JP2011111435A - Therapeutic agent for neuroblastoma or glioblastoma - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent for neuroblastoma and/or glioblastoma, a diagnostic agent and a diagnostic kit for neuroblastoma or glioblastoma, a diagnostic agent and a diagnostic kit for risk factors of neuroblastoma and/or glioblastoma, and a method for screening materials associated with active components of therapeutic or prophylactic agents for neuroblastoma and/or glioblastoma. <P>SOLUTION: The pharmaceutical agent of the invention can be effectively used for treating neuroblastoma and glioblastoma based on a new action mechanism by bringing the pharmaceutical agent to contain a material inhibiting functions of YB-1 gene and/or YB-1(Y box-binding protein-1) as the active components. Neuroblastoma and/or glioblastoma and the risk factors thereof can be also diagnosed by measuring expression of YB-1 gene or production amount of YB-1, or influence thereto as an index, and materials associated with the active components of the prophylactic or therapeutic agents for neuroblastoma and/or glioblastoma can be screened. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for neuroblastoma and / or glioblastoma, diagnostic agent and diagnostic kit for neuroblastoma and / or glioblastoma, diagnostic agent and diagnostic kit for neuroblastoma and / or glioblastoma The present invention also relates to a method for screening a substance relating to an active ingredient of a therapeutic or prophylactic agent for neuroblastoma and / or glioblastoma.

  Neuroblastoma, a neuroendocrine tumor, is the most common extracranial solid cancer in childhood and the most common cancer in childhood. This is the fourth most common childhood malignancy following leukemia, central nervous system tumors, and lymphomas. Neuroblastoma is one of the three leading causes of cancer death in children and is the largest cause of cancer death in infants. In the United States, there are about 650 new cases of neuroblastoma per year. Approximately 50% of neuroblastoma cases occur in children younger than 2 years. As a characteristic of neuroblastoma with a poor prognosis, it is known that the N-myc gene is amplified (Non-Patent Documents 1, 2, and 3).

  On the other hand, malignant glioma is the most common brain tumor in adults. Multiple glioblastoma (GBM) is the most common, most lethal, and most malignant type of glioma and is one of the highest fatalities among human cancers . Chemotherapy such as temozolomide, carboplatin, and cisplatin has only a modest effect, and there is a need to develop effective molecular targeted therapies as another means to treat malignant glioma. Examples of molecular target therapy include epidermal growth factor receptor (EGFR), EGFRvIII, HER2, bcr-abl, vascular endothelial growth factor (VEGF), mammalian rapamycin target, platelet-derived growth factor, and histone deacetylase. Targets are being tried.

  The reappearance of highly proliferative embryonic cells, neuroblasts and glioblasts, leads to neuroblastoma and glioblastoma. This is associated with the reappearance of various proteins in these cells that should diminish or disappear after birth or adulthood. For example, the reappearance of Y box binding protein 1 (YB-1) is associated with glioblastoma (Non-Patent Document 4).

  Y box binding protein 1 (YB-1) is a DNA / RNA binding protein that acts as a regulator of mRNA transcription and translation. YB-1 is the main part of the transcription mechanism. We have shown that YB-1 is involved early in the development of the central nervous system in mice and humans. YB-1 is also highly expressed in several human malignant tumors such as ovarian cancer and breast cancer (Non-patent Documents 5 and 6). However, the relationship between neuroblastoma and YB-1 has not been reported so far. In addition, molecular targeted therapy for malignant tumors targeting YB-1 has not been established.

