US20100222424A1 - Method of Screening Drug with the Use of 67 kDa Laminin Receptor and Drug Obtained Thereby - Google Patents

Method of Screening Drug with the Use of 67 kDa Laminin Receptor and Drug Obtained Thereby Download PDF

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US20100222424A1
US20100222424A1 US12/760,772 US76077210A US2010222424A1 US 20100222424 A1 US20100222424 A1 US 20100222424A1 US 76077210 A US76077210 A US 76077210A US 2010222424 A1 US2010222424 A1 US 2010222424A1
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Hirofumi Tachibana
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G01N2333/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49

Definitions

  • the present invention relates to a screening-method for drugs using a 67 kDa laminin receptor, and to a drug obtained by it.
  • 67 kDa laminin receptor (hereinafter it may be referred to as “67LR”) is a protein of 67 kDa, which is derived from a 37 kDa precursor protein translated from mRNA that codes for 295 amino acids, through intracellular acylation polymerization of the precursor protein by fatty acid for homo-dimerization or hetero-dimerization thereof, and only when it moves onto the surface of a cell membrane together with integlins, it functions as a laminin receptor ( Biochemistry, 1995, 34: 11276-11287, T. H. Landowski et al.; J. Cell. Biochem, 1998, 69: 244-251, S. Buto et al.).
  • the 37 kDa precursor protein participates in protein synthesis as a ribosome-related protein p40 and it is the same as that reported as a multidrug resistance-related protein (MGr1-Ag) ( Cell. Mal. Life Sci., 2002, 59: 1577-1583, Y. Shi at al.).
  • Mr1-Ag multidrug resistance-related protein
  • the laminin receptor is considered to be an oncofetal antigen as an immunogen for T cells, or that is, as a general tumor-specific transplantation antigen (Anticancer Research, 1999, 19: 5535-5542, J. H. Coggin, Jr., et al.).
  • 67LR a dozen or so types of laminin receptors have been already reported, and of those, the relationship between 67LR, and cancer is strongly suggested.
  • 67LR is known as an important prognostic factor in many types of cancers that indicates the degree of malignancy of human cancer patients ( Breast Cancer Research and Treatment, 1998, 52: 137-145, S. Menard, et al.; Clinical Cancer Research, 1997, 3: 227-231, G. Fontanini, et al.; Clinical Cancer Research, 1996, 2: 1777-1780, F. Basolo, et al.; J. Natl. Cancer Inst., 1991, 83: 29-36, V. Coice, et al.). In animal models, it is suggested that 67LR participates in proliferation, movement, invasion and metastasis of cancer cells.
  • VEGF vascular endothelial growth factor
  • 67LR acts as a receptor for a prion protein, which is considered as a cause for Creutzfelds-Jakob disease, and prion binding for internalization and further the binding and the internalization are inhibited by secretion of a membrane domain-deficient mutant 67LR (EMBO J., 2001, 20: 5863-5875, S. Gauczynski, at al.).
  • 67LR is expressed in a subset group of CD4+CD8 ⁇ or CD4 ⁇ CD8+ of CD45RO+/CD45RA ⁇ memory cells that are a subset of T cells, and an effect of 67LR to the immune system is suggested (J. Immunol., 1999, 163: 3430-3440, S. M. Canfield, et al.).
  • tea catechins include epigallocatechin, epicatechin gallate and epicatechin (hereinafter these may be referred to as “EGC”, “ECG” and “EC”, respectively).
  • tea has been used as a drug from the past, and recently, tea has been analyzed for the relation between its efficacy and its components.
  • EGCG is a component discovered by A. Bradfield, et al. in 1947 (J. Chem. Soc., 1947, 32: 2249, A. E. Bradfield, et al.).
  • tea catechins including EGCG have been reported, for example, antioxidation, anticancer, suppression of plasma cholesterol increase, suppression of blood pressure increase, platelet aggregation inhibition, suppression of blood sugar increase, dementia prevention, antiulcer, antiinflammation, antiallergy, antibacterial, anticaries, antiviral, detoxication, enteroflora improvement, deodorization ( Functions of Tea , edited by Keiichiro Muramatsu, Society Publishing Center, 2002).
