JP6046618B2 - Novel antitumor agent and screening method for novel antitumor agent - Google Patents

Description

  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.

  This application claims Japanese application Japanese Patent Application No. 2011-138981 priority used here by reference.

  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 (hereinafter sometimes simply referred to 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). .

  Unlike the inside of normal tissue, the inside of a solid tumor is composed of 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 is changed by anticancer drugs and radiation therapy, and HIF-1 is activated to induce the expression of Vascular Endothelial Growth Factor (VEGF). 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.

  In a hypoxic environment resident in a solid tumor (hypoxic region in a solid tumor), the radiosensitivity is remarkably reduced. Therefore, 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 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.

  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.

  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.

Cancer Sci. 94: 1021-1028. 2003 Br J Cancer, 100: 747-757. 2009 Current Signal Transduction Therapy. 5: 188-196. 2010 J. Biol. Chem., 277 (33), 29936-29944 (2002) Acta Neuropathol., 116, 597-602 (2008) Am. J. Pathol., 174, 1149-1153 (2009) N. Engl. J. Med., 360, 765-773 (2009)

International publication WO2010028099 pamphlet

  It is an object of the present invention 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.

  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.

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 tumor treatment method 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.

  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.

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) 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 | action of the factor (for example, IDH3 (alpha)) which affects HIF-1 activity. (Reference Example 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) 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) 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) 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) 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) 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) 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) 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 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 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 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 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 | mouth. (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 | mouth, and having quantified the blood vessel density in the formed tumor. (Example 3)

  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”). .

  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 represented by the same Accession Number: NM_005530.2.

Specific examples of the IDH3 inhibitory substance of the present invention include shRNA that can suppress the expression of IDH3α. Specific 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)

  In the present invention, to suppress IDH3 and to suppress HIF-1 means to suppress 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 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. Many reports on the relationship between HIF-1 and cancer are as described in the Background section.

  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.

  The IDH3 inhibitor contained in the novel antitumor agent of the present invention as an active ingredient may exert an antitumor effect by a mechanism different from the mechanism of action resulting from the relationship between IDH3 and HIF-1.

  The novel antitumor agent or HIF-1 inhibitor of the present invention may contain an additive generally used in medicine in addition to an effective amount of an IDH3 inhibitory substance as an active ingredient. 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.

  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, nicotinamide, and the like. 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.

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, the screening method 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.

  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.

  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.

  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.

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. .

  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. 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.

  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.

(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.

  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).

  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.

  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.

(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 the 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.

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 transfected 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)

  As a result, it was found 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 same Accession Number: NM_005530.2. it can.

(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.

  As a result, when IDH3α was strongly expressed under both conditions of normoxia (20%) and hypoxia (0.02%), a higher luciferase activity was observed compared to the negative control group (see FIG. 7). This suggests that IDH3α can stabilize HIF-1α protein.

  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.

  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.

(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 1-3), 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).

  Cell extracts were obtained from the IDH3α forced expression cell line (HeLa / IDH3α1-3) and negative control cell line (HeLa / EV1-3) described above, and the expression of IDH3α derived from the 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 / IDH3α1-3, whereas the expression of endogenous IDH3α was detected regardless of the presence or absence of 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.

(Example 1) Effect of shRNA on IDH3α gene
A cell line (HeLa / shIDH3α A2, A5, B2, B4) in which expression of endogenous IDH3α is constitutively knocked down by stably incorporating shRNA (A) to (C) expression plasmids against IDH3α into the HeLa cell line. , C1, C2). 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.

  In order to examine 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 IDH3α gene expression most effectively.

(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. .

(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.

  As described above in detail, 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 (9)

An antitumor agent comprising an isocitrate dehydrogenase 3 (IDH3) activity inhibitor as an active ingredient, and the IDH3 activity inhibitor as an active ingredient is selected from antisense nucleic acids, ribozyme nucleic acids, siRNA, shRNA and antibodies A novel antitumor agent, which is at least one IDH3α expression inhibitor and / or IDH3α function inhibitor . Antitumor agent suppressing IDH3 activity, characterized in that it have the inhibitory action of hypoxia-inducible factor 1 (HIF-1) activity, novel anti-tumor agent according to claim 1. The novel antitumor agent according to claim 2 , wherein the suppression of HIF-1 activity is due to suppression of expression or function of HIF-1α. The novel antitumor agent according to any one of claims 1 to 3 , wherein the tumor is an intractable malignant tumor. The novel antitumor agent according to claim 4 , wherein the refractory malignant tumor is a radiation therapy resistant cancer and / or an anticancer drug resistant cancer. Look including the IDH3 active inhibitor as an active ingredient, IDH3 activity inhibitor as an active ingredient, an antisense nucleic acid, at least one expression inhibitor of IDH3α selected from a ribozyme nucleic acids, siRNA, shRNA and antibodies and / or IDH3α An inhibitor of HIF-1 activity , which is a functional inhibitor of A screening method for a novel antitumor agent using inhibition of IDH3 activity as an index. The screening method of the novel antitumor agent of Claim 7 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. Cell lines capable of expressing a IDH3α is a HIF-1-dependent manner a cell line expressing a reporter gene, due to the measurement of the activity of the expression level or IDH3 of IDH3α is, measures the reporter gene product, to claim 8 A screening method for the described novel antitumor agent.