JP2018128461A - Detection method and kit for phospholipid hydroperoxide-dependent cell death, phospholipid hydroperoxide-dependent cell death inhibitor, and preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection method and kit for phospholipid hydroperoxide-dependent cell death, a phospholipid hydroperoxide-dependent cell death inhibitor, and a preventive or therapeutic agent for diseases by clarifying a factor involved in a cell death pathway caused by PHGPx depletion.SOLUTION: A detection method for phospholipid hydroperoxide-dependent cell death having a step of detecting activation of generation of phospholipid hydroperoxide and ERK, in a cell sample.SELECTED DRAWING: Figure 27

Description

  The present invention relates to a method and kit for detecting phospholipid hydroperoxide-dependent cell death, an inhibitor of phospholipid hydroperoxide-dependent cell death, and a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  The phospholipid constituting the biological membrane has an unsaturated fatty acid at the 2-position. As shown in FIG. 1, it is known that a phospholipid hydroperoxide is produced when a hydroxy radical generated from superoxide or hydrogen peroxide reacts with the unsaturated fatty acid by a Fenton reaction via iron.

  Phospholipid hydroperoxide glutathione peroxidase (hereinafter sometimes referred to as “PHGPx”) is an enzyme that directly reduces the produced phospholipid hydroperoxide and converts it into a phospholipid hydroxy form using glutathione as a coenzyme. is there. This PHGPx gene knockout mouse was known to be embryonic lethal (see, for example, Non-Patent Document 1). However, the mechanism of cell death due to PHGPx deficiency is unknown.

Imai H, et al., Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene, Biochem Biophys Res Commun., 305, 278-86, 2003.

  The present invention clarifies factors involved in the pathway of cell death caused by PHGPx deficiency, and a method and kit for detecting the cell death (hereinafter referred to as “phospholipid hydroperoxide-dependent cell death”), phospholipid hydroperoxide dependency The object is to provide proteins and nucleic acids involved in cell death, inhibitors of phospholipid hydroperoxide-dependent cell death, and preventive or therapeutic agents for diseases associated with phospholipid hydroperoxide-dependent cell death.

The present invention is as follows.
(1) Detection of phospholipid hydroperoxide-dependent cell death having a step of detecting an activation state of at least one factor selected from the group consisting of CDK4, MEK, ERK, Cdadc1 homolog, C87436 and Abhd2 in a cell Method.
(2) The detection includes measuring the localization state of the factor in cells, measuring the expression level of the factor at the mRNA level, measuring the expression level of the factor at the protein level, The method according to (1), wherein the method is performed by at least one selected from the group consisting of measuring a phosphorylation rate of a factor and measuring suppression of cell death due to suppression of expression of the factor.
(3) antibodies to phosphorylated or non-phosphorylated CDK4, MEK, ERK, Cdadc1 homolog, C87436 or Abhd2; primers for CDK4, MEK, ERK, Cdadc1 homolog, C87436 or Abhd2 mRNA amplification; or CDK4, MEK, A kit for detecting phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid against ERK, Cdadc1 homolog, C87436 or Abhd2.
(4) a protein comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and A protein that suppresses lipid hydroperoxide-dependent cell death.
(5) Nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 2 or having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 2 and reducing the expression level suppresses phospholipid hydroperoxide-dependent cell death. Nucleic acid encoding a protein.
(6) A protein comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3, and when the expression level is reduced, A protein that suppresses lipid hydroperoxide-dependent cell death.
(7) Nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 4 or having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 4 and reducing the expression level suppresses phospholipid hydroperoxide-dependent cell death. Nucleic acid encoding a protein.
(8) A protein comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 5 and when the expression level is reduced, phosphorous A protein that suppresses lipid hydroperoxide-dependent cell death.
(9) Nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 or having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 6 and reducing the expression level suppresses phospholipid hydroperoxide-dependent cell death. Nucleic acid encoding a protein.
(10) A phospholipid hydroperoxide-dependent cell death inhibitor comprising vitamin E or a derivative thereof as an active ingredient.
(11) A phospholipid hydroperoxide-dependent cell death inhibitor comprising, as an active ingredient, a siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4.
(12) A phospholipid hydroperoxide-dependent cell death inhibitor comprising, as an active ingredient, siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK.
(13) A phospholipid hydroperoxide-dependent cell death inhibitor comprising, as an active ingredient, siRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK.
(14) An inhibitor of phospholipid hydroperoxide-dependent cell death comprising siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient for Cdadc1 homolog, C87436 or Abhd2.
(15) A preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising vitamin E or a derivative thereof as an active ingredient.
(16) A prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising a CDK4 inhibitor or siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 as an active ingredient.
(17) A prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against MEK inhibitor or MEK.
(18) A prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising, as an active ingredient, siRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK.
(19) A preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against Cdadc1 homolog, C87436 or Abhd2.
(20) The preventive or therapeutic agent according to any one of (15) to (19), wherein the disease associated with phospholipid hydroperoxide-dependent cell death is heart failure.

  According to the present invention, a method and a kit for detecting phospholipid hydroperoxide-dependent cell death can be provided. In addition, proteins and nucleic acids involved in phospholipid hydroperoxide-dependent cell death can be provided. In addition, an inhibitor of phospholipid hydroperoxide-dependent cell death can be provided. In addition, a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death can be provided.

It is a figure explaining a biological membrane new lipid oxidation reaction and its metabolic pathway. It is a photograph which shows the result of the western blotting which detects PHGPx protein. It is a graph which shows a time-dependent change of the quantity of the phospholipid hydroperoxide (The hydroperoxide derived from 18: 0, 20: 4 phosphatidylcholine) produced | generated in PHGPx deficient MEF cell. It is a graph which shows the result of the MTT assay in which PHGPx-deficient MEF cells are lethal when tamoxifen is added. (A) is a graph showing the results of cell viability (%) showing the inhibitory effect of phospholipid hydroperoxide-dependent cell death when an apoptosis inhibitor is used. (B) is a photograph showing the result of fluorescent immunostaining of an index of apoptosis during staurosporine treatment and tamoxifen treatment. (A) is a photograph showing the result of fluorescent immunostaining of HMGB1 30 minutes after addition of tert-butyl hydroperoxide, and FIG. 6 (b) is a photograph showing the result of fluorescent immunostaining of HMGB1 48 hours after addition of tamoxifen. It is. (A) is a graph showing the results of cell viability (%) for phospholipid hydroperoxide-dependent cell death by knockdown of autophagy-related factor ATG5. (B) is a photograph showing the results of suppression of ATG5 expression by RT-PCR. (A) is a graph showing the results of cell viability (%) for phospholipid hydroperoxide-dependent cell death by knockdown cells of necrotosis-related factor Rip1. (B) is a photograph showing the result of Rip1 expression reduction by Western blotting. (A) is a graph showing the results of suppression of phospholipid hydroperoxide-dependent cell death using 3-ATA. (B) is a graph showing the results of suppression of phospholipid hydroperoxide-dependent cell death using U-0126. (C) is a graph showing the results of examining in vitro phospholipid autoxidation-inhibiting ability of 3-ATA, U-0126 and vitamin E. (A) is a graph showing the results of cell viability (%) for phospholipid hydroperoxide-dependent cell death by CDK4 knockdown. (B) is a photograph showing the result of CDK4 expression by Western blotting. (A) is a graph showing the results of cell viability (%) for phospholipid hydroperoxide-dependent cell death by MEK1 knockdown. (B) is a photograph showing the results of MEK1 expression by Western blotting. (A) And (b) is a photograph which shows the result of having examined the time-dependent change of the phosphorylation of MEK and ERK in the case of phospholipid hydroperoxide dependent cell death by Western blotting. (A) is a graph showing the results of cell viability (%) against phospholipid hydroperoxide-dependent cell death due to ERK2 knockdown. (B) is a photograph showing the result of ERK2 expression by Western blotting. (A) is a photograph showing the results of examining the effects of Trolox and 3-ATA on the phosphorylation of MEK and ERK by Western blotting. (B) is a model diagram showing a part of a phospholipid hydroperoxide-dependent cell death pathway. It is a graph which shows the result examined about the inhibitory effect of the phospholipid oxidation using the flow cytometry in the knockdown cell of CDK4 and MEK1. It is a model figure of the phospholipid hydroperoxide dependent cell death pathway clarified from the screening of a compound library. It is a figure which shows the outline | summary of the screening procedure of shRNA library. It is a graph which shows the result arranged about 151 candidate genes in order with the high inhibitory effect of phospholipid hydroperoxide dependence cell death by knockdown. (A) is a figure which shows the structure of Cdadc1 homolog gene and Cdadc1 variant-2 (Cdadc1 tv2) gene. (B) is a graph showing the results of cell viability (%) against phospholipid hydroperoxide-dependent cell death by knockdown of Cdadc1 homolog. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of C87436. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of ABHD2. (A) And (b) is a graph which shows the result of the MTT assay which examined the inhibitory effect with respect to the apoptosis and necrosis by knockdown of Cdadc1 homolog, C87436, ABHD2. (A) is a graph showing the results of measuring the change in the percentage (%) of cells with enhanced phosphorylation of ERK in phospholipid hydroperoxide-dependent cell death by Cdadclomolog, C87436, ABHD2 knockdown cells. (B) is a model diagram showing a part of a phospholipid hydroperoxide-dependent cell death pathway. (A) is a graph showing the results of investigating the inhibitory effect of phospholipid hydroperoxide production on phospholipid hydroperoxide-dependent cell death by Cdadclomolog, C87436, ABHD2 knockdown cells by flow cytometry. (B) is a model diagram showing a part of a phospholipid hydroperoxide-dependent cell death pathway. (A) And (b) is a graph which shows the result of having investigated the inhibitory effect of the iron chelator with respect to phospholipid hydroperoxide production | generation in phospholipid hydroperoxide dependent cell death by flow cytometry. (A) is a model diagram showing the action points of Mn-TBAP, NAC, deferoxamine, and vitamin E in the production of phospholipid hydroperoxide. (B) is a graph showing the results of cell viability (%) showing the inhibitory effect of Mn-TBAP, NAC, deferoxamine, and vitamin E on phospholipid hydroperoxide-dependent cell death. It is a model diagram of a novel signal pathway of the phospholipid hydroperoxide-dependent cell death pathway revealed in this study. (A) is a photograph of wild-type mice (Control) and cardiac-specific PHGPx-deficient mice (KO) on development day 17.5 and 18.5. (B) is a photograph showing the result of DAPI and TUNEL staining (TUNEL) of a tissue section of the heart of a mouse on the 17.5th day of development. (C) is a photograph showing the result of detecting the expression of PHGPx protein in the heart of a mouse on the 17.5th day of development by Western blotting. (A)-(c) is a photograph of a mouse fetus on the developmental process 18.5 days when the effect of 3-ATA was examined. (A) is an explanatory diagram of a model mouse of sudden arrhythmic death (heart failure) caused by a decrease in the amount of vitamin E in the diet, and shows that sudden death occurs in about 10 days when a vitamin E-added diet is changed to a normal diet It is a graph which shows the survival rate of a mouse | mouth. (B) is a graph showing the results of measuring the amount of vitamin E in the heart. (C) is a graph showing an electrocardiogram of a mouse. It is the graph which showed the inhibitory effect of 3-ATA with respect to the sudden death mouse model by the heart failure caused by the vitamin E fall. It is the graph which showed the inhibitory effect of U-0126 with respect to the sudden death mouse model by the heart failure caused by the vitamin E fall. (A) is a figure which shows the structure of a piMG-drU6 vector. (B) is a model diagram showing a process in which shRNA is expressed, siRNA is formed, and target RNA is destroyed. It is a figure which shows the structure of a pMXs-IR vector.

[Method for detecting phospholipid hydroperoxide-dependent cell death]
In one embodiment, the present invention comprises detecting the activation state of at least one factor selected from the group consisting of CDK4, MEK, ERK, Cdadc1 homolog, C87436 and Abhd2 in a cell. A method of detecting dependent cell death is provided.

