WO2015119362A1 - Anticancer adjuvant composition containing rip3 expression promoter as active ingredient, method for screening for anticancer adjuvant enhancing sensitivity of anticancer drug by promoting rip3 expression, and method for monitoring sensitivity of anticancer drug - Google Patents

Abstract

The present invention relates to an anticancer adjuvant pharmaceutical composition containing a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator as an active ingredient. In addition, the present invention provides a method for enhancing cancer cell death, by administering, to cancer cells, a RIP3 protein expression promoter or activator and an anticancer drug in combination. Additionally, the present invention relates to: a method for screening for an anticancer adjuvant which enhances the sensitivity of an anticancer drug by promoting RIP3 expression; and a method for monitoring the sensitivity of an anticancer drug through whether RIP3 is expressed. Therefore, it is expected that pretreating a demethylating agent so as to induce RIP3 expression and then using a conventional chemotherapeutic agent in a patient lacking RIP3 expression could be an effective treatment strategy. In addition, it is expected that monitoring the sensitivity of an anticancer drug and screening for an anticancer adjuvant enhancing the sensitivity of an anticancer drug in cancer treatment could be an effective strategy.

Description

Anticancer adjuvant composition comprising RIP3 expression promoter as an active ingredient, anticancer adjuvant screening method for promoting RIP3 expression and anticancer agent susceptibility monitoring method

The present invention relates to an anticancer adjuvant composition comprising a RIP3 expression promoter as an active ingredient and a method of combined administration with an anticancer agent. The present invention also relates to an anticancer adjuvant screening method for promoting RIP3 expression and enhancing anticancer drug sensitivity and an anticancer drug sensitivity monitoring method through RIP3 expression. The present invention also provides a method for providing anti-cancer drug sensitivity diagnostic biomarker composition comprising the RIP3 gene or a protein expressed from the gene and information necessary for anticancer drug susceptibility prognosis.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an important protein for apoptosis and plays a role in apoptosis by death receptors or apoptosis by other cellular stresses. . These apoptosis signals are via complexation with RIP1, which is dependent on phosphorylation or deacetylation, and by binding to a mixed lineage kinase domain-like protein (MLKL), a protein present in the mitochondria. It is known to be involved. The regulated mechanism of this signal transduction system is driven by apoptosis-regulating proteins, which regulate cell death and immune responses in lymphocytes, keratinocytes, and intestinal epithelial cells. Regulated necrosis plays a role in many etiological processes, such as degenerative, immune, and infectious and ischemic damage.

Disclosure of Invention An object of the present invention is to provide an anticancer adjuvant pharmaceutical composition comprising a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or an activator and an anticancer agent and a method for enhancing cancer cell death, which is used in combination with an anticancer agent. have.

Disclosure of Invention An object of the present invention is to provide an anticancer adjuvant screening method for promoting RIP3 (Receptor-interacting protein kinase-3) expression to enhance anticancer drug sensitivity and an anticancer drug sensitivity monitoring method through RIP3 expression.

Another object of the present invention is to provide an anticancer drug sensitivity diagnostic biomarker composition comprising a RIP3 gene or a protein expressed from the gene.

Still another object of the present invention is to provide a kit for predicting and diagnosing a cancer drug susceptibility diagnostic kit comprising a primer for amplifying the RIP3 gene or an antibody or aptamer specifically binding to a protein expressed from the gene. To provide.

Still another object of the present invention is to provide information necessary for anticancer susceptibility prognosis and anticancer agent reactivity, including measuring the expression level of RIP3 from a cancer patient sample.

In order to achieve the above object, the present invention provides a pharmaceutical composition for adjuvant anticancer comprising a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator.

In addition, the present invention is a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator; And it provides a method for enhancing cancer cell death, characterized in that the anticancer agent in combination with cancer cells.

In addition, the present invention comprises the steps of contacting the test substance to cancer cells; Measuring the level of expression or activity of a receptor-interacting protein kinase-3 (RIP3) protein in cancer cells in contact with the test substance; And it provides a method for screening an anticancer adjuvant comprising the step of selecting a test substance with increased expression or activity of the RIP3 protein compared to the control sample.

