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WO2012176651A1 - Novel anti-tumor agent, and method for screening for novel anti-tumor agent - Google Patents

Novel anti-tumor agent, and method for screening for novel anti-tumor agent Download PDF

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WO2012176651A1
WO2012176651A1 PCT/JP2012/064964 JP2012064964W WO2012176651A1 WO 2012176651 A1 WO2012176651 A1 WO 2012176651A1 JP 2012064964 W JP2012064964 W JP 2012064964W WO 2012176651 A1 WO2012176651 A1 WO 2012176651A1
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hif
idh3
idh3α
tumor
novel
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Japanese (ja)
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原田　浩
平岡　真寛
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国立大学法人 京都大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01041—Isocitrate dehydrogenase (NAD+) (1.1.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01042—Isocitrate dehydrogenase (NADP+) (1.1.1.42)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a novel antitumor agent and to a screening method for a novel antitumor agent. Furthermore, it is related with the novel treatment method of a malignant tumor.
  • hypoxia-inducible factor 1 (Hypoxia Inducible Factor-1: hereinafter sometimes referred to simply as “HIF-1”) is activated in a hypoxic environment within a malignant solid tumor, and treatment resistance and metastasis of cancer cells.
  • Non-patent Document 1 invasive ability and angiogenesis are enhanced.
  • HIF-1 is activated in response to changes in the tumor microenvironment after an anticancer agent or radiotherapy, and the treatment resistance of cancer is enhanced.
  • the solid tumor has a very different microenvironment.
  • hypoxic and hypotrophic environment where sufficient oxygen and nutrient sources are not provided at the distal end of the tumor blood vessels.
  • an acidic (low pH) environment exists in a region where ATP production by glycolysis and lactic acid fermentation is popular.
  • Cancer cells induce the expression of genes necessary to survive these harsh microenvironments.
  • cancer cells that exist in a hypoxic environment activate a transcription factor called HIF-1 to produce energy in the hypoxic environment (transformation of glucose metabolic pathway), improve the hypoxic environment (angiogenesis), hypoxia Avoid the environment (metastasis / infiltration).
  • Non-patent Document 3 the tumor microenvironment changes due to anticancer drugs and radiotherapy, and activation of HIF-1 induces the expression of vascular endothelial growth factor (Vascular Endothelial Growth Factor: VEGF), and VEGF induces tumor blood vessels Effect of reducing radiation damage to endothelial cells, preventing tumor vasculature from collapsing, ensuring supply of oxygen and nutrients to cancer cells, and finally ensuring survival of cancer cells after radiation therapy Works.
  • VEGF Vascular Endothelial Growth Factor
  • hypoxia may promote tumor growth, angiogenesis, and metastasis by inducing specific gene expression.
  • a strong correlation was reported between the number of hypoxia fractions and therapeutic effects and life prognosis. ing.
  • HIF-1 is a protein that is induced when oxygen supply to cells is deficient or when cells are exposed to excessive reactive oxygen species, and functions as a transcription factor.
  • nutrient deficiencies, decreased extracellular pH, and insufficient oxygen supply (hypoxia) due to insufficient blood flow are observed, but in order for cancer cells to survive, the lesion is formed by forming a new vascular network. It is necessary to secure blood flow to the limbs and escape from hypoxia.
  • HIF-1 is a transcription factor induced under hypoxic conditions so as to have a function for that purpose, and enhances transcription of various genes. HIF-1 is involved in the control of various gene expressions, such as genes involved in angiogenesis, cell proliferation, sugar metabolism, pH regulation, apoptosis, and the like.
  • HIF-1 examples include erythropoietin and VEGF.
  • HIF-1 is composed of two subunits, ⁇ and ⁇ .
  • HIF-1 ⁇ is said to regulate various genes such as adrenomedullin, matrix metalloproteinases (MMPs), endothelin (ET) -1, and nitric oxide synthase (NOS) 2.
  • MMPs matrix metalloproteinases
  • ETS endothelin
  • NOS nitric oxide synthase
  • an antitumor agent that suppresses HIF-1 is being developed.
  • examples include antisense oligonucleotides against the HIF-1 gene (Enzon, National Cancer Institute, Santaris Pharma) and low molecular weight compounds that suppress HIF-1 activity (Abbot).
  • HSP90 inhibitor etc. are also reported as a HIF-1 inhibitor (nonpatent literature 4).
  • HIF-1 is activated, where HIF-1-positive cancer cells involved in treatment resistance are located in the tumor, and what pathology and behavior are shown This is a major obstacle to achieving complete cure for cancer.
  • IDH1 isocitrate dehydrogenase
  • TCA cycle citrate cycle
  • IDH1 is known as an enzyme involved.
  • Patent Literature 1 Non-patent literature 5-7
  • An object of the present invention is to provide a novel antitumor agent and to provide a screening method for a novel antitumor agent. Furthermore, it aims at providing the novel treatment method of a malignant tumor.
  • the present inventors have focused on the fact that cancer treatment resistance is enhanced through the activation of HIF-1, and the activation of HIF-1 As a result of searching for substances that can be controlled, it was found for the first time that IDH3, a kind of isocitrate dehydrogenase, contributes to the activation of HIF-1, and the present invention was completed.
  • this invention consists of the following. 1. A novel antitumor agent comprising an IDH3 inhibitor as an active ingredient. 2. 2. The novel antitumor agent according to item 1 above, wherein the IDH3 suppression is IDH3 ⁇ expression suppression or function suppression. 3. 3. The novel antitumor agent according to 1 or 2 above, which suppresses IDH3 and suppresses HIF-1. 4). 4. The novel antitumor agent according to item 3 above, wherein the suppression of HIF-1 is suppression of expression or function of HIF-1 ⁇ . 5. 5. The novel antitumor agent according to any one of items 1 to 4, wherein the tumor is an intractable malignant tumor. 6). 6.
  • novel antitumor agent according to 5 above, wherein the refractory malignant tumor is a radiation therapy resistant cancer and / or an anticancer drug resistant cancer. 7.
  • An HIF-1 inhibitor comprising an IDH3 inhibitor as an active ingredient. 8).
  • the cell line capable of expressing IDH3 ⁇ is a cell line that expresses a reporter gene in an HIF-1-dependent manner, and the measurement of IDH3 ⁇ expression level or IDH3 activity is performed by measuring the reporter gene product. Screening method for novel antitumor agents. 11. A novel method for treating a tumor, comprising inhibiting at least one of the subunits constituting IDH3 as a target factor. 12 12. The novel method for treating a tumor according to item 11, wherein at least one of the subunits constituting IDH3 is IDH3 ⁇ . 13. 13. The novel method for treating a tumor according to item 11 or 12, wherein suppressing at least one of the subunits constituting IDH3 as a target factor is suppression of expression or function of IDH3 ⁇ . 14 14.
  • the IDH3 inhibitory substance of the present invention has an antitumor effect. Further, the IDH3 inhibitory substance of the present invention has a HIF-1 inhibitory effect. From these actions, an effective antitumor agent can be provided by screening a substance capable of suppressing the activity of IDH3.
  • FIG. 1 It is a figure which shows the antibiotic sensitivity of the cell in a normoxic condition and a hypoxic condition about the cell strain which integrated stably "the gene which expresses an antibiotic resistance gene under control of a HIF-1 responsive promoter.”
  • Reference Example 1 It is a conceptual diagram which shows the system for confirming the effect
  • Example 1 It is the figure which transplanted the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse
  • Example 1 It is the photograph which confirmed the magnitude
  • Example 1 It is the figure which transplanted the cancer cell line which each integrated stably the shRNA expression vector with respect to IDH3 (alpha) or HIF-1 (alpha) to an immunodeficient mouse, and confirmed subsequent tumor growth.
  • Example 2 It is a photograph figure which shows the intravascular tumor blood vessel which transplanted the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse
  • Example 3 It is a figure which shows the result of having transplanted the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse
  • IDH3 means isocitrate dehydrogenase 3 (Isocitrate Dehydrogenase 3).
  • the present invention relates to a novel antitumor agent comprising an IDH3 inhibitor as an active ingredient.
  • IDH3 is a heterotetramer composed of two molecules of ⁇ subunit (hereinafter “IDH3 ⁇ ”), one molecule of ⁇ subunit (hereinafter “IDH3 ⁇ ”) and one molecule of ⁇ subunit (hereinafter “IDH3 ⁇ ”). .
  • the “IDH3 inhibitory substance” may be any substance that can suppress IDH3 activity, and in order to suppress IDH3 activity, a substance that targets at least one subunit among the subunits constituting IDH3 If it is.
  • the target subunit is particularly preferably IDH3 ⁇ .
  • the expression suppression of IDH3 ⁇ or the function of IDH3 ⁇ may be suppressed.
  • suppression of IDH3 ⁇ expression refers to suppression of expression of IDH3 ⁇ biosynthetic gene (hereinafter simply referred to as “IDH3 ⁇ gene”), and any of the gene expression control processes including transcription and translation. It means the action of inhibiting the stage.
  • suppression of the function of IDH3 ⁇ refers to suppression of the function of IDH3 on isocitrate dehydrogenase activity by inhibiting part or all of IDH3 ⁇ .
  • the IDH3 inhibitor include antisense nucleic acids, ribozyme nucleic acids, siRNA, shRNA, low molecular compounds that suppress IDH3 ⁇ expression or IDH3 ⁇ function, peptides, enzymes, antibodies, and the like that can suppress IDH3 ⁇ expression.
  • the IDH3 inhibitory substance of the present invention includes pharmaceutically acceptable salts thereof in addition to the above substances.
  • IDH3 ⁇ can be identified by the amino acid sequence represented by GenBank Accession Number: NP_005521.1, and mRNA can be designated by the same Accession Number: NM_005530.2.
  • IDH3 inhibitory substance of the present invention include shRNA that can suppress the expression of IDH3 ⁇ .
  • sequences targeted by the shRNA include (A) to (C) shown in the following SEQ ID NOs: 1 to 3.
  • C) TCACC CATCT ATGAA TTTAC T SEQ ID NO: 3
  • suppressing IDH3 and suppressing HIF-1 means suppressing HIF-1 activity through suppressing IDH3 described above.
  • HIF-1 means hypoxia-inducible factor 1 (Hypoxia-Inducible Factor 1).
  • HIF-1 is a protein that is activated when oxygen supply to cells falls short or when cells are exposed to excessive reactive oxygen species. It is a functional substance. HIF-1 forms a heterodimer of HIF-1 ⁇ and HIF-1 ⁇ , and can exert a function of enhancing transcription of various genes as a transcription factor.
  • an oxygen-dependent degradation domain (Oxygen-dependent) degradation domain: ODD) exists in the approximate center of the HIF-1 ⁇ protein.
  • ODD oxygen-dependent degradation domain
  • HIF-1 ⁇ expression refers to suppression of HIF-1 ⁇ biosynthetic gene expression (hereinafter simply referred to as “HIF-1 ⁇ gene”), and gene expression including transcription and translation. It means an action that inhibits any step of the control process.
  • the suppression of the function of HIF-1 ⁇ refers to the suppression of the function exerted on the activity of HIF-1 by inhibiting part or all of HIF-1 ⁇ . Suppressing HIF-1 effectively exerts an antitumor effect against refractory malignant tumors such as radiation resistant cancer and chemotherapeutic resistant cancer such as chemotherapeutic agents. sell.
  • the present invention also extends to an HIF-1 inhibitor containing an IDH3 inhibitor as an active ingredient. That is, a drug containing an effective amount of the IDH3 inhibitory substance of the present invention is not limited to an antitumor agent as long as functions other than the antitumor effect can be exhibited by the HIF-1 inhibitory action.
  • the IDH3 inhibitor contained in the novel antitumor agent of the present invention as an active ingredient may exert an antitumor action by a mechanism different from the action mechanism resulting from the relationship between IDH3 and HIF-1.
  • the novel antitumor agent or HIF-1 inhibitor of the present invention contains an effective amount of an IDH3 inhibitory substance as an active ingredient, and may also contain additives generally used in medicine.
  • Additives include excipients, binders, lubricants, disintegrants, colorants, flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, isotonic agents, buffers, preservatives Agents, antioxidants, stabilizers, absorption promoters and the like, and these can be used in appropriate combinations as desired.
  • excipient examples include lactose, sucrose, glucose, corn starch, mannitol, sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, light anhydrous silicic acid, aluminum silicate, calcium silicate, and metasilicate.
  • examples thereof include magnesium aluminate acid and calcium hydrogen phosphate.
  • the binder include polyvinyl alcohol, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, macrogol and the like.
  • Examples of the lubricant include magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, polyethylene glycol, colloidal silica and the like.
  • Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl Examples include starch and sodium carboxymethyl starch.
  • Examples of the colorant include those allowed to be added to pharmaceuticals such as iron sesquioxide, yellow sesquioxide, carmine, caramel, ⁇ -carotene, titanium oxide, talc, riboflavin sodium phosphate, yellow aluminum lake, etc. Can be mentioned.
  • Examples of the flavoring agent include cocoa powder, mint brain, aroma powder, mint oil, menthol, dragon brain, and cinnamon powder.