Kohl NE, Kanda N, Schreck RR, et al. Transposition and amplification of oncogene-related sequences in human neuroblastomas.Cell 1983; 35: 359-67. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM.Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage.Science (New York, NY 1984; 224: 1121-4. Seeger RC, Brodeur GM, Sather H, et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas.The New England journal of medicine 1985; 313: 1111-6. Faury D, Nantel A, Dunn SE, et al. Molecular profiling identifies prognostic subgroups of pediatric glioblastoma and shows increased YB-1 expression in tumors.J Clin Oncol 2007; 25: 1196-208. Kohno K, Uchiumi T, Niina I, et al. Transcription factors and drug resistance. Eur J Cancer 2005; 41: 2577-86. Yahata H, Kobayashi H, Kamura T, et al. Increased nuclear localization of transcription factor Yb-1 in acquired cisplatin-resistance ovarian cancer.J Cancer Res Clin Oncol 2002; 128 (11): 621-6.

  The present invention relates to a therapeutic agent for neuroblastoma and / or glioblastoma, diagnostic agent and diagnostic kit for neuroblastoma and / or glioblastoma, diagnostic agent and diagnostic kit for neuroblastoma and / or glioblastoma Another object of the present invention is to provide a method for screening a substance relating to an active ingredient of a therapeutic or prophylactic agent for neuroblastoma and / or glioblastoma.

  The present inventors have found that YB-1 is detected in embryonic nerve cells and glial cells in the human fetal brain, but YB-1 is not detected in the brain of a healthy adult. We found that YB-1 reappears in malignant tumors such as blastoma and glioblastoma. The present inventors further found for the first time a correlation between the expression of N-myc, which is known as a marker of neuroblastoma with a poor prognosis, and the expression of YB-1. These findings suggested that YB-1 would be a new molecular target for the diagnosis and treatment of malignancies such as neuroblastoma and glioblastoma. As a result of extensive studies, the present inventors have found that YB-1 in neuroblastoma cells is effectively down-regulated by administration of YB-1 siRNA, and that down-regulation of YB-1 We found that the proliferation of tumor cells was significantly suppressed, and that the inhibitory effect of neuroblastoma cells by YB-1 downregulation was comparable to that of N-myc, and the same effect was observed in glioblastoma cells. As a result, the present invention has been completed.

That is, the present invention includes the following [1] to [8].
[1] A therapeutic agent for treating neuroblastoma or glioblastoma, comprising a Y box-binding protein 1 (YB-1) gene and / or a substance that inhibits the function of YB-1 as an active ingredient Drug.
[2] The active ingredient is selected from the group consisting of siRNA targeting the Y box binding protein 1 (YB-1) gene, antisense nucleic acids, vectors expressing these, and YB-1 neutralizing antibodies. The drug according to [1], which is characterized in that it exists.
[3] The drug according to [1] or [2], wherein the active ingredient is siRNA targeting the Y box binding protein 1 (YB-1) gene or a vector expressing the same.
[4] The drug according to any one of [1] to [3], wherein the siRNA targeting the Y box binding protein 1 (YB-1) gene is as shown in SEQ ID NO: 1.
[5] A diagnostic agent or diagnostic kit for neuroblastoma and / or glioblastoma comprising means for measuring the expression level of the Y box binding protein 1 (YB-1) gene and / or the production level of YB-1 as an index .
[6] A neuroblastoma comprising a means for measuring a mutation in one or more regions selected from the group consisting of a YB-1 gene, a gene of a factor that activates or inactivates YB-1, and a control region thereof And / or a risk factor diagnostic agent or diagnostic kit for glioblastoma.
[7] A method for screening a substance that can be used as a preventive or therapeutic agent for neuroblastoma and / or glioblastoma,
(1) a step of contacting a test substance with a transformed cell or a test tube expression system having a transcriptional regulatory region of the YB-1 gene and the YB-1 gene or reporter gene, and (2) a YB-1 gene or reporter gene Detecting a YB-1 or reporter protein production level to obtain a substance that suppresses YB-1 gene expression or YB-1 production,
Including methods.
[8] A method for screening a substance that can be used as a preventive or therapeutic agent for neuroblastoma and / or glioblastoma,
(1) The step of contacting the test substance with YB-1 or YB-1 target molecule, and (2) the amount of YB-1 or YB-1 target molecule bound or not to the test substance Detecting and obtaining a substance that suppresses the function of YB-1;
Including methods.

