KR101816593B1

KR101816593B1 - Method for preparing colorectal cancer cell line with anticancer drug resistance induced by ribosome-inactivating stress and method for screening inhibitors against anticancer drug resistance of colorectal cancer patients using the same

Info

Publication number: KR101816593B1
Application number: KR1020160026309A
Authority: KR
Inventors: 문유석; 이승준; 오창규
Original assignee: 부산대학교 산학협력단
Priority date: 2016-03-04
Filing date: 2016-03-04
Publication date: 2018-01-10
Also published as: KR20170104076A

Abstract

The present invention relates to a method for producing a ribosome-inhibiting stress-induced anticancer drug resistant colorectal cancer cell line and a method for screening an anticancer drug resistance-inhibiting agent in a colon cancer patient using the same, wherein the cancer cell exposed to the ribosome- And the sensitivity to the anticancer drug was deteriorated. It is anticipated that the potential resistance effect of anticancer drugs in anticancer therapy for colorectal cancer will be considered in advance and it will help to establish economical and effective anticancer therapy strategy while overcoming the problems of existing anticancer therapy.

Description

TECHNICAL FIELD The present invention relates to a method for preparing a ribosome-inhibiting stress-inducing anticancer drug-resistant colorectal cancer cell line and a method for screening anticancer drug resistance inhibitor against colorectal cancer patients using the same resistance to colorectal cancer patients using the same < RTI ID = 0.0 >

The present invention relates to a method for producing a ribosome-inactivating stress (RIS) -induced anticancer-resistant colon cancer cell line and a method for screening an anticancer drug resistance-inhibiting agent in a colon cancer patient using the same.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in developed countries and has a high mortality rate. Colorectal cancer can progress to other organs with a very high chance of metastasis, which is one of the major causes of early treatment, such as surgical resection, chemotherapy, and radiation therapy for colorectal cancer patients. Although chemotherapy is the most common and effective treatment strategy for metastatic colorectal cancer, chemical resistance to anticancer agents is a stumbling block to successful treatment.

Fluorouracil (5-FU) and cisplatin are first-line chemotherapeutic agents in the treatment of metastatic colorectal cancer. 5-FU is a pyrimidine derivative that induces DNA damage and eventually induces apoptosis. This is a representative compound that induces tumor suppressor p53. p53 is a well-known tumor suppressor that is activated in response to oxidative stress, osmotic stress and DNA damage, and plays an important role in DNA repair, cell cycle arrest, and apoptosis. On the other hand, macrophage inhibitory cytokine-1 (MIC-1) is an important target of p53. MIC-1 is known to play an important role in the regulation of cell death and metastasis in colon cancer cells.

CRC is an environmental disease that is primarily affected by accumulated epithelial stress from a variety of environmental factors including ribosome-inactivating stress (RIS). Exposure to RIS in the gastrointestinal tract is thought to affect the malignancy of epithelial tumor cells and the response to anticancer agents. Ribosomal inactivation is known to cause acute and chronic mucosal inflammation and is a pathological factor of human intestinal epithelial inflammatory disease including IBD. However, there are few studies related to anticancer drug resistance.

Korean Patent Publication No. 10-2016-0014565 (published Feb. 11, 2016)

The present invention provides a method for producing a RIS-induced anticancer-resistant colon cancer cell line comprising the step of exposing a colon cancer cell line to a ribosome-inactivating stress (RIS), and a method for screening an anticancer drug resistance-inhibiting agent in a colon cancer patient using the same do.

In order to solve the above-mentioned problems, the present invention provides a method for producing a RIS-induced cancer-resistant colon cancer cell line comprising the step of exposing a colon cancer cell line to ribosome-inactivating stress (RIS).

(1) preparing a RIS-induced cancer-resistant colon cancer cell line by exposing a colon cancer cell line to ribosome-inactivating stress (RIS); (2) contacting the test substance to the RIS-induced anticancer drug resistant colorectal cancer cell line; (3) measuring the sensitivity of the anticancer drug to the RIS-induced anticancer cancer-resistant colon cancer cell line in contact with the test substance; And (4) selecting a test substance having increased sensitivity to the anticancer drug in the colorectal cancer cell line as compared with the control sample.

The present invention also provides a composition for diagnosing cancer resistance of colon cancer patients, which comprises a preparation capable of measuring the expression level of p53 or macrophage inhibitory cytokine-1 (MIC-1) as an active ingredient do.

The present invention relates to a method for producing a ribosome-inhibiting stress-induced anticancer cancer-resistant colon cancer cell line and a method for screening anticancer drug resistance-inhibiting agent for colon cancer patients using the same, wherein the anticancer drug resistance of cancer cells is a major factor . Exposure to various ribosomal inhibitory stresses in the everyday environment may impair the sensitivity of anticancer drugs to colon cancer cells. By monitoring the p53-MIC-1 apoptosis signal change in advance, the potential tolerance effect of anticancer drugs in colon cancer chemotherapy can be considered in advance. It is expected that it will help to establish economical and effective anti-cancer treatment strategy while overcoming the problems of existing anticancer treatment.

