WO2011102461A1

WO2011102461A1 - Protein expressed specifically in ovarian clear cell adenocarcinoma and use applications thereof

Info

Publication number: WO2011102461A1
Application number: PCT/JP2011/053497
Authority: WO
Inventors: 憲昭荒川; 有佑増石; 結子山中; 平野　久; 博史川崎; 史樹平原; 悦子宮城
Original assignee: 公立大学法人横浜市立大学
Priority date: 2010-02-22
Filing date: 2011-02-18
Publication date: 2011-08-25
Also published as: JP5224308B2; JP5224309B2; JPWO2011102461A1; JP2013079979A

Abstract

A protein produced specifically by an ovarian clear cell adenocarcinoma cell is searched, and the use applications of the protein are established. Specifically disclosed are: a method for evaluating and/or identifying clear cell adenocarcinoma, which comprises measuring the expression of at least one protein selected from the group consisting of tissue factor pathway inhibitor-2, ceruloplasmin, insulin-like growth factor binding protein-1, doublecortin-containing protein-2, N-Myc down-regulation gene-1, ribonuclease-T2, growth differentiation factor-15, versican core protein, laminin subunit gamma-1, sponging-1, insulin-like growth factor binding protein-7, interleukin-6 and osteopontin in a biological sample; a kit for the evaluation and/or identification of clear cell adenocarcinoma; a method for identifying a substance that is effective for the treatment and/or prevention of clear cell adenocarcinoma; and a method for identifying a substance that can reduce the anticancer agent resistance of clear cell adenocarcinoma.

Description

Proteins specifically expressed in ovarian clear cell adenocarcinoma and their applications

The present invention relates to a protein specifically expressed in ovarian clear cell adenocarcinoma and its application. More specifically, the present invention relates to a protein specifically produced by high-grade ovarian clear cell adenocarcinoma cells as a diagnostic marker. And technology used as a drug discovery target.

Ovarian cancers are mostly epithelial tumors and are the most varied group of tumors histologically. Although the treatment outcome of ovarian cancer has been improving with the introduction of platinum and taxane preparations, it is still said to be poor. This is because early detection is difficult, and there are many advanced cases, and there exists a tissue type showing resistance to antineoplastic drugs. Therefore, recently, the necessity of treatment according to tissue type has also been proposed. Clear cell adenocarcinoma in particular is a highly malignant tissue type that exhibits marked resistance to antineoplastic drugs and is prone to blood metastasis. Although the frequency of clear cell adenocarcinoma in Japan has been on the rise, there is still no probable tumor marker specific to clear cell adenocarcinoma. The therapy remains unknown.
Since the tissue cell morphology of clear cell adenocarcinoma is rich in variation, it often takes a morphology similar to other tissue types and requires accurate diagnosis. Clinicopathologists determine the histological type of ovarian cancer, but there are few cases of clear cell carcinoma, and further studies are needed, and specific markers for clear cell adenocarcinoma have not been clarified.
Clear cell adenocarcinoma is currently resistant to platinum and taxane preparations primarily used in chemotherapy. The details of the resistance mechanism have yet to be clarified. Development of new antineoplastic drugs is essential.

An object of the present invention is to search for and use proteins specifically produced by ovarian clear cell adenocarcinoma cells.

The present inventors searched for proteins specifically produced by ovarian clear cell adenocarcinoma cells using a proteomic method, and found several marker candidate proteins and novel drug target candidate proteins. More specifically, we performed a proteomic analysis using clear cell adenocarcinoma cell lines OVISE and OVTOKO and the mucinous adenocarcinoma cell line MCAS as a comparative control to comprehensively identify proteins expressed specifically in clear cell adenocarcinoma cells 25 kinds of proteins that have not been reported so far were detected. These proteins are novel diagnostic markers for clear cell adenocarcinoma. Moreover, when the expression of 7 types of the said protein was suppressed by RNAi, remarkable growth inhibition of clear cell adenocarcinoma cells was observed in the expression suppression of 2 types of protein. It will be possible to develop new ovarian cancer drugs targeting these. The present invention has been completed based on these findings.
The gist of the present invention is as follows.

