JP2008000003A - Pim-1 ACTIVITY/PROTEIN INHIBITING PHARMACEUTICAL - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a cancer prophylactic/therapeutic agent inhibiting Pim-1 protein function and tumor angiogenesis and exhibiting anticancer activity, a cancer prophylactic/therapeutic agent through Pim-1 production inhibition by using an antisense DNA or a short interfering RNA and a cancer prophylactic/therapeutic agent through an increase of Pim-1 protein decomposition by activation of proteolytic decomposition system by ubiquitin-proteasome and to provide a method for screening a cancer prophylactic/therapeutic agent for preventing/treating cancers by Pim-1 production inhibition. <P>SOLUTION: Protein (serine/threonine kinase Pim-1) developed and increased in substantially all solid cancer cells in low oxygen has been found. Further, it has been found that the protein is decomposed by a ubiquitin-proteasome system, and that any tumor angiogenesis is inhibited by dominant negative Pim-1. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a therapeutic / prophylactic agent for cancer, and more particularly to a therapeutic / prophylactic agent for cancer that is effective against cancer cells and solid cancers in which the anticancer drug has stopped working.

  Currently, cancer deaths are the second leading cause of heart disease. Cancer is usually treated by surgery, radiation therapy, chemotherapy, immunotherapy, or hyperthermia. These cancer therapies are believed to be achieved through the removal of all cancer cells or death. If surgery is determined to be impossible, radiotherapy or chemotherapy is selected. Many types of anticancer agents currently used for chemotherapy are known. However, many of the anticancer drugs used clinically have many problems for solid cancer. It has been pointed out that anticancer drug-resistant cancer cells appear that once effective anticancer drugs are no longer effective, or that they are not effective for solid cancer from the beginning. It has been reported that anti-cancer drugs are less effective for cancers with lower oxygen partial pressure than before, but the state of hypoxia inside the cancer is thought to be the cause of solid cancers less effective for anti-cancer drugs.

  In advanced cancer, since the growth rate inside cancer cells is faster than that of surrounding normal cells, supply of newly generated blood vessels is insufficient, blood supply is insufficient, and hypoxia is assumed. For example, in Non-Patent Documents 1 to 6, cancer cells in a hypoxic state are more resistant to chemotherapy and radiotherapy than cancer cells in a high oxygen state, Inducing drug resistance in solid cancer cells is disclosed.

  On the other hand, Pim-1 (amino acid sequence / SEQ ID NO: 1, nucleotide sequence / SEQ ID NO: 2), which is a serine / threonine kinase, is inserted into a leukemia virus in a T cell lymphoma initially caused by murine leukemia virus (MuLV). Is a serine / threonine kinase identified as a gene that is often activated by (Non-patent Document 7). It has also been reported that Pim-1 in the cytoplasm functions as a factor for inhibiting apoptosis in various hematopoietic cells (Non-patent Document 8). However, the function of Pim-1 in solid cancer is not known.

Prior art document information related to the invention of the present application is shown below.
Teicher, BA Hypoxia and drug resistance.Cancer Metastasis Rev., 13: 139-168, 1994 Brown, JM & Giaccia, AJ The unique physiology of solid tumors: opportunities (and problems) for cancer therapy.Cancer Res., 58: 1408-1416, 1998 Brown, JM Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies.Mol. Med. Today, 6: 157-162, 2000 Luk, CK, Veinot-Drebot, L., Tjan, E. & Tannock, IF Effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines.J Natl. Cancer Inst., 82: 684-692, 1990 Sakata, K., Kwok, TT, Murphy, BJ, Laderoute, KR, Gordon, GR, Sutherland, RM Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance. Br. J Cancer, 64: 809-814 , 1991 Sanna, K. & Rofstad, EK Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro.Int.J Cancer, 58: 258-262, 1994 Cuypers, HT, Selten, G., Quint, W., Zijlstra, M., Maandag, ER, Boelens, W., van Wezenbeek, P., Melief, C., Berns, A. Murine leukemia virus-induced T- cell lymphomagenesis: integration of proviruses in a distinct chromosomal region.Cell, 37: 141-150, 1984 Selten, G., Cuypers, HT & Berns, A. Proviral activation of the putative oncogene Pim-1 in MuLV induced T-cell lymphomas.EMBO J, 4: 1793-1798, 1985

  An object of the present invention is to provide a prophylactic / therapeutic agent for cancer that inhibits the function of Pim-1 protein, inhibits tumor angiogenesis, and exhibits anticancer activity. We also provide cancer preventive and therapeutic agents through suppression of Pim-1 production using antisense DNA and short interfering RNA. The present invention also provides a preventive / therapeutic agent for cancer through the enhancement of Pim-1 proteolysis by activating the proteolytic system by ubiquitin / proteasome. Another object of the present invention is to provide a screening method for a cancer preventive / therapeutic agent for preventing / treating cancer by inhibiting Pim-1 production.

  As a result of intensive studies to achieve the above object, the present inventor has found a protein (serine / threonine kinase Pim-1) whose expression is increased in almost all solid cancer cells under hypoxia, It was found that it was degraded in the ubiquitin-proteasome system, and further, it was found that tumor angiogenesis was suppressed by dominant negative Pim-1, and the present invention was completed based on this finding.

