WO2011108699A1 - Gène lié à l'action des isoflavones - Google Patents

Gène lié à l'action des isoflavones Download PDF

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WO2011108699A1
WO2011108699A1 PCT/JP2011/055057 JP2011055057W WO2011108699A1 WO 2011108699 A1 WO2011108699 A1 WO 2011108699A1 JP 2011055057 W JP2011055057 W JP 2011055057W WO 2011108699 A1 WO2011108699 A1 WO 2011108699A1
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papd5
equol
cells
expression
cancer
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PCT/JP2011/055057
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Japanese (ja)
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立花　宏文
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国立大学法人九州大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a gene on which isoflavones, particularly equol, depend upon acting on a subject. More particularly, the present invention relates to Papd5. According to the present invention, Papd5 or a product thereof can be used as a biomarker related to cancer. The present invention is useful in the fields of medicine, alternative medicine, and research and development of pharmaceuticals, foods and cosmetics.
  • Isoflavones are a kind of flavonoid contained in soybean, mainly soybean germ, and there are three kinds of genistein, daidzein and glycitein.
  • soybean fermented foods such as miso and natto, these are contained in large amounts as aglycones, but in most foods, they are contained in the state of glycosides.
  • Glycosides are hydrolyzed by digestive enzymes and ⁇ -glucosidase of enteric bacteria to produce aglycones.
  • Daidzein is also metabolized to equol by certain intestinal bacteria. About half of the Japanese and about 20% of Westerners are said to have enteric bacteria that convert daidzein to equol.
  • Isoflavones have a variety of physiological functions and have been reported to improve menopause and osteoporosis (non-patent document 4) in postmenopausal women. For cancer, it is widely known that it exhibits anticancer activity against hormone-dependent cancers such as breast cancer (Non-patent document 5) and prostate cancer (Non-patent document 6). It has been. Isoflavones are similar in structure to estrogen, which is a type of steroid hormone, and therefore, when they bind to estrogen-receptor (ER) in vivo, they exhibit estrogen-like action or antagonize estrogen and exert antiestrogenic action. It is considered.
  • estrogen estrogen-receptor
  • Non-patent Document 7 genistein exhibits an anticancer action against hormone-independent cancers such as gastric cancer, melanoma, and pancreatic cancer by inhibiting tyrosine kinase and topoisomerase.
  • Non-patent Document 8 a part of its action is an ER-independent action.
  • Non-Patent Document 9 an antiallergic function
  • Non-Patent Document 10 a vascular relaxation function
  • Non-patent Document 5 As an in vitro report on isoflavones, it has been reported to induce apoptosis and inhibit growth of human cancer cells (Non-Patent Document 5), while suggesting an effect of promoting carcinogenesis (Non-Patent Document 11). ). In in vivo animal experiments, some organs have been reported to have a carcinogenic promoting effect (Non-patent Documents 12 and 13).
  • Soy isoflavone genistein modulates cell cycle progression and induces apoptosis in HER-2 / neu oncogene expressing human breast epithelial cells.
  • 67 kDa laminin receptor (67 LR) as a gene (EGCG sensing gene) expressed on the side of cancer cells indispensable for its anticancer activity.
  • 67 LR 67 kDa laminin receptor
  • the present inventor found that equol, a kind of isoflavones, suppresses the growth of the prostate cancer cell line PC-3 and the human cervical cancer cell line HeLa.
  • the present invention provides the following.
  • detecting the biomarker according to [1] including oligonucleotide, peptide, antibody, for PCR, DNA array, ELISA, protein array, mass spectrometry, or immunochromatography , Product.
  • a method for screening for an effective treatment for cancer comprising a step of detecting whether the amount of transcription or expression of Papd5 changes.
  • FIG. 1 is a graph showing the cell growth inhibitory activity and ER dependence of equol against various cancer cells.
  • Human cervical cancer cell line HeLa or human prostate cancer cell line PC-3 was seeded in a 24-well culture plate at 2 ⁇ 10 4 cells / mL and pre-cultured for 24 hours. After treatment with a medium supplemented with 1 mM of ICI182,780, which is an ER antagonist, for 30 minutes, equol was added and cultured for 72 hours, and the number of cells was counted.
  • FIG. 2 is a graph showing the cytostatic activity of equol against mouse melanoma cell line B16.
  • FIG. 3 is a graph showing the effect of ICI on the equol sensitivity of B16 cells.
  • B16 cells were seeded in a 24-well plate at 2 ⁇ 10 4 cells / mL, pretreated for 30 minutes with a medium supplemented with ICI182,780 at a final concentration of 1 mM, and equol at each concentration was then added. After culturing for 72 hours, the number of cells was measured.
  • ICI ER inhibitor ICI182,780 FIG.
  • FIG. 4 is a graph showing the involvement of Papd5 expression on the equol sensitivity of HeLa cells.
  • b) HeLa cells transfected with Scramble-shRNA or Papd5-shRNA expression vector were seeded in a 24-well culture plate at 2 x 10 4 cells / mL, and cultured for 72 hours in a medium supplemented with each concentration of equol. It was measured.
  • FIG. 5 is a graph showing the involvement of Papd5 expression on the equol sensitivity of B16 cells. a) RNA was collected from Scramble-shRNA expression vector-introduced B16 cells or Papd5-shRNA expression vector-introduced B16 cells, and after cDNA synthesis, the expression level of Papd5 mRNA was examined by RT-PCR.
  • FIG. 6 is a graph showing the involvement of Papd5 expression in the cancer cell growth inhibitory action of functional food factors. Each concentration of equol, genistein, zedaidzein, and EGCG was added to HeLa cells into which Scramble-shRNA or Papd5-shRNA expression vector was introduced, and the number of cells was measured after culturing for 72 hours.