  • anticancer effect that includes anti-mutation effect, anti-carcinogenic promotion effect, antitumor proliferation-inhibiting effect, anti-invasion/metastasis inhibiting effect, anti-neovascularization inhibiting effect.
  • Recent reports say that EGCG inhibits DNA synthesis in leukemia cells, thereby inducing apoptosis ( Int. J Mol. Med., 2001, 7: 645-652, D. M. Smith, et al.), and that GCG inhibits the growth of breast cancer cells ( J. Cell. Biochem., 2001, 82: 387-398, K. T. Kavanagh, et al.).
  • EGCG inhibits the proliferation of cancer cells more strongly than that of normal cells ( Arch. Biochem. Biophys., 2000, 376: 338-346, N. Ahmad, et al.).
  • catechins including EGCG inhibit neovascularization which has close relationship with tumor growth. As indicated, the mechanism is that catechins inhibit the phosphorylation of VEGFR-1, a receptor for VEGF, which is a growth factor for angioendothelial cells. It is reported that this does not depend on the antioxidation and anti-radical activity of catechins ( Cancer Res., 2002, 62: 381-385, S. Lamy, et al.).
  • catechins inhibit the phosphorylation of PDGF-R-beta by another growth factor, PDGF-BB in vascular smooth muscle cells, thereby inhibiting the hypertrophy of blood vessels ( FASEB J., 2002, 16: 893-895, A. Sachinidis, et al.).
  • catechins have various physiological effects in addition to the antitumor effect thereof. It is reported that EGCG inhibits glucose production in hepatic cells and promotes tyrosine phosphorylation of the insulin receptor and IRS-1, and is therefore effective against diabetes (J. Biol. Chem., 2002, 277: 34933-34940, M. E. Waltner-Law, et al.).
  • Non-Patent Reference 3 (Non-Patent Reference 3)
  • Non-Patent Reference 4 (Non-Patent Reference 4)
  • Non-Patent Reference 5 (Non-Patent Reference 5)
  • Non-Patent Reference 6 (Non-Patent Reference 6)
  • Non-Patent Reference 7 (Non-Patent Reference 7)
  • Non-Patent Reference 8 (Non-Patent Reference 8)
  • Non-Patent Reference 9 (Non-Patent Reference 9)
  • Non-Patent Reference 9 (Non-Patent Reference 9)
  • Non-Patent Reference 10 (Non-Patent Reference 10)
  • 67LR can be used as a target for drugs having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect, and have completed the present invention.
  • the invention is as follows:
  • a method of screening a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect which comprises a step of qualitatively or quantitatively determining the degree of binding of a test compound to a 67 kDa laminin receptor, and, when the test compound binds to the 67 kDa laminin receptor from the test data, then judging that the test compound is a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • [8] The drug of any of [3] to [6], which is used for a disease capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect and/or the cancer cell metastasis activity-inhibiting effect thereof.
  • a method for producing a pharmaceutical composition which comprises a step of producing the drug of any of [3] to [9] by chemical synthesis, and a step of adding a pharmaceutically acceptable carrier thereto.
  • a screening method for a drug which comprises a step of qualitatively or quantitatively determining the degree of binding of a compound having a galloyl group and a test compound to a 67 kDa laminin receptor, and, when the degree of binding of the test compound with the 67 kDa laminin receptor is higher than that of binding of the compound having a galloyl group to the 67 kDa laminin receptor from the test data, then judging that the test compound is a drug having the same pharmacological effect as that of catechins.
  • a screening method for a drug which comprises a step of making competition between the binding of a compound having a galloyl group to a 67 kDa laminin receptor and the binding of a test compound to the 67 kDa laminin receptor, and as a result of the competition, when the site at which the test compound has bound with the 67 kDa laminin receptor is the same as the site at which the compound having a galloyl group has bound with the 67 kDa laminin receptor, then judging that the test compound is a drug having the same pharmacological effect as that of the compound having a galloyl group.
  • [20] The drug of [18] or [19], which is used for a disease capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect and/or the cancer cell metastasis activity-inhibiting effect thereof.
