JP6637092B2 - Method and kit for detecting phospholipid hydroperoxide-dependent cell death, inhibitor of phospholipid hydroperoxide-dependent cell death, and agent for preventing or treating heart failure or sudden arrhythmic death - Google Patents

Description

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

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

  Phospholipid hydroperoxide glutathione peroxidase (hereinafter sometimes referred to as "PHGPx") is a phospholipid hydroperoxide that is directly reduced to a phospholipid hydroperoxide by using glutathione as a coenzyme and converted into a phospholipid hydroxy form. is there. This knockout mouse of the PHGPx gene was known to be fetal lethal (for example, see Non-Patent Document 1). However, the mechanism of cell death due to PHGPx deficiency was 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 due to PHGPx deficiency, and provides a method and kit for detecting the above cell death (hereinafter, referred to as “phospholipid hydroperoxide-dependent cell death”). An object of the present invention is to provide proteins and nucleic acids involved in cell death, inhibitors of phospholipid hydroperoxide-dependent cell death, and agents for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death.

The present invention is as follows.
(1) Detection of phospholipid hydroperoxide-dependent cell death, which comprises a step of detecting the activation state of at least one factor selected from the group consisting of CDK4, MEK, ERK, Cdadclhomolog, C87436, and Abhd2 in cells. Method.
(2) the detecting includes measuring a localization state of the factor in a cell, measuring an expression level of the factor at an mRNA level, measuring an expression level of the factor at a protein level, The method according to (1), wherein the method is performed by at least one selected from the group consisting of measuring the phosphorylation rate of a factor and measuring the suppression of cell death by suppressing the expression of the factor.
(3) an antibody against CDK4, MEK, ERK, Cdadc1homolog, C87436 or Abhd2, which is phosphorylated or unphosphorylated; a primer for amplifying mRNA of CDK4, MEK, ERK, Cdadc1homolog, C87436 or Abhd2; A kit for detecting phospholipid hydroperoxide-dependent cell death, comprising an siRNA, shRNA, ribozyme or antisense nucleic acid against ERK, Cdadclhomolog, 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; A protein that suppresses lipid hydroperoxide-dependent cell death.
(5) a nucleic acid comprising 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 A protein that suppresses lipid hydroperoxide-dependent cell death.
(7) a nucleic acid comprising 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 consisting of 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 the expression level is reduced. A protein that suppresses lipid hydroperoxide-dependent cell death.
(9) Having a homology of 80% or more with the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 or 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) An inhibitor of phospholipid hydroperoxide-dependent cell death, comprising vitamin E or a derivative thereof as an active ingredient.
(11) A CDK4 inhibitor or an inhibitor of phospholipid hydroperoxide-dependent cell death, which comprises an siRNA, shRNA, ribozyme or antisense nucleic acid for CDK4 as an active ingredient.
(12) 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.
(13) An ERK inhibitor or an inhibitor of phospholipid hydroperoxide-dependent cell death, comprising an siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against ERK.
(14) An inhibitor of phospholipid hydroperoxide-dependent cell death, comprising as an active ingredient siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1homolog, C87436 or Abhd2.
(15) An agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising vitamin E or a derivative thereof as an active ingredient.
(16) An agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising a CDK4 inhibitor or an 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 a MEK inhibitor or an siRNA, shRNA, ribozyme or antisense nucleic acid for MEK as an active ingredient.
(18) An agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising an ERK inhibitor or an siRNA, an shRNA, a ribozyme or an antisense nucleic acid as an active ingredient against ERK.
(19) An agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, which comprises as an active ingredient siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadcl1 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. Further, an agent for inhibiting phospholipid hydroperoxide-dependent cell death can be provided. In addition, a preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death can be provided.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram for 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 the time-dependent change of the amount of the phospholipid hydroperoxide (18: 0, 20: 4 hydroperoxide derived from phosphatidylcholine) produced in PHGPx-deficient MEF cells. It is a graph which shows the result of the MTT assay in which PHGPx deficiency MEF cell is lethal by addition of tamoxifen. (A) is a graph showing the results showing the inhibitory effect of phospholipid hydroperoxide-dependent cell death when an apoptosis inhibitor was used in terms of cell viability (%). (B) is a photograph showing the results of fluorescent immunostaining for 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 fluorescence immunostaining of HMGB1 48 hours after addition of tamoxifen. It is. (A) is a graph showing the results of cell viability (%) against phospholipid hydroperoxide-dependent cell death due to 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 (%) against phospholipid hydroperoxide-dependent cell death by knockdown cells of necroptosis-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 the ability of 3-ATA, U-0126 and vitamin E to inhibit phospholipid autoxidation in vitro. (A) is a graph showing the results of cell viability (%) against phospholipid hydroperoxide-dependent cell death due to knockdown of CDK4. (B) is a photograph showing the result of CDK4 expression by Western blotting. (A) is a graph showing the results of cell viability (%) against phospholipid hydroperoxide-dependent cell death due to knockdown of MEK1. (B) is a photograph showing the result of MEK1 expression by western blotting. (A) and (b) are photographs showing the results of examining the time course of MEK and ERK phosphorylation during 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 knockdown of ERK2. (B) is a photograph showing the result of ERK2 expression by Western blotting. (A) is a photograph showing the result of examining the effect of Trolox and 3-ATA on the phosphorylation of MEK and ERK by Western blotting. (B) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway. It is a graph which shows the result of having examined about the inhibitory effect of phospholipid oxidation using the flow cytometry in the knockdown cell of CDK4 and MEK1. FIG. 2 is a model diagram of a phospholipid hydroperoxide-dependent cell death pathway revealed from screening of a compound library. FIG. 2 is a diagram showing an outline of a screening procedure for an shRNA library. It is a graph which shows the result of having arrange | positioned the phospholipid hydroperoxide-dependent cell death suppression effect by knockdown about 151 candidate genes in descending order. (A) is a diagram showing the structures of the Cdadc1 homolog gene and the 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 Cdadcl1 homolog. It is a graph which shows the result of cell viability (%) with respect to the phospholipid hydroperoxide-dependent cell death by knockdown of C87436. It is a graph which shows the result of cell viability (%) with respect to the phospholipid hydroperoxide-dependent cell death by knockdown of ABHD2. (A) and (b) are graphs showing the results of an MTT assay examining the suppressive effects on apoptosis and necrosis by knockdown of Cdadcl1 homolog, C87436, ABHD2. (A) is a graph showing the results of measuring the change in the percentage (%) of ERK phosphorylation-enhanced cells in phospholipid hydroperoxide-dependent cell death due to Cdadcl1 homolog, C87436, and ABHD2 knockdown cells. (B) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway. (A) is a graph showing the results of examining the effect of suppressing the production of phospholipid hydroperoxide in phospholipid hydroperoxide-dependent cell death by knockdown cells of Cdadcl1 homolog, C87436, and ABHD2 by flow cytometry. (B) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway. (A) and (b) are graphs showing the results of examining the inhibitory effect of iron chelators on phospholipid hydroperoxide production 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 inhibitory effect of Mn-TBAP, NAC, deferoxamine, and vitamin E on phospholipid hydroperoxide-dependent cell death in terms of cell viability (%). 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 a wild-type mouse (Control) and a heart-specific PHGPx-deficient mouse (KO) on day 17.5 and day 18.5 of the development process. (B) is a photograph showing the results of DAPI and TUNEL staining (TUNEL) of a tissue section of a mouse heart at 17.5 days of development. (C) is a photograph showing the result of detecting the expression of PHGPx protein in the heart of a mouse at 17.5 days of development by Western blotting. (A) to (c) are photographs of mouse embryos at 18.5 days of development when the effect of 3-ATA was examined. (A) is an illustration of a model mouse with arrhythmic sudden death (heart failure) caused by a decrease in the amount of vitamin E in the diet, showing that sudden death occurs in about 10 days when the diet is switched from a diet supplemented with vitamin E to a normal diet. 4 is a graph showing the survival rate of failed mice. (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. Fig. 4 is a graph showing the inhibitory effect of 3-ATA on a mouse model of sudden death due to heart failure caused by a decrease in vitamin E. Fig. 4 is a graph showing the inhibitory effect of U-0126 on a mouse model of sudden death due to heart failure caused by a decrease in vitamin E. (A) is a diagram showing 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 provides a phospholipid hydroperoxide, comprising detecting the activation state of at least one factor selected from the group consisting of CDK4, MEK, ERK, Cdadclhomolog, C87436 and Abhd2 in a cell. Methods for detecting dependent cell death are provided.

