KR20180125906A - Pharmaceutical composition for enhancing effect of anti-cancer treating - Google Patents

Abstract

The present invention relates to a composition for enhancing chemotherapeutic effects of anticancer agents for gastric cancer, containing an interleukin-6 (IL-6) inhibitor as an active ingredient.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR ENHANCING EFFECT OF ANTI-CANCER TREATING [0002]

The present invention relates to a pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer and a method of screening for anticancer adjuvant for improving or treating gastric cancer.

Cancer is one of the greatest threats to human health, a disease that occurs when cells undergo a series of mutagenic processes that multiply and are immortalized in an unlimited, unregulated way. Causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital gene mutations.

The three major therapies for treating cancer are surgery, chemotherapy, and radiation therapy. The surgical procedure is effective in removing cancer cells that have not been metastasized because of the invasive method, but its effect is limited for metastatic cancer. In addition, the chemotherapy can not be completely effective because cancer cells having drug resistance may be produced.

On the other hand, gastric cancer is one of the most frequent carcinomas in the world, and its incidence is low in the US and Europe, while it is significantly higher in the Far East including Korea. In particular, gastric cancer is known to have poor efficacy of chemotherapy, so development of factors that can enhance the anticancer effect is required. Therefore, various auxiliary compositions have been developed for the treatment of cancer as in the Korean Patent No. 10-0597535, but it has not been recognized that the effect is still definite.

It is an object of the present invention to provide a composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer.

It is another object of the present invention to provide a screening method of a cancer-preventing adjuvant for the improvement or treatment of gastric cancer.

It should be understood, however, that the technical scope of the embodiments of the present invention is not limited to the above-described technical problems, and other technical problems may exist.

As a technical means for achieving the above technical object, the first aspect of the present invention provides a pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer, comprising an interleukin-6 (IL-6) .

According to one embodiment of the present invention, the gastric cancer may include cancer-associated fibroblasts (CAFs) in cancer cells, but the present invention is not limited thereto.

According to one embodiment of the disclosure, the interleukin-6 (IL-6) inhibitor is an interleukin-6 self-inhibitor; SiRNA capable of inhibiting the expression of interleukin-6; An antibody that specifically binds to interleukin-6; Antisense oligonucleotides that specifically bind to interleukin-6, and combinations thereof, but are not limited thereto.

According to one embodiment of the present invention, the anticancer agent is selected from the group consisting of 5-FU, acute thrombocytopenia, doxorubicin, paclitaxel, mitomycin, docetaxel, cisplatin, irinotecan, epothar, capecitabine, oxaliplatin, But are not limited thereto.

As a technical means for achieving the above technical object, the second aspect of the present invention provides a method for treating cancer, comprising the steps of: treating a test compound in a gastric cancer cell line mixed with gastric cancer tissue or cancer-related fibroblast containing cancer-associated fibroblast; Measuring the expression level of mRNA or protein of interleukin-6 in the treated gastric cancer tissue or gastric cancer cell line; And selecting a test compound that reduces the expression level of mRNA or protein of interleukin-6 in comparison with the untreated control group.

According to one embodiment of the present invention, the level of the interleukin-6 mRNA is determined by RT-PCR, real time quantitative RT-PCR, RNase But is not limited to, one of the methods selected from the group consisting of RNase protection method, Northern blotting and DNA chip technology assay.

According to one embodiment of the present invention, the expression of the interleukin-6 protein is detected by any one method selected from the group consisting of Western blotting, enzyme immunoassay (ELISA), immunohistochemistry, and flow cytometry (FACS) But is not limited thereto.

The above-described task solution is merely exemplary and should not be construed as limiting the present disclosure. In addition to the exemplary embodiments described above, there may be additional embodiments in the drawings and the detailed description of the invention.

According to the present invention, the present invention provides a pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer, or a method for screening anticancer adjuvant for improving or treating gastric cancer.

