JP2008214201A - Esophagus cancer cell proliferation inhibitor and method for screening the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new esophagus cancer cell proliferation inhibitor, and to provide a method for screening the same. <P>SOLUTION: This esophagus cancer cell proliferation inhibitor contains a compound for inhibiting calcium/calmodulin-dependent kinase II as an active ingredient. The method for screening the esophagus cancer cell proliferation inhibitor uses a change in the inhibition or gene expression of the activation of the calcium/calmodulin-dependent kinase II as an indicator. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an agent for inhibiting the growth of esophageal cancer cells comprising a compound that inhibits calcium / calmodulin-dependent kinase II. The present invention also relates to a method for screening for an inhibitor of proliferation of esophageal cancer cells using as an index the inhibition of the activation of calcium / calmodulin-dependent kinase II or the change in the expression level of the calcium / calmodulin-dependent kinase II inhibitor 1 gene.

  The esophagus is a luminal organ that connects the pharynx and stomach, mostly in the thoracic cavity and partly in the neck and abdominal cavity. The upper part of the thoracic cavity is between the trachea and the spine, and the lower part is surrounded by the heart, aorta and lungs. The esophagus serves to send food from the mouth to the stomach.

  The death rate from Japanese cancer in 2001 is 238.8 out of 100,000. The proportion of deaths due to esophageal cancer is increasing year by year, and in 2001, 5.0% of all cancer deaths were in men and 1.4% in women. The peak age of onset of esophageal cancer is in the 60s to 70s, and men often have onset. Also, environmental factors such as smoking, drinking, and hot foods are closely related to the occurrence. Furthermore, since the esophageal wall and its surroundings are rich in blood vessels and lymphatic vessels, it is known that there are many metastases of the developed cancer.

  Treatment of esophageal cancer is determined in consideration of the degree of progression (Clinical / Pathology of Esophageal Disease Study Group, 1999, Esophageal Cancer Handling Regulations 1999), metastasis, and general condition. Standard treatment methods for esophageal cancer are described in “Esophageal Cancer Treatment Guidelines” (2002) edited by the Japan Esophageal Disease Study Group. The most common therapy at present is surgical treatment, and the esophagus including cancer and surrounding tissues including lymph nodes are removed (lymphadenectomy), and the esophagus is reconstructed using other organs such as the stomach. However, surgical therapy, especially wide-area lymph node dissection, places a heavy burden on patients and requires consideration for lowering QOL after surgery. Endoscopic mucosal resection may be possible for early cancers that remain within the mucosa. Radiation may be performed from both radical treatment and symptomatic treatment. In addition, chemotherapy is performed in combination with surgery and radiation therapy. Currently, the combination therapy of 5-fluorouracil and cisplatin is considered to be the most effective for chemotherapy.

  The gene therapy for esophageal cancer has been carried out by injecting a vector incorporating the tumor suppressor gene p53 directly into the affected area by endoscopic surgery, but there has been a report that the progression has stopped for a certain period of time. There has been no report that has shrunk or disappeared to less than half. In recent years, there are few effective therapeutic agents for esophageal cancer, which are increasing, and it is desired to provide more effective therapeutic methods.

  Calcium is an important molecule responsible for controlling biological phenomena as a second messenger in cells. Within the cell, there are enzymes that are activated as the intracellular calcium concentration increases, and signals are transmitted to various molecules involved in cell proliferation, cell cycle, cell adhesion, movement, secretion, and the like. The calcium / calmodulin-dependent kinase family is known as a multifunctional protein kinase involved in calcium ion-dependent signal transduction. Calcium / calmodulin-dependent kinase II (CaMKII) is the most advanced calcium / calmodulin-dependent kinase family molecule, and there are four isoforms: CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. In the nervous system, CaMKIIα and CaMKIIβ are remarkably expressed, and are known to be involved in long-term memory in the hippocampus. CaMKIIγ and CaMKIIδ are known to exhibit ubiquitous expression, and there is a report that CaMKIIδ is particularly involved in myocyte differentiation (Non-patent Document 1). In recent years, it has been reported that the MAP kinase pathway, which is deeply involved in cell proliferation, is regulated by calcium / calmodulin-dependent kinase II, and calcium / calmodulin-dependent kinase II is expected to be involved in canceration of cells ( Non-patent document 2).

  Recently, a calcium / calmodulin-dependent kinase II inhibitor gene (CaMKIIN) has been identified as a molecule that binds to activated calcium / calmodulin-dependent kinase II and suppresses its function (Patent Document 1, Non-Patent Documents 3, 4). ). There are two types of calcium / calmodulin-dependent kinase II inhibitor genes: calcium / calmodulin-dependent kinase II inhibitor 1 (CaMKIINα) and calcium / calmodulin-dependent kinase II inhibitor 2 (CaMKIINβ). There is 40.9% homology in the sequence and 64.5% in the amino acid sequence.