(1) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Evaluation of clear cell adenocarcinoma comprising measuring expression in a biological sample for at least one protein selected from the group consisting of 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin; / Or discrimination method.
(2) The method according to (1), wherein expression in a biological sample is measured at a protein level.
(3) The method according to (1), wherein expression in a biological sample is measured at the nucleic acid level.
(4) The method according to any one of (1) to (3), wherein the biological sample is a cell, tissue or body fluid obtained from a subject.
(5) The method according to (4), wherein the body fluid is blood.
(6) The method according to (5), wherein the blood is whole blood, serum, plasma or plasma exchange fluid.
(7) When the expression in the cell, tissue or body fluid obtained from the subject is confirmed to be 2 or more times higher than the expression in the cell, tissue or body fluid obtained from the healthy subject, The method according to any one of (1) to (6), wherein the method is evaluated as suffering from a cell adenocarcinoma or a cancer cell type being clear cell adenocarcinoma.
(8) When the expression in the cell, tissue or body fluid obtained from the subject is confirmed to be 2 or more times higher at the nucleic acid level than the expression in the cell, tissue or body fluid obtained from the healthy subject, The method according to any one of (1) to (6), wherein the method is evaluated as suffering from a cell adenocarcinoma or a cancer cell type being clear cell adenocarcinoma.
(9) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Clear cell adenocarcinoma comprising a reagent capable of measuring expression in a biological sample for at least one protein selected from the group consisting of 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin Kit for evaluation and / or differentiation.
(10) The kit according to (9), wherein the reagent capable of measuring expression in a biological sample is any of the following (i), (ii), or (iii).
(i) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Antibody capable of specifically recognizing a protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin
(ii) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1, a nucleic acid probe capable of specifically hybridizing with mRNA encoding a protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin
(iii) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1, at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using mRNA encoding a protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin ( 11) A method for identifying a substance effective in the treatment and / or prevention of clear cell adenocarcinoma, comprising the following steps:
(a) contacting the test substance with clear cell adenocarcinoma cells,
(b) a step of culturing the clear cell adenocarcinoma cells contacted with the test substance in the step (a) for a predetermined time,
(c) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Measuring the expression in clear cell adenocarcinoma cells cultured in step (b) for at least one protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin ,as well as
(d) Evaluating the effect of the test substance on the expression in clear cell adenocarcinoma cells by comparing the expression in clear cell adenocarcinoma cells measured in step (c) with the expression in control cells not contacted with the test substance Said method comprising the steps.
(12) The method according to (11), further comprising a step of examining the effect of the test substance on clear cell adenocarcinoma cell proliferation.
(13) A method for identifying a substance that reduces anticancer drug resistance of clear cell adenocarcinoma, comprising the following steps:
(a) contacting the test substance with clear cell adenocarcinoma cells,
(b) a step of culturing the clear cell adenocarcinoma cells contacted with the test substance in the step (a) for a predetermined time,
(c) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Measuring the expression in clear cell adenocarcinoma cells cultured in step (b) for at least one protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin ,as well as
(d) Evaluating the effect of the test substance on the expression in clear cell adenocarcinoma cells by comparing the expression in clear cell adenocarcinoma cells measured in step (c) with the expression in control cells not contacted with the test substance Said method comprising the steps.
(14) The method according to (13), further comprising the step of examining the effect of the test substance on the anticancer drug resistance of clear cell adenocarcinoma cells.
(15) The method according to (1), wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.
(16) The kit according to (9), wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.
(17) The method according to (11), wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.
(18) The method according to (13), wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.

By using the protein detected and identified by the present inventors as a diagnostic marker, it becomes possible to accurately diagnose ovarian clear cell adenocarcinoma. In addition, by using a drug that inhibits the function of the protein or suppresses the expression thereof, it becomes possible to treat clear cell adenocarcinoma that has been impossible until now.

This specification includes the contents described in the specification and / or drawings of the Japanese Patent Application, Japanese Patent Application No. 2010-035737, which is the basis of the priority of the present application.

Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein in various ovarian cancer cell lines, Gene expression levels of laminin subunit gamma 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6, and osteopontin. The gene expression level of the protein group was measured by real-time-RT-PCR method. Protein amounts of laminin subunit gamma 1, N-Myc downstream regulatory gene 1, doublecortin-containing protein 2, sponge 1, osteopontin, and ribonuclease T2 in various ovarian cancer cell lines. The expression level of the protein group was analyzed by Western blotting. Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin in various ovarian tissue samples Gene expression levels of subunit gamma 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6, and osteopontin. The gene expression level of the protein group was measured by real-time-RT-PCR method. Detection and quantification of growth differentiation factor 15, osteopontin, and insulin-like growth factor binding protein 1 in serum of ovarian cancer patients.

Hereinafter, embodiments of the present invention will be described in more detail.
The present invention includes tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit Evaluation of clear cell adenocarcinoma comprising measuring the expression in a biological sample of at least one protein selected from the group consisting of gamma 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin And / or providing a discrimination method. The present inventors have found that the above protein is a cell line derived from ovarian clear cell adenocarcinoma cells compared to non-clear cell line cancer cell lines (OVCAR-3, OVSAHO, OVKATE, RMUG-S or MCAS) and tissues. (OVTOKO, OVISE, RMG-I, RMG-II, OVMANA or OVSAYO) and the expression were confirmed to be increased more than 2-fold in tissues (Examples 2, 3, and 4 described later). Furthermore, it was confirmed that the concentrations of growth differentiation factor 15, osteopontin, and insulin-like growth factor binding protein 1 in the serum of ovarian cancer patients were higher than those in the control group (healthy people, benign ovarian tumor patients) (described later). Example 5).

Tissue factor pathway inhibitor 2 has a molecular weight of 26,934, exhibits inhibitory activity against proteases such as trypsin, plasmin, factor VIIa / tissue factor, and is thought to be involved in matrix remodeling. High expression in the placenta is secreted extracellularly. UniProtKB / Swiss-Prot registration number P48307 (TFPI2_HUMAN).
Ceruloplasmin has a molecular weight of 122,205 and binds to 6-7 atoms of copper ion per molecule of the protein. In addition, it has ferroxidase activity that oxidizes 2 cargo iron ions into 3 iron iron. It is widely expressed in various tissues and abundantly present in blood. UniProtKB / Swiss-Prot registration number P00450 (CERU_HUMAN).
Insulin-like growth factor-binding protein 1 It has a molecular weight of 27,904 and binds to insulin-like growth factor I and II and is contained in blood. Many are present in placenta and amniotic fluid. UniProtKB / Swiss-Prot registration number P08833 (IBP1_HUMAN).
Doublecortin domain-containing protein 2 has a molecular weight of 52834 and its function is unknown. It is widely expressed in various tissues. SwissProt registration number Q9UHG0 (DCDC2_HUMAN).
N-Myc downstream regulated gene 1 protein, molecular weight 42835, function unknown. It is widely expressed in various tissues. SwissProt registration number Q92597 (NDRG1_HUMAN).
Ribonuclease T2 (Ribonuclease T2 precursor) has a molecular weight of 29481 and has ribonuclease activity. It is widely expressed in various tissues. SwissProt registration number O00584 (RNT2_HUMAN).
Growth / differentiation factor 15 (Mrowth / differentiation factor 15) molecular weight 34,154, strongly expressed in placenta, weakly expressed in prostate and large intestine. It is secreted extracellularly and functions as a cytokine and growth factor. UniProtKB / Swiss-Prot registration number Q99988 (GDF15_HUMAN).
Versican core protein molecular weight 372,820, high expression in brain, thyroid, testis, uterus. It is a component of the extracellular matrix and is thought to be involved in cell-to-cell information transmission. UniProtKB / Swiss-Prot registration number P13611 (CSPG2_HUMAN).
Laminin subunit gamma-1 precursor has a molecular weight of 177602 and is a component of the extracellular matrix and is involved in cell proliferation and migration. It is widely expressed in various tissues. SwissProt registration number P11047 (LAMC1_HUMAN).
Spondin-1 molecular weight 90973, a component of the extracellular matrix. Widely expressed in various tissues. SwissProt registration number Q9HCB6 (SPON1_HUMAN).
Insulin-like growth factor-binding protein 7 has a molecular weight of 29,130 and a low binding affinity with insulin-like growth factor I and II. Promotes prostacyclin synthesis. UniProtKB / Swiss-Prot registration number Q16270 (IBP7_HUMAN).
Interleukin-6 (molecular weight 23,718), secreted extracellularly and functions as a cytokine and growth factor. UniProtKB / Swiss-Prot P05231 (IL6_HUMAN).
Osteopontin molecular weight 35,423, secreted extracellularly, functions as a cytokine. Produced in various tissues. UniProtKB / Swiss-Prot registration number P10451 (OSTP_HUMAN).