  As described later, the inventors have found cell death (phospholipid hydroperoxide-dependent cell death) by a novel pathway distinct from apoptosis, necrosis, autophagic cell death, and necrosis. Furthermore, it has been clarified that CDK4, MEK, ERK, Cdadc1 homolog, C87436 and Abhd2 are included in the execution factors of phospholipid hydroperoxide-dependent cell death.

  Therefore, phospholipid hydroperoxide-dependent cell death can be detected by detecting the activation state of these factors. According to the method of this embodiment, it is possible to detect whether or not phospholipid hydroperoxide-dependent cell death is in progress, and after cell death has occurred, the cell death is a phospholipid hydroperoxide-dependent cell death pathway. It is also possible to determine whether or not it was through.

  Detection of the activation state of the factor includes, for example, measuring the intracellular localization state of the factor, measuring the expression level of the factor at the mRNA level, and determining the expression level of the factor at the protein level. It can be performed by at least one selected from the group consisting of measuring, measuring the phosphorylation rate of the factor, and measuring suppression of cell death due to suppression of the expression of the factor.

  The intracellular localization state can be measured, for example, by fixing a target cell, staining with a fluorescently labeled antibody against the above factor, and observing with a fluorescent microscope. Alternatively, the localization state of the factor in the cell can be measured by expressing a fusion protein of the factor and the fluorescent protein in the target cell and observing the fluorescence of the fusion protein with a fluorescence microscope. Here, examples of the fluorescent protein include green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and red fluorescent protein (RFP).

  As a result of measuring the localization state of the factor in the cell, for example, when a change is observed in the localization state of the factor compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not in progress, Phospholipid hydroperoxide-dependent cell death can be detected.

  The expression level of the above factor at the mRNA level can be measured, for example, by real-time PCR, Northern blotting or the like. For example, phospholipid hydroperoxide-dependent cell death is detected when there is a change in the expression level of the above factor at the mRNA level compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not ongoing be able to.

  The expression level of the factor at the protein level can be measured, for example, by Western blotting using an antibody against the factor. For example, phospholipid hydroperoxide-dependent cell death is detected when there is a change in the expression level of the above factors at the protein level compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not ongoing be able to.

  The phosphorylation rate of the factor can be measured by, for example, Western blotting using an antibody specific for the phosphorylated factor. For example, phospholipid hydroperoxide-dependent cell death can be detected when there is a change in the phosphorylation rate of the above factors compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not ongoing .

  Moreover, when cell death can be suppressed by suppressing the expression of the factor, it can be determined that the factor is involved in the cell death. Therefore, in such a case, phospholipid hydroperoxide-dependent cell death can be detected.

  Suppression of the expression of the factor can be performed, for example, by introducing siRNA, shRNA, ribozyme, antisense nucleic acid or the like against the factor into the cell. Moreover, the measurement of the suppression of cell death can be performed, for example, by measuring the survival rate of cells when the expression of the above factor is suppressed or not. The cell viability can be measured by, for example, calculating the change in the number of living cells by MTT assay or microscopic photography.

  siRNA (small interfering RNA) is a small double-stranded RNA of 21 to 23 base pairs used for gene silencing by RNA interference. The siRNA introduced into the cell binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.

  For siRNA, sense strand and antisense strand oligonucleotides are respectively synthesized by a DNA / RNA automatic synthesizer and denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, and then about 30 minutes. It can be prepared by annealing at ˜70 ° C. for about 1-8 hours.

  shRNA (short hairpin RNA) is a hairpin RNA sequence used for gene silencing by RNA interference. shRNA may be introduced into cells by a vector and expressed by U6 promoter or H1 promoter, or an oligonucleotide having shRNA sequence may be synthesized by a DNA / RNA automatic synthesizer and self-annealed in the same manner as siRNA. May also be prepared. The shRNA hairpin structure introduced into the cell is cleaved into siRNA and binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.

  Ribozymes are RNAs that have catalytic activity. Although some ribozymes have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA. The ribozyme may be a group I intron type, a size of 400 nucleotides or more such as M1 RNA contained in RNaseP, or may be about 40 nucleotides called a hammerhead type, a hairpin type, or the like.

  An antisense nucleic acid is a nucleic acid that is complementary to a target sequence. Antisense nucleic acid inhibits transcription initiation by triplex formation, suppresses transcription by hybridization with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridization with RNA that is being synthesized, Inhibition of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of transition from nucleus to cytoplasm by hybridization with mRNA, capping site and poly (A) addition site Suppression of splicing by hybridization with a protein, suppression of translation initiation by hybridization with a translation initiation factor binding site, suppression of translation by hybridization with a ribosome binding site near the initiation codon, translation region of mRNA and polysome binding site Hybridization outgrowth inhibitory peptide chain by the, by gene silencing due hybridization interaction site between a nucleic acid and a protein, it is possible to suppress the expression of a target gene.

  In this embodiment, siRNA, shRNA, ribozyme, and antisense nucleic acid may contain various chemical modifications in order to improve stability and activity. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue may be substituted with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, and the like. Moreover, at least one part may be comprised with nucleic acid analogs, such as a peptide nucleic acid (PNA).

[Kit for detection of phospholipid hydroperoxide-dependent cell death]
In one embodiment, the invention provides antibodies for CDK4, MEK, ERK, Cdadc1 homolog, C87436 or Abhd2; phosphorylated or non-phosphorylated primers; Alternatively, a kit for detecting phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4, MEK, ERK, Cdadc1 homolog, C87436 or Abhd2 is provided.

  Antibodies against the above-mentioned factors that are phosphorylated or non-phosphorylated are suitable for measuring the intracellular localization of the factors, measuring the expression level of the factors at the protein level, measuring the phosphorylation rate of the factors, etc. It is.

  The primer for mRNA amplification of the factor is suitable for measuring the expression level of the factor at the mRNA level.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against the above factor is suitable for suppressing the expression of the above factor.

  By using the kit of the present embodiment, phospholipid hydroperoxide-dependent cell death in a target cell can be easily detected.

[Cdadc1 homolog]
In one embodiment, the present invention comprises a protein consisting of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and the expression Reducing the amount provides a protein in which phospholipid hydroperoxide-dependent cell death is suppressed. Here, 1 or several means, for example, 1 to 20, 1 to 10, or 1 to 5.

  SEQ ID NO: 1 shows the amino acid sequence of Cdadc1 homolog. As will be described later, the inventors have revealed that Cdadc1 homolog is a novel protein encoded by a novel gene. Further, as described later, the inventors have found that Cdadc1 homolog is an execution factor for phospholipid hydroperoxide-dependent cell death, which is a novel cell death.

  Therefore, phospholipid hydroperoxide-dependent cell death can be controlled by suppressing or promoting the activity of Cdadc1 homolog protein in cells. Inhibition or promotion of the activity of the Cdadc1 homolog protein can be performed, for example, by decreasing or increasing the expression level of the Cdadc1 homolog protein, or by modifying the amino acid sequence of the Cdadc1 homolog protein.

  The decrease in the expression level of the Cdadc1 homolog protein can be performed, for example, by deleting the Cdadc1 homolog protein in the cell. Alternatively, it can be carried out by introducing siRNA, shRNA, ribozyme or antisense nucleic acid targeting the Cdadc1 homolog gene into cells. Moreover, the increase in the expression level of Cdadc1 homolog protein can be carried out by introducing an expression vector of Cdadc1 homolog protein into the cells.

  In another embodiment, the present invention relates to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2 or a phospholipid hydroperoxide having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 2 and reducing the expression level. Nucleic acids encoding proteins in which dependent cell death is suppressed are provided.

  SEQ ID NO: 2 shows the base sequence encoding Cdadc1 homolog. The nucleic acid of this embodiment may have the base sequence of SEQ ID NO: 2. In addition, the nucleic acid of this embodiment may have a base sequence different from the base sequence of SEQ ID NO: 2 as long as it encodes a protein whose phospholipid hydroperoxide-dependent cell death is suppressed when the expression level is reduced. Good. In this case, the base sequence of SEQ ID NO: 2 preferably has 80% or more homology, more preferably 85% or more, more preferably 90% or more, and more preferably 95%. It is particularly preferable to have the above homology.

  The nucleic acid of this embodiment may be artificially recombined. For example, the form incorporated in the expression vector may be sufficient.

[C87436]
In one embodiment, the present invention relates to a protein consisting of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3, and the expression Reducing the amount provides a protein in which phospholipid hydroperoxide-dependent cell death is suppressed. Here, 1 or several means, for example, 1 to 20, 1 to 10, or 1 to 5.

  SEQ ID NO: 3 shows the amino acid sequence of C87436. As described below, the inventors have found that C87436 is an executor of phospholipid hydroperoxide-dependent cell death, a novel cell death.

  Therefore, phospholipid hydroperoxide-dependent cell death can be controlled by suppressing or promoting the activity of C87436 protein in the cell. Inhibition or promotion of the activity of the C87436 protein can be performed, for example, by decreasing or increasing the expression level of the C87436 protein, modifying the amino acid sequence of the C87436 protein, or the like.

  The reduction of the expression level of C87436 protein can be performed, for example, by deleting the C87436 gene in the cell. Alternatively, it can be performed by introducing siRNA, shRNA, ribozyme or antisense nucleic acid targeting the C87436 gene into cells. In addition, the expression level of C87436 protein can be increased by introducing an expression vector for C87436 protein into cells.

  In another embodiment, the present invention relates to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4, or a phospholipid hydroperoxide having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 4 and decreasing the expression level. Nucleic acids encoding proteins in which dependent cell death is suppressed are provided.

  SEQ ID NO: 4 shows the base sequence encoding C87436. The nucleic acid of this embodiment may have the base sequence of SEQ ID NO: 4. In addition, the nucleic acid of this embodiment may have a base sequence different from the base sequence of SEQ ID NO: 4 as long as it encodes a protein whose phospholipid hydroperoxide-dependent cell death is suppressed when the expression level is reduced. Good. In this case, it is preferable to have 80% or more homology with the base sequence of SEQ ID NO: 4, more preferably 85% or more, more preferably 90% or more, and more preferably 95%. It is particularly preferable to have the above homology.

  The nucleic acid of this embodiment may be artificially recombined. For example, the form incorporated in the expression vector may be sufficient.

[Abhd2]
In one embodiment, the present invention comprises a protein comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 5, and expression Reducing the amount provides a protein in which phospholipid hydroperoxide-dependent cell death is suppressed. Here, 1 or several means, for example, 1 to 20, 1 to 10, or 1 to 5.

  SEQ ID NO: 5 shows the amino acid sequence of Abhd2. As described below, the inventors have found that Abhd2 is an executor of phospholipid hydroperoxide-dependent cell death, a novel cell death.

  Therefore, phospholipid hydroperoxide-dependent cell death can be controlled by suppressing or promoting the activity of Abhd2 protein in the cell. The suppression or promotion of the activity of the Abhd2 protein can be performed, for example, by reducing or increasing the expression level of the Abhd2 protein, or by modifying the amino acid sequence of the Abhd2 protein.

  The expression level of the Abhd2 protein can be reduced, for example, by deleting the Abhd2 gene in the cell. Alternatively, it can be carried out by introducing siRNA, shRNA, ribozyme or antisense nucleic acid targeting the Abhd2 gene into cells. Further, the expression level of Abhd2 protein can be increased by introducing an expression vector of Abhd2 protein into the cells.

  In another embodiment, the present invention relates to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6, or a phospholipid hydroperoxide having 80% or more homology with the nucleotide sequence of SEQ ID NO: 6 and reducing the expression level. Nucleic acids encoding proteins in which dependent cell death is suppressed are provided.

  SEQ ID NO: 6 shows the base sequence encoding Abhd2. The nucleic acid of this embodiment may have the base sequence of SEQ ID NO: 6. In addition, the nucleic acid of this embodiment may have a base sequence different from the base sequence of SEQ ID NO: 6 as long as it encodes a protein whose phospholipid hydroperoxide-dependent cell death is suppressed when the expression level is reduced. Good. In this case, it is preferable to have 80% or more homology with the base sequence of SEQ ID NO: 6, more preferably 85% or more, more preferably 90% or more, and more preferably 95% It is particularly preferable to have the above homology.

