JP2012225822A - Diagnostic, treatment method of colon cancer using apc-binding protein eb1 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide diagnostic and treatment methods of colon cancer, more specifically, to provide an inspection method of colon cancer using APC-binding protein EB1, or to provide a treatment method by targeting APC-binding protein EB1.SOLUTION: APC-binding protein EB1 is used as a marker of an inspection method for diagnosing colon cancer. As a result of investigating presence/absence of APC-binding protein EB1, appearance of APC-binding protein EB1 is remarkably high in colon cancer cells, and the appearance of APC-binding protein EB1 is remarkably low in colon normal mucous cells. Detection or quantity determination of APC-binding protein EB1 is performed by an immunological technique. Otherwise, growth of the colon cancer cells is suppressed by suppressing the appearance of APC-binding protein EB1. When the appearance of APC-binding protein EB1 is suppressed by the colon cancer cells, remarkable appearance suppression is observed.

Description

  The present invention relates to a marker for diagnosing a malignant tumor and a method for treating a malignant tumor using the marker. More specifically, the present invention relates to a test method and a treatment method for diagnosis of colorectal cancer using the marker.

APC-binding protein EB1 is a known protein discovered as a protein that binds to the tumor suppressor gene product APC (adenomatous polyposis coli) (Non-patent Document 1; LK Su et al 1995). So far, EB1 is a hepatocellular carcinoma tissue (Non-patent Document 2; Fujii)
et al 2005), esophageal cancer tissue (Non-Patent Document 3; Wang et al 2005), and gastric cancer tissue (Non-Patent Document 4; Nishigaki et al 2005) are reported to be highly expressed. However, regarding the diagnosis of colorectal cancer, use of EB1 as a single marker has not been reported. In particular, nothing related to the usefulness of EB1 in colorectal cancer has been reported.

Colorectal cancer is one of the most common and high-grade malignant tumors in the world and is the third leading cause of cancer death, which is a major health problem. Also, according to recent studies, the incidence of colorectal cancer seems to be increasing.

  In addition to diagnostic imaging such as X-ray CT and MRI, methods for detecting cancer markers that are specifically expressed in specific cancers, cancer markers that leak into the blood, and tissues are also widely used for cancer diagnosis. ing. Examples of cancer markers include diagnostic markers, prognostic prediction markers, and therapeutic response prediction markers.

  Examples of diagnostic markers for colorectal cancer that are used clinically include CEA and CA19-1, but there are no single tumor markers specific to colorectal cancer, so several types of tumor markers are measured. A lot of things.

  For the treatment of colorectal cancer, resection is the first choice if it is early. Chemotherapy has been tried as an adjuvant therapy. In particular, various molecular targeted therapies have been developed against the background of recent developments in molecular biology. However, the prognosis of patients who have started treatment after becoming advanced cancer is extremely poor.

Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, Trent J, Vogelstein B, Kinzler KW. APC binds to the novel protein EB1 Cancer Res. 1995 Jul 15; 55 (14): 2972-7. Fujii K, Kondo T, Yokoo H, Yamada T, IwatsukiK, Hirohashi S. Proteomic study of human hepatocellular carcinoma usingtwo-dimensional difference gel electrophoresis with saturation cysteine dye.Proteomics. 2005 Apr; 5 (5): 1411-22. Wang Y, Zhou X, Zhu H, Liu S, Zhou C, Zhang G, Xue L, Lu N, Quan L, Bai J, Zhan Q, Xu N. Overexpression of EB1 in humanesophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin / TCF pathway. Oncogene. 2005 Oct 6; 24 (44): 6637-45. Nishigaki R, Osaki M, Hiratsuka M, Toda T, Murakami K, Jeang KT, Ito H, Inoue T, Oshimura M. Proteomic identification of differentially-expressed genes in human gastric carcinomas. Proteomics. 2005Aug; 5 (12): 3205-13 .

  An object of the present invention is to provide a marker for predicting the prognosis of a malignant tumor and a method for examining a malignant tumor using the marker. More specifically, it is an object to provide a test method for diagnosis of colorectal cancer using the marker, or to provide a method for treating colorectal cancer.

In order to solve the above problems, the present inventors have conducted extensive research and extracted proteins from tissues containing malignant tumors obtained from biological specimens, and then prognostically using fluorescence two-dimensional electrophoresis and mass spectrometry. Related proteins were analyzed. As a result of the analysis, we focused on APC-binding protein EB1,
As a result of investigating the relationship between colon cancer tumor tissue and normal colon mucosa tissue of protein EB1, APC-binding
It was confirmed that the expression of protein EB1 was significantly higher in colon cancer tumor tissues. APC-binding
The present inventors have found that suppression of protein EB1 expression suppresses the growth of colon cancer cells, thereby completing the present invention.