  The drug of the present invention contains a substance that inhibits the function of the YB-1 gene and / or YB-1 protein as an active ingredient, thereby exhibiting an excellent antitumor effect based on a novel mechanism of action. It can be used effectively for the treatment of malignant tumors such as tumors and glioblastomas.

The diagnostic agent and diagnostic kit of the present invention can effectively diagnose neuroblastoma and / or glioblastoma by measuring the expression level of YB-1 gene or the production level of YB-1 as an index. it can.
In addition, the risk factor diagnostic agent and diagnostic kit of the present invention comprise a YB-1 gene, a gene for a factor that activates or inactivates YB-1, and one or more regions selected from the group consisting of control regions thereof. By measuring the mutation, the risk factor of neuroblastoma and / or glioblastoma can be effectively diagnosed.

  Further, the screening method of the present invention is a method in which a substance obtained by a synthetic or genetic recombination technique, a substance derived from nature or a derivative thereof is used for expression and / or function of YB-1 gene. By using as an index whether or not to suppress the disease, a substance relating to the active ingredient of a therapeutic or prophylactic agent for neuroblastoma and / or glioblastoma can be effectively screened.

FIG. 1 shows the expression of YB-1 and N-myc in a neuroblastoma cell line. FIG. 2 shows the cell growth inhibitory effect of YB-1 downregulation in the Kelly neuroblastoma cell line. FIG. 3 shows the expression of YB-1 in a glioblastoma cell line. FIG. 4 shows YB-1 downregulation at the protein and mRNA levels in the T98G cell line. FIG. 5 shows the effect of YB-1 downregulation on cell proliferation of the T98G cell line.

Hereinafter, embodiments of the present invention will be described in detail.
Drug for treating neuroblastoma or glioblastoma The drug of the present invention has a novel mechanism of action by containing a substance that inhibits the function of YB-1 gene and / or YB-1 as an active ingredient. Based on this, it exhibits an excellent antitumor effect, particularly an antitumor effect against neuroblastoma and glioma, and the most excellent antitumor effect against neuroblastoma and glioblastoma.

Neuroblastoma, also called neuroblastoma, refers to a malignant tumor derived from neuroblastoma. Glioma is a malignant tumor derived from glial cells (also called glial cells), and ciliary cell astrocytoma (WHO grade I), well-differentiated astrocytoma (WHO grade II), not yet Astrocytoma such as differentiated astrocytoma (WHO grade III), glioblastoma multiforme (WHO grade IV), mixed cell glioma such as ependymoma, oligodendroma, oligoastrocytoma However, it is not limited to these.
Substance that inhibits the function of Y box-binding protein 1 (YB-1) gene and / or YB-1 The agent of the present invention inhibits the function of Y box-binding protein 1 (YB-1) gene and / or YB-1 Contains substances as active ingredients.

  The method of inhibiting the function of the YB-1 gene may be a method of suppressing transcription of the YB-1 gene into mRNA, or translation of YB-1 protein by cleaving or destroying the mRNA after transcription. The method of suppressing this may be used. Examples of substances that can be used in the method of inhibiting the function of the YB-1 gene include, but are not limited to, siRNA, antisense nucleic acids, and vectors that express these siRNAs or antisense nucleic acids.

  Examples of the method for inhibiting the function of YB-1 include a method for binding to YB-1 and a method for destroying YB-1. An example of a substance that can be used in a method for inhibiting the function of YB-1 includes, but is not limited to, a neutralizing antibody for YB-1.

The above substances will be described in more detail below.
siRNA
As a gene-specific post-transcriptional repression method, there is a method using small interfering RNA (siRNA). This is specific to the target gene mRNA by introducing a short double-stranded RNA consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence into the cell. It is a method for efficiently inhibiting and suppressing the expression of a target gene by inducing destruction by binding selectively and selectively and cleaving the target gene.