  As described below, the present inventors have found apoptosis, necrosis, autophagic cell death, and cell death by a novel pathway distinct from necrotosis (phospholipid hydroperoxide-dependent cell death). Furthermore, it was revealed that the execution factors of phospholipid hydroperoxide-dependent cell death include CDK4, MEK, ERK, Cdadclhomolog, C87436 and Abhd2.

  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 also possible to detect whether or not phospholipid hydroperoxide-dependent cell death is in progress, and to determine whether the cell death occurs after the cell death occurs, Can be determined.

  The activation state of the factor is detected, for example, by measuring the localization state of the factor in a cell, measuring the expression level of the factor at the mRNA level, and measuring 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 above factor, and measuring the suppression of cell death by suppressing the expression of the above factor.

  The localization state in a cell can be measured, for example, by fixing a target cell, staining the cell with a fluorescent-labeled antibody against the above-described factor, and observing the cell with a fluorescence microscope. Alternatively, a fusion protein of the above-described factor and a fluorescent protein is expressed in a target cell, and the localization state of the above-mentioned factor in the cell can be measured by 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 measurement of the intracellular localization of the factor, for example, compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not progressing, when a change in the localization of the factor is observed, Phospholipid hydroperoxide-dependent cell death can be detected.

  The expression level of the above factors 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 a change in the expression level of the above factors at the mRNA level is observed as compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not in progress. be able to.

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

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

  When cell death can be suppressed by suppressing the expression of the above factor, it can be determined that the above 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 above factors can be performed, for example, by introducing siRNA, shRNA, ribozyme, antisense nucleic acid, etc. to the above factors into cells. In addition, the measurement of the suppression of cell death can be performed, for example, by measuring the survival rate of cells in the case where the expression of the above factor is suppressed and in the case where the expression is not performed. The cell survival rate can be measured, for example, by calculating the change in the number of living cells by MTT assay or microscopic imaging.

  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 and cleaves mRNA having a sequence complementary to the siRNA. This suppresses gene expression in a sequence-specific manner.

  The siRNA is prepared by synthesizing sense and antisense strand oligonucleotides with an automatic DNA / RNA synthesizer, for example, after denaturing in an appropriate annealing buffer at about 90 to about 95 ° C. for about 1 minute, then about 30 minutes. It can be prepared by annealing at ７０70 ° C. for about 1 to 8 hours.

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

  Ribozymes are RNAs having catalytic activity. There are ribozymes having various activities. Research on ribozymes as RNA-cleaving enzymes has enabled the design of ribozymes for 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 RNase P, or a ribozyme of about 40 nucleotides called a hammerhead type, a hairpin type, or the like.

  Antisense nucleic acids are nucleic acids that are complementary to a target sequence. Antisense nucleic acids inhibit transcription initiation by triplex formation, transcription suppression by hybridization with a site where an open loop structure is formed locally by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, Inhibition of splicing by hybridization at the junction between introns and exons, suppression of splicing by hybridization with spliceosome formation sites, suppression of translocation from the nucleus to cytoplasm by hybridization with mRNA, capping sites and poly (A) addition sites Suppression of splicing by hybridization with a translation initiation factor binding site, suppression of translation initiation by hybridization with a ribosome binding site near the initiation codon, mRNA translation region 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 the present embodiment, the siRNA, shRNA, ribozyme, and antisense nucleic acid may contain various chemical modifications in order to improve stability and activity. For example, a phosphate residue may be replaced with a chemically modified phosphate residue such as, for example, phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by a hydrolase such as nuclease. Further, at least a part thereof may be constituted by a nucleic acid analog such as a peptide nucleic acid (PNA).

[Kit for detecting phospholipid hydroperoxide-dependent cell death]
In one embodiment, the present invention provides an antibody against CDK4, MEK, ERK, Cdadc1homolog, C87436 or Abhd2, phosphorylated or unphosphorylated; a primer for mRNA amplification of CDK4, MEK, ERK, Cdadc1homolog, C87436 or Abhd2; Alternatively, the present invention provides a kit for detecting phospholipid hydroperoxide-dependent cell death, which comprises siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4, MEK, ERK, Cdadclhomoglog, C87436 or Abhd2.

  Antibodies to the phosphorylated or non-phosphorylated factor are suitable for measuring the localization state of the factor in cells, measuring the expression level of the factor at the protein level, measuring the phosphorylation rate of the factor, and the like. It is.

  The primer for amplifying mRNA of the above factor is suitable for measuring the expression level of the above factor at the mRNA level and the like.

  SiRNAs, shRNAs, ribozymes or antisense nucleic acids against the above factors are suitable for suppressing the expression of the above factors.

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

[Cdadc1homolog]
In one embodiment, the present invention relates to 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 expressed. Reduced amounts provide proteins that inhibit phospholipid hydroperoxide-dependent cell death. Here, one or several means, for example, 1 to 20, 1 to 10, 1 to 5 pieces.

  SEQ ID NO: 1 shows the amino acid sequence of Cdadcl1 homolog. As described below, the present inventors have revealed that Cdadcl1 homolog is a novel protein encoded by a novel gene. In addition, as described later, the present inventors have found that Cdadcl1 homolog is an execution factor of 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 Cdadcl1 homolog protein in cells. The suppression or promotion of the activity of the Cdadcl1 homolog protein can be performed, for example, by decreasing or increasing the expression level of the Cdadcl1 homolog protein, or by modifying the amino acid sequence of the Cdadcl1 homolog protein.

  The expression level of the Cdadc1homolog protein can be reduced, for example, by deleting the Cdadc1homolog gene in the cell. Alternatively, it can be carried out by, for example, introducing an siRNA, shRNA, ribozyme or antisense nucleic acid targeting the Cdadcl1 homolog gene into cells. In addition, the expression level of Cdadc1homolog protein can be increased by introducing an expression vector of Cdadc1homolog protein into cells.

  In another embodiment, the present invention relates to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 2, and having a reduced expression level. Provided is a nucleic acid encoding a protein in which dependent cell death is suppressed.

  SEQ ID NO: 2 shows a nucleotide sequence encoding Cdadcl1 homolog. The nucleic acid of the present embodiment may have the base sequence of SEQ ID NO: 2. In addition, the nucleic acid of the present embodiment may have a nucleotide sequence different from the nucleotide sequence of SEQ ID NO: 2 as long as the nucleic acid encodes a protein whose phospholipid hydroperoxide-dependent cell death is suppressed when the expression level is reduced. Good. In this case, it preferably has 80% or more homology with the nucleotide sequence of SEQ ID NO: 2, more preferably has 85% or more homology, further preferably has 90% or more homology, and has 95% or more homology. It is particularly preferred to have the above homology.

  The nucleic acid of the present embodiment may be artificially recombined. For example, it may be in a form incorporated into an expression vector.

[C87436]
In one embodiment, the present invention relates to 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 Reduced amounts provide proteins that inhibit phospholipid hydroperoxide-dependent cell death. Here, one or several means, for example, 1 to 20, 1 to 10, 1 to 5 pieces.

  SEQ ID NO: 3 shows the amino acid sequence of C87436. As described below, the inventors have found that C87436 is a determinant 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 cells. Suppression 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, and the like.

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

  In another embodiment, the present invention relates to a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having the homology of 80% or more with the nucleotide sequence of SEQ ID NO: 4, and having reduced expression level. Provided is a nucleic acid encoding a protein in which dependent cell death is suppressed.

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

  The nucleic acid of the present embodiment may be artificially recombined. For example, it may be in a form incorporated into an expression vector.

[Abhd2]
In one embodiment, the present invention relates to 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 expressed Reduced amounts provide proteins that inhibit phospholipid hydroperoxide-dependent cell death. Here, one or several means, for example, 1 to 20, 1 to 10, 1 to 5 pieces.

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

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

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

  In another embodiment, the present invention relates to a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 6, and reducing the expression level. Provided is a nucleic acid encoding a protein in which dependent cell death is suppressed.

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

  The nucleic acid of the present embodiment may be artificially recombined. For example, it may be in a form incorporated into an expression vector.

[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 below, the present inventors have found that administration of vitamin E or a derivative thereof in vitro or in vivo can suppress phospholipid hydroperoxide-dependent cell death. Therefore, vitamin E or a derivative thereof can be used as an inhibitor of phospholipid hydroperoxide-dependent cell death.

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

  When added to the culture medium of cultured cells, the dose of vitamin E or a derivative thereof is preferably, for example, 50 to 1000 μM, and more preferably, for example, 150 to 400 μM. When administered to animals, it is preferable to administer, for example, 50 to 260 mg / kg body weight per day in several divided doses.