  • a method for producing a pharmaceutical composition which comprises a step of producing the drug of any of [18] to [21] by chemical synthesis, and a step of adding a pharmaceutically-acceptable carrier thereto.
  • FIG. 1 is a diagrammatic representation of FIG. 1 :
  • FIG. 2
  • FIG. 3 is a diagrammatic representation of FIG. 3 :
  • FIG. 4
  • FIG. 5
  • FIG. 6 is a diagrammatic representation of FIG. 6 :
  • FIG. 7
  • FIG. 8
  • the invention provides a novel screening methods for drugs, using 67LR as a target.
  • One embodiment of the invention is a screening method for drugs having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect, which comprises a step of qualitatively or quantitatively determining the degree of binding of a test compound to a 67 kDa laminin receptor, and, when the test compound binds to the 67 kDa laminin receptor from the test data, then judging that the test compound is a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibi
  • 67LR used in the present invention itself is a known protein, and, for example, based on GenBank Accession No. NM-002295 registered cDNA sequence, cDNA of 67LR can be readily obtained according to an ordinary process by using the PCR with a template of various libraries to sandwich the sequence which encoding the present protein therebetween.
  • the cDNA thus obtained may be inserted into various commercially-available vectors in the form that enables protein expression, whereby it is easy to construct a cell line capable of expressing the present protein and to obtain the present protein itself.
  • cDNA production and protein expression Proc. Natl. Acad. Sci. U.S.A., 1988, 85: 6394, H. You, et al.; British Journal of Cancer, 1999, 80: 1115-1122, K. Satoh, et al.; Biochemistry, 1995, 34: 11276-11287, T. H. Landowski, et al.).
  • 67LR is a 40 S ribosome-binding protein of 37 kDa, but it is known that when the protein is expressed in membranes, it has 67 kDa.
  • any and every protein that has 67 kDa when expressed in membranes and has the ability of adhesion to bind to laminin is defined as 67LR for use in the present invention.
  • 67LR may be used in any form of, for example, a purified protein, or a soluble protein, or a protein bonded to a carrier, or a protein fused with any other protein.
  • a test compound is bound with 67LR whereby drugs are screened for those having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • the test compound in this step is not particularly limited and may have any form of cell extracts, cell culture supernatants, fermented microorganism products, organism extracts, plant extracts, purified or crude proteins, peptides, non-peptidic compounds, synthetic low-molecular compounds, natural compounds, gene libraries and others that are used in ordinary drug screening.
  • any suitable mode may be selected in accordance with the form of the test compound used.
  • a method of adding a test compound to a culture of 67LR expressing cells may be used.
  • the degree of binding of a test compound to 67LR is determined.
  • the method for the determination to be employed herein may be any of a qualitative method or a quantitative method.
  • One example of the method of determining the binding degree comprises using a surface plasmon resonance sensor as demonstrated in Examples given hereinunder.
  • the test compound is judged to be a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • the screening method of the invention is favorable for drugs having a cell growth-inhibiting effect, a neovascularization-inhibiting effect and/or a cancer cell metastasis activity-inhibiting effect.
  • a compound having a galloyl group which is suggested to have a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect has bound with 67LR and has extremely inhibited the cell growth.
  • a compound having a galloyl group may function as a cell growth factor via 67LR, further indicating that, like the compound having a galloyl group, a substance capable of binding to 67LR may have the same effects as those of the compound having a galloyl group, which are a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • a test compound capable of binding to 67LR is a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and a Creutzfelds-Jakob disease infection-inhibiting effect.
  • the compound having a galloyl group mentioned above includes galloyl group-having catechins such as epigallocatechin gallate and its 3-methyl-substituted derivative, and epicatechin gallate; galloyl group-having polyphenols; and trigalloylglucose, pentagalloylglucose, strictinin, pyrogallol. Preferred is epigallocatechin gallate.
  • the invention provides a screening method for drugs, using a compound having a galloyl group in addition to 67LR.
  • the screening method for drugs comprises a step of qualitatively or quantitatively determining the degree of binding of a compound having a galloyl group and a test compound to 67LR, and, when the degree of binding of the test compound to 67LR is higher than that of binding of the compound having a galloyl group to 67LR from the test data, then judging that the test compound is a drug having the same pharmacological effect as that of the compound having a galloyl group.