In addition, the present invention comprises the steps of measuring the expression or activity of the RIP3 protein in cancer cells; Measuring the expression or activity of RIP3 protein in normal tissue cells; And if the expression or activity of the RIP3 protein measured in the normal tissue cells compared to the expression or activity of the RIP3 protein is low The anti-cancer drug susceptibility monitoring method comprising the step of determining the resistance to cancer.

In addition, the present invention comprises the steps of treating the cancer cells RIP3 protein expression promoter or activator; Measuring the expression or activity of RIP3 protein in the treated cancer cells; And when the expression or activity of the RIP3 protein after the treatment is increased by 50 to 100% compared to the control sample before the treatment provides an anticancer drug sensitivity enhancement method comprising the step of determining that the anticancer drug sensitivity.

In addition, the present invention provides a biomarker composition for diagnosing anticancer drug sensitivity comprising the RIP3 gene or a protein expressed from the gene. The biomarker may predict cancer in tissues.

In addition, the present invention provides a kit for diagnosing anticancer agent sensitivity comprising a primer for amplifying the RIP3 gene or an antibody or aptamer specifically binding to a protein expressed from the gene. In addition, the kit may provide information necessary for predictive diagnosis of cancer in the tissue.

In addition, the present invention comprises the steps of measuring the expression level of RIP3 in a cancer patient sample; Measuring the expression level of RIP3 in the normal control sample; And determining that the anti-cancer drug resistance is low when the expression level of the RIP3 protein measured in the cancer patient sample is lower than the expression level of the RIP3 protein measured in the normal control sample. To provide.

The present invention relates to an anticancer adjuvant composition comprising a RIP3 expression promoter as an active ingredient and a method of coadministration with an anticancer agent. In the case of a triple negative (ER, PR, Her2 negative) patient presenting a problem in cancer treatment, 90 RIP3 expression was found to be low in%, indicating a significant decrease in RIP3 expression in cancer tissues compared to normal tissues of the same patient, suggesting that RIP3 is selectively lacking during tumor development and growth. In patients with deficiency, pretreatment with demethylating agents to induce RIP3 expression, followed by conventional chemotherapy would be an effective treatment strategy. In addition, the present invention relates to an anticancer adjuvant screening method that promotes RIP3 expression and enhances anticancer drug susceptibility and an anticancer drug susceptibility monitoring method through RIP3 expression. In 90% of patients, low RIP3 expression was found, and it was confirmed that RIP3 expression regulation affects the anticancer drug resistance of cancer cells.In particular, when RIP3 expression is inhibited, cancer cells are resistant to anticancer drugs, and thus the activity of the anticancer agent is decreased. In contrast, when RIP3 is expressed, it was confirmed that cancer cell death was increased depending on anticancer agent concentration. This is expected to be an effective strategy for screening anticancer adjuvants to monitor the sensitivity of anticancer drugs and to enhance the anticancer drug sensitivity in chemotherapy.

1 shows the results of RIP3 expression analysis in normal breast and breast cancer cell lines.

2 is a result of RIP3 expression by 5-aza-2'-deoxycytidine (5-AD) as a demethylating agent.

Figure 3 is the result of RIP3 expression by the demethylating agent 5-azacytidine (5-AZA).

4 is a result of cancer cell line death sensitization by the combination of the demethylating agent (5-AD) and the anticancer agent.

5 is a result of the cancer cell line death sensitization by the combination of the demethylating agent (5-AD and 5-AZA) and the anticancer agent.

Fig. 6 shows the results of inhibition of cancer cell line killing sensitization effect of demethylating agent (5-AD) by inhibition of RIP3 expression.

7 is an immunostaining picture of RIP3 of representative normal breast tissue and breast cancer tissue.

8 and 9 are representative H-score schematics for immunostaining of RIP3 in normal breast and breast cancer tissues.

10 is a result of HT-29 survival rate according to the treatment according to the concentration of the anticancer agent in the cells inhibited RIP3 expression.

11 shows the survival rate of T47D according to the concentration-specific treatment of the anticancer agent in cells in which RIP3 expression is suppressed.

12 is a graph of metastatic relapse-free survival for 10 years of 1,166 breast cancer patients.

