JPWO2006022114A1 - Brain tumor detection method, brain tumor detection substance used therefor, and pharmaceutical composition - Google Patents

Abstract

A method for detecting a brain tumor capable of reliably and early detecting a brain tumor and a brain tumor detection material used therefor are provided. Further provided are pharmaceutical compositions for treating brain tumors. Detection of a brain tumor based on detection of an N-linked sugar chain A2G2F of a glycoprotein specifically expressed in the brain tumor is beneficial for early detection and treatment of the brain tumor. Furthermore, a brain tumor can be reliably and early detected using a lectin having binding specificity for the N-linked sugar chain A2G2F. Moreover, since the brain tumor detection substance has a lectin that specifically binds to the N-linked sugar chain A2G2F of the glycoprotein, the brain tumor can be detected early and easily based on the sugar chain A2G2F. Furthermore, since the pharmaceutical composition containing a lectin having binding specificity for the N-linked sugar chain A2G2F of the glycoprotein as an active ingredient induces apoptosis of brain tumor cells, the prevention of diseases caused by brain tumor cells and It is effective for treatment.

Description

  The present invention relates to a brain tumor detection method, a brain tumor detection substance used therefor, and a pharmaceutical composition.

  In recent years, it has become known that sugar chains are bound to many proteins, and sugar chains bound to proteins play an important role in cell-to-cell recognition, cell adhesion, and infection of viruses and microorganisms to hosts. In addition to the above, it is becoming clear that it is also acting as a signal for protein transport and an auxiliary factor in the formation of protein three-dimensional structure.

  In addition, the sugar chain of glycoconjugates (glycoproteins, glycolipids, proteoglycans) is attracting attention as a third life chain after proteins and nucleic acids. In particular, it has been reported that glycoprotein sugar chains are essential for the functional expression of the glycoprotein. For example, a glycoprotein called P0 in the peripheral nervous system myelin functions as an intercellular adhesion factor, and it is known that the intercellular adhesion activity is lost when a modification is made to the sugar chain binding site of P0. This is not because sugar chains had adhesive activity, but because the sugar chains that existed to support the protein structure on the cell membrane disappeared, P0 could not maintain the conformation necessary for adhesion. It is considered. In this way, molecules with glycoprotein sugar chains play an extremely important role in the development of the nervous system and the recognition between cells in various other regions. Sex is recognized. However, purification of glycoprotein sugar chains requires a huge amount of time, and there is no systematic analysis method for glycoprotein sugar chains expressed on cell surfaces and tissues. The elucidation has not progressed much.

Therefore, Ikenaka et al., One of the present inventors, developed a systematic analysis method for glycoprotein sugar chains by automation and simplification using an HPLC system. And it discovered that Triantennary trigalactosylated structure with one outer arm fucosylation (A3G3F0) which is one kind of sugar chain increased in lung cancer patient serum by this method (Non-patent Documents 1 and 2). Patent Document 1 discloses a cancer detection method for detecting cancer, particularly lung cancer based on the N-linked sugar chain A3G3F0 of glycoprotein, and a cancer detection substance used therefor. Furthermore, Non-Patent Document 3 describes the expression of ST8Sia II (STX) gene that regulates polysialic acid, which is highly expressed in metastatic lung cancer when the expression of polysialic acid present on nerve cell adhesion molecule (N-CAM) is high. It has been reported that there is a correlation between the malignancy of tumors.
JP 2001-289860 A J. et al. Biochem. 129: 537-542, 2001 Anal. Biochem. 267: 336-343, 1999 Cancer Res., 61: 1666-1670, 2001

  However, the above-mentioned patent documents and non-patent documents disclose only glycoprotein sugar chains specific to lung cancer, and none of the glycoprotein sugar chains in cancers of other organs. In particular, tumors in the brain, unlike cancers of other organs such as the lungs, exist in a special organ called the brain. Depending on the site of occurrence and occupation, the function of the brain, which has an important center for living, is impaired. Was causing serious symptoms. From such a background, there has been a demand for a method for analyzing a sugar chain abnormality due to a disease state in the brain and detecting a brain tumor reliably and at an early stage.

  Diagnosis of a brain tumor is performed by image diagnosis such as a CT scan or a nuclear magnetic resonance apparatus, and the discovery of a detection substance that plays a role as a tumor marker specific to a brain tumor has been expected.