  The present invention has been made on the basis of the above findings, and is a partial peptide of serine / threonine kinase Pim-1 or a salt thereof (hereinafter referred to as “dominant negative Pim-1” as a preventive / therapeutic agent for cancer). (Also called). Also provided is naked DNA, which is an expression vector in which a partial peptide of serine / threonine kinase Pim-1 or a salt thereof is incorporated into a plasmid vector. We also provide antisense DNA and short interfering RNA that can be used to prevent or treat cancer through suppression of Pim-1 production. It also contains Pim-1 protein expression decrease, Pim-1 neutralizing antibody and cancer chemotherapeutic agent that solves the enhancement of Pim-1 proteolysis by activation of proteolytic systems such as heat shock protein, ubiquitin and proteasome A solid cancer preventive and therapeutic agent is provided. Also provided is a screening method for Pim-1 production suppression preventive and therapeutic agents.

The present invention also provides a screening kit for a preventive / therapeutic agent for inhibiting Pim-1 production.
The present invention also provides a prophylactic / therapeutic agent for cancer comprising a polypeptide having the same or substantially the same amino acid sequence as the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3.
The present invention also provides a method for screening an apoptosis-inducing agent, characterized by using the promoter activity of serine / threonine kinase Pim-1.
The present invention also provides a screening kit for an apoptosis-inducing agent comprising the promoter activity of serine / threonine kinase Pim-1.
The present invention also provides an apoptosis inducer obtained using the screening method or screening kit.
The present invention also relates to a preventive / therapeutic agent for cancer comprising a polypeptide having the same or substantially the same amino acid sequence as the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 having an apoptosis-inducing effect. provide.
The present invention also provides an apoptosis inducer comprising an antibody against serine / threonine kinase Pim-1 or a partial peptide thereof or a salt thereof.
The present invention also provides a screening method for an anticancer agent potentiator characterized by using serine / threonine kinase Pim-1, a partial peptide thereof, or a salt thereof.
The present invention also provides a screening kit for an anticancer agent potentiator comprising serine / threonine kinase Pim-1, a partial peptide thereof, or a salt thereof.
The present invention also provides an anticancer agent enhancer obtained using the screening method or screening kit.
The present invention also relates to a preventive / therapeutic agent for cancer comprising a polypeptide having the same or substantially the same amino acid sequence as the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 that acts as an anticancer agent enhancer. provide.
The present invention also provides a polynucleotide comprising a nucleotide sequence having at least 95% homology with the following polynucleotides (a) and (b).
(A) a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 4 or a polynucleotide capable of hybridizing to a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 2;
(B) a polynucleotide comprising a base sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3, or the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 3 A polynucleotide which is a cDNA capable of hybridizing to a polynucleotide comprising a base sequence encoding a polypeptide having the same amino acid sequence.
The present invention also provides a recombinant vector containing the polynucleotide.
The present invention also provides a host cell that retains the expression vector.
In addition, the present invention is the same as the amino acid sequence represented by SEQ ID NO: 3, comprising the steps of culturing the above host cell under conditions suitable for polypeptide expression and recovering the polypeptide from the obtained culture. A method for producing a polypeptide having substantially the same amino acid sequence or a salt thereof is provided.
The present invention also provides a preventive / therapeutic agent for cancer comprising the polynucleotide or the recombinant vector.
The present invention also provides an apoptosis inducer comprising the polynucleotide or the recombinant vector.
The present invention also provides an anticancer agent enhancer comprising the polynucleotide or the recombinant vector.
Further, the present invention relates to antisense DNA that leads to suppression of Pim-1 protein production represented by SEQ ID NO: 11-13, which acts as a preventive / therapeutic agent for cancer through suppression of PIm-1 protein production, and SEQ ID NO: 5 A short interfering RNA represented by -10 is provided.
The present invention also provides the above antisense DNA and short interfering RNA that act as apoptosis inducers.
The present invention also provides the above antisense DNA and short interfering RNA that act as an anticancer agent enhancer.
In addition, since proteasome inhibitors induce PIm-1 protein expression in the present invention, proteolytic systems such as proteasomes are expected to be important for protein expression. However, heat shock proteins, ubiquitin / proteasome systems, etc. A prophylactic / therapeutic agent for cancer comprising the activation of the proteolytic system and a Pim-1 neutralizing antibody and a chemotherapeutic agent.
The present invention also provides a prophylactic / therapeutic method for cancer using activation of proteolytic systems such as heat shock protein and ubiquitin / proteasome system, and a combination of Pim-1 neutralizing antibody and a chemotherapeutic agent.
The present invention also provides an apoptosis inducer comprising activation of a proteolytic system such as heat shock protein and ubiquitin / proteasome system, and a Pim-1 neutralizing antibody and a chemotherapeutic agent.
The present invention also provides an anticancer agent enhancer comprising a proteolytic system such as heat shock protein or ubiquitin / proteasome system, a Pim-1 neutralizing antibody and a chemotherapeutic agent.

By inhibiting the function of the Pim-1 protein or the Pim-1 gene, it is possible to exert cancer treatment, apoptosis induction, and an anticancer agent enhancing effect, and therefore, the functions of dominant negative Pim-1 and Pim-1 And compounds that inhibit protein expression are effective in the treatment of cancer.

The present invention will be described below.
In the present invention, “serine / threonine kinase Pim-1” (hereinafter also referred to as “Pim-1” in the present specification) is a polypeptide having serine / threonine kinase activity. Pim-1 has also been identified as a gene that is activated by insertion of MuLV in T cell lymphoma caused by murine leukemia virus (MuLV) (Cuypers, HT, Selten, G., Quint, W., Zijlstra, M., Maandag, ER, Boelens, W., van Wezenbeek, P., Melief, C., Berns, A. Murine leukemia virus-induced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region. 37: 141-150, 1984).
A compound that inhibits the activity of Pim-1 is a candidate for a drug that induces apoptosis, and can be applied to treatment / prevention of cancer and an enhancer of an anticancer agent.