  Vitamin E or its derivative may be administered as it is, or may be administered as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive.

(Pharmaceutical composition)
Examples of the pharmaceutical composition include those which are orally used as tablets, capsules, elixirs, and microcapsules, if necessary, which are sugar-coated. In addition, those used parenterally in the form of a sterile solution of water or a pharmaceutically acceptable liquid other than water, or a suspension can be used. Further, a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavor, excipient, vehicle, preservative Formulations may be made by appropriately combining with agents, binders, and the like, and mixing in a unit dosage form generally required for pharmaceutical practice.

  Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, and alginic acid. Agents, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry. When the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated using vehicles such as distilled water for injection, according to standard formulation practice.

  Aqueous solutions for injection include, for example, physiological saline, dextrose, and other adjuvants such as isotonic solutions containing D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents, such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, non-ionic surfactants such as Polysorbate 80 ™, HCO-50 may be used in combination.

  The oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. It may also be combined with buffers such as phosphate buffers and sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol and antioxidants. The prepared injection is usually filled into an appropriate ampoule.

  Administration to patients is performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. If the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. The dose and administration method vary depending on the patient's body weight, age, symptoms and the like, but can be appropriately selected by those skilled in the art.

  The dose of the compound varies depending on the condition, but in the case of oral administration, generally, in 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 believed to be 0-20 mg.

  In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, and administration method. It may be preferable to administer about 0.01-30 mg, about 0.1-20 mg, about 0.1-10 mg per day by intravenous injection.

  Preferable examples in which a therapeutic effect of the inhibitor of the present embodiment is expected include heart failure, arrhythmic sudden death, and the like.

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

  As described below, the present inventors have revealed that CDK4 is involved in the execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, a CDK4 inhibitor or siRNA, shRNA, ribozyme or antisense nucleic acid against 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 for CDK4 is not particularly limited as long as it can suppress CDK4 expression.

  When the dose of the CDK4 inhibitor is added to the culture medium of cultured cells, for example, the dose is preferably 5 to 20 μM. When administered to an animal, it is preferable to administer, for example, 2 to 10 mg / kg of body weight at a time, once a day.

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

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

  The CDK4 inhibitor or the siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 may be administered as such, or may be formulated as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. 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 an siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against MEK.

  As described below, the present inventors have revealed that MEK is involved in the execution factor of phospholipid hydroperoxide-dependent cell death. Thus, siRNAs, shRNAs, ribozymes or antisense nucleic acids against MEK inhibitors or MEK can be used as inhibitors of phospholipid hydroperoxide dependent cell death.

  Examples of MEK inhibitors include, for example, MEK inhibitor I ((2Z) -3-amino-3-[(2-aminophenyl) sulfanyl] -2- {3- [hydroxy (pyridin-4-yl) methyl] phenyl}. Prop-2-ennitrile), 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 for MEK is not particularly limited as long as it can suppress the expression of MEK.

  When the dose of the MEK inhibitor is added to the culture medium of cultured cells, it is preferably, for example, 1 to 100 μM, and more preferably, for example, 5 to 20 μM. When administered to an animal, it is preferable to administer, for example, 2 to 10 mg / kg of body weight at a time, once a day.

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

  The siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK may be administered as such, or may be formulated as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. 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 as an active ingredient against ERK. In the present embodiment, the ERK may be ERK1 or ERK2.

  As described below, the present inventors have revealed that ERK is involved in the execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, siRNAs, shRNAs, ribozymes or antisense nucleic acids against ERK inhibitors or ERK can be used as inhibitors of phospholipid hydroperoxide dependent cell death.

  Examples of the ERK inhibitor include, for example, 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 for ERK is not particularly limited as long as it can suppress ERK expression.

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

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

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

(Cdadc1homolog, C87436 or Abhd2 expression inhibitor)
In one embodiment, the present invention provides an inhibitor of phospholipid hydroperoxide-dependent cell death, comprising as an active ingredient siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadcl1 homolog, C87436 or Abhd2.

  As described below, the present inventors have revealed that Cdadclhomolog, C87436, and Abhd2 are included in the execution factors of phospholipid hydroperoxide-dependent cell death. Thus, siRNAs, shRNAs, ribozymes or antisense nucleic acids against Cdadc1homolog, C87436 or Abhd2 can be used as inhibitors of phospholipid hydroperoxide-dependent cell death.

  The siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1homolog, C87436 or Abhd2 is not particularly limited as long as it can suppress the expression of Cdadc1homolog, C87436 or Abhd2.

  The dose of siRNA, shRNA, ribozyme or antisense nucleic acid to Cdadcl1 homolog, C87436 or Abhd2 is preferably, for example, 50 nM to 10 μM, and more preferably, for example, 50 to 200 nM when added to the culture medium of cultured cells. When administered to an animal, it is preferable to administer, for example, 50 to 500 mg / kg body weight once a day once a day.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against Cdadcl1 homolog, C87436 or Abhd2 may be administered as such, or may be formulated as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

[Preventive 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 a disease 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 a disease 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 to a patient in need thereof an effective amount of vitamin E or a derivative thereof. I will provide a.

  As described below, the present inventors have found that administration of vitamin E or a derivative thereof in vitro or in vivo can suppress phospholipid hydroperoxide-dependent cell death. Therefore, vitamin E or a derivative thereof can be used as an agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death.

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

  When administering vitamin E or a derivative thereof, it is preferable to administer, for example, 50 to 260 mg / kg body weight per day in several divided doses.

  Vitamin E or its derivative may be administered as it is, 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 CDK4 inhibitor or an agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 as an active ingredient. I do.

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

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

  As described below, the present inventors have revealed that CDK4 is involved in the execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, a CDK4 inhibitor or an siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 can be used as a preventive or therapeutic agent for a disease 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 for CDK4 is not particularly limited as long as it can suppress CDK4 expression.

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

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

  The CDK4 inhibitor or the siRNA, shRNA, ribozyme or antisense nucleic acid against CDK4 may be administered as such, or may be formulated as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. 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 agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against MEK. I do.

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

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

  Examples of MEK inhibitors include, for example, MEK inhibitor I ((2Z) -3-amino-3-[(2-aminophenyl) sulfanyl] -2- {3- [hydroxy (pyridin-4-yl) methyl] phenyl}. Prop-2-ennitrile), 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 for MEK is not particularly limited as long as it can suppress the expression of MEK.

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

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

  The siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK may be administered as such, or may be formulated as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. 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 agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising an siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against ERK. I do.

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

  As described below, the present inventors have revealed that ERK is involved in the execution factor of phospholipid hydroperoxide-dependent cell death. Therefore, an ERK inhibitor or siRNA, shRNA, ribozyme or antisense nucleic acid against ERK can be used as an agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death.

  Examples of the ERK inhibitor include, for example, 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 for ERK is not particularly limited as long as it can suppress ERK expression.

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

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

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

(Cdadc1homolog, C87436 or Abhd2 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 Cdadcl1 homolog, C87436 or Abhd2. I do.

  In another embodiment, the present invention provides an siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadclhomolog, C87436 or Abhd2 for the prevention or treatment of a disease associated with phospholipid hydroperoxide-dependent cell death.

  In another embodiment, the present invention relates to the production of an siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadclhomoglog, 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 relates to a phospholipid hydroperoxide-dependent method, comprising administering to a patient in need of said treatment an effective amount of an siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1homolog, C87436 or Abhd2. Provided is a method for preventing or treating a disease associated with cell death.

  As described below, the present inventors have revealed that Cdadclhomolog, C87436, and Abhd2 are included in the execution factors of phospholipid hydroperoxide-dependent cell death. Therefore, siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1homolog, C87436 or Abhd2 can be used as an agent for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death.

  The siRNA, shRNA, ribozyme or antisense nucleic acid against Cdadc1homolog, C87436 or Abhd2 is not particularly limited as long as it can suppress the expression of Cdadc1homolog, C87436 or Abhd2.

  When administering siRNA, shRNA, ribozyme or antisense nucleic acid to Cdadcl1 homolog, C87436 or Abhd2, it is preferable to administer, for example, 50 to 500 mg / kg body weight once a day once a day.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against Cdadcl1 homolog, C87436 or Abhd2 may be administered as such, or may be formulated as a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. May be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

  The above-mentioned diseases associated with phospholipid hydroperoxide-dependent cell death include, for example, heart failure and sudden arrhythmic death. As described below, the present inventors have shown in an in vivo experiment using mice that the above preventive or therapeutic agent can prevent heart failure (sudden arrhythmic death).

  Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

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

Specifically, Tg (loxP-PHGPx): PHGPx ^{− / −} mouse in which a PHGPx gene (loxP-PHGPx) flanked by loxP sequences was transfected (Tg) into a PHGPx gene knockout mouse (PHGPx ^{− / −} ). And PHGPx ^+/− mice were bred. Subsequently, mouse fetal fibroblasts (MEF) were prepared from 13.5 day embryos whose genotype was Tg (loxP-PHGPx): PHGPx ^{− / −} . Subsequently, the cells were immortalized by introducing the T antigen gene of SV40 virus into the obtained cells. Subsequently, the tamoxifen-induced CreERT2 gene was introduced into the obtained cells. As a result, a tamoxifen-induced PHGPx-deficient MEF cell line was obtained.

  In addition, 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 medium of a tamoxifen-induced PHGPx-deficient MEF cell line, tamoxifen binds to the estrogen receptor portion of CreER2, exposing a nuclear translocation signal of the estrogen receptor. As a result, CreER2 translocates into the nucleus, and the PHGPx genomic gene flanked by loxP is destroyed. The concentration of tamoxifen to be added may be about 1 μM final concentration.

[Experimental example 2]
(Administration of Tamoxifen to Tamoxifen-Induced PHGPx-Deficient MEF Cell Line)
Tamoxifen at a final concentration of 1 μM was added to the medium of the tamoxifen-induced PHGPx-deficient MEF cell line. Subsequently, cells were collected at 0, 24 and 48 hours after the addition of tamoxifen, 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 result of Western blotting for detecting PHGPx protein. As a result, it was confirmed that the expression level of PHGPx protein was reduced within 24 hours after tamoxifen administration.

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

  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 CYX) was used for mass spectrometry. As a column for liquid chromatography, an ACQUITY UPLC (trademark) BEH C18 column (0.17 μm, 150 mm × 1.0 mm) was used.

  Samples of the total phospholipid fraction extracted from each cell at 0, 12, 24, 36 and 48 hours after the 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: 100: 0 (0-5 minutes), 50:50 of mobile phase B (isopropanol, 0.1% boric acid, 0.028% ammonia) (5-25 minutes), 50:50 (25-49 minutes), 100: 0 (59-60 minutes) and 100: 0 (60-75 minutes) step gradient, flow rate 70 μL / min, column temperature 30 ° C. Separated under conditions.

  The MS / MS analysis was performed in a negative ion mode using the technique of MRM (Multi Reaction Monitoring). The ion spray voltage was set to -4500V. Nitrogen gas was used as barrier gas and collision gas. In the detection of oxidized phospholipids, the collision energy was set at 20-65 eV. The scan range of the apparatus was set at m / z 50 to 950, and the scan speed was 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 at -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 ms and a declustering potential of -80V. The resolution of Q1 and O3 was set to the unit, Q1 was set to 886 m / z, 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 at a final concentration of 1 μM to the culture medium of the tamoxifen-induced PHGPx-deficient MEF cell line, the cells at 0, 12, 24, 36, and 48 hours were 18: 0 (stearic acid), 20: 4 is a graph showing the amount of phospholipid hydroperoxide (PCOOH), which is an oxidized primary product generated by oxidation from phosphatidylcholine (PC) having 4 (arachidonic acid), over time. At 24 hours after the addition of tamoxifen, phospholipid hydroperoxide, which is a primary oxidation product of phospholipid containing arachidonic acid, was produced. Similar results were 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 at a final concentration of 1 μM was added to the medium of the tamoxifen-induced PHGPx-deficient MEF cell line, and the cell viability was measured by the MTT assay. In addition, the same examination was performed for a vitamin E-added group having a final concentration of 200 μM at the same time as tamoxifen.

  More specifically, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) was added to the medium of the cells 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 became clear that cell death occurred between 48 hours and 72 hours after the addition of tamoxifen. Moreover, it became clear that cell death was completely suppressed by the 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 mediated through the cell death pathway by apoptosis.

  In the culture medium of a tamoxifen-induced PHGPx-deficient MEF cell line, a caspase inhibitor, Z-VAD-FMK, was added at a final concentration of 50 μM, a mitochondrial protein apoptosis-inducing factor (AIF) release inhibitor, DHIQ, at a final concentration of 300 μM, A trolox, which is an E derivative, to which a final concentration of 400 μM was added, and cells to which nothing was added as a control were prepared. Tamoxifen at a final concentration of 1 μM was added simultaneously to each cell. Three days after the addition of tamoxifen and the inhibitor, 20 photos were randomly taken with a Keyence all-in-one microscope, and the number of viable cells attached was counted. Observation was performed at 10 × magnification using a Plan Flour ELWD 20 × 0.45 (Nicon) as an objective lens (one field of view: 877.5 μm × 661.2 μm). The cell viability (%) was expressed assuming that the number of cells when cultured for 3 days without adding tamoxifen was 100%.

  The results are shown in FIG. Caspase inhibitors and AIF-based 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 culture medium of a tamoxifen-induced PHGPx-deficient MEF cell line was prepared by adding a staurosporine, an apoptosis-inducing reagent, to a final concentration of 0.5 μM and a tamoxifen at a final concentration of 1 μM. After 5 hours, 24 hours and 48 hours, each cell was subjected to immunofluorescent staining to stain for cytochrome C and activated caspase-3. In addition, DNA fragmentation characteristic of apoptosis was detected by the TUNEL (TdT-mediated dUTP nickel end labeling) method.

  The results are shown in FIG. In the staurosporine treatment, cytochrome C release was observed 5 hours later, caspase 3 was activated, and TUNEL-positive cell death (apoptosis) was observed 24 hours later. On the other hand, in the phospholipid hydroperoxide-dependent cell death, release of cytochrome C and activation of caspase 3 were not observed (FIG. 48 shows the cells after 48 hours, but not after 60 hours). With TUNEL staining only, 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)
Whether the phospholipid hydroperoxide-dependent cell death was due to necrosis was examined in detail.

  Tamoxifen was added to the culture medium of 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, at the moment of cell death, no cell swelling, characteristic of necrosis, was observed. Immediately before cell death, the cells deformed shape, and finally detached from the petri dish and suddenly died (not shown).

  Subsequently, tamoxifen-inducible PHGPx-deficient MEF cell line culture medium was prepared by adding tert-butyl hydroperoxide as a necrosis inducer at a final concentration of 300 mM, and tamoxifen at a final concentration of 1 μM. After 30 minutes (tert-butyl hydroperoxide added group) and 48 hours later (tamoxifen added group), each cell was subjected to fluorescent immunostaining, and the release of HMGB1 (High Mobility Group Box1) protein, which is a characteristic of necrosis, to the cytoplasm. Was observed.

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

  As a result, in cell death due to 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 no release to the cytoplasm was 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 or not 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, and cells in which ATG5 expression was knocked down were prepared. As the ATG5-specific shRNA, two types of shRNA, ATG5 shRNA-3 (SEQ ID NO: 7) and ATG5 shRNA-4 (SEQ ID NO: 8), were used. As a control, a cell into which only the shRNA expression vector was introduced was 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 photographed with a Keyence all-in-one microscope, and 20 photographs were taken. The number of living cells was counted. The cell viability (%) was expressed as the cell viability (%), where the number of cells 24 hours after the addition of tamoxifen was 100 (%), and the ratio of the viable cell number at each time was changed.

  The results are shown in FIGS. 7 (a) and 7 (b). Knockdown of ATG5 expression failed to suppress phospholipid hydroperoxide-dependent cell death. In addition, as shown in FIG. 7 (b), the results of RT-PCR confirmed that the introduction of shRNA knocked down the expression of the ATG5 gene.

  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 nectosis)
It was examined in detail whether phospholipid hydroperoxide-dependent cell death was due to necrosis. Nectosis is a form of cell death known as programmed necrosis. It is also 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, and cells in which Rip1 expression was knocked down were prepared. Rip1 shRNA-4 (SEQ ID NO: 9) was used as Rip1 specific shRNA. As controls, cells into which only the shRNA expression vector was introduced and cells into which Trolox was added at a final concentration of 400 μM in a medium were prepared. Tamoxifen was added to the culture medium of these cells at a final concentration of 1 μM, and the living cells after 24 hours and 72 hours were randomly photographed with a Keyence all-in-one microscope to obtain 20 live cells. Was counted. The cell viability (%) was expressed as the cell viability (%), where the number of cells 24 hours after the addition of tamoxifen was 100 (%) and the number of viable cells adhered 72 hours later.