The inventors of the present application have found that when a culture solution in which cancer-associated fibroblast (CAF), which is a main cell constituting a tumor matrix in gastric cancer, is cultured and co-cultured with gastric cancer cells, the anticancer effect is decreased Respectively. In addition, IL-6 is secreted in CAF, and the anticancer effect is reduced. Therefore, it has been confirmed that a large therapeutic effect can be obtained in the treatment of gastric cancer having anticancer drug resistance by inhibiting expression of IL-6 mRNA or protein targeting IL-6 expression.

Accordingly, the present invention provides a pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer, comprising an interleukin-6 (IL-6) inhibitor as an active ingredient. In addition, it is possible to provide a screening method of a cancer-improving adjuvant for the treatment or improvement of gastric cancer by selecting a test compound capable of inhibiting the expression of mRNA or protein of IL-6.

FIG. 1 is a schematic diagram of a conditioned medium in which gastric cancer-associated fibroblast (NAF) or cancer-associated fibroblast (CAF) is cultured in gastric cancer cells according to an embodiment of the present invention. , CM) was added to determine the change in the anticancer activity (IC50).
FIG. 2 is a photograph showing the increase in the inhibitory effect of 5-FU of an anticancer agent by CAF on gastric cancer cell lines MKN-45 and MKN-1 according to one embodiment of the present invention.
FIG. 3 shows the results of western blot analysis of changes in cell death markers (cleaved caspase 3 & PARP) upon addition of anticancer drugs when co-culturing gastric cancer cells with NAF or CAF.
FIG. 4 shows the results of confirming activation of signal transduction pathway of JAK-STAT3 (signal transducer and activator of transcription 3) in the signal transduction pathway of IL-6 when CAF of CMC was added to gastric cancer cells according to one embodiment of the present invention .
FIG. 5 (A) is a result of confirming that cytokines such as IL-6 and IL-8 are increased when CAF is co-cultured with the gastric cancer cell line MKN-45 according to an embodiment of the present invention. B) shows that JAK-STAT3 (signal transducer and activator of transcription 3) signaling pathway is activated in the signal transduction pathway of IL-6 when CAF is co-cultured with the gastric cancer cell line MKN-45 according to one embodiment of the present invention to be.
FIG. 6 shows the results of RT-qPCR analysis showing that the expression of IL6 was increased in CAF as compared to NAF according to one embodiment of the present invention. Although the expression of IL-6 was little in gastric cancer cells, it was mainly expressed in fibroblasts, And the expression level in CAF was also high in protein level by RT-PCR and ELISA.
FIG. 7 shows that when IL-6-inhibited CAF, in which expression of IL-6 is inhibited by tetracycline, was co-cultured with gastric cancer cells and suppressed the expression of IL-6 according to one embodiment of the present invention , And the increase in the expression of cell death markers suppressed by CAF.
FIG. 8 shows that IL-6-inhibited CAF, in which expression of IL-6 is inhibited by a monoclonal antibody, was co-cultured with gastric cancer cells and suppressed the expression of IL-6 according to one embodiment of the present invention , And the increase in the expression of cell death markers suppressed by CAF.
FIG. 9 is a graph showing changes in the STAT3 signaling pathway and IL-6 receptor antagonist effect on CM-induced anticancer drug response by administration of IL-6 and IL-6 receptor antibodies in gastric cancer cells according to one embodiment of the present invention The results are shown in Fig.
FIG. 10 is a graph showing the effect of 5-FU alone or actemra, an IL-6 receptor inhibitor, on the mouse model of gastric cancer or gastric cancer cells and CAFs injected according to an embodiment of the present invention Results.
11 shows the results of confirming the effect of 5-FU which is an anticancer agent according to one embodiment of the present invention.
12 shows the results of transcript analysis for NAF and CAF according to one embodiment of the present application.
FIG. 13 shows results of gene expression in NAF and CAF according to one embodiment of the present invention by RT-PCR and ELISA.
FIG. 14 shows the results of cell activity according to the presence or absence of CAF-CM, monoclonal antibody, and IgG in cancer cell lines according to one embodiment of the present invention.
FIG. 15 shows the results of analysis of the difference in gene expression in the biopsy tissue before gastrectomy chemotherapy by the nCounter method in gastric cancer patients who underwent preoperative chemotherapy according to one embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention.