  The nucleic acid of this embodiment may be artificially recombined. For example, the form incorporated in the expression vector may be sufficient.

[Inhibitor of phospholipid hydroperoxide-dependent cell death]
(Vitamin E or its derivatives)
In one embodiment, the present invention provides a phospholipid hydroperoxide-dependent cell death inhibitor comprising vitamin E or a derivative thereof as an active ingredient.

  As described later, the inventors have found that phospholipid hydroperoxide-dependent cell death can be suppressed by administering vitamin E or a derivative thereof in vitro or in vivo. Therefore, vitamin E or a derivative thereof can be used as an inhibitor of phospholipid hydroperoxide-dependent cell death.

  Derivatives of vitamin E include Trolox (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid), troglitazone (5-[[4-[(3,4-dihydro-6- Hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) methoxy] -phenyl] methyl] -2,4-thiazolidinedione) and the like.

  When the dosage of vitamin E or a derivative thereof is added to the culture medium of cultured cells, for example, 50 to 1000 μM is preferable, and for example, 150 to 400 μM is preferable. Moreover, when administering to an animal, it is preferable to divide, for example, 50-260 mg / kg body weight per day into several times.

  Vitamin E or a derivative thereof may be administered per se, or may be administered as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive.

(Pharmaceutical composition)
Examples of formulation in a pharmaceutical composition include those used orally as tablets, capsules, elixirs, and microcapsules with sugar coating as necessary. Moreover, what is used parenterally in the form of a sterile solution with water or a pharmaceutically acceptable liquid other than water, or an injection of suspension. Furthermore, a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative And those formulated by mixing in a unit dosage form generally required for pharmaceutical practice, in appropriate combination with agents, binders and the like.

  Additives that can be mixed into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, swelling such as corn starch, gelatin, and alginic acid Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, the above material may further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

  Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, glucose, and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethyleneglycol, nonionic surfactant, for example, polysorbate 80 (trademark), HCO-50.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, transdermal, or oral administration to a patient are performed by methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

  The dose of the compound varies depending on the symptom, but in the case of oral administration, generally for an adult (with a body weight of 60 kg), about 0.1 to 100 mg, about 1.0 to 50 mg, about 1. It is considered to be 0-20 mg.

  When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, and administration method. For example, in the form of an injection, it is usually 1 for an adult (with a body weight of 60 kg). It may be preferred to administer about 0.01-30 mg, about 0.1-20 mg, about 0.1-10 mg per day by intravenous injection.

  Suitable examples of the therapeutic effect expected from the inhibitor of this embodiment include heart failure and sudden arrhythmic death.

(CDK4 inhibitor or CDK4 expression inhibitor)
In one embodiment, the present invention provides a phospholipid hydroperoxide-dependent cell death inhibitor comprising, as an active ingredient, a siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4.

  As will be described later, the inventors have revealed that CDK4 is contained in an execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4 can be used as an inhibitor of phospholipid hydroperoxide-dependent cell death.

  Examples of the CDK4 inhibitor include 3-ATA (3-amino-9-thio [10H] -acridone), NSC-625987 (1,4-dimethoxy-9-thio [10H] -acridone) and the like. The siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 is not particularly limited as long as it can suppress the expression of CDK4.

  The dose of the CDK4 inhibitor is preferably, for example, 5 to 20 μM when added to the culture medium of cultured cells. Moreover, when administering to an animal, it is preferable to administer 2-10 mg / kg body weight once a day, for example.

  For example, 3-ATA may be administered at 1 to 50 mg / kg body weight, more preferably 2 to 10 mg / kg body weight once a day.

  The dosage of siRNA, shRNA, ribozyme or antisense nucleic acid for CDK4 is preferably 50 nM to 10 μM, for example, preferably 50 to 200 nM, when added to the culture medium of cultured cells. Moreover, when administering to an animal, it is preferable to administer 50-500 mg / kg body weight once a day, for example.

  The siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4 may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

(MEK inhibitor or MEK expression inhibitor)
In one embodiment, the present invention provides a MEK inhibitor or an inhibitor of phospholipid hydroperoxide-dependent cell death comprising siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against MEK.

  As will be described later, the inventors have revealed that MEK is contained in an execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK can be used as an inhibitor of phospholipid hydroperoxide-dependent cell death.

  Examples of MEK inhibitors include MEK inhibitor I ((2Z) -3-amino-3-[(2-aminophenyl) sulfanyl] -2- {3- [hydroxy (pyridin-4-yl) methyl] phenyl}. Prop-2-enenitrile), U-0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-amino-phenylthio] butadiene) and the like. The siRNA, shRNA, ribozyme or antisense nucleic acid against MEK is not particularly limited as long as it can suppress the expression of MEK.

  When added to the culture medium of cultured cells, the dosage of the MEK inhibitor is preferably, for example, 1-100 μM, for example, 5-20 μM. Moreover, when administering to an animal, it is preferable to administer 2-10 mg / kg body weight once a day, for example.

  The dosage of siRNA, shRNA, ribozyme or antisense nucleic acid to MEK is preferably 50 nM to 10 μM, for example, preferably 50 to 200 nM, when added to the culture medium of cultured cells. Moreover, when administering to an animal, it is preferable to administer 50-500 mg / kg body weight once a day, for example.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

(ERK inhibitor or ERK expression inhibitor)
In one embodiment, the present invention provides an ERK inhibitor or an inhibitor of phospholipid hydroperoxide-dependent cell death comprising an siRNA, shRNA, ribozyme or antisense nucleic acid against ERK as an active ingredient. In the present embodiment, ERK may be ERK1 or ERK2.

  As will be described later, the inventors have revealed that ERK is contained in an execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK can be used as an inhibitor of phospholipid hydroperoxide-dependent cell death.

  Examples of ERK inhibitors include ERK Inhibitor ({3- (2-aminoethyl) -5-[(4-ethoxyphenyl) methylene] -2,4-thiazolidinedione, HCl}), ERK Inhibitor II ({5 -(2-phenyl-pyrazolo [1,5-a] pyridin-3-yl) -1H-pyrazolo [3,4-c] pyridazin-3-ylamine}) and the like. The siRNA, shRNA, ribozyme or antisense nucleic acid against ERK is not particularly limited as long as it can suppress the expression of ERK.

  When the dosage of the ERK inhibitor is added to the culture medium of cultured cells, for example, 1 to 100 μM is preferable, and for example, 5 to 20 μM is preferable. Moreover, when administering to an animal, it is preferable to administer 2-10 mg / kg body weight once a day, for example.

  The dosage of siRNA, shRNA, ribozyme or antisense nucleic acid against ERK is preferably 50 nM to 10 μM, for example, preferably 50 to 200 nM, when added to the culture medium of cultured cells. Moreover, when administering to an animal, it is preferable to administer 50-500 mg / kg body weight once a day, for example.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

(Cdadc1 homolog, C87436 or Abhd2 expression inhibitor)
In one embodiment, the present invention provides a phospholipid hydroperoxide-dependent cell death inhibitor comprising, as an active ingredient, siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2.

  As described later, the inventors have clarified that Cdadc1 homolog, C87436, and Abhd2 are included in the execution factors of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 can be used as an inhibitor of phospholipid hydroperoxide-dependent cell death.

  The siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 is not particularly limited as long as it can suppress the expression of Cdadc1 homomolog, C87436 or Abhd2.

  The dose of siRNA, shRNA, ribozyme or antisense nucleic acid for Cdadc1 homolog, C87436 or Abhd2 is, for example, preferably 50 nM to 10 μM, for example 50 to 200 nM, when added to the culture medium of cultured cells. Moreover, when administering to an animal, it is preferable to administer 50-500 mg / kg body weight once a day, for example.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

[Prophylactic or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death]
(Vitamin E or its derivatives)
In one embodiment, the present invention provides a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death, comprising vitamin E or a derivative thereof as an active ingredient.

  In another embodiment, the present invention provides vitamin E or a derivative thereof for the prevention or treatment of diseases associated with phospholipid hydroperoxide-dependent cell death.

  In another embodiment, the present invention provides the use of vitamin E or a derivative thereof for the manufacture of a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death.

  In another embodiment, the present invention provides a method for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising administering an effective amount of vitamin E or a derivative thereof to a patient in need of treatment. I will provide a.

  As described later, the inventors have found that phospholipid hydroperoxide-dependent cell death can be suppressed by administering vitamin E or a derivative thereof in vitro or in vivo. Therefore, vitamin E or a derivative thereof can be used as a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  Derivatives of vitamin E include Trolox (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid), troglitazone (5-[[4-[(3,4-dihydro-6- Hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) methoxy] -phenyl] methyl] -2,4-thiazolidinedione) and the like.

  When vitamin E or a derivative thereof is administered, for example, 50 to 260 mg / kg body weight per day is preferably divided into several times.

  Vitamin E or a derivative thereof may be administered per se, or may be administered as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. Preferred forms of the pharmaceutical composition are the same as those described above.

(CDK4 inhibitor or CDK4 expression inhibitor)
In one embodiment, the present invention provides a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising a siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against CDK4 inhibitor or CDK4. To do.

  In another embodiment, the present invention provides a siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4 for the prevention or treatment of diseases associated with phospholipid hydroperoxide-dependent cell death.

  In another embodiment, the present invention relates to the use of siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4 for the manufacture of a prophylactic or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death. I will provide a.

  In another embodiment, the present invention provides a phospholipid hydroperoxide-dependent method comprising administering an effective amount of a siRNA, shRNA, ribozyme or antisense nucleic acid to a CDK4 inhibitor or CDK4 to a patient in need of treatment. A method for preventing or treating a disease associated with cell death is provided.

  As will be described later, the inventors have revealed that CDK4 is contained in an execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4 can be used as a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  Examples of the CDK4 inhibitor include 3-ATA (3-amino-9-thio [10H] -acridone), NSC-625987 (1,4-dimethoxy-9-thio [10H] -acridone) and the like. The siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 is not particularly limited as long as it can suppress the expression of CDK4.

  When a CDK4 inhibitor is administered, for example, 1 to 50 mg / kg body weight per dose, more preferably 2 to 10 mg / kg body weight is preferably administered once a day. For example, 3-ATA may be administered at a dose of 0.5 to 10 mg / kg body weight, more preferably 1 to 2 mg / kg body weight once a day.

  As for the dose of siRNA, shRNA, ribozyme or antisense nucleic acid for CDK4, for example, 50 to 500 mg / kg body weight per dose is preferably administered once a day.

  The siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 inhibitor or CDK4 may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

(MEK inhibitor or MEK expression inhibitor)
In one embodiment, the present invention provides a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising an siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against a MEK inhibitor or MEK. To do.

  In another embodiment, the present invention provides a siRNA, shRNA, ribozyme or antisense nucleic acid against a MEK inhibitor or MEK for the prevention or treatment of diseases associated with phospholipid hydroperoxide-dependent cell death.

  In another embodiment, the present invention relates to the use of siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK for the manufacture of a prophylactic or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death. I will provide a.

  In another embodiment, the invention provides a phospholipid hydroperoxide-dependent comprising administering to a patient in need of treatment an effective amount of siRNA, shRNA, ribozyme or antisense nucleic acid against a MEK inhibitor or MEK. A method for preventing or treating a disease associated with cell death is provided.

  As will be described later, the inventors have revealed that MEK is contained in an execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK can be used as a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  Examples of MEK inhibitors include MEK inhibitor I ((2Z) -3-amino-3-[(2-aminophenyl) sulfanyl] -2- {3- [hydroxy (pyridin-4-yl) methyl] phenyl}. Prop-2-enenitrile), U-0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-amino-phenylthio] butadiene) and the like. The siRNA, shRNA, ribozyme or antisense nucleic acid against MEK is not particularly limited as long as it can suppress the expression of MEK.