In the present invention, the biological sample may be a sample derived from a subject, such as cells, tissues, body fluids obtained from the subject, specifically, tissue collected or excised from the ovary of the subject, blood of the subject (for example, Examples thereof include whole blood, serum, plasma, plasma exchange fluid, and the like. Whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample.
In the method of the present invention, expression in a biological sample may be measured at the protein level or at the nucleic acid level. For example, it can be measured by Northern blotting, RT-PCR, Western blotting, immunohistochemical analysis and the like. Alternatively, measurement may be performed using a cDNA microarray, ELISA method or the like.

In order to measure the expression of the protein at the protein level, an antibody that specifically recognizes the protein may be used. The antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by a known method. When measured by Western blotting, the antibody is secondarily detected using ¹²⁵ I-labeled protein A, peroxidase-conjugated IgG, or the like. When measurement is performed by immunohistochemical analysis, the antibody may be labeled with a fluorescent dye, ferritin, enzyme, or the like.

In order to measure the expression of the protein at the nucleic acid level, a nucleic acid probe capable of specifically hybridizing with the mRNA of the protein may be used (when measured by Northern blotting). Alternatively, at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the mRNA of the protein as a template may be used (when measured by RT-PCR method). Nucleic acid probe and nucleic acid primer are gene information of the above proteins (NCBI / GenBank registration number of nucleoprotein gene is tissue factor pathway inhibitor 2 (NM_006528.2), ceruloplasmin (NM_000096.3), insulin-like growth factor binding protein 1 (NM_000596.2), doublecortin-containing protein 2 (NM_016356.3), N-Myc downstream regulatory gene 1 (NM_001135242.1), ribonuclease T2 (NM_003730.4), growth differentiation factor 15 (NM_004864.2), versican Core protein (NM_001126336.2), laminin subunit gamma 1 (NM_002293.3), sponge 1 (NM_006108.2), insulin-like growth factor binding protein 7 (NM_001553.1), interleukin 6 (NM_000600.3), osteopontin (NM_000582.2)). The nucleic acid probe is usually about 15 to 1500 bases. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme, or the like. The nucleic acid primer is usually about 15 to 30 bases. The nucleic acid primer may be labeled with a radioactive element, fluorescent dye, enzyme or the like.

In the present invention, the presence or absence of expression (protein level or nucleic acid level) of the protein in a biological sample may be detected, or the expression level may be measured. The presence or absence of the expression of the protein and / or its mRNA can be confirmed by the presence or absence of the appearance of spots or bands at predetermined positions. The expression level of the protein and / or its mRNA can be measured by the staining intensity of spots and bands. Alternatively, the protein and / or its mRNA may be quantified.

The protein or gene whose expression is to be measured may be one kind or plural kinds. More accurate evaluation may be possible by referring to a plurality of gene expressions and a plurality of protein expression data. In order to detect multiple gene expression and multiple protein expression at the same time, DNA array (probe is fixed to the substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, DECEMBER 2002, 951-960), protein chip (antibody substrate) It is preferable to use a detection method such as (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, SEPTEMBER 2002, 683-695), Luminex (NATURE REVIEWS, DRUG DISCOVERY, VOLUME 1, JUUNE 2002, 447-456).

The test subject is a patient suspected of suffering from clear cell adenocarcinoma (particularly ovarian clear cell adenocarcinoma), but may be any human subject who is considered to be at risk.

As one example of the present invention, clear cell adenocarcinoma (particularly ovarian clear cell adenocarcinoma) can be evaluated and differentiated according to the following criteria.
As a result of measuring the expression of at least one of the above proteins in cells, tissues or body fluids obtained from subjects and comparing it with cells, tissues or body fluids obtained from healthy subjects, it was confirmed that the increase in protein level was more than 2-fold. In some cases, it is evaluated that the patient is suffering from clear cell adenocarcinoma or the cancer tissue type is clear cell adenocarcinoma. The greater the number of proteins that have been found to increase expression by a factor of 2 or more at the protein level, the greater the certainty of the assessment that the patient is suffering from clear cell adenocarcinoma or that the cancer tissue type is clear cell adenocarcinoma. it is conceivable that.
As another example of the present invention, evaluation and differentiation of clear cell adenocarcinoma (particularly ovarian clear cell adenocarcinoma) can be performed according to the following criteria.
As a result of measuring the expression of at least one of the above proteins in cells, tissues or body fluids obtained from subjects and comparing it with cells, tissues or body fluids obtained from healthy subjects, it was confirmed that there was a two-fold increase in nucleic acid level. In some cases, it is evaluated that the patient is suffering from clear cell adenocarcinoma or the cancer tissue type is clear cell adenocarcinoma. The greater the number of proteins that have been found to increase expression by a factor of 2 or more at the nucleic acid level, the greater the certainty of the assessment that the patient is suffering from clear cell adenocarcinoma or that the cancer tissue type is clear cell adenocarcinoma. it is conceivable that.

