

Although apoptosis increased even when only TNF-α+IFN-γ was treated, it can be confirmed that rapid apoptosis occurred when pLTA and TNF-α+IFN-γ were treated together.

Referring to FIG. 6 , DNA laddering, a representative evidence of apoptosis, was increased by pLTA and TNF-α+IFN-γ.

Referring to Figure 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 Tunnel assay.

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

Example 7: Mouse anticancer experiment

To confirm the anticancer effect of the composition of the present invention, CT-26 (5x105 cells/200 μL), a mouse colorectal cancer cell line, was subcutaneously injected into the flank of Balb/c mice (n=3).

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

After 2 days, the experiment was performed for 3 weeks by repeatedly injecting pLTA and TNF-α+IFN-γ. The control group was subcutaneously injected with PBS every 3 days.

In the pLTA group, only 100 mg/kg of pLTA was injected subcutaneously every 3 days, and in the TNF-α+IFN-γ group, only 50 μg/kg of TNF-α+IFN-γ was subcutaneously injected every 3 days.

Referring to FIG. 9 , the growth of cancer cells in the pLTA+TNF-α+IFN-γ experimental group was inhibited by TNF-α+IFN-γ compared to the control and pLTA alone treatment. On the last day of the experiment, cancer cells were isolated from mice and the weight and size of the cancer cells were measured ( FIGS. 10 and 12 ).

Compared to the control group, the growth of cancer cells was partially inhibited when pLTA was treated alone.

On the other hand, in the case of the experimental group treated with TNF-α+IFN-γ, the weight and size of cancer cells were significantly reduced, which is presumed to have slightly higher concentrations of TNF-α+IFN-γ used in the experiment.

In addition, in the case of the pLTA+TNF-α+IFN-γ treatment group, the growth of cancer cells was inhibited the most.

Referring to FIG. 12 , cancer cells not treated with pLTA and TNF-α+IFN-γ 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 for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified 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 illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

Claims (10)

As a pharmaceutical composition for preventing or treating colon cancer comprising lipoteichoic acid, TNF-α and IFN-γ as active ingredients,
The lipoteichoic acid: TNF-α: IFN-γ is 1: 0.00005 to 0.0001: 0.00005 to 0.0001 will be included in a concentration ratio of, colon cancer prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is a pharmaceutical composition that induces apoptosis of cancer cells within 24 hours. The method of claim 1,
The lipoteichoic acid is a pharmaceutical composition derived from the cell wall of lactic acid bacteria. The method of claim 1,
When the lipoteichoic acid is 100 μg / mL,
The TNF-α is 5 ng / mL to 10 ng / mL,
The IFN-γ is 5 ng / mL to 10 ng / mL, the pharmaceutical composition. delete The method of claim 1,
The lipoteichoic acid, TNF-α and IFN-γ are simultaneously (simultaneous), separately (separate), or a pharmaceutical composition, characterized in that administered sequentially (sequential). The method of claim 1,
The lipoteichoic acid, TNF-α and IFN-γ is a pharmaceutical composition that exhibits anticancer activity through a synergistic action in the body. As a health functional food for preventing or improving colon cancer comprising lipoteichoic acid, TNF-α and IFN-γ as active ingredients,
The lipoteichoic acid: TNF-α: IFN-γ is 1: 0.00005 to 0.0001: 0.00005 to 0.0001 will be included in a concentration ratio of, colon cancer prevention or improvement health functional food. As an anticancer adjuvant composition for the prevention or treatment of colorectal cancer comprising the composition of any one of claims 1 to 4, 6 and 7,
The lipoteichoic acid: TNF-α: IFN-γ is 1: 0.00005 to 0.0001: to be included in a concentration ratio of 0.00005 to 0.0001, an anticancer adjuvant composition for the prevention or treatment of colorectal cancer. A method for treating colon cancer in animals other than humans, comprising administering lipoteichoic acid, TNF-α and IFN-γ simultaneously (simultaneous), separately (separate), or sequentially (sequential),
The lipoteichoic acid: TNF-α: IFN-γ is 1: 0.00005 to 0.0001: 0.00005 to 0.0001 will be included in a concentration ratio of, colon cancer treatment method.

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