JP2009268381A - Cancer biomarker targeting cancer-specific acetic acid metabolism, method for examination, and therapeutic agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for examination in order to search for a new cancer-specific metabolic pathway and for cancer diagnosis targeting the same, and to provide a therapeutic agent, a method for screening thereof, and a method for evaluating treatment. <P>SOLUTION: The method for determining whether or not an individual is suffering from cancer includes measuring the expression level of acetyl-CoA synthase 2 or the amount of produced acetic acid under hypoxic conditions in a sample collected from the individual. The method for treating the cancer includes inhibiting the expression of the acetyl-CoA synthase 2 gene or an activity of the product thereof. A method for screening a substance for treating the cancer includes bringing a cell expressing the acetyl-CoA synthase 2 into contact with a test substance, measuring the expression of the acetyl-CoA synthase 2 gene in the cell, and selecting the substance inhibiting the expression or the activity of the acetyl-CoA synthase 2 in comparison with a control cell without contacting the test substance as a substance can treate the cancer. The method for evaluating anti-cancer treating effects on a patient suffering from the cancer includes evaluating the change of the expression of the acetyl-CoA synthase 2 in a sample containing a cancer site, and collected from the patient suffering from the cancer during treatment or undergoing the treatment with time. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cancer therapeutic agent targeting a cancer-specific acetate metabolism or a gene encoding acetyl-CoA synthetase 2, a therapeutic agent screening method, a test method, and a therapeutic evaluation method.

In general, glycolysis is enhanced in cancer cells, thereby preferentially obtaining energy. Therefore, enhanced glucose metabolism has become a good biomarker for cancer cells. For example, positron emission tomography (FDG-PET) using ¹⁸ F-labeled 2-fluor-2-deoxyglucose (FDG) has been established as a useful cancer cell diagnostic method. (Non-Patent Documents 1 and 2). However, the differentiation of cancer cells using glucose metabolism as an index is indistinguishable from normal cells and inflammatory cells with active glucose metabolism, and it is difficult to differentiate in cancers of some tissues. Patent Document 3).
Israel, O.M. et al. J Nucl Med 45, 2045-2051 (2004) Dang, C.I. V. & Semenza, G .; L. Trends in biochemical sciences 24, 68-72 (1999) Takashi Terauchi et al: Indications and limitations of PET screening. Consensus Cancer Treatment 2006 VOL. 5 NO. 3 Lotus Publishing

  An object of the present invention is to search for a novel cancer-specific metabolic pathway, and to provide a test method, a therapeutic agent, a screening method thereof, and a therapeutic evaluation method for cancer diagnosis targeting this.

The present inventors have found that a specific acetate metabolic pathway catalyzed by cytoplasmic acetyl-CoA synthetase 2 (hereinafter sometimes abbreviated as “Acss2”) is enhanced in cancer cells.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A method for determining whether or not the individual suffers from cancer, comprising measuring the expression level of acetyl CoA synthetase 2 in a sample collected from the individual. [2] The following (a) or (b) :
(A) a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of acetyl CoA synthetase 2; and (b) an expression level is measured using an antibody that specifically recognizes acetyl CoA synthetase 2. The method [3] of the above [1], comprising evaluating the change in the expression of acetyl-CoA synthetase 2 in a sample containing a cancer site collected from a cancer patient during or after treatment. Method for evaluating cancer therapeutic effect [4] (a) or (b) below:
(A) Nucleic acid probe or nucleic acid primer capable of specifically detecting the transcription product of acetyl CoA synthetase 2 (b) Whether the individual suffers from cancer, comprising an antibody that specifically recognizes acetyl CoA synthetase 2 Agent for determining whether or not [5] A method for determining whether or not the individual suffers from cancer, comprising measuring the amount of acetic acid produced in a sample collected from the individual [6] Hypoxic conditions The method [7] of [5] above, wherein the amount of acetic acid produced is measured in (a), (b) or (c) below:
(A) Antisense nucleic acid against mRNA of acetyl CoA synthetase 2 gene (b) siRNA against mRNA of acetyl CoA synthetase 2 gene
(C) A therapeutic agent for cancer comprising any of an expression vector capable of generating an antisense nucleic acid or siRNA against mRNA of acetyl CoA synthetase 2 gene [8] contacting a test substance with acetyl CoA synthetase 2 expressing cells Measuring the expression of acetyl-CoA synthetase 2 in the cells, and selecting a substance that suppresses the expression of acetyl-CoA synthetase 2 compared to a control cell not contacted with the test substance as a substance capable of treating cancer A method for screening a substance capable of treating cancer [9] contacting a test substance with acetyl CoA synthetase 2, measuring the activity of acetyl CoA synthetase 2, and not contacting the test substance In comparison, the activity of acetyl CoA synthetase 2 was suppressed. A method for screening a substance capable of treating cancer, comprising selecting the obtained substance as a substance capable of treating cancer [10] Determination of cancer comprising measuring the amount of radiolabeled acetic acid taken up by cells of an individual Method [11] A reagent for detecting cancer cells and the like containing radioactively labeled acetic acid is provided.

  By examining the expression variation of cancer-specific acetyl-CoA synthetase 2, rapid and simple diagnosis of cancer becomes possible. In particular, cancer-specific acetyl-CoA synthetase 2 has a markedly different expression level compared to normal cells under hypoxic conditions, so that cancer can be determined more easily. Furthermore, cancer can be treated by controlling the expression of acetyl-CoA synthetase 2 in cancer. Further, by using this gene or an expression product thereof and selecting a substance that controls the expression of the gene or the activity of the product, a novel therapeutic agent for cancer can be searched. Alternatively, by monitoring the expression of acetyl CoA synthetase 2, it is possible to evaluate the therapeutic effect on cancer.

  The present invention relates to a method for determining whether an individual suffers from cancer, which comprises measuring the expression level of acetyl-CoA synthetase 2 in a sample collected from the individual. It is known that acetyl CoA synthetase 2 whose expression level is measured in the determination method of the present invention catalyzes the reaction of ATP + acetate + CoA⇔AMP + diphosphatate + acetyl-CoA and participates in acetic acid metabolism in the cytoplasm. The human nucleotide sequence (amino acid sequence) of the acetyl-CoA synthetase 2 has Genbank accession numbers NM_001076552 [corresponding to SEQ ID NO. 1] (NP_001070020 [corresponding to SEQ ID NO. 2]) and NM_018867 [corresponding to SEQ ID NO. 3] (NP_061147 [ The mouse nucleotide sequence is registered as accession number NM — 019811 [corresponding to SEQ ID NO: 5] (NP — 062785 [corresponding to SEQ ID NO: 6]).

  In the present specification, the type of cancer is not particularly limited, and examples of the cancer include lung cancer, skin cancer, colon cancer, and breast cancer. The determination method of the present invention is suitably used for the above cancer.

  The individual to which the determination method of the present invention can be applied is not particularly limited, for example, an individual who may be suffering from cancer, an individual suspected of suffering from, or an individual who is actually suffering from cancer, Examples thereof include mammals such as humans, monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats and the like. Preferably, it is a human.

  The sample used in the determination method of the present invention is a cell collected from the individual to be tested and can contain a gene product (eg, RNA, protein, degradation product thereof, etc.) to be detected. Or it will not be restrict | limited especially if it is the structure | tissue containing a cell. For example, the brain, each part of the brain (eg, olfactory bulb, amygdaloid nucleus, basal cerebral sphere, hippocampus, hypothalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, Thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint Adipose tissue, skeletal muscle, and the like.

  Expression of acetyl CoA synthetase 2 gene in a sample collected from an individual is prepared by preparing a RNA (eg, total RNA, mRNA) fraction from the sample and detecting a transcription product of acetyl CoA synthetase 2 gene contained in the fraction. It can be examined by doing. The RNA fraction can be prepared using a known method such as guanidine-CsCl ultracentrifugation or AGPC, but using a commercially available RNA extraction kit (eg, RNeasy Mini Kit; manufactured by QIAGEN, etc.) High-purity total RNA can be prepared quickly and easily from a small amount of sample. As a means for detecting the transcript of the acetyl CoA synthetase 2 gene in the RNA fraction, for example, a method using hybridization (Northern blot, dot blot, DNA chip analysis, etc.) or PCR (RT-PCR, competitive PCR, And a method using real-time PCR and the like. Quantitative PCR methods such as competitive PCR and real-time PCR are preferred because they can detect changes in the expression of the acetyl-CoA synthetase 2 gene quickly and easily from a small amount of sample with high quantitativeness.