That is, this invention consists of the following.
1. A method for examining colorectal cancer, wherein APC-binding protein EB1 is detected as a marker.
2. Inspection method including the following steps:
(1) A step of detecting or quantifying APC-binding protein EB1 in a biological sample collected from a living body;
(2) Process of determining the presence of a malignant tumor by the detected or quantified APC-binding protein EB1. A test method for detecting or quantifying APC-binding protein EB1 by an immunological technique.
4). 5. The examination method according to item 4 above, wherein the immunological technique is immunohistological staining.
5. APC-binding protein EB1 is used as a molecule that inhibits the growth of colon cancer cells

According to the test method of the present invention, specifically, immunohistological staining, APC-binding protein
Cells with high expression of EB1 were diagnosed morphologically and pathologically as colon cancer tumor cells.

Moreover, according to the test method for colorectal cancer of the present invention, for example, APC-binding protein EB1-positive cells were 100% colorectal cancer, whereas APC-binding protein EB1-negative cells were 100%, and APC-binding
Protein EB1-positive cells were diagnosed as colorectal cancer.

  According to the test method for colorectal cancer of the present invention using APC-binding protein EB1 as a single marker, it is possible to accurately diagnose colorectal cancer. Colorectal cancer can be determined by examining a sample excised with an endoscope or the like by the examination method of the present invention. That is, by using APC-binding protein EB1 as a single marker, it is possible to diagnose colorectal cancer practically, simply, rapidly and at low cost.

It is a figure which shows the protein spot of about 3500 by fluorescence two-dimensional electrophoresis. (Reference example) It is the figure which compared the expression of APC-binding proteinEB1 in a colon large mucosa and a colon cancer tumor tissue by the fluorescence two-dimensional electrophoresis method (a) and the western blotting method (b). Example 1 It is the figure which confirmed the expression of APC-binding protein EB1 in a colon cancer tumor tissue by immunohistochemical staining. (Example 2) It is a figure which shows that the proliferation of the tumor cell fell by suppressing the expression of APC-binding protein EB1 in a colon cancer cell. (Example 3)

The present invention relates to the diagnosis of malignant tumors that detect APC-binding protein EB1 as a marker. The amino acid sequence of APC-binding protein EB1 in the present invention is the Swisssprot at the time of filing.
Although what is described in detabase Accession No.Q15691 can be mention | raise | lifted as an example, if it is what is called APC-binding protein EB1 protein, it will not be limited to the protein which consists of the above-mentioned arrangement | sequence.

  In the present invention, diagnosis of colorectal cancer means detection of colorectal cancer tumor cells from a biological sample containing colorectal cancer tumor cells.

  In the present invention, the treatment of colorectal cancer means that the proliferation of colorectal cancer tumor cells is significantly reduced. Examples of the corresponding reduction in the living body include a clinical treatment reduction effect and a tumor reduction effect determined by image diagnosis based on an evaluation standard called RECIST.

The prognostic prediction test of the present invention can be performed by a method including the following steps.
(1) A step of detecting or quantifying APC-binding protein EB1 in a biological sample collected from a living body;
(2) A step of diagnosing colorectal cancer with detected or quantified APC-binding protein EB1.

  In this specification, a biological specimen refers to a specimen obtained from a living body, and a specimen pretreated for use in various tests and tests is referred to as a sample. The biological specimen for use in the test method of the present invention is not particularly limited as long as it is derived from the tissue of a malignant tumor patient, but preferably a specimen containing malignant tumor cells. The biological specimen is a specimen obtained from a living body, for example, a patient, and may be a specimen obtained for the examination method of the present invention, such as a specimen obtained for use in another examination or a specimen collected by surgery. There may be. For example, when a specimen is subjected to an immunohistochemical staining test, a paraffin section prepared from a specimen obtained from a patient can be used as a sample to be subjected to the examination. For example, when the specimen is subjected to Western blotting or RT-PCR, a protein extract or mRNA extract prepared from a specimen obtained from a patient can be used as a sample to be tested.

In the test method of the present invention, the method for detecting or quantifying APC-binding protein EB1 as a marker is not particularly limited as long as it can confirm APC-binding protein EB1 in a biological sample. APC-binding protein EB1 in each biological specimen can be detected or quantified by any method exemplified below. The detection or quantification of APC-binding protein EB1 is simply APC-binding protein.
The presence or absence of protein EB1 may be detected, and the expression level of APC-binding protein EB1 may be determined relative or absolute. APC-binding protein EB1 expression may be detected or quantified at the protein level, or may be detected or quantified at the mRNA level.