The siRNA used in the present invention is not particularly limited as long as it targets a gene encoding YB-1, but preferably has a length of about 15 to 30 bases, and has a length of about 18 to 23 bases. Is more preferable. A person skilled in the art can design an siRNA used in the present invention using any region as a target candidate based on the human YB-1 gene sequence represented by SEQ ID NO: 7, for example. Specifically, sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 can be given as preferable examples. The sequence represented by SEQ ID NO: 1 is most preferred. The siRNA designed in this way may be appropriately synthesized by a method commonly used by those skilled in the art, or may be synthesized using a general contracted synthesis service.
Antisense nucleic acid In the present specification, the nucleic acid means RNA or DNA. A method using an antisense nucleic acid is also known as a gene-specific post-transcriptional suppression method. This is a method of inhibiting the translation of a target mRNA into a protein by introducing an antisense nucleic acid having a sequence complementary to the target mRNA into a cell or the like.

The antisense nucleic acid used in the present invention is not particularly limited as long as it targets YB-1 mRNA, but is preferably 50 bases or less, and more preferably 25 bases or less. preferable. A person skilled in the art can prepare an antisense nucleic acid to be used in the present invention by appropriately selecting an arbitrary region based on, for example, the sequence of human YB-1 mRNA (SEQ ID NO: 7). The antisense nucleic acid used in the present invention is preferably a sequence complementary to the target sequence of the mRNA of YB-1, but may not be completely complementary as long as gene expression can be effectively suppressed, The complementarity is preferably 90% or more, and most preferably 95% or more. The antisense nucleic acid used in the present invention may have an arbitrary non-complementary region in addition to a region complementary to the target sequence of YB-1 mRNA. In addition, the antisense nucleic acid used in the present invention may have been arbitrarily modified.
Expression Vector In the present invention, as a substance that inhibits the function of the Y box binding protein 1 (YB-1) gene and / or YB-1, a vector that expresses the above siRNA or antisense nucleic acid can also be used. The expression vector used in the present invention may be of any origin as long as it can transform animal cells, and can be appropriately selected from, for example, adenovirus, retrovirus, herpesvirus and the like.

A person skilled in the art can appropriately prepare a vector for expressing siRNA or antisense nucleic acid targeting the YB-1 gene using these vectors.
Neutralizing antibody In the present invention, a neutralizing antibody can also be used as a substance that inhibits the function of YB-1. The neutralizing antibody used in the present invention may be any antibody as long as it can bind to YB-1 and inhibit its function, and may be, for example, a monoclonal antibody or a polyclonal antibody. Further, not only complete antibodies but also fragments (Fab's) that retain specific antigen-binding properties are also included in the neutralizing antibody of the present invention.
Drug The drug of the present invention may be in the form of a Y box-binding protein 1 (YB-1) gene and / or a substance that inhibits the function of YB-1 as it is, or a drug containing a pharmaceutically acceptable additive. It may be in the form of a composition.

  The drug of the present invention can be formulated according to the purpose by a general method together with a pharmacologically acceptable carrier, diluent or excipient. Examples of diluents and carriers include liquid diluents such as water, ethanol, propylene glycol and glycerin, solid diluents and excipients such as glucose, sucrose, dextrin, cyclodextrin and gum arabic. In addition, emulsifiers, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, antioxidants, and the like that are generally used in formulation can be appropriately blended.

  The pharmaceutical composition containing the agent of the present invention may contain other Y-binding protein 1 (YB-1) gene and / or a substance that inhibits the function of YB-1 as necessary, as long as it does not cause an undesirable interaction. These drugs can be mixed or used in combination with other drugs.