  • FIG. 6 is a graph showing the involvement of Papd5 expression in the cancer cell growth inhibitory action of functional food factors. Each concentration of equol, genistein, zedaidzein, and EGCG was added to HeLa cells into which Scramble-shRNA or Papd5-shRNA expression vector was introduced, and the number of cells was measured after culturing for 72 hours.
  • FIG. 7 is a graph showing the effect of oral intake of equol on tumors knocked down by Papd5 expression. Cancer was transplanted by subcutaneously injecting cells into which the Papd5-shRNA5- expression vector had been introduced into the back of the mouse, and then equal was orally administered, and the change in tumor volume over time was measured.
  • FIG. 8 is a graph showing the cell growth promoting effect of equol on cancer cells knocked down by Papd5pd expression. Each concentration of equal was added to B16 cells (siPapd5) introduced with Papd5-shRNA expression vector or B16 cells (scramble) introduced with Scramble-shRNA, and the number of cells was counted.
  • FIG. 1 is a graph showing the effect of oral intake of equol on tumors knocked down by Papd5 expression. Cancer was transplanted by subcutaneously injecting cells into which the Papd5-shRNA5- expression vector had been introduced into the back of the mouse,
  • FIG. 9A is a view showing nucleotide sequences and amino acid sequences related to the present invention.
  • FIG. 9B shows the nucleotide sequence and amino acid sequence related to the present invention.
  • FIG. 9C is a view showing a nucleotide sequence and an amino acid sequence related to the present invention.
  • the present invention is a biomarker for diagnosing the effectiveness of Papd5 or a product thereof in a subject of an anticancer substance selected from the group consisting of equol, daidzein and analogs thereof. Regarding use.
  • Papd5 is used in the meaning of the Papd5 gene unless otherwise specified.
  • the polynucleotide comprising the nucleotide sequence represented as SEQ ID NO: 1, 3 or 5 in the sequence listing, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4 or 6, or any of them refers to a homolog. More specifically, it is one of the following (a), (b), (c), (d), (e) or (f).
  • A a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 5 (more specifically, DNA);
  • B a polynucleotide that hybridizes with a polynucleotide comprising a sequence complementary to the polynucleotide of (a) under stringent conditions and can exhibit cytostatic activity in the presence of equol or daidzein (more specific Then DNA);
  • C a polynucleotide (more specifically, DNA) having high sequence identity with the polynucleotide of (a) and capable of exhibiting cytostatic activity in the presence of equol or daidzein;
  • D a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, or 6 (more specifically, DNA);
  • E The amino acid sequence of SEQ ID NO: 2, 4 or 6 encodes an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and inhibit
  • nucleotide sequence of human Papd5 is shown as SEQ ID NO: 1
  • amino acid sequence of human Papd5 protein is shown as SEQ ID NO: 2.
  • SEQ ID NO: 3 nucleotide sequence of mouse Papd5, as SEQ ID NO: 4, amino acid sequence of mouse Papd5 protein, as SEQ ID NO: 5, as nucleotide sequence of rat Papd5, as SEQ ID NO: 6, as rat The amino acid sequence of Papd5 protein is shown.
  • Papd5 is preferably derived from a mammal, more preferably from a human.
  • the term “product” of Papd5 refers to an antibody that specifically recognizes the above-mentioned Papd5 transcription product (mRNA), expression product (protein), or any one of them unless otherwise specified. .
  • mRNA consisting of the nucleotide sequence represented as SEQ ID NO: 1, 3 or 5 or a homologue thereof. However, in SEQ ID NO: 1, 3, or 5, t is replaced with u.
  • SEQ ID NO: 1, 3, or 5 the same applies to SEQ ID NO: 1, 3, or 5 as the mRNA sequence.
  • mRNA or a homologue thereof is any of the following (a ′), (b ′), (c ′), (d ′), (e ′), or (f ′).
  • a ′ a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, 3, or 5 (more specifically, RNA);
  • B ′ a polynucleotide that hybridizes with a polynucleotide comprising a sequence complementary to the polynucleotide of (a ′) under stringent conditions and can exhibit cell growth inhibitory activity in the presence of equol or daidzein ( More specifically, RNA);
  • C ′ a polynucleotide (more specifically, RNA) having high sequence identity with the polynucleotide of (a ′) and capable of exhibiting cytostatic activity in the presence of equol or daidzein;
  • D ′ a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, or 6 (more specifically, RNA);
  • E ′ encoding an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and /
  • a product is a protein consisting of 2, 4 or 6 amino acid sequences or a homologue thereof. Specifically, it is one of the following (d ′′), (e ′′) or (f ′′).
  • (D ′′) a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 6;
  • E ′′) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, 4 or 6, and cell growth inhibitory activity in the presence of equol or daidzein
  • stringent conditions refers to the conditions of 6M urea, 0.4% SDS, 0.5 ⁇ SSC or a hybridization condition equivalent thereto, unless otherwise specified. May be applied under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 ⁇ SSC or equivalent hybridization conditions. Under each condition, the temperature can be about 40 ° C. or higher, and if higher stringency conditions are required, for example, about 50 ° C. or about 65 ° C. may be used.
  • the number of amino acids to be substituted when “one or more amino acids are substituted, deleted, inserted, and / or added” is the number of amino acids to be replaced or the protein encoding the protein.
  • the nucleotide is not particularly limited as long as it has a desired function, but it is about 1 to 9 or 1 to 4.