  • Examples of the emulsifier or surfactant include stearyl triethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, glyceryl monostearate, sucrose fatty acid ester, and glycerin fatty acid ester.
  • solubilizer examples include polyethylene glycol, propylene glycol, benzyl benzoate, ethanol, cholesterol, triethanolamine, sodium carbonate, sodium citrate, polysorbate 80, and nicotinamide.
  • suspending agent examples include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose, in addition to the surfactant.
  • examples of the isotonic agent include glucose, sodium chloride, mannitol, sorbitol and the like.
  • buffer examples include buffer solutions such as phosphate, acetate, carbonate, citrate.
  • Examples of the preservative include methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • Examples of the antioxidant include sulfite, ascorbic acid, ⁇ -tocopherol and the like.
  • Examples of the stabilizer include ascorbic acid, sodium edetate, erythorbic acid, tocopherol and the like.
  • Examples of the absorption promoter include isopropyl myristate, tocopherol, calciferol and the like.
  • the present invention also extends to a method for screening a novel antitumor agent using IDH3 activity suppression as an index.
  • the screening method for a novel antitumor agent of the present invention is not particularly limited as long as it is a system that can evaluate the activity of IDH3.
  • it can be performed by a method including the following steps. 1) a step of treating a cell line capable of expressing IDH3 ⁇ with a candidate substance; 2) A step of measuring IDH3 ⁇ expression level or IDH3 activity for the above cell lines before and after treatment with a candidate substance, and evaluating IDH3 activity suppression from IDH3 ⁇ expression level or IDH3 activity.
  • the cell line capable of expressing IDH3 ⁇ is preferably a cell line that expresses a reporter gene in an HIF-1-dependent manner.
  • the reporter gene is stably integrated downstream of the HIF-1-responsive promoter.
  • a cell line capable of expressing IDH3 ⁇ may contain a fusion gene of a reporter gene and a gene encoding the ODD region of HIF-1 ⁇ instead of the HIF-1 responsive promoter.
  • a cell line prepared in advance may be used, or a step of preparing the cell line may be included in the step of the screening method of the present invention.
  • the reporter gene a gene known per se can be used. Although not particularly limited, it is preferable to use a gene capable of emitting light or fluorescence such as luciferase or GFP.
  • a cell line that can be used in the screening method of the present invention can be constructed by the following method. First, a cell line that stably incorporates a reporter gene downstream of the HIF-1-responsive promoter is established. Furthermore, a vector capable of expressing IDH3 ⁇ in large quantities is introduced into the cells. Since IDH3 ⁇ activates HIF-1, a cell line incorporating the reporter gene emits intense luminescence when an IDH3 mass expression vector is introduced. Examples of cell lines that can be used include human cervical cancer-derived cell line HeLa, human alveolar carcinoma-derived cell line A549, and human colon cancer-derived cell line Colon 26.
  • the novel anti-tumor of the present invention is obtained by culturing the cells established as described above or already established in a medium containing a candidate substance, and measuring an indicator derived from a reporter gene, such as luminescence and fluorescence, for the cultured cells. Agents can be screened. Specifically, the selected substance can be the antitumor agent of the present invention when the candidate substance shows a decrease in the amount of luminescence or fluorescence, or a decrease in the expression level of IDH3 ⁇ .
  • screening may be performed by the following method, for example. 1) a step of treating a cell line capable of expressing IDH3 ⁇ with a candidate substance; 2) Obtain an extract from cells treated with the candidate substance, Western blotting or ELISA using IDH3 ⁇ antibody or HIF-1 ⁇ antibody, and antibodies against downstream HIF-1 genes such as VEGF, etc. To evaluate IDH3 activity suppression by confirming the expression level of IDH3 ⁇ , HIF-1 ⁇ or HIF-1 downstream gene and the activity of HIF-1 or HIF-1 downstream gene, etc. .
  • the candidate substances screened above are HIF-1 responsive reporter genes, for example, the tumor cells obtained by transplanting cancer cells stably incorporating a reporter gene downstream of the HIF-1 responsive promoter into immunodeficient mice.
  • the cancer mouse can be evaluated by observing, visualizing, and quantifying a reporter substance such as a luminescent protein or a fluorescent protein that is expressed in an HIF-1-dependent manner after the cancer mouse is treated with the screened candidate substance.
  • the IDH3 inhibitor contained as an active ingredient in the novel antitumor agent of the present invention was achieved as a result of repeated investigations by selecting target substances by focusing on the activity of HIF-1 by the method of Reference Example 1 below. It is a thing.
  • a new antitumor agent can be screened by a screening method focusing on the activity of HIF-1.
  • the novel antitumor agent of the present invention contains an IDH3 inhibitory substance as an active ingredient, and IDH3 ⁇ is cited as the target substance. It is done. In this reference example, the reason why IDH3 ⁇ has been selected as a target substance will be described.
  • HIF-1 which is a kind of hypoxia-inducible factor
  • the present inventors have reported that cancer treatment resistance and malignancy are enhanced through activation of HIF-1.
  • the mechanism by which HIF-1 is activated and where the HIF-1-positive cancer cells involved in treatment resistance are located in the tumor and what kinetics / behavior is shown. was not revealed. Therefore, the present inventor conducted an experiment to exhaustively search for factors (genes) that activate HIF-1.
  • FIG. 2 shows the cell viability when the cells were cultured under normoxic and hypoxic conditions using a medium containing antibiotics at various concentrations.
  • a cDNA library is appropriately introduced into the cells established above using a retroviral vector, 20%) cells were cultured in a medium containing blasticidin, and cells that formed colonies were selected (see FIG. 1). In the surviving cells, HIF-1 was activated by the function of the gene integrated (incorporated) into the genomic DNA, and it was expected that the expression of the blasticidin resistance gene was induced as a result. Therefore, the cDNA fragment was amplified by performing a PCR reaction using “genomic DNA purified from living cells” as a template and further using a “primer sandwiching the inserted cDNA”.
  • This DNA fragment was subcloned into the EcoRV site of a general-purpose plasmid vector (specifically, pBlueScript II SK +).
  • a general-purpose plasmid vector specifically, pBlueScript II SK +.
  • HeLa / 5HRE-Luc cells are introduced with the respective shRNA (A)-(C) expression plasmids or shRNA (Scr) expression plasmids for negative control, and under normal oxygen (20%) or hypoxia (0.02%) environment After culturing in, a luciferase assay was performed. (See Figure 5)
  • IDH3 ⁇ can be identified by the amino acid sequence represented by GenBank Accession Number: NP_005521.1, and mRNA can be identified by the Accession Number: NM_005530.2. it can.
  • HIF-1 protein is composed of HIF-1 ⁇ and HIF-1 ⁇ subunits.
  • HIF-1 ⁇ one of the subunits, undergoes oxygen-dependent ubiquitination and is rapidly degraded after translation in normoxic cells.
  • ODD oxygen-dependent degradation domain
  • Cell extract was obtained from the above-mentioned IDH3 ⁇ forced expression cell line (HeLa / IDH3 ⁇ 1-3) and negative control cell line (HeLa / EV31-3), and the expression of IDH3 ⁇ derived from pcDNA4A / IDH3 ⁇ plasmid was fused. Detected with antibody against myc tag. On the other hand, by using an antibody against IDH3 ⁇ , the expression of endogenous IDH3 ⁇ was confirmed together with the IDH3 ⁇ protein fused with the myc tag derived from the pcDNA4A / IDH3 ⁇ plasmid. Both proteins could be detected independently with or without myc tag.
  • Example 1 Effect of shRNA on IDH3 ⁇ gene
  • Cell line in which expression of endogenous IDH3 ⁇ is constitutively knocked down by stably incorporating each expression plasmid of shRNA (A) to (C) against IDH3 ⁇ into HeLa cell line (HeLa / shIDH3 ⁇ A2, A5, B2, B4, C1, C2) was established.
  • cell lines (HeLa / shRNA (Scr) N5, N9) stably incorporating the shRNA (Scr) expression plasmid were established. After culturing these cell lines under normoxic conditions (20%) or hypoxic conditions (0.02%), cell extracts were obtained, and Western blotting was performed using anti-HIF-1 ⁇ antibody and anti-IDH3 ⁇ antibody.
  • Example 2 Anti-tumor effect by knockdown of IDH3 ⁇ gene or HIF-1 ⁇ gene shRNA (B) expression plasmid for IDH3 ⁇ gene, shRNA expression plasmid for HIF-1 ⁇ gene, and shRNA (Scr) expression plasmid of negative control HeLa cells stably incorporating each of these were transplanted into immunodeficient mice, and the tumor size was measured. As a result, more effective tumor growth inhibitory effect was observed in the tumor in which the IDH3 ⁇ gene was knocked down compared to the tumor in which the HIF-1 ⁇ gene was knocked down (FIG. 15). From the above, it was confirmed that suppressing IDH3 ⁇ can more effectively suppress tumor growth than directly suppressing HIF-1 ⁇ .
  • IDH3 ⁇ has actions other than stabilizing HIF-1 ⁇ .
  • a substance capable of suppressing IDH3, specifically, at least one of the subunits constituting IDH3, specifically, a substance capable of suppressing IDH3 ⁇ can exert an effect as an antitumor agent. .
  • Example 3 Action of shRNA on IDH3 ⁇ gene “Cell HeLa / shIDH3 ⁇ B2 in which expression of endogenous IDH3 ⁇ is constantly knocked down” established in Example 1, and “negative control cell HeLa / shRNA (Scr) N9” "And 10 million and 1 million cells, respectively, were transplanted into immunodeficient mice.
  • the resulting solid tumors have the same size, i.e., 45 days after transplantation for HeLa / shIDH3 ⁇ B2 tumors, 37 days after transplantation for HeLa / shRNA (Scr) N9 tumors, the sections are removed from vascular endothelial cells. Were stained with a marker (CD31 antibody).
  • the IDH3 inhibitor of the present invention has an antitumor effect. Further, the IDH3 inhibitory substance of the present invention has a HIF-1 inhibitory effect. From these actions, an effective antitumor agent can be provided by screening a substance capable of suppressing the activity of IDH3. Since the novel antitumor agent containing the IDH3 inhibitory substance of the present invention as an active ingredient can control the activity of HIF-1, it is useful for intractable tumors such as radiotherapy resistant cancer and anticancer drug resistant cancer. Even if it is effective, it can be effective.

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Abstract

Provided are: a novel anti-tumor agent; a method for screening for a novel anti-tumor agent; and a novel method for treating a malignant tumor. The novel anti-tumor agent comprises a substance capable of inhibiting isocitrate dehydrogenase 3 (IDH3) as an active ingredient. The method for screening for a novel anti-tumor agent employs the inhibition of the activity of IDH3 as a measure. The novel method for treating a malignant tumor comprises inhibiting at least one of IDH3-constituting subunits as a target factor. Attention is focused on a fact that the resistance to a cancer therapy is increased through the activation of hypoxia-inducible factor 1 (HIF-1), which is a factor occurring normally in a solid tumor, under low-oxygen environments, and a substance capable of controlling the activation of HIF-1 is searched for. As a result, it is newly found that IDH3 is involved in the activation of HIF-1. This finding leads to the accomplishment of the present invention.

Description

新規抗腫瘍剤及び新規抗腫瘍剤のスクリーニング方法Novel antitumor agent and screening method for novel antitumor agent
　本発明は、新規抗腫瘍剤に関し、また新規抗腫瘍剤のスクリーニング方法に関する。さらに悪性腫瘍の新規治療方法に関する。 The present invention relates to a novel antitumor agent and to a screening method for a novel antitumor agent. Furthermore, it is related with the novel treatment method of a malignant tumor.
　本出願は、参照によりここに援用されるところの日本出願特願２０１１－１３８９８１号優先権を請求する。 This application claims the priority of Japanese Patent Application No. 2011-133891 incorporated herein by reference.
　厚生労働省が公表した資料によると、日本人男性の約２人に１人、女性の約３人に１人が一生のうちに"がん"と診断され、肝臓がん、すい臓がん、肺がん患者の５年生存率はわずか２０％にも満たないことが報告されている。生命予後不良の主因は、腫瘍内の不均一（ヘテロ）な微小環境下で、一部のがん細胞が抗がん剤や放射線治療へ抵抗性を獲得することにあると考えられている。例えば悪性固形腫瘍内の低酸素環境下では低酸素誘導因子１（Hypoxia Inducible Factor-1：以下、単に「HIF-1」という場合もある。）が活性化し、がん細胞の治療抵抗性、転移・浸潤能、血管新生が亢進することが知られている（非特許文献１）。また、抗がん剤や放射線治療後の腫瘍内微小環境変化に応答してHIF-1が活性化し、がんの治療抵抗性が亢進することが報告されている（非特許文献２、３）。 According to data released by the Ministry of Health, Labor and Welfare, about 1 in 2 Japanese men and 1 in 3 women are diagnosed with "cancer" in their lifetime, liver cancer, pancreatic cancer, lung cancer It has been reported that the 5-year survival rate of patients is less than 20%. It is considered that the main cause of poor life prognosis is that some cancer cells acquire resistance to anticancer drugs and radiation therapy in a heterogeneous micro environment within the tumor. For example, hypoxia-inducible factor 1 (Hypoxia Inducible Factor-1: hereinafter sometimes referred to simply as “HIF-1”) is activated in a hypoxic environment within a malignant solid tumor, and treatment resistance and metastasis of cancer cells. -It is known that invasive ability and angiogenesis are enhanced (Non-patent Document 1). In addition, it has been reported that HIF-1 is activated in response to changes in the tumor microenvironment after an anticancer agent or radiotherapy, and the treatment resistance of cancer is enhanced (Non-patent Documents 2 and 3). .