  When administering a MEK inhibitor, it is preferable to administer, for example, 2 to 10 mg / kg body weight once a day.

  When administering siRNA, shRNA, ribozyme or antisense nucleic acid against MEK, it is preferable to administer 50 to 500 mg / kg body weight once a day, for example.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

(ERK inhibitor or ERK expression inhibitor)
In one embodiment, the present invention provides an ERK inhibitor or a preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising an siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against ERK. To do.

  In another embodiment, the present invention provides an ERK inhibitor or a siRNA, shRNA, ribozyme or antisense nucleic acid against ERK for the prevention or treatment of diseases associated with phospholipid hydroperoxide dependent cell death.

  In another embodiment, the present invention relates to the use of an siRNA, shRNA, ribozyme or antisense nucleic acid against an ERK inhibitor or ERK for the manufacture of a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death. I will provide a.

  In another embodiment, the invention provides a phospholipid hydroperoxide-dependent comprising administering to a patient in need of treatment an effective amount of siRNA, shRNA, ribozyme or antisense nucleic acid against an ERK inhibitor or ERK. A method for preventing or treating a disease associated with cell death is provided.

  As will be described later, the inventors have revealed that ERK is contained in an execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK can be used as a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  Examples of ERK inhibitors include ERK Inhibitor ({3- (2-aminoethyl) -5-[(4-ethoxyphenyl) methylene] -2,4-thiazolidinedione, HCl}), ERK Inhibitor II ({5 -(2-phenyl-pyrazolo [1,5-a] pyridin-3-yl) -1H-pyrazolo [3,4-c] pyridazin-3-ylamine}) and the like. The siRNA, shRNA, ribozyme or antisense nucleic acid against ERK is not particularly limited as long as it can suppress the expression of ERK.

  When administering an ERK inhibitor, it is preferable to administer, for example, 2 to 10 mg / kg body weight once a day.

  When administering siRNA, shRNA, ribozyme or antisense nucleic acid against ERK, it is preferable to administer 50 to 500 mg / kg body weight once a day, for example.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

(Cdadc1 homolog, C87436 or Abhd2 expression inhibitor)
In one embodiment, the present invention provides a preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against Cdadc1 homolog, C87436 or Abhd2. To do.

  In another embodiment, the present invention provides siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 for the prevention or treatment of diseases associated with phospholipid hydroperoxide dependent cell death.

  In another embodiment, the present invention relates to siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 for the manufacture of a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death. Provide use.

  In another embodiment, the present invention provides a phospholipid hydroperoxide-dependent, comprising administering to a patient in need of treatment an effective amount of siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2. A method for preventing or treating a disease associated with cell death is provided.

  As described later, the inventors have clarified that Cdadc1 homolog, C87436, and Abhd2 are included in the execution factors of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 can be used as a prophylactic or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  The siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 is not particularly limited as long as it can suppress the expression of Cdadc1 homomolog, C87436 or Abhd2.

  When siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 is administered, it is preferable to administer 50 to 500 mg / kg body weight once a day, for example.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1 homolog, C87436 or Abhd2 may be administered as such or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

  Examples of the above-mentioned diseases associated with phospholipid hydroperoxide-dependent cell death include heart failure and sudden arrhythmic death. As will be described later, the inventors have clarified that the above-mentioned preventive or therapeutic agent can prevent heart failure (arrhythmic sudden death) in an in vivo experiment using mice.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Experimental Example 1]
(Establishment of Tamoxifen-induced PHGPx-deficient MEF cell line)
In order to elucidate the mechanism of PHGPx-deficient cell death, a tamoxifen-induced PHGPx-deficient MEF cell line was established. In this cell, the PHGPx genomic gene is deleted by the Cre-loxP system within 24 hours when tamoxifen is added to the medium.

Specifically, a PHGPx gene (loxP-PHGPx) sandwiched between loxP sequences was introduced (Tg) into a PHGPx gene knockout mouse (PHGPx ^{− / −} ), and Tg (loxP-PHGPx): PHGPx ^{− / −} mouse And PHGPx ^+/− mice were mated. Subsequently, mouse embryo fibroblasts (MEF) were prepared from 13.5 day embryos whose genotype was Tg (loxP-PHGPx): PHGPx ^{− / −} . Subsequently, the obtained cells were immortalized by introducing a T antigen gene of SV40 virus. Subsequently, the tamoxifen-inducible CreERT2 gene was introduced into the obtained cells. Thereby, a tamoxifen-induced PHGPx-deficient MEF cell line was obtained.

  CreERT2 is obtained by mutating Cre (CreER), which is a fusion protein with an estrogen receptor, so that it does not react with estrogen but reacts with tamoxifen. When tamoxifen is added to the tamoxifen-induced PHGPx-deficient MEF cell line medium, tamoxifen binds to the estrogen receptor portion of CreER2, and the nuclear translocation signal of the estrogen receptor is exposed. As a result, CreER2 moves into the nucleus and the PHGPx genomic gene sandwiched between loxPs is destroyed. The concentration of tamoxifen to be added may be about 1 μM final concentration.

[Experiment 2]
(Administration of Tamoxifen to Tamoxifen-Induced PHGPx-Deficient MEF Cell Line)
Tamoxifen with a final concentration of 1 μM was added to the medium of the Tamoxifen-induced PHGPx-deficient MEF cell line. Subsequently, cells at 0, 24 and 48 hours after tamoxifen addition were collected, and the amount of PHGPx protein in each cell sample was measured by Western blotting using an anti-PHGPx antibody.

  FIG. 2 is a photograph showing the results of Western blotting for detecting PHGPx protein. As a result, it was confirmed that the expression level of PHGPx protein decreased within 24 hours after tamoxifen administration.

[Experiment 3]
(Detection of phospholipid hydroperoxide in PHGPx-deficient cells)
Tamoxifen at a final concentration of 1 μM was added to the tamoxifen-induced PHGPx-deficient MEF cell line medium, and the phospholipid hydroperoxide produced was detected. For detection, liquid chromatography (LC) -electrospray ionization mass spectrometry (ESI-MS) / mass spectrometry (MS) was used.

  An ACQUITY UPLC apparatus (Waters) was used for liquid chromatography, and a quadrupole linear ion trap mass spectrometry system (trade name 4000 Q-TRAP, AB Sciex) was used for mass analysis. An ACQUITY UPLC ™ BEH C18 column (0.17 μm, 150 mm × 1.0 mm) was used as the liquid chromatography column.

  Samples of the total phospholipid fraction extracted from each cell at 0, 12, 24, 36 and 48 hours after addition of tamoxifen were subjected to an autosampler, and mobile phase A (acetonitrile / methanol / water = 2: 2: 1 ( v / v), 0.1% boric acid, 0.028% ammonia): mobile phase B (isopropanol, 0.1% boric acid, 0.028% ammonia) 100: 0 (0-5 min), 50:50 (5-25 min), 50:50 (25-49 min), 100: 0 (59-60 min) and 100: 0 (60-75 min) step gradient, flow rate 70 μL / min, column temperature 30 ° C. Separated by condition.

  The MS / MS analysis was performed in a negative ion mode using a MRM (Multi Reaction Monitoring) technique. The ion spray voltage was set to -4500V. Nitrogen gas was used as a barrier gas and a collision gas. In the detection of oxidized phospholipids, the collision energy was set to 20-65 eV. The scan range of the apparatus was set to m / z 50-950 and a scan speed of 1000 Th / sec. Q0 trapping was turned on and the linear ion trap filter time was set to 10 milliseconds. The declustering potential was set to -105V. The resolution of Q1 was set to “unit”. The characteristic fragmentation pattern of phosphatidylcholine hydroperoxide (from 18: 0: 20: 4) was set at a dwell time of 50 milliseconds and a declustering potential of −80V. The resolution of Q1 and O3 was set to unit, Q1 was set to 886 m / z, and O3 was set to 283 m / z (collision energy −60 eV) and 335 m / z (collision energy −45 eV).

  FIG. 3 shows that after adding tamoxifen having a final concentration of 1 μM to the tamoxifen-induced PHGPx-deficient MEF cell line, cells at 0, 12, 24, 36, and 48 hours, 18: 0 (stearic acid), 20: It is the graph which showed the production amount of phospholipid hydroperoxide (PCOOH) which is an oxidation primary product produced | generated by oxidation from phosphatidylcholine (PC) which has 4 (arachidonic acid) with time. Phospholipid hydroperoxide, a primary oxidation product of phospholipid containing arachidonic acid, was produced at a peak 24 hours after the addition of tamoxifen. Similar results have been obtained with phospholipid hydroperoxide, which is a primary oxidation product of phospholipids containing DHA (not shown).

[Experimental Example 4]
(MTT assay of PHGPx-deficient cells)
Tamoxifen having a final concentration of 1 μM was added to the tamoxifen-induced PHGPx-deficient MEF cell line, and cell viability was measured by MTT assay. The same study was performed for the vitamin E addition group having a final concentration of 200 μM simultaneously with tamoxifen.

  More specifically, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazole bromide) is added to the cell culture medium 24, 48 and 72 hours after the addition of tamoxifen, After 4 hours, each cell was dissolved in dimethyl sulfoxide (DMSO), and the absorbance at 540 nm was measured.

  FIG. 4 is a graph showing the results of the MTT assay. It was revealed that cell death occurred between 72 hours and 48 hours after the addition of tamoxifen. Moreover, it became clear that cell death was completely suppressed by addition of vitamin E.

[Experimental Example 5]
(Comparison between phospholipid hydroperoxide-dependent cell death and apoptosis)
It was examined in detail whether phospholipid hydroperoxide-dependent cell death (cell death due to PHGPx deficiency) is via a cell death pathway due to apoptosis.

  Tamoxifen-induced PHGPx-deficient MEF cell line has a final concentration of 50 μM Z-VAD-FMK, a caspase inhibitor, and a final concentration of 300 μM, DHIQ, a release inhibitor of apoptosis-inducing factor (AIF), a mitochondrial protein. An E derivative, Trolox, having a final concentration of 400 μM and a cell to which nothing was added were prepared as controls. Tamoxifen with a final concentration of 1 μM was simultaneously added to each cell. Three days after the addition of tamoxifen and inhibitors, 20 photographs were taken at random with an all-in-one microscope from Keyence, and the number of viable cells attached was counted. Observation was performed at 10 times using Plan Floor ELWD 20 × 0.45 (Nicon) as an objective lens (one visual field: 877.5 μm × 661.2 μm). The cell viability (%) was expressed as 100% when the number of cells cultured for 3 days without adding tamoxifen.

  The results are shown in FIG. Caspase inhibitors and AIF inhibitors could not suppress phospholipid hydroperoxide-dependent cell death. On the other hand, Trolox, a vitamin E derivative, was able to suppress cell death.

  Next, a tamoxifen-inducible PHGPx-deficient MEF cell line was prepared by adding an apoptosis-inducing reagent, staurosporine, at a final concentration of 0.5 μM, and tamoxifen at a final concentration of 1 μM. After 5 hours, 24 hours, and 48 hours, each cell was fluorescently immunostained to stain cytochrome C and activated caspase-3. Further, DNA fragmentation, which is a feature of apoptosis, was detected by the TUNEL (TdT-mediated dUTP nick end labeling) method.

  The results are shown in FIG. In staurosporine treatment, cytochrome C release was observed after 5 hours, caspase 3 was activated, and TUNEL-positive cell death (apoptosis) was observed after 24 hours. On the other hand, no release of cytochrome C or activation of caspase 3 was observed in phospholipid hydroperoxide-dependent cell death (the figure shows 48 hours later, but was not observed even after 60 hours). Only in TUNEL staining, dot-like staining was observed in the nucleus, but no DNA ladder was detected.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from typical apoptosis.

[Experimental Example 6]
(Comparison between phospholipid hydroperoxide-dependent cell death and necrosis)
We examined in detail whether phospholipid hydroperoxide-dependent cell death was due to necrosis.