  The form of the pharmaceutical composition containing the drug of the present invention is not particularly limited, and is, for example, a solid form such as a powder, a granule, or a tablet; a liquid such as a solution, emulsion, or dispersion; or It can adjust to arbitrary forms, such as semi-solid forms, such as paste form. As oral preparations, dosage forms such as powders, granules, fine granules, tablets, pills, troches, capsules (including soft capsules and hard capsules), chewables, and solutions can be used. Moreover, as a parenteral formulation, it can be set as dosage forms, such as an injection, an instillation, an external medicine, an inhalant, or a suppository.

  The pharmaceutical composition containing the drug of the present invention preferably contains a Y-binding protein 1 (YB-1) gene as an active ingredient and / or a substance that inhibits the function of YB-1, preferably 0.001 to 95% by weight, more preferably Can be used as a form containing 0.01 to 95% by weight, more preferably 0.1 to 95% by weight.

The dose and mode of administration of the drug of the present invention may be appropriately selected depending on the subject, pathological condition and its progress, administration route, dosage form and other conditions. For example, the dose of siRNA of the present invention is usually 1 day. 0.001-100 mg / kg body weight per day, preferably 0.01-10 mg / kg per day.
Diagnostic agent and diagnostic kit
The present inventors have revealed that the expression level of YB-1 gene and / or YB-1 is increased in neuroblastoma and / or glioblastoma. Therefore, it is possible to know the degree of deterioration of neuroblastoma and / or glioblastoma by measuring the expression level of YB-1 gene and / or YB-1 using a biopsy sample of a patient.

  When measuring the expression level of the YB-1 gene, for example, RNA is extracted from a biopsy sample of a patient, cDNA synthesis is performed by a reverse transcription reaction, and measurement is performed using a real-time PCR method using it as a template. it can. In addition, using the same prepared cDNA as a template, a suitable primer is used to amplify the YB-1 gene fragment by a normal PCR reaction, and the intensity of the band corresponding to the YB-1 gene fragment is determined by gel electrophoresis. The expression level of the YB-1 gene can also be measured by the determination method. In addition, any method can be used as a method for measuring the expression level of the YB-1 gene, as long as it is a quantitative method for RNA or DNA, such as the Northern hybridization method or cDNA array method.

  In addition, by measuring the concentration of YB-1 using a biopsy sample of a patient suffering from neuroblastoma and / or glioblastoma, the degree of deterioration of neuroblastoma and / or glioblastoma is known. You can also As a measuring method, for example, an ELISA or RIA method using an antibody against YB-1, a quantification method by HPLC or mass spectrometry can be used. At this time, YB-1 does not need to be in a complete form, and may be fragmented if it can be measured.

The present invention includes a diagnostic agent or diagnostic kit for neuroblastoma and / or glioblastoma comprising a means for measuring the expression level of YB-1 gene or the production amount of YB-1 using the above-described measurement means or the like.
Risk factor diagnostic agent and diagnostic kit According to the present invention, it was shown that the YB-1 gene can be used as a diagnostic marker for neuroblastoma and / or glioblastoma. Therefore, a risk factor can be diagnosed by detecting a gene mutation that increases the expression level of YB-1 gene or the production amount of YB-1 or activates YB-1. In addition, the risk of neuroblastoma and / or glioblastoma is low if gene mutations that reduce the expression level of YB-1 gene, YB-1 production, or inactivate YB-1 are detected. there is a possibility. Examples of a region where such a gene mutation may exist include a regulatory region of the YB-1 gene, a gene of a factor that activates or inactivates YB-1, and a regulatory region thereof. As a method for knowing mutations in these genes, for example, DNA is isolated from a biological sample such as a blood sample of a patient according to a conventional method, and its base sequence is determined by a method commonly used by those skilled in the art and compared with a normal sequence. Can be done. Once the relationship between the mutation and neuroblastoma and / or glioblastoma is clarified, the risk factor diagnosis of neuroblastoma and / or glioblastoma can be achieved by DNA chip detection or SNP analysis that detects only the mutation. It can be performed. One or more genes may be detected.