  Detection or quantification of APC-binding protein EB1 expression at the protein level is preferably performed by an immunological technique and is suitable. For example, immunostaining method (including fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, radioisotope labeled antibody method), separation by electrophoresis and detection or quantification by fluorescence, enzyme, radioisotope, etc. (Including Western blotting and fluorescence two-dimensional electrophoresis), enzyme immunoassay adsorption (ELISA), dot blotting and the like. The detection or quantification at the mRNA level can be performed by, for example, RT-PCR (preferably real-time RT-PCR), Northern blotting, Branched DNA assay, and the like.

  The immunohistological staining method can employ a method known per se and is not particularly limited, but specific examples thereof are shown below. A biological specimen isolated from a malignant patient is fixed in formalin by a conventional method, embedded in paraffin, sliced into a tissue piece of about 4 μm in thickness with a microtome, and pasted on a slide glass as a section sample use. The section sample is completely treated with xylene to remove paraffin, passed through an alcohol solution gradually reduced in concentration from 100% to be hydrophilized, and washed with water. Thereafter, in order to increase the permeability of the antibody, the section sample is dipped in a pH 6.0 citrate buffer solution placed in a heat-resistant glass container, and the antigen is activated by heat treatment at 121 ° C. for 10 minutes in an autoclave. Allow to cool to room temperature, wash the buffer with running water, and then perform immunohistochemical staining. After blocking endogenous peroxidase activity and non-specific reaction, anti-APC-binding protein EB1 antibody is added dropwise to the section sample and allowed to react overnight at room temperature. After washing, each is reacted for 30 minutes using an HRP-labeled anti-rabbit antibody (DAKO). After washing, color is developed using DAB solution (3,3'-diaminobenzidine tetrahydrochloride) (DAKO). For cleaning, TBST (DAKO) is used. After washing with running water, the cell nucleus of the specimen is stained with a hematoxylin solution. After washing with running water, the alcohol solution and then the xylene solution are passed through and dehydrated. The mounting medium is dropped on the specimen, covered with a cover glass, and observed with a microscope. Under the microscope, the APC-binding protein EB1 protein in hepatocellular carcinoma cells is observed as a brown color. As described below, positive / negative determination can be performed by the color development.

  The results of detection or quantification of APC-binding protein EB1 expression can be classified into two stages (positive and negative). The classification of APC-binding protein EB1 expression is preferably performed by a pathologist, clinician, laboratory technician or laboratory with sufficient experience, depending on the detection or quantification method. For example, APC-binding protein EB1 expression can be classified by a pathologist when using an immunohistochemical staining method, or by a laboratory technician when using Western blotting or RT-PCR.

The classification of APC-binding protein EB1 expression is based on the expression level of APC-binding protein EB1 in biological specimens from patients.
It is preferably performed by comparing with the expression level of protein EB1. APC-binding protein
Depending on the number of stages that classify the results of EB1 expression, multiple controls are preferably used. For example, APC-binding
When classifying the results of protein EB1 expression into two types (positive and negative), two types of controls corresponding to each step (APC-binding protein EB1 positive control and APC-binding)
protein EB1 negative control) is preferably used. Moreover, it is preferable to use a control derived from a healthy person as one of the controls.

When the results of APC-binding protein EB1 expression are classified into two stages, positive and negative, by the immunohistochemical staining method, the results of immunohistochemical staining can be determined as follows, for example.
a. “Strongly dyeable”
b. "Stain is weakly dyed"
c. "No dyeing"
And the ratio of a, b, c to the whole malignant cell is measured.

The present invention extends to a test reagent and a test kit for detecting or quantifying APC-binding protein EB1 in a biological sample. With this kit, the expression of APC-binding protein EB1 in a biological specimen obtained from a patient can be detected or quantified. That is, as a test reagent kit for detecting or quantifying the expression of APC-binding protein EB1 at the protein level, a test kit used for immunological techniques such as immunohistological staining and Western blotting can be mentioned. When the test is performed by an immunological technique, at least anti-APC-binding protein EB1 antibody is contained in the test reagent. Anti-APC-binding
The protein EB1 antibody is not particularly limited as long as it can detect the expression of APC-binding protein EB1. For example, monoclonal and polyclonal antibodies, labeled antibodies, chimeric antibodies, humanized antibodies, and binding activity fragments thereof Is mentioned. Moreover, the test reagent kit may contain a label used for detection in addition to the antibody. The kit can contain a buffer, a chromogenic substrate, a secondary antibody, a reagent such as a blocking agent, instruments and controls necessary for the test, and the like.