The present invention includes tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit A clear cell gland comprising a reagent capable of measuring expression in a biological sample for at least one protein selected from the group consisting of gamma 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin Kits for cancer assessment and / or differentiation are also provided.
As one example, the kit of the present invention contains an antibody capable of specifically recognizing the protein as a reagent. The antibody may be immobilized on a substrate (in the case of a protein chip). The kit may further include an instrument for collecting a biological sample, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like. In addition to the method of using the kit, the instruction manual may describe the evaluation and / or differentiation criteria for clear cell adenocarcinoma.

As another example, the kit of the present invention contains a nucleic acid probe capable of specifically hybridizing with the mRNA of the protein as a reagent. The nucleic acid probe may be immobilized on a substrate (in the case of a DNA array). The kit may further include an instrument for collecting a biological sample, an anticoagulant, a reagent for extracting RNA from the biological sample, a reagent for detecting RNA, an instruction manual, and the like. . In addition to the method of using the kit, the instruction manual may describe the evaluation and / or differentiation criteria for clear cell adenocarcinoma.

As yet another example, the kit of the present invention contains at least one pair of nucleic acid primers that can specifically amplify cDNA synthesized using the mRNA of the protein as a template as a reagent. The kit may further include an instrument for collecting a biological sample, an anticoagulant, reagents for extracting RNA from the biological sample, reagents for detecting RNA, instruction manuals, and the like. In addition to the method of using the kit, the instruction manual may describe the evaluation and / or differentiation criteria for clear cell adenocarcinoma.

The present invention also provides a method for identifying a substance effective for the treatment and / or prevention of clear cell adenocarcinoma. This method comprises the following steps:
(a) contacting the test substance with clear cell adenocarcinoma cells,
(b) a step of culturing the clear cell adenocarcinoma cells contacted with the test substance in the step (a) for a predetermined time,
(c) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. measuring at least one protein selected from the group consisting of insulin-like growth factor binding protein 7, interleukin 6 and osteopontin, in a clear cell adenocarcinoma cell cultured in step (b), and
(d) Evaluating the effect of the test substance on the expression in clear cell adenocarcinoma cells by comparing the expression in clear cell adenocarcinoma cells measured in step (c) with the expression in control cells not contacted with the test substance Process.

The test substance may be any substance, such as protein, peptide, vitamin, hormone, polysaccharide, oligosaccharide, monosaccharide, low molecular weight compound, nucleic acid (DNA, RNA, oligonucleotide, mononucleotide, etc.), lipid, other than the above Natural compounds, synthetic compounds, plant extracts, fractions of plant extracts, mixtures thereof and the like.
Clear cell adenocarcinoma cells used in the method of the present invention are tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15. Expression of at least one protein selected from the group consisting of versican core protein, laminin subunit gamma 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin at the protein level or nucleic acid level As long as it is observed, it may be derived from any organism, and examples include those derived from mammals such as humans, pigs, monkeys, chimpanzees, dogs, cows, rabbits, rats, and mice. Yes, but clear cell adenocarcinoma of human origin (In particular, cells that have been established cell line, specifically, OVTOKO, OVISE, OVMANA, OVSAYO, RMG-I, RMG-II (available from JCRB)) be used preferably.

The contact between the test substance and the clear cell adenocarcinoma cell may be by any method, and examples thereof include a method of adding the test substance to the clear cell adenocarcinoma cell. Alternatively, the test substance may be administered after transplanting clear cell adenocarcinoma cells into a living body such as a mammal other than a human (eg, mouse, rat, guinea pig, rabbit, pig, etc.).
The culture time of clear cell adenocarcinoma cells after contact with the test substance is not particularly limited as long as the test substance has an effect on the expression of the protein in the clear cell adenocarcinoma cells.

The control cells that were not contacted with the test substance to be compared may be clear cell adenocarcinoma cells before the test substance is contacted, or the same treatment was performed except that the test substance was not contacted. It may be a cell adenocarcinoma cell.
As one example of the present invention, in clear cell adenocarcinoma cells contacted with a test substance, the expression level of the protein is decreased at the protein level or nucleic acid level as compared to the control cell, and the test substance is If the test substance can be evaluated to have an effect of decreasing the expression at the protein level or the nucleic acid level, the test substance can be identified as a substance effective for the treatment and / or prevention of clear cell adenocarcinoma.
Furthermore, a step of examining the effect of the test substance on clear cell adenocarcinoma cell proliferation may be included. The effect of the test substance on clear cell adenocarcinoma cell proliferation can be examined, for example, by contacting the test substance with a clear cell adenocarcinoma cell and culturing for a predetermined time, and then measuring the number of living cells.
As one example of the present invention, in a clear cell adenocarcinoma cell contacted with a test substance, when the proliferation of the clear cell adenocarcinoma cell is inhibited as compared with a control cell, the test substance is a clear cell gland. It is believed that the certainty that is effective in the treatment and / or prevention of cancer is increased.

The present invention also provides a method for identifying a substance that decreases the resistance to an anticancer drug of clear cell adenocarcinoma. This method comprises the following steps:
(a) contacting the test substance with clear cell adenocarcinoma cells,
(b) a step of culturing the clear cell adenocarcinoma cells contacted with the test substance in the step (a) for a predetermined time,
(c) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Measuring the expression in clear cell adenocarcinoma cells cultured in step (b) for at least one protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin ,as well as
(d) Evaluating the effect of the test substance on the expression in clear cell adenocarcinoma cells by comparing the expression in clear cell adenocarcinoma cells measured in step (c) with the expression in control cells not contacted with the test substance Process.