Thus, the inventors confirmed that RIP3-dependent apoptosis may have an effect on cytotoxicity in chemotherapeutic agents. In many cancer cell lines, RIP3 expression is inhibited, and this inhibition of RIP3 expression is not only resistant to apoptosis by death receptors, but also to a variety of standard anticancer therapies such as chemotherapeutic agents, particularly DNA damaging drugs or taxanes. It could be confirmed that it gives resistance. The expression of RIP3 by 5-aza-2'-deoxycytidine (5-AD), which is a demethylating agent used in the present invention, was restored and the sensitivity to the chemotherapeutic agent was increased. These results confirmed that in patients with lack of RIP3 expression, pretreatment with demethylating agent induces RIP3 expression, followed by the use of conventional chemotherapeutic agents. The invention has been completed.

In addition, the present inventors were able to confirm that the regulation of RIP3 expression affects the resistance to anticancer drugs of cancer cell lines.In particular, when RIP3 expression is inhibited, cancer cells become resistant to anticancer drugs and the activity of the anticancer drugs is inhibited, whereas RIP3 is expressed. When it was confirmed that the cancer cell death is increased depending on the anticancer agent concentration was completed the present invention.

The present invention provides a pharmaceutical composition for adjuvant anticancer comprising a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or an activator.

Specifically, the RIP3 protein expression promoter or activator may be a compound, peptide, peptide mimetics, aptamers, antibodies and natural products that specifically bind to the gene expression control region of RIP3. Specifically, the composition can induce demethylation of RIP3 protein.

Preferably, the cancer may be, but is not limited to, breast cancer, cervical cancer, liver cancer or colon cancer.

Preferably, the RIP3 protein may be a protein from all eukaryotic organisms having RIP3, including mammals such as humans, cattle, goats, sheep, pigs, mice, rabbits, for example human RIP3 (NCBI accession no. NP_006862.2).

The term "peptide mimetics" in the present invention is a peptide or nonpeptide that inhibits the binding domain of the RIP3 protein leading to RIP3 activity.

In the present invention, the term "Aptamer" is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure by itself and having a characteristic that can bind with high affinity and specificity to a target molecule. Aptamers are compared to single antibodies because of their inherent high affinity (usually pM levels) and their specificity to bind to target molecules, and thus have high potential as alternative antibodies, particularly as "chemical antibodies."

"Antibodies" of the present invention can be used both those prepared through RIP3 injection or commercially available. In addition, the antibodies include polyclonal antibodies, monoclonal antibodies, fragments capable of binding epitopes, and the like. Polyclonal antibodies can be produced by conventional methods of injecting the RIP3 into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like. It can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology.

The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisenses, siRNA oligonucleotides, and natural extracts as active ingredients. The pharmaceutical compositions or complex preparations of the present invention may be prepared using pharmaceutically acceptable and physiologically acceptable auxiliaries in addition to the active ingredients, which may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants. Solubilizers such as lubricants and flavoring agents can be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Pharmaceutical formulation forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be. The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to prevent or treat a disease. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. For example, in adults, when administered once or several times a day, the inhibitor of the present invention is administered once or several times a day, when the compound is 0.1ng / kg to 10g / kg, a polypeptide, In the case of proteins or antibodies, 0.1ng / kg ~ 10g / kg, antisense nucleotides, siRNA, shRNAi, miRNA can be administered at a dose of 0.01ng / kg ~ 10g / kg.

In addition, the present invention is a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator; And it provides a method for enhancing cancer cell death, characterized in that the anticancer agent in combination with cancer cells.

Specifically, treating cancer cells with a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator; And it may comprise the step of administering an anticancer agent to the treated cancer cells.

Preferably, the cancer cells may be breast cancer cells, cervical cancer cells, liver cancer cells or colorectal cancer cells, and the anticancer agent may be doxorubicin or etoposide, but is not limited thereto.

The present invention comprises the steps of contacting a test substance to cancer cells; Measuring the level of expression or activity of a receptor-interacting protein kinase-3 (RIP3) protein in cancer cells in contact with the test substance; And it provides a method for screening an anticancer adjuvant comprising the step of selecting a test substance with increased expression or activity of the RIP3 protein compared to the control sample.

Preferably, the expression or activity level of the RIP3 protein is reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting and flow cytometry. It may be measured by any one selected from the group consisting of assays (FACS), but is not limited thereto.