It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

It will be appreciated that throughout the specification it will be understood that when a member is located on another member "top", "top", "under", "bottom" But also the case where there is another member between the two members as well as the case where they are in contact with each other.

Throughout this specification, when an element is referred to as " including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

As used herein, the terms " about, " " substantially, " and the like are used herein to refer to or approximate the numerical value of manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to prevent unauthorized exploitation by unauthorized intruders of the mentioned disclosure. Also, throughout the present specification, the phrase " step " or " step " does not mean " step for.

Throughout this specification, the term " combination thereof " included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

 Throughout this specification, the description of "A and / or B" means "A, B, or A and B".

Hereinafter, the pharmaceutical composition for enhancing the anticancer therapeutic effect of the anticancer agent for gastric cancer and the screening method for the anticancer adjuvant for improving or treating gastric cancer will be described in detail with reference to the embodiments, examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

The first aspect of the present invention relates to a pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer, comprising an inhibitor of the action of interleukin-6 (IL-6) as an active ingredient.

The inventors of the present application have found that when a culture solution in which cancer-associated fibroblast (CAF), which is a main cell constituting a tumor matrix in gastric cancer, is cultured and co-cultured with gastric cancer cells, the anticancer effect is decreased Respectively. In addition, IL-6 is secreted in CAF, and the anticancer effect is reduced. Therefore, if the expression of IL-6 mRNA or protein is inhibited or the inhibition of IL-6-induced gastric cancer cell activation is targeted by the expression of IL-6, a great therapeutic effect can be obtained in the treatment of gastric cancer having anticancer drug resistance .

Therefore, according to one embodiment of the present invention, the gastric cancer may include, but is not limited to, cancer-associated fibroblasts (CAFs) in cancer cells.

The term " cancer-associated fibroblasts " (CAF) refers to fibroblasts present in tissues surrounding cancer or malignant tumors. The cancer-associated fibroblast (CAF) can be easily separated, for example, from specimens obtained by excising certain malignant tumor tissues. It is preferable that the malignant tumor tissue to be the isolation source of CAF used in the present invention is gastric cancer tissue.

The interleukin-6 is a glycoprotein having a molecular weight of 210,000, which is abbreviated as IL-6 and isolated by B cell stimulating factor 2 (BSF-2), which induces terminal differentiation of B cells into antibody producing cells. It is a cytokine produced by several cells such as T lymphocytes, B lymphocytes, macrophages, and fibroblasts, and is the same molecule as interferon β2 (IFN-β2). Immune responses, proliferation and differentiation of hematopoietic and neural cells, and acute response.

Human IL-6 is composed of 212 amino acid residues containing 28 signal peptides. The overproduction of IL-6 is known to be closely related to the development of various immunological disorders, inflammatory diseases, and lymphoid tumors.

According to one embodiment of the disclosure, the interleukin-6 (IL-6) inhibitor is an interleukin-6 self-inhibitor; SiRNA capable of inhibiting the expression of interleukin-6; An antibody that specifically binds to interleukin-6; Antisense oligonucleotides that specifically bind to interleukin-6, and combinations thereof, but are not limited thereto.

 The antibody is a polyclonal antibody or a monoclonal antibody.

The polyclonal antibody can be prepared by injecting an external host with a protein or fragment thereof expressed by the gene as an immunogen according to methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep or rabbits. The immunogen is injected intraperitoneally, intraperitoneally or subcutaneously, and is generally administered with an adjuvant to increase the antigenicity. Serum is collected periodically from an external host to collect the sera showing improved titer and specificity for the antigen or isolate and purify the antibody therefrom.