  On the other hand, brain tumor treatment is based on the complete removal of the entire tumor, but in malignant brain tumors, the boundaries are not clear, so it is often impossible to remove the tumor completely, and combined with radiotherapy, chemotherapy, and immunotherapy. is doing. Antibiotics such as mitomycin C and bleomycin are known as agents that inhibit the growth of tumor cells and treat diseases caused by tumor cells. Thus, it causes necrosis, that is, necrosis, and eliminates pathological cells. However, this cell death due to necrosis is not sufficiently effective due to strong side effects on normal cells or living organisms, and there is a serious defect that normal cells also cause new diseases. On the other hand, cell death, or apoptosis, programmed to actively evoke cells under physiological conditions, without affecting the surrounding cells, cell surface curvature, nuclear chromatin condensation, chromosomal DNA It is a phenomenon in which cells die due to unique morphological abnormalities such as fragmentation, and if this can be induced, it is very advantageous in the treatment of diseases such as malignant tumors.

  Therefore, an object of the present invention is to provide a brain tumor detection method capable of reliably and early detecting a brain tumor and a brain tumor detection material used therefor. Another object of the present invention is to provide a pharmaceutical composition for inducing apoptosis of brain tumor cells and effectively preventing and treating brain tumors.

  As a result of intensive studies in view of the above problems, it has been reported that the expression pattern of N-linked sugar chains in normal brain tissue is preserved without any solid difference (Non-Patent Document 1). When the expression patterns of N-linked sugar chains with glioma cells were compared, it was noted that specific ones of N-linked sugar chains of glycoproteins increased in glioma cells. A conjugated sugar chain was identified. The inventors have found that the identified N-linked sugar chain A2G2F is not expressed in normal tissues but is expressed only in glioma cells. Furthermore, the inventors have found that lentil (LCA) lectin induces apoptosis of glioma cells and arrived at the present invention.

  The method for detecting a brain tumor according to claim 1 of the present invention is characterized in that a brain tumor is detected based on detection of an N-linked sugar chain A2G2F of a glycoprotein.

  The method for detecting a brain tumor according to claim 2 of the present invention is characterized in that, in claim 1, the lectin having binding specificity to the N-linked sugar chain A2G2F is used.

  The brain tumor detection method according to claim 3 of the present invention is characterized in that, in claim 2, the lectin is a lentil lectin.

  The method for detecting a brain tumor according to claim 4 of the present invention is characterized in that, in claim 1, the brain tumor is a glioma.

  The brain tumor detection substance according to claim 5 of the present invention is characterized by containing a lectin having binding specificity for the N-linked sugar chain A2G2F of glycoprotein.

  The brain tumor detection substance according to claim 6 of the present invention is characterized in that, in claim 5, the lectin is a lentil lectin.

  The pharmaceutical composition according to claim 7 of the present invention is a pharmaceutical composition for inducing apoptosis of brain tumor cells, and has a binding specificity for N-linked sugar chain A2G2F of glycoprotein. As an active ingredient.

  The pharmaceutical composition according to claim 8 of the present invention is characterized in that, in claim 7, the lectin is a lentil lectin.

  The pharmaceutical composition according to claim 9 of the present invention is characterized in that, in claim 7, the brain tumor cell is a glioma cell.

  According to the method for detecting a brain tumor described in claim 1 of the present invention, the brain tumor can be reliably and early detected based on the N-linked sugar chain A2G2F of the glycoprotein specifically expressed in the brain tumor. Can do. Furthermore, it is useful for the early detection and treatment of brain tumors.

  According to the method for detecting a brain tumor according to claim 2 of the present invention, a brain tumor is reliably and early detected using the lectin having binding specificity to the N-linked sugar chain A2G2F. be able to. Furthermore, it is useful for the early detection and treatment of brain tumors.

  According to the method for detecting a brain tumor according to claim 3 of the present invention, glioma can be detected using the lentil lectin having binding specificity to the N-linked sugar chain A2G2F. Furthermore, it is useful for the early detection and treatment of glioma.

  According to the method for detecting a brain tumor according to claim 4 of the present invention, glioma can be reliably and early detected based on the N-linked sugar chain A2G2F.