So far, it has been reported that calcium / calmodulin-dependent kinase II inhibitor 1 and cancer are found to suppress cell growth in colon cancer cell lines, but the detailed molecular mechanism is unknown. is there. (Non-patent document 5).
JP-T-2004-506442 Gaertner TR, Kolodziej SJ, Wang D, Kobayashi R, Koomen JM, Stoops JK, Waxham MN. kinase II., "" J Biol Chem. "279: 12484-12494 (2004) Illario M, Cavallo AL, Bayer KU, Di Matola T, Fenzi G, Rossi G, Vitale M., "Calcium / calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation.,""J Biol Chem. '' 278: 45101-45108 (2003) Chang BH, Mukherji S, Soderling TR., "Calcium / calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain.,""Neuroscience". 102: 767-777 (2001) Chang BH, Mukherji S., Soderling TR, "Characterization of a calmodulin kinase II inhibitor protein in brain.,""Proc Natl Acad Sci US A.". 95: 10890-10895 (1998) Zhang J, Li N, Yu J, Zhang W, Cao X., "Molecular cloning and characterization of a novel calcium / calmodulin-dependent protein kinase II inhibitor from human dendritic cells.,""Biochem Biophys Res Commun." 229-234 (2001)

  Development of an effective treatment method for esophageal cancer is highly necessary because the number of patients has increased in recent years, and the physical burden at the time of treatment is large and refractory. At present, new combinations of anticancer drugs and gene therapy targeting specific molecules have been attempted, but no significant effect has been achieved. An object of the present invention is to provide compounds having an effect of suppressing the growth of esophageal cancer cells, and to provide a screening method for these compounds.

  As a result of DNA microarray analysis and RT-PCR analysis, the present inventors significantly reduced the expression of calcium / calmodulin-dependent kinase II inhibitor 1 gene (CaMKIINα) in esophageal cancer tissues compared to normal esophageal tissues. I found out. Furthermore, in esophageal cancer cells, the calcium / calmodulin-dependent kinase II inhibitor 1 gene suppresses the transcriptional activity of the transcription factor activator protein 1 (AP-1), which is known as a signal transduction molecule involved in cancer development. And the present invention has been completed.

  The present invention relates to an agent for suppressing the growth of esophageal cancer cells comprising a compound that inhibits the activation of calcium / calmodulin-dependent kinase II.

  In a preferred embodiment of the present invention, the compound that inhibits activation of calcium / calmodulin-dependent kinase II is (1) a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, (2) SEQ ID NO: A polynucleotide having 80% or more identity with the nucleotide sequence of 1 or SEQ ID NO: 2, and deletion or substitution of one to several bases in the nucleotide sequence of the polynucleotide of (3) (1) or (2) Alternatively, it is a growth inhibitor of esophageal cancer cells, which is a polynucleotide selected from added polynucleotides. Furthermore, the present invention includes an agent for suppressing the growth of esophageal cancer cells in which these polynucleotides are incorporated into a vector.

  In another preferred embodiment of the present invention, the compound that inhibits activation of calcium / calmodulin-dependent kinase II is a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, SEQ ID NO: 3 or SEQ ID NO: A growth inhibitor of esophageal cancer cells, which is a variant of a polypeptide represented by the amino acid sequence of 4, or a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a fragment of the variant of the polypeptide It is.

  In another preferred embodiment of the present invention, the compound that inhibits activation of calcium / calmodulin-dependent kinase II is represented by the formulas (I) to (III):

It is related with the growth inhibitor of the esophageal cancer cell which is any compound represented by these.

  Furthermore, the present invention relates to an agent for inhibiting the growth of esophageal cancer cells, which uses as an index the inhibition of the activation of calcium / calmodulin-dependent kinase II or the change (increase or decrease) in the expression level of calcium / calmodulin-dependent kinase II inhibitor 1 gene. A method for screening is provided. A preferred embodiment is a method of screening an esophageal cancer cell growth inhibitor by measuring inhibition of calcium / calmodulin-dependent kinase II activation using the transcriptional activity of activator protein 1 as an index.

  The present invention provides an esophageal cancer cell growth inhibitor and a screening method therefor. As specifically demonstrated in the examples below, inhibition of calcium / calmodulin-dependent kinase II activation significantly suppresses proliferation of esophageal cancer cells. Therefore, calcium / calmodulin-dependent kinase II is useful as a molecular target for the treatment of esophageal cancer, and an agent for inhibiting the growth of esophageal cancer cells comprising a compound that inhibits the activation of calcium / calmodulin-dependent kinase II provided by the present invention Is useful as a therapeutic agent for esophageal cancer.

  The present invention is an esophageal cancer cell growth inhibitor comprising a compound that inhibits activation of calcium / calmodulin-dependent kinase II (CaMKII). Here, the activation of calcium / calmodulin-dependent kinase II means an increase in the enzyme activity of calcium / calmodulin-dependent kinase II, which is a phosphorylating enzyme.

  The enzyme activity of calcium / calmodulin-dependent kinase II can be measured using the amount of phosphorylation of a serine or threonine residue of a protein or peptide that can be a substrate of the enzyme as an index. In addition, the transcriptional activity of activator protein 1 can be measured using a plasmid containing a reporter gene sequence having a binding sequence of activator protein 1 as a transcription factor as a promoter site.

  Calcium / calmodulin-dependent kinase II is a kind of multifunctional protein kinase with a wide substrate specificity, and four isoforms, namely CaMKIIα (SEQ ID NO: 5), CaMKIIβ (SEQ ID NO: 6), and CaMKIIγ (SEQ ID NO: 7). ), CaMKIIδ (SEQ ID NO: 8). That is, the calcium / calmodulin-dependent kinase II in the present invention includes the four isoforms represented by the amino acid sequences of SEQ ID NOs: 5 to 8.

  A cell line refers to a cell that is collected from a living body and is capable of proliferating almost infinitely when cultured under appropriate culture conditions. Appropriate culture conditions here refer to conditions that provide temperature, nutrients, growth factors, attachment surfaces, etc. according to each cell.