Such monoclonal antibodies can be produced by immortalized cell line generation techniques by fusion as known to those of skill in the art. For example, a protein expressed by the gene is immunized with a mouse or a peptide is synthesized and bound to bovine serum albumin and immunized with a mouse. An antigen-producing B lymphocyte isolated from a mouse is fused with a myeloma of a human or a mouse to generate an immortalized hybridoma, and an enzyme-linked immunosorbent assay (ELISA) linked immunoabsorbent assays (ELISA) to identify monoclonal antibodies. After positive clones were selected, the antibodies were isolated and purified, or injected into the abdominal cavity of rats, and the ascites was collected to obtain monoclonal antibodies have.

The monoclonal antibody can be quantitatively assayed by using a secondary antibody to which an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) is conjugated, or a substrate thereof, Or may be quantitatively analyzed using a monoclonal antibody against the protein directly coupled with an AP or HRP enzyme or the like.

The reaction of the protein with the antibody can be carried out using a protein identification test such as western blot, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA) and immunohistochemistry (IHC) However, the present invention is not limited thereto.

According to one embodiment of the present invention, the anticancer agent can be used without limitation as long as it is a known anticancer agent that can be used for gastric cancer treatment. For example, the anticancer agent may be selected from the group consisting of 5-FU (Fluorouracil), actemra, doxorubicin, paclitaxel, mitomycin, Docetaxel, cisplatin, irinotecan But are not limited to, those selected from the group consisting of epoxides, epoxides, capecitabine, oxaliplatin, derivatives thereof, and combinations thereof.

The anti-cancer therapy using the above-mentioned anticancer drug is a therapy that can show effects on the whole body. However, when the cancer cells do not respond to the anti-cancer drugs at the time of treatment, or when the tumor is initially effectively reduced, . In this case, since it shows so-called multi-drug resistance (MDR) which is resistant to not only the anticancer agent used for the treatment but also the other anticancer agent, once the cancer cell acquires resistance to the anticancer agent, , And they suffer from great difficulties in cancer treatment. Therefore, it is very important to develop a method that can reduce the resistance of cancer cells to anticancer drugs.

The compositions according to the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose, lactose, or gelatin.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The amount of the pharmaceutical composition to be used may vary depending on the age, sex, and body weight of the patient, and may be administered once to several times per day so that the effect can be sufficiently exhibited.

In addition, the dosage of such a pharmaceutical composition may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, nasal inhalation, intra-uterine dermal or intracerebroventricular injections.

A second aspect of the present invention is a method for treating cancer, comprising the steps of: treating a test compound in a gastric cancer cell line mixed with a gastric cancer tissue or a cancer-associated fibroblast containing cancer-associated fibroblast; Measuring the expression level of mRNA or protein of interleukin-6 in the treated gastric cancer tissue or gastric cancer cell line; And selecting a test compound that reduces the expression level of mRNA or protein of interleukin-6 in comparison with the untreated control group. The present invention also relates to a method for screening a cancer-preventing adjuvant for improving or treating gastric cancer.

According to one embodiment of the present invention, gastric cancer including CAFs has resistance to an anticancer agent, and CAFs secretes IL-6. In particular, since it has been confirmed that the expression of IL-6 increases the resistance of cancer cells to anticancer agents, it is possible to provide a method of screening a test compound capable of reducing expression of IL-6 by 4.

According to one embodiment of the present invention, the level of the interleukin-6 mRNA is determined by RT-PCR, real time quantitative RT-PCR, RNase But is not limited to, one of the methods selected from the group consisting of RNase protection method, Northern blotting and DNA chip technology assay.

According to one embodiment of the present invention, the expression of the interleukin-6 protein is detected by any one method selected from the group consisting of Western blotting, enzyme immunoassay (ELISA), immunohistochemistry, and flow cytometry (FACS) But is not limited thereto.