  The results are shown in FIGS. 8 (a) and 8 (b). Knockdown of Rip1 expression failed to suppress cell death due to PHGPx deficiency. In addition, as shown in FIG. 8 (b), the results of Western blotting confirmed that the expression of Rip1 protein was knocked down by the introduction of shRNA.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from necrosis. That is, it was revealed 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)
A tamoxifen-induced PHGPx-deficient MEF cell line using a library of inhibitors for about 300 known enzymes (Kakenhi, MEXT, New Academic Area, Cancer Support, Chemotherapy Support Team, Standard Inhibitor Kit) A compound that can suppress cell death after 72 hours when tamoxifen was added at a final concentration of 1 μM was screened. As a result, 3-ATA and NSC625959, which are inhibitors of cyclin-dependent kinase 4 (CDK4), and U-0126 and MEK inhibitor 4, which are inhibitors of mitogen-activated protein kinase kinase (MEK1 / 2). Was found to be able to suppress phospholipid hydroperoxide-dependent cell death. On the other hand, p38 and JNK inhibitors could not 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, which is a CDK4 inhibitor. FIG. 9 (b) is a graph showing the results of suppression of phospholipid hydroperoxide-dependent cell death using U-0126 which is an inhibitor of MEK1 / 2.

  To the medium of the tamoxifen-induced PHGPx-deficient MEF cell line, 3-ATA or U-0126 at each concentration and tamoxifen at a final concentration of 1 μM were added, and 24 hours and 72 hours later, the live cells were analyzed using a Keyence All-in-One microscope. Twenty photographs were taken at random, and the number of viable cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the number of cells 24 hours after the addition of tamoxifen was 100 (%) and the number of viable cells adhered 72 hours later.

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

  FIG. 9 (c) is a graph showing the results of examining the ability of 3-ATA, U-0126 and vitamin E to inhibit phospholipid autoxidation in vitro. To a solution of dilinoleoyl phosphatidylcholine in ethanol was added 3-ATA, U-0126 and vitamin E at each concentration, and 50 μl of each amount was added to Imlon 1 plate (Thermo Fisher Scientific). The mixture was allowed to dry at room temperature and left standing overnight for auto-oxidation. For measurement of phospholipid hydroperoxide generated by auto-oxidation, dihydrorhodamine 123 (DHR) (Molecular Probes), which is a probe that emits fluorescence by reacting with phospholipid hydroperoxide, is reacted at 1 μg / ml for 30 minutes, and the fluorescence is measured. 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, whereas 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. This suggests that 3-ATA and U-0126 have no antioxidant ability and inhibit CDK4 and MEK1 / 2 activities, thereby suppressing phospholipid hydroperoxide-dependent cell death. .

[Experimental example 11]
(Kinase activity of CDK4 is required for phospholipid hydroperoxide-dependent cell death)
By expressing shRNA in tamoxifen-induced PHGPx-deficient MEF cells in a retrovirus infection system, knockdown cells of CDK4 were prepared, and it was examined whether phospholipid hydroperoxide-dependent cell death could be suppressed.

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

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

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in CDK4 knockdown cells (Cdk4 KD / mock). Further, phospholipid hydroperoxide-dependent cell death was restored in cells in which the human CDK4 gene was re-introduced after knocking down CDK4 (Cdk4 KD / hCdk4). On the other hand, phospholipid hydroperoxide-dependent cell death was not restored in cells (Cdk4 KD / hCdk4D158N) into which CDK4 was knocked down and the human CDK4 gene in which kinase activity was disrupted was introduced. In addition, the star in the figure shows that there is a significant difference at 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 phospholipid hydroperoxide-dependent cell death requires CDK4 kinase activity.

(Preparation of cells knocking down CDK4)
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 a 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, using the retrovirus infection system, the shRNA expression cassette was integrated into the genome of the target cell, CDK4-specific shRNA was expressed, and CDK4 knocked down cells were obtained.

  FIG. 33 (b) is a model diagram showing a process in which siRNA is formed from the expressed shRNA and the target RNA is destroyed. The shRNA vector was provided by Dr. Kenzo Hirose, Faculty of Medicine, University of Tokyo and Dr. Kotaro Sugo, Faculty of Medicine, Nagoya University. Cells in which genes other than CDK4 described below were knocked down, and ATG5 and Rip1 knockdown cells used in Experimental Examples 7 and 8 were also prepared by the same method.

(Reexpression 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 provided by Dr. Toshio Kitamura, Institute of Medical Science, The University of Tokyo. Expression of genes other than CDK4, which will be described later, was also performed by the same method.

(Gene introduction into PlatE cells)
PlatE cells were used for retrovirus preparation from shRNA vectors and pMXs-IR vectors. PlatE cells are cells in which the gag-pol gene and the env gene have been incorporated into 293T cells, which are human embryonic kidney epithelial cells.

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

PlatE cells were seeded on a 6 cm Petri dish (CORNING) at 5 × 10 ⁵ cells / dish and cultured for 1 day. To a 1.5 mL tube, 200 μL of DMEM FCS (−), 2 μg / 2 μL of the generated plasmid DNA (shRNA vector or pMXs-IR vector incorporating a target gene), and 8 μL of Plus Reagent (invitrogen, product number 11514-015) are added. 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 added to the above reaction solution, and left 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 the cells were allowed to stand at 37 ° C. for 3 hours in a CO ₂ incubator to perform gene transfer. Thereafter, the reaction solution was replaced with 6 mL of DMEM 10% FCS, and the cells were 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.

(Viral infection)
On the day before virus infection, tamoxifen-induced PHGPx-deficient cells were seeded on a 6 cm Petri dish (CORNING) at 1 × 10 ⁵ cells / dish and cultured for one day. 5 mL of a virus solution to which Polybrene ™ (Hexadimethrine bromide, SIGMA, Catalog No. H9268) was added to a final concentration of 8 μg / mL was infected by replacing the cell culture medium, and the culture medium was replaced after 24 hours of culture. . Polybrene (trademark) was dissolved in sterile water to a concentration of 10 mg / mL in a clean bench, filter sterilized, and diluted at the time of use.

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

  FIG. 11A is a graph showing the examination result. First, MEK1-specific shRNA (SEQ ID NO: 11) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line, and a cell in which MEK1 was knocked down was prepared (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 in which kinase activity was disrupted (hMek1S222A), 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 photographs were taken at random of all living cells 24 hours and 72 hours after the addition of tamoxifen with an all-in-one microscope of Keyence, and the number of living cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the number of cells 24 hours after the addition of tamoxifen was 100 (%) and the number of viable cells adhered 72 hours later.

  The introduced human MEK1 gene (SEQ ID NO: 17) was resistant to the above-mentioned mouse MEK1-specific shRNA. 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 was restored in cells into which the human MEK1 gene was introduced after MEK1 was knocked down (MEK1 KD / hMek1). On the other hand, phospholipid hydroperoxide-dependent cell death was not restored in cells into which the human MEK1 gene in which kinase activity was disrupted after MEK1 was knocked down (MEK1 KD / hMek1S222A). In addition, the star in the figure shows that there is a significant difference at a risk rate of less than 5%.

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

  These results revealed that MEK1 kinase activity is required for phospholipid hydroperoxide-dependent cell death.

[Experimental example 13]
(Phosphorylation of MEK1 / 2 and ERK is enhanced 36 hours after tamoxifen addition)
We examined when phosphorylation of MEK and phosphorylation of its substrate, ERK, occur during the process of phospholipid hydroperoxide-dependent cell death.

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

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

[Experimental example 14]
(Knockdown of ERK2 suppresses phospholipid hydroperoxide-dependent cell death)
By expressing shRNA with a retrovirus, knockdown cells of ERK2 were prepared, and it was examined whether phospholipid hydroperoxide-dependent cell death can be suppressed.

  FIG. 13A is a graph showing the examination result. First, ERK2-specific shRNA (SEQ ID NO: 12) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line, and a cell in which ERK2 was knocked down was prepared (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 photographs were taken at random of all living cells 24 hours and 72 hours after the addition of tamoxifen with an all-in-one microscope of Keyence, and the number of living cells attached was counted. The cell viability (%) was expressed as the cell viability (%), where the number of cells 24 hours after the addition of tamoxifen was 100 (%) and the number of viable cells adhered 72 hours later.

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

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

  These results suggest that ERK2 is involved in phospholipid hydroperoxide-dependent cell death downstream of MEK.