The control cells that were not contacted with the test substance to be compared may be clear cell adenocarcinoma cells before the test substance is contacted, or the same treatment was performed except that the test substance was not contacted. It may be a cell adenocarcinoma cell.

As one example of the present invention, in clear cell adenocarcinoma cells contacted with a test substance, the expression level of the protein is decreased at the protein level or nucleic acid level as compared to the control cell, and the test substance is If the test substance can be evaluated to have an effect of decreasing the expression at the protein level or the nucleic acid level, the test substance can be identified as a substance that reduces the resistance to anticancer drugs of clear cell adenocarcinoma.

Furthermore, a step of examining the effect of the test substance on the anticancer drug resistance of clear cell adenocarcinoma cells may be included. The effect of the test substance on the resistance of the clear cell adenocarcinoma cells to the anticancer drug is measured, for example, before or after contacting the clear cell adenocarcinoma cells with the test substance, after adding the anticancer drug and culturing for an appropriate period of time. It can be examined by doing. If the number of viable cells of the clear cell adenocarcinoma cells contacted with the test substance is less than the number of viable cells of the control cells, it can be considered that the resistance of the clear cell adenocarcinoma cells is decreased. it can.
As one example of the present invention, in a clear cell adenocarcinoma cell contacted with a test substance, when the anticancer drug resistance of the clear cell adenocarcinoma cell is reduced as compared with a control cell, the test substance is a clear cell. It is believed that the certainty of reducing the resistance to anticancer drugs in adenocarcinoma will increase.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[Example 1] Proteomic analysis of ovarian clear cell adenocarcinoma cells
Methods In order to search for proteins specifically producing ovarian clear cell adenocarcinoma, first, comprehensive analysis of intracellular proteins was performed using cultured ovarian cancer cells. Ovarian clear cell adenocarcinoma cell lines OVTOKO and OVISE, and mucinous adenocarcinoma cell line MCAS as a comparative control were cultured in RPMI1640 medium containing 10% fetal bovine serum, and each cell was cultured in 7 M urea, 2 M thiourea, 4% Protein was extracted by dissolving in 30 mM Tris-HCl buffer (pH 8.5) containing CHAPS. The obtained protein extract was concentrated using a Microsep 3 K OMEG ultrafiltration membrane (Daiichi Kagaku) and replaced with a 0.5 M trimethylammonium dicarbonate solution. Next, reductive alkylation was performed using 50 mM tris- (2-carboxyethyl) phosphine) and 200 mM methylmethanethiosulfonic acid, followed by trypsin digestion at 37 ° C. for 16 hours. Each obtained tryptic peptide was labeled with iTRAQ reagent (Applied Biosystems). The combination of the mass of each iTRAQ tag and the cells was labeled 115 for the peptide obtained from OVTOKO, 116 for the peptide obtained from OVISE, and 114 and 117 for the peptide obtained from MCAS. After mixing each sample, it was purified by a Sep-Pak C18 column and subjected to analysis by a mass spectrometer. After concentrating the peptide sample labeled with iTRAQ tag with a centrifugal evaporator, cation exchange column (Hi Still SCX 0.8 mm ID x 35 mm, KYA) and reverse layer column (HiQ Still C18-3 W-3 0.15 mm ID x 35) mm, KYA) using 2D nano LC system (Dina-2A, KYA) and direct nano LC / MALDI plate spotting system (Dina Map System, KYA) connected to the target plate simultaneously with sample separation Was dropped. Separation using a cation exchange column is a stepwise method using ammonium formate at concentrations of 25, 50, 75, 100, 150, 200, 300, and 500 mM. Adsorption was performed with 2% acetonitrile containing fluoroacetic acid, followed by a linear concentration gradient of 2.0-80% acetonitrile. Α-Cyano-4-hydroxycinnamic acid was used as a matrix. The peptide mixed with the matrix was dropped onto the target plate, and the protein was identified and quantified using a MALDI-TOF / TOF mass spectrometer AB4800 (Applied Biosystems) and ProteinPilot analysis software.
The protein contained in the culture supernatant was also analyzed in the same manner. Cells were cultured in RPMI1640 medium containing 10% fetal bovine serum in 150 mm culture dishes. After 24 hours, the medium was added to 20 mL of RPMI1640 medium (serum-free medium) containing 4.0 nM epidermal growth factor (Sigma). Exchanged. After further culturing for 2 days, the medium was filtered through a 0.22 μm filter and freeze-dried. The obtained culture supernatant powder was dissolved in 30 mM Tris-HCl buffer (pH 8.5) containing 7 M urea, 2 M thiourea and 4% CHAPS, and the culture supernatant was desalted and concentrated by acetone precipitation. It redissolved in 10 mM triethanolammonium dicarbonate buffer (pH 8.5, Sigma) containing 1% of the surfactant RapiGest. The obtained protein sample was analyzed with a mass spectrometer in the same manner as described above.

Experimental results A total of 1020 proteins were identified from protein extracts from cells. Among them, cell lines derived from clear cell adenocarcinoma compared to MCAS, a non-clear cell adenocarcinoma cell line (OVTOKO) And OVISE) were able to identify 37 proteins that were more than twice as expressed. In addition, as a result of examining the proteins contained in the culture supernatant of these cells, a total of 280 types of proteins were detected and identified, and there were 58 types of proteins detected and identified only from clear cell adenocarcinoma cell groups, Twenty-eight types of proteins classified as extracellular secretion type and cell membrane bound type were included. In order to confirm whether these proteins are actually up-regulated in clear cell adenocarcinoma, we next performed expression analysis using real-time RT-PCR.