  According to the substance for detecting brain tumor according to claim 5 of the present invention, since the lectin having binding specificity to the N-linked sugar chain A2G2F of glycoprotein is contained, the brain tumor is detected based on the sugar chain A2G2F. It can be detected. Moreover, a brain tumor can be detected earlier and more easily by using the detection substance of the present invention.

  According to the brain tumor detection substance according to claim 6 of the present invention, since it contains lentil lectin having binding specificity to the N-linked sugar chain A2G2F of the glycoprotein, the brain tumor is based on the sugar chain A2G2F. Can be detected. Moreover, a brain tumor can be detected earlier and more easily by using the detection substance of the present invention.

  According to the pharmaceutical composition of claim 7 of the present invention, since it has a cell growth inhibitory activity based on the induction of apoptosis, only the growth of brain tumor cells can be selectively performed without affecting surrounding normal cells. Since it can inhibit, it is effective in the prevention and treatment of diseases caused by brain tumor cells.

  According to the pharmaceutical composition of claim 8 of the present invention, apoptosis can be selectively induced to brain tumor cells.

  According to the pharmaceutical composition of claim 9 of the present invention, glioma cells can be selectively eliminated by induction of apoptosis.

2 is a two-dimensional map of mannose units versus glucose units of a standard sugar chain. It is a figure which shows the structural formula of a sugar chain. GluNAc is an abbreviation for N-acetylglucosamine, Man is mannose, Gal is galactose, and Fuc is fucose. In addition, the meaning of each symbol in the structural formula is as follows: An: number of GlcNAc antennas binding to the M3 sugar chain structure, Gn: number of galactose residues attached to the non-reducing end, F: reduction One having fucose bonded to the terminal GluNAc residue, F0: one having fucose α1-3 bonded outside GlcNAc, B: one having GlcNAc bonded to the middle mannose of the M3 sugar chain structure. It is a reverse phase HPLC elution pattern figure in Example 1 of this invention. A shows normal tissue and B shows glioblastoma. In addition, the numbers described in the peaks in the figure correspond to the sugar chain numbers described in FIGS. It is a microscope picture which shows the result of the immunohistochemical dyeing | staining in Example 2 of this invention. A shows a glioma cell line and B shows a glioblastoma tissue. It is a graph which shows the result of the average survival rate of U251, T98G, ON-12 cell when LCA lectin in Example 3 of this invention is added. It is a microscope picture which shows the result of having observed the aggregation of the chromatin of the apoptotic cell in Example 3 of this invention under the fluorescence microscope. A shows no addition of LCA lectin, and B shows addition of LCA lectin. It is a graph which shows the detection result of hypodiploid DNA by the flow cytometry method in Example 3 of this invention. A shows no addition of LCA lectin, and B shows addition of LCA lectin. It is a graph which shows the detection result of the apoptotic cell by the flow cytometry method using the MEBSTAIN apoptosis kit in Example 3 of this invention. A shows no addition of LCA lectin, and B shows addition of LCA lectin. It is a graph which shows the detection result of the activated caspase-3 by the flow cytometry method in Example 3 of this invention. A shows T98G cells, B shows U251 cells, and C shows ON12 cells.

  Hereinafter, the present invention will be described in detail.

  A2G2F in the present invention is a glycoprotein sugar chain (Galβ1,4-GlcNAcβ1,2-Manα1,3-) (Galβ1,4-GlcNAcβ1,2-Manα1,6-) Manβ1,4-GlcNAcβ1,4- (Fucα1 , 6-) GlcNAc, and the meaning of each symbol A2G2F is as follows. An: number of GlcNAc antennas that bind to the M3 sugar chain structure, Gn: number of galactose residues attached to the non-reducing end, and F: fucose attached to the GluNAc residue at the reducing end.

  The brain tumor that can be detected by the detection method of the present invention is a tumor that occurs in a tissue inside the skull, such as the brain and surrounding tissues of the brain, and a “primary brain tumor” that occurs from the brain tissue itself. Examples include “metastatic brain tumors” in which cancers of other organs metastasize to the brain. Examples of primary brain tumors include glioma, which accounts for about 30% of primary brain tumors, and glioblastoma with the highest malignancy among gliomas.