In the present invention, mutations in one or more regions selected from the group consisting of a YB-1 gene, a gene of a factor that activates or inactivates YB-1, and a control region thereof using the above-described analysis means are performed. It includes a neuroblastoma and / or glioblastoma risk factor diagnostic agent or diagnostic kit, including means for measuring.
Screening method Examples of a screening method for substances that can be used as the active ingredient of the drug according to the present invention include a method using the function of suppressing the expression of YB-1 gene as an index, and a method using the function suppression effect of YB-1 as an index Can be mentioned.

  In the method using the expression suppression function of the YB-1 gene as an index, for example, (1) expression of a test substance in a transformed cell having a transcription control region of the YB-1 gene and a YB-1 gene or a reporter gene or in vitro And (2) suppressing the expression of YB-1 gene or the production of YB-1 by detecting the expression level of YB-1 gene or reporter gene or the production level of YB-1 or reporter protein. A substance can be obtained.

  In the method using the function-suppressing effect of YB-1 as an index, for example, (1) a test substance is brought into contact with YB-1 or a target molecule of YB-1, and (2) is bound to the test substance. A substance that suppresses the function of YB-1 can be obtained by detecting the amount of YB-1 or the target molecule of YB-1.

Here, the test substance may be a substance obtained by a synthetic or genetic recombination technique, a naturally derived substance, or a derivative thereof.
The YB-1 gene or YB-1 or derivatives thereof used in the screening method of the present invention may be derived from any species, for example, the human YB-1 gene (accession number: NM_004559, SEQ ID NO: 7 ) And its translation product (SEQ ID NO: 8), rat YB-1 gene (accession number: NM_031563.3, SEQ ID NO: 9) and its translation product (SEQ ID NO: 10), mouse YB-1 gene (accession) No .: NM — 011732.2, SEQ ID NO: 11) and translation products (SEQ ID NO: 12) derived from mammals. From these, an appropriate one may be selected according to the subject of drug administration and the purpose of research and development.

  The target molecule of YB-1 may be any molecule as long as it interacts with YB-1 or a molecule in which expression of YB-1 directly affects the expression level. Alternatively, it may be a polymer such as a protein.

  The transformed cells used in the screening method of the present invention may be derived from prokaryotic cells or eukaryotic cells, and for example, Escherichia coli, yeast, insect cells, mammalian cells and the like can be used.

  The in vitro expression system used in the screening method of the present invention may be any system as long as it is a cell-free protein expression or synthesis system. For example, for in vitro protein translation from mRNA. A reaction solution containing necessary reagents and, if necessary, reagents necessary for in vitro transcription of mRNA from a gene, those skilled in the art can select an appropriate system according to the purpose.

  As the reporter gene used in the screening method of the present invention, for example, chloramphenicol acetyltransferase (CAT), β-galactosidase (β-Gal), luciferase, green fluorescent protein (GFP) and the like can be used. Not.

The amount of the reporter protein that changes with the addition of the test substance can be measured as an enzyme activity, or can be measured using an antibody or the like.
The expression level of the YB-1 gene or reporter gene, which changes with the addition of the test substance, and the production amount of the target molecule of YB-1 or YB-1 are measured by the above-mentioned RNA, DNA, and protein detection and quantification methods. can do.

By using these methods, not only therapeutic agents for neuroblastoma and / or glioblastoma but also active ingredients of prophylactic agents can be screened.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