  • Means for preparing a polynucleotide according to such an amino acid sequence include, for example, the site-directed mutagenesis method (Kramer W & Fritz H-J: Methods Enzymol 154: 350, 1987).
  • nucleotides when “identity” is “high”, it means sequence identity of at least 80% or more, preferably 90% or more, more preferably 95% or more.
  • identity when referring to amino acid sequences, identity refers to sequence identity of at least 80% or more, preferably 90% or more, more preferably 95% or more. Nucleotide or amino acid sequence identity is determined using the algorithm BLAST by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) it can.
  • polynucleotide refers to DNA or RNA unless otherwise specified. Which one is indicated will be clear to the skilled person from the context. DNA can be single-stranded or double-stranded.
  • Biomarker is objectively measured as an index of a pharmacological response to a normal biological process, pathological process, or therapeutic intervention, unless otherwise specified. It is used to refer to a substance (tangible) that can be evaluated.
  • a biomarker comprising Papd5 or a product thereof includes not only the whole (full length) of Papd5 or a product thereof but also a part thereof, unless otherwise specified. This “part” is a part that is long enough that the presence of Papd5 or its product can be inferred.
  • Papd5 or a product thereof is referred to as “use as a biomarker”, not only when all of Papd5 or a product thereof is used, but also a part of Papd5 or a product thereof, and Papd5 or a product thereof. It also includes the case where a part having such a length that the existence of the above can be estimated is used.
  • use as a biomarker includes use for detection of SNP, use for detection of gene mutation, use for detection of localization change.
  • Anti-cancer substances equal, daidzein
  • Compounds used in or related to the present invention include the following:
  • genistein and EGCG inhibit the growth of HeLa cells in the same way as equol.
  • the effect was not affected by the decrease in Papd5 expression.
  • the cell growth inhibitory action of daidzein was inhibited in the cells in which the expression of Papd5 was reduced, like equol (Example and FIG. 6).
  • the term “anticancer substance” means a general component effective for the treatment of cancer, unless otherwise specified.
  • the anticancer substance may be a food component or a medicine (anticancer agent).
  • an anticancer substance selected from the group consisting of equol, daidzein and analogs thereof is used. “These analogs” have anti-cancer activity (more specifically, cancer cell growth-inhibitory activity) similar to that of daidzein glycosides, equol or daidzein, but the effect is due to decreased expression of Papd5. Substances that are inhibited are included. Such substances may act in an ER-independent manner.
  • the human cervical cancer cell line HeLa and the human prostate cancer cell line PC-3 were inhibited by equol and treated with ER antagonist ICI182,780 (ICI). Even in the case of the action, the action was not inhibited. From this, it can be said that equol exhibits cytostatic activity against these cancer cell lines by an action mechanism independent of ER (FIG. 1).
  • estrogen receptor has the ability to bind to estrogen (estrone (E1), estradiol (E2) and estriol (E3)), unless otherwise specified, and accepts steroids.
  • estrogen estrone
  • E2 estradiol
  • E3 estriol
  • the estrogen receptor has two isoforms, ER ⁇ (NR3A1, 595 amino acid residues) and ER ⁇ (NR3A2, 530 residues).
  • Estrogens have the function of promoting the formation of reproductive functions and cell proliferation.
  • ER-independent or “ER-independent” means acting on a target without depending on ER, unless otherwise specified. Whether or not the action of a substance is “ER-independent” can be appropriately determined by those skilled in the art. For example, due to the presence of an antagonist of ER, the effect to be exerted is unaffected or can be neglected (eg, effects within ⁇ 30% compared to the effect in the absence) Can be judged using the index of For more detailed conditions, reference can be made to the description of the examples in the present specification.
  • a disease or condition that is improved by the action of equol or daidzein unless otherwise specified, cancer, particularly cervical cancer, endometrial cancer, prostate cancer; stomach cancer, melanoma, Pancreatic cancer (Non-patent document 7); Thymic hypertrophy or atrophy (Non-patent document 8); Allergy (Non-patent document 9), vascular stenosis or relaxation (Non-patent document 10).
  • cancer is cancer.
  • treatment when “treatment” is concerned with cancer, unless otherwise specified, it prevents cancer, reduces the risk of developing cancer, treats cancer, and suppresses progression of cancer. Including doing.
  • oligonucleotides, peptides, antibodies, PCR (RT-PCR, real-time PCR), DNA array, ELISA, protein array, or immunochromatography Products (kits, etc.) for graphy are provided.
  • Such products specifically amplify all or part of the appropriate length of the oligonucleotide probe, Papd5 or its transcript, which can be detected by specifically hybridizing with Papd5 or its transcript.
  • An antibody (which can be expressed in Escherichia coli) and may contain an oligonucleotide primer of an appropriate length that can specifically recognize Papd5, its transcription product or its expression product.
  • a Papd5-specific antibody can be obtained by immunization with a highly antigenic peptide derived from Papd5), and may contain a SELEX product or the like.
  • SELEX product or the like.
  • the product including the above-described primers can be a PCR kit, a DNA chip (microarray), or a protein array.
  • a group consisting of equol or daidzein and analogs thereof comprising a step of detecting transcription or expression of Papd5 in a subject or a sample collected from the subject (usually not assumed to be returned to the subject).
  • Methods for determining the effectiveness of a more selected anticancer substance in a subject are provided.
  • the method used is preferably a PCR method, a DNA array method, an ELISA method, a protein array method, or an immunochromatography method.
  • the effectiveness of the anticancer substance selected from the group consisting of equol or daidzein and their analogs is low or ineffective It can be judged.
  • anything assumed as a cause of suppression of gene transcription or expression is considered to affect the transcription or expression of Papd5.