  The cancer preventive / therapeutic agent, apoptosis-inducing agent, and anticancer agent-enhancing agent of the present invention comprises a compound that inhibits the activity of a partial peptide of Pim-1 or a salt thereof, or a salt thereof (SEQ ID NO: 3). Polypeptide having the same or substantially the same amino acid sequence as the polypeptide having the amino acid sequence). The polypeptide having the amino acid sequence represented by SEQ ID NO: 3 is a polypeptide lacking the kinase active domain of Pim-1 having the amino acid sequence represented by SEQ ID NO: 1, A polypeptide in which the amino acid residue is deleted. The polypeptide having the amino acid sequence represented by SEQ ID NO: 3 is a polypeptide lacking the kinase active domain, and in the presence of this polypeptide, the activity of Pim-1 is inhibited. As a result, the polypeptide It can be used as a cancer treatment / prevention agent, apoptosis inducer, anticancer agent enhancer.

  In addition, as a compound that inhibits the activity of Pim-1, double-stranded RNA against a gene encoding Pim-1, that is, a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 (hereinafter referred to as short interfering in the present specification). (Also referred to as RNA) (SEQ ID NO: 5-10) and antisense nucleotides (SEQ ID NO: 11-13). The sequence lists representative examples, including short interfering RNA and antisense that inhibit Pim-1 expression even if the sequence is different.

  As the polypeptide having the amino acid sequence represented by SEQ ID NO: 3, the polypeptide substantially identical to the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 may be a synthetic polypeptide.

  The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 is about 50% or more, preferably about 60% or more, more preferably about 70% or more, with the amino acid sequence represented by SEQ ID NO: 3. More preferred is an amino acid sequence having a homology of about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.

  Examples of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 include one or more (for example, about 1 to 50, preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 3. ), A protein having an amino acid sequence from which amino acids are deleted, 1 or 2 (for example, about 1 to 100, preferably about 1 to 30) amino acids are added to the amino acid sequence represented by SEQ ID NO: 1. A protein having an amino acid sequence, a protein having an amino acid sequence in which one or more (for example, about 1 to 100, preferably about 1 to 30) amino acids are inserted into the amino acid sequence represented by SEQ ID NO: 1, 1 or 2 (for example, about 1 to 100, preferably about 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1 Proteins and the like containing the amino acid sequence of a combination peptide, or those having the amino acid sequence are substituted with amino acids are also included. When the amino acid is inserted, substituted or deleted, the position of the insertion, substitution or deletion is not particularly limited.

In the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, the C-terminus is any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Also good. Examples of R in the ester include alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and n-butyl, and cyclohexane having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl. An alkyl group, an aryl group having 6 to 12 carbon atoms such as phenyl and α-naphthyl, a phenyl-alkyl group such as benzyl and phenethyl, an α-naphthyl-alkyl group such as α-naphthylmethyl, and a carbon number of 7 to 14 Individual aralkyl groups, pivaloyloxymethyl groups, and the like. When the protein represented by SEQ ID NO: 1 has a carboxyl group (or carboxylate) other than the C-terminus, the carboxyl group may be amidated or esterified. Examples of the ester in this case include the above-described C-terminal ester. Furthermore, in the protein represented by SEQ ID NO: 3, the amino group of the N-terminal amino acid residue (eg, methionine residue) is a protective group (alkanoyl having 1 to 6 carbon atoms such as formyl group and acetyl group) Are protected with an acyl group having 1 to 6 carbon atoms, etc., N-terminal glutamine residues generated by cleavage in vivo are pyroglutamine oxidized, on the side chain of amino acids in the molecule Of the substituents (-OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, alkanoyl groups having 1 to 6 carbon atoms such as formyl group, acetyl group, etc.) It may be a protein protected with an acyl group having 1 to 6 carbon atoms, or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.

  The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 may be in the form of a salt. Examples of such a salt include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases. (Eg, alkali metal salts) and the like are used, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid). The polypeptide used in the present invention or a salt thereof can also be produced from a human or warm-blooded animal cell or tissue as described above by a known protein purification method. It can also be produced by culturing a transformant containing a DNA encoding a protein (for example, a DNA comprising the base sequence represented by SEQ ID NO: 4). Moreover, it can also manufacture according to the well-known peptide synthesis method. When producing from human or mammalian tissues or cells, homogenize human or mammalian tissues or cells, and then perform extraction using acid, etc., and use the resulting extract for reverse phase chromatography or ion exchange chromatography. Etc. can be purified and isolated by combining the chromatography.

  The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 can also be synthesized by a known peptide synthesis method. For peptide synthesis, commercially available resins for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid in which the α-amino group and the side chain functional group are appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein or partial peptide is excised from the resin, and at the same time, various protecting groups are removed to obtain the target protein or partial peptide or a salt thereof. For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide, and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reactions. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reactions, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.

Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like. The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like. The hydroxyl group of serine can be protected by esterification or etherification. Examples of the group suitable for esterification are derived from a lower (1 to 6 carbon atoms) alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, a carbonic acid group such as a benzyloxycarbonyl group and an ethoxycarbonyl group. Substituents and the like can be used. Moreover, as a substituent suitable for etherification, a benzyl group, a tetrahydropyranyl group, t-butyl group etc. are mentioned, for example. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like. Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

  Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol (pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl Alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOBt)] and the like can be used. Examples of the activated amino group of the raw material include a corresponding phosphoric acid amide. Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, or the like can be used. Such elimination reaction by acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1 It is effective to add a cation scavenger such as 1,4-butanedithiol, 1,2-ethanedithiol and the like. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the 1,2-ethanedithiol or 1,4-butane described above. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the elimination of the protecting group, and the activation of the functional group involved in the reaction can be appropriately selected from known substituents or known means. As another method for obtaining an amide form of a protein or partial peptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is formed on the amino group side to the desired chain length. After the extension, a protein or partial peptide from which only the N-terminal α-amino group protecting group of the peptide chain is removed and a protein or partial peptide from which only the C-terminal carboxyl group protecting group has been removed are produced, and these The protein or peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein or peptide. This crude protein or peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide form of the desired protein or peptide. In order to obtain a protein or peptide ester, for example, the α-carboxyl group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein or peptide amide is obtained in the same manner as the protein or peptide amide. An ester of a peptide can be obtained.

  The partial peptide of Pim-1 or a salt thereof used in the present invention can be produced according to a known peptide synthesis method. Alternatively, it can be produced by cleaving Pim-1 used in the present invention with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, a partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protective group, the target peptide can be produced by removing the protective group. it can.

  It can also be produced by cleaving the above-described Pim-1 with an appropriate peptidase.

  The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is a DNA encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 (for example, the DNA consisting of the base sequence represented by SEQ ID NO: 4). It can also be produced by culturing a transformant transformed with an expression vector containing. Examples of the DNA include genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA. Bacteriophages, plasmids, cosmids, phagemids and the like are used as vectors used for the library. Moreover, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.

  As a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, DNA containing the base sequence represented by SEQ ID NO: 4 or DNA having the base sequence represented by SEQ ID NO: 4 Any DNA may be used as long as it contains a base sequence that hybridizes under highly stringent conditions.

  Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 4 under highly stringent conditions include, for example, about 50% or more, preferably about 60% or more, with the base sequence represented by SEQ ID NO: 4. More preferred is DNA containing a base sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. Hybridization can be performed according to a known method or a method analogous thereto. For example, it can be performed according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions. The highly stringent conditions are, for example, conditions in which the sodium concentration is 19 to 40 mM, preferably 19 to 20 mM, and the temperature is 50 to 70 ° C., preferably 60 to 65 ° C. In particular, the case where the sodium concentration is 19 mM and the temperature is 65 ° C. is most preferable.

As a means for cloning DNA that completely encodes the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, synthetic DNA having a part of the base sequence encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 A DNA fragment or synthetic DNA encoding a part or the entire region of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 may be amplified by a PCR method using a primer, or DNA incorporated into an appropriate vector. It can be selected by hybridization with the one labeled with the one used. The hybridization method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. The DNA base sequence is converted by PCR, a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LAPCR method, Gapped duplex The method can be carried out according to a known method such as the Kunkel method or a method analogous thereto. The DNA encoding the cloned protein can be used as it is or after digestion with a restriction enzyme or addition of a linker if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA, or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

  A recombinant vector containing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, for example, a polynucleotide consisting of the base sequence represented by SEQ ID NO: 4, is used as a promoter of a promoter in an appropriate expression vector. It can manufacture by connecting downstream. Examples of the vector include plasmids derived from E. coli (for example, pBR322, pBR325, pUC18 or pUC118), plasmids derived from Bacillus subtilis (for example, pUB110, pTP5 or pC194), yeast-derived plasmids (for example, pSH19 or pSH15), and bacteriophages such as λ phage. In addition to animal viruses such as phages, retroviruses, vaccinia viruses or baculoviruses, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when the host is Escherichia coli, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, T3 promoter, araBAD promoter, etc., and when the host is Bacillus, SPO1 promoter, penP Promoter, XYL promoter, HWP promoter, CWP promoter and the like are preferable. When the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP Promoters, ADH promoters and the like are preferable. When animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like are preferably used. When insect cells are used as hosts, polyhedrin promoter, OplE2 promoter and the like are used.

If desired, an enhancer, splicing signal, poly A addition signal, selection marker, SV40 replication origin (hereinafter sometimes abbreviated as SV40orgdi) and the like known in the art can be added to the recombinant vector. If necessary, the protein encoded by the DNA of the present invention can be expressed as a fusion protein with other proteins (for example, glutathione S transferase and protein A). Such fusion proteins can be cleaved using site-specific proteases and separated into their respective proteins. Examples of the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance. gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance) and the like. In particular, when a dhfr gene-deficient Chinese hamster cell is used and the dhfr gene is used as a selection marker, the target gene can also be selected by a medium not containing thymidine.
Examples of host cells that can be used include Escherichia, Bacillus, yeast, insect cells, insects, animal cells, and the like. Specific examples of the genus Escherichia include Escherichia coli K12 / DH1 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)), JM103 (Nucleic Acids Research, 9, 309 ( 1981)), JA221 (Journal of Molecular Biology, 120, 517 (1978)), HB101 (Journal of Molecular Biology, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954), DH5α and Examples of the Bacillus genus include Bacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 [Journal of Biochemistry, 95, 87 (1984)]. And Bacillus brevis, etc. Examples of yeast include Saccharomyces cerevisiae AH2. 2, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccaromyces pombe NCYC1913, NCYC2036, Pichia pastoris M71, and Hansenula polymorpha. Examples of the cells include, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, and High Five ^™ cells derived from eggs of Trichoplusia ni. , Cells derived from Mamestra brassicae or cells derived from Estigmena acrea, etc. When the virus is BmNPV, cocoon-derived cell lines (Bombyx mori N cells; BmN cells), etc. are used. Sf9 cells (ATCC CRL1711), Sf21 cells (above, Vaughn, JL et al., In. In Vivo, 13, 213-217, (1977)), etc. As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985). )]. Examples of mammalian cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mice, and the like. L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used. If necessary, a polynucleotide encoding a signal sequence suitable for the host cell may be added to the 5 ′ end of the polynucleotide encoding dominant negative Pim-1. When using Escherichia as a host cell, PhoA signal sequence, OmpA, signal sequence, etc. are used. When using Bacillus as a host cell, α-amylase signal sequence, subtilisin signal sequence, etc. are used. When yeast is used as the host cell, MFα signal sequence, SUC2 signal sequence, etc. are used. When animal cells are used as the host cell, insulin signal sequence, α-interferon signal sequence, antibody molecule A signal sequence such as a signal sequence is used. Using the expression vector containing the polynucleotide encoding the polypeptide of the present invention thus constructed, a transformant can be produced.