In detail, the anticancer adjuvant may enhance the sensitivity of the anticancer agent. More specifically, the anticancer agent is preferably doxorubicin, etoposide, or taxol, but is not limited thereto.

As used to refer to the screening method of the present invention, the term "test material" refers to an unknown candidate used in screening to examine whether it affects the expression level of a gene or affects the expression or activity of a protein. do. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

In addition, the present invention comprises the steps of measuring the expression or activity of the RIP3 protein in cancer cells; Measuring the expression or activity of RIP3 protein in normal tissue cells; And if the expression or activity of the RIP3 protein measured in the normal tissue cells compared to the expression or activity of the RIP3 protein is low The anti-cancer drug susceptibility monitoring method comprising the step of determining the resistance to cancer.

Preferably, the cancer cells may be breast cancer cells, cervical cancer cells, liver cancer cells or colorectal cancer cells, and the anticancer agent may be, but is not limited to, doxorubicin, etoposide, or taxol. .

In addition, the present invention comprises the steps of treating the cancer cells RIP3 protein expression promoter or activator; Measuring the expression or activity of RIP3 protein in the treated cancer cells; And when the expression or activity of the RIP3 protein after the treatment is increased by 50 to 100% compared to the control sample before the treatment provides an anticancer drug sensitivity enhancement method comprising the step of determining that the anticancer drug sensitivity.

In addition, the present invention provides a biomarker composition for diagnosing anticancer drug sensitivity comprising the RIP3 gene or a protein expressed from the gene.

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or as long as a person has a particular disease or condition. Determining the prognosis of the object, or therametrics (eg, monitoring the condition of the object to provide information about treatment efficacy).

In addition, the present invention provides a kit for diagnosing anticancer agent sensitivity comprising a primer for amplifying the RIP3 gene or an antibody or aptamer specifically binding to a protein expressed from the gene.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3-terminal hydroxyl group, which can form complementary templates and base pairs and act as a starting point for template strand copying. Refers to a nucleic acid sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer lengths may be appropriately selected according to techniques known in the art.

In addition, the kit of the present invention is an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that is developed by reaction with a substrate, a color substrate solution to be reacted with the label, a washing solution, and an enzyme. It may include a reaction stop solution and the like, and may be prepared in a number of separate packaging or compartments containing the reagent components used.

The label of the secondary antibody conjugate is preferably a conventional coloring agent that performs a color reaction, horseradish peroxidase (HRP), basic dephosphatase (alkaline phosphatase), colloidal gold (coloid gold), poly L-lysine-fluorescein (FITC) isothiocyanate, fluorescent materials such as rhodamine-B-isothiocyanate (RITC), dyes, and the like.

In addition, the present invention comprises the steps of measuring the expression level of RIP3 in a cancer patient sample; Measuring the expression level of RIP3 in the normal control sample; And determining that the anti-cancer drug resistance is low when the expression level of the RIP3 protein measured in the cancer patient sample is lower than the expression level of the RIP3 protein measured in the normal control sample. To provide.

Specifically, the expression level of the RIP3 may be measured through an antigen-antibody reaction, and more specifically, the antigen-antibody response may be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format includes enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, and flow cytometry (flow). cytometry), fluorescence activated cell sorting (FACS), enzyme substrate coloration, and antigen-antibody aggregation.

As used herein, the term “patient sample” includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ from the normal control in the expression level of RIP3, a cancer marker susceptibility diagnostic biomarker. However, it is not limited thereto.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Experimental Example

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

1. Reagent

RIP3 antibodies were purchased from Abcam. Actin antibody, Doxorubicin, etoposide, 5-AD, 5-AZA were obtained from Sigma-Aldrich.

2. Cell Culture

Various cancer cell lines were cultured in media presented by ATCC. DLD1, HeLa, MCF7 were incubated in DMEM with 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin and 100 ug / mL streptomycin. HCC1937, BT-549, MDA-MB231, MDA-MB468, SK-BR3, ZR75-1, T47D contains 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin And RPMI with 100 / mL streptomycin added.

3. Normal human cells

Mammary epithelial cells (HME) were produced by Clonetics Corp. (San Diego, CA). HMLE is a normal mammary epithelial cell, immortalized with hTERT and also infected by retrovirus with SV40 large and small T antigens.