  In the case of Northern blot or dot blot hybridization, detection of the acetyl CoA synthetase 2 gene can be performed using, for example, a nucleic acid probe that can specifically detect the transcription product of acetyl CoA synthetase 2. In one preferred embodiment, the nucleic acid probe used in the determination method of the present invention has about 15 bases or more, preferably about 18 to about, contained in the nucleotide sequence represented by any of SEQ ID NOs: 1, 3, or 5. A polynucleotide comprising a continuous nucleotide sequence of 500 bases, more preferably from about 18 to about 200 bases, and even more preferably from about 18 to about 50 bases, or a complementary sequence thereof. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The nucleic acid used as the probe may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, an antisense strand can be used.

  The nucleic acid probe used in the determination method of the present invention is a polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence represented by any of SEQ ID NOs: 1, 3, or 5. Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. As stringent conditions, for example, a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by one or more times at 65 ° C. in 0.2 × SSC / 0.1% SDS Cleaning and the like. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted. The length of the nucleic acid probe is usually about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and further preferably about 18 to about 50 bases.

  A nucleic acid that functions as a probe capable of detecting the expression of the acetyl CoA synthetase 2 gene uses a primer set described later that can amplify a part or all of the transcription product of the gene, and is used for an individual (eg, human, monkey, mouse, rat) , Dogs, cows, horses, pigs, sheep, goats, cats, rabbits, hamsters, guinea pigs, etc.) [eg, hepatocytes, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, myocytes, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, obesity) Cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells Or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells exist [eg, brain, brain regions (eg, olfactory bulb, amygdala, basal sphere, hippocampus) , Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (eg, large intestine, small intestine) , Blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue, skeletal muscle, etc.] The above acetyl-CoA synthetase is amplified by colony or plaque hybridization from the above-mentioned cDNA or genomic DNA library derived from cells or tissues. It can be obtained by cloning the gene 2 gene or cDNA and, if necessary, using a restriction enzyme or the like to obtain a fragment of an appropriate length. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (described above). Alternatively, the nucleic acid is a commercially available DNA / RNA automatic synthesizer that converts part or all of the base sequence and / or its complementary strand sequence based on the base sequence information shown in any of SEQ ID NOs: 1, 3, or 5. It can also be obtained by chemically synthesizing using the above. Alternatively, a chip on which the nucleic acid is immobilized can be produced by directly synthesizing the nucleic acid in situ (on chip) on a solid phase such as silicon or glass.

  A nucleic acid that functions as a primer capable of amplifying a part or all of the transcript of the acetyl CoA synthetase 2 gene is based on the nucleotide sequence information shown in SEQ ID NO: 1, 3, or 5 and its complementary sequence A part of the strand sequence can be obtained by chemically synthesizing using a commercially available DNA / RNA automatic synthesizer or the like.

The nucleic acid is preferably labeled with a labeling agent in order to enable detection and quantification of the target nucleic acid. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ³² P], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, biotin- (strept) avidin can also be used for the binding between the probe and the labeling agent.

  In the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis, then transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, and prepared as described above. After specifically hybridizing in the hybridization buffer containing the labeled probe, the amount of labeled acetyl CoA synthetase 2 gene is measured by measuring the amount of label bound to the membrane for each band by an appropriate method. can do. Also in the case of dot blotting, the expression level of the acetyl CoA synthetase 2 gene can be measured by subjecting the membrane spotted with the RNA fraction to a hybridization reaction in the same manner and measuring the labeled amount of the spot.

  In the case of DNA chip analysis, for example, cDNA in which an appropriate promoter such as T7 promoter is introduced is synthesized from the RNA fraction prepared as described above by reverse transcription, and further cRNA is synthesized using RNA polymerase (this) Sometimes a labeled cRNA is obtained by using a mononucleotide labeled with biotin or the like as a substrate). The labeled cRNA is brought into contact with the above-described probe on which the probe is immobilized and subjected to a hybridization reaction, and the amount of label bound to each probe on the solid phase is measured to measure the expression level of the acetyl CoA synthetase 2 gene. can do.

According to another preferred embodiment, a quantitative PCR method is used as a method for measuring the expression of the acetyl CoA synthetase 2 gene. Examples of quantitative PCR include competitive PCR and real-time PCR.
As a set of oligonucleotides used as primers in PCR, for example, a nucleic acid primer capable of specifically detecting the transcription product of acetyl CoA synthetase 2 can be mentioned. In one preferred embodiment, the nucleic acid primer used in the determination method of the present invention is, for example, about 15 bases or more, preferably about 15 contained in the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, or 5. Designed to amplify a DNA fragment of about 100 bp to several kbp with a length of continuous nucleotide sequence of about 50 bases, more preferably about 15 to about 30 bases, more preferably about 15 to about 25 bases A set of polynucleotides that are hybridizable to a polynucleotide that is complementary to the polynucleotide (sense strand) sequence and a polynucleotide having a nucleotide sequence complementary to the polynucleotide sequence (antisense strand). Can be mentioned.

  Competitive RT-PCR is a method in which a known amount of another template nucleic acid that can be amplified by a set of primers that can amplify a target DNA is allowed to coexist in a reaction solution as a competitor to cause a competitive amplification reaction. A method for calculating the amount of target DNA by comparing the amounts. Therefore, in the case of competitive RT-PCR, in addition to the primer set described above, it can be amplified with the primer set, and can be distinguished from the amplification product of the target nucleic acid (ie, the transcription product of the acetyl CoA synthetase 2 gene) after amplification ( For example, a known amount of competitor nucleic acid is used (for example, different amplification sizes, different migration patterns of restriction enzyme-treated fragments). Since the target nucleic acid and the competitor nucleic acid compete for the primers and amplification occurs competitively, the quantity ratio of the amplified products reflects the quantity ratio of the original template. The competitor nucleic acid may be DNA or RNA. In the case of DNA, cDNA may be synthesized by reverse transcription reaction from the RNA fraction prepared as described above, and then PCR may be performed in the presence of the primer set and the competator. In the case of RNA, the competor is added to the RNA fraction. May be added to perform reverse transcription reaction, and the primer set may be further added to perform PCR. In the latter case, since the efficiency of the reverse transcription reaction is taken into consideration, the absolute amount of the original mRNA can be estimated.

On the other hand, real-time PCR is a method for monitoring the amount of amplification in real time using a fluorescent reagent, and requires an apparatus in which a thermal cycler and a spectrofluorometer are integrated. Such devices are commercially available. There are several methods depending on the fluorescent reagent to be used, and examples include the intercalator method, the TaqMan ^TM probe method, the Molecular Beacon method, and the like. In either case, after synthesizing cDNA from the RNA fraction prepared as described above by reverse transcription reaction, a reagent that emits fluorescence by binding to the above primer set and double-stranded DNA such as SYBR Green I, ethidium bromide ( Intercalator), a nucleic acid that can be used as the probe (however, the nucleic acid hybridizes to the target nucleic acid in the amplification region) and a fluorescent substance (eg, FAM, HEX, TET, FITC, etc.) and a quencher (Example: TAMRA, DABCYL, etc.) A fluorescent reagent (probe) such as a modified one (TaqMan ^TM probe or Molecular Beacon probe) is added to the PCR reaction system. Since the intercalator binds to the synthesized double-stranded DNA and emits fluorescence when irradiated with excitation light, the amount of amplification product generated can be monitored by measuring the fluorescence intensity, thereby increasing the amount of original template cDNA. Can be estimated. The TaqMan ^TM probe is an oligonucleotide that can be hybridized to the target nucleic acid amplification region with both ends modified with a fluorescent substance and a quenching substance, and hybridizes to the target nucleic acid during annealing, but does not emit fluorescence due to the presence of the quenching substance In the extension reaction, fluorescence is emitted by being decomposed by the exonuclease activity of DNA polymerase and releasing a fluorescent substance. Therefore, the amount of amplification product produced can be monitored by measuring the fluorescence intensity, thereby estimating the amount of the original template cDNA. The Molecular Beacon probe is an oligonucleotide that can be hybridized to the target nucleic acid amplification region with both ends modified with a fluorescent substance and a quenching substance, and can take a hairpin secondary structure, and is quenched when it has a hairpin structure. Fluorescence is not emitted due to the presence of the substance, but is emitted when the distance between the fluorescent substance and the quenching substance is increased by hybridization to the target nucleic acid during annealing. Therefore, the amount of amplification product produced can be monitored by measuring the fluorescence intensity, thereby estimating the amount of the original template cDNA. Since real-time RT-PCR can monitor the amount of PCR amplification in real time, electrophoresis is unnecessary, and the expression of the acetyl CoA synthetase 2 gene can be analyzed more rapidly.