[Example 2]
Gene expression levels of identified proteins in clear cell adenocarcinoma-derived cell lines
Experimental method In order to examine the gene expression level of the protein whose expression level was high in clear cell adenocarcinoma cell line group, quantitative RT-PCR analysis was performed. 3 types of cell serous cells (OVCAR-3, OVSAHO, OVKATE), 2 types of mucous (RMUG-S, MCAS), 6 types of clear cells (OVTOKO, OVISE, RMG-I, RMG-II, OVMANA, OVSAYO) Total RNA was extracted from ovarian cancer cell lines using RNeasy spin column (Qiagen). Then, 1.0 μg of total RNA was reverse-transcribed with a random hexamer primer using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), and the resulting cDNA was used with SYBR premix ExTaq II reagent (Takara Bio). An amplification reaction was performed. A real-time PCR analyzer (Agilent P3000) was used for quantification. The primer sequences used are shown below. Tissue factor pathway inhibitor 2 (sense primer: 5'-TTGTTAGCAGGGAGGATTGC-3 '(SEQ ID NO: 1), antisense primer: 5'-TCCGGATTCTACTGGCAAAG-3' (SEQ ID NO: 2)), ceruloplasmin (sense primer: 5'-TGTGGAGAGGAGAACGGAGA -3 ′ (SEQ ID NO: 3), antisense primer: 5′-CTTCATGCCGCCTGTGTAAT-3 ′ (SEQ ID NO: 4)), insulin-like growth factor binding protein 1 (sense primer: 5′-CTGCCAAACTGCAACAAGAA-3 ′ (SEQ ID NO: 5) , Antisense primer: 5'-TATCTGGCAGTTGGGGTCTC-3 '(SEQ ID NO: 6)), doublecortin-containing protein 2 (sense primer: 5'-AGTCTCAAGGAGCTGGCAGTG-3' (SEQ ID NO: 7), antisense primer: 5'-CTAAGCCACGGCAGCATAGTC-3 '(SEQ ID NO: 8)), N-Myc downstream control gene 1 (sense primer: 5'-GTGGAGGGCCTTGTCCTTATC-3' (SEQ ID NO: 9), antisense primer: 5'-TTGATGAACAGGTGCAGGTTG-3 '( SEQ ID NO: 10)), ribonuclease T2 (sense primer: 5'-CGTGACAACCATGAGTGGAA-3 '(SEQ ID NO: 11), antisense primer: 5'-TCGGGCCATAGTCCATGTAT-3' (SEQ ID NO: 12)), growth differentiation factor 15 (sense primer) : 5'-CTCCAGATTCCGAGAGTTGC-3 '(SEQ ID NO: 13), antisense primer: 5'-AGAGATACGCAGGTGCAGGT-3' (SEQ ID NO: 14)), versican core protein (sense primer: 5'-CGTGAGACAGGATGCTTGTG-3 '(SEQ ID NO: 13) 15), antisense primer: 5'-GGTGCACTTTGTGAGCAAGA-3 '(SEQ ID NO: 16)), laminin subunit gamma 1 (sense primer: 5'-CAGCCTTCTTGACCGACTAC-3' (SEQ ID NO: 17), antisense primer: 5'- AGACGCACGTAAGTGATGTC-3 ′ (SEQ ID NO: 18)), Spongin 1 (sense primer: 5′-TCCCAGTGGTCGGAATGTAAC-3 ′ (SEQ ID NO: 19), antisense primer: 5′-CTCCAGCGTAGCTTTTG GATG-3 ′ (SEQ ID NO: 20)), insulin-like growth factor binding protein 7 (sense primer: 5′-CATCCAATTCCCAAGGACAG-3 ′ (SEQ ID NO: 21), antisense primer: 5′-TATAGCTCGGCACCTTCACC-3 ′ (SEQ ID NO: 22) )), Interleukin 6 (sense primer: 5'-TGCCAGTATTCCCAGGAGTC-3 '(SEQ ID NO: 23), antisense primer: 5'-CTCCAGCGTAGCTTTTGGATG-3' (SEQ ID NO: 24)), osteopontin (sense primer: 5'-GCCGAGGTGATAGTGTGGTT -3 ′ (SEQ ID NO: 25), antisense primer: 5′-TGAGGTGATGTCCTCGTCTG-3 ′ (SEQ ID NO: 26)), annexin 4 (sense primer: 5′-TGGCAACCAAAGGAGGTACTG-3 ′ (SEQ ID NO: 27), antisense primer: 5′-TGGTGCTCTTGTAGGCTGTCC-3 ′ (SEQ ID NO: 28)).

Experimental results Among the six types of clear cell adenocarcinoma cell lines, the expression level of five or more cells is higher than that of four or more non-clear cell adenocarcinoma-derived cell lines. 13 proteins, tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin sub Unit gamma 1, sponge 1, insulin-like growth factor binding protein 7, interleukin 6, and osteopontin were newly found (FIG. 1).

[Example 3]
Comparison of expression levels of laminin subunit gamma 1, N-Myc downstream regulatory gene 1, doublecortin-containing protein 2, sponge 1, osteopontin, and ribonuclease T2 by Western blotting
Experimental method To confirm whether the identified protein is really up-regulated in clear cell adenocarcinoma cell lines, laminin subunit gamma 1, N-Myc downstream regulatory gene 1, doublecortin-containing protein 2, sponge 1 The protein expression levels of osteopontin and ribonuclease T2 were examined by Western blotting. The cell extract was developed on SDS-PAGE with an acrylamide concentration of 12.5% and then transferred to a PVDF membrane. After blocking with a blocking one reagent (Nacalai Tesque), it was reacted with an antibody against the target protein. After reacting with a secondary antibody labeled with horseradish peroxidase, the protein-antibody complex was detected with ECL plus reagent. The primary antibodies used are shown below. Anti-doublecortin-containing protein 2 goat antibody (IMGENEX), anti-N-Myc downstream regulatory gene 1 rabbit antibody (ZYMED), anti-ribonuclease T2 rabbit antibody (Proteintech), anti-laminin subunit gamma 1 goat antibody (Santa Cruz), anti-osteopontin mouse Antibody (R & D Systems).