  The growth inhibitor of esophageal cancer cells of the present invention inhibits or suppresses the proliferation of esophageal cancer cells by adding it to the culture solution when culturing cell lines or living cells under the above-mentioned appropriate culture conditions. Or a compound that induces cell death. Inhibition, suppression, and cell death of esophageal cancer cells were observed by visual observation, detection of changes in cell surface antigens by flow cytometry, detection of dye uptake into cells, or tritium thymidine added to the culture medium. It can be confirmed by detecting the amount of intracellular uptake.

  The esophageal cancer cell growth inhibitor of the present invention is a compound that inhibits the activation of calcium / calmodulin-dependent kinase II, and has the effect of suppressing the increase in the enzyme activity of calcium / calmodulin-dependent kinase II as described above. It contains a compound. Examples of the compound having an action of suppressing an increase in the enzyme activity of calcium / calmodulin-dependent kinase II include a low molecular weight organic compound, a polynucleotide, a polypeptide and the like having the action.

  The esophageal cancer cell growth inhibitor of the present invention is preferably a calcium / calmodulin-dependent kinase II inhibitor gene, that is, a calcium / calmodulin-dependent kinase II inhibitor, as a compound that inhibits activation of calcium / calmodulin-dependent kinase II. 1 (CaMKIINα) and calcium / calmodulin-dependent kinase II inhibitor 2 (CaMKIINβ). Specifically, the esophageal cancer cell growth inhibitor of the present invention is more preferably (1) a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and (2) SEQ ID NO: 1 or SEQ ID NO: A polynucleotide having 80% or more identity with the base sequence of 2, or one to several bases in the base sequence of the polynucleotide of (3) (1) or (2) has been deleted, substituted or added It contains a polynucleotide.

  In the present invention, “polynucleotide” means a nucleic acid including both polyribonucleotide (RNA) and polydeoxynucleotide (DNA). The DNA includes cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, and synthetic RNA.

  The polynucleotide that inhibits the activation of calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention preferably has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and 80% or more, preferably 85% or more, preferably Polynucleotides having a percent identity of 90% or greater, more preferably 95% or greater are included. Here, “% identity” can be determined by introducing a gap using a BLAST or FASTA protein or gene search system (Karlin, S. et al., 1993, Proceedings of the National Academic. Sciences USA, 90, p. 5873-5877; Altschul, SF, et al., 1990, Journal of Molecular Biology, 215, p. 403-410; Et al., 1988, Proceedings of the National Academic Sciences USA, Vol. 85, pp. 2444-2448).

  The polynucleotide that inhibits the activation of calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention includes the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 1 or SEQ ID NO: Polynucleotides in which one to several bases in the base sequence of any one of the polynucleotides having the nucleotide sequence of No. 2 and 80% or more identity are deleted, substituted or added are included. Here, “several” means an integer of about 5, 4, 3, or 2 or less. “Addition” means adding another base sequence to the base sequence of the original polynucleotide, and the site to be added is the 3 ′ end or the 5 ′ end of the original polynucleotide. Or may be added to several sites in the polynucleotide to the same or different degrees.

  The polynucleotide that inhibits the activation of the calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention may be incorporated into an expression vector so that the polynucleotide can be expressed. Cell growth can be suppressed by introducing an expression vector incorporating the polynucleotide into esophageal cancer cells. Methods for incorporating these polynucleotides into expression vectors include chemical / physical means such as the calcium phosphate method, DEAE-dextran method, electroporation method, liposome method, and microinjection method.

A viral vector is used as an expression vector into which the polynucleotide that inhibits the activation of the calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention is incorporated, and the polynucleotide incorporated in the viral vector is used as an esophageal cancer cell. Alternatively, it can be introduced into a living body. As a viral vector, in addition to an adenovirus vector that has been clinically used by introducing a tumor suppressor gene such as the p53 gene, a retrovirus vector, an adeno-associated virus vector, an adenovirus / adeno-associated virus hybrid vector, a lenti Examples thereof include virus vectors and vaccinia virus vectors. In introducing a viral vector into esophageal cancer cells, it is desirable that infection of viral particles of 100 or less, preferably 20 or less, more preferably 5 or less per cell occurs. In addition, in the introduction of a viral vector into a living body, infectious virus particles of 1 × 10 ¹¹ or less, preferably 1 × 10 ¹⁰ or less, preferably 1 × 10 ⁹ or less, more preferably 1 × 10 ⁷ or less per dosage unit. It is desirable to administer to esophageal cancer tissue.

  The compound that inhibits the activation of calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention includes a calcium / calmodulin-dependent kinase II inhibitor gene, ie, calcium / calmodulin-dependent kinase II inhibitor 1 (CaMKIINα) or Polypeptides encoded by calcium / calmodulin-dependent kinase II inhibitor 2 (CaMKIINβ) are included. Specifically, a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 is included. In addition, the esophageal cancer cell growth inhibitor of the present invention is a variant of the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 as long as it has an action of inhibiting the activation of calcium / calmodulin-dependent kinase II Or a fragment of the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Moreover, the polypeptide by which the said polypeptide, its variant, or its fragment was modified is also contained.

  Here, in the “variant” of the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, one or several amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 3 or 4 Polypeptide. Further, a polypeptide in which one or several amino acids are added to the amino terminus, carboxyl terminus, or polypeptide of the polypeptide is also included. These mutations may be present in the same or different degrees at several sites. Furthermore, the variants of the polypeptide include the partial sequence and about 80% or more, about 85% or more, preferably about 90% or more, more preferably about 95% or more, about 97% or more, about 98% or more, about Polypeptides that show 99% or more percent identity are also included. Such mutants include, for example, natural mutants such as homologues of mammal species different from humans, and variants based on polymorphic mutations between mammals of the same species (eg, race).