　正常組織内部と異なり、固形腫瘍内部は極めて異質な微小環境で構成されている。腫瘍血管の遠位には十分な酸素や栄養源が供給されない低酸素、低栄養な環境が存在する。その一方で、解糖系や乳酸発酵によるATP産生が盛んな領域には酸性（低pH）環境が存在する。がん細胞はこれら過酷な微小環境を生き延びるために必要な遺伝子の発現を誘導する。例えば、低酸素環境に存在するがん細胞は、HIF-1という転写因子を活性化して低酸素環境でのエネルギー産生（グルコース代謝経路の変換）、低酸素環境の改善（血管新生）、低酸素環境からの回避（転移・浸潤）を図る。このような、がん細胞の環境応答システムが、がんの治療抵抗性においても重要な役割を果たしていることが報告されている（非特許文献３）。すなわち、抗がん剤や放射線治療によって腫瘍内微小環境が変化し、HIF-1が活性化することで、血管内皮増殖因子（Vascular Endothelial Growth Factor：VEGF）の発現を誘導し、VEGFが腫瘍血管内皮細胞の放射線障害を軽減し、腫瘍血管系が崩壊を免れ、がん細胞への酸素と栄養源の供給が保障され、最終的に放射線治療後のがん細胞の生存が保障されるという作用がはたらく。 Unlike the normal tissue, the solid tumor has a very different microenvironment. There is a hypoxic and hypotrophic environment where sufficient oxygen and nutrient sources are not provided at the distal end of the tumor blood vessels. On the other hand, an acidic (low pH) environment exists in a region where ATP production by glycolysis and lactic acid fermentation is popular. Cancer cells induce the expression of genes necessary to survive these harsh microenvironments. For example, cancer cells that exist in a hypoxic environment activate a transcription factor called HIF-1 to produce energy in the hypoxic environment (transformation of glucose metabolic pathway), improve the hypoxic environment (angiogenesis), hypoxia Avoid the environment (metastasis / infiltration). It has been reported that such an environmental response system for cancer cells plays an important role in cancer treatment resistance (Non-patent Document 3). In other words, the tumor microenvironment changes due to anticancer drugs and radiotherapy, and activation of HIF-1 induces the expression of vascular endothelial growth factor (Vascular Endothelial Growth Factor: VEGF), and VEGF induces tumor blood vessels Effect of reducing radiation damage to endothelial cells, preventing tumor vasculature from collapsing, ensuring supply of oxygen and nutrients to cancer cells, and finally ensuring survival of cancer cells after radiation therapy Works.
　固形腫瘍内に常在する低酸素環境下（固形腫瘍内低酸素領域）では、放射線感受性が著しく低下するため、腫瘍低酸素はがんの放射線治療成績不良の一因とされている。また近年、低酸素は特異的な遺伝子発現を誘導することにより、腫瘍増殖、血管新生、転移を促進する可能性が指摘されている。事実、子宮頸がん、頭頚部がん等の腫瘍内酸素分圧測定を施行した国内外の臨床研究において、低酸素分画の多寡と治療効果や生命予後との間に強い相関が報告されている。 In a hypoxic environment that is resident in a solid tumor (hypoxic region in a solid tumor), radiosensitivity is remarkably reduced, and thus tumor hypoxia is considered to be a cause of poor radiotherapy results for cancer. Recently, it has been pointed out that hypoxia may promote tumor growth, angiogenesis, and metastasis by inducing specific gene expression. In fact, in domestic and overseas clinical studies that performed intratumoral oxygen partial pressure measurements of cervical cancer, head and neck cancer, etc., a strong correlation was reported between the number of hypoxia fractions and therapeutic effects and life prognosis. ing.
　HIF-1は、細胞に対する酸素供給が不足状態に陥った時や細胞が過剰な活性酸素種にさらされた時に誘導されてくるタンパク質であり、転写因子として機能する。がんの病巣においては栄養不足や細胞外pHの低下、血流不足による酸素供給不足(低酸素)状態が認められるが、がん細胞が生き延びるためには新たに血管網を形成することにより病巣への血流を確保し、低酸素状態を脱する必要がある。そのための機能を担うべく低酸素条件において誘導される転写因子がHIF-1であり、種々の遺伝子の転写を亢進させる。HIF-1は様々な遺伝子発現の制御に関与しており、たとえば血管新生や細胞増殖、糖代謝、pH調節やアポトーシスなどに関わる遺伝子が挙げられる。HIF-1による発現制御を受ける遺伝子としてエリスロポエチンやVEGF等が挙げられる。HIF-1は、αとβの２つのサブユニットから構成されている。HIF-1αはアドレノメデュリンやマトリックスメタロプロテアーゼ（MMPs）、エンドセリン（ET）-1、一酸化窒素合成酵素（NOS）2など様々な遺伝子の制御を行っているといわれている。HIF-1αは有酸素状態の細胞内でユビキチン化を受け、タンパク質分解酵素複合体である26Sプロテアソームにより速やかに分解される。 HIF-1 is a protein that is induced when oxygen supply to cells is deficient or when cells are exposed to excessive reactive oxygen species, and functions as a transcription factor. In cancer lesions, nutrient deficiencies, decreased extracellular pH, and insufficient oxygen supply (hypoxia) due to insufficient blood flow are observed, but in order for cancer cells to survive, the lesion is formed by forming a new vascular network. It is necessary to secure blood flow to the limbs and escape from hypoxia. HIF-1 is a transcription factor induced under hypoxic conditions so as to have a function for that purpose, and enhances transcription of various genes. HIF-1 is involved in the control of various gene expressions, such as genes involved in angiogenesis, cell proliferation, sugar metabolism, pH regulation, apoptosis, and the like. Examples of genes that are regulated by HIF-1 include erythropoietin and VEGF. HIF-1 is composed of two subunits, α and β. HIF-1α is said to regulate various genes such as adrenomedullin, matrix metalloproteinases (MMPs), endothelin (ET) -1, and nitric oxide synthase (NOS) 2. HIF-1α undergoes ubiquitination in aerobic cells and is rapidly degraded by the 26S proteasome, a proteolytic enzyme complex.
　上記メカニズムに鑑み、HIF-1を抑制する抗腫瘍剤について開発が進められている。例えば、HIF-1遺伝子に対するアンチセンスオリゴヌクレオチド（Enzon、National Cancer Institute、Santaris Pharma）やHIF-1活性を抑制する低分子化合物（Abbot社）が挙げられる。またHIF-1抑制剤として、HSP90阻害剤等も報告されている（非特許文献４）。しかしながら、HIF-1がどのような機序で活性化するのか、また治療抵抗性に関わるHIF-1陽性がん細胞が腫瘍内のどこに局在し、どの様な病態・挙動を示すのかは明らかにされておらず、がんの完治を目指すうえで大きな障害となっている。 In view of the above mechanism, an antitumor agent that suppresses HIF-1 is being developed. Examples include antisense oligonucleotides against the HIF-1 gene (Enzon, National Cancer Institute, Santaris Pharma) and low molecular weight compounds that suppress HIF-1 activity (Abbot). Moreover, HSP90 inhibitor etc. are also reported as a HIF-1 inhibitor (nonpatent literature 4). However, it is clear how HIF-1 is activated, where HIF-1-positive cancer cells involved in treatment resistance are located in the tumor, and what pathology and behavior are shown This is a major obstacle to achieving complete cure for cancer.
　イソクエン酸脱水素酵素（isocitrate dehydrogenase：以下、単に「IDH」という場合もある。）にはいくつかのアイソフォームがあり、クエン酸回路（TCA回路）において、イソクエン酸からαケトグルタル酸への代謝に関わる酵素としてIDH1が公知である。IDHとがんの関係では、IDH1遺伝子やIDH2遺伝子に特定の変異がある場合にIDH1やIDH2の正常な機能が抑制され、腫瘍の悪性化に寄与することが報告されている（特許文献１、非特許文献５－７）。しかしながら、変異型でないIDHについて腫瘍の悪性化に関わる報告はなく、IDH1は腫瘍に対してむしろ良好な予後因子であると考えられている。 There are several isoforms in isocitrate dehydrogenase (hereinafter sometimes referred to simply as “IDH”). In the citrate cycle (TCA cycle), the metabolism of isocitrate to α-ketoglutarate is possible. IDH1 is known as an enzyme involved. Regarding the relationship between IDH and cancer, it has been reported that when there are specific mutations in the IDH1 gene and IDH2 gene, normal functions of IDH1 and IDH2 are suppressed, contributing to tumor malignancy (Patent Literature 1, Non-patent literature 5-7). However, there is no report regarding tumor malignancy for non-mutated IDH, and IDH1 is considered to be a rather good prognostic factor for tumors.
国際公開WO2010028099号パンフレットInternational publication WO2010028099 pamphlet
　本発明は、新規抗腫瘍剤を提供することを課題とし、また新規抗腫瘍剤のスクリーニング方法を提供することを課題とする。更に悪性腫瘍の新規治療方法を提供することを課題とする。 An object of the present invention is to provide a novel antitumor agent and to provide a screening method for a novel antitumor agent. Furthermore, it aims at providing the novel treatment method of a malignant tumor.
　本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、HIF-1の活性化を介してがんの治療抵抗性が亢進することに着目し、HIF-1の活性化を制御しうる物質を探索したところ、イソクエン酸脱水素酵素の一種であるIDH3がHIF-1の活性化に寄与していることを初めて見出し、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors have focused on the fact that cancer treatment resistance is enhanced through the activation of HIF-1, and the activation of HIF-1 As a result of searching for substances that can be controlled, it was found for the first time that IDH3, a kind of isocitrate dehydrogenase, contributes to the activation of HIF-1, and the present invention was completed.
　即ち本発明は、以下よりなる。
１．IDH3抑制物質を有効成分として含む、新規抗腫瘍剤。
２．IDH3抑制が、IDH3αの発現抑制又は機能抑制である、前項１に記載の新規抗腫瘍剤。
３．IDH3を抑制し、HIF-1を抑制することを特徴とする、前項１又は２に記載の新規抗腫瘍剤。 
４．HIF-1を抑制することが、HIF-1αの発現抑制又は機能抑制である前項３に記載の新規抗腫瘍剤。 
５．腫瘍が、難治性悪性腫瘍である、前項１～４のいずれか１に記載の新規抗腫瘍剤。
６．難治性悪性腫瘍が、放射線治療抵抗性がん及び／又は抗がん剤抵抗性がんである、前項５に記載の新規抗腫瘍剤。
７．IDH3抑制物質を有効成分として含む、HIF-1抑制剤。
８．IDH3の活性抑制を指標とする、新規抗腫瘍剤のスクリーニング方法。
９．以下の工程を含む、前項８に記載の新規抗腫瘍剤のスクリーニング方法：
１）IDH3αを発現しうる細胞株を候補物質で処理する工程；
２）候補物質で処理した前後で、上記細胞株についてIDH3αの発現量又はIDH3の活性を測定し、IDH3αの発現量又はIDH3の活性からIDH3の活性抑制を評価する工程。
１０．IDH3αを発現しうる細胞株が、HIF-1依存的にレポーター遺伝子を発現する細胞株であり、IDH3αの発現量又はIDH3の活性の測定が、レポーター遺伝子産物を計測することによる、前項９に記載の新規抗腫瘍剤のスクリーニング方法。
１１．IDH3を構成するサブユニットの少なくとも一つを標的因子として抑制することを含む、腫瘍の新規治療方法。
１２．IDH3を構成するサブユニットの少なくとも一つがIDH3αである、前項１１に記載の腫瘍の新規治療方法。
１３．IDH3を構成するサブユニットの少なくとも一つを標的因子として抑制することが、IDH3αの発現抑制又は機能抑制である、前項１１又は１２に記載の腫瘍の新規治療方法。
１４．腫瘍が、難治性悪性腫瘍である、前項１１～１３のいずれか１に記載に腫瘍の新規治療方法。
１５．難治性悪性腫瘍が、放射線治療抵抗性がん及び／又は抗がん剤抵抗性がんである、前項１４に記載の腫瘍の新規治療方法。 That is, this invention consists of the following.
1. A novel antitumor agent comprising an IDH3 inhibitor as an active ingredient.
2. 2. The novel antitumor agent according to item 1 above, wherein the IDH3 suppression is IDH3α expression suppression or function suppression.
3. 3. The novel antitumor agent according to 1 or 2 above, which suppresses IDH3 and suppresses HIF-1.
4). 4. The novel antitumor agent according to item 3 above, wherein the suppression of HIF-1 is suppression of expression or function of HIF-1α.