  The above-described host cell transformation can be performed according to a method known in the art. For example, methods for transforming host cells are described in the following documents. Proc. Natl. Acad. Sci. USA, 69, 2110 (1972); Gene, 17, 107 (1982); Molecular & General Genetics, 168, 111 (1979); Methods in Enzymology, 194, 182- 187 (1991); Proc. Natl. Acad. Sci. USA), 75, 1929 (1978); 263-267 (1995) (published by Shujunsha); and Virology, 52, 456 (1973).

  The method for introducing a recombinant vector into a bacterium such as E. coli is not particularly limited as long as it can introduce DNA into the bacterium. For example, a method using calcium ions (Cohen, SN et al .: Proc . Natl. Acad. Sci., USA, 69: 2110 (1972), electroporation method and the like.

  When yeast is used as the host, the method for introducing the recombinant vector into the yeast is not particularly limited as long as it can introduce DNA into the yeast. For example, the electroporation method, the spheroplast method, lithium acetate Law.

  When an animal cell is used as a host, the method for introducing the recombinant vector into the animal cell is not particularly limited as long as it can introduce DNA into the animal cell. For example, electroporation method, calcium phosphate method, lipofection Law.

  When an insect cell is used as a host, the method for introducing the recombinant vector into the insect cell is not particularly limited as long as it can introduce DNA into the insect cell. For example, the calcium phosphate method, lipofection method, electroporation Law.

  As a method for confirming whether or not a gene has been incorporated into a host, for example, a PCR method, a Southern hybridization method, a Northern hybridization method or the like can be used. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. The amplified product is then subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide, SYBR Green solution, etc., and then the amplified product is detected as a single band and transformed. Can be confirmed. PCR may be performed using a primer previously labeled with a fluorescent dye or the like to detect the amplification product. Furthermore, after the amplification product is bound to a solid phase such as a microplate, a method of confirming the amplification product using fluorescence or an enzyme reaction can also be used.

  A polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3, or a salt thereof, is obtained by culturing the above host cell under conditions suitable for expression of the polypeptide, and a culture obtained Recovering the polypeptide from.

  Specifically, after homogenizing the above-described host cells, extraction with an acid or the like is performed, and the extracted solution is subjected to a known protein purification method by combining chromatography such as reverse phase chromatography or ion exchange chromatography. can do.

  When the obtained polypeptide is a free form, it can be converted into an appropriate salt by a known method. Moreover, when obtained as a salt, it can be converted into a free form or other salt by a known method.

  A polynucleotide comprising a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 of the present invention, or a cDNA capable of hybridizing to the polynucleotide. The polynucleotide or the recombinant vector containing the polynucleotide can be used as a cancer preventive / therapeutic agent, apoptosis inducer, and anticancer agent enhancer.

  Moreover, as antisense nucleotide (SEQ ID NO: 11-13) and double-stranded RNA, 21-23 base pair short interfering RNA (SEQ ID NO: 5-10) is a cancer preventive / therapeutic agent, apoptosis inducer, And can be used as an anticancer agent enhancer. Double-stranded RNA against the polynucleotide consisting of the base sequence represented by SEQ ID NO: 2 suppresses the expression of the gene having the same sequence, and therefore suppresses the expression of the polynucleotide composed of the base sequence represented by SEQ ID NO: 2. Therefore, the expression of Pim-1 is suppressed and the activity of Pim-1 can be inhibited. As such a double-stranded RNA, a short interference RNA of 21 to 23 base pairs is preferable. As a method for preparing double-stranded RNA, a conventionally known method can be used without particular limitation, and for example, it can be produced using a Silencer si RNA construction kit (manufactured by Ambion).

Examples of the double-stranded RNA for the polynucleotide having the base sequence represented by SEQ ID NO: 2 include, for example, a polynucleotide ( ^5′- aaugaugaagucgaagagaucccugucucc-3 ^′ ) having the sequence represented by SEQ ID NO: 5, and SEQ ID NO: 6 in polynucleotide (5 ^{'-aagaucucuucgacuucaucaccugucuc-3')} consisting of a double-stranded RNA consisting of the sequence represented SEQ ID NO: a polynucleotide consisting of the sequence 7 and ^{(5 '-aaaucuaaugagaugcugacaccugucuc-3')} , SEQ ID NO: A double-stranded RNA consisting of a polynucleotide represented by the sequence represented by 8 ( ^5′- aaugucagcaucucauuagauccugucuc-3 ^′ ), a polynucleotide comprising the sequence represented by SEQ ID NO: 9 ( ^5′- aaauccauggaugguucuggaccacccucuc-3 ^′ ), SEQ ID NO: consisting of a polynucleotide consisting of the sequence ^{10 (5 '-aauccagaaccauccauggauccugucuc-3'} ) double-stranded R A, and the like.