  In the method for detecting a brain tumor of the present invention, an N-linked sugar chain of an extracted brain tissue is analyzed by a glycoprotein sugar chain system using HPLC, and an N-linked sugar chain A2G2F of a glycoprotein peculiar to a brain tumor is detected. Is a method of detecting a brain tumor based on the above. Further, the present invention is a method for detecting a brain tumor using a lectin having binding specificity for the N-linked sugar chain A2G2F.

  The lectin used in the brain tumor detection method of the present invention is a substance having a specific binding activity to sugar among proteins or glycoproteins present in plants, animals and microorganisms. As the lectin used in the brain tumor detection method of the present invention, for example, a lentil (LCA) lectin is preferable. This lentil lectin is known to specifically bind to α-Fuc bound to the C-6 position of the sugar chain (Asn type (N type) sugar chain) GlcNAc residue bound to the asparagine of the polypeptide chain. Yes. In glioblastoma cells and glioblastoma tissues, only A2G2F has a fucosylated α1-6GlcNAc structure to which LCA lectin specifically binds among the sugar chain structures present in 0.1% or more. .

  By the method for detecting a brain tumor of the present invention, a glioma can be detected with high accuracy, and further, a brain tumor such as glioma or glioblastoma by using a lectin (LCA lectin) having binding specificity to A2G2F. Can be detected earlier and more easily. In addition, brain tumors such as glioma and glioblastoma may be detected earlier and easily by using an antibody that specifically recognizes A2G2F.

  The substance for detecting a brain tumor of the present invention is not particularly limited as long as it contains a lectin that specifically binds to the N-linked sugar chain A2G2F of glycoprotein. Examples of the lectin that specifically binds to the N-linked sugar chain A2G2F include LCA lectin. Furthermore, the substance for detecting a brain tumor of the present invention may comprise an antibody that specifically recognizes an N-linked sugar chain A2G2F. Examples of antibodies that recognize A2G2F include monoclonal antibodies and polyclonal antibodies. These antibodies can be produced by administering A2G2F sugar chains to experimental animals using conventional protocols.

  In addition, a lectin that specifically binds A2G2F or an antibody that specifically recognizes A2G2F can be labeled with a chemical fluorescent reagent, a radioisotope, or the like. Examples of the chemical fluorescent reagent include fluorescein, rhodamine and luminol, and examples of the radioisotope include 3H, 14C, 35S, 33P, 32P and 125I. With this label, A2G2F can detect or diagnose brain tumors early and reliably with high accuracy by using diagnostic methods based on image diagnosis such as conventional CT scans and nuclear magnetic resonance devices as brain tumor markers, for example. It is.

  The pharmaceutical composition of the present invention is a pharmaceutical composition that induces apoptosis of brain tumor cells, and contains, as an active ingredient, a lectin having binding specificity for the N-linked sugar chain A2G2F of glycoprotein. The lectin used as an active ingredient of the present invention is a substance having a specific binding activity to sugar among proteins or glycoproteins present in plants, animals and microorganisms. Moreover, as a lectin used as an active ingredient of this invention, a lentil (LCA: Lens culinaris Agglutinin) lectin is preferable.

  The pharmaceutical composition of the present invention may be used as a pharmaceutical composition for inducing apoptosis in combination with a pharmaceutical agent or a pharmaceutical ingredient depending on the purpose of use. Examples of the pharmaceutical agent used in combination include an anticancer agent, an antiviral agent, and an antiautoimmune disease agent. Thus, the pharmaceutical composition of the present invention is effective for the prevention and treatment of diseases caused by brain tumor cells by encapsulating a pharmaceutical agent such as an anticancer agent in the pharmaceutical composition, thereby concentrating the drug locally.