  Tamoxifen was added to the tamoxifen-induced PHGPx-deficient MEF cell line at a final concentration of 1 μM, and the moment of cell death was examined by time-lapse analysis. However, the swelling of cells characteristic of necrosis at the moment of cell death was not observed. The cell deformed just before the cell death, and finally it was peeled off from the petri dish and suddenly lethal (not shown).

  Subsequently, a tamoxifen-induced PHGPx-deficient MEF cell line was prepared by adding a necrosis-inducing agent, tert-butyl hydroperoxide, at a final concentration of 300 mM and a tamoxifen-added tamoxifen at a final concentration of 1 μM. After 30 minutes (tert-butyl hydroperoxide addition group) and 48 hours (tamoxifen addition group), each cell was fluorescent immunostained, and the release of HMGB1 (High Mobility Group Box 1) protein, which is characteristic of necrosis, into the cytoplasm Was observed.

  FIG. 6 (a) is a photograph showing the result 30 minutes after the addition of tert-butyl hydroperoxide, and FIG. 6 (b) is a photograph showing the result 48 hours after the addition of tamoxifen. In FIGS. 6A and 6B, the distribution of HMGB1 was stained with red and the nuclei were stained with Hoechst (blue).

  As a result, in the cell death caused by the addition of tert-butyl hydroperoxide, release of HMGB1, which is an indicator of necrosis, from the nucleus to the cytoplasm was observed, whereas in phospholipid hydroperoxide-dependent cell death, HMGB1 remained in the nucleus. And release into the cytoplasm was not observed.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from necrosis.

[Experimental Example 7]
(Comparison between phospholipid hydroperoxide-dependent cell death and autophagic cell death)
Whether phospholipid hydroperoxide-dependent cell death was due to autophagic cell death was examined in detail. It is known that the ATG5 gene is essential for causing autophagic cell death.

  ATG5-specific shRNA (short hairpin RNA) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line to prepare cells in which the expression of ATG5 was knocked down. As the ATG5-specific shRNA, two types of shRNAs, ATG5 shRNA-3 (SEQ ID NO: 7) and ATG5 shRNA-4 (SEQ ID NO: 8), were used. As a control, cells into which only the shRNA expression vector was introduced were prepared. Tamoxifen was added to the culture medium of these cells at a final concentration of 1 μM, and the cells after 24, 36, 48, 60 and 72 hours were randomly taken with a Keyence all-in-one microscope and 20 photographs were attached. The number of live cells that have been counted. The cell viability (%) was expressed as the cell viability (%), with the number of cells 24 hours after adding tamoxifen being 100 (%) and the change in the ratio of the number of viable cells at each time.

  The results are shown in FIGS. 7 (a) and 7 (b). Even when ATG5 expression was knocked down, phospholipid hydroperoxide-dependent cell death could not be suppressed. Moreover, as shown in FIG.7 (b), it was confirmed from the result of RT-PCR that the expression of ATG5 gene was knocked down by introduction of shRNA.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from autophagic cell death.

[Experimental Example 8]
(Comparison between phospholipid hydroperoxide-dependent cell death and necrosis)
We examined in detail whether phospholipid hydroperoxide-dependent cell death was due to necrosis. Necrosis is a form of cell death known as programmed necrosis. In addition, it is known that knockdown of receptor-interacting protein kinase 1 (Rip1) significantly suppresses necrosis.

  Rip1-specific shRNA (small hairpin RNA) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line to prepare cells in which Rip1 expression was knocked down. Rip1 shRNA-4 (SEQ ID NO: 9) was used as the Rip1 specific shRNA. Further, as a control, cells into which only the shRNA expression vector was introduced and cells in which Trolox was added at a final concentration of 400 μM in the medium were prepared. Tamoxifen was added to the culture medium of these cells at a final concentration of 1 μM, and after 24 hours, 72 hours later, the living cells were randomly taken with an all-in-one microscope from Keyence, and 20 attached living cells. Counted the number of. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  The results are shown in FIGS. 8 (a) and 8 (b). Even when Rip1 expression was knocked down, cell death due to PHGPx deficiency could not be suppressed. Moreover, as shown in FIG.8 (b), it was confirmed from the result of Western blotting that the expression of Rip1 protein was knocked down by introduction of shRNA.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from necrosis. That is, it has been clarified that phospholipid hydroperoxide-dependent cell death is a novel cell death different from apoptosis, necrosis, autophagic cell death, and necrosis.

[Experimental Example 9]
(Screening of compounds capable of suppressing phospholipid hydroperoxide-dependent cell death)
Using a library of inhibitors to about 300 known enzymes (Ministry of Education, Culture, Sports, Science and Technology, New Academic Area, Cancer Support, Chemotherapy Foundation Support Activity Group, Standard Inhibitor Kit), Tamoxifen-induced PHGPx-deficient MEF cell line A compound capable of suppressing cell death after 72 hours when tamoxifen was added at a final concentration of 1 μM was screened. As a result, U-126 and MEK inhibitor I, which are inhibitors of 3-ATA and NSC625987, which are inhibitors of cyclin-dependent kinase 4 (CDK4), and inhibitors of mitogen-activated protein kinase kinase (MEK1 / 2), and MEK inhibitor I. It was found that this compound can suppress phospholipid hydroperoxide-dependent cell death. On the other hand, inhibitors of p38 and JNK were unable to suppress phospholipid hydroperoxide-dependent cell death.

[Experimental Example 10]
(Suppression of phospholipid hydroperoxide-dependent cell death using inhibitors of CDK4 and MEK1 / 2)
FIG. 9A is a graph showing the results of suppression of phospholipid hydroperoxide-dependent cell death using 3-ATA, an inhibitor of CDK4. FIG. 9B is a graph showing the results of suppression of phospholipid hydroperoxide-dependent cell death using U-0126, which is an inhibitor of MEK1 / 2.

  Tamoxifen-induced PHGPx-deficient MEF cell line was supplemented with 3-ATA or U-0126 at each concentration and 1 μM final concentration of tamoxifen. After 24 hours and 72 hours, viable cells were observed with an all-in-one microscope from Keyence. Twenty pictures were taken at random, and the number of living cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  As a result, it was revealed that both 3-ATA and U-0126 suppress phospholipid hydroperoxide-dependent cell death in a concentration-dependent manner.

  FIG. 9 (c) is a graph showing the results of examining the in vitro phospholipid autoxidation inhibiting ability of 3-ATA, U-0126 and vitamin E. Each concentration of 3-ATA, U-0126 and Vitamin E was added to an ethanol solution of dilinoleoylphosphatidylcholine, and 50 μl of each was added to Imron 1 plate (Thermo Fisher Scientific), 37 The mixture was dried to dryness and left to stand overnight for autooxidation. For the measurement of phospholipid hydroperoxide generated by auto-oxidation, dihydrorhodamine 123 (DHR) (Molecular Probes), which is a probe that reacts with phospholipid hydroperoxide to emit fluorescence, is reacted at 1 μg / ml for 30 minutes, and fluorescence is obtained. The amount of fluorescence was measured with a plate reader (excitation wavelength: 485 nm, fluorescence wavelength: 530 nm).

  Vitamin E inhibits phospholipid autoxidation in vitro, but 3-ATA and U-0126 do not inhibit phospholipid autoxidation in vitro at concentrations that can inhibit phospholipid hydroperoxide-dependent cell death It became clear. From this, it was considered that 3-ATA and U-0126 have no antioxidant ability and inhibit phospholipid hydroperoxide-dependent cell death by inhibiting CDK4 and MEK1 / 2 activities. .

[Experimental Example 11]
(CDK4 kinase activity is required for phospholipid hydroperoxide-dependent cell death)
By expressing shRNA in tamoxifen-induced PHGPx-deficient MEF cells in a retrovirus-infected system, a knockdown cell of CDK4 was created to examine whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 10A is a graph showing the examination results. First, a CDK4-specific shRNA (SEQ ID NO: 10) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which CDK4 was knocked down (Cdk4 KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, a human CDK4 expression vector (hCdk4), a human CDK4 expression vector with a disrupted kinase activity (hCdk4D158N), and a vector alone (mock) were introduced into a cell line in which CDK4 was knocked down. Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 72 hours later, the live cells were randomly photographed with a Keyence all-in-one microscope, and the number of viable cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  The human CDK4 gene (SEQ ID NO: 16) introduced here was resistant to the mouse CDK4-specific shRNA described above. That is, the human CDK4 gene was not knocked down by the mouse CDK4-specific shRNA described above.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in CDK4 knockdown cells (Cdk4 KD / mock). Moreover, in cells (Cdk4 KD / hCdk4) in which human CDK4 gene was reintroduced after knocking down CDK4, phospholipid hydroperoxide-dependent cell death was recovered. On the other hand, phospholipid hydroperoxide-dependent cell death was not recovered in cells (Cdk4 KD / hCdk4D158N) into which CDK4 was knocked down and into which a human CDK4 gene having a kinase activity destroyed was introduced. In addition, the asterisk in the figure indicates that there is a significant difference with a risk rate of less than 5%.

  FIG. 10 (b) shows the result of confirming the expression of CDK4 protein by Western blotting.

  The above results revealed that CDK4 kinase activity is required for phospholipid hydroperoxide-dependent cell death.

(Preparation of cells in which CDK4 is knocked down)
In the above experimental example, tamoxifen-induced PHGPx-deficient MEF cells in which CDK4 was knocked down were prepared as follows.

  First, a DNA fragment encoding CDK4-specific shRNA (SEQ ID NO: 10) was incorporated downstream of the U6 promoter of the shRNA vector (piMG-drU6) shown in FIG. 33 (a). Subsequently, the retrovirus infection system was used to integrate the above-mentioned shRNA expression cassette into the genome of the target cell, to express CDK4-specific shRNA, and to obtain cells in which CDK4 was knocked down.

  FIG. 33 (b) is a model diagram showing a process in which siRNA is formed from expressed shRNA and target RNA is destroyed. shRNA vectors were obtained from Prof. Kenzo Hirose, School of Medicine, The University of Tokyo, and Prof. Atsutaro Sugao, School of Medicine, Nagoya University. Cells in which genes other than CDK4 described later were knocked down, and ATG5 and Rip1 knockdown cells used in Experimental Examples 7 and 8 were also prepared in the same manner.

(Re-expression of CDK4)
The pMXs-IR vector shown in FIG. 34 was used as a high expression vector of the retrovirus infection system used in the reexpression experiment. The pMXs-IR vector was obtained from Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo. The expression of genes other than CDK4, which will be described later, was also performed in the same manner.

(Gene transfer to PlatE cells)
PlatE cells were used for the preparation of retroviruses from shRNA and pMXs-IR vectors. The Plat E cell is a cell in which the gag-pol gene and the env gene are incorporated into 293T cells, which are human embryonic kidney epithelial cells.

PlatE cells were cultured at 37 ° C. using Dulbecco's modified Eagle medium (DMEM, Nissui Pharmaceutical) containing 10% FCS, 2 mM glutamine (GIBCO), 100 units / ml penicillin (GIBCO), 100 μg / ml streptomycin (GIBCO). And cultured in a CO ₂ incubator.

PlatE cells were seeded in a 6 cm petri dish (CORNING) with 5 × 10 ⁵ cells / dish and cultured for 1 day. Add 200 μL of DMEM FCS (−) to the 1.5 mL tube, 2 μg / 2 μL of the generated plasmid DNA (shRNA vector or pMXs-IR vector incorporating the target gene), and 8 μL of Plus Reagent (invitrogen, product number 11514-015). And left at room temperature for 15 minutes. Next, 50 μL of DMEM FCS (−) and 12 μL of Lipofectamine Reagent (Invitrogen, product number 18324-020) were mixed in advance and the mixture was allowed to stand at room temperature for 15 minutes. The cells were washed with a phosphate buffer (PBS), and 1.728 mL of DMEM FCS (−) was added. Subsequently, the above reaction solution was added to the cells and allowed to stand in a CO ₂ incubator at 37 ° C. for 3 hours for gene transfer. Thereafter, the reaction solution was replaced with 6 mL of DMEM 10% FCS, and cultured at 37 ° C. for about 48 hours.