  Alternatively, the expression of the acetyl CoA synthetase 2 gene in a sample collected from an individual is prepared by preparing a protein fraction from the sample and detecting a translation product of the gene contained in the fraction (ie, acetyl CoA synthetase 2). Can be examined. Detection of acetyl CoA synthetase 2 can be performed by an immunoassay (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody that specifically recognizes acetyl CoA synthetase 2, or acetyl CoA The acetic acid metabolic activity possessed by synthetase 2 can also be measured by measuring using a known technique. Alternatively, detection of acetyl CoA synthetase 2 can also be performed using a mass spectrometry method such as MALDI-TOFMS.

An antibody specifically recognizing acetyl CoA synthetase 2 is an acetyl CoA synthetase 2 polypeptide or a partial peptide having antigenicity thereof, specifically, the entire peptide sequence shown in any one of SEQ ID NOs: 2, 4, or 6. Alternatively, a partial peptide having a portion corresponding to an epitope can be used as an immunogen and can be produced by an existing general production method. In the present specification, the antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody or a single chain antibody, and binding properties thereof. Fragments are included, but are not limited to these. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) ₂ , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.

  In applying each immunological measurement method to the test method of the present invention, special conditions, operations and the like are not required to be set. A measurement system for acetyl CoA synthetase 2 may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and procedures in each method. For details of these general technical means, it is possible to refer to reviews, books and the like. For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62)), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Technologies (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Technologies (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technologies and Monoclonal Antibodies)) (published by Academic Press).

  The determination method of the present invention determines whether or not an individual is suffering from cancer by measuring the expression level of the acetyl CoA synthetase 2 gene whose expression varies in correlation with cancer by the above method. As shown in Examples described later, the expression level of the acetyl CoA synthetase 2 gene is higher in cancer cells than in normal cells. Therefore, there is a positive correlation between the expression level of the acetyl CoA synthetase 2 gene and the cancer incidence of the individual, and it can be determined that the higher the expression level of the gene, the higher the probability of being cancerous.

  For example, normal cells (tissues) and cancer cells from an individual confirmed to have no cancer (negative control) and an individual who is clinically judged to have cancer (positive control) The (tissue) is removed, and the expression level of the acetyl CoA synthetase 2 gene in the cell (tissue) suspected of being cancer removed from the target individual is compared with that of the positive control and the negative control. Alternatively, a correlation diagram between the expression level of the acetyl CoA synthetase 2 gene in the sample and the rate clinically determined to be cancer is prepared in advance, and the expression of the acetyl CoA synthetase 2 gene in cancer removed from the subject individual The level may be compared to its correlation diagram. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.

  Then, from the comparison result of the expression level of the acetyl CoA synthetase 2 gene, when the expression level of the acetyl CoA synthetase 2 gene to be measured is relatively high, it can be determined that the probability of cancer is relatively high. . Conversely, when the expression level of the acetyl CoA synthetase 2 gene to be measured is relatively low, it can be determined that the probability of cancer is relatively low.

  By applying the above cancer determination method to patients who have been treated for cancer or experimental animals (eg, monkeys, dogs, rats, mice, etc.) that have been administered cancer therapeutic drug candidate compounds, the therapeutic effect can be evaluated. It can be carried out. That is, the expression of the acetyl-CoA synthetase 2 gene in a sample containing a cancer site collected over time from a cancer patient who is undergoing treatment or treatment is measured and compared, and acetyl-CoA synthetase 2 after treatment is compared with that before treatment. If a tendency to suppress the increase in gene expression is shown, it can be determined that a therapeutic effect is observed.

The present invention also provides an agent for determining cancer, which can be preferably used in the determination method of the present invention. The agent for the determination is the above (a) or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of acetyl CoA synthetase 2; and (b) an antibody that specifically recognizes acetyl CoA synthetase 2. When the agent for determination includes two or more of the above-described nucleic acids and / or antibodies, each nucleic acid or antibody specifically recognizes a different part of the base sequence of the acetyl CoA synthetase 2 gene from each other, or of acetyl CoA synthetase 2 Different epitopes can be specifically recognized.

  When the agent of the present invention includes the reagent containing the nucleic acid (a) as a constituent, examples of the nucleic acid include the nucleic acid for probe or the oligonucleotide for primer described above in the determination method of the present invention.

Nucleic acid capable of detecting the expression of acetyl CoA synthetase 2 gene can be provided as a solid in a dry state or in an alcohol precipitate state, or dissolved in water or a suitable buffer (eg, TE buffer). Can also be provided. When used as a labeled probe, the nucleic acid can be provided in a state of being previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substance and can be used after labeling.
Alternatively, the nucleic acid can be provided in a state immobilized on an appropriate solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like. As immobilization means, a functional group such as an amino group, an aldehyde group, an SH group, or biotin is introduced into a nucleic acid in advance, and a functional group capable of reacting with the nucleic acid on a solid phase (eg, aldehyde) Group, amino group, SH group, streptavidin, etc.), and the solid phase and the nucleic acid are cross-linked by covalent bond between the two functional groups, or the polyanionic nucleic acid is coated with the polycation and the static phase is coated. Examples of the method include immobilization of nucleic acid using electric coupling, but are not limited thereto.

  The nucleic acid contained in the agent for determination is constructed so that the expression of the acetyl CoA synthetase 2 gene can be detected by the same method (eg, Northern blot, dot blot, DNA array technology, quantitative RT-PCR, etc.) It is particularly preferred that

  When the agent of the present invention contains the reagent containing the antibody (b) as a component, examples of the antibody include the antibodies described above in the determination method of the present invention.

  The reagent constituting the agent of the present invention is not only a nucleic acid or antibody capable of detecting the expression of the acetyl CoA synthetase 2 gene, but also other substances necessary in the reaction for detecting the expression of the gene, It is possible to further contain a substance that does not adversely influence the reaction by storing in the above. Alternatively, the reagent may be provided with a separate reagent containing other substances necessary in the reaction for detecting the expression of the acetyl CoA synthetase 2 gene. For example, when the reaction for detecting the expression of the acetyl CoA synthetase 2 gene is PCR, examples of the other substance include a reaction buffer, dNTPs, and a heat-resistant DNA polymerase. When competing PCR or real-time PCR is used, it can further include a competitor nucleic acid, a fluorescent reagent (such as the above intercalator and fluorescent probe), and the like. When the reaction for detecting the expression of the acetyl CoA synthetase 2 gene is an antigen-antibody reaction, examples of the other substance include a reaction buffer, a competent antibody, and a labeled secondary antibody (for example, a primary antibody). In the case of rabbit anti-human acetyl CoA synthetase 2 antibody, mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), blocking solution and the like can be mentioned.

  The present invention also relates to a method for determining whether or not an individual suffers from cancer, comprising measuring the amount of acetic acid produced in a sample collected from the individual. Individuals and samples to which the determination method of the present invention can be applied are the same as described above.

  The amount of acetic acid produced in a sample collected from an individual can be examined, for example, by quantifying acetic acid released into the medium by culturing the sample in the medium. The amount of acetic acid can be measured using a known method such as thin layer chromatography (TLC, Thin Layer Chromatography) or high performance liquid chromatography (HPLC, High Performance Liquid Chromatography). The amount of acetic acid can be quantified quickly and easily using a kit for use (eg, F-kit; manufactured by R-Biopharm AG, etc.).

Cancer-specific acetic acid metabolism is not just a change in the amount of metabolism in cancer cells compared to normal cells, but, for example, by placing cancer cells and normal cells collected from individuals under hypoxic conditions, It is characterized by a remarkable difference in the transition of production. Specifically, as shown in the Examples below, since the amount of acetic acid produced in cancer cells is higher in hypoxic conditions than in normoxic conditions, the difference in the amount of acetic acid produced between cancer cells and normal cells. Is greater under hypoxic conditions. Therefore, in the method of the present invention, by measuring the amount of acetic acid produced under hypoxic conditions, it can be more clearly determined whether or not the individual is suffering from cancer. Here, the “normal oxygen condition” refers to a condition close to the atmospheric oxygen concentration. In the examples, the conditions of 95% air and 5% CO ₂ (corresponding to 20% O _2, 74% N _2, 5% CO ₂ ) prepared using a CO ₂ incubator were used as normal oxygen conditions. The “low oxygen condition” refers to a condition in which the oxygen concentration is lower than the normal oxygen condition (for example, a concentration below 20%, preferably 1.0-1.5%). In the examples, the conditions of 1.2% O _2, 93.8% N _{2, and} 5% CO ₂ prepared using a Personal Multi Gas Incubator (Astec) were defined as low oxygen conditions.