The second aspect of the present invention relates to a method for screening anticancer adjuvants based on the modulation of IL-6 expression in gastric cancer including CAFs, and a detailed description thereof is omitted for the parts overlapping with the first aspect of the present invention. Although the description of the first aspect is omitted from the second aspect of the present application, the same can be applied.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example  One] Cancer-associated fibroblasts  Confirmed reduction of the effect of anticancer drug by cultured culture

(1). The presence of cancer-associated fibroblast (CAF), which is directly isolated from stomach cancer tissues in patients with gastric cancer, is classified as normal gastric tissue-associated fibroblast (NAF) The gastric cancer cell lines MKN-1 and MKN-45 (purchased from Korean Cell Line Bank) were cultured using conditioned medium (CM), and 5-FU, an anti-cancer drug, The reactivity was confirmed.

FIG. 1 is a graph showing the effect of a conditioned media (culture medium) on gastric cancer-associated fibroblast (NAF) and cancer-associated fibroblast (CAF) in gastric cancer cells according to an embodiment of the present invention , CM) was added to determine the change in the anticancer activity (IC50).

As a result, as shown in FIG. 1, when CM containing cultured NAF was added, it was confirmed that cells were killed by increasing 5-FU concentration. However, when CM containing CAFs was added, I can confirm that it is small. In addition, in the case of IC50, the addition of CAFs was about three times or more higher than that in the case of adding NAF-cultured CM. Thus, CAFs decreased the effect of the anticancer drug.

FIG. 2 is a photograph showing the increase in the inhibitory effect of 5-FU of an anticancer agent by CAF on gastric cancer cell lines MKN-45 and MKN-1 according to one embodiment of the present invention.

Specifically, according to the results shown in FIG. 2, it can be confirmed that the cell number is decreased by the anticancer agent 5-FU in the gastric cancer cell lines (MKN-45 and MKN-1) without CAF, 5-FU was inhibited and there was no change in cell number.

[ Example  2] CAF  Confirmation of the effect of anticancer drugs by

NAFs and CAFs co-cultured were found to be affected by the anticancer effect of gastric cancer cell line MKN45. For this purpose, MKN45 was cultured in a medium containing 10% fetal bovine serum (FBS), and a transwell device with a pore size of 0.4 μm was placed on top of the culture dish and co-cultured with NAF or CAF And 5 uM of 5-FU was treated for 72 h.

Then, expression of cleaved caspase-3 or PARP (poly-ADP ribose polymerase) protein, which is a marker of apoptosis, was confirmed by western blotting to confirm the effect of NAF and CAF co-culture Respectively.

Cells of the culture dish were collected for the Western blot and then the cells were lysed. Proteins were extracted and quantified. Proteins were separated using an electrophoresis apparatus, and the separated proteins were transferred to a poly membrane. Then, expression of each protein was confirmed by attaching a secondary antibody for expression of an antibody and a fluorescent substance to the proteins.

It was confirmed that the substance secreted from CM was IL-6.

FIG. 3 shows the results of western blot analysis of changes in cell death markers (Cleaved caspase 3 & PARP) upon addition of an anticancer agent when co-culturing gastric cancer cells with NAF or CAF according to an embodiment of the present invention.

In addition, as shown in FIG. 3, it was confirmed that the cut-off caspase-3 and PARP, which are apoptosis markers after 5-FU treatment, were increased and then decreased again by co-culture with NAF and CAF.

[ Example  3] IL-6 signaling pathway confirmation

It was confirmed that JAK-STAT3, a signal pathway of IL-6, was activated after 5-FU-induced gastric cancer cell line stimulation using CAF-CM.

MKN45 cell line was cultured on a culture dish and 5-FU was administered and CAF-CM or IL-6 was administered for 72 hours. Protein was extracted from each cell and the activation of Jak-STAT3 was quantitatively confirmed by Western blotting.

FIG. 4 shows the results of confirming activation of signal transduction pathway of JAK-STAT3 (signal transducer and activator of transcription 3) in the signal transduction pathway of IL-6 when CAF of CMC was added to gastric cancer cells according to one embodiment of the present invention .

As a result, JAK (Janus kinase) and STAT3 (signal transducer and STAT3), which are one of the IL-6 signaling pathways, were identified by CAF-CM based on a known document (Mihara M et al. activator of transcription 3) was phosphorylated.