(Preparation of virus solution)
Subsequently, the culture supernatant was transferred to a 15 mL tube (CORNING) and centrifuged at 3,000 rpm for 5 minutes at 4 ° C. The supernatant was transferred to a new tube and used as a virus solution.

(Virus infection)
The day before virus infection, tamoxifen-inducible PHGPx-deficient cells were seeded in a 6 cm petri dish (CORNING) at 1 × 10 ⁵ cells / dish and cultured for 1 day. 5 mL of virus solution to which Polybrene ™ (Hexadimethine bromide, SIGMA, catalog number H9268) was added to a final concentration of 8 μg / mL was infected by replacing the cell culture medium, and the culture medium was changed after 24 hours of culture. . In addition, Polybrene (trademark) was dissolved in sterile water so as to be 10 mg / mL in a clean bench, sterilized by filter, and diluted at the time of use.

[Experimental example 12]
(The kinase activity of MEK1 is required for phospholipid hydroperoxide-dependent cell death)
By expressing shRNA with a retrovirus, a knockdown cell of MEK1 was prepared, and it was examined whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 11A is a graph showing the examination results. First, MEK1-specific shRNA (SEQ ID NO: 11) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which MEK1 was knocked down (MEK1 KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, a human MEK1 expression vector (hMek1), a human MEK1 expression vector (hMek1S222A) in which the kinase activity was disrupted, and a vector alone (mock) as a control were introduced into cells in which MEK1 was knocked down. Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 72 hours later, the live cells were randomly photographed with a Keyence all-in-one microscope, and the number of viable cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  The human MEK1 gene (SEQ ID NO: 17) introduced here was resistant to the mouse MEK1-specific shRNA described above. That is, the human MEK1 gene was not knocked down by the mouse MEK1-specific shRNA described above.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in MEK1 knockdown cells (MEK1 KD / mock). In addition, phospholipid hydroperoxide-dependent cell death recovered in cells (MEK1 KD / hMek1) into which MEK1 gene was introduced after knocking down MEK1. On the other hand, phospholipid hydroperoxide-dependent cell death was not recovered in cells (MEK1 KD / hMek1S222A) into which human kinase kinase activity was disrupted after MEK1 was knocked down. In addition, the asterisk in the figure indicates that there is a significant difference with a risk rate of less than 5%.

  FIG. 11 (b) shows the result of confirming the expression of MEK1 protein by Western blotting.

  From the above results, it became clear that kinase activity of MEK1 is necessary for phospholipid hydroperoxide-dependent cell death.

[Experimental Example 13]
(The phosphorylation of MEK1 / 2 and ERK increases after 36 hours after tamoxifen addition)
We examined when phosphorylation of MEK and its substrate, ERK, occurred during phospholipid hydroperoxide-dependent cell death.

  Tamoxifen was added to the tamoxifen-induced PHGPx-deficient MEF cell line at a final concentration of 1 μM. Subsequently, 24, 27, 30, 33, 36, 39, 42, 45, and 48 hours after the addition of tamoxifen, cell samples were used, and anti-MEK antibody, anti-phosphorylated MEK antibody, anti-ERK antibody and anti-phosphorus were used. Western blotting using oxidized ERK antibody was performed.

  12 (a) and 12 (b) are photographs showing the results of Western blotting. As a result, it became clear that phosphorylation of MEK1 / 2 was enhanced 36-45 hours after the addition of tamoxifen, and phosphorylation of ERK2 was enhanced 36-48 hours after the addition of tamoxifen. There was no increase in phosphorylation of ERK1. When tamoxifen was not added, phosphorylation of MEK and ERK2 was not observed at this time (not shown).

[Experimental Example 14]
(ERK2 knockdown suppresses phospholipid hydroperoxide-dependent cell death)
By expressing shRNA with a retrovirus, ERK2 knockdown cells were created to investigate whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 13A is a graph showing the examination results. First, an ERK2-specific shRNA (SEQ ID NO: 12) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which ERK2 was knocked down (Erk2 shRNA-2). As a control, cells into which only the vector was introduced were prepared (mock). Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 72 hours later, the live cells were randomly photographed with a Keyence all-in-one microscope, and the number of viable cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  As a result, in ERK2 knockdown cells (Erk2 shRNA-2), suppression of phospholipid hydroperoxide-dependent cell death was observed, and when ERK2 was knocked down, phospholipid hydroperoxide-dependent cell death was suppressed. It became.

  FIG. 13 (b) shows the results of confirming the expression of ERK1 and ERK2 proteins by Western blotting.

  From the above results, it was considered that ERK2 is involved downstream of MEK in phospholipid hydroperoxide-dependent cell death.

[Experimental Example 15]
(In the phospholipid hydroperoxide-dependent cell death pathway, phospholipid hydroperoxide production and CDK4 function upstream of MEK1 / 2)
It was investigated whether MEK and ERK phosphorylation occurring 39 hours after tamoxifen addition functioned downstream of phospholipid hydroperoxide formation and CDK4 activation occurring 24 hours after tamoxifen addition. Specifically, in a medium of a Tamoxifen-induced PHGPx-deficient MEF cell line, a lipid oxidation inhibitor, Trolox of vitamin E derivative (final concentration 400 μM), and a CDK4 inhibitor 3-ATA (final concentration 10 μM) Was added simultaneously with tamoxifen at a final concentration of 1 μM, and phosphorylation of MEK and ERK2 occurring 39 hours after the addition of tamoxifen was examined by Western blotting.

  FIG. 14A is a photograph showing the results of Western blotting. As a result, the increase in phosphorylation of MEK and ERK2 was suppressed by the addition of Trolox and 3-ATA, and therefore, it became clear that the MEK-ERK2 pathway is located downstream of lipid oxidation and CDK4 activation.

  FIG. 14 (b) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway that has been clarified from the above results.

[Experimental Example 16]
(In the phospholipid hydroperoxide-dependent cell death pathway, CDK4 functions downstream of the production of phospholipid hydroperoxide)
Whether the activation of CDK4 and MEK1 functions downstream of the production of phospholipid hydroperoxide was examined by flow cytometry.

  FIG. 15 is a graph showing the results of flow cytometry. A cell (Cdk4 KD) in which a CDK4-specific shRNA (SEQ ID NO: 10) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell, and a cell (Mek1 KD) into which a MEK1-specific shRNA (SEQ ID NO: 11) was introduced were prepared. Further, as a control, a cell (Control) into which only the vector was introduced was prepared. Each cell was incubated for 24 hours in the presence or absence of tamoxifen at a final concentration of 1 μM, then stained with H2DCFDA, a fluorescent dye capable of detecting the production of phospholipid hydroperoxide, and measured by flow cytometry.

  As a result, it was confirmed that the addition of tamoxifen also increased the phospholipid hydroperoxide in cells in which CDK4 or MEK1 was knocked down. From this result, it became clear that the activation of CDK4 and MEK1 functions downstream of the production of phospholipid hydroperoxide.

  FIG. 16 shows a model diagram of the phospholipid hydroperoxide-dependent cell death pathway, which was clarified from the above results. When PHGPx is deficient, phospholipid hydroperoxide, which is a primary oxidative product of phospholipid, rises with a peak after 24 hours. Downstream of that, CDK4 is activated, and after 36 hours, MEK and ERK of the MAP kinase pathway are phosphorylated, and cell death occurs between 48 hours and 72 hours.

[Experimental Example 17]
(Identification of execution factors for phospholipid hydroperoxide-dependent cell death using lentiviral shRNA library)
In order to identify an execution factor of phospholipid hydroperoxide-dependent cell death, screening using a lentivirus comprehensive shRNA library (SBI) was performed. In this system, since the sequence of shRNA is created to bind to Genechip (trade name, Affymetrix), Genechip can be used to identify the sequence of shRNA.

  First, tamoxifen-induced PHGPx-deficient MEF cells were infected with a pool-type lentivirus capable of expressing exhaustive shRNA to produce shRNA-expressing cells. Tamoxifen having a final concentration of 1 μM was added to the medium of the shRNA-expressing cells, and normally, cells that survived 96 hours after complete cell death were collected. RNA was extracted from the collected cells, cDNA containing the shRNA sequence that had been introduced into the cells was amplified several times, a Genechip probe was prepared, and microarray analysis was performed.

  FIG. 17 is a diagram showing an outline of a screening procedure for an shRNA library. According to two experiments, the shRNA sequences contained in the two lentivirus-infected cells that survived 96 hours after addition of tamoxifen and all the cells that survived 96 hours in the absence of tamoxifen. Genechip was performed using a probe obtained by amplifying, and a probe enriched in tamoxifen-added cells as compared to unadded cells was detected. As a result of trying to identify common cell death performing factors concentrated in two experiments, 151 genes were obtained as candidates for cell death performing factors.

[Experiment 18]
(Determination of candidate genes for execution factors of phospholipid hydroperoxide-dependent cell death by expression of single shRNA using a retrovirus infection system)
Each of the identified candidate genes 151 of exemplifying factors for phospholipid hydroperoxide-dependent cell death was knocked down, and the inhibitory effect on phospholipid hydroperoxide-dependent cell death was examined.

  One type of shRNA expression vector of a candidate gene was introduced into tamoxifen-inducible PHGPx-deficient MEF cells one by one using a retrovirus infection system to prepare cells in which each candidate gene was knocked down. Tamoxifen with a final concentration of 1 μM was added to the medium of each cell, and the effect of suppressing cell death after 96 hours was measured. Specifically, 24 hours after addition of tamoxifen, 96 hours later, 20 cells were randomly taken with an all-in-one microscope from Keyence, and the number of viable cells attached was counted. The cell viability (%) was calculated as the cell viability (%) by setting the number of cells 96 hours after addition of tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  FIG. 18 shows the results obtained by arranging the ratio of the number of cells (cell viability (%)) after 96 hours from the addition of tamoxifen to 151 candidate genes in descending order of the number of cells after 24 hours from the addition of tamoxifen. It is a graph to show.

  We found 17 genes that could confirm the suppression of target gene expression by real-time PCR, and could strongly suppress phospholipid hydroperoxide-dependent cell death more than CDK4 knockdown cells. In addition, 20 genes can be confirmed by real-time PCR to suppress the expression of the target gene, and the effect of suppressing phospholipid hydroperoxide-dependent cell death is higher than that of MEK1 knockdown cells and lower than that of CDK4 knockdown cells. I found it.

  These 37 genes did not contain any genes involved in known cell death such as apoptosis. This result further supports that phospholipid hydroperoxide-dependent cell death is a novel cell death.

[Experimental Example 19]
(Cdadc1 homolog is an executioner of phospholipid hydroperoxide-dependent cell death)
From the above candidate genes, the Cdadc1 homolog gene (SEQ ID NO: 2) was found. This gene was a novel gene. From the results of the homology search, it was predicted that Cdadc1 homolog is a new variant of Cdcadc1. However, Cdadc1 was also a gene with unknown function. Cdadc1 was presumed to have two cytidine dCMP deaminase domains, whereas Cdadc1 homolog was presumed to have only one cytidine dCMP deaminase domain. Cdadc1 homolog had the same amino acid sequence as Cdadc1 except for the five amino acids at the C-terminus. The amino acid sequence of Cdadc1 homolog is shown in SEQ ID NO: 1. FIG. 19 (a) is a diagram showing the structures of the Cdadc1 homolog gene and the Cdadc1 variant-2 (Cdadc1 tv2) gene.