Experimental results Double-cortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, laminin subunit gamma 1, sponge 1, and osteopontin protein levels are similar to those of annexin 4 known so far, clear cell adenocarcinoma cells A significant increase was observed in the strain group (Fig. 2).

Example 4
Gene expression levels of 13 protein groups in clear cell adenocarcinoma tissues
Experimental method In order to confirm whether 13 types of proteins that were highly expressed in cell lines derived from cell adenocarcinomas were also expressed in ovarian cancer tissues, 5 clear cell adenocarcinomas, 4 non-clear cell line carcinomas Total RNA was extracted from cases, 4 benign tumors, and tissue samples using an RNeasy spin column (Qiagen). In addition, total RNA from 2 healthy ovarian tissues was purchased from Ambion and Ziagen. Then, 1.0 μg of total RNA was reverse-transcribed with a random hexamer primer using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), and the resulting cDNA was used with SYBR premix ExTaq II reagent (Takara Bio). An amplification reaction was performed. A real-time PCR analyzer (Agilent P3000) was used for quantification. The primers used in Example 2 were used.

Experimental results As previously reported, the expression level of annexin A4 gene was confirmed to be very high in clear cell adenocarcinoma tissues compared to normal tissues, benign tumor tissues, and non-clear cell line cancer tissues. Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit Gamma 1, insulin-like growth factor binding protein 7, and osteopontin are also found to be significantly higher expressed in clear cell adenocarcinoma tissue type specimens than in healthy, benign tumors, and non-clear cell carcinoma ovarian tissues. It was. Moreover, about interleukin 6, it was shown that the expression level in clear cell adenocarcinoma is significantly high compared with non-clear cell line carcinoma.

Example 5
Detection and quantification of growth differentiation factor 15, osteopontin, and insulin-like growth factor binding protein 1 in serum of ovarian cancer patients

Experimental method Among the identified proteins, serum of ovarian cancer patients with growth differentiation factor 15, osteopontin, and insulin-like growth factor binding protein 1 (obtained from the Yokohama City University Hospital and Genomic Collaborative Research Human Sample Bank) The concentration was measured using a commercially available ELISA kit. 12 control groups (5 healthy subjects, 7 benign ovarian tumors), 5 non-clear cell adenocarcinoma patient groups (serous 3 adenocarcinoma, 2 mucinous adenocarcinoma), 5 clear cell adenocarcinoma patients A total of 22 cases in the group were included. Quantkine Human GDF-15 Immunoassay Kit (R & D Systems) for growth differentiation factor 15, Human Osteopontin N-Half Assay Kit (IBL) for osteopontin, and IGFBP-1 ELISA kit (Mediagnost) for insulin-like growth factor binding protein 1 Measured and compared between groups.

The experimental result is shown in FIG. Growth differentiation factor 15 was relatively high in the clear cell patient group compared to the control group (Control), and a higher tendency was observed compared to the non-clear cell adenocarcinoma patient group. The concentration of osteopontin was very high in the non-clear cell adenocarcinoma patient group, but the clear cell adenocarcinoma patient group also showed a relatively high tendency compared to the control group. As for insulin-like growth factor binding protein 1, patients detected at very high values were present in both groups of clear cell adenocarcinoma and non-clear cell adenocarcinoma patients. From these results, the protein group can be used as a marker for diagnosing clear cell adenocarcinoma. In particular, growth differentiation factor 15 is effective in identifying clear cell adenocarcinoma patients among ovarian cancer patients. It was shown that.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

The clear cell adenocarcinoma evaluation and / or differentiation method of the present invention can be used for diagnosis of clear cell adenocarcinoma. In addition, the substance identification method of the present invention can be used for searching for therapeutic agents for clear cell adenocarcinoma.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the DNA sequence of the tissue factor pathway inhibitor 2 sense primer.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the DNA sequence of the tissue factor pathway inhibitor 2 antisense primer.
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the DNA sequence of the ceruloplasmin sense primer.
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the DNA sequence of ceruloplasmin antisense primer.
<SEQ ID NO: 5>
SEQ ID NO: 5 shows the DNA sequence of insulin-like growth factor binding protein 1 sense primer.
<SEQ ID NO: 6>
SEQ ID NO: 6 shows the DNA sequence of the insulin-like growth factor binding protein 1 antisense primer.
<SEQ ID NO: 7>
SEQ ID NO: 7 shows the DNA sequence of doublecortin-containing protein 2 sense primer.
<SEQ ID NO: 8>
SEQ ID NO: 8 shows the DNA sequence of doublecortin-containing protein 2 antisense primer.
<SEQ ID NO: 9>
SEQ ID NO: 9 shows the DNA sequence of the N-Myc downstream control gene 1 sense primer.
<SEQ ID NO: 10>
SEQ ID NO: 10 shows the DNA sequence of the N-Myc downstream control gene 1 antisense primer.
<SEQ ID NO: 11>
SEQ ID NO: 11 shows the DNA sequence of ribonuclease T2 sense primer.
<SEQ ID NO: 12>
SEQ ID NO: 12 shows the DNA sequence of ribonuclease T2 antisense primer.
<SEQ ID NO: 13>
SEQ ID NO: 13 shows the DNA sequence of growth differentiation factor 15 sense primer.
<SEQ ID NO: 14>
SEQ ID NO: 14 shows the DNA sequence of growth differentiation factor 15 antisense primer.
<SEQ ID NO: 15>
SEQ ID NO: 15 shows the DNA sequence of a versican core protein sense primer.
<SEQ ID NO: 16>
SEQ ID NO: 16 shows the DNA sequence of a versican core protein antisense primer.
<SEQ ID NO: 17>
SEQ ID NO: 17 shows the DNA sequence of laminin subunit gamma 1 sense primer.
<SEQ ID NO: 18>
SEQ ID NO: 18 shows the DNA sequence of laminin subunit gamma 1 antisense primer.
<SEQ ID NO: 19>
SEQ ID NO: 19 shows the DNA sequence of sponge 1 sense primer.
<SEQ ID NO: 20>
SEQ ID NO: 20 shows the DNA sequence of the sponge 1 antisense primer.
<SEQ ID NO: 21>
SEQ ID NO: 21 shows the DNA sequence of insulin-like growth factor binding protein 7 sense primer.
<SEQ ID NO: 22>
SEQ ID NO: 22 shows the DNA sequence of insulin-like growth factor binding protein 7 antisense primer.
<SEQ ID NO: 23>
SEQ ID NO: 23 shows the DNA sequence of the interleukin 6 sense primer.
<SEQ ID NO: 24>
SEQ ID NO: 24 shows the DNA sequence of the interleukin 6 antisense primer.
<SEQ ID NO: 25>
SEQ ID NO: 25 shows the DNA sequence of osteopontin sense primer.
<SEQ ID NO: 26>
SEQ ID NO: 26 shows the DNA sequence of osteopontin antisense primer.
<SEQ ID NO: 27>
SEQ ID NO: 27 shows the DNA sequence of annexin 4 sense primer.
<SEQ ID NO: 28>
SEQ ID NO: 28 shows the DNA sequence of annexin 4 antisense primer.