FIG. 5 (A) is a result of confirming that cytokines such as IL-6 and IL-8 are increased when CAF is co-cultured with the gastric cancer cell line MKN-45 according to an embodiment of the present invention. B) shows that JAK-STAT3 (signal transducer and activator of transcription 3) signaling pathway is activated in the signal transduction pathway of IL-6 when CAF is co-cultured with the gastric cancer cell line MKN-45 according to one embodiment of the present invention to be.

The MKN45 cell line was cultured on a culture dish and 5-FU was administered and CAF was administered for 6 hours and 24 hours. Protein was extracted from each cell and the activation of Jak-STAT3 was quantitatively confirmed by Western blotting.

5, cytokines such as IL-6 and IL-8 did not increase when the gastric cancer cell line MKN-45 was alone, whereas when the gastric cancer cell line MKN-45 and CAF were co-cultured, IL-6 , IL-8, and other cytokines. In addition, we confirmed that JAK-STAT3 phosphorylation signaling system is increased by CAF.

[ Example  4] CAF  Specific IL-6 overexpression confirmed

IL-6 was mainly expressed in fibroblasts, and CAF was overexpressed in comparison with NAF.

For this, gastric cancer cells (KATO-Ⅲ, MKN-28, and MKN-45) not containing CAF were purchased from the Korean Cell Line Bank, and CAF-containing gastric cancer cells were prepared by co- Respectively.

IL-6 expression in various gastric cancer cell lines and CAF and NAF was assessed at RNA and protein levels.

For this purpose, RNA was extracted from 13 pairs of NAF and CAF and qRT-PCR was used to quantitatively measure the expression of IL-6.

In addition, RNA was extracted from KATO-Ⅲ, MKN28, MKN45 and various NAF and CAF pairs, and the expression of IL-6, IL-6R and gp130 was confirmed by RT-PCR.

In addition, the expression of IL-6 at the protein level of NAF and CAF cultured in culture medium alone or in the culture medium co-cultured with MKN45 and various cultured gastric cancer cell lines was confirmed by ELISA method.

FIG. 6 shows the results of RT-qPCR analysis showing that the expression of IL6 was increased in CAF as compared to NAF according to one embodiment of the present invention. Although the expression of IL-6 was little in gastric cancer cells, it was mainly expressed in fibroblasts, And the expression level in CAF was also high in protein level by RT-PCR and ELISA.

As a result, as shown in FIG. 6, IL-6 gene was significantly expressed in CAF as compared with NAF, and IL-6 was not expressed in gastric cancer cell line. there was. In addition, it was confirmed that secretion of IL-6 was higher in CAF than in NAF and secreted out of the cell. In gastric cancer cell lines, it was confirmed that there was almost no such secretion.

[ Example  5] From CAF  The inhibition of IL-6 expression and the inhibition of IL-6-induced cancer cell stimulation,

It was confirmed that the resistance to the anticancer agent was decreased when co-cultured with CAF inhibited the expression of IL-6.

For this, tet-on IL-6 shRNA vector was prepared to inhibit the expression of IL-6 upon tetracycline administration, and the inhibition of IL-6 was confirmed by RT-PCR after injection into CAF.

The cell line thus prepared was co-cultured with MKN-45 in the same manner as in Example 2, and tetracycline was administered to inhibit the expression of IL-6, and the change in cell death markers was confirmed by Western blotting.

In addition, IL-6 receptor-mediated inhibition of the gastric cancer cell line by IL-6 receptor monoclonal antibody and the effect of increasing the susceptibility to chemotherapy were also confirmed.

For this, western blotting was carried out in the same manner as in Example 3 above. In particular, the monoclonal antibody to IL-6 (purchased from Sino Biological, Anti-IL6R AB) was administered to the medium at a concentration of 800ng / ml to confirm the anticancer activity upon inhibition of IL-6 signaling. (800 ng / ml) was also confirmed.

FIG. 7 shows that when IL-6-inhibited CAF, in which expression of IL-6 is inhibited by tetracycline, was co-cultured with gastric cancer cells and suppressed the expression of IL-6 according to one embodiment of the present invention , And the increase in the expression of cell death markers suppressed by CAF.