  In addition, the pharmaceutical composition of the present invention is used in various dosage forms conventionally used in this field as a pharmaceutical preparation. For example, it can be formulated in the form of powder, tablets, pills, solutions, suspensions, emulsions, granules, capsules, injections (solutions, suspensions, etc.), and solubilizing agents as desired Additives commonly used in injection solutions such as buffers, isotonic agents, stabilizers, preservatives, soothing agents, fillers, extenders, binders, moisturizers, disintegrants, surface active agents, Or an excipient | filler etc. may be used together. Furthermore, the pharmaceutical composition of the present invention is administered orally or parenterally. The dosage is increased or decreased depending on the degree of brain tumor to be treated and is not particularly limited.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

(Expression analysis of N-linked glycoprotein sugar chain in glioblastoma tissue and glioma cell line)
Glycoprotein sugar chain analysis was performed using 3 normal brain tissues, 15 glioblastoma tissues, and 3 glioma cell lines. Three normal brain tissues (male: female = 2: 1, mean age 55.2) as controls were collected with informed consent from patients undergoing epileptic surgery. In addition, 15 glioblastoma tissues were collected by obtaining informed consent from patients with glioblastoma (see Table 1) at Niigata University Hospital.

First, as pretreatment of the specimen, brain tissue was perfused with cold PBS, and its weight was measured. Then, 9 times the amount of acetone at −20 ° C. was added. PBS prepared by dissolving NaCl 8 g, KCl 0.2 g, Na ₂ HPO ₄ 1.44 g, KH ₂ PO ₄ 0.24 g in 1 liter of deionized water to a pH of 7.4 was used. The brain tissue was homogenized in 9 times amount of cold acetone using a polytron homogenizer, left at −20 ° C. for 60 minutes, and then centrifuged at 4 ° C. and 8000 G for 20 minutes. After removing the supernatant, the same operation was performed again, and then the precipitate was lyophilized to obtain a specimen. A sample having a dry weight of 2 mg was subjected to hydrazine decomposition using GlycoPrep ^™ 1000 (Oxford Glycosystems, Oxford, UK) to separate sugar chains from the protein. Next, after the separated sugar chain is fluorescently labeled by pyridylamination using GlycoTag ^™ (Takara, Tokyo, Japan), the sugar chain is neuraminidase (derived from Arthrobacter ureafaciens, Nacalai Tesque, Kyoto, Japan). To cleave the sialic acid in the side chain. As a result of the cleavage of the charged sialic acid, neutral sugar chains present in the brain tissue of the specimen could be obtained.

  In order to classify sugar chains of various sizes according to their size, samples containing the above-mentioned neutral sugar groups labeled with fluorescence were analyzed by normal phase HPLC (Asahipak NH2P-50 size-fractionation column (4.6 × 50 mm; Shodex, Tokyo, Japan)), and the amount of sugar chain was detected by a fluorescence detector and measured. The measurement conditions of normal phase HPLC are described in detail. The flow rate is 0.6 ml / min, the column temperature is 30 ° C., the solvent A contains 20% acetonitrile and 0.3% acetic acid, and the pH is adjusted to 7.0 with triethylamine. As water, solvent B contains 93% acetonitrile and 0.3% acetic acid, and water adjusted to pH 7.0 with triethylamine was first started at a solvent A: solvent B ratio of 97: 3. Was increased linearly to 19% after 1 minute and 57% after 35 minutes. The fluorescently labeled sugar chains eluted from this are detected at an excitation wavelength of 310 nm and a detection wavelength of 380 nm, and standard sugar chains (PA-sugar chains M2A, M3B, M4B, M5A, M6B, M7A, M8A, M9A (Takara, Tokyo, The fractions M2 to M10 were collected according to the elution time of Japan)). The M10 elution time was set as M9A elution time +1.5 minutes.

  In order to classify the saccharides separated by size by normal phase HPLC, this time according to the structural characteristics of the glycan molecule, Cosmosil 5C18-P reversed-phase column (4.6 × 150 mm; Nacali Tesque) ) As the HPLC system, PU-2089 plus HPLC pump (Nihon Bunkou, Tokyo, Japan), FP-2055 plus fluorescence detector (Nihon Bunkou), and AS-2055 plus autosampler (Nihon Bunkou) were used. Jasco Bowin (Nihon Bunkou) was used for the control system and the analysis of peak elution time and relative content. The measurement conditions of the reverse phase HPLC are described in detail. The flow rate is 1.5 ml / min, the column temperature is 30 ° C., the solvent C is 0.1 M ammonium acetate buffer (pH 4.0), and the solvent D is 0 in the solvent C. Assuming 5% 1-butanol was added, the solvent C: solvent D ratio was initially started at 94: 6, and solvent D was increased linearly to 80% 45 minutes after loading the sample. . The fluorescently labeled sugar chains eluted from this were detected at an excitation wavelength of 320 nm and a detection wavelength of 400 nm, and the elution patterns were compared and examined by the following procedure to determine the structure and content of sugar chains in the specimen.