  For example, for a cancer patient or the like, (a) an expression vector under the control of a promoter capable of functioning in the target cell is administered to the patient to express the polypeptide of the present invention in vivo. Thus, (b) the polynucleotide of the present invention is introduced into the removed cells in the same manner as described above, and after the polypeptide is expressed, the cells are transplanted into a patient to exert the above-described effects. When the above renucleotide is used for the above purpose, the polynucleotide may be administered alone or after being inserted into a suitable vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then administered according to conventional means. .

  As shown in Example 1, the ubiquitin / proteasome proteolytic system is involved in the regulation of the expression level of Pim-1 protein, and the compound that activates the ubiquitination and proteasome proteolytic system is a PIm-1 protein. Induces the disappearance of In addition, the treatment and prevention of cancer using a combination of chemotherapeutic agents and proteolytic system activation such as Geldanamycin, an inhibitor of heat shock protein, in which Pim-1 protein disappears, is effective.

  The method for screening for a prophylactic / therapeutic agent for cancer of the present invention is characterized by using the promoter region of Pim-1 (SEQ ID NO: 14). Specifically, the promoter region of Pim-1 is linked to a reporter gene expression vector and introduced into a host cell. This method comprises a step of detecting a Pim-1 production inhibitor by culturing this transformed cell with a test substance under hypoxia and measuring reporter activity.

  The reporter gene is not particularly limited, but a reporter gene that is stable and whose activity can be easily quantified is preferable. Examples of such a reporter gene include DNAs encoding luciferase, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, peroxidase, HIS3 gene, green fluorescence protein (GFP) and the like. It is not limited to.

  That is, in the screening method described above, when the test substance has an activity of suppressing Pim-1 production, the expression of the reporter gene is suppressed or inhibited, so that by detecting the expression of the reporter gene, It becomes possible to detect whether the test substance promotes or inhibits the activity of Pim-1 production.

  Screening is performed by confirming the presence of the active substance compared to the activity in the absence of the test substance. By this method, it is possible to screen for an apoptosis-inducing agent.

  Examples of test samples used in the screening method of the present invention include cell extracts, plant extracts, purified or crude proteins, peptides, non-peptidic compounds, synthetic low molecular compounds, natural compounds, gene libraries and the like. Can be mentioned.

Next, the screening kit of the present invention will be described.
The screening kit of the present invention contains cells in which the promoter region of Pim-1 is linked to a reporter gene expression vector and introduced into a host cell. The screening kit of the present invention is used to screen for compounds that inhibit Pim-1 production.

  The compound that inhibits the activity of Pim-1, such as the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3, as described above is used as it is or together with a physiologically recognized carrier such as an adjuvant for promoting intake. It can be formulated and administered. When the polypeptide is used as a cancer prevention / treatment, apoptosis induction, or anticancer agent enhancer, it is preferably purified to 90%, more preferably 95% or more, more preferably 98% or more, and most preferably 99% or more. It is preferred to use the prepared polypeptide. The above polypeptide is, for example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating as required, or in the form of aerosolized inhalants, or water or other pharmaceuticals It can be used parenterally in the form of an injectable solution such as a sterile solution with an acceptable liquid or a suspension.

  For example, the polypeptide of the present invention in a generally accepted unit dosage form required for practicing formulation with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. It can be produced by mixing. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained. Examples of additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid, Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, red oil and cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type material. The injection is prepared by dissolving, suspending or emulsifying the polypeptide of the present invention in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. Buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), A preservative (for example, benzyl alcohol, phenol, etc.), an antioxidant and the like may be blended. The prepared injection solution is usually filled in a suitable ampoule. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose.

  A recombinant vector into which the polynucleotide has been inserted is also formulated in the same manner as described above, and is usually used parenterally. Since the preparation thus obtained is safe and has low toxicity, for example, a warm-blooded animal (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey) , Chimpanzee, etc.) and can be used as a cancer preventive / therapeutic agent, apoptosis inducer, or anticancer agent enhancer.

  Examples of cancers targeted for the above cancer treatment / prevention agent and anticancer agent enhancer include pancreatic cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testis Examples include cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, and the like, and are particularly effective for solid cancer in which intracellular oxygen concentration is reduced.

  When using the cancer therapeutic / preventive agent, apoptosis-inducing agent, or anticancer agent-enhancing agent of the present invention, in addition to direct administration to a patient, it can be formulated and administered by a known pharmaceutical method. For example, a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer and the like may be formulated and administered as appropriate. it can. Administration to a patient can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, and the like, as well as intranasally, transbronchially, intramuscularly, or orally by methods known to those skilled in the art. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited thereto.

Example 1
It was investigated whether Pim-1 was decomposed by protease under normoxic partial pressure. PCI-43 cells were cultured under low oxygen partial pressure and normoxic partial pressure. The cells cultured under a low oxygen partial pressure were sampled before culturing, after 4 hours, 12 hours and 24 hours from the start of culturing, and were subjected to the same operation as in Example 5 for Western blot analysis. In the culture under normoxic partial pressure, the medium was added with a proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) at a concentration of 50 μM. In culture under normoxic partial pressure, sampling was performed before the start of culture, 6 hours after the start of culture, and 12 hours after the start, and Western blot analysis was performed. The results are shown in FIG. As shown in FIG. 1, in the culture under a low oxygen partial pressure, the amount of Pim-1 increases with the passage of time after exposure to the low oxygen partial pressure. Along with this, the amount of Pim-1 was increased. This indicates that Pim-1 protein is degraded in the absence of proteasome inhibitors.