FIG. 8 shows that IL-6-inhibited CAF, in which expression of IL-6 is inhibited by a monoclonal antibody, was co-cultured with gastric cancer cells and suppressed the expression of IL-6 according to one embodiment of the present invention , And the increase in the expression of cell death markers suppressed by CAF.

7 and 8, it was confirmed that STAT3 was activated by IL-6 and the response to the anticancer agent was reduced. On the other hand, when IL-6 was inhibited by using the antibody, STAT3 activation was decreased and the reactivity to the anticancer agent was increased according to the concentration of the antibody.

[ Example  6] Expression of IL-6 in animal models

After xenografts were transplanted into BALB / c nude mice, tumorigenesis and anticancer effects were examined when mixed with CAF.

Tumors were formed by injecting MKN45 cell line (1 × 10 ⁷ ) alone or with CAFs ( ⁵ × 10 ⁵ ) subcutaneously in 12 BALB / c nude mice at 6 to 8 weeks of age and then administered 5-FU at a dose of 25 mg / kg three times per week Intraperitoneal administration was performed and the size change was measured for 3 weeks.

FIG. 9 is a graph showing changes in the STAT3 signaling pathway and IL-6 receptor antagonist effect on CM-induced anticancer drug response by administration of IL-6 and IL-6 receptor antibodies in gastric cancer cells according to one embodiment of the present invention The results are shown in Fig.

As a result, it was found that the effect of 5-FU was decreased when CAFs were injected as compared with the single injection of gastric cancer cell line, as shown in FIG.

[ Example  7] Effect of IL-6 Inhibition in Animal Model

5-FU was administered alone in the same manner as in Example 6 after subcutaneously injecting MKN1 cell line alone (1x10 ⁶ ) or CAFs (5x10 ⁵ ) into subcutaneously 6 to 8 weeks of BALB / c nude mice, The effect of IL-6 inhibition was confirmed by administering IL-6R antibody (Anti-IL-6 Ab) to part of mice injected with CAFs.

FIG. 10 is a graph showing the effect of 5-FU alone or actemra, an IL-6 receptor inhibitor, on the mouse model of gastric cancer or gastric cancer cells and CAFs injected according to an embodiment of the present invention Results.

As a result, as shown in FIG. 10, it was confirmed that the effect of the anticancer agent decreased by CAFs was increased again when the IL6 inhibitor was inhibited by the antibody against the IL-6 receptor, and the tumor size and weight were decreased. Particularly, when the anticancer drug is administered, the body weight can be reduced. In the case of inhibiting IL-6, the body weight is heavier than the case of administration alone with an anticancer drug or CAF. In addition, immunochemical staining of tissues resected after 3 weeks of treatment showed that the number of cells expressing alpha-smooth muscle actin, a fibroblast marker, was increased in tumors formed by injection with CAF, The expression of caspase-3, a markers of apoptosis, was significantly decreased upon injection with CAF, and then increased again after administration of Actemra.

11 shows the results of confirming the effect of 5-FU which is an anticancer agent according to one embodiment of the present invention.

Specifically, according to the results shown in FIG. 11, the inhibitory effect of 5-FU, an anticancer agent, was increased by CAF, compared to NAF, in gastric cancer cell line MKN-1. In particular, accumulation of fibroblasts in tumors increased in tumors mixed with CAF and immunochemical staining revealed that the expression of apoptotic markers was decreased upon treatment with 5-FU anticancer agents.

[ Example  8] Expression of IL-6

The expression of IL-6 was increased in CAF, but not in cancer cells.

12 shows the results of transcript analysis for NAF and CAF according to one embodiment of the present application.

12, genes associated with JAK-STAT3 signaling among genes that increase in CAF are LIF, IL-6, IL-12, IL-24, IL-15-RA, IL- And so on.

FIG. 13 shows results of gene expression in NAF and CAF according to one embodiment of the present invention by RT-PCR and ELISA.