4. Human Breast Cancer Tissue Manufacturing

Human breast cancer and comparative normal samples were obtained from Yonsei University College of Medicine (Seoul, South Korea). In all cases, informed consent was obtained from all participants, and this study was approved by the Yonsei Institutional Review Board (IRB).

5. Lentiviral shRNA Experiment

MISSION short-hairpin RNA (shRNA) plasmids targeting the coding site or 3 'UTR of hRIP3 mRNA (NM_006871) and non-target control sequences (NM-027088) were obtained from Sigma-Aldrich. Lentivirus plasmids were transfected into 293T cells (System Biosciences, LV900A-1) using Lipofectamine 2000 (Invitrogen, 11668019). Pseudoviral particles were collected two days after lentiviral plasmid transfection and infected with various cancer cells in the presence of polybrene (10 μg / mL). Two days after infection, infected cells were selected with puromycin and RIP3 knockdown was confirmed by immunoblotting. Cells not previously expressing endogenous RIP3 were treated with 5-AD for 4 days later, and then analyzed by immunoblotting.

6. Western Blot Analysis (Immunoblotting)

Cells were lysed in M2 buffer. Equal amounts of cell extracts were analyzed via SDS-PAGE and Western blot and visualized with increased chemiluminescence (ECL, Amersham).

7. Cytotoxicity Assay

Apoptosis was measured using a tetrazolium dye colorimetric test [MTT Assay] and measured at 570 nm.

8. Immunohistochemistry Analysis

Immunohistochemical analysis was performed using UltraVision LP Detection System TL-060-HD (Thermo Scientific, Bioanalytica) according to the manufacturer's instructions. Thin paraffin sections (4.5 μm) were removed with xylene and rehydrated in various concentrations of aqueous ethanol. Antigen recovery was performed by heating the slides for 15 minutes with a microwave in 10 mM citrate buffer (pH 6.0). The endogenous peroxidase activity was blocked by reacting with 3% hydrogen peroxide dissolved in TBS for 10 minutes and reacted overnight at 4 ° C. with an anti-RIP3 antibody diluted 1: 300. Chromogen was reacted for 5 minutes with 3, 3'-diaminobenzidine (TL-015-HD, Thermo Scientific, Bioanalytica, Greece) solution and counterstained with Meyer's hematoxylin. . Immunohistochemical staining was determined based on percentage of stained cells and immunostaining intensity. The present invention used H-score, which was obtained by multiplying the percentage of stained cells and the staining intensity [0 (negative), 1 (weak), 2 (medium) or 3 (strong)]. H-scores range from 0 to 300. Staining was performed at the same time for each sample in tumor and normal tissue. Staining results were interpreted by a professional pathologist who did not know the clinical data.

9. Statistical Analysis

Data is shown as mean ± SD. Statistical analysis was performed using ANOVA and unpaired Student's t-test. For P-values of 0.01 or lower, it was judged to have statistical significance. Statistical calculations were performed using SPSS software of Windows Version 12.0 (SPSS, Chicago, IL).

Example 1 RIP3 Expression Analysis in Breast Cancer Cell Lines

After lysis of the cancer cell lines to separate proteins, Western blotting was performed using SDS-PAGE. As a result of analyzing the RIP3 expression pattern of the breast cancer cell line, it was confirmed that RIP3 is not expressed in 60% or more cell lines. It was confirmed that RIP3 is inhibited by a specific mechanism in cancer cell lines (FIG. 1).

Example 2 RIP Expression Analysis by Demethylating Agent

The cancer cell lines were cultured at 10-20% density, treated with 5-AD twice for 4 days, and then RIP3 expression patterns were analyzed using Western blotting. When the demethylating agent (5-AD, 2uM) was treated to three cancer cell lines (HeLa, MDA-MB231, BT549) that do not express RIP3, expression of RIP3 was induced. This means that the expression of RIP3 is suppressed (FIG. 2).

 In addition, after incubating cancer cell lines with a density of 10 to 20%, treated with 5-AZA twice for 4 days, RIP3 expression patterns were analyzed using Western blotting technique. When the concentration of 5-AZA similar to 5AD was treated in DLD-1 colorectal cancer cell line that does not express RIP3, it was confirmed that RIP3 expression was induced, and these results showed that RIP3 inhibited expression in cancer cell line by methylation. It is disproving that it is (FIG. 3).