  • disease risk factors such as congenital factors, oxidative stress, genetic mutations due to carcinogenic factors, diet, lifestyle (smoking, alcohol consumption, exercise, etc.)
  • obesity hypertension, hyperglycemia and hyperlipidemia
  • Papd5 Transcription or expression can be suppressed.
  • the term “subject” refers to cancer animals, healthy animals, cancers, unless otherwise specified. Includes patients and healthy individuals.
  • the term “subject” when it is ⁇ applied '' with respect to anticancer substances and natural products that are candidates for anticancer substances, in addition to testing on samples, administration of pharmaceuticals to individuals, intake of food, This includes applying cosmetics, including when it is done for others.
  • the “medicine” referred to in the present invention includes animal drugs in addition to drugs for humans, unless otherwise specified, and the “food” referred to in the present invention includes not only solid foods, unless otherwise specified. Including beverages and for humans as well as for animals, such as pet food, feed.
  • the “cosmetics” as used in the present invention refers to those used for the purpose of cosmetics, which are not intended for treatment of illness, except when specifically described.
  • the screening method of the present invention may comprise the following steps: 1) A step of applying a test product which is a natural product, food, cosmetic, pharmaceutical product or compound to a subject, a sample collected from the subject or a cell having Papd5 and capable of transcription or expression of Papd5; 2) detecting whether or not the amount of transcription or expression of Papd5 in the subject or sample changes; and 3) if the amount of transcription or expression increases, the test substance is effective for treating cancer.
  • the process of selecting as a simple thing.
  • the screening method of the present invention is particularly suitable for screening an object suitable for application to a subject in combination with equol, daidzein or an analog thereof.
  • the presence or absence of mutation of Papd5, or the amount of transcription or expression, as an index, according to the individuality of the subject, a pharmaceutical, food or cosmetic for treatment of a disease or condition improved by the action of equol or daidzein, Or the application plan can be designed.
  • a pharmaceutical, food or cosmetic for treatment of a disease or condition improved by the action of equol or daidzein Or the application plan can be designed.
  • the effect of the anticancer drug camptothecin is reduced (Non-patent Document 14), but it seems that the action of equol or daidzein can be expected.
  • Implementation of the present invention including actions such as “treatment”, “application”, and “design” includes medical actions by doctors and actions that do not rely on doctors. *
  • experiments using human-derived cells 1, 5 and 6 are genes and sequences that cause transcription of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 in the sequence listing (mRNA of human Papd5).
  • the human cervical cancer cell line HeLa used for the measurement of equol's cytostatic activity and ER-dependent cell count on various human cancer cells is DMEM medium supplemented with 10% fetal bovine serum (FCS) (BIOLOGICAL).
  • FCS fetal bovine serum
  • the human prostate cancer cell line PC-3 was subcultured and maintained in RPMI1640 medium supplemented with 10% FCS at 37 ° C. under 5% CO 2 with water vapor saturation. Cells were maintained in culture in the logarithmic growth phase.
  • the DMEM medium used for the culture was Dulbecco's MEM medium (Cosmo Bio Co., Ltd.) 13.38 g per 1 L of dH 2 O, 5.958 g of HEPES (Wako Pure Chemical Industries, Ltd.), 200,000 units of penicillin G for injection (Meiji Seika Co., Ltd.) Company) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd.) 0.1 vial, NaHCO 3 (nacalai tesque) 3.7 g were suspended and then sterilized with a 0.22 ⁇ m filter.
  • RPMI-1640 medium is 10.4 g of RPMI-1640 medium (Nissui Pharmaceutical) per 1 L of dH 2 O, 2.38 g of 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane sulfonic acid (HEPES), 100 U / penicillin. mL, streptomycin 100 mg / L, and NaHCO 3 (Wako Pure Chemical Industries, Ltd.) 2.0 g were suspended, and the pH was adjusted to 6.8 to 7.4 by blowing carbon dioxide, and then sterilized by filtration through a 0.22 ⁇ m filter. Then, 10% of fetal calf serum (FCS) was added and used for cell culture.
  • FCS fetal calf serum
  • PBS suspends 8.0 g of NaCl (nacalai tesque) per 1 L of dH 2 O, 0.2 g of KCl (nacalai tesque), 1.15 g of Na 2 HPO 4 (Wako Pure Chemical Industries, Ltd.), 0.2 g of KH 2 PO 4 (nacalai tesque).
  • the trypsin solution was prepared by suspending 0.05 g of EDTA ⁇ 2Na (Wako Pure Chemical Industries, Ltd.) and 0.02 g of trypsin (nacalai tesque) per 100 mL of PBS, and sterilizing the filter.
  • Equol (Funakoshi)
  • Daidzein (Funakoshi)
  • Genistein (Funakoshi)
  • DMSO nacalai tesque
  • ICI182,780 invitrogen was dissolved in DMSO to a concentration of 10 mM and stored at ⁇ 80 ° C.
  • each cell was adjusted to 2 ⁇ 10 4 cells / mL, seeded in a 24-well plate (nunc TM ), and precultured in a medium containing 10% FCS for 24 hours. Thereafter, the medium was replaced with 2% FCS-containing medium supplemented with ICI182,780 to 1 ⁇ M, treated for 30 minutes, and equol (LC Labolatories) was added to each concentration. After culturing for 72 hours, the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.
  • mouse melanoma cell line B16 American Type Culture Collection
  • the mouse melanoma cell line B16 used for the measurement of the cell proliferation inhibitory activity cell number and cell viability of equol against the mouse melanoma cell line B16 was 5% FCS-added DMEM medium at 37 ° C with water vapor saturation. Passaged and maintained under% CO 2 conditions. Cells were maintained in culture in the logarithmic growth phase.