The results shown in Fig. 13 confirm that IL-6 is the only gene that increases in CAF as compared to NAF. In particular, it was confirmed that expression of IL-6 is increased in CAF, which is not a cancer cell.

[ Example  9] The inhibitory effect of IL-6 on gastric cancer cell line

It was confirmed that the resistance to the anticancer agent was decreased when co-cultured with CAF inhibited the expression of IL-6.

For this purpose, monoclonal antibody against IL-6 (purchased from Sino Biological, Anti-IL6R AB) was administered to the medium at a concentration of 400 ng / ml or 800 ng / ml, As a control experiment, the effect of antibodies against IgG (400 ng / mg or 800 ng / ml) was also confirmed.

FIG. 14 shows the results of cell activity according to the presence or absence of CAF-CM, monoclonal antibody, and IgG in cancer cell lines according to one embodiment of the present invention.

14, when the IL-6 was inhibited with the monoclonal antibody, the cytotoxicity of the cancer cell lines MKN-1 and MKN-45 was found to be decreased upon administration of the anticancer agents 5-FU and cisplatin Respectively. Specifically, when the concentration of monoclonal antibody was 800 ng / ml in MKN-45, the cell activity of cancer cell line MKN-45 decreased, whereas when the concentration of antibody against IgG increased, the cell activity of cancer cell line MKN-45 Did not change. That is, the monoclonal antibody effectively inhibited IL-6 and inhibited the IL-6, thereby increasing the effect of the anticancer agent and decreasing the cell activity of the cancer cell.

[ Example  10] From CAF  Generated Extracellular  Of substrate-related genes Confirmation of response to anticancer drugs

The anticancer response was confirmed by the presence or absence of an increase in the extracellular matrix-related genes mainly produced in CAF.

FIG. 15 shows the results of analysis of the difference in gene expression in the biopsy tissue before gastrectomy chemotherapy by the nCounter method in gastric cancer patients who underwent preoperative chemotherapy according to one embodiment of the present invention.

The results shown in FIG. 15 show that the increase in the extracellular matrix-related genes mainly generated in CAF, including IL-6, decreases the response to the anticancer agent.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (7)

A pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer, comprising an inhibitor of interleukin-6 (IL-6) as an active ingredient. The method according to claim 1,
Wherein the gastric cancer comprises cancer-associated fibroblasts (CAFs) in cancer cells. The method according to claim 1,
The interleukin-6 (IL-6) inhibitor may be an interleukin-6 inhibitor; SiRNA capable of inhibiting the expression of interleukin-6; An antibody that specifically binds to interleukin-6; Antisense oligonucleotides specifically binding to interleukin-6, and combinations thereof. The pharmaceutical composition for enhancing the anticancer therapeutic effect of an anticancer agent for gastric cancer. The method according to claim 1,
Wherein the anticancer agent is selected from the group consisting of 5-FU, attemera, doxorubicin, paclitaxel, mitomycin, docetaxel, cisplatin, irinotecan, epothar, capecitabine, oxaliplatin, derivatives thereof, A pharmaceutical composition for enhancing the effect of an anticancer agent for cancer therapy. Treating the test compound with a gastric cancer cell line mixed with a gastric cancer tissue or a cancer-related fibroblast containing cancer-associated fibroblast;
Measuring the expression level of mRNA or protein of interleukin-6 in the treated gastric cancer tissue or gastric cancer cell line; And
Selecting a test compound that reduces the expression level of mRNA or protein of the interleukin-6 in comparison with the untreated control group. 6. The method of claim 5,
The level of the interleukin-6 mRNA may be determined by RT-PCR, real time quantitative RT-PCR, RNase protection method, Wherein the measurement is performed by any one method selected from the group consisting of Northern blotting and DNA chip technology assay. 6. The method of claim 5,
Wherein the expression of the interleukin-6 protein is measured by any one method selected from the group consisting of Western blotting, enzyme immunoassay (ELISA), immunohistochemistry, and flow cytometry (FACS) Screening method for therapeutic anti-cancer adjuvant.

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