<Example 3> Sensitization of Cancer Cell Lines by Combination of Demethylating Agent and Anticancer Agent

The cancer cell lines were initially cultured at a density of 10-20%, and then treated with 5-AD or 5-AZA twice for 4 days to induce the expression of RIP3. After culturing the same number of drug-treated HeLa cancer cell lines, the same concentration of anticancer agent was treated to analyze the sensitization effect by the demethylating agent.

After inducing the expression of RIP3 by 5-AD, and confirming the effect that the death of cancer cell line is sensitized when combined with the anticancer agent, it was confirmed that RIP3 is associated with the death of cancer cell line by the anticancer agent (Fig. 4).

In addition, in the case of 5-AZA, which induces the expression of RIP3 in addition to 5-AD, the sensitizing effect of cancer cell lines was confirmed when inducing RIP3 expression and in combination with anticancer agents (FIG. 5).

Example 4 Inhibition of Cancer Cell Line Death Sensitization Effect of Demethylating Agent by RIP3 Inhibition

 The Lenti-virus system was used to create stable cells that continuously inhibit RIP3 expression in cervical cancer cell lines (HeLa cell lines). In contrast to non-target shRIP3 cell lines, expression of RIP3 is inhibited by shRNA despite 5-AD treatment. Therefore, the function of RIP3 could be confirmed in the sensitizing effect of the combination of anticancer and demethylating agents. Non-target cell lines and shRIP3 cell lines were initially cultured at 10-20% density, and then treated with 5-AD twice for 4 days to determine the expression of RIP3. And the sensitizing effect by the combined treatment with the anti-cancer drug cultured the same number was analyzed using a cell death experiment (MTT assay) (Fig. 6).

Demethylating agents (5-AD, 5-AZA) are not drugs specific to specific proteins, and therefore may cause expression of various proteins in addition to RIP3. Therefore, in order to determine whether apoptosis sensitization effect by the combination of anticancer agent and demethylating agent is effected by proteins other than RIP3, experiments were performed using shRIP3 cell line that specifically inhibits RIP3 expression. In non-target cell lines, 5-AD treatment produces a sensitizing effect in combination with anticancer agents by the expression of RIP3, but 5-AD in shRIP3 cell lines that specifically inhibit RIP3 expression. RIP3 is not expressed by the shRNA system even after treatment. Based on these results, in the apoptosis experiment, in the shRIP3 cell line that does not express RIP3, the sensitizing effect was suppressed, and the sensitizing effect in the combination of demethylating agent and anticancer agent was confirmed that RIP3 is an important molecule. It also suggests a new anticancer strategy that can increase the death of cancer cell lines by promoting the expression of RIP3.

Example 5 Immunostaining Analysis of Normal Breast and Breast Cancer Tissues

Paraffin blocks were prepared by separating tumor tissue from non-tumor tissue from 132 breast cancer patients. The prepared paraffin block was cut to a thickness of 4.5 μm and used for plating on slides. After deparaffinization with xylene and hydration with an aqueous solution of ethanol-water concentration, the non-specific enzyme reaction is removed with hydrogen peroxide, and then the hidden antigen is disassembled using a citric acid solvent. Next, the diluted normal serum is reacted for 20 minutes to block the nonspecific reaction, followed by 24 hours reaction with RIP3 (1: 300). After washing with water, the secondary antibody conjugated with biotinin is washed with water for 30 minutes. After reacting with avidin-biotin complex for 30 minutes, it is washed with water, and then dyed for 5 minutes with DAB colorant. After staining the nucleus with hematoxylin, it is washed with water and enclosed.

The intensity of color development with DAB was set to 0 (no color development), 1 (weak color development), 2 (medium color), 3 (strong color development), and the range of the dyed area and the intensity was expressed as H-score. Staining results were analyzed by a pathologist.

 The experimental results showed immunostaining pictures of RIP3 of representative normal breast tissue and breast cancer tissue, and the results were plotted with H-score (FIGS. 7, 8 and 9). It was confirmed that the expression of RIP3 is significantly decreased in cancer tissues compared to normal breast tissues.