Figure 1 shows colon cancer cell lines that are highly sensitive to changes in susceptibility to anticancer drugs when exposed to ribosomal inhibitory stress.
FIG. 2 shows various colon cancer cell lines showing a sensitive sensitivity to chemotherapeutic agents due to exposure to ribosomal inhibitory stress.
FIG. 3 shows the inhibition effect of the anticancer drug cell death induced by exposure to ribosomal inhibitory stress.
FIG. 4 shows the inhibition of cancer cell apoptosis (nuclear nucleation) induced by exposure to ribosomal inhibitory stress.
Figure 5 shows the results of reduced susceptibility to p53 and MIC-1 dependent ribosomal inhibitory stress-induced chemotherapeutic agents.
FIG. 6 shows the results of inhibiting 5-FU susceptibility of anticancer drug induced by ribosomal inhibitory stress and confirming the relation of p53-MIC-1 cell death factor.
FIG. 7 shows the results of confirming the inhibition of paclitaxel sensitivity to anticancer drug induced by ribosomal inhibitory stress and confirming the relation of p53-MIC-1 cell death factor.

Accordingly, the present inventors have scientifically verified that a cancer cell exposed to ribosomal inhibitory stress is susceptible to an anticancer drug by causing an abnormality in an apoptosis signal activated by an anticancer drug. Inhibition of p53, MIC-1 cell death signal activated by 5-FU and paclitaxel when exposed to ribosomal inhibitory stress results in decreased susceptibility to anticancer drugs. The inventors of the present invention have completed the present invention by confirming that the increase in tolerance to the anticancer drug induced by ribosomal inhibition stress is markedly higher than that in various cancer cells.

The present invention provides a method for producing an RIS-induced cancer-resistant colon cancer cell line, which comprises exposing a colon cancer cell line to ribosome-inactivating stress (RIS).

Preferably, the step of exposing the colorectal cancer cell line to the RIS may be, but is not limited to, treatment of anisomycin (ANS) or deoxynivalenol (DON).

Preferably, the anticancer agent may be fluorouracil (5-FU) or paclitaxel, but is not limited thereto.

Also, the present invention provides the RIS-induced anticancer drug-resistant colon cancer cell line prepared above.

(1) preparing a RIS-induced cancer-resistant colon cancer cell line by exposing a colon cancer cell line to ribosome-inactivating stress (RIS); (2) contacting the test substance to the RIS-induced anticancer drug resistant colorectal cancer cell line; (3) measuring the sensitivity of the anticancer drug to the RIS-induced anticancer cancer-resistant colon cancer cell line in contact with the test substance; And (4) selecting a test substance having increased sensitivity to the anticancer drug of the colorectal cancer cell line as compared with the control sample.

The term "test substance" used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening in order to examine whether it affects the expression amount of a gene or affects the expression or activity of a protein. do. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

Specifically, the agent capable of measuring the expression level of p53 or MIC-1 may be a primer or a probe specifically binding to the p53 or MIC-1 gene, a primer or a probe specifically binding to the p53 or MIC-1 protein Antibodies, peptides, aptamers or compounds, but are not limited thereto.

The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).

In addition, the present invention provides a kit for diagnosing cancer resistance in a colorectal cancer patient comprising the composition.

As used herein, the term "primer" refers to a short nucleic acid that can form base pairs with a complementary template with a nucleic acid sequence having a short free 3 'hydroxyl group and serves as a starting point for template strand replication It refers to the sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to an mRNA. The presence or absence of a specific mRNA, You can check the amount. The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, the term "antibody" means a specific immunoglobulin as indicated in the art and directed against an antigenic site. An antibody in the present invention means an antibody that specifically binds to IL-22 of the present invention, and the antibody can be produced according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.

In addition, the kit of the present invention comprises an antibody specifically binding to a marker component, a secondary antibody conjugate conjugated with a label that develops upon reaction with the substrate, a coloring substrate solution to be colored with the label, A reaction stop solution, and the like, and may be manufactured from a number of separate packaging or compartments including the reagent components used.

As used herein, the term "peptide" has a high binding capacity to the target material and does not cause denaturation during thermal / chemical treatment. Also, because of its small size, it can be used as a fusion protein by attaching it to other proteins. It can be used as a diagnostic kit and a drug delivery material because it can be specifically attached to a polymer protein chain.