5. 5. The novel antitumor agent according to any one of items 1 to 4, wherein the tumor is an intractable malignant tumor.
6). 6. The novel antitumor agent according to 5 above, wherein the refractory malignant tumor is a radiation therapy resistant cancer and / or an anticancer drug resistant cancer.
7. An HIF-1 inhibitor comprising an IDH3 inhibitor as an active ingredient.
8). A screening method for a novel antitumor agent using inhibition of IDH3 activity as an index.
9. The method for screening for a novel antitumor agent according to item 8, which comprises the following steps:
1) a step of treating a cell line capable of expressing IDH3α with a candidate substance;
2) A step of measuring IDH3α expression level or IDH3 activity for the above cell lines before and after treatment with a candidate substance, and evaluating IDH3 activity suppression from IDH3α expression level or IDH3 activity.
10. 10. The cell line capable of expressing IDH3α is a cell line that expresses a reporter gene in an HIF-1-dependent manner, and the measurement of IDH3α expression level or IDH3 activity is performed by measuring the reporter gene product. Screening method for novel antitumor agents.
11. A novel method for treating a tumor, comprising inhibiting at least one of the subunits constituting IDH3 as a target factor.
12 12. The novel method for treating a tumor according to item 11, wherein at least one of the subunits constituting IDH3 is IDH3α.
13. 13. The novel method for treating a tumor according to item 11 or 12, wherein suppressing at least one of the subunits constituting IDH3 as a target factor is suppression of expression or function of IDH3α.
14 14. The novel method for treating a tumor according to any one of items 11 to 13, wherein the tumor is an intractable malignant tumor.
15. 15. The novel method for treating a tumor according to item 14, wherein the refractory malignant tumor is a radiation therapy resistant cancer and / or an anticancer drug resistant cancer.
　本発明のIDH3抑制物質は抗腫瘍効果を有する。また、本発明のIDH3抑制物質はHIF-1抑制効果を有する。これらの作用から、IDH3の活性を抑制しうる物質をスクリーニングすることで、効果的な抗腫瘍剤を提供しうる。 The IDH3 inhibitory substance of the present invention has an antitumor effect. Further, the IDH3 inhibitory substance of the present invention has a HIF-1 inhibitory effect. From these actions, an effective antitumor agent can be provided by screening a substance capable of suppressing the activity of IDH3.
HIF-1応答性プロモーターの制御下で抗生物質耐性遺伝子を発現しうる細胞株を用いてHIF-1活性を"正"に制御する因子（遺伝子）を網羅的に探索する（スクリーニングする）方法を示す概念図である。（参考例１）A method for exhaustively searching (screening) factors (genes) that positively control HIF-1 activity using cell lines that can express antibiotic resistance genes under the control of HIF-1-responsive promoters FIG. (Reference Example 1) "HIF-1応答性プロモーターの制御下で抗生物質耐性遺伝子を発現する遺伝子"を安定に組み込んだ細胞株について、常酸素条件及び低酸素条件での細胞の抗生物質感受性を示す図である。（参考例１）It is a figure which shows the antibiotic sensitivity of the cell in a normoxic condition and a hypoxic condition about the cell strain which integrated stably "the gene which expresses an antibiotic resistance gene under control of a HIF-1 responsive promoter." (Reference Example 1) HIF-1活性に影響を及ぼす因子（例えばIDH3α）の作用を確認するための系を示す概念図である。（参考例２）It is a conceptual diagram which shows the system for confirming the effect | action of the factor (for example, IDH3 (alpha)) which affects HIF-1 activity. (Reference Example 2) IDH3αの強制発現が細胞のHIF-1活性に及ぼす影響を、常酸素（20%）及び低酸素（0.02%）条件下で確認した図である。（参考例２）It is the figure which confirmed the influence which forced expression of IDH3 (alpha) exerts on the HIF-1 activity of a cell under normoxic (20%) and hypoxic (0.02%) conditions. (Reference Example 2) がん細胞での内在性のIDH3αの発現をshRNAで抑制（ノックダウン）した時のHIF-1活性に及ぼす影響を、常酸素（20%）及び低酸素（0.02%）条件下で確認した図である。（参考例２）The figure which confirmed the influence on the HIF-1 activity when the expression of endogenous IDH3α in cancer cells was suppressed (knocked down) with shRNA under normoxic (20%) and hypoxic (0.02%) conditions. It is. (Reference Example 2) HIF-1αのODD領域とルシフェラーゼとの融合遺伝子を発現するレポーター遺伝子を用いて、IDH3αの強制発現がHIF-1αタンパク質の安定性に及ぼす影響を確認するための系を示す概念図である。（参考例３）It is a conceptual diagram which shows the system for confirming the influence which forced expression of IDH3 (alpha) exerts on the stability of HIF-1 (alpha) protein using the reporter gene which expresses the fusion gene of the ODD area | region of HIF-1 (alpha) and luciferase. (Reference Example 3) HIF-1αのODD領域とルシフェラーゼとの融合遺伝子を発現するレポーター遺伝子を用いて、IDH3αの強制発現がHIF-1αタンパク質の安定性に及ぼす影響を、常酸素（20%）及び低酸素（0.02%）条件下で確認した図である。（参考例３）Using a reporter gene that expresses the fusion gene of HIF-1α ODD region and luciferase, the effect of forced expression of IDH3α on the stability of HIF-1α protein was examined using normal oxygen (20%) and hypoxia (0.02%). ) It is the figure confirmed under conditions. (Reference Example 3) HIF-1αのODD領域とルシフェラーゼとの融合遺伝子を発現するレポーター遺伝子を用いて、内在性のIDH3αの発現をノックダウンした場合のHIF-1αタンパク質の安定性に及ぼす影響を、常酸素（20%）及び低酸素（0.02%）条件下で確認した図である。（参考例３）The effect on the stability of HIF-1α protein when knocking down the expression of endogenous IDH3α using a reporter gene that expresses a fusion gene between the ODD region of HIF-1α and luciferase was measured using normoxia (20% ) And low oxygen (0.02%) conditions. (Reference Example 3) IDH3αの一過的な強制発現が、常酸素（20%）及び低酸素（0.02%）条件下でのHIF-1α発現量に及ぼす影響をウェスタンブロッティングにより確認した図である。（参考例３）It is the figure which confirmed the influence which transient forced expression of IDH3 (alpha) exerts on the expression level of HIF-1 (alpha) under normoxic (20%) and hypoxia (0.02%) conditions. (Reference Example 3) IDH3α発現ベクター又はその空ベクターを安定に導入したがん細胞株を、それぞれ免疫不全マウスに移植し、その後の腫瘍増殖を確認した図である。（参考例４）It is the figure which transplanted the cancer cell strain which introduce | transduced IDH3 (alpha) expression vector or its empty vector stably into an immunodeficient mouse | mouth, respectively, and confirmed subsequent tumor growth. (Reference Example 4) IDH3α発現ベクター又はその空ベクターを安定に導入したがん細胞株でのIDH3αの発現をウェスタンブロッティングにより確認した図である。（参考例４）It is the figure which confirmed the expression of IDH3 (alpha) in the cancer cell strain which introduce | transduced IDH3 (alpha) expression vector or its empty vector stably by Western blotting. (Reference Example 4) IDH3αに対するshRNA発現ベクター又は陰性対照用shRNA発現ベクターをがん細胞株に安定に組み込み、それらを常酸素（20%）及び低酸素（0.02%）条件で処理したときのIDH3α及びHIF-1αの発現を、ウェスタンブロッティングにより確認した図である。（実施例１）IDH3α and HIF-1α expression when shRNA expression vector against IDH3α or shRNA expression vector for negative control is stably incorporated into cancer cell lines and treated under normoxic (20%) and hypoxic (0.02%) conditions Is a figure confirmed by Western blotting. Example 1 IDH3αに対するshRNA発現ベクター又は陰性対照用shRNA発現ベクターを安定に組み込んだがん細胞株を免疫不全マウスに移植し、その後の腫瘍増殖を確認した図である。（実施例１）It is the figure which transplanted the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse | mouth, and confirmed subsequent tumor growth. Example 1 IDH3αに対するshRNA発現ベクター又は陰性対照用shRNA発現ベクターを安定に組み込んだがん細胞株を免疫不全マウスに移植し、形成された腫瘍の大きさを確認した写真図である。（実施例１）It is the photograph which confirmed the magnitude | size of the formed tumor by transplanting the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse | mouth. Example 1 IDH3α又はHIF-1αに対するshRNA発現ベクターを、各々安定に組み込んだがん細胞株を免疫不全マウスに移植し、その後の腫瘍増殖を確認した図である。（実施例２）It is the figure which transplanted the cancer cell line which each integrated stably the shRNA expression vector with respect to IDH3 (alpha) or HIF-1 (alpha) to an immunodeficient mouse, and confirmed subsequent tumor growth. (Example 2) IDH3αに対するshRNA発現ベクター又は陰性対照用shRNA発現ベクターを安定に組み込んだがん細胞株を免疫不全マウスに移植し、形成された腫瘍内血管を示す写真図である。（実施例３）It is a photograph figure which shows the intravascular tumor blood vessel which transplanted the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse | mouth. Example 3 IDH3αに対するshRNA発現ベクター又は陰性対照用shRNA発現ベクターを安定に組み込んだがん細胞株を免疫不全マウスに移植し、形成された腫瘍内の血管密度を定量した結果を示す図である。（実施例３）It is a figure which shows the result of having transplanted the cancer cell line which integrated stably the shRNA expression vector with respect to IDH3 (alpha), or the shRNA expression vector for negative controls to the immunodeficient mouse | mouth, and having quantified the blood vessel density in the formed tumor. Example 3
　本明細書において「IDH3」とは、イソクエン酸脱水素酵素３（Isocitrate Dehydrogenase 3）を意味する。本発明は、IDH3抑制剤を有効成分として含む新規抗腫瘍剤に関する。IDH3はαサブユニット（以下、「IDH3α」）２分子、βサブユニット（以下、「IDH3β」）１分子及びγサブユニット（以下、「IDH3γ」）１分子で構成されるヘテロ４量体である。 In the present specification, “IDH3” means isocitrate dehydrogenase 3 (Isocitrate Dehydrogenase 3). The present invention relates to a novel antitumor agent comprising an IDH3 inhibitor as an active ingredient. IDH3 is a heterotetramer composed of two molecules of α subunit (hereinafter “IDH3α”), one molecule of β subunit (hereinafter “IDH3β”) and one molecule of γ subunit (hereinafter “IDH3γ”). .
　本発明において「IDH3抑制物質」とは、IDH3活性を抑制しうる物質であればよく、IDH3活性を抑制するために、IDH3を構成するサブユニットのうち少なくとも１種のサブユニットを標的とする物質であればよい。本発明において、標的とされるサブユニットとしては、特に好ましくはIDH3αである。ここで、IDH3を抑制するために、IDH3αの発現抑制又はIDH3αの機能を抑制すればよい。ここにおいて、IDH3αの発現抑制とはIDH3αの生合成遺伝子（以下、単に「IDH3α遺伝子」という。）の発現が抑制されることをいい、転写と翻訳をはじめとする遺伝子発現制御過程のいずれかの段階を阻害する作用を意味する。また、IDH3αの機能抑制とは、IDH3αの一部又は全部を阻害することによるIDH3のイソクエン酸脱水素酵素活性に及ぼす機能を抑制することをいう。IDH3抑制物質として、例えばIDH3αの発現を抑制しうるアンチセンス核酸、リボザイム核酸、siRNA、shRNAや、IDH3αの発現若しくはIDH3αの機能を抑制する低分子化合物、ペプチド、酵素や抗体などが挙げられる。本発明のIDH3抑制物質は、上記物質の他、それらの薬学的に許容しうる塩も含まれる。ここにおいて、IDH3αは、GenBank Accession Number：NP_005521.1で示されるアミノ酸配列で特定することができ、mRNAは、同Accession Number：NM_005530.2で示すことができる。 In the present invention, the “IDH3 inhibitory substance” may be any substance that can suppress IDH3 activity, and in order to suppress IDH3 activity, a substance that targets at least one subunit among the subunits constituting IDH3 If it is. In the present invention, the target subunit is particularly preferably IDH3α. Here, in order to suppress IDH3, the expression suppression of IDH3α or the function of IDH3α may be suppressed. Here, suppression of IDH3α expression refers to suppression of expression of IDH3α biosynthetic gene (hereinafter simply referred to as “IDH3α gene”), and any of the gene expression control processes including transcription and translation. It means the action of inhibiting the stage. In addition, suppression of the function of IDH3α refers to suppression of the function of IDH3 on isocitrate dehydrogenase activity by inhibiting part or all of IDH3α. Examples of the IDH3 inhibitor include antisense nucleic acids, ribozyme nucleic acids, siRNA, shRNA, low molecular compounds that suppress IDH3α expression or IDH3α function, peptides, enzymes, antibodies, and the like that can suppress IDH3α expression. The IDH3 inhibitory substance of the present invention includes pharmaceutically acceptable salts thereof in addition to the above substances. Here, IDH3α can be identified by the amino acid sequence represented by GenBank Accession Number: NP_005521.1, and mRNA can be designated by the same Accession Number: NM_005530.2.