<Example 6> Survival analysis of HT-29 according to the treatment according to the concentration of anticancer agent in the cells inhibited RIP3 expression

HT-29 cells (American Tissue Culture Collection) were cultured in a 37 ° C. incubator using DMEM medium supplemented with penicillin-streptomycin (10 IU / ml) and 10% FBS. The shRNAi double helix used in the present invention was commercially synthesized from Sigma-Aldrich. The shRNA used in the present invention was designed for the targeting of the coding region of the human RIPK3 mRNA sequence (NCBI Reference sequence NM_006871).

Firstly cultured HT-29 cells were dispensed 2 × 10 ⁵ in 35 mm dish. The next day the cells were transformed with shRNA particles with polybrene (10ug / ml) according to the protocol instructions. The control group used shRNAs that did not target specific proteins (NCBI Reference sequence NM_027088).

In order to measure the amount of RIP3 protein produced in the transformed cells, the HT-29 cells transformed using PBS were first washed, and then treated with lysis buffer to obtain a supernatant, followed by Western Blot of BIO-RARD. Proteins were separated using KIT. The isolated protein reacted with the titration antibody (anti-β-actin (1: 5,000, Sigma), anti-RIP3 (1: 1,000, Abcam) and secondary HRP-conjugated antibody (Jackson) HRP were immunobilon western Detected using Chemiluminescent HRP substrate KIT (Thermo).

As a result, as shown in FIG. 10, almost no RIP3 protein was detected in HT-29 cells transformed with RIP3 shRNA, and the transformed RIP3 shRNA effectively knocked down the RIP3 gene. I could confirm that.

To determine whether the RIP3 gene is related to anticancer susceptibility, we measured the cell survival rate of the cancer cells after treatment with doxorubicin and etoposide in HT-29 transformed with RIP3 shRNA. . Specifically, HT-29 cells transformed with RIP3 shRNA were incubated for 48 hours after 2.5uM of doxorubicin, 5uM and 50uM of etoposide and 100uM, and the test was performed with 0.1mg of MTT (3-([ Replace with fresh medium containing 4,5-dimethylthiazol-2-yl0] -2,5-diphenyltetrazolium bromide) and incubate for another 2 hours.Terazolium salt with live cells dissolved in purple formazan Precipitates made by reduction with crystals were subjected to color analysis (colormatric), after which the medium was removed and the resulting formazan crystals were dissolved in 500 ul of DMSO and absorbance was measured using an ELISA reader (at 570 nm). Cell viability is expressed as relative percentage according to the control defined as 100% viability.

As a result, as shown in FIG. 10 (right), the control group reduced cell viability depending on the concentration of Doxorubicin and Etoposide, whereas the experimental group knocked down RIP3 using shRNA cells compared to the control group. It was confirmed that the survival rate was increased.

<Example 7> Survival Analysis of T47D by Anti-cancer Agents in RIP3-Inhibited Cells

In order to confirm that RIP3 also affects breast cancer cells, T47D cells expressing RIP3 were used. First, RIP3 was transformed by the same method described in Example 6.

Through Western blotting method, almost no RIP3 protein was detected in T47D transformed with RIP3 shRNA, and it was confirmed that the RIP3 gene was effectively knocked down (FIG. 11). MTT analysis was performed to determine whether RIP3 increases anticancer drug sensitivity in breast cancer cell T47D. Doxorubicin, etoposide, and Taxol were reacted for 48 hours at the concentrations shown in FIG. According to the MTT analysis, the control group was killed according to the concentration of anticancer agent, whereas the cell line transformed with RIP3 significantly increased the anticancer drug resistance, especially in the experimental group treated with etoposide.

Through this, it could be confirmed that RIP3 expression regulation affects the resistance to cancer cell lines. In particular, when RIP3 expression is inhibited, cancer cells become resistant to anticancer drugs and the activity of anticancer drugs is inhibited, whereas RIP3 is expressed. Cancer cell death was increased depending on the concentration of anticancer drugs.