  • the invention also provides a screening method for drugs, which comprises a step of making competition between the binding of a compound having a galloyl group to a 67 kDa laminin receptor and the binding of a test compound to the 67 kDa laminin receptor, and as a result of the competition, when the site at which the test compound has bound to the 67 kDa laminin receptor is the same as the site at which the compound having a galloyl group has bound to the 67 kDa laminin receptor, then judging that the test compound is a drug having the same pharmacological effect as that of the compound having a, galloyl group.
  • a monoclonal antibody to 67LR interferes with the binding of a compound having a galloyl group to 67LR, therefore interfering with the cell growth-inhibiting effect of a compound having a galloyl group.
  • a compound having a galloyl group interferes with the binding of an anti-67LR antibody to 67LR.
  • the binding site of a compound having a galloyl group overlaps with the antibody-recognition site thereof.
  • test compound in competitive reaction of a compound having a galloyl group and a test compound with 67LR, when the test compound has bound at the same site as that at which the compound having a galloyl group has bound, then the test compound may be considered to have the same effect as that of the compound having a galloyl group.
  • the screening method of the other embodiment of the invention not using a compound having a galloyl group has been described in detail hereinabove, and its description shall apply to the embodiments of the invention using a compound having a galloyl group.
  • the compound having a galloyl group as referred to herein for use in the screening method of these embodiments of the invention may be suitably diluted with a buffer such as PBS before its use, as in Examples given hereinunder.
  • a buffer such as PBS
  • the order of adding the test compound and the compound having a galloyl group thereto is not specifically limited.
  • the drugs screened as above are usable as those for diseases capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect, the cancer cell metastasis activity-inhibiting effect, the neuroprotective effect, the anti-allergic effect, the anti-arteriosclerotic effect and/or the Creutzfelds-Jakob disease infection-inhibiting effect thereof.
  • the invention is the most suitable for cancer.
  • a drug may then be produced through ordinary chemical synthesis.
  • a pharmaceutically-acceptable carrier may be added thereto. Accordingly, a method for producing a pharmaceutical composition which comprises a step of producing the drug obtainable in the above-mentioned screening method, through chemical synthesis, and a step of adding a pharmaceutically-acceptable carrier thereto; and the pharmaceutical composition obtainable according to the production method are within the scope of the invention.
  • the pharmaceutically-acceptable carrier therein includes, for example, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer.
  • the drug may be suitably combined with any of these and may be formulated into a pharmaceutical composition. It may be administered to patients in any mode of, for example, intra-arterial injection, intravenous injection, subcutaneous injection or oral administration. Depending on the drug and on the body weight, the age and the condition of the patient, the administration route may be suitably selected. Similarly, the does may be suitably selected and determined depending on the drug and on the body weight, the age and the condition of the patient.
  • a test compound may be encoded by a DNA thereof, then the DNA can be inserted into a gene therapy vector for carrying out gene therapy with it.
  • a compound capable of binding to 67LR at a site thereof that is the same as the site at which a compound having a galloyl group binds to 67LR is also within the scope of the invention. As demonstrated in Examples given hereinunder, this is well supported by the following: A monoclonal antibody to 67LR interferes with the binding of a compound having a galloyl group to 67LR, therefore interfering with the cell growth-inhibiting effect of a compound having a galloyl group. Contrary to this, a compound having a galloyl group interferes with the binding of an anti-67LR antibody to 67LR. Specifically, the binding site of a compound having a galloyl group overlaps with the antibody-recognition site thereof.
  • the compound of the type is considered to have the same effect as that of a compound having a galloyl group, it is usable as a cell growth inhibitor, a neovascularization inhibitor and a cancer cell metastasis activity inhibitor., or that is, as an anticancer agent.