  A polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a fragment of a variant of the polypeptide includes a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 3 or 4 or a partial sequence thereof. Polypeptides comprising a sequence of at least 50, preferably 30, 20, more preferably 10 consecutive amino acids in which one or several amino acids are deleted, substituted or added are included. .

  In addition, the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a variant thereof, or a fragment thereof is “modified polypeptide” includes a peptide bond or a modified peptide bond (ie, Polypeptides comprising two or more amino acids linked together by peptide isosteres) are included. The polypeptide can contain amino acids that are different from the 20 amino acids encoded by the gene. The modified polypeptide also includes a polypeptide that has been modified by either a natural modification such as a post-translational modification or a chemical modification method well known in the art. Modifications may be made at any position within the polypeptide including the peptide backbone, amino acid side chains and the amino terminus or carboxyl terminus. The same type of modification may be present in the same or varying degrees at several sites in the polypeptide. Different types of modifications may also be included. The polypeptide may be branched as a result of, for example, ubiquitination or may be cyclic. Cyclic, branched and branched cyclic polypeptides may result from natural modifications following translation or may be made by synthetic methods.

  Cell proliferation is increased by introducing the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a variant of the polypeptide, or a fragment of the polypeptide or a variant of the polypeptide into an esophageal cancer cell. Can be inhibited. Examples of the introduction into esophageal cancer cells include a method using cellular protein transport, a liposome method, a method of binding the peptide with a molecule having high affinity on the cell surface, and the like. Furthermore, esophageal cancer can be treated by administering these polypeptides to a patient.

  The above-mentioned polypeptide can be produced, for example, by chemical synthesis or DNA recombination techniques that are conventional techniques in the art. In the chemical synthesis of the peptide, for example, it can be synthesized using a commercially available peptide synthesizer such as Applied Biosystems or Shimadzu Corporation.

  Furthermore, the polypeptide can be obtained from the cell or culture supernatant by incorporating the polynucleotide described above into an expression vector and culturing a prokaryotic or eukaryotic host cell transformed or transfected with the vector.

  Examples of host cells in this case include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis), yeast (eg, Saccharomyces cerevisiae), insect cells (eg, Sf9 cells), mammalian cells (eg, COS, CHO, BHK), and the like. Can be used. In addition to DNA encoding the polypeptide, the vector can contain regulatory elements such as promoters, enhancers, polyadenylation signals and ribosome binding sites, replication origins, terminators, selectable markers, and the like.

  In order to facilitate the purification of the polypeptide, it can be produced and produced in the form of a fusion protein in which a labeled peptide is added to the C-terminus or N-terminus of the peptide. Representative labeled peptides include histidine repeats of 6 to 10 residues, FLAG, myc peptides, GFP polypeptides, etc., but the labeled peptides are not limited to these. For purification of a polypeptide to which a labeled peptide is added, affinity chromatography suitable for each labeled peptide that is generally used can be used.

  When the polypeptide is produced without a labeled peptide, for example, a purification method can be exemplified by a method by ion exchange chromatography. In addition to this, a method of combining gel filtration, hydrophobic chromatography, isoelectric point chromatography and the like may be used.

  The compound that inhibits the activation of calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention includes a low-molecular-weight organic compound that has an action of suppressing an increase in the enzyme activity of calcium / calmodulin-dependent kinase II. It is. Examples thereof include compounds that suppress phosphorylation leading to activation of calcium / calmodulin-dependent kinases, dephosphorylate phosphorylation sites, and inhibit this kinase activity itself. Preferred low molecular weight organic compounds having the action of suppressing the increase in the enzyme activity of calcium / calmodulin-dependent kinase II contained in the growth inhibitor of the present invention are represented by the following chemical formulas (I) to (III):

It is any compound represented by these.

  The compound (KN-62) represented by the chemical formula (I) is commercially available (available from Seikagaku Corporation, SIGMA-ALDRICH, etc.) or existing synthetic methods (for example, J. Med. Chem., 37: 4079-4084 (1994)). In addition, the compound (KN-93) represented by the chemical formula (II) is commercially available (available from Calbiochem, SIGMA-ALDRICH, Seikagaku Corporation, etc.) or an existing synthesis method (for example, Biochem. Biophys. Res. Commun., 181: 968-975 (1991)) can be used. Furthermore, the compound represented by the chemical formula (III) (lavendastin C) is commercially available (available from Calbiochem, SIGMA-ALDRICH, Wako Pure Chemical Industries, etc.) or existing synthetic methods (for example, Biochemistry). , 23: 5036-5041 (1984)).

  Among these, KN-93 represented by the chemical formula (II) is a cell-permeable calcium / calmodulin-dependent kinase II inhibitor. KN-93 is a protein kinase similar in properties to calcium / calmodulin-dependent kinase II, and acts on protein kinase A that is activated by cyclic AMP to phosphorylate serine or threonine residues of the protein. Instead, it acts on calcium / calmodulin-dependent kinase II activated by calcium or calmodulin.