　本発明のIDH3抑制物質として、具体的にはIDH3αの発現を抑制しうるshRNAを例示することができる。当該shRNAが標的とする具体的な配列としては、以下の配列番号１～３に示す（A）～（C）が挙げられる。
（A）TGACT TGTGT GCAGG ATTGAT　（配列番号１）
（B）AGATG GTATT GGCCC AGAAA T　（配列番号２）
（C）TCACC CATCT ATGAA TTTAC T　（配列番号３） Specific examples of the IDH3 inhibitory substance of the present invention include shRNA that can suppress the expression of IDH3α. Specific examples of sequences targeted by the shRNA include (A) to (C) shown in the following SEQ ID NOs: 1 to 3.
(A) TGACT TGTGT GCAGG ATTGAT (SEQ ID NO: 1)
(B) AGATG GTATT GGCCC AGAAA T (SEQ ID NO: 2)
(C) TCACC CATCT ATGAA TTTAC T (SEQ ID NO: 3)
　本発明において、IDH3を抑制しHIF-1を抑制するとは、上述のIDH3を抑制することを介してHIF-1活性を抑制することを意味する。ここにおいて、「HIF-1」とは、低酸素誘導因子１（Hypoxia-Inducible Factor 1）を意味する。HIF-1は、背景技術の欄でも説明したように、細胞に対する酸素供給が不足状態に陥った時、もしくは細胞が過剰な活性酸素種に曝された時に活性化するタンパク質であり、転写因子として機能する物質である。HIF-1は、HIF-1αとHIF-1βとのヘテロ２量体を形成し、転写因子として種々の遺伝子の転写を亢進させる機能を発揮しうる。HIF-1の低酸素依存的活性を制御している部位として、HIF-1αタンパク質のほぼ中央にある酸素依存的分解ドメイン(Oxygen-dependent degradation domain: ODD)が存在する。HIF-1とがんとの関係について多く報告されていることは、背景技術の欄で説明したとおりである。 In the present invention, suppressing IDH3 and suppressing HIF-1 means suppressing HIF-1 activity through suppressing IDH3 described above. Here, “HIF-1” means hypoxia-inducible factor 1 (Hypoxia-Inducible Factor 1). As explained in the background section, HIF-1 is a protein that is activated when oxygen supply to cells falls short or when cells are exposed to excessive reactive oxygen species. It is a functional substance. HIF-1 forms a heterodimer of HIF-1α and HIF-1β, and can exert a function of enhancing transcription of various genes as a transcription factor. As a site controlling the hypoxia-dependent activity of HIF-1, an oxygen-dependent degradation domain (Oxygen-dependent) degradation domain: ODD) exists in the approximate center of the HIF-1α protein. Many reports on the relationship between HIF-1 and cancer are as described in the Background section.
　しかしながら、本発明においてIDH3とHIF-1の活性との関係を初めて見出した。IDH3とHIF-1活性との関係を初めて見出した経緯については、後述の参考例において、詳細に説明する。ここで、HIF-1活性を抑制するために、HIF-1αの発現抑制若しくは機能を抑制、又はHIF-1αとHIF-1βの結合を阻害すればよい。ここにおいて、HIF-1αの発現抑制とはHIF-1αの生合成遺伝子（以下、単に「HIF-1α遺伝子」という。）の発現が抑制されることをいい、転写と翻訳をはじめとする遺伝子発現制御過程のいずれかの段階を阻害する作用を意味する。また、HIF-1αの機能抑制とは、HIF-1αの一部又は全部を阻害することによるHIF-1の活性に及ぼす機能を抑制することをいう。HIF-1を抑制することで、腫瘍のうち難治性悪性腫瘍、例えば放射線抵抗性がんや化学療法剤などの抗がん剤抵抗性がんに対して、効果的に抗腫瘍効果を発揮しうる。また、本発明は、IDH3抑制物質を有効成分として含むHIF-1抑制剤にも及ぶ。即ち、HIF-1抑制作用によって、抗腫瘍効果以外の機能を発揮しうるのであれば、本発明のIDH3抑制物質を有効量含む薬剤は抗腫瘍剤に限定されるものではない。 However, in the present invention, the relationship between IDH3 and HIF-1 activity was found for the first time. The background of finding the relationship between IDH3 and HIF-1 activity for the first time will be described in detail in Reference Examples described later. Here, in order to suppress the HIF-1 activity, the expression suppression or function of HIF-1α may be suppressed, or the binding between HIF-1α and HIF-1β may be inhibited. Here, suppression of HIF-1α expression refers to suppression of HIF-1α biosynthetic gene expression (hereinafter simply referred to as “HIF-1α gene”), and gene expression including transcription and translation. It means an action that inhibits any step of the control process. Further, the suppression of the function of HIF-1α refers to the suppression of the function exerted on the activity of HIF-1 by inhibiting part or all of HIF-1α. Suppressing HIF-1 effectively exerts an antitumor effect against refractory malignant tumors such as radiation resistant cancer and chemotherapeutic resistant cancer such as chemotherapeutic agents. sell. The present invention also extends to an HIF-1 inhibitor containing an IDH3 inhibitor as an active ingredient. That is, a drug containing an effective amount of the IDH3 inhibitory substance of the present invention is not limited to an antitumor agent as long as functions other than the antitumor effect can be exhibited by the HIF-1 inhibitory action.
　本発明の新規抗腫瘍剤に有効成分として含まれるIDH3抑制物質は、IDH3とHIF-1との関係に起因する作用機序とは異なる機序によって抗腫瘍作用を発揮することもあり得る。 The IDH3 inhibitor contained in the novel antitumor agent of the present invention as an active ingredient may exert an antitumor action by a mechanism different from the action mechanism resulting from the relationship between IDH3 and HIF-1.
　本発明の新規抗腫瘍剤又はHIF-1抑制剤は、有効成分としてのIDH3抑制物質を有効量含む他、一般に医薬に使用される添加剤を含んでいてもよい。添加剤としては、賦形剤、結合剤、滑沢剤、崩壊剤、着色剤、矯味矯臭剤、乳化剤、界面活性剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、防腐剤、抗酸化剤、安定化剤、吸収促進剤等を挙げることができ、所望により、これらを適宜組み合わせて使用することもできる。 The novel antitumor agent or HIF-1 inhibitor of the present invention contains an effective amount of an IDH3 inhibitory substance as an active ingredient, and may also contain additives generally used in medicine. Additives include excipients, binders, lubricants, disintegrants, colorants, flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, isotonic agents, buffers, preservatives Agents, antioxidants, stabilizers, absorption promoters and the like, and these can be used in appropriate combinations as desired.
　具体的には、上記賦形剤としては、例えば乳糖、白糖、ブドウ糖、コーンスターチ、マンニトール、ソルビトール、デンプン、α化デンプン、デキストリン、結晶セルロース、軽質無水ケイ酸、ケイ酸アルミニウム、ケイ酸カルシウム、メタケイ酸アルミン酸マグネシウム、リン酸水素カルシウム等を挙げることができる。上記結合剤としては、例えばポリビニルアルコール、メチルセルロース、エチルセルロース、アラビアゴム、トラガント、ゼラチン、シェラック、ヒドロキシプロピルメチルセルロース、ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、ポリビニルピロリドン、マクロゴール等を挙げることができる。上記滑沢剤としては、例えばステアリン酸マグネシウム、ステアリン酸カルシウム、フマル酸ステアリルナトリウム、タルク、ポリエチレングリコール、コロイドシリカ等を挙げることができる。上記崩壊剤としては、例えば結晶セルロース、寒天、ゼラチン、炭酸カルシウム、炭酸水素ナトリウム、クエン酸カルシウム、デキストリン、ペクチン、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、クロスカルメロースナトリウム、カルボキシメチルスターチ、カルボキシメチルスターチナトリウム等を挙げることができる。上記着色剤としては、例えば三二酸化鉄、黄色三二酸化鉄、カルミン、カラメル、β－カロチン、酸化チタン、タルク、リン酸リボフラビンナトリウム、黄色アルミニウムレーキ等、医薬品に添加することが許可されているものを挙げることができる。上記矯味矯臭剤としては、例えばココア末、ハッカ脳、芳香散、ハッカ油、メントール、竜脳、桂皮末等を挙げることができる。上記乳化剤又は界面活性剤としては、例えばステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、モノステアリン酸グリセリン、ショ糖脂肪酸エステル、グリセリン脂肪酸エステル等を挙げることができる。上記溶解補助剤としては、例えばポリエチレングリコール、プロピレングリコール、安息香酸ベンジル、エタノール、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、ポリソルベート８０、ニコチン酸アミド等を挙げることができる。上記懸濁化剤としては、前記界面活性剤のほか、例えばポリビニルアルコール、ポリビニルピロリドン、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース等の親水性高分子を挙げることができる。上記等張化剤としては、例えばブドウ糖、塩化ナトリウム、マンニトール、ソルビトール等を挙げることができる。上記緩衝剤としては、例えばリン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液を挙げることができる。上記防腐剤としては、例えばメチルパラベン、プロピルパラベン、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸等を挙げることができる。上記抗酸化剤としては、例えば亜硫酸塩、アスコルビン酸、α－トコフェロール等を挙げることができる。上記安定化剤としては、アスコルビン酸、エデト酸ナトリウム、エリソルビン酸、トコフェロール等を挙げることができる。上記吸収促進剤としては、ミリスチン酸イソプロピル、トコフェロール、カルシフェロール等を挙げることができる。 Specific examples of the excipient include lactose, sucrose, glucose, corn starch, mannitol, sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, light anhydrous silicic acid, aluminum silicate, calcium silicate, and metasilicate. Examples thereof include magnesium aluminate acid and calcium hydrogen phosphate. Examples of the binder include polyvinyl alcohol, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, macrogol and the like. Examples of the lubricant include magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, polyethylene glycol, colloidal silica and the like. Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl Examples include starch and sodium carboxymethyl starch. Examples of the colorant include those allowed to be added to pharmaceuticals such as iron sesquioxide, yellow sesquioxide, carmine, caramel, β-carotene, titanium oxide, talc, riboflavin sodium phosphate, yellow aluminum lake, etc. Can be mentioned. Examples of the flavoring agent include cocoa powder, mint brain, aroma powder, mint oil, menthol, dragon brain, and cinnamon powder. Examples of the emulsifier or surfactant include stearyl triethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, glyceryl monostearate, sucrose fatty acid ester, and glycerin fatty acid ester. Examples of the solubilizer include polyethylene glycol, propylene glycol, benzyl benzoate, ethanol, cholesterol, triethanolamine, sodium carbonate, sodium citrate, polysorbate 80, and nicotinamide. Examples of the suspending agent include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose, in addition to the surfactant. Examples of the isotonic agent include glucose, sodium chloride, mannitol, sorbitol and the like. Examples of the buffer include buffer solutions such as phosphate, acetate, carbonate, citrate. Examples of the preservative include methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like. Examples of the stabilizer include ascorbic acid, sodium edetate, erythorbic acid, tocopherol and the like. Examples of the absorption promoter include isopropyl myristate, tocopherol, calciferol and the like.
　本発明は、IDH3の活性抑制を指標とする新規抗腫瘍剤のスクリーニング方法にも及ぶ。本発明の、新規抗腫瘍剤のスクリーニング方法は、IDH3の活性を評価可能な系であればよく、特に限定されないが、例えば以下の工程を含む方法により行うことができる。
１）IDH3αを発現しうる細胞株を候補物質で処理する工程；
２）候補物質で処理した前後で、上記細胞株についてIDH3αの発現量又はIDH3の活性を測定し、IDH3αの発現量又はIDH3の活性からIDH3の活性抑制を評価する工程。 The present invention also extends to a method for screening a novel antitumor agent using IDH3 activity suppression as an index. The screening method for a novel antitumor agent of the present invention is not particularly limited as long as it is a system that can evaluate the activity of IDH3. For example, it can be performed by a method including the following steps.
1) a step of treating a cell line capable of expressing IDH3α with a candidate substance;
2) A step of measuring IDH3α expression level or IDH3 activity for the above cell lines before and after treatment with a candidate substance, and evaluating IDH3 activity suppression from IDH3α expression level or IDH3 activity.
　IDH3αを発現しうる細胞株は、HIF-1依存的にレポーター遺伝子を発現する細胞株であることが好ましく、例えばHIF-1応答性プロモーターの下流にレポーター遺伝子が安定に組み込まれているのが好適である。IDH3αを発現しうる細胞株には、HIF-1応答性プロモーターの代わりに、レポーター遺伝子とHIF-1αのODD領域をコードする遺伝子の融合遺伝子を含めてもよい。係る細胞株は、予め作製した細胞株を用いてもよいし、本発明のスクリーニング方法の工程に細胞株を準備する工程を含めてもよい。レポーター遺伝子としては、自体公知の遺伝子を使用することができ、特に限定されないがルシフェラーゼやGFP等のように発光や蛍光を発しうる遺伝子を用いるのが好適である。 The cell line capable of expressing IDH3α is preferably a cell line that expresses a reporter gene in an HIF-1-dependent manner.For example, it is preferable that the reporter gene is stably integrated downstream of the HIF-1-responsive promoter. It is. A cell line capable of expressing IDH3α may contain a fusion gene of a reporter gene and a gene encoding the ODD region of HIF-1α instead of the HIF-1 responsive promoter. As such a cell line, a cell line prepared in advance may be used, or a step of preparing the cell line may be included in the step of the screening method of the present invention. As the reporter gene, a gene known per se can be used. Although not particularly limited, it is preferable to use a gene capable of emitting light or fluorescence such as luciferase or GFP.