[Experimental example 15]
(In the phospholipid hydroperoxide-dependent cell death pathway, phospholipid hydroperoxide production and CDK4 function upstream of MEK1 / 2)
It was examined whether the phosphorylation of MEK and ERK occurring 39 hours after the addition of tamoxifen functions downstream of the production of phospholipid hydroperoxide and activation of CDK4 occurring 24 hours after the addition of tamoxifen. Specifically, in the culture medium of a tamoxifen-induced PHGPx-deficient MEF cell line, the lipid oxidation inhibitor, vitamin E derivative trolox (final concentration: 400 μM), and the CDK4 inhibitor 3-ATA (final concentration: 10 μM) Was added simultaneously with tamoxifen at a final concentration of 1 μM, and the 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 result of Western blotting. As a result, the enhancement of the phosphorylation of MEK and ERK2 was suppressed by the addition of Trolox and 3-ATA, which revealed that the MEK-ERK2 pathway was 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 clarified from the above results.

[Experimental example 16]
(In the phospholipid hydroperoxide-dependent cell death pathway, CDK4 functions downstream of phospholipid hydroperoxide production)
Whether or not CDK4 and MEK1 activation functioned downstream of phospholipid hydroperoxide production was examined by flow cytometry.

  FIG. 15 is a graph showing the results of flow cytometry. Cells in which CDK4-specific shRNA (SEQ ID NO: 10) was introduced into tamoxifen-induced PHGPx-deficient MEF cells (Cdk4 KD) and cells in which MEK1-specific shRNA (SEQ ID NO: 11) was introduced (Mek1 KD) were prepared. 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, and 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 addition of tamoxifen increased phospholipid hydroperoxide even in cells in which CDK4 or MEK1 had been knocked down. The results revealed that CDK4 and MEK1 activation functioned downstream of phospholipid hydroperoxide production.

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

[Experimental example 17]
(Identification of Executing Factors for Phospholipid Hydroperoxide-Dependent Cell Death Using Lentivirus shRNA Library)
In order to identify the execution factor of phospholipid hydroperoxide-dependent cell death, screening using a lentivirus comprehensive shRNA library (SBI) was performed. In this system, since the shRNA sequence is created so as to bind to Genechip (trade name, Affymetrix), Genechip can be used for identification of the shRNA sequence.

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

  FIG. 17 is a diagram showing an outline of a procedure for screening an shRNA library. According to two experiments, the shRNA sequences contained in the two lentivirus-infected cells that survived 96 hours after the addition of tamoxifen and the shRNA sequences contained in all the cells that survived 96 hours after the addition of tamoxifen were not added. Genechip was carried out using the probe obtained by amplifying, and a probe more concentrated in the tamoxifen-added cells than in the non-added cells was detected. Attempts to identify a common cell death executive factor enriched in the two experiments resulted in the acquisition of 151 genes as candidate cell death executive factors.

[Experimental example 18]
(Identification of candidate genes for the execution factor of phospholipid hydroperoxide-dependent cell death by expression of single shRNA using retrovirus infection system)
Each of the identified 151 candidate genes for the phospholipid hydroperoxide-dependent cell death executive factor was knocked down, and the effect of suppressing phospholipid hydroperoxide-dependent cell death was examined.

  A shRNA expression vector of a candidate gene was introduced into tamoxifen-induced PHGPx-deficient MEF cells one by one using a retrovirus infection system, and cells in which each candidate gene was knocked down were prepared. Tamoxifen at a final concentration of 1 μM was added to the medium of each cell, and the inhibitory effect on cell death after 96 hours was measured. Specifically, 20 photographs were taken at random of the living cells 24 hours and 96 hours after the addition of tamoxifen using an all-in-one microscope of Keyence, and the number of viable cells attached was counted. The cell viability (%) was calculated assuming that the number of cells 96 hours after the addition of tamoxifen was 100 (%) and the ratio of the number of viable cells adhered 96 hours after the addition of tamoxifen was the cell viability (%).

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

  By real-time PCR, expression suppression of the target gene was confirmed, and 17 genes were found which could more strongly suppress phospholipid hydroperoxide-dependent cell death than CDK4 knockdown cells. In addition, the suppression of expression of the target gene can be confirmed by real-time PCR, 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 include 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]
(Cdadcl1 homolog is an executive of phospholipid hydroperoxide-dependent cell death)
Among the candidate genes described above, the Cdadcl1 homolog gene (SEQ ID NO: 2) was found. This gene was a novel gene. From the results of the homology search, Cdadcl1 homolog was predicted to be a new variant of Cdcadc1. However, Cdadc1 was also a gene of unknown function. Cdadc1 was presumed to have two cytidine dCMP deaminase domains, whereas Cdadc1homolog was presumed to have only one cytidine dCMP deaminase domain. Cdadc1homolog had the same amino acid sequence as Cdadc1 except for the five C-terminal amino acids. The amino acid sequence of Cdadcl1 homolog is shown in SEQ ID NO: 1. FIG. 19A is a diagram showing the structure of the Cdadc1homolog gene and the Cdadc1 variant-2 (Cdadc1 tv2) gene.

  By expressing shRNA in a retrovirus infection system, knockdown cells of Cdadc1homolog were prepared, and it was examined whether phospholipid hydroperoxide-dependent cell death could be suppressed.

  FIG. 19B is a graph showing the result of the study. First, Cdadc1homolog-specific shRNA (SEQ ID NO: 13) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line, and cells in which Cdadc1homolog was knocked down were prepared (Cdadc1h KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, a Cdadc1homolog expression vector (Cdadc1h), a Cdadc1 variant-2 expression vector (Cdadc1tv2), and a vector alone (mock) as a control were introduced into these cells.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty photographs were taken at random of the living cells 24 hours and 96 hours after the addition of tamoxifen with a Keyence all-in-one microscope, and the number of living cells attached was counted. The cell viability (%) was calculated assuming that the number of cells 96 hours after the addition of tamoxifen was 100 (%) and the ratio of the number of viable cells adhered 96 hours after the addition of tamoxifen was the cell viability (%).

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Cdadc1homolog knockdown cells (Cdadc1h KD / mock). In addition, in the cells into which the Cdadc1homolog gene was introduced after knocking down the Cdadc1homolog (Cdadc1h KD / Cdadc1h), phospholipid hydroperoxide-dependent cell death was recovered. On the other hand, in the cells into which the Cdadc1 variant-2 gene was introduced after knocking down Cdadc1homolog (Cdadc1h KD / Cdadc1tv2), phospholipid hydroperoxide-dependent cell death was not recovered.

  From the above results, it was revealed that Cdadcl1 homolog is an execution factor of phospholipid hydroperoxide-dependent cell death. In addition, it was revealed that Cdadcl1 homolog has a variant-selective function in phospholipid hydroperoxide-dependent cell death.

[Experimental example 20]
(C87436 is an executive of phospholipid hydroperoxide-dependent cell death)
C87436 gene (SEQ ID NO: 4) was found among the candidate genes described above. The C87436 gene was a gene of unknown function whose cDNA was only reported.

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

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

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty photographs were taken at random of the living cells 24 hours and 96 hours after the addition of tamoxifen with a Keyence all-in-one microscope, and the number of living cells attached was counted. The cell viability (%) was calculated assuming that the number of cells 96 hours after the addition of tamoxifen was 100 (%) and the ratio of the number of viable cells adhered 96 hours after the addition of tamoxifen was the cell viability (%).

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

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

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

  Abhd2 knockdown cells were prepared by expressing shRNA in a retrovirus infection system, 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, and cells in which Abhd2 was knocked down were prepared (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) as a control were introduced into these cells, respectively. The Abhd2 cDNA incorporated into the Abhd2 expression vector used had a silent mutation introduced so as not to be knocked down by Abhd2-specific shRNA.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty photographs were taken at random of the living cells 24 hours and 96 hours after the addition of tamoxifen with a Keyence all-in-one microscope, and the number of living cells attached was counted. The cell viability (%) was calculated assuming that the number of cells 96 hours after the addition of tamoxifen was 100 (%) and the percentage of viable cells adhered 96 hours after the addition of tamoxifen was the cell viability (%).

  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 restored 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]
(Cdadcl1 homolog, C87436 and Abhd2 are not involved in apoptosis and necrosis)
It was examined whether or not Cdadc1homolog knockdown cells, C87436 knockdown cells and Abhd2 knockdown cells in which phospholipid hydroperoxide-dependent cell death was suppressed can suppress apoptosis or necrosis.

  FIG. 22A is a graph showing the result of examining apoptosis. Knockdown cells of Cdadc1homolog (Cdadc1h KD), knockdown cells of C87436 (C87436 KD), knockdown cells of Abhd2 (Abhd2 KD), and control cells (Control) were prepared. Staurosporine, an apoptosis-inducing reagent, was added to the medium of each cell to a final concentration of 0.01 to 1.50 μM. Subsequently, after incubation for 24 hours, the cell viability (%) of each cell was measured by the MTT assay.