The term "aptamer " as used herein refers to a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid having a stable tertiary structure by itself and having a characteristic capable of binding with high affinity and specificity to a target molecule ). &Lt; / RTI > As described above, since the aptamer is composed of a polynucleotide which is capable of specifically binding to an antigenic substance like the antibody and is more stable than the protein, has a simple structure, and is easy to synthesize, .

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example 1> Reduction of cytotoxic susceptibility to anticancer drugs when ribosomal inhibitory stress is exposed

The HCT-116 p53 gene-deficient cell line and its control, the same genotype HCT-116 colon cancer cell line, were distributed from the Bert Vogelstein research team of Johns Hopkins University, and the lung cancer cell line A549, ovarian cancer cell line A2780, hepatoma cell line HepG2, Various colon cancer cell lines such as HCT-8, DLD-1, LOVO, SW480, LS174T, HCT15 and HT29 were purchased from ATCC (Manassas, USA), a cell line distribution company. Cell culture was performed in RPMI (Weljin, Daegu, Republic of Korea) on the basis of culture medium containing 10% fetal bovine serum (FBS) and 5% mixed antibiotics (50 units / mL penicillin, 50 mg / mL streptomycin) % CO ₂ , 37 캜 constant-humidity incubator. To induce ribosome inhibitory stress in various cancer cells, 50 ng / mL of anisomycin (ANS; Sigma, USA) or deoxynivalenol (DON; Sigma, USA) for 24 hours, and further treated with 375 μM 5-FU for 48 hours. To confirm the cytotoxicity against 5-FU of chemotherapeutic agent, the detached apoptotic cells were treated with trypan blue trypan blue) and hemocytometer. In the lung cancer cell line A549, liver cancer cell line HepG2, and ovarian cancer cell line A2780, it was confirmed that the cytotoxic effect on the 5-FU of the anticancer drug was reduced when the cells were exposed to the DON and ANS inhibitory ribosomal action (FIG. 1). In addition, it was found that the susceptibility to cytotoxic effects of anticancer agents was significantly higher in HCT-116 colon cancer cell lines than in other cancer cells.

To confirm this, LOVO, DLD-1, SW480, LS174T, HCT15 and HT29 were exposed to DON (500 ng / mL) for 24 hours and 375 μM 5-FU were further treated for 48 hours Changes in cell cytotoxicity were calculated by trypan blue staining and hemocytometer, and the relative cytotoxicity was quantified. As shown in FIG. 2, the relative cytotoxic susceptibility to 5-FU of the anticancer agent was remarkably reduced to a similar pattern (FIG. 2).

In order to clarify the direct relationship between the exposure of ribosomal inhibitory stress and the change of anticancer drug susceptibility, the change of apoptotic effect was confirmed. HCT-116 colon cancer cell line (8 × 10 ⁵ ) was exposed to DON (500 ng / mL) or ANS (50 ng / mL) for 24 hours and 375 μM 5-FU for another 48 hours. All of the detached cells and adherent cells were recovered by trypsin treatment, washed with phosphate-based saline (PBS), resuspended in Annexin V binding buffer, and resuspended in Annexin V- Cells killed with FITC antibody (BD phamigen ^™ ; 1:40) and propidium iodide (PI, 100 μg / mL) were labeled and analyzed with a flow cytometer (FACS Canto II; Becton Dickinson, And PI positive-labeled cells were measured and quantified. (4 quadrants) and late apoptotic cells (1 quadrant) than the control group exposed only to 5-FU at the time of exposure to ribosomal inhibitory stress (FIG. 3).

In order to confirm the inhibition of cytotoxic effect of cancer cells exposed to ribosomal inhibitory stress, immunohistochemical staining was used to stain nuclei to directly observe segmented nuclei and to quantify the number of cells with segmented nuclei, Respectively. The cells were exposed to DON (500 ng / mL) or ANS (50 ng / mL) for 24 h in HCT-116 colon cancer cell line (1x10 ⁵ ), treated with 375 μM 5-FU for additional 48 h, After immobilization with paraformaldehyde, non-specific antibody binding was reduced with 3% bovine serum albumin (BSA) and 0.2% triton X-100 was treated to improve antibody and dye permeability. , And 6-diamidino-2-phenylindole (DAPI). Cellular outline was stained with phalloidine (1: 1,000) with FITC fluorescent protein binding. The degree of segmentation of nuclei was observed with a confocal microscope (FV-1000, Olympus, Japan). As shown in FIG. 4, when exposed to DON and ANS, the number of cells with nuclei segmented by 5-FU decreased.

A series of results suggests that the cytotoxicity of anticancer drugs may directly affect the death signal of colon cancer cells when exposed to the ribosomal inhibitory environment.