  • mouse melanoma cell line B16 was adjusted to 2 ⁇ 10 4 cells / mL, seeded in a 24-well plate, and pre-cultured in DMEM medium containing 5% FCS for 24 hours.
  • the cells were counted for 72 hours or 24, 48, 72, 96 hours in a 2% FCS-containing DMEM medium containing equol having a final concentration of 0, 1, 5, 10, 25, 50 ⁇ M, and the number of cells was counted using a cell counter. Student's t test was used for statistical processing of the experimental results.
  • B16 cells were adjusted to 2 ⁇ 10 4 cells / mL, seeded in 24-well plates, and pre-cultured in DMEM medium containing 5% FCS for 24 hours. After treatment with 2% FCS-containing DMEM medium containing ICI 182,780 at a final concentration of 1 ⁇ M for 30 minutes, equol was added to a final concentration of 0, 1, 5, 10, 25 ⁇ M. After culturing for 72 hours, the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.
  • Mouse Embryo cDNA library ML8000BB (Clontech, Mountain View, CA) was fragmented with restriction enzymes EcoRI and Sph I, and introduced into pLPCX-modified retroviral vector FGS library (MFL-ESP) (1.0 ⁇ g / ⁇ L) 3 ⁇ L, pVSV-G vector (Takara Bio Inc.) (1.0 ⁇ g / ⁇ L) 3 ⁇ L and FuGENE 6 Transfection Reagent (Roche) 6 ⁇ L were mixed and introduced into each packaging cell.
  • MFL-ESP pLPCX-modified retroviral vector FGS library
  • pVSV-G vector (Takara Bio Inc.)
  • FuGENE 6 Transfection Reagent (Roche) 6 ⁇ L were mixed and introduced into each packaging cell.
  • the packaging cell culture supernatant is passed through a filter (0.22 ⁇ m), and polybrene (Hexadimethrine bromide) (SIGMA-ALDRICH) is added to a final concentration of 8 ⁇ g / mL, and then adjusted to 1 ⁇ 10 4 cells / mL the previous day. Then, the cells were seeded in a 5 mL dish and sprinkled on B16 cells that had been cultured in a DMEM medium containing 5% FCS to infect the virus in the culture supernatant. To the packaging cells, a fresh 10% FCS-containing DMEM medium was added, and the culture was continued. This operation was performed four times every 12 hours.
  • polybrene Hexadimethrine bromide
  • the infected B16 cells were adjusted to 1 ⁇ 10 4 cells / mL, seeded in a 96-well plate (nunc TM ), and recovered for 24 hours in 2% FCS-containing DMEM medium. After recovery culture, the cells were cultured in a DMEM medium containing 1% FCS in a medium containing 80 ⁇ M equol to obtain equol-resistant B16 cells.
  • pLPCX-S (5'-GAT CCG CTA GCG CTA CCG GAC TCA GAT-3 ', SEQ ID NO: 7) and pLPCX-A (5'-CTT TCA TTC CCC CCT TTT TCT GGA GAC-3 ', And the primer set of SEQ ID NO: 8) was used by adjusting to 20 ⁇ M with TE buffer (10 mM Tris (nacalai tesque) -HCl, 1 mM EDTA, pH 8.0). The same applies to the primers used in the following PCR.
  • the introduced cDNA fragment was amplified by PCR using TE buffer.
  • TGRADIENT Biometra (registered trademark ) , Germany) or PC320 (ASTEC, Fukuoka) was used.
  • PCR is 0.5 ⁇ L of each primer (sense primer and antisense primer) per sample, dNTP mix 1 ⁇ L, 10 x buffer 1 ⁇ L, Ex taq 0.1 ⁇ L and dH 2 O 5.9 ⁇ L purchased from Takara Bio Inc. (Shiga) 1 ⁇ L of the template was suspended, initial denaturation at 94 ° C. for 2 minutes, denaturation reaction at 94 ° C. for 30 seconds, annealing at 67.1 ° C.
  • the agarose gel was prepared by dissolving Agarose S (Wako Pure Chemical Industries, Ltd.) at a concentration of 1 to 1.5% in TAE buffer.
  • TAE buffer 242 g of Tris (nacalaitesque) -HCl per 1 L, 37.2 g of EDTA 2Na, 57.1 mL of glacial acetic acid (nacalai tesque) are dissolved in dH 2 O, adjusted to pH 8.5, and then filled up to 1 L with dH 2 O.
  • the mixture was sterilized by autoclaving to prepare a 50 ⁇ TAE buffer, which was diluted 50 times with dH 2 O and used.
  • GelMate (TOYOBO) was used for the electrophoresis tank.
  • SOB is 3.0 g of Bacto Tryptone (Wako Pure Chemical Industries, Ltd.), 0.75 g of Bacto Yeast Extract (Wako Pure Chemical Industries, Ltd.), 0.078 g of NaCl, 0.027 g of KCl, and dH 2 O per 150 ml. Added to 148.5 ml. After cooling the solution to room temperature autoclaved, which 2M Mg 2+ solution separately autoclaved and (12.324g MgSO 4 ⁇ 7H 2 O of (Wako Pure Chemical Industries, Ltd.) + 10.165g of MgCl 2 ⁇ 6H 2 1.5 ml of O (nacalai tesque) was mixed with water to make 50 ml and sterilized by autoclaving).