  Hereinafter, the details of the present invention will be described in the examples with reference to the background that led to the completion of the present invention, and the present invention will be described more specifically. However, the present invention is not limited thereto, and the present invention Various applications are possible without departing from the technical idea of the present invention.

(Example 1) Analysis of protein by two-dimensional electrophoresis In the present invention, the background of focusing on APC-binding protein EB1 is as follows. Colon cancer tumor tissue and normal colon mucosal epithelial cells were collected, and proteins were extracted. Thereafter, proteins with different expression were analyzed in colorectal cancer tumor tissue cells and colorectal normal mucosal epithelial cells by fluorescence two-dimensional electrophoresis (2D-DIGE) (see FIG. 1) and mass spectrometer. Surgical specimens of 59 cases of colorectal cancer used for research with the approval of the Ethics Committee from the National Cancer Center Central Hospital were used. APC-binding protein EB1

1) Sample preparation The surgical specimen was crushed into powder by Multi-Bead Shocker ^™ (Yasui Kikai, Osaka). Protein extraction buffer (6M
Urea, 2M thiourea, 3% CHAPS, 1% TritonX-100) were added to extract the protein. The extracted protein was labeled with a fluorescent dye (Saturation Dy Cy5 ^™ , GE). The labeling was performed as follows. (1) Tris buffer at pH 8.0 was added to a final concentration of 30 mM, then (2) 1 nmol of TECP (Tris (2-carboxyethyl) phosphine hydrochloride, Sigma) was added, and (3) 37 ° C. For 60 minutes. Next, (4) 4 nmol of Cy5 fluorescent dye was added and treated at 37 ° C. for 30 minutes. An equal amount of protein sample was collected from the protein sample used in this experiment and mixed to obtain an internal control sample. The internal control sample was labeled with a fluorescent dye (Saturation Dy Cy3 ^™ , GE) as described above. Individual samples labeled with Cy5 and internal control samples labeled with Cy3 were mixed and brought to a final volume of 420 μl with urea solubilizer. At that time, dithiothreitol (DDT) was added to a final concentration of 65 mM, and Ampholine ^™ (GE Healthcare Biosciences) was added to a concentration of 2%. A sample in which an individual sample labeled with Cy5 and an internal control sample labeled with Cy3 were mixed was run on a single two-dimensional electrophoresis gel.

2) Two-dimensional electrophoresis First, the first-dimension electrophoresis is immobiline gel (24cm, pI
4-7, GE) and Multiphor II ^™ (GE Healthcare Biosciences). Immobiline gels were swollen overnight at room temperature with the protein samples to be run. Electrophoresis was performed at 40000 Vh. The second-dimensional electrophoresis used a 9-15% polyacrylamide gradient gel and a two-dimensional electrophoresis apparatus. Electrophoresis was performed at 15 ° C. for 10 hours at 18 W per electrophoresis apparatus.

3) Protein detection After the completion of electrophoresis, the gel in a state of being sandwiched between glass plates is detected with a laser scanner (Typhoon) for the purpose of detecting proteins.
(Trio ^™ , GE) and scanned.

4) Expression analysis The read image is image analysis software Progenesis SameSpots software
(Nonlinear Dynamics, Newcastle, UK).

5) Protein identification a. In-gel digestion Spots were collected from the gel in 96-well plates using a fully automated spot collection device ProHunter ^™ (AsOne). The gel was thoroughly washed with methanol and treated with proteolytic enzyme (trypsin) at 37 ° C. overnight. This treatment results in peptide formation of the protein. The obtained peptide was recovered by washing the gel with 60% acetonitrile.
b. Mass spectrometry LTQ ^™ (Thermo Electron) was used to measure the mass of the peptide. MasCot ^™ was used to search the database for protein identification.

6) Results As a result of performing the two-dimensional electrophoresis by the above method, from the approximately 3500 protein spots, in the comparison between each group, (1) Wilcoxon test p <0.01, (2) The average value is more than doubled. 110 protein spots were selected on the basis of the difference. As a result of protein identification using a mass spectrometer, protein spots derived from APC-binding protein EB1 were contained in 110 spots (FIG. 2).

(Example 2) Confirmation of APC-binding protein EB1 by Western blotting For the purpose of verifying a reference example, the expression of APC-binding protein EB1 was examined by Western blotting (see Fig. 2).

  APC-binding protein EB1 was hardly detected in normal colorectal mucosal epithelial cells, and high expression was observed in colorectal cancer tumor tissues.