  By expressing shRNA in a retrovirus infection system, a knockdown cell of Cdadc1 homolog was created, and it was examined whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 19B is a graph showing the examination results. First, Cdadc1 homolog-specific shRNA (SEQ ID NO: 13) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare a cell in which Cdadc1 homolog was knocked down (Cdadc1h KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, a Cdadc1 homolog expression vector (Cdadc1h), an expression vector for Cdadc1 variant-2 (Cdadc1tv2), and a vector alone (mock) were introduced into these cells, respectively.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) by setting the number of cells 96 hours after addition of tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Cdadc1 homolog knockdown cells (Cdadc1h KD / mock). In addition, phospholipid hydroperoxide-dependent cell death was recovered in cells (Cdadc1h KD / Cdadc1h) into which Cdadc1 homomolog gene was introduced after knocking down Cdadc1 homomolog. On the other hand, phospholipid hydroperoxide-dependent cell death was not recovered in cells in which Cdadc1 homomollog was knocked down and the Cdadc1 variant-2 gene was introduced (Cdadc1h KD / Cdadc1tv2).

  From the above results, it was revealed that Cdadc1 homolog is an execution factor of phospholipid hydroperoxide-dependent cell death. Moreover, it was revealed that Cdadc1 homolog has a variant-selective function in phospholipid hydroperoxide-dependent cell death.

[Experiment 20]
(C87436 is an executioner of phospholipid hydroperoxide-dependent cell death)
Among the candidate genes described above, the C87436 gene (SEQ ID NO: 4) was found. The C87436 gene was a gene with unknown function for which only cDNA was reported.

  By expressing shRNA with a retrovirus, knockdown cells of C87436 were prepared, and it was examined whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 20 is a graph showing the examination results. First, C87436-specific shRNA (SEQ ID NO: 14) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which C87436 was knocked down (C87436 KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, the C87436 expression vector (C87436) and the vector alone (mock) were introduced into these cells, respectively. The C87436 cDNA incorporated into the C87436 expression vector used was introduced with a silent mutation so that it would not be knocked down by the C87436-specific shRNA.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) by setting the number of cells 96 hours after addition of tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in C87436 knock-down cells (C87436 KD / mock). In addition, phospholipid hydroperoxide-dependent cell death was restored in the cells into which C87436 gene was introduced after knocking down C87436 (C87436 KD / C87436).

  From the above results, it was revealed that C87436 is an execution factor of phospholipid hydroperoxide-dependent cell death.

[Experiment 21]
(Abhd2 is an executioner of phospholipid hydroperoxide-dependent cell death)
Among the candidate genes described above, the Abhd2 gene was found. The Abhd2 gene is thought to be a hydrolase having an alpha / beta hydrolase domain structure, but its substrate has not been clarified.

  By expressing shRNA in a retrovirus-infected system, Abhd2 knockdown cells were prepared, and it was examined whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 21 is a graph showing the examination results. First, Abhd2-specific shRNA (SEQ ID NO: 15) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which Abhd2 was knocked down (Abhd2 KD). As a control, cells into which only the vector was introduced were prepared (Control). Further, an Abhd2 expression vector (Abhd2) and a vector alone (mock) were introduced into these cells, respectively. As the Abhd2 cDNA incorporated into the Abhd2 expression vector, a cDNA having a silent mutation introduced so as not to be knocked down by Abhd2-specific shRNA was used.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) by setting the number of cells 96 hours after addition of tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Abhd2 knockdown cells (Abhd2 KD / mock). In addition, phospholipid hydroperoxide-dependent cell death was recovered in cells (Abhd2 KD / Abhd2) into which Abhd2 gene was introduced after Abhd2 was knocked down.

  From the above results, it was revealed that Abhd2 is an execution factor of phospholipid hydroperoxide-dependent cell death.

[Experimental example 22]
(Cdadc1 homolog, C87436 and Abhd2 are not involved in apoptosis or necrosis)
It was investigated whether Cdadclomomol knockdown cells, C87436 knockdown cells and Abhd2 knockdown cells in which phospholipid hydroperoxide-dependent cell death was suppressed were able to suppress apoptosis or necrosis.

  FIG. 22A is a graph showing the results of studying apoptosis. A Cdadc1 homolog knockdown cell (Cdadc1h KD), a C87436 knockdown cell (C87436 KD), an Abhd2 knockdown cell (Abhd2 KD), and a control cell (Control) were prepared. Staurosporine, an apoptosis-inducing reagent, was added to the medium of each cell so that the final concentration was 0.01 to 1.50 μM. Subsequently, after 24 hours of incubation, the cell viability (%) of each cell was measured by MTT assay.

  As a result, it was revealed that Cdadc1 homolog, C87436 and Abhd2 have no inhibitory effect on apoptosis due to addition of staurosporine.

  FIG.22 (b) is a graph which shows the result of having examined about necrosis. A Cdadc1 homolog knockdown cell (Cdadc1h KD), a C87436 knockdown cell (C87436 KD), an Abhd2 knockdown cell (Abhd2 KD), and a control cell (Control) were prepared. To each cell medium, tert-butyl hydroperoxide, a necrosis-inducing reagent, was added to a final concentration of 2.3 to 300.0 mM. The final concentration of 300 mM is a condition for inducing necrosis. Subsequently, after incubation for 30 minutes, the cell viability (%) of each cell was measured by MTT assay.

  As a result, it was revealed that Cdadc1 homolog, C87436, and Abhd2 did not show an inhibitory effect on necrosis caused by the addition of 300 mM tert-butyl hydroperoxide.

  However, the addition of tert-butyl hydroperoxide in a low concentration region was confirmed to suppress necrosis, although it was weak in C87436 and Abhd2 gene knockdown cells. Lipid oxidation is induced by the addition of tert-butyl hydroperoxide, suggesting that the phospholipid hydroperoxide-dependent cell death pathway functions simultaneously under the conditions where tert-butyl hydroperoxide is added at a low concentration. It was.

[Experimental example 23]
(Cdadc1 homolog and C87436 function upstream of ERK phosphorylation and Abhd2 functions downstream of ERK phosphorylation)
When Tamoxifen is added to tamoxifen-induced PHGPx-deficient MEF cell line and tamoxifen is stained with anti-phosphorylated ERK antibody, cells with enhanced ERK phosphorylation are detected 36 hours after addition of tamoxifen. be able to.

  Thus, Cdadclomomol, C87436 or Abhd2 of tamoxifen-induced PHGPx-deficient MEF cell lines was knocked down, and enhancement of ERK phosphorylation when tamoxifen having a final concentration of 1 μM was added to the medium was examined.

  Tamoxifen-induced PHGPx-deficient MEF cells knocked down Cdadc1 homomol (Cdadc1h KD), Tamoxifen-induced PHGPx-deficient MEF cells knocked down C87436 (C87436 KD), Tamoxifen-induced PHGPb dh MEb cells deficient Abhd2 As a control, Tamoxifen-induced PHGPx-deficient MEF cells (Control) were prepared.

  Subsequently, tamoxifen having a final concentration of 1 μM was added to the medium of each cell, and unadded cells were prepared. After 36 hours, the cells were fluorescently stained with an anti-phosphorylated ERK antibody. Subsequently, the number of cells in which ERK was phosphorylated was counted by fluorescence microscopy.

  FIG. 23 (a) is a graph showing the results of measuring the percentage (%) of cells with enhanced ERK phosphorylation. The graph shows the rate of increase in the number of cells with phosphorylated ERK when treated with tamoxifen, assuming that the number of cells in which ERK phosphorylation is observed in tamoxifen-free cells is 100, and It is expressed as a percentage (%).

  As a result, enhancement of ERK phosphorylation was confirmed only in Abhd2 gene knockdown cells. From these results, it was considered that Cdadc1 homolog and C87436 function upstream of ERK phosphorylation, and Abhd2 functions downstream of ERK phosphorylation. FIG. 23 (b) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway that has been clarified from the above results.

[Experimental Example 24]
(Cdadc1 homolog is involved in the production of phospholipid hydroperoxide by PHGPx deficiency)
Tampofen-induced PHGPx-deficient MEF cell lines Cdadc1 homolog, C87436 or Abhd2 were knocked down, and the production of phospholipid hydroperoxide by PHGPx deficiency when tamoxifen at a final concentration of 1 μM was added to the medium was examined.

  FIG. 24 (a) is a graph showing the results of detecting the production of phospholipid hydroperoxide by flow cytometry. Specifically, first, Tamoxifen-induced PHGPx-deficient MEF cells (Cdadc1h KD) in which Cdadclomomol was knocked down, Tamoxifen-inducible PHGPx-deficient MEF cells (C87436 KD) in which C87436 was knocked down, Tamoxifen-induced PH in which Abhd2 was knocked down A deficient MEF cell (Abhd2 KD) and a tamoxifen-induced PHGPx deficient MEF cell (Control) as a control were prepared.

  Subsequently, tamoxifen having a final concentration of 1 μM was added to the culture medium of these cells. After incubation for 26 hours, the cells were stained with H2DCFDA, which is a fluorescent dye capable of detecting the formation of phospholipid hydroperoxide, and measured by flow cytometry.

  As a result, it was revealed that the production of phospholipid hydroperoxide due to PHGPx deficiency was strongly suppressed in Cdadc1 homolog knockdown cells.

  From this, it was considered that Cdadc1 homolog is involved in the production of phospholipid hydroperoxide by PHGPx deficiency. FIG. 24 (b) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway that has been clarified from the above results.

[Experiment 25]
(The generation of phospholipid hydroperoxide by PHGPx deficiency does not involve iron)
Conventionally, it has been considered that the oxidation reaction of phospholipid is carried out through a hydroxy radical generated by the Fenton reaction through iron. Therefore, it was examined whether or not the production of phospholipid hydroperoxide due to PHGPx deficiency can be suppressed using deferoxamine, an iron chelator.

  For the study, H2DCFDA, which is a fluorescent dye capable of detecting the production of phospholipid hydroperoxide, and flow cytometry were used. A tamoxifen-induced PHGPx-deficient MEF cell line was prepared by adding deferoxamine (DFO) having a final concentration of 100 μM or vitamin E having a final concentration of 200 μM. Tamoxifen at a final concentration of 1 μM was further added to each of these cells. As a control, tamoxifen-induced PHGPx-deficient MEF cells incubated in the presence (1 μM) or absence of tamoxifen were used. 26 hours after the addition of tamoxifen, each cell was stained with H2DCFDA and analyzed by flow cytometry.

  FIG. 25A and FIG. 25B are graphs showing the results of flow cytometry. The production of phospholipid hydroperoxide due to PHGPx deficiency could not be suppressed by deferoxamine, an iron chelator, but could be suppressed by vitamin E. From this result, it was shown that the production of phospholipid hydroperoxide by PHGPx deficiency does not involve iron.

[Experiment 26]
(Effect of inhibition of Fenton reaction on phospholipid hydroperoxide-dependent cell death)
Manganese (III) tetrakis (4-carbophenyl) porphyrin (Mn-TBAP), N-acetylcysteine (NAC), deferoxamine (DFO), which is thought to suppress the Fenton reaction, is added to the medium of Tamoxifen-induced PHGPx-deficient MEF cell line It was investigated whether or not phospholipid hydroperoxide-dependent cell death could be suppressed.

  Mn-TBAP has an action of eliminating superoxide, NAC has an action of eliminating hydrogen peroxide, and deferoxamine has an action of chelating iron. FIG. 26 (a) is a model diagram showing the action points of Mn-TBAP, NAC, deferoxamine, and vitamin E in the production of phospholipid hydroperoxide.

  A medium in which Mn-TBAP (final concentration 100 μM), NAC (final concentration 5 mM), deferoxamine (final concentration 100 μM) or vitamin E (final concentration 200 μM) was prepared in the medium of the Tamoxifen-induced PHGPx-deficient MEF cell line. As a control, tamoxifen-inducible PHGPx-deficient MEF cells without any added medium were prepared.

  Tamoxifen with a final concentration of 1 μM was added to the medium of each cell. Twenty (24) hours after the addition of tamoxifen, 72 hours later, the live cells were randomly photographed with a Keyence all-in-one microscope, and the number of viable cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  FIG. 26 (b) is a graph showing the results of measuring the survival rate of each cell. As a result, addition of Mn-TBAP, NAC, and deferoxamine (DFO) failed to suppress phospholipid hydroperoxide-dependent cell death. From these results, it was considered that the generation of phospholipid hydroperoxide due to PHGPx deficiency may be carried out in a system involving Cdadc1 homolog without involving the Fenton reaction.