  A calcium / calmodulin-dependent kinase II inhibitor, which is a low molecular weight organic compound such as the compounds represented by the chemical formulas (I) to (III), is added to the culture supernatant of esophageal cancer cells, thereby proliferating the cells. Can be suppressed. Furthermore, in vivo, since it is permeable to the cell membrane, it can be administered directly to the cancer site by endoscopic procedures or the like, thereby suppressing the proliferation of esophageal cancer cells and treating esophageal cancer.

  The esophageal cancer cell growth inhibitor of the present invention can include any pharmacologically acceptable pharmaceutical additive, carrier or carrier. The term “pharmacologically acceptable” means that the dose or concentration used is non-toxic to the living body or cells derived from the living body.

  Examples of pharmacologically acceptable pharmaceutical additives include those that can provide effects as buffering agents, stabilizers, and excipients. Specifically, buffers such as phosphoric acid, citric acid, other organic acids or their salts, antioxidants including ascorbic acid, tween (registered trademark), pluronic (registered trademark), nonionic such as PEG Surfactant, chelating agent such as EDTA, low molecular weight (less than 10 residues) polypeptide, protein such as serum albumin, gelatin, immunoglobulin, hydrophilic polymer such as polyvinylpyrrolidone, glycine, glutamine, asparagine, Examples thereof include amino acids such as arginine and lysine, monosaccharides such as glucose, mannose, dextrin and dextran, disaccharides or other carbohydrates, sugar alcohols such as mannitol and sorbitol, salt-forming counterions such as sodium ions, and the like.

  Moreover, as a pharmacologically acceptable carrier or carrier, a cationic lipid or a polycationic lipid can be used. Examples of the cationic lipid or polycationic lipid include Lipofectin (registered trademark), Lipofectamine (registered trademark), DOTMA, DOGS (Transfectam; Dioctadecylamidoglycylspermine), DOPE (1,2-dioleoyl- sn-glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane), DDAB (dimethyldioctadecylammonium bromide), polybrene, biocompatible polymer or copolymer nanoparticles, or , Poly (ethyleneimine) (PEI), polylysine, protamine, pK17, peptide K8, peptide p2, and the like.

  The esophageal cancer cell growth inhibitor of the present invention can be administered by intravenous injection or infusion, and can also be administered orally, transdermally, transmucosally, vaginally, subcutaneously, instilled, intramuscularly, intrarectally and the like. . Furthermore, it can also be directly injected into the affected area where esophageal cancer has occurred by esophageal endoscope technology.

  The present invention suppresses the growth of esophageal cancer cells using as an index the change (increase or decrease) of the expression level of calcium / calmodulin-dependent kinase II inhibitor 1 in a sample sample or the inhibition of the activation of calcium / calmodulin-dependent kinase II. Methods for screening agents are provided.

  Examples of the specimen sample used in the screening method of the present invention include biological tissues such as esophageal tissue and surrounding tissues, esophageal cells, cell lines derived from normal esophageal tissues, cell lines derived from esophageal cancer, blood, serum, Body fluids such as plasma and urine can be used. The biological tissue of the subject can be collected by a biopsy method or obtained by surgery. The subject is a mammal, preferably a human.

  Methods for detecting or measuring the expression of the calcium / calmodulin-dependent kinase II inhibitor 1 gene or the calcium / calmodulin-dependent kinase II inhibitor 2 gene include the expression or expression level of mRNA that is a transcription product of the gene, Similarly, there is a method for detecting or measuring the presence or amount of a protein or a fragment of the protein that is a translation product of the gene. The transcription product of the gene can be detected or measured according to a known method for specifically detecting the expression of a specific gene, such as Northern blotting, RT-PCR, in situ hybridization, or DNA macroarray.

  As a method for detecting the expression or measuring the expression level of a calcium / calmodulin-dependent kinase II inhibitor 1 gene-derived polypeptide or a calcium / calmodulin-dependent kinase II inhibitor 2 gene-derived polypeptide, a conventional enzyme or fluorophore can be used. A step of contacting a specimen sample with an antibody or a fragment thereof that recognizes a calcium / calmodulin-dependent kinase II inhibitor 1 gene-derived polypeptide or a calcium / calmodulin-dependent kinase II inhibitor 2 gene-derived polypeptide that is optionally labeled; And qualitatively or quantitatively measuring the antibody complex can be used. Detection or measurement is, for example, a method for detecting the presence of a calcium / calmodulin-dependent kinase II inhibitor 1-derived polypeptide or a calcium / calmodulin-dependent kinase II inhibitor 2 gene-derived polypeptide or measuring an expression level by immunoelectron microscopy, an enzyme antibody This is performed by a method for detecting the presence of the polypeptide or measuring the expression level by a conventional method such as ELISA (for example, ELISA), fluorescent antibody method, radioimmunoassay method, homogeneous method, heterogeneous method, solid phase method, sandwich method, etc. be able to.

  The activity of calcium / calmodulin-dependent kinase II can be evaluated by measuring the efficiency of donating phosphate groups to a molecule that can serve as a substrate within a unit time under appropriate conditions in a test tube. For example, the method described in Biochemical Journal, 378: 391-397 (2004) can be applied.

  Examples of a method for measuring the degree of inhibition of calcium / calmodulin-dependent kinase II activation in esophageal cancer cells include a method for measuring the transcriptional activity of various transcriptional regulatory factors by a reporter assay. Examples of transcriptional regulatory factors to be measured include activator protein 1, CREB, Gli, NFκB, Smad, β-catenin and the like.