1) Protein recovery Each cell or xenograft tissue was crushed into powder by Multi-Bead Shocker ^™ (Yasui Kikai, Osaka). Protein was extracted by adding a protein extraction buffer to the powdered tissue in the same manner as in the Reference Example.

2) Western blotting 5 μg of the extracted protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Mouse anti-APC-binding protein EB1 (SantaCruz) was used as the primary antibody, and anti-mouse antibody (GE) labeled with peroxidase was used as the secondary antibody. The primary antibody was diluted 500 times and the secondary antibody was diluted 1000 times. An ECL Plus kit (GE) was used for detection. The intensity of the detected band is the Lumino Image Analyzer LAS-3000 ^TM
(Fuji Film) and ImageQuant ^TM software (GE
Healthcare).

3) Results As a result of analysis by Western blotting, strong expression of APC-binding protein EB1 was confirmed in colon cancer tumor tissues (FIG. 2).

(Example 3) Confirmation of APC-binding protein EB1 by immunohistochemical staining
Immunohistochemical staining using APC-binding protein EB1 antibody was performed.

Immunohistochemical staining was performed on paraffin-embedded tissue section samples. Section samples deparaffinized according to a conventional method were autoclaved at 121 ° C. for 10 minutes in 10 mM citrate buffer (pH 6.0). Section samples were labeled with a 500-fold diluted mouse anti-APC-binding protein EB1 antibody (SantaCruz) and HRP-labeled anti-mouse antibody (DAKO). DAB solution (3,3'-diaminobenzidine
Tetrahydrochloride) (DAKO) was used for coloring, and TBST (DAKO) was used for washing. After washing with running water, the cell nuclei of the specimen were stained with a hematoxylin solution.

  The results of APC-binding protein EB1 staining by immunohistochemical staining are shown in FIG. As a result, the colon cancer tumor cell Colo320 was positive for APC-binding protein EB1 staining, and the colon normal mucosal epithelial cells were negative for APC-binding protein EB1, similar to the results of spot detection by fluorescence two-dimensional electrophoresis. .

(Example 4) APC-binding protein EB1 as a therapeutic target
In order to verify the usefulness of APC-binding protein EB1 as a therapeutic target, an experiment was conducted on colon cancer cells to suppress APC-binding protein EB1 by siRNA method.

  In cells in which the expression of APC-binding protein EB1 was forcibly reduced, proliferation was significantly suppressed.

  Based on the above, it is considered that APC-binding protein EB1 is useful as a biomarker for colorectal cancer detection and can be a molecular target for colorectal cancer.

  As described in detail above, according to the prognostic test method of the present invention, specifically, the spot concentration by fluorescent two-dimensional electrophoresis or Western blotting, the expression of APC-binding protein EB1 is specific to tumor tissue for colorectal cancer. It was the target.

  Immunostaining confirmed that APC-binding protein EB1 is a powerful marker for the diagnosis of colon cancer cells.

In addition, according to experiments using siRNA, APC-binding
It was found that suppressing the expression of protein EB1 suppresses the growth of colon cancer cells.

  Further, according to the present invention, APC-binding protein EB1 was suggested to be useful as a molecular target for colorectal cancer.

  According to the test method of the present invention using APC-binding protein EB1 as a single marker, the test method of the present invention is used when a malignant tumor tissue is collected by biopsy or the like for a case suspected of having colon cancer. By testing, colorectal cancer can be accurately diagnosed, and an appropriate treatment method can be selected. In addition, the treatment results for colorectal cancer can be improved by performing treatment targeting APC-binding protein EB1.

Claims (9)

A diagnostic test method for colorectal cancer which detects APC-binding protein EB1 as a marker. The test method according to claim 1, wherein colorectal cancer is diagnosed. Inspection method including the following steps:
(1) A step of detecting or quantifying APC-binding protein EB1 in a biological sample collected from a living body;
(2) A step of diagnosing colorectal cancer with detected or quantified APC-binding protein EB1. The test method according to claim 3, wherein the biological specimen is a biological specimen containing a malignant tumor tissue composed of colorectal cancer. A test method for detecting or quantifying APC-binding protein EB1 by an immunological technique. The prognostic test method according to claim 4, wherein the immunological technique is immunohistological staining. The marker of the malignant tumor which consists of APC-binding protein EB1 used for the test | inspection method in any one of Claims 1-6. A method for treating colorectal cancer using APC-binding protein EB1 as a growth suppression target for colorectal cancer cells. The method for inhibiting cell proliferation according to claim 7, wherein the method for treating colorectal cancer.