  The determination method of the present invention determines whether or not an individual is suffering from cancer by measuring the amount of acetic acid produced whose expression varies in correlation with cancer by the above method. As mentioned above, cancer cells produce more acetic acid than normal cells. Therefore, there is a positive correlation between the amount of acetic acid produced and the cancer incidence of the individual, and it can be determined that the higher the amount of acetic acid produced, the higher the probability of cancer.

  For example, normal cells (tissues) and cancer cells from an individual confirmed to have no cancer (negative control) and an individual who is clinically judged to have cancer (positive control) (Tissue) is removed, and the amount of acetic acid produced in cells (tissue) suspected of being cancer removed from the target individual is compared with that of the positive control and negative control. Alternatively, a correlation diagram between the amount of acetic acid produced in cancer and the rate clinically judged to be cancer can be prepared in advance, and the amount of acetic acid produced in cancer removed from the target individual can be compared with that correlation diagram. Good. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.

  And from the comparison result of the acetic acid production amount, when the acetic acid production amount to be measured is relatively high, it can be determined that the probability of cancer is relatively high. Conversely, when the amount of acetic acid produced as a measurement target is relatively low, it can be determined that the probability of cancer is relatively low.

As described above, in the acetyl CoA synthetase 2 gene of the present invention, a remarkable difference is observed in the transition of gene expression between cancer cells and normal cells. Therefore, these genes are not just diagnostic markers for cancer, but also promising therapeutic target candidates for cancer.
That is, the present invention also provides a method for treating cancer by suppressing the expression of acetyl CoA synthetase 2 gene or the activity of its product in an individual.

Specifically, the method comprises administering to an individual in need of cancer treatment an effective amount of a substance that inhibits the expression of the acetyl CoA synthetase 2 gene or the activity of their products. Accordingly, the present invention also provides the following (a), (b) or (c):
(A) Antisense nucleic acid against mRNA of acetyl CoA synthetase 2 gene (b) siRNA against mRNA of acetyl CoA synthetase 2 gene
(C) A therapeutic agent for cancer, comprising either an antisense nucleic acid or an expression vector capable of generating siRNA against mRNA of acetyl CoA synthetase 2 gene.

(A) Antisense nucleic acid against mRNA of acetyl CoA synthetase 2 gene In the present invention, the antisense nucleic acid against mRNA of acetyl CoA synthetase 2 gene is a base sequence complementary or substantially complementary to the base sequence of the mRNA or A nucleic acid containing a part of the nucleic acid has a function of suppressing protein synthesis by forming a specific and stable double strand with the target mRNA and binding it. Here, “substantially complementary” has a complementarity of about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more between base sequences. That means. In one preferred embodiment, the antisense nucleic acid against mRNA of acetyl CoA synthetase 2 gene in the present invention is a polynucleotide comprising any of the following nucleotide sequences:
(1) A nucleotide sequence complementary to the nucleotide sequence represented by any of SEQ ID NOs: 1, 3 or 5 (2) Complementary to the nucleotide sequence of the initial transcript that yields the nucleotide sequence of (1) above by post-transcriptional processing Nucleotide sequence (3) Nucleotide sequence which is a partial sequence of (1) or (2) above 15 nucleotide sequence Antisense nucleic acid is polydeoxyribonucleotide containing 2-deoxy-D-ribose, D-ribose Other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) Or other polymers containing special bonds (However, the polymer contains nucleotides having an arrangement that allows base pairing and base attachment as found in DNA and RNA). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things, eg proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signals Peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides, etc.) having side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, Containing metal, radioactive metal, boron, oxidizing metal, etc.), containing an alkylating agent, or having a modified bond (eg, α-anomeric nucleic acid, etc.) Also good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

  As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the acetyl CoA synthetase 2 gene. Since the position of the acetyl-CoA synthetase 2 gene on the chromosome has been clarified in humans and mice, the genomic sequence of the region where the gene is present and the cDNA (or EST) base sequence of the acetyl-CoA synthetase 2 gene are BLAST. Intron sequences can be determined by comparison using a homology search program such as FASTA.

The target region of the antisense nucleic acid of the present invention is particularly limited in length as long as the antisense nucleic acid is hybridized and as a result, translation into a protein that is an acetyl CoA synthetase 2 gene product is inhibited. However, it may be the entire sequence or a partial sequence of mRNA encoding these proteins, and includes a short sequence of about 10 bases and a long sequence of mRNA or the initial transcript. In view of easiness of synthesis, antigenicity, intracellular migration, and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but not limited thereto. Specifically, 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, translation initiation codon, protein coding region, ORF translation stop codon, 3 ′ end of acetyl CoA synthetase 2 gene An untranslated region, 3 ′ end palindromic region, 3 ′ end hairpin loop or the like may be selected as a preferred target region of an antisense nucleic acid, but is not limited thereto.
Although the nucleotide sequence of the portion that hybridizes with the target mRNA in the antisense nucleic acid varies depending on the base composition of the target sequence, it can hybridize with the mRNA of the acetyl CoA synthetase 2 gene under physiological conditions. Those having about 90% or more (preferably 95% or more, most preferably 100%) identity to the complementary sequence. The identity in the nucleotide sequence is determined using, for example, the homology calculation algorithm NCBI BLAST-2 (National Center for Biotechnology Information Local Alignment Search Tool) and the following conditions (gap open = 5; gap extension = 2; x_dropoff = 50) Expected value = 10; filtering = ON).

  Furthermore, the antisense nucleic acid of the present invention not only hybridizes with the mRNA of the acetyl CoA synthetase 2 gene and the initial transcription product to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA. It may be one that forms a triplex and can inhibit transcription to RNA (antigene).

The nucleotide molecules constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the hydroxyl group at the 2 ′ position of the sugar (ribose) of each nucleotide is represented by —OR (R is, for example, CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN and the like may be substituted). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl, etc. Is mentioned.

  The conformation of the sugar part of RNA is dominated by C2′-endo (S type) and C3′-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to N-type by crosslinking the 2 ′ oxygen and the 4 ′ carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleos). Nucleotides Nucleic Acids, 22, 1619-21, 2003) can also be preferably used.

  The antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of acetyl CoA synthetase 2 gene, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems). , Beckman, etc.) and can be prepared by synthesizing a sequence complementary thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.

(B) siRNA against mRNA of acetyl CoA synthetase 2 gene
When a short double-stranded RNA is introduced into a cell, a phenomenon called RNA interference (RNAi), in which mRNA complementary to the RNA is degraded, has been known in nematodes, insects, plants, etc. Since it has been confirmed that this phenomenon occurs widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme. siRNA can be designed as appropriate using commercially available software (eg, RNAi Designer; Invitrogen) based on the base sequence information of the target mRNA.

  The siRNA is typically a double-stranded oligo RNA consisting of RNA having a sequence complementary to the nucleotide sequence of mRNA of the target gene or a partial sequence thereof (hereinafter referred to as target nucleotide sequence) and its complementary strand. A single-stranded RNA in which a sequence complementary to a target nucleotide sequence (first sequence) and its complementary sequence (second sequence) are linked via a hairpin loop portion, and the hairpin loop type By adopting the structure, RNA in which the first sequence forms a double-stranded structure with the second sequence (small hairpin RNA: shRNA) is also a preferred embodiment of siRNA. One preferred embodiment of the siRNA of the present invention includes a polynucleotide comprising the nucleotide sequence shown in any of SEQ ID NOs: 7, 9, 11 and 13 (the sequence is a sequence as shRNA), The nucleotide sequences set forth in SEQ ID NOs: 8, 10, 12, and 14 serve as target sequences for the RNAi effect. Therefore, more preferred siRNA of the present invention includes a polynucleotide comprising a nucleotide complementary to the nucleotide sequence shown in any of SEQ ID NOs: 8, 10, 12, and 14. The RNA sequences shown in SEQ ID NOs: 7 to 14 are described as DNA for convenience.