Claims

Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1, spongin 1. Evaluation and / or differentiation of clear cell adenocarcinoma comprising measuring expression in a biological sample for at least one protein selected from the group consisting of insulin-like growth factor binding protein 7, interleukin 6 and osteopontin Method.
The method according to claim 1, wherein expression in the biological sample is measured at the protein level.
The method according to claim 1, wherein the expression in the biological sample is measured at the nucleic acid level.
The method according to any one of claims 1 to 3, wherein the biological sample is a cell, tissue or body fluid obtained from a subject.
The method according to claim 4, wherein the body fluid is blood.
6. The method according to claim 5, wherein the blood is whole blood, serum, plasma, or plasma exchange fluid.
Clear cell adenocarcinoma when expression in cells, tissues or body fluids obtained from a subject is confirmed to be at least a 2-fold increase in protein level compared to expression in cells, tissues or body fluids obtained from healthy subjects The method according to any one of claims 1 to 6, wherein the method is evaluated as having clear cell adenocarcinoma or having a cancer tissue type.
Clear cell adenocarcinoma when expression in a cell, tissue or body fluid obtained from a subject is confirmed to be at least twice as high at the nucleic acid level as compared to expression in a cell, tissue or body fluid obtained from a healthy subject The method according to any one of claims 1 to 6, wherein the method is evaluated as having clear cell adenocarcinoma or having a cancer tissue type.
Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1, spongin 1. Evaluation of clear cell adenocarcinoma comprising a reagent capable of measuring expression in a biological sample for at least one protein selected from the group consisting of insulin-like growth factor binding protein 7, interleukin 6 and osteopontin; // Kit for identification.
The kit according to claim 9, wherein the reagent capable of measuring expression in a biological sample is any of the following (i), (ii), or (iii).
(i) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Antibody capable of specifically recognizing a protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin
(ii) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1, a nucleic acid probe capable of specifically hybridizing with mRNA encoding a protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin
(iii) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1, at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using mRNA encoding a protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin
A method for identifying a substance effective for the treatment and / or prevention of clear cell adenocarcinoma comprising the following steps:
(a) contacting the test substance with clear cell adenocarcinoma cells,
(b) a step of culturing the clear cell adenocarcinoma cells contacted with the test substance in the step (a) for a predetermined time,
(c) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Measuring the expression in clear cell adenocarcinoma cells cultured in step (b) for at least one protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin ,as well as
(d) Evaluating the effect of the test substance on the expression in clear cell adenocarcinoma cells by comparing the expression in clear cell adenocarcinoma cells measured in step (c) with the expression in control cells not contacted with the test substance Said method comprising the steps.
The method according to claim 11, further comprising the step of examining the effect of the test substance on clear cell adenocarcinoma cell proliferation.
A method for identifying a substance that reduces anticancer drug resistance of clear cell adenocarcinoma comprising the following steps:
(a) contacting the test substance with clear cell adenocarcinoma cells,
(b) a step of culturing the clear cell adenocarcinoma cells contacted with the test substance in the step (a) for a predetermined time,
(c) Tissue factor pathway inhibitor 2, ceruloplasmin, insulin-like growth factor binding protein 1, doublecortin-containing protein 2, N-Myc downstream regulatory gene 1, ribonuclease T2, growth differentiation factor 15, versican core protein, laminin subunit gamma 1. Measuring the expression in clear cell adenocarcinoma cells cultured in step (b) for at least one protein selected from the group consisting of sponge 1, insulin-like growth factor binding protein 7, interleukin 6 and osteopontin ,as well as
(d) Evaluating the effect of the test substance on the expression in clear cell adenocarcinoma cells by comparing the expression in clear cell adenocarcinoma cells measured in step (c) with the expression in control cells not contacted with the test substance Said method comprising the steps.
Furthermore, the method of investigating the effect of the to-be-tested substance with respect to the anticancer agent tolerance of clear cell adenocarcinoma cell.
The method of claim 1, wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.
The kit according to claim 9, wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.
12. The method of claim 11, wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.
14. The method of claim 13, wherein the protein is selected from the group consisting of growth differentiation factor 15, osteopontin and insulin-like growth factor binding protein 1.