  As a result, it became clear that Cdadc1homolog, C87436 and Abhd2 did not show any inhibitory effect on apoptosis caused by the addition of staurosporine.

  FIG. 22 (b) is a graph showing the result of examining necrosis. Knockdown cells of Cdadc1homolog (Cdadc1h KD), knockdown cells of C87436 (C87436 KD), knockdown cells of Abhd2 (Abhd2 KD), and control cells (Control) were prepared. Tert-butyl hydroperoxide, a necrosis-inducing reagent, was added to the medium of each cell to a final concentration of 2.3 to 300.0 mM. A final concentration of 300 mM is a condition for inducing necrosis. Subsequently, after incubating for 30 minutes, the cell viability (%) of each cell was measured by the MTT assay.

  As a result, it was revealed that Cdadc1homolog, C87436 and Abhd2 did not show an inhibitory effect on necrosis caused by the addition of 300 mM tert-butyl hydroperoxide.

  However, when tert-butyl hydroperoxide was added in the low concentration region, the effect of suppressing necrosis was confirmed in C87436 and Abhd2 gene knockdown cells, although weak. 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. Was.

[Experimental example 23]
(Cdadcl homolog and C87436 function upstream of ERK phosphorylation, Abhd2 functions downstream of ERK phosphorylation)
When tamoxifen at a final concentration of 1 μM is added to a tamoxifen-induced PHGPx-deficient MEF cell line and fluorescent staining is performed with an anti-phosphorylated ERK antibody, cells with enhanced ERK phosphorylation are detected 36 hours after the addition of tamoxifen. be able to.

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

  Tamoxifen-induced PHGPx-deficient MEF cells (Cdadcl1h KD) knocked down Cdadclhomolog, tamoxifen-induced PHGPx-deficient MEF cells (C87436 KD) knocked down C87436, and tamoxifen-induced PHGPxDhd MEF cells knocked down Abhd2 (C87436 KD) And a tamoxifen-induced PHGPx-deficient MEF cell (Control) as a control.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to the culture medium of each cell, and cells not added were prepared. After 36 hours, the cells were stained with anti-phosphorylated ERK antibody and stained with fluorescence. Subsequently, the number of ERK phosphorylated cells was counted by fluorescence microscopy.

  FIG. 23 (a) is a graph showing the results of measuring the percentage (%) of cells in which ERK phosphorylation was enhanced. The graph calculates the rate of increase in the number of cells having phosphorylated ERK upon tamoxifen treatment when the number of cells showing ERK phosphorylation in tamoxifen-free cells was 100, and calculated the percentage of cells with increased ERK phosphorylation when treated with tamoxifen. It is expressed as a ratio (%).

  As a result, enhancement of ERK phosphorylation was confirmed only in the Abhd2 gene knockdown cells. These results suggest that Cdadcl1 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 clarified from the above results.

[Experimental example 24]
(Cdadcl1 homolog is involved in the production of phospholipid hydroperoxide due to PHGPx deficiency)
Cdadclhomolog, C87436 or Abhd2 of the tamoxifen-induced PHGPx-deficient MEF cell line was knocked down, and the production of phospholipid hydroperoxide due to PHGPx deficiency when tamoxifen at a final concentration of 1 μM was added to the medium was examined.

  FIG. 24A is a graph showing the result of detecting the production of phospholipid hydroperoxide by flow cytometry. Specifically, first, a tamoxifen-induced PHGPx-deficient MEF cell (Cdadc1h KD) in which Cdadc1homolog was knocked down, a tamoxifen-induced PHGPx-deficient MEF cell (C87436 KD) in which C87436 was knocked down, and a tamoxifen-induced PH in which Abhd2 was knocked down. Defective MEF cells (Abhd2 KD) and tamoxifen-induced PHGPx-deficient MEF cells (Control) were prepared as controls.

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

  As a result, it was revealed that the generation of phospholipid hydroperoxide due to PHGPx deficiency was strongly suppressed in the knockdown cells of Cdadcl1 homolog.

  From this, it was considered that Cdadcl1 homolog was involved in the production of phospholipid hydroperoxide due to PHGPx deficiency. FIG. 24 (b) is a model diagram showing a part of the phospholipid hydroperoxide-dependent cell death pathway clarified from the above results.

[Experimental example 25]
(Production of phospholipid hydroperoxide due to PHGPx deficiency is not mediated by iron)
Heretofore, it has been considered that the oxidation reaction of phospholipids is mediated by a hydroxyl radical generated by the Fenton reaction via iron. Therefore, it was examined whether or not the production of phospholipid hydroperoxide due to PHGPx deficiency can be suppressed using deferoxamine, which is an iron chelator.

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

  FIGS. 25A and 25B are graphs showing the results of flow cytometry. The production of phospholipid hydroperoxide due to PHGPx deficiency could not be suppressed by deferoxamine, which is an iron chelator, but could be suppressed by vitamin E. The results indicated that the production of phospholipid hydroperoxide due to PHGPx deficiency was not mediated by iron.

[Experimental example 26]
(Effect of suppression of Fenton reaction on phospholipid hydroperoxide-dependent cell death)
Manganase (III) tetrakis (4-carboxyphenyl) porphyrin (Mn-TBAP), N-acetylcysteine (NAC), and deferoxamine (DFO), which are thought to suppress the Fenton reaction, are added to the tamoxifen-induced PHGPx-deficient MEF cell line medium. It was examined whether this could suppress phospholipid hydroperoxide-dependent cell death.

  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. 26A is a model diagram showing the action points of Mn-TBAP, NAC, deferoxamine, and vitamin E in the production of phospholipid hydroperoxide.

  A medium obtained by adding Mn-TBAP (final concentration: 100 μM), NAC (final concentration: 5 mM), deferoxamine (final concentration: 100 μM), or vitamin E (final concentration: 200 μM) to the medium of a tamoxifen-induced PHGPx-deficient MEF cell line was prepared. As a control, tamoxifen-induced PHGPx-deficient MEF cells to which nothing was added to the medium were prepared.

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

  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) could not suppress phospholipid hydroperoxide-dependent cell death. From these results, it was considered that the generation of phospholipid hydroperoxide due to PHGPx deficiency may be performed in a system involving Cdadcl1 homolog without involving the Fenton reaction.

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

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

  When PHGPx is deficient, phospholipid hydroperoxide rises after 24 hours. Downstream, CDK4 and C87436 function. After 36 hours, MEK and ERK of the MAP kinase pathway are phosphorylated. Abhd2 functions downstream of ERK phosphorylation, causing cell death between 48 and 72 hours. Examples of the case where PHGPx is deficient include a case where the PHGPx gene is damaged, a case where intracellular glutatin is depleted, a case where vitamin E is deficient, an inactivation of an enzyme by a drug targeting PHGPx, and the like.

[Experimental example 27]
(Myocardium-specific PHGPx-deficient mice are lethal by cardiomyocyte death in 17.5 days of development)
It is known that PHGPx-deficient mice are lethal in 7.5 days of development. Here, mice in which PHGPx was specifically deficient in the heart were produced, and the effects thereof were analyzed. First, mating between a PHGPx ^+/− mouse and a mouse (Cre ^{+ / +} ) having a Cre gene downstream of a muscle creatine kinase promoter (Muscle creatine kinase), which is a heart-specific promoter, was repeated, and Cre ^{+ / +} PHGPx ^{+ / −} Mice were obtained.

Subsequently, by crossing Cre ^{+ / +} PHGPx ^+/− mice and the above-mentioned Tg (loxP-PHGPx) ^{+ / +} : PHGPx ^{− / −} mice, Cre ^+/− Tg (loxP-PHGPx) ^{+ / -} : PHGPx ^{− / −} mice were obtained. This mouse is deficient in heart-specific PHGPx.

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

  FIG. 28 (b) is a photograph showing the results of DAPI staining and TUNEL staining (TUNEL) of a tissue section of the heart of a mouse at 17.5 days of development. In the heart tissue of the heart-specific PHGPx-deficient mouse (KO), a large number of TUNEL-staining positive cells (cell death) were observed. When the mother was fed a diet supplemented with vitamin E daily (50 mg vitamin E / 100 g diet), cell death of cardiac tissue of cardiac-specific PHGPx-deficient mice (KO) on day 17.5 of development was completely suppressed and normal. A heart-specific PHGPx-deficient mouse was born. Caspase-3 activation and DNA ladder were not observed in cardiomyocyte death that occurred in fetal heart-specific PHGPx-deficient mouse embryos on day 17.5 when mother mice were bred on a normal diet. Oxidant accumulation was observed (not shown). This suggests that cardiomyocyte death also induces new cell death through lipid oxidation.