  The length of the portion complementary to the target nucleotide sequence contained in the siRNA is usually about 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more (typically about 21 to 23 bases in length). The length is not particularly limited as long as RNA interference can be caused. If the siRNA is longer than 23 bases, the siRNA is degraded in the cell to produce siRNA of about 20 bases, so theoretically, the upper limit of the length of the portion complementary to the target nucleotide sequence is It is the full length of the nucleotide sequence of the target gene mRNA (mature mRNA or early transcript). However, considering the ease of synthesis and antigenicity problems, the length of the complementary portion is, for example, about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less. That is, the length of the complementary portion is, for example, about 15 bases or more, preferably about 18 to about 200 bases, more preferably about 20 to about 50 bases, and further preferably about 20 to about 30 bases.

  Further, the total length of siRNA is usually about 18 bases or more, for example, about 20 bases or more (typically about 21 to 23 bases in length), but is not particularly limited as long as it can cause RNA interference. In theory, there is no upper limit on the length of siRNA. However, considering the ease of synthesis, antigenic problems, etc., the length of siRNA is, for example, about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less. That is, the length of siRNA is, for example, about 18 bases or more, preferably about 18 to about 200 bases, more preferably about 20 to about 50 bases, and further preferably about 20 to about 30 bases. In addition, the length of shRNA shall be shown as the length of the double-stranded part when taking a double-stranded structure.

  The target nucleotide sequence and the sequence complementary to that contained in the siRNA are preferably completely complementary. However, with respect to base mutations at positions off the center of siRNA (which may be within the range of identity of at least 90% or more, preferably 95% or more), the cleavage activity due to RNA interference is not completely lost. Partial activity may remain. On the other hand, the mutation of the base at the center of siRNA has a large influence, and the cleavage activity of mRNA due to RNA interference can be extremely reduced.

  The siRNA may have additional bases that do not form base pairs at the 5 'or 3' ends. The length of the additional base is usually 5 bases or less. The additional base may be DNA or RNA, but the use of DNA can improve the stability of siRNA. Examples of such an additional base sequence include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and tttt-3. Examples of the sequence include ', uuuu-3', but are not limited thereto.

  The length of the loop portion of the shRNA hairpin loop is not particularly limited as long as it can cause RNA interference, but is usually about 5 to 25 bases. The nucleotide sequence of the loop portion is not particularly limited as long as it can form a loop and shRNA can cause RNA interference.

  The above-mentioned polynucleotide determines the target sequence based on the mRNA sequence of the acetyl CoA synthetase 2 gene (for example, the nucleotide sequence represented by any one of SEQ ID NOs: 1, 3 or 5) or the chromosomal DNA sequence, and commercially available DNA / It can be prepared by synthesizing a complementary nucleotide sequence using an automatic RNA synthesizer (Applied Biosystems, Beckman, etc.). For siRNA, the sense strand and the antisense strand were respectively synthesized by a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, and then about 30 to about 70 ° C. For about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to overlap each other, annealing them, and ligating with ligase. It can also be prepared by synthesizing a short hairpin RNA (shRNA) serving as a precursor of siRNA and cleaving it with a dicer.

(C) Expression vector capable of producing antisense nucleic acid or siRNA against mRNA of acetyl CoA synthetase 2 gene The therapeutic agent of the present invention expresses any polynucleotide selected from the above (a) or (b). The obtained (encoding) expression vector can be used as an active ingredient. In one preferred embodiment, the expression vector capable of producing an antisense nucleic acid or siRNA against the mRNA of the acetyl CoA synthetase 2 gene in the present invention is an expression vector comprising a polynucleotide comprising any of the following nucleotide sequences;
(1) A nucleotide sequence complementary to the nucleotide sequence represented by any of SEQ ID NOs: 1, 3 or 5 (2) Complementary to the nucleotide sequence of the initial transcript that yields the nucleotide sequence of (1) above by post-transcriptional processing Nucleotide sequence (3) nucleotide sequence (4) which is a partial sequence of (1) or (2) above 15 nucleotide sequences and complementary to the nucleotide sequence shown in any of SEQ ID NOs: 8, 10, 12 and 14 Nucleotide sequence In the expression vector, the above-mentioned polynucleotide or a nucleic acid encoding it (preferably DNA) exhibits promoter activity in cells (for example, cancer cells) of an individual (preferably human) to be administered. Is operably linked to the resulting promoter.

  The promoter used is not particularly limited as long as it can function in the cells of the individual to be administered. As the promoter, a pol I promoter, pol II promoter, pol III promoter, or the like can be used. Specifically, SV40-derived early promoter, viral promoter such as cytomegalovirus LTR, mammalian constituent protein gene promoter such as β-actin gene promoter, and RNA promoter such as tRNA promoter are used.

  When the expression of siRNA is intended, it is preferable to use a pol III promoter as the promoter. Examples of the polIII promoter include U6 promoter, H1 promoter, tRNA promoter and the like.

  The expression vector of the present invention preferably contains a transcription termination signal, that is, a terminator region, downstream of the above-mentioned polynucleotide or the nucleic acid encoding it. Furthermore, a selection marker gene for selecting transformed cells (a gene that imparts resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, and the like) can be further contained.

  The type of vector used for the expression vector in the present invention is not particularly limited, and examples of suitable vectors for administration to mammals such as humans include viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses. Among these, adenovirus has advantages such as extremely high gene transfer efficiency and can be introduced into non-dividing cells. However, since integration of the transgene into the host chromosome is extremely rare, gene expression is transient and usually lasts only about 4 weeks. Considering the persistence of the therapeutic effect, use of an adeno-associated virus that has relatively high gene transfer efficiency, can be introduced into non-dividing cells, and can be integrated into the chromosome via an inverted terminal repeat (ITR) Also preferred.

  As another preferred example of a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the acetyl CoA synthetase 2 gene or a part thereof, the mRNA is specifically cleaved within the coding region. The ribozyme obtained is mentioned. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA since it only uses RNA as a substrate. Acetyl-CoA synthetase 2 gene When mRNA has a double-stranded structure by itself, the target sequence is single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res. , 29 (13): 2780-2788 (2001)].

  Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of acetyl CoA synthetase 2 gene or a part thereof is provided in a special form such as liposome or microsphere, or applied to gene therapy Or can be given in an added form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside linkage. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

  The protein expression inhibitory activity of these nucleic acids can be examined using a transformant introduced with the acetyl CoA synthetase 2 gene, a gene expression system in vivo or in vitro, or a protein translation system in vivo or in vitro.

  The substance that suppresses the expression of the acetyl CoA synthetase 2 gene in the present invention is a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of mRNA of the acetyl CoA synthetase 2 gene as described above or a part thereof. Without limitation, other substances such as low molecular weight compounds may be used as long as they directly or indirectly inhibit the production of acetyl CoA synthetase 2 gene protein. Such a substance can be obtained, for example, by the screening method of the present invention described later.

In the present invention, the “substance that suppresses the activity of the acetyl CoA synthetase 2 gene product” may be any substance as long as it suppresses the physiological function of the acetyl CoA synthetase 2 gene product once functionally produced. Specifically, examples of the substance that suppresses the activity of the acetyl CoA synthetase 2 gene product include neutralizing antibodies against proteins that are acetyl CoA synthetase 2 gene products. The antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known antibody or antiserum production methods.
In a preferred embodiment, since the antibody against the acetyl CoA synthetase 2 gene product is used as a pharmaceutical for human administration, the antibody (preferably a monoclonal antibody) has a risk of showing antigenicity when administered to a human. Are specifically human antibodies, humanized antibodies, mouse-human chimeric antibodies, and particularly preferably fully human antibodies. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods. In addition, a fully human antibody can be produced from a human-human (or mouse) hybridoma, but in order to provide a large amount of antibody stably and at low cost, a human antibody-producing mouse or a phage display method is used. It is desirable to manufacture using.

  In another preferred embodiment, the substance that inhibits the activity of the acetyl CoA synthetase 2 gene product is a low molecular weight compound that exhibits antagonist activity against the product. Such a compound can be obtained, for example, using the screening method of the present invention described later.