  For example, in the case of the activity evaluation of activator protein 1, calcium is measured by measuring the change in the transcriptional activity of activator protein 1 after the compound to be evaluated is introduced into the cell or added to the cell culture medium. / The activity of calmodulin-dependent kinase II can be evaluated. By using this method, it is possible to evaluate whether the compound to be evaluated has an esophageal cancer cell growth inhibitory effect. That is, by measuring the inhibition of the activation of calcium / calmodulin-dependent kinase II using the transcriptional activity of activator protein 1 as an index, an esophageal cancer cell growth inhibitor can be screened. In this case, the compound to be evaluated may be a gene incorporated into an expression vector, a protein subjected to cell membrane permeability treatment, or a low molecular organic compound.

  Specifically, in the reporter assay method, for example, primary cells derived from human esophageal cancer, esophageal cancer-derived cell lines such as KYSE series, or mammalian cell lines such as HEK293 and CHO are used. In addition, a reporter plasmid vector comprising a reporter gene sequence such as luciferase, β-galactosidase, Green Fluorescent Protein (GFP) and the like, and further binding a transcriptional regulatory region sequence capable of binding to a transcription factor to be measured to the promoter region of the reporter, It introduce | transduces into said cell with the expression vector which integrated the gene made into the evaluation object. After the introduction of both vectors, the cells are cultured for 6 to 48 hours, preferably 12 to 48 hours, more preferably 24 to 48 hours, and then a reporter protein substrate is added to generate luminescence generated by the substrate conversion. Then, reporter activity is measured using signals such as fluorescence and color development as indicators. Furthermore, by detecting that the reporter activity in the cell incorporating the expression vector incorporating the gene to be evaluated is significantly changed compared to the cell having the expression vector not containing the gene to be evaluated, The transcription factor regulatory activity of the gene to be evaluated can be evaluated.

  In the reporter assay, luciferase, β-galactosidase, Green Fluorescent Protein (GFP), etc. may be used as the reporter gene as described above. In addition, the origin may be derived from any organism such as firefly and Renilla, as long as it is luciferase. The substrate is not particularly limited as long as the translation product of the reporter gene can be used. Many measurement kits (Bright-Glo (registered trademark) Luciferase Assay System (manufactured by Promega), Luciferase, etc.) are available from each company. Assay Kit (manufactured by Stratagene) is commercially available, and any of them may be used.

Example 1: DNA microarray analysis of esophageal cancer tissue 1) Clinicopathological findings of the experimenter From the 119 esophageal cancer patients who obtained informed consent, the esophageal excised tissue at the time of esophageal cancer removal surgery or esophageal biopsy Got. Esophageal cancer tissue was determined macroscopically and / or histopathologically for the excised tissue piece, and the esophageal cancer lesion and normal tissue were separated and immediately frozen and stored in liquid nitrogen.

2) Total RNA extraction and cDNA preparation As samples, tissues of esophageal cancer lesions in esophageal tissues of patients with esophageal cancer and non-cancerous tissues (normal tissues) in the same esophageal tissues were used. Total RNA was prepared from each tissue using Trizol reagent (Invitrogen) according to the recommended protocol.

  About 1 μg of totalRNA obtained by the above method, using oligo (dT) primer and random nonamer together, reverse transcription reaction with Cyscribe First-Strand cDNA Labeling Kit (manufactured by GE Healthcare) using the protocol recommended by the manufacturer. went. Recommended by the manufacturer by adding Cy3-dUTP (manufactured by GE Healthcare) to total RNA derived from normal tissue or esophageal cancer tissue, and Cy5-dUTP (manufactured by GE Healthcare) to reference totalRNA (manufactured by Stratagene). The cDNA was labeled during the reverse transcription reaction according to the protocol. The labeled cDNA was purified by QIA quick PCR purification Kit (manufactured by QIAGEN) and then used for hybridization.

3) Preparation of oligo DNA microarray As an oligo DNA microarray, a DNA chip prepared according to “GeneChip” (registered trademark) (Human Genome U133 A) manufactured by Affymetrix or the method described in this specification was used.

  A method for producing a DNA chip is shown below. First, in order to determine the type of oligo DNA to be mounted, genes were narrowed down using “GeneChip”. The operation of “GeneChip” was performed based on the procedures established by the company such as “Complete GeneChip” (registered trademark) Instrument System. As a result of analysis using “Complete GeneChip”, a total of 8961 genes were extracted that might change expression due to esophageal cancer and genes that could serve as experimental controls.

  With respect to the extracted 8961 genes, sequences 60-70 residues at sites with high sequence specificity were selected and synthesized so as not to cause sequence duplication. MATUNAMI-DNA microarray coat of 8961 kinds of synthetic oligo DNAs containing oligo DNA of SEQ ID NO: 9 dissolved in Solution I (Takara Bio Inc.) diluted 4 times and containing 30 μM A spotter (GMS417arrayer, manufactured by Affymetrix) was spotted on a glass DMSO-compatible Type I amino-modified oligo DNA fixed coat (manufactured by Matsunami Glass Industrial Co., Ltd.) in a humidity environment of 50-60%.

4) Hybridization 1 μg of labeled cDNA was dissolved in an antisense oligo cocktail (manufactured by QIAGEN), applied to a DNA chip on which a Gap cover glass (manufactured by Matsunami Glass Industrial Co., Ltd.) was mounted, and hybridized at 42 ° C. for 16 hours. It was. After completion of hybridization, the DNA chip was washed successively with 2 × SSC / 0.1% SDS, 1 × SSC, and 0.2 × SSC.