< Example 2> Ribosome Inhibition Stress-Induced Anticancer Drug Sensitivity Decreased and Relevance of Cell-killing Factor p53 and MIC-1 Mediated Signal Suppression

In order to elucidate the specific mechanism of inhibition of anticancer drug susceptibility upon exposure to ribosomal inhibitory stress, attention was paid to p53 and MIC-1 cancer cell death signals activated by anticancer drugs from existing research and accumulated research literature and research results. Colon cancer cells exposed to anticancer drugs are induced to a high level of p53 and MIC-1, activating the cell death signal. (100, 300, 500 ng / mL) and ANS (100, 300, 500 ng / mL) in the control colon cancer cell line, the p53 deficient colorectal cancer cell line and the MIC-1 inhibitory colorectal cancer cell line to confirm the direct correlation between p53 and MIC- 25, 50, and 100 ng / mL) were pretreated for 24 hours and treated with 375 μM 5-FU for 48 hours. The cells were then stained with trypan blue and quantitated using a hemocytometer Respectively. The cytotoxicity of the anticancer drug 5-FU was decreased according to the concentrations of DON and ANS exposed. However, p53-deficient cells and MIC-1-deficient cells did not show cytotoxic susceptibility inhibition tendency (FIG. 5). These results suggest that apoptotic factors p53 and MIC-1 play an important role in inhibiting cytotoxic susceptibility of anticancer drugs induced by ribosomal inhibitory stress.

To confirm the direct relationship between the inhibitory effect of ribosomal function and p53 and MIC-1-dependent cell death signal, shRNA was introduced into cells pretreated with DON (500 ng / mL) for 24 hours, p53 deficient cells and MIC-1 MIC-1-deficient cells were treated with anticancer agents 5-FU (100 μM) and paclitaxel (100 nM) for 48 hours, respectively. The recovered cells were sonicated for 30 seconds by adding protein extraction buffer (lysis buffer: 1% [w / v] SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris pH 7.4). The extracted proteins were quantitated by WelProt protein assay (Weljin, Daegu, Korea) and separated by PAGE electrophoresis (Bio-Rad gel mini electrophoresis; Bio-Rad, Hercules, CA). Polyvinylidene fluoride fluoride membranes (PVDF, Amersham Bioscience, Piscataway, NJ). MIC-1, p53 and poly (ADP-ribose) polymerase-1 (PARP-1) antibodies were attached at 4 ° C for at least 12 hours after blocking non-specific antibody binding with TBST buffer solution containing 5% defatted milk powder. Subsequently, the secondary antibody conjugated with horseradish peroxidase was attached for 2 hours, washed with TBST buffer, and each protein was detected and quantified with ECL substrate reaction solution (ELPIS Biotech, Taejon, Korea). The degree of apoptosis was determined by quantifying the detected poly (ADP-ribose) polymerase-1 (PARP-1) protein fragment. Cells killed with propidium iodide (PI, 100 μg / mL) were labeled and the PI positive-labeled cells were measured and quantified using a flow cytometer (FACS Canto II; Becton Dickinson, USA) . As shown in FIG. 6, the cytotoxic susceptibility to 5-FU of the anticancer agent (PARP1 / 2 fragment) was decreased and the p53-deficient cell and the MIC-1-deficient cell showed similar patterns when the ribosome inhibitory stress was exposed. Increased cell death (increased PI-positive cell count) for 5-FU on chemotherapy was not observed in all cases where DON was pretreated, p53 deficient or MIC-1 deficient. These results were also observed in the other anticancer drug paclitaxel (Fig. 7).

Having described the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Comprising exposing a colon cancer cell line to ribosome-inactivating stress (RIS) by treating anisomycin (ANS) or deoxynivalenol (DON), wherein the p53 and macrophage inhibition (5-FU) or paclitaxel (5-FU), wherein the chemotherapeutic agent is a chemotherapeutic agent selected from the group consisting of fluorouracil (5-FU) and paclitaxel Resistant RIS-induced anticancer drug resistant colon cancer cell line.

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A RIS-induced cancer-resistant colon cancer cell line inhibited in expression of p53 and MIC-1 prepared according to claim 1.

(1) Expression of p53 and MIC-1 by exposing the colon cancer cell line to ribosome-inactivating stress (RIS) by treating anisomycin (ANS) or deoxynivalenol (DON) Producing the suppressed RIS-induced anticancer drug resistant colorectal cancer cell line;
(2) contacting the test substance to the RIS-induced anticancer drug resistant colorectal cancer cell line;
(3) measuring the sensitivity of the anticancer drug to the RIS-induced anticancer cancer-resistant colon cancer cell line in contact with the test substance; And
(4) A method for screening a test substance having increased sensitivity to an anticancer drug of a colon cancer cell line as compared to a control sample, wherein the anticancer drug is fluorouracil (5-FU) Or paclitaxel (paclitaxel) in colorectal cancer patients.

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