Example 2
To elucidate the role of Pim-1, a dominant negative Pim-1 transfectant was established. The Pim-1 transfectant is a peptide that lacks the kinase activity domain of wild-type Pim-1, that is, the peptide represented by SEQ ID NO: 3.
The dominant negative Pim-1 cDNA lacking the kinase active domain was amplified from the RT product of mRNA purified from PCI-10 cells and cloned into PCR4-TOPO. Plasmid sequencing was performed using the DyeDeoxy Terminator kit (Perkin-Elmer) using an ABI377 automated sequencing device (Applied Biosystems). Ligated to the cloned fragment (Invitrogen). The RT-PCR method will be briefly described below.

37 ° C in a reaction mixture containing 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM each dNTP, 2 μM random primer, and 1000 U AMLV reverse transcriptase (Gibco BRL) By incubating for 1 hour, cDNA was amplified from each RNA sample (5 μg). PCR amplification of cDNA consisted of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl ₂ , 0.1% Triton X-100, 200 μM each dNTP, 10 μM each specific primer, and 1 U Taq polymerase (Gibco BrL In the reaction mixture. PCR was performed in a DNA thermal cycler (Barnstead / Thermolyne) for 35 cycles (94 ° C., 1 minute, 60 ° C., 1 minute, 72 ° C., 2 minutes).

The following PCR primers were used.
dnpim-1 forward: 5'-GTAGAATTCGCCACCATGCCTGCCTAATGGCACTCGAGTG-3 '(SEQ ID NO: 15)
Reverse: 5'-GTACTATTTGCTGGGCCCCGGCGAC-3 '(SEQ ID NO: 16)

The resulting vector was transduced into PCI-43 cells using Lipofectamine (Life Technologies) into the expression vector. Transfectants were selected with 1,200 μg / ml G-418 and then cloned by limiting dilution to obtain dominant negative transfectants dnp3, dnp4 and dnp10. The transfectants were then maintained in the presence of 600 μg / ml G-418.
The obtained transformed cells were subjected to protein electrophoresis. Samples were prepared using 1% NP-40 lysis buffer (50 μM Tris PH 7.5, 150 nM NaCl, 2 μM EDTA, 1 μM, EGTA, 50 μM NaF, 1 μM Na ₃ VO ₄ , 1 μM PMSF).
As a control, a vector transformed was also used.

  The results are shown in FIG. FIG. 2 shows the results of protein electrophoresis of transformed cells. As shown in FIG. 2, dnp3, dnp4, and dnp10 detected peaks indicating the presence of Pim-1 lacking the kinase domain. This peak was not detected in the case of transforming only the vector (v3 and v4).

Example 3
5 × 10 ⁶ V3, dnp3, dnp4 and dnp10 were subcutaneously injected into the right flank of SCID mice. After subcutaneous injection, the tumor size was observed every 3 days until day 21. The tumor size was measured by measuring the short diameter and the long diameter with a caliper and calculating the volume by the following calculation formula to obtain the tumor size.
(Minor axis) x (minor axis) x (major axis) /
Tumor cells were excised from mice 6 days after subcutaneous injection, and immunohistochemical staining was performed to determine the number of CD31 positive cells. The results are shown in FIG.

  In FIG. 3, a is an anti-CD31 stained image of a tumor tissue of a mouse subcutaneously injected with V3, and b is an anti-CD31 stained image stained image of a tumor tissue of a mouse subcutaneously injected with dnP4. As shown in FIG. 3, CD3 staining positive cells of v3 were observed, but CD31 staining positive cells of dnP4 were not observed.

Example 4
5 × 10 ⁶ pancreatic cancer cell lines BxPc3 were subcutaneously injected into the right flank of SCID mice. After subcutaneous injection, the expression vector was administered into the thigh muscle every 14 days from the 5th day, and the size of the tumor was observed until the 35th day. The tumor size was measured by measuring the short diameter and the long diameter with a caliper and calculating the volume by the following calculation formula to obtain the tumor size.
(Minor axis) x (minor axis) x (major axis) / 2
The results are shown in FIG. FIG. 4 is a graph showing changes in tumor size when various vectors are administered.

In the graph of FIG. 4, the horizontal axis is the number of days elapsed after subcutaneous injection, and the vertical axis is the tumor size (mm ³ ). The graph of FIG. 4 shows the average and standard deviation in an experiment conducted using five SCID mice. As shown in FIG. 4, in the group to which v3 was administered, the tumor size increased with the passage of days. On the other hand, in the group administered with the dominant negative Pim-1, the tumor size decreased from the 20th day after the administration. This shows that dominant negative Pim-1 lacks tumorigenicity.

Example 5
FIG. 5 is a diagram showing a screening method. As shown in the upper part of the figure, the Pim-1 promoter region is incorporated into a reporter speech vector such as luciferase, expressed in cells, seeded at 10,000 cells / well, added with 1-10 μM of the test substance, and cultured for 24 hours. . On the other hand, 293 normal cell lines are seeded at 10,000 cells / well as shown in the lower stage, and the test substance is added in the same manner and cultured. A test substance added in a well in which a decrease in reporter activity is recognized becomes a candidate substance. On the other hand, substances showing toxicity to normal cells are excluded from candidate substances.