  • TB is mixed with 0.3 g of PIPES (Wako Pure Chemical Industries, Ltd.), 0.22 g of CaCl 2 (Wako Pure Chemical Industries, Ltd.) and 1.86 g of KCl in 100 ml of water per 100 ml, and then the pH is adjusted to KOH (nacalai tesque) adjusted to 6.7. After adding 1.09 g of MnCl 2 .4H 2 O (Wako Pure Chemical Industries, Ltd.), the total amount was adjusted to 100 ml, filter sterilized (0.22 ⁇ m), and stored at 4 ° C. E. coli was cultured using ampicillin-containing liquid LB medium or ampicillin-containing LB plates.
  • Ampicillin-containing liquid LB medium was filled with 1 g of Bacto Tryptone 10 g, Bacto Yeast Extract 5 g, and NaCl 10 g to 1 L with dH 2 O to a pH of 7.0, and autoclaved. Before use, 150 mg / ml ampicillin was added at a 1000-fold dilution.
  • the LB plate was added with 2 g of Bacto Tryptone, 1 g of Bacto Yeast Extract, 2 g of NaCl, and 5 g of Bacto Agar (Wako Pure Chemical Industries, Ltd.) per 200 ml, filled up to 200 ml with dH2O and sterilized by autoclave.
  • SOC 100 mL SOB per 100 mL, 1 mL of 2M glucose solution (18.016 g mixed in water and autoclaved to 50 mL), dispensed, and stored at room temperature or 4 ° C. It was centrifuged (1000 ⁇ g) for 5 minutes at room temperature to remove 900 ⁇ L of supernatant.
  • the Escherichia coli was applied to an ampicillin-containing LB plate and cultured at 37 ° C. overnight, and the colonies that had risen were applied to an ampicillin-containing LB plate coated with x-gal and IPTG for blue-white determination.
  • the plate was used by applying 25 ⁇ L of 20 mg / ml X-gal and 25 ⁇ L of 0.1M IPTG to an LB plate containing ampicillin.
  • 20 mg / ml X-gal 100 mg of X-gal was dissolved in 5 ml of NN-dimethyl-formamide (Wako Pure Chemical Industries, Ltd.) and stored at -20 ° C.
  • IPTG For 0.1M IPTG, 1 g of IPTG (Wako Pure Chemical Industries, Ltd.) was dissolved in 4.2 ml of dH 2 O and stored at ⁇ 20 ° C. After overnight culture at 37 ° C, a white colony that has risen as a template, 0.5 ⁇ L each of primer set of pLPCX-S and pLPCX-A, 2.5 mM dNTP mix 1 ⁇ L, 25 mM MgCl 2 purchased from Fermentas UAB 1 ⁇ L of 10 ⁇ buffer with (NH 4 ) 2 SO 4 , 0.1 ⁇ L of Taq DNA Polymerase (Recombinant) and 4.9 ⁇ L of dH 2 O were suspended in appropriate amounts.
  • PCR was performed at an initial denaturation of 94 ° C. for 2 minutes, a denaturation reaction at 94 ° C. for 30 seconds, an annealing at 67.1 ° C. for 30 seconds, an extension reaction at 72 ° C. for 2 minutes, and a denaturation reaction, annealing, and extension reaction were performed for 30 cycles. After PCR, 1 ⁇ L of BPB was added to each sample, and electrophoresis was performed on a 1.5% agarose gel.
  • Plasmid DNA was recovered from the clones in which the insert was confirmed using LaboPass TM Mini (LaboPass) and suspended in TE buffer. Using the recovered plasmid DNA as a template, an extension reaction for sequencing was performed. The reaction was performed using the BigDye Terminator v3.1Cycle Sequencing Kit according to the Big Dye Terminator cycle sequencing protocol. The reaction solution was mixed with 3.5 ⁇ L of buffer, 3.2 pmol of primer, appropriate amount of template, 1.0 ⁇ L of BigDye Terminator v3.1Cycle Sequencing Kit (premix), and adjusted to 20 ⁇ L with dH 2 O. The reaction was performed at initial denaturation 96 ° C. for 1 minute, denaturation reaction 96 ° C.
  • T7 (5'-CTA ATA CGA CTC ACT ATA GGG C-3 ', SEQ ID NO: 9)
  • LacZ (5'-AGA TAT GAC CAT GAT TAC GCC-3', SEQ ID NO: 10) are used as primers.
  • the buffer was adjusted to 1.0 pmol / ⁇ L before use. Thereafter, the base sequence was determined with an ABI3130xl DNA sequencer. The equol resistance causative gene was identified from the nucleotide sequence information.
  • HeLa cells in which expression of Papd5 was knocked down Construction of human cervical cancer cell line HeLa in which Papd5 was knocked down was performed as follows.
  • a vector shPapd5-RNA
  • a pLKO.1-puro vector SIGMA-ALDRICH
  • SIGMA-ALDRICH 5′-GCCACATATAGAGATTGGATA-3
  • a vector was added to the cell, and the cell was collected 48 hours later, seeded in a 96-well plate, and selected with puromycin (invivogen).
  • the reagents used were those prepared at the time of identification of equol's anticancer activity-related gene. 1 mL of TRIzol® Reagent was added to the cells and left at room temperature for 5 minutes. Next, 200 ⁇ L of chloroform was added and stirred, allowed to stand at room temperature for 3 minutes, and then centrifuged (12000 ⁇ g) at 4 ° C. for 15 minutes.
  • a primer for amplifying Papd5, Papd5-F (5'-CACAAAGTCGCAGATGAGGA-3 ', SEQ ID NO: 12), Papd5-R (5'-TGGACTGTGTGGCAGAAGAG-3', SEQ ID NO: 13) becomes 10 mM.