  Before the sample is put into the reverse phase HPLC, a glucose oligomer (standard sugar chain to which 3 to 22 glucoses are bound) is put in advance and its elution time is measured, and the internal standard of the sample elution time is measured. And Calculate how much the peak of the sample is comparable to the internal standard and express it as a glucose unit (GU). For each peak observed in reverse phase HPLC, the sugar chain contained in that peak is collected and once again put into normal phase HPLC. In the same way as GU, this time calculate mannose unit (MU), and for each peak recognized by reversed-phase HPLC, GU on the horizontal axis and MU on the vertical axis using two indicators, GU and MU. Plot it on a two-dimensional graph. This method of distinguishing the characteristics of sugar chains in two dimensions is performed on sugar chains of known structure (purchased from Takara, Seikagaku Corporation (Tokyo, Japan), Oxford GlycoSystems), and then sugar chains derived from brain tissue. The structure was identified by whether or not the points overlap on the two-dimensional map. FIG. 1 is a two-dimensional map of standard sugar chains, where the horizontal axis indicates glucose unit values and the vertical axis indicates mannose unit values. FIG. 2 shows the sugar chain structural formulas of the numbers plotted on the graph of FIG. That is, number 1 is A0G0F, 2 is A2G1F0 (3) FB, 3 is A2G1F0 (6) FB, 4 is A2G2, 5 is A2G2F, 6 is A3G3, 7 is BA2, 8 is M2B, 9 is M3B, 10 is M4B , 11 is M5A, 12 is M6B, 13 is M7A, 14 is M7B, 15 is M8A, and 16 is M9A.

  FIG. 3 shows the elution patterns of all fractions (M2 to M11) in reversed-phase HPLC analysis of N-linked sugar chains of human normal brain tissue (A) and glioblastoma tissue (B). Peak 5 was confirmed only in the M6 and M7 fractions of glioblastoma tissue (B) and not in human normal brain tissue (A). This peak 5 was identified as sugar chain A2G2F by the two-dimensional map shown in FIG. From this, it was found that sugar chain A2G2F is a glycoma-specific sugar chain that is not expressed in normal brain tissue but is expressed only in glioma cells.

  In addition, separation of normal brain tissue (Normal Brain), glioblastoma tissue (GBM), and glioma cell line (Cell Line), and the content (%) of N-linked sugar chain of the identified glycoprotein Table 2 shows.

  In normal brain tissue, an average of 46.4% of all N-linked sugar chains was identified. In addition, when the number of branches was taken into consideration, sugar chains having a 2-branched structure were most frequently observed at 6.27%, but hardly branched sugar chains having 3-branched structures and 4-branched structures were observed. . In the oligomannose series, M5A was observed most frequently, followed by M6B and M9A. Furthermore, 2.78% was observed in A2G2.

  A2G2F was not found in normal brain tissue, but was found in glioblastoma cell lines and glioblastoma tissue (p <0.01). Moreover, it confirmed that it became a single peak by co-injection with a standard sample (A2G2F). A2G2F was contained in 2.9 ± 1.93% in glioblastoma tissues and 5.60 ± 1.14% in glioma cell lines. Ion exchange HPLC was performed on two glioblastoma tissues, and the sialylation rate of A2G2F was determined to be 62.20% and 56.19%. A2G2 was found to be 12.66 ± 8.33% in glioblastoma tissue and was significantly higher than 2.78 ± 0.60% in normal brain tissue. A3G3 was found to be 1.00 ± 0.79% in glioblastoma tissue and 3.51 ± 2.13% in glioma cell lines. The hyperbranched glycan observed in lung cancer and hepatocellular carcinoma was not found in the glioblastoma tissue.

(Lectin staining)
As a result of HPLC, lectin staining was performed on A2G2F that was contained in glioblastoma and was not detected in normal brain. Since the fucosylated α1-6GlcNAc structure to which LCA lectin specifically binds in cells was only A2G2F among the sugar chain structures present in 0.1% or more, it was changed to the core α1,6-fucosylation structure. LCA (Lens bean lectin; Seikagaku Corporation, Tokyo, Japan) that specifically binds was used.