5) Measurement of gene expression level The DNA chip hybridized by the above-described method was scanned using an Agilent microarray scanner (manufactured by Agilent), and an image was obtained to quantify the fluorescence intensity. Statistical processing is described in Speed T. et al. By “Statistical analysis of gene expression microarray data” (Chapman & Hall / CRC), C. Et al., “A beginner's guide Microarray gene expression data analysis” (published by Blackwell publishing). That is, the logarithm values of the data obtained from the image analysis after the hybridization were taken, smoothed by global normalization and LOWESS (locally weighted scatter plot smoother), and normalized by the scaling process by MAD.

  As a result, calcium / calmodulin-dependent kinase II inhibitor 1 was expressed as a gene (p <6.2E-13, by Student's t-test) whose expression level in esophageal cancer lesions was significantly less than in non-cancerous tissue parts. I was able to find it.

Example 2: Measurement of expression level of calcium / calmodulin-dependent kinase II inhibitor 1 gene in esophagus and esophageal cancer tissue by quantitative RT-PCR method 1) Preparation of RNA Total RNA derived from normal tissue or esophageal cancer tissue is BioChain Institute. , Inc. Purchased from the company. The RNA of the KYSE-180 cell line was extracted as follows. The collected cells were suspended using a Homogenizer (manufactured by Invitrogen), and then extracted using a Micro-to Midi Total RNA Purification System (manufactured by Invitrogen). The extraction method followed the company's recommended protocol. The extracted RNA was dissolved in 60 μl of Nuclease free water, and the amount of RNA was measured with NanoDrop (manufactured by NanoDrop Technologies Inc.).

2) Quantitative RT-PCR
About 1 μg of the above total RNA, an oligo (dT) primer was used and a reverse transcription reaction was performed based on the protocol recommended by the company using SuperScript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen). 80 μl of TE was added to the synthesized cDNA to make a final volume of 100 μl.

  Quantitative RT-PCR was performed with reference to the methods described in “First-time real-time PCR”, “Basic knowledge of real-time PCR”, and “Real-time PCR practice” published by Takara Bio.

  The calibration curve was prepared by diluting cDNA synthesized from Human reference RNA for GAPDH as an internal standard, and PCR fragment for calcium / calmodulin-dependent kinase II inhibitor 1 to arbitrary concentrations. The composition and reaction conditions were SYBR Premix ExTaq (manufactured by Takara Bio Inc.), and an amplification reaction was performed with ABI PRISM 7000. Using 1 μl of the synthesized cDNA (corresponding to 10 ng of total RNA), the primer was 0.2 M (final concentration) and 45 cycles of amplification reaction were performed.

  As a result, it was confirmed that the expression level of the calcium / calmodulin-dependent kinase II inhibitor 1 gene was reduced to about 1 / 7.7 in normal esophageal tissues in esophageal cancer tissues. In addition, the expression of KYSE-180, which is a cell line derived from esophageal cancer, was reduced to about 1 / 9.1 of normal esophageal tissue.

Example 3
In esophageal cancer cell lines, the effect of the calcium / calmodulin-dependent kinase II inhibitor 1 gene on the activator protein 1 pathway, which is a cancer signaling pathway, was examined. KYSE-180 was used as an esophageal cancer cell line.

1) Confirmation of calcium / calmodulin-dependent kinase II inhibitor 1 gene expression by RT-PCR method pAP-1 (PMA) -TA-Luc (manufactured by CLONTECH), a vector incorporating 0.7 μg of activator protein 1 reporter gene ) And pME18SFL3 vector (manufactured by TOYOBO) containing 0.18 to 1.7 μg of calcium / calmodulin-dependent kinase II inhibitor 1 gene, 5 μl of lipofectamine (manufactured by Invitrogen) and Plus reagent (manufactured by Invitrogen) 5 At the same time, μl was introduced into the KYSE-180 cell line. The introduction method followed the instructions for use of lipofectamine. Total RNA was extracted from the KYSE-180 cell line after 48 hours from introduction of the gene. Of these, 1 μg was used, and the reverse transcription reaction was performed by SuperScript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) using oligo (dT) primer. The method followed the company's recommended protocol. 80 μl of TE was added to the synthesized cDNA to make a final volume of 100 μl. In the PCR reaction, 1 μl of synthesized cDNA (corresponding to 20 ng of total RNA) was amplified using TaKaRA Taq (Takara Bio). The composition was according to the company's recommended protocol, the primer was 0.2M (final concentration), and the cycle number was 23 cycles.

  As a result, expression of the calcium / calmodulin-dependent kinase II inhibitor 1 gene according to the amount of the transgene was confirmed in the cell line into which the calcium / calmodulin-dependent kinase II inhibitor 1 gene was introduced.

2) Reporter assay pAP-1 (PMA) -TA-Luc (manufactured by CLONTECH), a vector incorporating 0.7 μg of activator protein 1 reporter gene, and 0.18 to 1.7 μg of calcium / calmodulin dependence The pME18SFL3 vector (manufactured by TOYOBO) incorporating the kinase II inhibitor 1 gene was simultaneously introduced into the cells using 5 μl of lipofectamine (manufactured by Invitrogen) and 5 μl of Plus reagent (manufactured by Invitrogen). The introduction method followed the instructions for use of lipofectamine. 24 hours after gene transfer, the cells were detached and seeded on 96-well plates at 6 × 10 ⁴ cells / well. Further, 24 hours later (48 hours after gene transfer), TPA (25 ng / ml) was added for stimulation, and 6 hours after addition of TPA, the culture supernatant was removed and washed, and then PLB (Promega ) The cells were disrupted with 50 μl to obtain a cell extract.