  As detailed above, the amount of Pim-1 was increased in various cancer cells exposed to low oxygen partial pressure, and Pim-1 was degraded under normal oxygen partial pressure, but proteolysis Inhibiting the system increased the amount of Pim-1 even under normoxic partial pressure. Inhibiting the function of the Pim-1 gene with dominant negative Pim-1 reduced tumor angiogenesis and decreased tumorigenicity.

  Thus, by inhibiting the function of the Pim-1 protein or the Pim-1 gene, it is possible to exert a cancer treatment, apoptosis induction, and an anticancer drug enhancing effect. Therefore, dominant negative Pim-1 and A compound that inhibits the function of Pim-1 or protein expression is effective in the treatment of cancer.

It is a photograph which shows the expression increase of Pim-1 protein in the presence of a proteasome inhibitor. It is a photograph which shows Dominant negative Pim-1 protein expression of a Dominant negative Pim-1 introduction strain. It is a photograph which shows the result of having dye | stained the vascular endothelial cell. It is a figure which shows the therapeutic effect by secretory type Dominant negative Pim-1. It is a figure which shows the screening method.

Claims (18)

Inhibition of Dominant negative Piom-1 and Pim-1 expression by removing Pim-1 N-terminal polypeptide used for cancer prevention and treatment by inhibiting serine / threonine kinase Pim-1 represented by SEQ ID NO: 3 An anti-sense DNA represented by SEQ ID NO: 11-13 and a short interference RNA represented by SEQ ID NO: 5-10. Anti-apoptotic inhibitory effect of serine / threonine kinase Pim-1 and 1-81 polypeptide of N-terminal part of serine / threonine kinase Pim-1 used for the prevention and treatment of cancer by inhibiting tumor blood vessel application Removed truncated polypeptide Dominant negative Piom-1 (SEQ ID NO: 4) and anti-sense DNA represented by SEQ ID NO: 11-13 that inhibit Pim-1 expression and SEQ ID NO: 5-10 short interference RNA. Naked DNA obtained by incorporating a gene sequence (SEQ ID NO: 3) encoding Dominant negative Piom-1 from which the N-terminal polypeptide of Pim-1 has been removed into a plasmid-type expression vector. Anti-apoptotic inhibitory effect of serine / threonine kinase Pim-1 and 1-81 polypeptide of N-terminal part of serine / threonine kinase Pim-1 used for cancer prevention / treatment by inhibiting tumor angiogenesis Naked DNA incorporated into a plasmid-type expression vector incorporating a gene sequence (SEQ ID NO: 3) encoding the removed truncated polypeptide Dominant negative Piom-1. Prophylactic / therapeutic agent for solid cancer by anti-sense DNA represented by SEQ ID NO: 11-13 and short interference RNA represented by SEQ ID NO: 5-10 that inhibits protein expression of serine / threonine kinase Pim-1 . Solid form through inhibition of tumor angiogenesis by anti-sense DNA represented by SEQ ID NO: 11-13 and short interference RNA represented by SEQ ID NO: 5-10 that inhibits protein expression of serine / threonine kinase Pim-1 Cancer preventive / therapeutic agent. Inhibits Pim-1 protein action by inhibitors of transcription factors that regulate the transcription of the serine / threonine kinase Pim-1 gene, heat shock proteins involved in Pim-1 protein degradation, and inhibitors of the ubiquitin / proteasome degradation system A prophylactic / therapeutic agent for cancer comprising a compound or a salt thereof. Cancers comprising peptides and inhibitors that inhibit the interaction with receptors on vascular endothelial cells that inhibit the tumor angiogenesis action of Pim-1 protein, compounds that inhibit signal transduction leading to angiogenesis, or salts thereof Preventive and therapeutic agent. The cancer preventive / therapeutic agent according to claim 5, 6, 7, and 8, wherein the cancer is pancreatic cancer. Anti-sense DNA that inhibits serine / threonine kinase Pim-1 protein expression by inhibiting the expression of serine / threonine kinase Pim-1 protein by compounds that inhibit Pim-1 protein action , Short interference RNA, inhibitors of transcription factors controlling transcription of serine / threonine kinase Pim-1 gene, heat shock proteins involved in Pim-1 protein degradation, inhibitors of ubiquitin / proteasome degradation system and cancer chemotherapeutic agents A solid cancer preventive and therapeutic agent comprising: Anti-sense DNA that inhibits serine / threonine kinase Pim-1 protein expression by inhibiting the expression of serine / threonine kinase Pim-1 protein by compounds that inhibit Pim-1 protein action , Short interference RNA, inhibitors of transcription factors controlling transcription of serine / threonine kinase Pim-1 gene, heat shock proteins involved in Pim-1 protein degradation, inhibitors of ubiquitin / proteasome degradation system and cancer chemotherapeutic agents A solid cancer prevention and treatment method comprising The preventive / therapeutic agent for cancer according to claim 10, wherein the cancer is pancreatic cancer. The method for preventing or treating cancer according to claim 11, wherein the cancer is pancreatic cancer. A screening method for a prophylactic / therapeutic agent for cancer using the search for an inhibitor of Pim-1 expression in which the promoter region of serine / threonine kinase Pim-1 is connected to a reporter expression vector. A prophylactic / therapeutic agent for cancer using a method in which the promoter region of serine / threonine kinase Pim-1 is connected to a reporter expression vector and a substance having an anti-angiogenic activity is searched for by searching for an inhibitor of Pim-1 protein expression Screening method. The kit used for the screening method of Claim 14 and 15. A preventive / therapeutic agent for cancer obtained using the screening kit according to claim 16. The cancer preventive / therapeutic agent obtained using the screening kit according to claim 16, wherein the cancer is pancreatic cancer.