  According to the following procedure, staining with LCA (lens bean lectin) was performed on glioblastoma tissue (n = 10), three types of glioma cell lines, and normal brain tissue (n = 3) as a control, and observed with a microscope. did. First, using frozen sections of each sample, LCA-FITC (Seikagaku Corporation, Tokyo, Japan; 500 μg / ml: diluted 1:50) labeled with a fluorescent reagent FITC was added, reacted for 20 minutes, and then fluorescence microscope Observed. In addition, non-immune mouse immunoglobulin was used as a negative control for sugar chain A2G2F.

  The results are shown in FIG. FIG. 4-A shows immunohistochemical staining of a glioma cell line, and FIG. 4-B shows immunohistochemical staining of glioblastoma tissue. Glioma cell lines stained very strongly with LCA (FIG. 4-A), and glioblastoma tissues also stained strongly with LCA (FIG. 4-B). In normal brain tissue, no clear staining was observed in the parenchyma (not shown). From this result, it was revealed that LCA lectin that specifically binds to A2G2F specifically binds to glioblastoma tissue and glioma cell line. Therefore, it is possible to detect a brain tumor using a lectin labeled with a fluorescent reagent having binding specificity for A2G2F expressed only in the brain tumor.

(Cell proliferation and apoptosis measurement)
U251, T98G (Riken Cell Bank, Tsukuba, Japan), and ON-12 glioma cell lines cultured from glioblastoma patient tissue were treated with 10% FCS, 50 mM 2-mercaptoethanol, 10 mM Hepes. ) (PH 7.4), cultured in MEM medium (Invitrogen, Tokyo, Japan) containing 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin was used as a sample.

(1) Each cell of U251, T98G, ON-12 was seeded at 5 × 10 ³ cells in a 96-well plate, 24 hours later, each concentration of LCA lectin was added, and the culture was further continued for 24 hours. Thereafter, 10 μl of TetraColor ONE reagent (Seikagaku Corporation, Tokyo, Japan) was added, and after 4 hours, the absorbance at 420 nm was measured, and the cell growth ability of each cell was examined.

(2) 1 × 10 ⁴ cells of U251, T98G, ON-12 were cultured on culture slides (BD Falcon, Bedford, Mass.), 25 μg / ml LCA lectin was added and cultured for 24 hours, and then Hoechst 33342 (bisbenzimide H33342 Fluorochrome: Calbiochem-Novabiochem. Co., CA) was added at 10 μl / ml, and nuclear chromatin was observed with a fluorescence microscope.

(3) 1 × 10 ⁶ cells of U251, T98G and ON-12 were added with 25 μg / ml LCA lectin, cultured for 24 hours, and then stained with 50 μg / ml propidium iodide (Sigma, Tokyo, Japan). The hypodiploid DNA was measured by flow cytometry.

  (4) DNA fragmentation is indicated by nick-end labeling of DNA ends with biotinylated dUTP and staining with FITC-labeled avidin using MEBSTAIN apoptosis kit (Medical & Biological Laboratories CO., Nagoya, Japan) In addition, cell death due to apoptosis was detected for each cell. Specifically, 25 μg / ml LCA lectin was added to each cell and cultured for 24 hours. The cells were collected by trypsin treatment, washed several times with PBS / 0.2% BSA (Bovine serum albumin), and 4% PFA. After fixing with (paraformaldehyde) for 30 minutes, after washing with PBS / 0.2% BSA, 1 ml of 70% ethanol was added and further fixed at −20 ° C. for 30 minutes. Next, after washing with PBS / 0.2% BSA, 30 μl of TdT (terminal deoxynucleotidyl transferase) reaction solution was added to the cell sediment and stirred, followed by reaction at 37 ° C. for 1 hour. Next, after washing with PBS / 0.2% BSA, the cell sediment was suspended in 500 μl of PBS / 0.2% BSA and measured by flow cytometry.