  An antisense nucleic acid, siRNA, or a nucleic acid capable of generating an antisense nucleic acid or siRNA against the mRNA of the acetyl CoA synthetase 2 gene of the present invention (see “Antisense nucleic acid, siRNA or anti-RNA against the mRNA of the acetyl CoA synthetase 2 gene of the present invention”). A “nucleic acid capable of producing a sense nucleic acid or siRNA” may be abbreviated as “an antisense nucleic acid of the present invention” hereinafter), or a neutralizing antibody against an acetyl CoA synthetase 2 gene product, or an acetyl CoA synthetase 2 gene product A pharmaceutical containing a low molecular weight compound exhibiting antagonistic activity against a human or other mammals (eg, mouse, rat, rabbit, etc.) has a low toxicity and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form. Hits , Swine, cattle, cats, dogs, oral or parenteral monkeys, etc.) (e.g., intravascular administration, such as subcutaneous administration) to.

When the antisense nucleic acid or the like of the present invention is used as a medicament such as the above therapeutic agent for cancer, it can be formulated and administered according to a method known per se. That is, the therapeutic agent of the present invention is inserted into a suitable expression vector for mammalian cells such as a retrovirus vector, adenovirus vector, adeno-associated virus vector or the like in a functional manner and then formulated according to conventional means. be able to. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving the pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .

  The antisense nucleic acid of the present invention or the like, or a neutralizing antibody against the acetyl CoA synthetase 2 gene product, or a medicine containing a low molecular weight compound exhibiting antagonist activity against the acetyl CoA synthetase 2 gene product may be administered as such. Or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration contains a neutralizing antibody or low molecular weight compound or a salt thereof such as the antisense nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient. It's okay. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, for example, the above-described antisense nucleic acid of the present invention, such as a neutralizing antibody or a low molecular weight compound or a salt thereof, is dissolved, suspended, or suspended in a sterile aqueous liquid or oily liquid usually used for injection. It can be prepared by emulsification. 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) additive of hydrogenated castor oil)), and the like. 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. The prepared injection solution is preferably filled in a suitable ampoule. A suppository used for rectal administration may be prepared by mixing a neutralizing antibody or a low molecular weight compound such as the above-described antisense nucleic acid with a normal suppository base.

  Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The neutralizing antibody or low molecular weight compound such as the antisense nucleic acid of the present invention of the present invention usually contains, for example, 5 to 500 mg per dosage unit dosage form, especially 5 to 100 mg for injections and 10 to 250 mg for other dosage forms. It is preferable.

  The dose of the above-mentioned pharmaceutical or veterinary drug containing a neutralizing antibody or low molecular weight compound such as the antisense nucleic acid of the present invention varies depending on the administration subject, symptoms, administration route, etc., for example, adult cancer patients In this case, the antisense nucleic acid of the present invention as a single dose is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight. Is conveniently administered by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

  Each of the above-described compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with a neutralizing antibody or a low molecular weight compound such as the antisense nucleic acid of the present invention.

As described above, there is a possibility that cancer can be treated by suppressing the expression of the acetyl CoA synthetase 2 gene or the activity of its product.
Therefore, the present invention also provides that a test substance is contacted with an acetyl CoA synthetase 2 expressing cell, the expression of the acetyl CoA synthetase 2 gene in the cell is measured, and compared with a control cell that is not contacted with the test substance. Provided is a method for screening a substance for treating cancer, comprising selecting a substance that suppresses the expression of acetyl-CoA synthetase 2 as a substance capable of treating cancer.

  The acetyl-CoA synthetase 2-expressing cell is not particularly limited as long as the cell itself or any cell containing it (eg, blood, tissue, organ, etc.). Blood, tissue, organ, etc. may be isolated from the living body and cultured, or the test substance may be administered to the living body itself and the biological sample may be isolated after a lapse of a certain time.

  Examples of test substances include proteins, peptides, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc., and these substances are novel. It may be a well-known thing.

  In the case of using isolated cells / tissues, the contact of the test substance with the cells is, for example, a medium suitable for culturing the cells / tissues (eg, a minimum containing about 5 to 20% fetal bovine serum). Essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI 1640 medium, 199 medium, F12 medium, etc.) and various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris hydrochloride buffer) A test substance is added to the solution, borate buffer solution, acetate buffer solution and the like, and the cells are incubated for a predetermined time. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of, for example, about 0.1 nM to about 100 nM. Examples of the incubation time include about 10 minutes to about 24 hours.

  The expression level of the acetyl CoA synthetase 2 gene in the acetyl CoA synthetase 2 expressing cells can be measured in the same manner as in the test method of the present invention, using the reagent contained in the test kit of the present invention described above.

  For example, in the above screening method, when the expression level (mRNA amount or protein amount) of the acetyl CoA synthetase 2 gene in the presence of the test substance is significantly inhibited compared to the expression level in the absence of the test substance, The test substance can be selected as a candidate for a substance having a cancer therapeutic effect.

  Symptoms of administration of an acetyl-CoA synthetase 2 gene expression inhibitor (either free or salt form) obtained using the screening method of the present invention to an individual who has developed cancer By examining the presence or absence of improvement, it is possible to confirm the presence or absence of an actual cancer therapeutic effect.

  When the substance obtained using the screening method of the present invention is used as a therapeutic agent for cancer, it can be formulated in the same manner as any substance that suppresses the expression of the acetyl-CoA synthetase 2 gene or the activity of its product. It can be administered orally or parenterally to humans or other mammals by similar routes and dosages.

  The present invention also includes contacting a test substance with an acetyl-CoA synthetase 2-expressing cell, measuring the activity of the acetyl-CoA synthetase 2 gene product in the cell, and comparing acetyl with respect to a control cell not contacted with the test substance. Provided is a method for screening a substance for treating cancer, comprising selecting a substance that suppresses the activity of the CoA synthetase 2 gene product as a substance capable of treating cancer. The cells used in this method, the type of test substance, the mode of contact between the test substance and the cells, and the like are the same as in the method using the expression of the acetyl CoA synthetase 2 gene as an indicator.

  The measurement of the activity of the acetyl CoA synthetase 2 gene product in the above screening method can be carried out by appropriately selecting a method known per se according to each protein. For example, when a substance (eg, ligand) that interacts with a gene product and exerts its physiological activity is known, the test substance is contacted with the immobilized acetyl-CoA synthetase 2 gene product in the presence of the labeled substance. And solid-liquid separation, and then measuring the amount of the label bound to the solid phase, thereby competitively binding to the ligand product (eg, agonist, antagonist), the gene product and the ligand A substance that changes the binding property with the substance can be selected. By simultaneously measuring the physiological activity (eg, enzyme activity) of the gene product, it is possible to identify whether the substance that binds to the gene product competitively with the ligand substance is an agonist or an antagonist.

  For example, in the above screening method, when the activity of the acetyl-CoA synthetase 2 gene product in the presence of the test substance is significantly inhibited compared to the activity in the absence of the test substance, the test substance is treated with cancer. It can be selected as a candidate for a substance having a therapeutic effect.

  A substance that suppresses the activity of the acetyl-CoA synthetase 2 gene (which may be a free form or a salt form) obtained by using the screening method of the present invention is administered to an individual who has developed cancer, and its symptoms By examining the presence or absence of improvement, it is possible to confirm the presence or absence of an actual cancer therapeutic effect.

  When the substance obtained using the screening method of the present invention is used as a therapeutic agent for cancer, it can be formulated in the same manner as any substance that suppresses the expression of the acetyl-CoA synthetase 2 gene or the activity of its product. It can be administered orally or parenterally to humans or other mammals by similar routes and dosages.

  The acetyl-CoA synthetase 2 of the present invention is also generally known as an enzyme that fixes acetic acid in cells, while it catalyzes the reversible reaction of ATP + acetate + CoA ＋ AMP + diphosphate + acetyl-CoA, so that uptake of acetic acid in cancer compared to normal cells The amount increases. Therefore, the present invention also relates to a method for determining cancer, comprising measuring the amount of radiolabeled acetic acid taken up by an individual cell.

  The individual subject to the determination method of the present invention is not particularly limited. For example, an individual who may be suffering from cancer, an individual suspected of suffering from cancer, or an individual who is actually suffering from cancer Examples thereof include mammals such as humans, monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep and goats. Preferably, it is a human.

  The cells used in the determination method of the present invention include cancer cells or cancer cells of individuals who are likely to suffer from cancer, suspected of suffering from cancer, or who are actually suffering from cancer. Tissues including, for example, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver , Gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, Examples thereof include bone, joint, adipose tissue, skeletal muscle, or cells thereof. In addition, these cells may be collected from an individual or may remain in the living body, that is, the method of the present invention may be applied in vitro or in vivo. The cancer to which the determination method of the present invention can be applied is not particularly limited, and examples thereof include lung cancer, skin cancer, colon cancer and breast cancer, and can be preferably applied.