Example 8 Metastatic Relapse-free Survival Analysis of 10 Years of Breast Cancer Patients

FIG. 12 shows a metastatic relapse-free survival graph for 10 years of 1,166 breast cancer patients. As a result of analyzing the survival rate with the gene expression level of RIP3 above 50% and below 50%, there was statistically significant difference of p <0.0085, demonstrating that the expression level of RIP3 affects the survival rate of patients. Results were analyzed using Breast Cancer Gene-Expression Miner v3.0 software designed by Jezequel et al. (Breast Cancer Research and Treatment 2012; 131: 765-75).

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (21)

1. Anti-cancer auxiliary pharmaceutical composition comprising a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator.
2. According to claim 1, wherein the RIP3 protein expression promoter or activator is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to RIP3 Auxiliary pharmaceutical composition.
3. According to claim 1, wherein the composition is an anticancer adjuvant pharmaceutical composition, characterized in that to induce demethylation of RIP3 protein.
4. According to claim 1, wherein the cancer cancer cancer, cervical cancer, liver cancer or colon cancer, characterized in that the auxiliary pharmaceutical composition for cancer.
5. Receptor-interacting protein kinase-3 (RIP3) protein expression promoters or activators; And a cancer cell death enhancing method comprising administering an anticancer agent to the cancer cells in combination.
6. The method of claim 5, further comprising: treating cancer cells with a receptor-interacting protein kinase-3 (RIP3) protein expression promoter or activator; And

   Cancer cell death enhancement method comprising the step of administering an anticancer agent to the treated cancer cells.
7. The method of claim 5, wherein the cancer cells are breast cancer cells, cervical cancer cells, liver cancer cells, or colon cancer cells.
8. The method of claim 5, wherein the anticancer agent is doxorubicin or etoposide.
9. Contacting the test substance with cancer cells;

   Measuring the level of expression or activity of a receptor-interacting protein kinase-3 (RIP3) protein in cancer cells in contact with the test substance; And

   A method for screening an anticancer adjuvant comprising screening a test substance with increased expression or activity of the RIP3 protein compared to a control sample.
10. 10. The method of claim 9, wherein the expression or activity of the RIP3 protein is Reverse Transcription-Polymerase chain Reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western Blotting And flow cytometry (FACS). The method for screening an anticancer adjuvant characterized in that it is measured by any one selected from the group consisting of.
11. The method of claim 9, wherein the cancer cells are breast cancer cells, cervical cancer cells, liver cancer cells, or colon cancer cells.
12. 10. The method of claim 9, wherein the anticancer adjuvant enhances the sensitivity of the anticancer agent.
13. The method of claim 12, wherein the anticancer agent is doxorubicin, etoposide, or taxol.
14. Measuring the expression or activity of RIP3 protein in cancer cells;

    Measuring the expression or activity of RIP3 protein in normal tissue cells; And

    Anticancer drug sensitivity monitoring method comprising the step of determining that the anti-cancer drug resistance when the expression or activity of the RIP3 protein measured in the cancer cells is low compared to the expression or activity of the RIP3 protein measured in the normal tissue cells.
15. 15. The method of claim 14, wherein the cancer cells are breast cancer cells, cervical cancer cells, liver cancer cells, or colon cancer cells.
16. The method of claim 14, wherein the anticancer agent is doxorubicin, etoposide, or taxol.
17. Treating the cancer cells with a RIP3 protein expression promoter or activator;

    Measuring the expression or activity of RIP3 protein in the treated cancer cells; And

    Anti-cancer drug sensitivity enhancement method comprising the step of determining that the anti-cancer drug sensitivity is increased when the expression or activity of the RIP3 protein after the treatment is increased by 50 to 100% compared to the control sample before the treatment.
18. Anti-cancer drug sensitivity diagnostic biomarker composition comprising the RIP3 gene or a protein expressed from the gene.
19. Anti-cancer drug sensitivity diagnostic kit comprising a primer for amplifying the RIP3 gene or an antibody or aptamer specifically binding to a protein expressed from the gene.
20. Measuring the expression level of RIP3 in a cancer patient sample;

    Measuring the expression level of RIP3 in the normal control sample; And

    A method for providing anti-cancer drug susceptibility prognosis comprising determining that the anti-cancer drug resistance is low when the expression level of the RIP3 protein measured in the cancer patient sample is lower than the expression level of the RIP3 protein measured in the normal control sample.
21. The method of claim 20, wherein the expression level of RIP3 is measured by an antigen-antibody response.

Images