  • a solution diluted with TE buffer was used. 2 ⁇ L of the prepared cDNA, 1 ⁇ L of each primer, and 12.5 ⁇ L of SYBR Premix Ex Taq II (2 ⁇ , SYBR PrimerScript RT-PCR Kit II) were mixed and set in the Thermal Cycler Dice Real Time System for reaction. PCR conditions were initial denaturation at 95 ° C. for 10 seconds, then 95 ° C. for 5 seconds and 60 ° C. for 20 seconds. Similarly, the expression level of GAPDH as an internal standard was examined in the same manner as before.
  • HeLa cells into which a control vector (scramble-shRNA) was constantly introduced were also prepared.
  • Papd5-shRNA-introduced HeLa cells and scramble-shRNA-introduced HeLa cells created in 5 of Papd5 on equol sensitivity of HeLa cells are subcultured and maintained in the same manner as WildType HeLa cells in DMEM medium containing 10% FCS. did.
  • HeLa cells were seeded in a 24-well plate at 2 ⁇ 10 4 cells / mL and pre-cultured in DMEM medium containing 10% FCS for 24 hours. Thereafter, the cells were replaced with 10% FCS-containing DMEM medium containing equol having a final concentration of 1,5,10,25 ⁇ M, cultured for 72 hours, and the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.
  • B16 cells in which expression of Papd5 was knocked down were performed as follows.
  • a vector for knocking down the expression of Papd5
  • a pLKO.1-puro vector SIGMA-ALDRICH
  • SIGMA-ALDRICH 5′-CTGACGAGGATTCCGTGAAAG-3 ′
  • B16 cells were seeded at 2 ⁇ 10 4 cells / mL, 24 hours later, 2 ⁇ g of vector and 6 ⁇ L of Fugene 6 were mixed with DMEM to a total volume of 200 ⁇ L and added to the cells.
  • a vector was added to the cell, and the cell was collected 48 hours later, seeded in a 96-well plate, and selected with puromycin (invivogen).
  • puromycin invivogen
  • knockdown of Papd5 was confirmed as follows.
  • the reagents used were those prepared at the time of identification of equol's anticancer activity-related gene. 1 mL of TRIzol (Registered Trademark) Reagent was added to the cells and left at room temperature for 5 minutes. Next, 200 ⁇ L of chloroform was added and stirred, allowed to stand at room temperature for 3 minutes, and then centrifuged (12000 ⁇ g) at 4 ° C. for 15 minutes.
  • RT-PCR is 10 mM of primers that amplify Papd5, Papd5-F (5′-GGAGGTAGTGAGCAGGATCG-3 ′, SEQ ID NO: 15), Papd5-R (5′-ATCCTCGTCAGCGACTTTGT-3 ′, SEQ ID NO: 16).
  • a solution diluted with TE buffer was used.
  • PCR was performed at an initial denaturation of 94 ° C. for 2 minutes, a denaturation reaction at 94 ° C. for 1 minute, an annealing at 60 ° C. for 1 minute, an extension reaction at 72 ° C. for 1 minute, and a denaturation reaction, annealing, and extension reaction were performed for 35 cycles.
  • ⁇ -Actin as an internal standard was examined in the same manner as before. After PCR, 1 ⁇ L of BPB was added, and electrophoresis was performed on a 1.5% agarose gel to which EtBr Solution (Wako Pure Chemical Industries, Ltd.) was added at a 10,000-fold dilution.
  • B16 cells into which a control vector (scramble-shRNA) was constantly introduced were also prepared.
  • B16 cells were seeded on a 24-well plate at 2 ⁇ 10 4 cells / mL and pre-cultured in DMEM medium containing 5% FCS for 24 hours. Thereafter, the medium was replaced with 2% FCS-containing DMEM medium containing equol having a final concentration of 10 ⁇ M and cultured for 72 hours, and the number of cells was counted with a cell counter. Student's t test was used for statistical processing of experimental results.
  • Functional food factors equol, genistein (Tokyo Kasei Kogyo), daidzein (Tokyo Kasei Kogyo), EGCG
  • Papd5-shRNA-introduced HeLa cells created in Papd5's involvement 5 on the cell growth inhibitory action of functional food factors The effect of was examined. Equol, genistein and daidzein were dissolved in DMSO to 100 mM and stored at -30 ° C.
  • EGCG Teavigo
  • HeLa cells were seeded on a 24-well plate at 2 ⁇ 10 4 cells / mL and pre-cultured in DMEM medium containing 10% FCS for 24 hours. Thereafter, the cells were replaced with 2% FCS-containing DMEM medium containing each sample at final concentrations of 1, 5, 10, and 20 ⁇ M, cultured for 72 hours, and the number of cells was counted with a cell counter. Student's t test was used for statistical processing of the experimental results.
  • GSE gene suppressor elements
  • the mouse melanoma cell line B16 was seeded on a 24-well culture plate at 2 ⁇ 10 4 cells / mL and cultured for 72 hours in a medium supplemented with equol or the number of cells was measured every 24 hours (FIG. 2).
  • this cell line was found to be a cancer cell line to which the GSE method can be applied because its growth was remarkably suppressed by equol (FIG. 2).
  • Papd5-shRNA Papd5-specific shRNA expression vector
  • scramble-shRNA human cervical cancer cell line HeLa into which expression was constantly introduced
  • HeLa cells introduced with Papd5-shRNA or scramble-shRNA were seeded in a 24-well culture plate at 2 x 10 4 cells / mL, cultured for 72 hours in a medium supplemented with equol of each concentration, the number of cells was measured, and equol The sensitivity to was investigated. As a result, HeLa cells into which scramble-shRNA had been introduced were inhibited by equol. On the other hand, the sensitivity to equol completely disappeared in HeLa cells in which Papd5-shRNA was introduced to reduce the amount of Papd5 expression (FIG. 4b).