The materials and methods used in this example are shown below.
Cell lines and reagents
IMR-32, SK-N-SH and Kelly neuroblastoma cell lines, and T98G and U251 cell lines derived from grade 4 glioblastoma were purchased from ATCC. IMR-32 and SK-N-SH cell lines were cultured in MEM and DMEM, respectively, and T98G, U251 and Kelly cell lines were cultured in RPM1. All media contained 10% FBS and 1% penicillin / streptomycin.
Antibody Rabbit anti-YB-1 antibody was purchased from Cell Signaling Technology (Denvers, Massachusetts, USA). Mouse anti-N-Myc antibody (C-19) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Immunohistochemistry
To assess YB-1 status in vitro, IMR-32, SK-N-SH and Kelly neuroblastoma cell lines, and T98G glioblastoma cell lines were evaluated by immunohistochemistry. Cells (1 × 10 5) are seeded on glass coverslips, washed with phosphate buffered saline (PBS), fixed with 2% formaldehyde for 20 minutes, washed twice with PBS, then 0.1% Incubated with PBS containing% saponin (Sigma) for 30 minutes. Next, the cover slip was washed with PBS, together with a rabbit anti-YB-1 antibody and a mouse anti-N-myc antibody (C-19) dissolved in a buffer containing 10% bovine serum albumin and 2% goat serum. Incubate for 1 hour at room temperature in a humidified container. After washing 3 times with PBS, slides were incubated with Alexa 488 anti-rabbit antibody and Alexa546 anti-mouse antibody for 1 hour, washed 3 times and then using VECTASHIELD® mounting medium (Vector Laboratories, California, USA) And mounted. 4,6-diamidino-2-phenylindole was used for nuclear staining.
Down-regulation of YB-1 or N-myc using siRNA Transformed Kelly, a representative neuroblastoma cell, and T98G, a representative glioblastoma cell line, with 50 nM YB-1 siRNA or control siRNA Converted. The Kelly cell line was also transformed with N-myc siRNA or control siRNA (AllStars Neg. Control siRNA) for N-myc downregulation studies. All siRNAs were purchased from Qiagen. Lipofectamin ™ RNAiMAX was used for siRNA transfection according to the manufacturer's (Invitrogen) directed protocol.
Proliferation assay
Cells treated with YB-1 siRNA, N-myc siRNA, and control siRNA were trypsinized and seeded again in a 24-well dish. Proliferation assays were performed by counting cell numbers at 0, 24, 48, and 72 hours using a Becton Coulter Counter.

《YB-1 targeting in neuroblastoma》
Expression analysis of YB-1 and N-myc in neuroblastoma cells As shown in FIG. 1, the IMR-32 and Kelly cell lines in which N-myc is amplified showed significantly more YB-1 expression. In many cells, expression of YB-1 in the cytoplasm was associated with N-myc accumulation in the nucleus.

Co-expression of N-myc and YB-1 was also observed in vivo in neuroblastoma patients (data not shown). Since YB-1 and N-myc expression are correlated both in vivo and in vitro, and YB-1 is evaluated in the same manner as N-myc, YB-1 may be considered as a prognostic marker. It is suggested.
Down-regulation of YB-1 in neuroblastoma cell lines Next, to evaluate the effect of YB-1 down-regulation on the proliferation of neuroblastoma cell lines, we used Kelly neuroblastoma cells using YB-1 siRNA The expression of YB-1 in the strain was suppressed. Here, the cell growth inhibitory effect by the down-regulation by N-myc and the cell growth inhibitory effect by the YB-1 down-regulation were compared and evaluated. The result is shown in FIG.

  Down-regulation of YB-1 significantly suppressed cell proliferation. Both YB-1 down-regulation and N-myc down-regulation suppressed the growth of Kelly, a neuroblastoma cell that amplifies N-myc. Interestingly, the growth inhibitory effect of YB-1 was comparable to N-myc.