Add 50 μl of luminescent substrate (20 mM Tricine, 2.67 mM MgSO ₄ , 0.1 mM EDTA, 33.3 mM DTT, 0.53 mM ATP, 0.22 mM Coenzyme-A, 0.047 mM D-Luciferin) to 10 μl of cell extract. The luminescence value of Luciferin was measured with a luminometer.

  The results are shown in FIGS. The graph of FIG. 1 shows the measurement results of AP-1 activity by the reporter assay when TPA stimulation is performed and the non-stimulated cells are used by the graph of FIG. In each graph, the horizontal axis represents the amount of calcium / calmodulin-dependent kinase II inhibitor 1 gene introduced (μg) or the amount of vector introduced (μg), and the vertical axis represents Luciferin luminescence value (intensity) depending on AP-1 activity. Represents. Each nucleic acid electrophoresis image is a nucleic acid electrophoresis image in which the expression level of GAPDH is detected as a calcium / calmodulin-dependent kinase II inhibitor 1 gene and a control gene. It was shown that the activator protein 1 signal activity was suppressed depending on the concentration and expression level of the calcium / calmodulin-dependent kinase II inhibitor 1 gene in both cases with and without TPA stimulation.

Example 4: Effect of calcium / calmodulin-dependent kinase II inhibitor on cell growth rate KN-93 (manufactured by Cosmo Bio), which is a calcium / calmodulin-dependent kinase II inhibitor, or a control compound has no inhibitory activity. The proliferation ability of KYSE-180 cells when KN-92 (manufactured by Cosmo Bio), which is a KN-93 derivative, was added to the culture medium was measured. The number of cells at the start of the culture was 60000 cells / well, and seeded on a 96-well plate. The number of cells after adding the compound to the culture solution and culturing at 37 ° C. in a CO ₂ incubator for 3 days was measured using Alamar Blue (manufactured by TREK Diagnostics Systems Lyd).

  90 μl of F12 / RPMI medium containing 5% FBS and penicillin and 10 μl of Alamar Blue were mixed to prepare a preparation solution. The supernatant of the cells cultured by the above method was removed, and 100 μl of the prepared solution was added per well. After adding the prepared solution, the mixture was cultured at 37 ° C. for 2 hours, and the absorbance at 570 nm (OD570) and the absorbance at 600 nm (OD600) were measured with a plate reader. The absorbance <OD570> and <OD600> values at 570 nm and 600 nm of the specimen and blank were substituted into the following calculation formula (1), and the reduction rate was calculated as an index of the number of cells.

  Cells cultured with the control compound KN-92 added to the culture showed the same growth ability as cells cultured without the compound. In contrast, when the calcium / calmodulin-dependent kinase II inhibitor KN-93 was added to the culture medium and cultured, the proliferation of KYSE-180 cells was remarkably suppressed (Table 1).

FIG. 1 is a graph showing TPA-stimulated (25 ng / ml, 6 hours) AP-1 activity-dependent Luciferin luminescence values in a KYSE-180 cell line transfected with a calcium / calmodulin-dependent kinase II inhibitor 1 gene, and It is the nucleic acid electrophoresis image which detected the expression level of GAPDH as a gene and a control gene. The horizontal axis of the graph shows the amount of calcium / calmodulin-dependent kinase II inhibitor 1 gene introduced, and the vertical axis shows the luminescence value of Luciferin. FIG. 2 is a graph showing AP-1 activity-dependent Luciferin luminescence values in unstimulated KYSE-180 cell lines into which the calcium / calmodulin-dependent kinase II inhibitor 1 gene was introduced, and GAPDH as a control gene. It is the nucleic acid electrophoresis image which detected the expression level. The horizontal axis of the graph shows the amount of calcium / calmodulin-dependent kinase II inhibitor 1 gene introduced, and the vertical axis shows the luminescence value of Luciferin.

Claims (7)

  An agent for suppressing the growth of esophageal cancer cells, comprising a compound that inhibits activation of calcium / calmodulin-dependent kinase II (CaMKII). The growth inhibitor of esophageal cancer cells according to claim 1, wherein the compound that inhibits activation of calcium / calmodulin-dependent kinase II is a polynucleotide selected from the following (1) to (3).
(1) A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
(2) A polynucleotide having 80% or more identity with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
(3) A polynucleotide in which one to several bases have been deleted, substituted or added in the base sequence of the polynucleotide of (1) or (2).   The growth inhibitor of esophageal cancer cells according to claim 3, wherein the polynucleotide is incorporated into a vector that can be expressed.   A compound that inhibits activation of calcium / calmodulin-dependent kinase II is a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 The growth inhibitor of esophageal cancer cells according to claim 1, which is a mutant, or a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a fragment of the polypeptide. Compounds that inhibit the activation of calcium / calmodulin-dependent kinase II are represented by the chemical formulas (I) to (III)
The growth inhibitor of esophageal cancer cells according to claim 1, which is any compound represented by the formula:   A method of screening for an inhibitor of proliferation of esophageal cancer cells using as an index inhibition of activation of calcium / calmodulin-dependent kinase II or change in the expression level of calcium / calmodulin-dependent kinase II inhibitor 1 gene.   The method for screening for an esophageal cancer cell growth inhibitor according to claim 6, wherein inhibition of activation of calcium / calmodulin-dependent kinase II is measured using transcriptional activity of activator protein 1 as an index.