　本発明のスクリーニング方法に使用可能な細胞株は、以下の方法により構築することができる。まずはじめに、HIF-1応答性プロモーターの下流にレポーター遺伝子を安定に組み込んだ細胞株を樹立する。さらにIDH3αを大量に発現しうるベクターを前記細胞に導入する。IDH3αはHIF-1を活性化するため、当該レポーター遺伝子を組み込んだ細胞株は、IDH3大量発現ベクターを導入した場合に強い発光を生じる。使用可能な細胞株としては、例えばヒト子宮頸がん由来細胞株HeLa、ヒト肺胞上皮がん由来細胞株A549、ヒト大腸がん由来細胞株Colon26などが挙げられる。 A cell line that can be used in the screening method of the present invention can be constructed by the following method. First, a cell line that stably incorporates a reporter gene downstream of the HIF-1-responsive promoter is established. Furthermore, a vector capable of expressing IDH3α in large quantities is introduced into the cells. Since IDH3α activates HIF-1, a cell line incorporating the reporter gene emits intense luminescence when an IDH3 mass expression vector is introduced. Examples of cell lines that can be used include human cervical cancer-derived cell line HeLa, human alveolar carcinoma-derived cell line A549, and human colon cancer-derived cell line Colon 26.
　上記で樹立された、又は既に樹立された細胞を、候補物質を含む培地で培養し、培養細胞についてレポーター遺伝子由来の指標、例えば発光量や蛍光量を測定することで、本発明の新規抗腫瘍剤をスクリーニングすることができる。具体的には、候補物質により発光量や蛍光量の低下、又はIDH3αの発現量の低下を認めた場合に、選別された物質は本発明の抗腫瘍剤となりえる。 The novel anti-tumor of the present invention is obtained by culturing the cells established as described above or already established in a medium containing a candidate substance, and measuring an indicator derived from a reporter gene, such as luminescence and fluorescence, for the cultured cells. Agents can be screened. Specifically, the selected substance can be the antitumor agent of the present invention when the candidate substance shows a decrease in the amount of luminescence or fluorescence, or a decrease in the expression level of IDH3α.
　上記の他、例えば以下の方法によりスクリーニングを行ってもよい。
１）IDH3αを発現しうる細胞株を候補物質で処理する工程；
２）候補物質で処理した細胞から抽出液を得、IDH3α抗体又はHIF-1α抗体、さらには例えばVEGFなどに代表されるHIF-1下流遺伝子に対する抗体などを利用したウェスタンブロッティングやELISA、もしくは対象とする遺伝子の活性測定などを行い、IDH3α、HIF-1α又はHIF-1下流遺伝子の発現量及びHIF-1やHIF-1下流遺伝子の活性などを確認することで、IDH3の活性抑制を評価する工程。 In addition to the above, screening may be performed by the following method, for example.
1) a step of treating a cell line capable of expressing IDH3α with a candidate substance;
2) Obtain an extract from cells treated with the candidate substance, Western blotting or ELISA using IDH3α antibody or HIF-1α antibody, and antibodies against downstream HIF-1 genes such as VEGF, etc. To evaluate IDH3 activity suppression by confirming the expression level of IDH3α, HIF-1α or HIF-1 downstream gene and the activity of HIF-1 or HIF-1 downstream gene, etc. .
　上記でスクリーニングされた候補物質は、HIF-1応答性レポーター遺伝子、例えばHIF-1応答性プロモーターの下流にレポーター遺伝子を安定に組み込こんだがん細胞を免疫不全マウスに移植し、得られた担がんマウスを前記スクリーニングされた候補物質で処理した後、HIF-1依存的に発現するレポーター物質、例えば発光タンパク質や蛍光タンパク質を観察・可視化・定量することで評価することができる。 The candidate substances screened above are HIF-1 responsive reporter genes, for example, the tumor cells obtained by transplanting cancer cells stably incorporating a reporter gene downstream of the HIF-1 responsive promoter into immunodeficient mice. The cancer mouse can be evaluated by observing, visualizing, and quantifying a reporter substance such as a luminescent protein or a fluorescent protein that is expressed in an HIF-1-dependent manner after the cancer mouse is treated with the screened candidate substance.
　本発明の新規抗腫瘍剤に有効成分として含有されるIDH3抑制剤は、下記の参考例１の方法によりHIF-1の活性に着目して標的物質を選別し、鋭意検討を重ねた結果達成されたものである。本発明のIDH3抑制剤のみならず、HIF-1の活性に着目した選別方法により、あらたな抗腫瘍剤をスクリーニングすることができる。 The IDH3 inhibitor contained as an active ingredient in the novel antitumor agent of the present invention was achieved as a result of repeated investigations by selecting target substances by focusing on the activity of HIF-1 by the method of Reference Example 1 below. It is a thing. In addition to the IDH3 inhibitor of the present invention, a new antitumor agent can be screened by a screening method focusing on the activity of HIF-1.
　本発明の理解を深めるために、以下に本発明の新規抗腫瘍剤を完成させるに至った経緯を参考例に示し、本発明を実施例に示してより具体的に説明するが、本発明はこれに限定されるものではないことはいうまでもない。 In order to deepen the understanding of the present invention, the background to the completion of the novel antitumor agent of the present invention will be shown in Reference Examples, and the present invention will be described in more detail with reference to Examples. Needless to say, the present invention is not limited to this.
（参考例１）本発明の新規抗腫瘍剤の標的物質を選別するに至った経緯
　本発明の新規抗腫瘍剤は、IDH3抑制物質を有効成分として含むものであり、その標的物質としてIDH3αが挙げられる。本参考例では、IDH3αが標的物質として選別されるに至った経緯を説明する。 (Reference Example 1) Background of selection of target substance of novel antitumor agent of the present invention The novel antitumor agent of the present invention contains an IDH3 inhibitory substance as an active ingredient, and IDH3α is cited as the target substance. It is done. In this reference example, the reason why IDH3α has been selected as a target substance will be described.
　低酸素誘導性因子の一種であるHIF-1については、HIF-1の活性化を介して、がんの治療抵抗性及び悪性化が亢進することが、本発明者らにより報告されている（非特許文献２、３）。しかしながら、HIF-1がどのような機序で活性化するのか、また治療抵抗性に関わるHIF-1陽性がん細胞が腫瘍内の何処に局在し、どのような動態・挙動を示すのかは、明らかにされていなかった。そこで、本発明者はHIF-1を活性化する因子（遺伝子）を網羅的に探索する実験を実施した。まず、血管内皮細胞増殖因子(VEGF)遺伝子のプロモーター由来のHIF-1応答性エンハンサーHREを含む人工的なHIF-1応答性プロモーターの制御下で、ブラストサイジンという抗生物質への耐性を担うタンパク質を発現する遺伝子を構築した。そして、当該遺伝子を安定に組み込んだ細胞株を樹立した（図１参照）。 Regarding HIF-1, which is a kind of hypoxia-inducible factor, the present inventors have reported that cancer treatment resistance and malignancy are enhanced through activation of HIF-1. Non-patent documents 2, 3). However, the mechanism by which HIF-1 is activated and where the HIF-1-positive cancer cells involved in treatment resistance are located in the tumor and what kinetics / behavior is shown. Was not revealed. Therefore, the present inventor conducted an experiment to exhaustively search for factors (genes) that activate HIF-1. First, a protein responsible for the resistance to an antibiotic called blasticidin under the control of an artificial HIF-1-responsive promoter, including the HIF-1-responsive enhancer HRE derived from the vascular endothelial growth factor (VEGF) gene promoter A gene that expresses was constructed. And the cell strain which integrated the said gene stably was established (refer FIG. 1).
　上記細胞株によるブラストサイジン耐性遺伝子の発現は、HIF-1応答性プロモーターの制御下にあるため、有酸素条件ではブラストサイジン耐性タンパク質の発現が認められない。その結果、当該細胞株は酸素存在下で薬剤に感受性を示して死滅する。逆に、低酸素刺激などによってHIF-1活性が亢進した場合に、当該細胞はブラストサイジン耐性タンパク質を発現するようになり、結果として薬剤耐性を示す。各濃度の抗生物質を含む培地を用いて上記細胞を常酸素条件及び低酸素条件で培養した際の細胞生存率を図２に示した。 Since the expression of the blasticidin resistance gene by the above cell line is under the control of the HIF-1 responsive promoter, the expression of the blasticidin resistant protein is not observed under aerobic conditions. As a result, the cell line is sensitive to the drug and killed in the presence of oxygen. On the contrary, when HIF-1 activity is enhanced by hypoxic stimulation or the like, the cell expresses a blasticidin resistant protein, and as a result, exhibits drug resistance. FIG. 2 shows the cell viability when the cells were cultured under normoxic and hypoxic conditions using a medium containing antibiotics at various concentrations.
　HIF-1活性化に影響を及ぼしうる遺伝子を網羅的に探索（スクリーニング）するために、上記で樹立した細胞に、レトロウィルスベクターを利用してcDNAライブラリーを適宜導入し、これを常酸素（20%）条件下にてブラストサイジンを含む培地で培養し、コロニーを形成する細胞を選択した（図１参照）。ここで生き残った細胞内では、ゲノムDNAにインテグレートされた（組込まれた）遺伝子の機能によってHIF-1が活性化し、結果としてブラストサイジン耐性遺伝子の発現が誘導されていることが期待できた。そこで"生存細胞から精製したゲノムDNA"を鋳型とし、さらに"挿入されたcDNAを挟みこむプライマー"を用いてPCR反応を行うことで、当該cDNA断片を増幅した。このDNA断片を汎用のプラスミドベクター（具体的にはpBlueScript II SK+）のEcoRV部位にサブクローニングした。T7プライマーとT3プライマーを用いて挿入されたcDNAの塩基配列を解析し、その情報を基にホモロジー検索を実施した結果、HIF-1を活性化する機能を持ち得る遺伝子としてIDH3αを同定するに至った。 In order to comprehensively search (screen) for genes that can affect HIF-1 activation, a cDNA library is appropriately introduced into the cells established above using a retroviral vector, 20%) cells were cultured in a medium containing blasticidin, and cells that formed colonies were selected (see FIG. 1). In the surviving cells, HIF-1 was activated by the function of the gene integrated (incorporated) into the genomic DNA, and it was expected that the expression of the blasticidin resistance gene was induced as a result. Therefore, the cDNA fragment was amplified by performing a PCR reaction using “genomic DNA purified from living cells” as a template and further using a “primer sandwiching the inserted cDNA”. This DNA fragment was subcloned into the EcoRV site of a general-purpose plasmid vector (specifically, pBlueScript II SK +). As a result of analyzing the nucleotide sequence of the inserted cDNA using the T7 primer and T3 primer and performing a homology search based on the information, IDH3α was identified as a gene capable of activating HIF-1. It was.
（参考例２）IDH3αによるHIF-1活性に及ぼす影響
　参考例１にて獲得したIDH3αがHIF-1を活性化する機能を有することを確認する目的で、以下の実験を行った（図３参照）。まずヒトIDH3α cDNAをpcDNA4/myc-His Aプラスミド（Invitrogen社）のEcoR1-Xho1部位に挿入し、IDH3α強制発現プラスミドpcDNA4A/IDH3αを構築した。当該強制発現プラスミド内では、IDH3α遺伝子の翻訳終結コドンが欠失されていること、またIDH3α構造遺伝子とその下流のmycタグがインフレームで融合されていることから、当該プラスミドはIDH3αとmycタグの融合タンパク質を強制発現する。HIF-1依存的にルシフェラーゼを発現する5HREp-lucレポーター遺伝子を安定に組み込んだヒト子宮頸がん由来細胞株HeLa/5HRE-Luc（Harada et al. Mol Imaging, 4: 182-193. 2005）に、当該pcDNA4A/IDH3αをトランスフェクトした。常酸素（20%）又は低酸素（0.02%）条件下で培養した後に細胞溶解用試薬（Passive Lysis buffer：Promega社）を用いて細胞抽出液を得、ルシフェラーゼアッセイキット（Promoga社）を用いて、当該細胞抽出液中のルシフェラーゼ活性を定量した。陰性対照実験として、IDH3α cDNAを組み込んでいないpcDNA4/myc-His Aプラスミド（Empty Vector：以下、単に「空ベクター」という。）を用いて同様の実験を行った。その結果、空ベクターを導入した場合と比較して、pcDNA4A/IDH3αを導入した場合に、酸素条件に関わらず有意にルシフェラーゼ活性が亢進することを確認した（図４参照）。この結果は、IDH3αの過剰発現によって、HIF-1が活性化したことを示している。 (Reference Example 2) Effect of IDH3α on HIF-1 Activity The following experiment was conducted to confirm that IDH3α obtained in Reference Example 1 has a function of activating HIF-1 (see FIG. 3). ). First, human IDH3α cDNA was inserted into EcoR1-Xho1 site of pcDNA4 / myc-His A plasmid (Invitrogen) to construct IDH3α forced expression plasmid pcDNA4A / IDH3α. In the forced expression plasmid, the translation termination codon of the IDH3α gene has been deleted, and the IDH3α structural gene and its downstream myc tag are fused in-frame. Forced expression of the fusion protein. To the human cervical cancer cell line HeLa / 5HRE-Luc (Harada et al. Mol Imaging, 4: 182-193. 2005) stably incorporating the 5HREp-luc reporter gene that expresses luciferase in a HIF-1-dependent manner The pcDNA4A / IDH3α was transfected. After culturing under normoxic (20%) or hypoxic (0.02%) conditions, a cell extract is obtained using a cell lysis reagent (Passive Lysis buffer: Promega), and a luciferase assay kit (Promoga) is used. The luciferase activity in the cell extract was quantified. As a negative control experiment, a similar experiment was performed using pcDNA4 / myc-His A plasmid (Empty Vector: hereinafter simply referred to as “empty vector”) in which IDH3α cDNA was not incorporated. As a result, it was confirmed that, when pcDNA4A / IDH3α was introduced, luciferase activity was significantly enhanced regardless of the oxygen condition, compared with the case where an empty vector was introduced (see FIG. 4). This result indicates that HIF-1 was activated by overexpression of IDH3α.