《YB-1 targeting in glioblastoma》
Expression analysis of YB-1 in glioblastoma cell line
The expression of YB-1 in T98G and U251 cell lines derived from WHO grade 4 glioblastoma multiforme was evaluated by both immunoblotting and immunohistochemistry. As shown in FIG. 3, YB-1 was highly expressed in both cells.
Downregulation of YB-1 in glioblastoma cell lines
YB-1 was down-regulated in the T98G cell line. Down-regulation of YB-1 was confirmed by immunoblotting and real-time PCR for mRNA expression. As shown in FIG. 4, the expression of YB-1 was significantly down-regulated when 50 nM YB-1 siRNA was used as compared to the case of transformation with control siRNA.
Effect of YB-1 downregulation on cell proliferation
In order to evaluate the effect on cell proliferation by down-regulating YB-1, the proliferation rate of cells down-regulated by YB-1 siRNA was evaluated. First, T98G cells were transformed with YB-1 siRNA. The growth rate of cells down-regulated by YB-1 siRNA was compared to cells transformed with control siRNA. As a result, as shown in FIG. 5, down-regulation of YB-1 significantly reduced cell proliferation of T98G cells.

  The drug of the present invention contains a substance that inhibits the function of the YB-1 gene and / or YB-1 protein as an active ingredient, thereby exhibiting an excellent antitumor effect based on a novel mechanism of action. It can be used effectively for the treatment of malignant tumors such as tumors and glioblastomas.

The diagnostic agent and diagnostic kit of the present invention can effectively diagnose neuroblastoma and / or glioblastoma by measuring the expression level of YB-1 gene or the production level of YB-1 as an index. it can.
In addition, the risk factor diagnostic agent and diagnostic kit of the present invention comprise a YB-1 gene, a gene for a factor that activates or inactivates YB-1, and one or more regions selected from the group consisting of control regions thereof. By measuring the mutation, the risk factor of neuroblastoma and / or glioblastoma can be effectively diagnosed.

  Further, the screening method of the present invention is a method in which a substance obtained by a synthetic or genetic recombination technique, a substance derived from nature or a derivative thereof is used for expression and / or function of YB-1 gene. By using as an index whether or not to suppress the disease, a substance relating to the active ingredient of a therapeutic or prophylactic agent for neuroblastoma and / or glioblastoma can be effectively screened.

Claims (8)

  A drug for treating neuroblastoma or glioblastoma, comprising a Y box-binding protein 1 (YB-1) gene and / or a substance that inhibits the function of YB-1 as an active ingredient.   The active ingredient is selected from the group consisting of siRNA targeting the Y box binding protein 1 (YB-1) gene, antisense nucleic acid, a vector expressing them, and a neutralizing antibody of YB-1. The medicament according to claim 1, characterized in that   The drug according to claim 1 or 2, wherein the active ingredient is siRNA targeting the Y box binding protein 1 (YB-1) gene or a vector expressing the same.   The drug according to any one of claims 1 to 3, wherein the siRNA targeting the Y box binding protein 1 (YB-1) gene is as shown in SEQ ID NO: 1.   A diagnostic agent or diagnostic kit for neuroblastoma and / or glioblastoma, comprising means for measuring the expression level of the Y box binding protein 1 (YB-1) gene and / or the production level of YB-1 as an index.   Neuroblastoma and / or comprising means for measuring mutations in one or more regions selected from the group consisting of the YB-1 gene, the gene of the factor that activates or inactivates YB-1, and their control regions Risk factor diagnostic agent or diagnostic kit for glioblastoma. A method for screening a substance that can be used as a preventive or therapeutic agent for neuroblastoma and / or glioblastoma,
(1) a step of contacting a test substance with a transformed cell or a test tube expression system having a transcriptional regulatory region of the YB-1 gene and the YB-1 gene or reporter gene, and (2) a YB-1 gene or reporter gene Detecting a YB-1 or reporter protein production level to obtain a substance that suppresses YB-1 gene expression or YB-1 production,
Including methods. A method for screening a substance that can be used as a preventive or therapeutic agent for neuroblastoma and / or glioblastoma,
(1) The step of contacting the test substance with YB-1 or YB-1 target molecule, and (2) the amount of YB-1 or YB-1 target molecule bound or not to the test substance Detecting and obtaining a substance that suppresses the function of YB-1;
Including methods.