  • B16 cells transfected with Papd5-shRNA or scramble-shRNA were seeded in a 24-well culture plate at 2 x 10 4 cells / mL, cultured for 72 hours in a medium supplemented with equol of each concentration, the number of cells was measured, and equol The sensitivity to was investigated. As a result, B16 cells into which scramble-shRNA had been introduced were inhibited by equol. On the other hand, in B16 cells in which Papd5-shRNA was introduced and the expression level of Papd5 was reduced, the sensitivity to equol completely disappeared (FIG. 5b).
  • Equol was found to suppress cell growth of human cervical cancer cell line HeLa, human prostate cancer cell line PC-3, and mouse melanoma cell line B16. Furthermore, it was revealed that ER is not involved in these cell growth inhibitory activities.
  • Papd5 was identified as a gene candidate responsible for equol's cytostatic activity from B16 cells, where Equol exhibits ER-independent growth-suppressing activity. Papd5 is a gene involved in functional expression of ER-independent equol. It was thought that. When the expression of Papd5 in B16 cells and HeLa cells was specifically reduced using RNA interference, the sensitivity of equol to cytostatic activity disappeared from both cancer cell lines.
  • Papd5 is an essential gene for functional expression of ER independent of equol. It was also revealed that Papd5 is an essential gene in the anticancer activity of daidzein, the precursor of equol.
  • a mouse melanoma cell line B16 cell in which expression of Papd5 was knocked down was obtained by introducing the Papd5-shRNA expression vector, and 5 x 10 5 cells / 100 mL PBS per mouse was added to C57BL. Cancer was transplanted by subcutaneous injection into the back of / 6J mice (male, 6 weeks old). Thereafter, equol 0.8 mg dissolved in 400 mL of 1% DMSO-H 2 O per mouse was forcibly orally administered once every two days, and changes in tumor volume over time were measured. On day 24 after cancer transplantation, the mice were sacrificed and the tumor weight was measured. The control group received 1% DMSO-H 2 O as a solvent.
  • Equal cell growth-promoting action on cancer cells knocked down in Papd5 expression In the same manner as in the previous example, a mouse melanoma cell line B16 cell or Scramble in which Papd5-shRNA expression vector was introduced and Papd5 expression was knocked down was introduced. -Equal concentrations of each concentration were added to shRNA-introduced B16 cells and cultured for 72 hours, and the number of cells was counted. The test group to which only the equal solvent (DMSO) was added was used as a control, and the relative fine number (%) was calculated with the number of control cells as 100%.
  • DMSO equal solvent

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Abstract

L'invention porte sur un gène (ou sur son produit), de protéine « 5 contenant un domaine associé à Pap » (Papd5), identifié en tant que gène nécessaire pour que l'équol présente une activité anti-cancéreuse, pour une utilisation en tant que biomarqueur pour le diagnostic de l'efficacité, chez un sujet, d'une substance anti-cancéreuse sélectionnée dans un groupe comprenant l'équol, la daïdzéine et leurs analogues. L'invention porte également sur un produit de détection d'un biomarqueur en utilisant la PCR, une puce à ADN, l'ELISA, une puce à protéines, la spectrométrie de masse ou une immunochromatographie. L'invention porte, en outre, sur un procédé pour la détermination de l'efficacité, chez un patient, d'une substance anti-cancéreuse sélectionnée dans un groupe comprenant l'équol, la daïdzéine et leurs analogues. Ledit procédé inclut une étape de détection de la transcription ou de l'expression de Papd5 dans un échantillon prélevé du sujet.
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017066796A3 (fr) * 2015-10-16 2017-06-22 The Children's Medical Center Corporation Modulateurs de maladies impliquant des télomères
WO2017066712A3 (fr) * 2015-10-16 2017-06-22 The Children's Medical Center Corporation Modulateurs de maladie de télomères
US11220689B2 (en) 2015-10-16 2022-01-11 Children's Medical Center Corporation Modulators of telomere disease
CN108350508A (zh) * 2015-10-29 2018-07-31 株式会社益力多本社 雌马酚产生能力的测定方法
CN109414448A (zh) * 2016-06-17 2019-03-01 豪夫迈·罗氏有限公司 用于减少PAPD5或PAPD7 mRNA治疗乙型肝炎感染的核酸分子
US11191775B2 (en) 2016-06-17 2021-12-07 Hoffmann-La Roche Inc. PAPD5 and PAPD7 inhibitors for treating a hepatitis B infection
US11534452B2 (en) 2016-06-17 2022-12-27 Hoffmann-La Roche Inc. Nucleic acid molecules for reduction of PAPD5 or PAPD7 mRNA for treating hepatitis B infection
US10953034B2 (en) 2017-10-16 2021-03-23 Hoffmann-La Roche Inc. Nucleic acid molecule for reduction of PAPD5 and PAPD7 mRNA for treating hepatitis B infection
US11484546B2 (en) 2017-10-16 2022-11-01 Hoffman-La Roche Inc. Nucleic acid molecule for reduction of PAPD5 and PAPD7 mRNA for treating hepatitis B infection
WO2019240872A1 (fr) * 2018-06-15 2019-12-19 The Board Of Regents Of The University Of Texas System Méthodes de traitement et de prévention du mélanome avec du s-équol
US11090286B2 (en) 2018-06-15 2021-08-17 The Board Of Regents Of The University Of Texas System Methods of treating and preventing breast cancer with S-equol

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