As the radioisotope in acetic acid to be radiolabeled, for example, [ ³ H], [ ¹⁸ O], [ ¹¹ C], [ ¹⁴ C] and the like are used. Preferably, [ ¹¹ C], [ ¹⁴ C]. ^Furthermore, it is also used labeled ^[18 F] fluoro acetic acid ^[18 F].

The determination method of the present invention includes positron emission tomography (PET), single photon emission computed tomography (SPECT), nuclides that do not emit positrons ([ ³ H], [ ¹⁴ C], etc.), liquid Scintillation counters and γ counters are used, but since a radiopharmaceutical labeled with the elements that make up the body, such as oxygen and carbon, is used, it is possible to reflect physiological biochemical functions. A photographic method (PET) is mentioned.

  The determination method of the present invention determines whether or not an individual suffers from cancer by measuring the amount of acetic acid taken up by cells whose expression varies in correlation with cancer by the above method. As shown in Examples described later, cancer cells have higher acetic acid uptake levels than normal cells. Therefore, in the above determination, there is a positive correlation between the acetic acid uptake level and the cancer incidence of the individual, and it can be determined that the higher the acetic acid uptake level, the higher the probability of cancer.

  For example, radiolabeled acetic acid is administered to a human suffering from or suspected of having cancer. By positron emission tomography (PET), the uptake level of acetic acid at a specific location can be compared with surrounding normal cells (tissues), and it can be determined whether or not it is cancer based on the results output in the video etc. . Comparison of uptake levels is preferably performed based on the presence or absence of significant differences.

  Then, from the comparison result of the acetic acid uptake level, when the uptake level of acetic acid to be measured is relatively high, it can be determined that the probability of cancer is relatively high. Conversely, when the uptake level of acetic acid to be measured is relatively low (a level comparable to that of the surrounding normal tissue), it can be determined that the probability of cancer is relatively low.

The present invention also provides a reagent for detecting cancer cells, which contains radiolabeled acetic acid, which can be preferably used in the determination method of the present invention.
In addition to radiolabeled acetic acid, the reagent further contains another substance necessary for the reaction for detecting the acetic acid, which does not adversely affect the reaction when stored in the coexisting state. Can do. For example, as a pharmaceutical carrier, a stabilizer such as ascorbic acid, a pH adjuster such as an acid or a base, a buffer such as a phosphate buffer, or an isotonic agent such as a physiological saline can be used. . Alternatively, the reagent may be provided with a separate reagent containing other substances necessary in the reaction to detect the acetic acid.

  The detection reagent of the present invention is optimally administered by intravenous injection, but can be administered by other general parenteral means (for example, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.). The dose is appropriately determined depending on the patient's weight, age, sex, and various conditions such as a measuring instrument represented by a detection device. Generally, in the case of an adult patient, the radioactivity is in the range of 10 MBq to 25 MBq, preferably 15 MBq to 20 MBq.

  The present invention will be described in more detail with reference to the following examples, but these are merely examples and do not limit the scope of the present invention.

Example 1 Metabolic Characteristics of Cancer Cells Mouse-derived cancer cells (Lung carcinoma, LLC1 (LLC); melanoma, B16; colon carcinoma, Colon-26 (Colon); The metabolite was measured using 3T3 clone A31 (3T3)). First, using a 24-well plate, 5 × 10 ⁴ cells per well were cultured in 1 mL of Dulbecco's modified Eagle's (DME) medium (10% Fetal bovine serum, antibiotics) under normoxic conditions (CO ₂ culture). And 5% CO ₂ in air, 37 ° C.) for 24 hours. Thereafter, the medium was replaced with a new DME medium, and cultured for 24 hours under normoxic and hypoxic conditions (Personal Multi Gas Incubator [Aspec], 1.2% O ₂ , 93.8% N ₂ , and 5% CO ₂ ). did. Culture medium was collected and ion chromatography (ion chromatography (Shimadzu); ICS Sep ION-300 ion-exclusion column (300 x 7.8 mm id, Transgenomic); flow rate 0.4 mL / min; temperature 70 ° C). , Eluent 0.0085N H ₂ SO ₄ ; ultraviolet detector (Shimadzu) 210 nm], after confirming the content of organic acid and sugar roughly, quantification of metabolites using F-kit (R-Biopharm AG) Analysis was performed.
The results are shown in FIG. Cancer cells were found to produce more acetic acid than normal cells. Cancer cells have also been shown to produce more acetic acid when placed under hypoxic conditions. On the other hand, normal cells hardly produced acetic acid under hypoxic conditions.

Example 2 Gene Expression Analysis of Cancer Cells Cells (LLC, B16, Colon, C127I, 3T3) (1-6 × 10 ⁶ cells) cultured under normoxic and hypoxic conditions in the same manner as in Example 1. ) Was collected, and RNA was purified using a Micro-to-Midi total RNA purification system (Invitrogen Life Technology). Using this, gene expression analysis was performed using a real-time quantitative RT-PCR method. The analysis, using the Comparative C _T method, the internal standard was used beta-actin. In addition, the reaction was carried out using ABI PRISM 7000 sequence detection system (Applied Biosystems), and Taqman One-Step RT-PCR Master mix reagents (Taqman gene expres- sion) was used as the reaction reagent.
The results are shown in FIG. Examination of gene expression of enzymes that are expected to be related to various acetate metabolisms revealed that cytoplasmic acetyl-coenzyme A (CoA) synthetase 2 is highly expressed in cancer cells and further improved in hypoxia. It became clear. Its expression pattern was similar to the pattern of acetic acid production.

Example 3 Role of Acss2 in Acetic Acid Production of Cancer Cells For the purpose of examining whether Acss2 contributes to acetic acid production in cancer cells (LLC, B16, Colon, C127I), an inhibition experiment of Acss2 was performed. First, in order to cause stable RNA interference (RNAi) in the cells, gene introduction was performed using Misslative transduction particles (SHVRS-NM 019811; Sigma) to create an Ass2 knockdown cancer cell line. As shRNA for Acss2, shRNA consisting of the sequences described in Sequence Listing 7, 9, 11 and 13 was used. In addition, Non-targeting shRNA (SHC002V, Sigma) was used as a control. Using Puromycin, the stably introduced cancer cells were selected. The suppression of gene expression of Asss2 was confirmed using a real-time quantitative RT-PCR method. Each shRNA showed an inhibitory effect on Acss2, but the inhibitory effect differed depending on the cell type. Specifically, a high inhibitory effect was observed on shRNA consisting of the sequence described in SEQ ID NO: 9 for LLC, SEQ ID NO: 11 for B16, SEQ ID NO: 13 for Colon, and SEQ ID NO: 7 for C127I. The RNAi cell line thus obtained was examined for acetic acid production according to the method of Example 1.
The results are shown in FIG. In all cancer cell lines in which Acss2 was knocked down, it was revealed that the gene expression of Acss2 was suppressed and acetic acid production decreased accordingly. Although Asss2 has generally been considered to be an enzyme that fixes acetic acid in cells, it has been reported that it enzymatically catalyzes a reversible reaction of [ATP + acetate + CoA⇔AMP + diphosphate + acetyl-CoA]. From this example, it was clarified that in Cancer cells, the reverse reaction of Asss2, which converts acetate-CoA to acetate, that is, acetate energy production reaction was observed.

Example 4 Role of Asss2 in Hypoxic Survival of Cancer Cells—Acs2 RNAi Inhibition of Cancer Hypoxic Survival—
Using the RNAi cell line prepared in Example 3, the role of Asss2 involved in hypoxic survival was examined. First, using a 24-well plate, 5 × 10 ⁴ cells per well were seeded in 1 mL DME medium and cultured under normoxic conditions for 24 h. After that, the medium was changed and transferred to a hypoxic condition (0 day). Thereafter, every other day, the medium was replaced with a DME medium previously treated with hypoxia and cultured for 7 days. The number of cells was counted every other day by a trypan blue dye-exclusion method. In addition, two-color fluorescence cell-viability assay was performed (ToxCount, Active Motif) for the purpose of confirming cell morphology and viability.
The results are shown in FIG. The Asss2 RNAi cell line shortened the hypoxic survival period compared to the control RNAi line. This indicates that acetate metabolism by Asss2 works favorably for hypoxic survival of cancer cells. Moreover, it has shown that the gene knockdown of Asss2 has the inhibitory effect with respect to the hypoxic survival of cancer. When cultured under normoxic conditions, there was no significant difference in cell growth.