  (5) Intracellular caspase-3 was examined using a FITC-conjugated rabbit anti-active caspase-3 monoclonal antibody (BD PharMingen, Tokyo, Japan). Specifically, 25 μg / ml of LCA lectin was added to each cell of U251, T98G, ON-12 and cultured for 24 hours or longer to recover the cell sediment, and then FITC-conjugated rabbit anti-activated caspase-3 monoclonal antibody ( (BD PharMingen, Tokyo, Japan) was added in an amount of 20 μl, stirred, and reacted at 4 ° C. for 1 hour. Next, after washing with PBS / 0.2% BSA, the cell sediment was suspended in 500 μl of PBS / 0.2% BSA and measured by flow cytometry.

  The results of the above-described cell proliferation or apoptosis measurements (1) to (5) are shown in FIGS. FIG. 5 is a graph showing the results of the average survival rate of U251, T98G and ON-12 cells when LCA lectin was added to U251, T98G and ON-12 cells, respectively. As can be seen from FIG. 5, in the cells of U251, T98G, ON-12, suppression of cell proliferation ability was recognized in a concentration-dependent manner by adding LCA lectin.

  FIG. 6 shows that the chromatin aggregates of apoptotic cells were fluorescent using the Hoechst 33342 fluorescent dye after culturing the T98G cells without LCA lectin (A) and those with LCA lectin added (B). It is a microscope picture which shows the result observed under the microscope. In the cells to which the LCA lectin of FIG. 6-B was added, dyes gathered at the chromatin aggregated portions (locations indicated by arrows), and strong blue fluorescent staining was observed. This result indicated that apoptosis was induced by LCA lectin since chromatin aggregation was one of the most characteristic morphological changes associated with apoptosis.

  FIG. 7 is a graph showing the detection results of hypodiploid DNA by flow cytometry. Fig. 7-A is a typical DNA histogram of a control nucleus without addition of LCA lectin to T98G cells, and Fig. 7-B is a DNA histogram of cells cultured with addition of LCA lectin to T98G cells. . As shown in FIG. 7, when hypodiploid DNA was examined by nuclear staining, it was 0.29% without treatment in T98G cells, but it was 31.99% when LCA lectin was added. An increase in hypodiploid DNA was observed.

  FIG. 8 is a graph showing the detection results of apoptotic cells by flow cytometry using MEBSTAIN apoptosis kit. In the observation of DNA fragmentation in the nucleosome by staining with the MEBSTAIN apoptosis kit, it was 2.08% in the case where the LCA lectin of T98G cells was not added (FIG. 8-A), but the LCA lectin was added. In the sample (FIG. 8B), it was 16.45%, and an increase in DNA fragmentation was observed by adding LCA lectin. From this result, it was found that apoptosis of glioma cells was induced by LCA lectin.

  FIG. 9 is a graph showing the detection result of activated caspase-3 by flow cytometry. 9-A, B, and C show the detection results of caspase-3 positive cells in T98G, U251, and ON12 cells, respectively. Moreover, the solid line in a figure shows what added LCA lectin, and a broken line shows what did not add LCA lectin. The cells of T98G, U251, and ON12 were 36.78%, 23.03%, and 20.86%, respectively, when LCA lectin was added, and activated caspases compared to those without LCA lectin. An increase of -3 was observed. As can be seen from these results, LCA lectin induced apoptosis in glioma cells, and it was revealed that it was caspase pathway-dependent.

Claims (9)

  A brain tumor detection method comprising detecting a brain tumor based on detection of an N-linked sugar chain A2G2F of a glycoprotein.   The method for detecting a brain tumor according to claim 1, wherein a lectin having binding specificity to the N-linked sugar chain A2G2F is used.   The brain tumor detection method according to claim 2, wherein the lectin is a lentil lectin.   The method for detecting a brain tumor according to claim 1, wherein the brain tumor is a glioma.   A substance for detecting a brain tumor comprising a lectin having binding specificity to an N-linked sugar chain A2G2F of a glycoprotein.   6. The substance for detecting a brain tumor according to claim 5, wherein the lectin is a lentil lectin.   A pharmaceutical composition for inducing apoptosis of brain tumor cells, comprising a lectin having binding specificity for N-linked sugar chain A2G2F of glycoprotein as an active ingredient.   The pharmaceutical composition according to claim 7, wherein the lectin is a lentil lectin.   The pharmaceutical composition according to claim 7, wherein the brain tumor cells are glioma cells.