　IDH3αが真にHIF-1を活性化しうるか否かを検討する目的で、IDH3α遺伝子内の以下の（A）～（C）の配列に対するshRNA発現プラスミド（SABiosciences社：品番KH15075P）を用いて実験を行った。陰性対照として、ヒト遺伝子の何れにも一致しない以下（D）の配列に対するshRNA(Scr)発現プラスミドを用いた。
（A）TGACT TGTGT GCAGG ATTGAT　（配列番号１）
（B）AGATG GTATT GGCCC AGAAA T　（配列番号２）
（C）TCACC CATCT ATGAA TTTAC T　（配列番号３）
（D）GGAAT CTCAT TCGAT GCATA C （配列番号４）
　HeLa/5HRE-Luc細胞に当該shRNA(A)～(C)各発現プラスミド、又は陰性対照用のshRNA(Scr)発現プラスミドを導入し、常酸素（20%）又は低酸素（0.02%）環境下で培養した後に、ルシフェラーゼアッセイを実施した。（図５参照） In order to investigate whether IDH3α can truly activate HIF-1, experiments were conducted using shRNA expression plasmids (SABiosciences: product number KH15075P) against the following sequences (A) to (C) in the IDH3α gene. went. As a negative control, an shRNA (Scr) expression plasmid against the following (D) sequence that does not match any of the human genes was used.
(A) TGACT TGTGT GCAGG ATTGAT (SEQ ID NO: 1)
(B) AGATG GTATT GGCCC AGAAA T (SEQ ID NO: 2)
(C) TCACC CATCT ATGAA TTTAC T (SEQ ID NO: 3)
(D) GGAAT CTCAT TCGAT GCATA C (SEQ ID NO: 4)
HeLa / 5HRE-Luc cells are introduced with the respective shRNA (A)-(C) expression plasmids or shRNA (Scr) expression plasmids for negative control, and under normal oxygen (20%) or hypoxia (0.02%) environment After culturing in, a luciferase assay was performed. (See Figure 5)
　上記の結果、陰性対照群と比較して、IDH3αの発現を抑制した場合に、低酸素刺激によるHIF-1活性化が抑制されることが明らかになった。このことより、IDH3αがHIF-1活性に関与することが示唆された。本参考例及び以降の参考例、実施例において、IDH3αは、GenBank Accession Number：NP_005521.1で示されるアミノ酸配列で特定することができ、mRNAは、同Accession Number：NM_005530.2で特定することができる。 As a result, it was clarified that HIF-1 activation by hypoxic stimulation is suppressed when the expression of IDH3α is suppressed as compared with the negative control group. This suggested that IDH3α is involved in HIF-1 activity. In this reference example and the following reference examples and examples, IDH3α can be identified by the amino acid sequence represented by GenBank Accession Number: NP_005521.1, and mRNA can be identified by the Accession Number: NM_005530.2. it can.
（参考例３）HIF-1αタンパク質の安定性に及ぼすIDH3αの影響
　HIF-1タンパク質は、HIF-1αとHIF-1βのサブユニットから構成されている。サブユニットの一つであるHIF-1αは酸素依存的なユビキチン化を受けて、常酸素状態の細胞内では、翻訳後速やかに分解される。HIF-1の低酸素依存的活性を制御している部位として、HIF-1αタンパク質のほぼ中央にある酸素依存的分解ドメイン(Oxygen-dependent degradation domain: ODD)が存在する。このODD領域とルシフェラーゼとの融合タンパク質をSV40プロモーターの制御下で発現するプラスミドpGL3/ODD-Luc（Wakamatsu et al. Eur J Pharmacol. 617:17-22. 2009）を安定に導入したHeLa/ODD-Luc細胞と、pcDNA4A/IDH3αプラスミドを利用して、IDH3αの強制発現がHIF-1αタンパク質の安定性に及ぼす影響をルシフェラーゼ活性としてモニターした（図６参照）。陰性対照として空ベクター（pcDNA4/myc-His Aプラスミド）を同様に細胞に導入した系を用いた。 (Reference Example 3) Effect of IDH3α on the stability of HIF-1α protein HIF-1 protein is composed of HIF-1α and HIF-1β subunits. HIF-1α, one of the subunits, undergoes oxygen-dependent ubiquitination and is rapidly degraded after translation in normoxic cells. As a site that controls the hypoxia-dependent activity of HIF-1, there is an oxygen-dependent degradation domain (ODD) in the middle of the HIF-1α protein. HeLa / ODD- stably transfected with plasmid pGL3 / ODD-Luc (Wakamatsu et al. Eur J Pharmacol. 617: 17-22. 2009) expressing this fusion protein of ODD region and luciferase under the control of SV40 promoter Using Luc cells and pcDNA4A / IDH3α plasmid, the effect of forced expression of IDH3α on the stability of HIF-1α protein was monitored as luciferase activity (see FIG. 6). As a negative control, a system in which an empty vector (pcDNA4 / myc-His A plasmid) was similarly introduced into cells was used.
　上記の結果、常酸素（20%）、低酸素（0.02%）のいずれの条件においてもIDH3αを強発現させた場合に、陰性対照群に比べて高いルシフェラーゼ活性を認めた（図７参照）。このことより、IDH3αはHIF-1αタンパク質を安定化しうることが示唆された。 As a result of the above, a high luciferase activity was observed compared to the negative control group when IDH3α was strongly expressed under both conditions of normoxia (20%) and hypoxia (0.02%) (see FIG. 7). This suggests that IDH3α can stabilize HIF-1α protein.
　HIF-1αの安定化が真にIDH3αによるものかを確認するために、上述のHeLa/ODD-Luc細胞に、参考例２で示したIDH3αに対するshRNA(A)～(C)各発現プラスミドを導入して、ルシフェラーゼ活性を指標にHIF-1αタンパク質の安定性をモニターした。陰性対照として、shRNA(Scr)発現プラスミドを用いた。その結果、陰性対照群と比較して、IDH3αの発現を抑制した場合に、低酸素刺激によるODD-Lucの安定化が抑制されることが明らかになった（図８参照）。このことより、IDH3αがHIF-1αタンパク質の安定性に関与することが示唆された。 In order to confirm whether the stabilization of HIF-1α is really due to IDH3α, the shRNA (A) to (C) expression plasmids for IDH3α shown in Reference Example 2 were introduced into the above HeLa / ODD-Luc cells. The stability of HIF-1α protein was monitored using luciferase activity as an index. As a negative control, an shRNA (Scr) expression plasmid was used. As a result, it was revealed that the stabilization of ODD-Luc by hypoxic stimulation was suppressed when the expression of IDH3α was suppressed as compared with the negative control group (see FIG. 8). This suggests that IDH3α is involved in the stability of HIF-1α protein.
　HeLa細胞にpcDNA4/myc-His Aプラスミド（EV:空ベクター）又はpcDNA4A/IDH3αプラスミドを導入し、常酸素（20%）又は低酸素（0.02%）の条件下で培養して得た細胞抽出液について、タンパク質の発現をウェスタンブロッティングにより確認した。その結果、IDH3αを強制発現させた場合に、HIF-1αの発現量が増加することを認めた（図９参照）。その傾向は低酸素条件下でより顕著であった。このことより、IDH3αはHIF-1αの発現において重要な役割を担う因子であることが示唆された。 Cell extract obtained by introducing pcDNA4 / myc-His A plasmid (EV: empty vector) or pcDNA4A / IDH3α plasmid into HeLa cells and culturing under normoxic (20%) or hypoxic (0.02%) conditions The protein expression was confirmed by Western blotting. As a result, it was confirmed that the expression level of HIF-1α was increased when IDH3α was forcibly expressed (see FIG. 9). The tendency was more prominent under hypoxic conditions. This suggests that IDH3α is a factor that plays an important role in the expression of HIF-1α.
（参考例４）IDH3αの有無による腫瘍増殖作用
　参考例１～３の結果より、IDH3αはHIF-1αの発現及びHIF-1活性に"正"の影響を与える因子であることが示唆された。IDH3αが腫瘍増殖に及ぼす影響を検討する目的で、HeLa細胞にIDH3α強制発現ベクターを安定に導入したIDH3α強制発現細胞（HeLa/IDH3α 1～3）を、免疫不全マウスに移植し、その後の腫瘍増殖速度を計測した。対照として空ベクターを導入したHeLa細胞（HeLa/EV 1～3）を用い、同様の実験を行った。上記の結果、IDH3αの強制発現によって腫瘍増殖速度が亢進することが確認された（図１０参照）。 (Reference Example 4) Tumor growth effect by presence or absence of IDH3α From the results of Reference Examples 1 to 3, it was suggested that IDH3α is a factor that positively affects the expression and HIF-1 activity of HIF-1α. For the purpose of examining the effects of IDH3α on tumor growth, IDH3α forced expression cells (HeLa / IDH3α 1 to 3) in which IDH3α forced expression vector is stably introduced into HeLa cells are transplanted into immunodeficient mice, and then tumor growth The speed was measured. A similar experiment was performed using HeLa cells (HeLa / EV 1 to 3) into which an empty vector was introduced as a control. As a result, it was confirmed that the tumor growth rate was enhanced by forced expression of IDH3α (see FIG. 10).
　上述のIDH3α強制発現細胞株（HeLa/IDH3α 1～3）及び陰性対照細胞株（HeLa/EV 1～3）より細胞抽出液を得、pcDNA4A/IDH3αプラスミドに由来するIDH3αの発現を、融合されているmycタグに対する抗体で検出した。一方、IDH3αに対する抗体を用いることで、pcDNA4A/IDH3αプラスミドに由来するmycタグと融合されたIDH3αタンパク質と共に、内在性IDH3αの発現を確認した。mycタグの有無により、両タンパク質を各々独立して検出することが出来た。pcDNA4A/IDH3αプラスミドに由来するIDH3αの発現はHeLa/IDH3α 1～3においてのみ検出されたのに対し、内在性のIDH3αの発現はpcDNA4A/IDH3αの有無に関わらず検出された（図１１参照）。このことより、内在性のIDH3αの存在も図１０にて測定した腫瘍増殖に寄与していることが懸念された。 Cell extract was obtained from the above-mentioned IDH3α forced expression cell line (HeLa / IDH3α 1-3) and negative control cell line (HeLa / EV31-3), and the expression of IDH3α derived from pcDNA4A / IDH3α plasmid was fused. Detected with antibody against myc tag. On the other hand, by using an antibody against IDH3α, the expression of endogenous IDH3α was confirmed together with the IDH3α protein fused with the myc tag derived from the pcDNA4A / IDH3α plasmid. Both proteins could be detected independently with or without myc tag. The expression of IDH3α derived from the pcDNA4A / IDH3α plasmid was detected only in HeLa / IDH3α 1 to 3, whereas the expression of endogenous IDH3α was detected with or without pcDNA4A / IDH3α (see FIG. 11). From this, it was feared that the presence of endogenous IDH3α also contributed to the tumor growth measured in FIG.