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

[Experimental example 28]
(Administration of the CDK4 inhibitor 3-ATA to the abdominal cavity of mother mice suppresses lethality of heart-specific PHGPx-deficient 18.5 day-old fetuses)
It was examined whether or not administration of the CDK4 inhibitor 3-ATA to the abdominal cavity of mother mice could suppress the lethality of heart-specific PHGPx-deficient mice.

  Developmental Process of Heart-Specific PHGPx-Deficient Mouse Embryo On days 14.5, 15.5, 16.5 and 17.5 days, 1.5 mg / kg body weight of 3-ATA was administered intraperitoneally to mother mice, Embryos at 18.5 days of development were observed.

  As a result, edema was suppressed in 8 out of 10 heart-specific PHGPx-deficient mice. Edema was observed in the remaining two animals. Two of the eight animals whose edema was suppressed were alive. The remaining six died.

  29 (a)-(c) are photographs of mouse embryos 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 heart-specific PHGPx-deficient mouse to which 3-ATA was not administered (heart PHGPx KO 3-ATA (−)). FIG. 29 (c) is a photograph of a heart-specific PHGPx-deficient mouse to which 3-ATA was administered (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 around 10 days after switching to a normal diet)
In the fetal period of heart-specific PHGPx-deficient mice, when mothers were fed a diet supplemented with vitamin E, lethality was completely suppressed. In addition, it was revealed that when the heart-specific PHGPx-deficient mice born were continuously fed daily with a vitamin E-added diet (50 mg vitamin E / 100 g diet), they grew normally.

  Here, the diet supplemented with vitamin E contained vitamin E in an amount of 130 mg / kg body weight per day. The normal diet also contained 13 mg / kg body weight of vitamin E per daily dose.

  FIG. 30 (a) is a graph showing the survival rate after changing the diet of heart-specific PHGPx-deficient mice that grew normally by feeding a diet supplemented with vitamin E to a normal diet. It was revealed that changing the diet of normally grown heart-specific PHGPx-deficient mice to a normal diet causes sudden death around 10 days before.

  FIG. 30 (b) shows heart-specific PHGPx-deficient mice fed a diet supplemented with vitamin E (a diet supplemented with vitamin E), and heart-specific PHGPx-deficient mice that died by changing the diet to a normal diet (KO (dead diet)). 7) and a graph showing the results of measuring the amount of vitamin E in the heart of a wild-type mouse fed with a normal diet (wild on a normal diet). Hearts of heart-specific PHGPx-deficient mice fed a vitamin E supplemented diet 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, a decrease in the amount of vitamin E in the heart induces cardiomyocyte death due to lipid oxidation (not shown).

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

[Experimental example 30]
(The CDK4 inhibitor 3-ATA can prolong the sudden death of heart-specific PHGPx-deficient mice caused by heart failure caused by changing from a diet supplemented with vitamin E to a normal diet)
The effects of administering the CDK4 inhibitor 3-ATA after changing the diet of heart-specific PHGPx-deficient mice fed a vitamin E-supplemented diet to a normal diet were examined.

  FIG. 31 shows mice (Vit.E-) in which the diet of heart-specific PHGPx-deficient mice was changed to normal diet, the diet of heart-specific PHGPx-deficient mice was changed to normal diet, and the diet was further changed to normal diet. Mice (Vit. E-, 3-ATA +) to which 2 mg / kg body weight of 3-ATA was intraperitoneally administered once a day from the day, and a heart-specific PHGPx-deficient mouse were fed a vitamin E-supplemented diet, followed by further experiments. It is a graph which shows the survival rate of the mouse (Vit.E +, 3-ATA +) which administered intraperitoneally 2 mg / kg body weight of 3-ATA once a day from the 4th day after starting.

  The median survival time from the start of the experiment of mice (Vit.E-) (n = 23) in which the diet of heart-specific PHGPx-deficient mice was changed to the normal diet was 10 days. Further, the diet of heart-specific PHGPx-deficient mice was changed to a normal diet, and a mouse (Vit) in which 2 mg / kg body weight of 3-ATA was intraperitoneally administered once a day from the fourth day after the diet was changed to the normal diet (Vit The median survival from the start of the experiment for .E-, 3-ATA +) (n = 6) was 18 days. These results showed a significant difference at a risk of less than 1%.

  The above results indicate that the CDK4 inhibitor 3-ATA can prolong the sudden death of heart-specific PHGPx-deficient mice caused by heart failure caused by changing from a diet supplemented with vitamin E to a normal diet.

[Experimental example 31]
(MEK inhibitor U-0126 can prolong the sudden death of heart-specific PHGPx-deficient mice caused by heart failure caused by changing from a diet supplemented with vitamin E to a normal diet)
After changing the diet of heart-specific PHGPx-deficient mice fed a vitamin E-added diet to a normal diet, the effect of administering the MEK inhibitor U-0126 was examined.

  FIG. 32 shows that the diet of heart-specific PHGPx-deficient mice was changed to normal diet (Vit. E-), the diet of heart-specific PHGPx-deficient mice was changed to normal diet, and the diet was further changed to normal diet. Mice (Vit. E-, U-0126 +) to which 3 mg / kg body weight of U-0126 was intraperitoneally administered once a day from the day and heart-specific PHGPx-deficient mice were fed a vitamin E-supplemented diet, and further tested. It is a graph which shows the survival rate of the mouse (Vit.E +, U-0126 +) which administered intraperitoneally 3 mg / kg body weight of U-0126 once a day from the 4th day after starting.

  The median survival time from the start of the experiment of mice (Vit.E-) (n = 23) in which the diet of heart-specific PHGPx-deficient mice was changed to the normal diet was 10 days. In addition, the diet of heart-specific PHGPx-deficient mice was changed to a normal diet, and a mouse in which 3 mg / kg body weight of U-0126 was intraperitoneally administered once a day from the fourth day after the diet was changed to a normal diet (Vit The median survival from the start of the experiment for .E-, U-0126 +) (n = 7) was 20 days. These results showed a significant difference at a risk of less than 1%.

  The above results showed that the MEK inhibitor U-0126 was able to prolong the sudden death of heart-specific PHGPx-deficient mice caused by heart failure caused by changing from a diet supplemented with vitamin E 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. Further, an agent for inhibiting phospholipid hydroperoxide-dependent cell death can be provided. In addition, a preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death can be provided.

Claims (4)

A method for detecting phospholipid hydroperoxide-dependent cell death,
Detecting the production of phospholipid hydroperoxide in the cell sample, measuring the expression level of MEK at the mRNA level, measuring the expression level of MEK at the protein level, measuring the phosphorylation rate of MEK And at least one step selected from the group consisting of measuring the suppression of cell death by suppressing the expression of MEK,
When the expression level of MEK at the mRNA level was measured, it was found that the expression level was increased as compared to normal cells in which phospholipid hydroperoxide-dependent cell death was not in progress. Indicates that death is in progress,
When the expression level of MEK at the protein level was measured, it was found that the expression level was increased as compared to normal cells in which phospholipid hydroperoxide-dependent cell death was not in progress. Indicates that death is in progress,
When the phosphorylation rate of MEK was measured, it was found that the phosphorylation rate was increased as compared to normal cells in which phospholipid hydroperoxide-dependent cell death was not in progress, indicating that phospholipid hydroperoxide-dependent cell death was observed. Indicates that it is in progress,
When the suppression of cell death due to the suppression of MEK expression was measured, cell death was suppressed by suppressing the expression of MEK, indicating that phospholipid hydroperoxide-dependent cell death is in progress. Detection method. A reagent for detecting the production of phospholipid hydroperoxide, an antibody against phosphorylated MEK and an antibody against unphosphorylated MEK; a primer for amplifying mRNA of MEK; or an siRNA, shRNA, ribozyme or antisense nucleic acid against MEK;
A kit for detecting phospholipid hydroperoxide-dependent cell death, comprising:   A MEK inhibitor or an inhibitor of phospholipid hydroperoxide-dependent cell death, comprising an siRNA, shRNA, ribozyme or antisense nucleic acid as an active ingredient against MEK. Heart failure caused by phospholipid hydroperoxide-dependent cell death or arrhythmic sudden death caused by phospholipid hydroperoxide-dependent cell death, comprising as an active ingredient siRNA, shRNA, ribozyme or antisense nucleic acid against MEK inhibitor or MEK Prophylactic or therapeutic agent.