Example 5 Role of Asss2 in Tumor Growth-Inhibition Effect of Access2 RNAi on Tumor Growth-
A transplantation experiment was conducted in which the RNAi cancer cell line prepared in Example 3 was planted in nude mice. For the experiment, 6-week-old BALB / c Slc-nu / nu male nude mice (Japan SLC) was used. RNAi cancer cells [8 × 10 ⁶ (Colon) or 1 × 10 ⁷ (LLC, B16, C127I)] suspended in PBS were transplanted subcutaneously into the mouse thigh. Tumor size was measured every 3 days for 12 days. Tumor volume (mm ³ ) was determined as (major axis) × (minor axis) ² × (6 / π). Animal experiments were conducted in accordance with Fukui University animal experiment guidelines.
The results are shown in FIG. In all cell lines examined, the tumor growth of the Asss2 RNAi cells was slower than that of the Control RNAi cells. This indicates that acetate metabolism by Asss2 is important for tumor growth. Moreover, it has shown that the gene suppression of Acss2 has an effect in tumor growth suppression.

Example 6 Contribution of Acss2 to Acetic Acid Tracer Uptake From the results of Example 3, it was revealed that Acss2 is highly expressed in cancer cells and there is a specific acetic acid metabolic pathway utilizing its reversibility. This means that Asss2 is a reversible enzyme that controls the dynamic equilibrium between forward and reverse reactions. Based on this, the inventors thought that by monitoring the uptake of radioactive acetic acid tracer, it was possible to monitor the increased expression of Asss2 in cancer cells. Therefore, the uptake of [ ¹⁴ C] acetic acid into cancer cells and the contribution of Acss2 to it were examined. First, the cells were seeded on a 24-well plate and pre-cultured for 24 hours. At this time, considering the growth rate, LLC, B16, and Colon were 5 × 10 ⁴ cells, C127I was 8 × 10 ⁴ cells, and 3T3 was 1 × 10 ⁵ cells per well. RNAi cells were seeded at 1 × 10 ⁵ cells. The cells were cultured under normoxia for 1 h after exchanging with 37 kBq [ ¹⁴ C] acetic acid-added DME medium. On the other hand, in the experiment under hypoxic conditions, after 2 h or 6 h of hypoxic treatment after pre-culture, the medium was replaced with a 37 kBq [ ¹⁴ C] acetic acid-added medium and cultured for 1 h under low oxygen. Thereafter, the medium was removed and washed twice with ice-cold PBS, 0.5 ml 0.2 N NaOH was added, and the cells were lysed at room temperature for 2 h. 9.5 mL ACSII (Amersham) was added to this, and the radioactivity was measured using the liquid scintillation counter (LSC-5000, Aloka).
As a result, the uptake pattern of the radioactive acetic acid tracer almost coincided with the expression pattern of Asss2 (FIG. 6). In addition, the uptake of radioactive acetic acid tracer was reduced in the Asss2 RNAi cells (FIGS. 7 and 8). From this, it became clear that Asss2 is also involved in the incorporation of radioactive acetic acid tracer. This means that by using a radioactive acetic acid tracer, the expression of Acss2 and the activity of cancer-specific acetic acid metabolism can be monitored. In general, the radioactive tracer can be detected even with a small amount of accumulation. Therefore, the radioactive tracer is useful for detection and imaging of cancer-specific acetic acid metabolism.

  The determination method of the present invention is useful as a determination method for quick and simple cancer diagnosis, particularly for cancer diagnosis that can be easily determined under hypoxic conditions, and as a means for monitoring the therapeutic effect on cancer treatment. In addition, the substance that suppresses the expression / activity of the product of the Asss2 gene of the present invention can be a promising candidate for a cancer therapeutic agent. Furthermore, the screening method of the present invention is useful for searching for novel cancer therapeutic agents.

It is the figure which quantitatively analyzed the amount of acetic acid in a culture medium under normoxia and hypoxia conditions using various cancer cells derived from mice and normal cells. It is the figure which analyzed quantitatively the expression level of the various genes estimated to be related to acetic acid metabolism under normoxic and hypoxic conditions using various cancer cells derived from mice and normal cells. In the figure, the left column corresponds to Asss2, Acot, and Aldh, respectively. Acss2, acetyl CoA synthetase 2; Acot, aldehyde dehydrogenase; Aldh, acetyl CoA hydrase It is the figure which analyzed quantitatively the expression level of the Asss2 gene by the real-time quantitative RT-PCR method and the acetic acid production amount in a culture medium by ion chromatography using the Asss2 knockdown cell line of various mouse-derived cancer cells. In the figure, the left column corresponds to Control RNAi, and the right column corresponds to Access2 RNAi. * P <0.01; ** P <0.005; *** P <0.001 a) It is a figure showing the result of having measured the number of viable cells every other day under hypoxic conditions using the Asss2 knockdown cell line of various cancer cells derived from mice. * P <0.005; ** P <0.001; ↓ Time when morphological observation was performed b) Results of two-color fluorescence cell-viability assay to confirm cell morphological observation and viability in a) FIG. It is a figure which shows the result of having transplanted the Asss2 knockdown cell line of various mouse-derived cancer cells to BALB / c Slc-nu / nu male nude mice, and measuring the tumor size every 3 days. * P <0.03; ** P <0.02; *** P <0.01 It is the figure which measured the uptake | capture of 37 kBq [< ¹⁴ > C] acetic acid by the liquid scintillation counter on normoxia and hypoxia conditions using various cancer cells derived from a mouse | mouth, and a normal cell. * P <0.02; ** P <0.01; *** P <0.001 vs 3T3 Normoxia It is the figure which measured the uptake | capture of 37 kBq [< ¹⁴ > C] acetic acid by the liquid scintillation counter on the normoxia and hypoxia conditions using the Asss2 knockdown cell strain of various cancer cells (LLC, B16) derived from a mouse | mouth. * P <0.01; ** P <0.001; *** P <0.0001 It is the figure which measured the uptake | capture of 37 kBq [< ¹⁴ > C] acetic acid by the liquid scintillation counter on the normoxia and hypoxia conditions using the Asss2 knockdown cell strain of various cancer cells (Colon, C127I) derived from a mouse. * P <0.01; ** P <0.001; *** P <0.0001

Claims (11)

A method for determining whether or not the individual suffers from cancer, comprising measuring the expression level of acetyl CoA synthetase 2 in a sample collected from the individual. Below (a) or (b):
(A) a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of acetyl CoA synthetase 2; and (b) an expression level is measured using an antibody that specifically recognizes acetyl CoA synthetase 2. The method of claim 1. A method for evaluating an anticancer therapeutic effect on a cancer patient, comprising evaluating a transition of expression of acetyl-CoA synthetase 2 in a sample containing a cancer site collected over time from a cancer patient being treated or treated. Below (a) or (b):
(A) Nucleic acid probe or nucleic acid primer capable of specifically detecting the transcription product of acetyl CoA synthetase 2 (b) Whether the individual suffers from cancer, comprising an antibody that specifically recognizes acetyl CoA synthetase 2 Agent for determining whether or not. A method for determining whether or not the individual suffers from cancer, comprising measuring the amount of acetic acid produced in a sample collected from the individual. The method according to claim 5, wherein the amount of acetic acid produced under hypoxic conditions is measured. (A), (b) or (c) below:
(A) Antisense nucleic acid against mRNA of acetyl CoA synthetase 2 gene (b) siRNA against mRNA of acetyl CoA synthetase 2 gene
(C) A therapeutic agent for cancer, comprising either an antisense nucleic acid or an expression vector capable of generating siRNA against the mRNA of the acetyl CoA synthetase 2 gene. Contacting a test substance with an acetyl-CoA synthetase 2-expressing cell, measuring the expression of acetyl-CoA synthetase 2 in the cell, and suppressing the expression of acetyl-CoA synthetase 2 compared to a control cell not contacting the test substance A method for screening a substance capable of treating cancer, comprising selecting the obtained substance as a substance capable of treating cancer. Contacting a test substance with acetyl-CoA synthetase 2, measuring the activity of acetyl-CoA synthetase 2, and a substance that suppresses the activity of acetyl-CoA synthetase 2 compared to the case where the test substance is not contacted with cancer A method for screening a substance capable of treating cancer, comprising selecting as a substance capable of treating cancer. A method for determining cancer, comprising measuring the amount of radiolabeled acetic acid taken up by cells of an individual. A reagent for detecting cancer cells, comprising radioactively labeled acetic acid.