（実施例１）IDH3α遺伝子に対するshRNAの作用
　HeLa細胞株に、IDH3αに対するshRNA(A)～(C)各発現プラスミドを安定に組み込むことによって、内在性IDH3αの発現を恒常的にノックダウンした細胞株（HeLa/shIDH3α A2, A5, B2, B4, C1, C2）を樹立した。陰性対照としてshRNA(Scr)発現プラスミドを安定に組み込んだ細胞株（HeLa/shRNA(Scr) N5, N9）を樹立した。これらの細胞株を常酸素条件（20%）又は低酸素条件（0.02%）で培養した後に細胞抽出液を得、抗HIF-1α抗体と抗IDH3α抗体を用いてウェスタンブロッティングを行った。その結果、陰性対照群（N5, N9細胞）ではIDH3αの発現が酸素条件にかかわらず認められ、HIF-1αの発現は低酸素条件下においてのみ認められた。一方、IDH3αをノックダウンした細胞株群では、HIF-1αの発現が低酸素環境においても認められなかった（図１２）。このことより、IDH3αが低酸素刺激によるHIF-1αの発現において決定的な役割を担っていることが確認された。 (Example 1) Effect of shRNA on IDH3α gene Cell line in which expression of endogenous IDH3α is constitutively knocked down by stably incorporating each expression plasmid of shRNA (A) to (C) against IDH3α into HeLa cell line (HeLa / shIDH3α A2, A5, B2, B4, C1, C2) was established. As a negative control, cell lines (HeLa / shRNA (Scr) N5, N9) stably incorporating the shRNA (Scr) expression plasmid were established. After culturing these cell lines under normoxic conditions (20%) or hypoxic conditions (0.02%), cell extracts were obtained, and Western blotting was performed using anti-HIF-1α antibody and anti-IDH3α antibody. As a result, in the negative control group (N5, N9 cells), IDH3α expression was observed regardless of oxygen conditions, and HIF-1α expression was observed only under hypoxic conditions. On the other hand, in the cell line group in which IDH3α was knocked down, expression of HIF-1α was not observed even in a hypoxic environment (FIG. 12). This confirmed that IDH3α plays a decisive role in the expression of HIF-1α by hypoxic stimulation.
　IDH3αの発現が腫瘍増殖に及ぼす影響を検討する目的で、内在性IDH3αの発現を恒常的にノックダウンさせた細胞株HeLa/shIDH3α A2, A5, B2, B4, C1, C2及び陰性対照細胞HeLa/shRNA(Scr) N5, N9を樹立し、各々30万個を免疫不全マウスに移植後、腫瘍の大きさを計測した。その結果、IDH3αの発現をノックダウンした場合に、腫瘍の増殖が有意に抑制されることが確認された（図１３）。また、移植４６日後の腫瘍サイズを目視した写真からも同様の結果が確認された（図１４）。また、IDH3α遺伝子の発現を最も効果的にノックダウンするshRNA (B)を用いた群においては、腫瘍形成そのものが抑制されうることも確認された。 In order to investigate the effect of IDH3α expression on tumor growth, cell lines HeLa / shIDH3α A2, A5, B2, B4, C1, C2 and negative control cells HeLa / shRNA (Scr) N5 and N9 were established and 300,000 each were transplanted into immunodeficient mice, and the size of the tumor was measured. As a result, it was confirmed that tumor growth was significantly suppressed when the expression of IDH3α was knocked down (FIG. 13). Moreover, the same result was confirmed also from the photograph which looked at the tumor size 46 days after transplantation (FIG. 14). It was also confirmed that tumor formation itself could be suppressed in the group using shRNA (B) that knocked down expression of IDH3α gene most effectively.
（実施例２）IDH3α遺伝子又はHIF-1α遺伝子のノックダウンによる抗腫瘍効果
　上記のIDH3α遺伝子に対するshRNA(B)発現プラスミド、HIF-1α遺伝子に対するshRNA発現プラスミド、及び陰性対照のshRNA(Scr)発現プラスミドを各々安定に組み込んだHeLa細胞を免疫不全マウスに移植し、腫瘍の大きさを計測した。その結果、IDH3α遺伝子をノックダウンした腫瘍のほうが、HIF-1α遺伝子をノックダウンした腫瘍に比べて、より効果的な腫瘍増殖抑制効果が認められた（図１５）。以上により、IDH3αを抑制したほうがHIF-1αを直接抑制するよりも、より効果的に腫瘍の増殖を抑制しうることが確認された。この結果は、IDH3αがHIF-1αを安定化させる以外の作用を有していることを示唆しているのかもしれない。以上、IDH3を抑制しうる物質、具体的にはIDH3を構成するサブユニットの少なくとも一つ、具体的にはIDH3αを抑制しうる物質は、抗腫瘍剤として効果を発揮しうることが確認された。 (Example 2) Anti-tumor effect by knockdown of IDH3α gene or HIF-1α gene shRNA (B) expression plasmid for IDH3α gene, shRNA expression plasmid for HIF-1α gene, and shRNA (Scr) expression plasmid of negative control HeLa cells stably incorporating each of these were transplanted into immunodeficient mice, and the tumor size was measured. As a result, more effective tumor growth inhibitory effect was observed in the tumor in which the IDH3α gene was knocked down compared to the tumor in which the HIF-1α gene was knocked down (FIG. 15). From the above, it was confirmed that suppressing IDH3α can more effectively suppress tumor growth than directly suppressing HIF-1α. This result may suggest that IDH3α has actions other than stabilizing HIF-1α. As described above, it was confirmed that a substance capable of suppressing IDH3, specifically, at least one of the subunits constituting IDH3, specifically, a substance capable of suppressing IDH3α, can exert an effect as an antitumor agent. .
（実施例３）IDH3α遺伝子に対するshRNAの作用
　実施例１で樹立した"内在性IDH3αの発現を恒常的にノックダウンさせた細胞HeLa/shIDH3α B2"、及び"陰性対照細胞HeLa/shRNA(Scr)N9" の懸濁液を準備し、各々1,000万個および100万個の細胞を免疫不全マウスに移植した。生じた固形腫瘍の大きさが同じになるタイミング、すなわちHeLa/shIDH3α B2腫瘍の場合は移植45日後、HeLa/shRNA(Scr)N9腫瘍の場合は移植37日後に摘出し、その切片を血管内皮細胞のマーカー（CD31抗体）で染色した。検出された腫瘍内血管の密度を定量したところ、IDH3α遺伝子をノックダウンした場合に血管密度が減少することが確認された（図１６、１７）。
　当該データは、IDH3αを抑制した場合に、HIF-1α発現量の低下を介して腫瘍血管密度を低下させ得ることを示唆している。 (Example 3) Action of shRNA on IDH3α gene “Cell HeLa / shIDH3α B2 in which expression of endogenous IDH3α is constantly knocked down” established in Example 1, and “negative control cell HeLa / shRNA (Scr) N9” "And 10 million and 1 million cells, respectively, were transplanted into immunodeficient mice. When the resulting solid tumors have the same size, i.e., 45 days after transplantation for HeLa / shIDH3α B2 tumors, 37 days after transplantation for HeLa / shRNA (Scr) N9 tumors, the sections are removed from vascular endothelial cells. Were stained with a marker (CD31 antibody). When the density of the detected intravascular tumor blood vessel was quantified, it was confirmed that the blood vessel density decreased when the IDH3α gene was knocked down (FIGS. 16 and 17).
The data suggests that when IDH3α is suppressed, tumor blood vessel density can be reduced through a decrease in HIF-1α expression.
　以上詳述したように、本発明のIDH3抑制物質は抗腫瘍効果を有する。また、本発明のIDH3抑制物質はHIF-1抑制効果を有する。これらの作用から、IDH3の活性を抑制しうる物質をスクリーニングすることで、効果的な抗腫瘍剤を提供しうる。本発明のIDH3抑制物質を有効成分として含む新規抗腫瘍剤は、HIF-1の活性を制御しうることから、難治性腫瘍、例えば放射線治療抵抗性がんや抗がん剤抵抗性がんに対しても有効に効果を発揮しうる。 As described in detail above, the IDH3 inhibitor of the present invention has an antitumor effect. Further, the IDH3 inhibitory substance of the present invention has a HIF-1 inhibitory effect. From these actions, an effective antitumor agent can be provided by screening a substance capable of suppressing the activity of IDH3. Since the novel antitumor agent containing the IDH3 inhibitory substance of the present invention as an active ingredient can control the activity of HIF-1, it is useful for intractable tumors such as radiotherapy resistant cancer and anticancer drug resistant cancer. Even if it is effective, it can be effective.

Claims (15)

  1. イソクエン酸脱水素酵素３（IDH3）抑制物質を有効成分として含む、新規抗腫瘍剤。 A novel antitumor agent comprising an isocitrate dehydrogenase 3 (IDH3) inhibitor as an active ingredient.
  2. IDH3抑制が、IDH3αの発現抑制又は機能抑制である、請求項１に記載の新規抗腫瘍剤。 The novel antitumor agent according to claim 1, wherein the IDH3 suppression is IDH3α expression suppression or function suppression.
  3. IDH3を抑制し、低酸素誘導因子１(HIF-1)を抑制することを特徴とする、請求項１又は２に記載の新規抗腫瘍剤。  The novel antitumor agent according to claim 1 or 2, which suppresses IDH3 and suppresses hypoxia-inducible factor 1 (HIF-1).
  4. HIF-1を抑制することが、HIF-1αの発現抑制又は機能抑制である請求項３に記載の新規抗腫瘍剤。  The novel antitumor agent according to claim 3, wherein the suppression of HIF-1 is suppression of expression or function of HIF-1α.
  5. 腫瘍が、難治性悪性腫瘍である、請求項１～４のいずれか１に記載の新規抗腫瘍剤。 The novel antitumor agent according to any one of claims 1 to 4, wherein the tumor is an intractable malignant tumor.
  6. 難治性悪性腫瘍が、放射線治療抵抗性がん及び／又は抗がん剤抵抗性がんである、請求項５に記載の新規抗腫瘍剤。 The novel antitumor agent according to claim 5, wherein the refractory malignant tumor is a radiation therapy resistant cancer and / or an anticancer drug resistant cancer.
  7. IDH3抑制物質を有効成分として含む、HIF-1抑制剤。 An HIF-1 inhibitor comprising an IDH3 inhibitor as an active ingredient.
  8. IDH3の活性抑制を指標とする、新規抗腫瘍剤のスクリーニング方法。 A screening method for a novel antitumor agent using inhibition of IDH3 activity as an index.
  9. 以下の工程を含む、請求項８に記載の新規抗腫瘍剤のスクリーニング方法：
    １）IDH3αを発現しうる細胞株を候補物質で処理する工程；
    ２）候補物質で処理した前後で、上記細胞株についてIDH3αの発現量又はIDH3の活性を測定し、IDH3αの発現量又はIDH3の活性からIDH3の活性抑制を評価する工程。     The screening method of the novel antitumor agent of Claim 8 including the following processes:
    1) a step of treating a cell line capable of expressing IDH3α with a candidate substance;
    2) A step of measuring IDH3α expression level or IDH3 activity for the above cell lines before and after treatment with a candidate substance, and evaluating IDH3 activity suppression from IDH3α expression level or IDH3 activity.
  10. IDH3αを発現しうる細胞株が、HIF-1依存的にレポーター遺伝子を発現する細胞株であり、IDH3αの発現量又はIDH3の活性の測定が、レポーター遺伝子産物を計測することによる、請求項９に記載の新規抗腫瘍剤のスクリーニング方法。 The cell line capable of expressing IDH3α is a cell line that expresses a reporter gene in an HIF-1-dependent manner, and the expression level of IDH3α or IDH3 activity is measured by measuring the reporter gene product. A screening method for the described novel antitumor agent.
  11. IDH3を構成するサブユニットの少なくとも一つを標的因子として抑制することを含む、腫瘍の新規治療方法。 A novel method for treating a tumor, comprising inhibiting at least one of the subunits constituting IDH3 as a target factor.
  12. IDH3を構成するサブユニットの少なくとも一つがIDH3αである、請求項１１に記載の腫瘍の新規治療方法。 The novel method for treating a tumor according to claim 11, wherein at least one of the subunits constituting IDH3 is IDH3α.
  13. IDH3を構成するサブユニットの少なくとも一つを標的因子として抑制することが、IDH3αの発現抑制又は機能抑制である、請求項１１又は１２に記載の腫瘍の新規治療方法。  The novel method for treating tumor according to claim 11 or 12, wherein suppressing at least one of the subunits constituting IDH3 as a target factor is suppression of expression or function of IDH3α.
  14. 腫瘍が、難治性悪性腫瘍である、請求項１１～１３のいずれか１に記載の腫瘍の新規治療方法。 The novel method for treating a tumor according to any one of claims 11 to 13, wherein the tumor is an intractable malignant tumor.
  15. 難治性悪性腫瘍が、放射線治療抵抗性がん及び／又は抗がん剤抵抗性がんである、請求項１４に記載の腫瘍の新規治療方法。 The novel treatment method for a tumor according to claim 14, wherein the refractory malignant tumor is a radiotherapy resistant cancer and / or an anticancer drug resistant cancer.
PCT/JP2012/064964 2011-06-22 2012-06-12 Novel anti-tumor agent, and method for screening for novel anti-tumor agent WO2012176651A1 (en)

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WO2010028099A1 (en) * 2008-09-03 2010-03-11 The Johns Hopkins University Genetic alterations in isocitrate dehydrogenase and other genes in malignant glioma

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WO2010028099A1 (en) * 2008-09-03 2010-03-11 The Johns Hopkins University Genetic alterations in isocitrate dehydrogenase and other genes in malignant glioma

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