  FIG. 27 shows a model diagram of the phospholipid hydroperoxide-dependent cell death pathway, which has been clarified from the above results.

  Cdadc1 homolog is involved in the process of producing phospholipid hydroperoxide (PCOOH) from phospholipids. The produced phospholipid hydroperoxide is normally reduced to phospholipid hydroxy form (PCOH) by PHGPx.

  When PHGPx is deficient, phospholipid hydroperoxide rises with a peak after 24 hours. CDK4 and C87436 are functioning downstream. After 36 hours, MEK and ERK in the MAP kinase pathway are phosphorylated. Abhd2 functions downstream of ERK phosphorylation and causes cell death between 48 and 72 hours later. Examples of cases where PHGPx is deficient include cases where the PHGPx gene is damaged, intracellular glutamine is depleted, vitamin E is deficient, enzyme is inactivated by drugs targeting PHGPx, and the like.

[Experiment 27]
(Myocardium-specific PHGPx-deficient mice are killed by cardiomyocyte death at 17.5 days of development)
PHGPx-deficient mice are known to be lethal at 7.5 days of development. Here, a mouse deficient in PHGPx in a heart-specific manner was prepared, and the effect was analyzed. First, mating of a PHGPx ^+/− mouse with a mouse (Cre ^{+ / +} ) having a Cre gene downstream of a muscle creatine kinase promoter which is a heart-specific promoter is repeated, and Cre ^{+ / +} PHGPx ^{+ //-} mice were obtained.

Subsequently, Cre ^{+ / +} PHGPx ^+/− mice and the above-described Tg (loxP-PHGPx) ^{+ / +} : PHGPx ^{− / −} mice are crossed to create Cre ^+/− Tg (loxP-PHGPx) ^{+ / −} : PHGPx ^{− / −} mice were obtained. This mouse is heart-specific and lacks PHGPx.

  When the heart-specific PHGPx-deficient mouse thus obtained was observed, it grew normally until 16.5 days of development, but the cardiomyocytes suddenly died on 17.5 days, and 18.5 days. Was fatal, causing edema. FIG. 28 (a) is a photograph of wild-type mice (Control) and heart-specific PHGPx-deficient mice (KO) at 17.5 days (17.5 dpc) and 18.5 days (18.5 dpc) during development. is there.

  FIG. 28 (b) is a photograph showing the results of DAPI staining and TUNEL staining (TUNEL) of a heart tissue section of a mouse on the 17.5th day of development. In the heart tissue of heart-specific PHGPx-deficient mice (KO), many cells (cell death) positive for TUNEL staining were observed. When the mother was given a dietary supplement with vitamin E (50mg vitamin E / 100g diet) every day, cardiac death of heart-specific PHGPx-deficient mice (KO) on development day 17.5 was completely suppressed and normal In addition, heartbeat-specific PHGPx-deficient mice were born. No caspase 3 activation or DNA ladder was observed in cardiomyocyte death that occurred in the fetus of a heart-specific PHGPx-deficient mouse on day 17.5 when a mother mouse was raised on a normal diet. Oxidant accumulation was observed (not shown). This suggests that this cardiomyocyte death is also induced by a novel cell death via lipid oxidation.

  FIG. 28 (c) is a photograph showing the result of detecting the expression of PHGPx protein in the heart of a mouse on the 17.5th day of development by Western blotting. It was confirmed that PHGPx protein is deficient in heart-specific PHGPx-deficient mice (KO).

[Experiment 28]
(Administration of CDK4 inhibitor 3-ATA to the abdominal cavity of mother mice suppresses death of heartbeat-specific PHGPx-deficient mice at 18.5 days)
It was examined whether or not lethality of heartbeat-specific PHGPx-deficient mice can be suppressed by administering CDK4 inhibitor 3-ATA into the abdominal cavity of mother mice.

  Developmental process of heartbeat-specific PHGPx-deficient mouse embryos 14.5 days, 15.5 days, 16.5 days, 17.5 days, 1.5 mg / kg body weight of 3-ATA was administered into the abdominal cavity of the mother mice, The fetus on the 18.5th day of the development process was observed.

  As a result, edema was suppressed in 8 out of 10 heartbeat-specific PHGPx-deficient mice. Edema was observed in the remaining two animals. Of the 8 animals in which edema was suppressed, 2 animals were alive. The remaining six were dead.

  FIGS. 29 (a)-(c) are photographs of mouse fetuses at 18.5 days of development. FIG. 29 (a) is a photograph of a wild type mouse, and FIG. 29 (b) is a photograph of a heartbeat-specific PHGPx-deficient mouse that was not administered with 3-ATA (heart PHGPx KO 3-ATA (−). FIG. 29 (c) is a photograph of a heartbeat-specific PHGPx-deficient mouse administered with 3-ATA (heart PHGPx KO 3-ATA (+)).

[Experimental example 29]
(Heart-specific PHGPx-deficient mice grow normally on a diet supplemented with vitamin E, but suddenly die about 10 days after changing to a normal diet.)
In the fetal stage of heartbeat-specific PHGPx-deficient mice, when the mother was fed a dietary supplement with vitamin E, lethality was completely suppressed. In addition, it was revealed that when a heartbeat-specific PHGPx-deficient mouse was continuously fed with a diet containing vitamin E (50 mg vitamin E / 100 g diet) every day, the mice grew normally.

  Here, the vitamin E supplemented diet contained 130 mg / kg body weight of vitamin E per day. The normal diet also contained 13 mg / kg body weight of vitamin E per day.

  FIG. 30 (a) is a graph showing the survival rate after changing the diet of heartbeat-specific PHGPx-deficient mice that have grown normally by giving a vitamin E-added diet to a normal diet. It was found that sudden death occurred around 10 days when the diet of normally grown heartbeat-specific PHGPx-deficient mice was changed to a normal diet.

  FIG. 30 (b) shows a heartbeat-specific PHGPx-deficient mouse fed with a vitamin E-added diet (vitamin E-added diet) and a heartbeat-specific PHGPx-deficient mouse that died by changing the diet to a normal diet (normal diet KO (dead)). ) And the results of measuring the amount of vitamin E in the heart of a wild-type mouse (normal diet wild) fed a normal diet. The heart of a heartbeat-specific PHGPx-deficient mouse fed a dietary supplement with vitamin E contained about 24 nmol of vitamin E per gram of heart. The normal diet contained about 4 nmol of vitamin E per gram of heart. In this mouse lethal model, cardiomyocyte death due to lipid oxidation is induced by a decrease in the amount of vitamin E in the heartbeat (not shown).

  FIG. 30 (c) shows a heartbeat-specific PHGPx-deficient mouse (vitamin E-added diet heart KO) fed with a vitamin E-added diet and a heartbeat-specific PHGPx-deficient mouse (normal diet) on the 10th day after changing the diet to a normal diet. It is a graph which shows the electrocardiogram just before death of heart KO). An arrhythmia was observed in the electrocardiogram of heartbeat-specific PHGPx-deficient mice in which the diet was changed to a normal diet, and sudden arrhythmic death (heart failure) was observed.

[Experiment 30]
(CDK4 inhibitor 3-ATA can prolong the sudden death due to heart failure in heartbeat-specific PHGPx-deficient mice, which is caused by changing from a vitamin E-added diet to a normal diet)
After changing the diet of heartbeat-specific PHGPx-deficient mice fed with a vitamin E-added diet to a normal diet, the effect of administering the CDK4 inhibitor 3-ATA was examined.

  FIG. 31 shows a case where the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet (Vit.E-), the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet, and the diet was changed to a normal diet. Vitamin E supplemented diet was given to mice (Vit.E−, 3-ATA +) and heartbeat-specific PHGPx-deficient mice administered intraperitoneally with 2 mg / kg body weight of 3-ATA once a day from the day. It is a graph which shows the survival rate of the mouse | mouth (Vit.E +, 3-ATA +) intraperitoneally administered 2 mg / kg body weight 3-ATA once a day from the 4th day after the start.

  The median survival time from the start of the experiment for mice (Vit. E−) (n = 23) in which the heartbeat-specific PHGPx-deficient mice were changed to a normal diet was 10 days. In addition, the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet, and the mice were administered intraperitoneally with 2 mg / kg body weight of 3-ATA once a day from the fourth day after changing the diet to a normal diet (Vit The median survival time from the start of the experiment for E-, 3-ATA +) (n = 6) was 18 days. These results were significantly different with a risk rate of less than 1%.

  From the above results, it was shown that the CDK4 inhibitor 3-ATA can prolong the sudden death due to heart failure in heartbeat-specific PHGPx-deficient mice caused by changing from a vitamin E-added diet to a normal diet.

[Experimental example 31]
(The MEK inhibitor U-0126 can prolong the sudden death due to heart failure in heartbeat-specific PHGPx-deficient mice that occur when a vitamin E-added diet is changed to a normal diet)
After changing the diet of heartbeat-specific PHGPx-deficient mice fed with a vitamin E-added diet to a normal diet, the effect of administering MEK inhibitor U-0126 was examined.

  FIG. 32 shows a case where the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet (Vit.E-), the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet, and the diet was changed to a normal diet. Vitamin E supplemented food was given to mice (Vit.E-, U-0126 +) and U.S. heart-specific PHGPx-deficient mice given 3 mg / kg body weight of U-0126 intraperitoneally once daily from the day. It is a graph which shows the survival rate of the mouse | mouth (Vit.E +, U-0126 +) which intraperitoneally administered U-0126 of 3 mg / kg body weight once a day from the 4th day after the start.

  The median survival time from the start of the experiment for mice (Vit. E−) (n = 23) in which the heartbeat-specific PHGPx-deficient mice were changed to a normal diet was 10 days. Furthermore, the heartbeat-specific PHGPx-deficient mice were changed from a normal diet to a normal diet, and mice were administered intraperitoneally with 3 mg / kg body weight of U-0126 once a day from the 4th day (Vit). .E-, U-0126 +) (n = 7) had a median survival time of 20 days from the start of the experiment. These results were significantly different with a risk rate of less than 1%.

  From the above results, it was shown that the MEK inhibitor U-0126 can prolong the sudden death due to heart failure in heartbeat-specific PHGPx-deficient mice, which are caused by changing from a vitamin E-added diet to a normal diet.

  According to the present invention, a method and a kit for detecting phospholipid hydroperoxide-dependent cell death can be provided. In addition, proteins and nucleic acids involved in phospholipid hydroperoxide-dependent cell death can be provided. In addition, an inhibitor of phospholipid hydroperoxide-dependent cell death can be provided. In addition, a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death can be provided.

Claims (6)

  A method for detecting phospholipid hydroperoxide-dependent cell death, comprising detecting phospholipid hydroperoxide production and ERK activation in a cell sample.   Detection of ERK activation includes measuring the intracellular localization of ERK, measuring the expression level of ERK at the mRNA level, measuring the expression level of ERK at the protein level, The method according to claim 1, wherein the method is performed by at least one selected from the group consisting of measuring an oxidation rate and measuring cell death suppression due to suppression of ERK expression. Reagents for detecting the production of phospholipid hydroperoxide, and antibodies against phosphorylated ERK and non-phosphorylated ERK; primers for ERK mRNA amplification; or siRNA, shRNA, ribozyme or antisense nucleic acid against ERK,
A kit for detecting phospholipid hydroperoxide-dependent cell death, comprising:   A phospholipid hydroperoxide-dependent cell death inhibitor comprising, as an active ingredient, siRNA, shRNA, ribozyme or antisense nucleic acid against ERK inhibitor or ERK.   A prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising an ERK inhibitor or an siRNA, shRNA, ribozyme or antisense nucleic acid against ERK as an active ingredient.   The prophylactic or therapeutic agent according to claim 5, wherein the disease associated with phospholipid hydroperoxide-dependent cell death is heart failure.