  • the human lung cancer cell line A549 (ATCC Number: CCL-185) used in this experiment were subcultivated and kept in an ERDF medium (Kyokutoh Pharmaceutical) supplemented with 10% fetal bovine serum (FBS) (Bio Source International, Camarillo, Calif.), at 37° C. in a water vapor-saturated 5% CO 2 condition. 1.125 g of NaHCO 3 (Wako Pure Chemicals, Osaka, Japan) was added to one liter of the ERDF medium. The cells were cultivated and kept in their logarithmic growth phase. Human Burkit's lymphoma cell line DND39 were subcultivated and kept in a 5% FBS-added RPMI-1640 medium (Nissui, Japan) at 37° C.
  • FBS fetal bovine serum
  • Green tea catechins, epigallocatechin-3-O-gallate (EGCG), epicatechin-3-O-gallate (ECG), epigallocatechin (EGC), epicatechin (EC), catechin (C) and epigallocatechin-3-(3-O-methyl)-gallate (EGCG3′′Me) were dissolved in a phosphate buffer (PBS) to have a concentration of 5 mM. Before use, it was suitably thawed.
  • PBS phosphate buffer
  • PBS was prepared by dissolving 8.0 g of NaCl (Nacalai Tesque, Inc.), 0.2 g of KCl (Nacalai Tesque, Inc.), 1.15 g of Na 2 HPO 4 (Nacalai Tesque, Inc.) and 0.2 g of KH 2 PO 4 (Nacalai Tesque, Inc.) in 1 liter of ultra-pure water.
  • Caffeine and quercetin were purchased from Nacalai Tesque, Inc., and the former was suspended in PBS while the latter was in dimethylsulfoxide (DMSO) (Nacalai Tesque, Inc.), each having a concentration of 5 mM.
  • DMSO dimethylsulfoxide
  • Tripanblue (Wako Pure Chemicals) was suspended in PBS to have a concentration of 1%, and sterilized in an autoclave at 121° C. for 20 minutes. All-trans-retionic acid (ATRA) was purchased from Sigma (St. Louis, Mo.), and dissolved in ethanol.
  • ATRA All-trans-retionic acid
  • TRIzol used for RNA extraction was purchased from Invitrogen (Carlsbad, Calif.).
  • Aqueous 0.1% diethyl pyrocarbonate (DEPC) was purchased from Sigma (St. Louis, Mo.).
  • a DEPC solution used for RNA dissolution was prepared by adding DEPC to distilled water to have a final concentration of 0.1%, then stirring it for 2 hours and autoclaving it.
  • Oligotex-dT30 and human placenta-derived RNase inhibitor were purchased from Takara (Kyoto, Japan); and Molony murine leukemia virus (MMLV)-reverse transferase was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).
  • pT ARGE TTM Mammalian Expression Vector System Promega, Madison, Wis.
  • QIAGEN Plasmid Midi Kit or EndoFree Plasmid Maxi Kit both QIAGEN.
  • An LB medium was prepared by dissolving 10 g of Bacto Tryptone (Becton Dickinson, Sunnyvale, Calif.), 5 g of Bacto Yeast Extract (DIFCO LABORATORIES, Detroit, Mich.) and 5 g of NaCl in 1 liter of ultra-pure water, and autoclaving it. After cooled to 60° C., 1000 ml of ampicillin (150 mg/ml) (prepared by dissolving ampicillin sodium (Wako Pure Chemicals) in ultra-pure water to have a concentration of 150 mg/ml and filtering it for sterilization) was added to it.
  • ampicillin 150 mg/ml
  • LB plate For an LB plate, 2 g of Bacto Tryptone, 1 g of Bacto Yeast Extract, 2 g of NaCl and 5 g of Bacto Agar (DIFCO) were dissolved in 200 ml of ultra-pure water and autoclaved. After cooled to 60° C., 200 ml of ampicillin (150 mg/ml) was added to it, and this was put into 10-ml dishes (Falcon), as divided into portions of 10 ml each. Isopropyl-b-D(-)-thiogalactopyranoside (IPTG) was purchased from Wako Pure Chemicals, and it was formulated to have a concentration of 0.1 M.
  • IPTG Isopropyl-b-D(-)-thiogalactopyranoside
  • ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan), in which was used ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kits Version 2.0 (Applied Biosystems).
  • TSR Template Suppression Reagent
  • FuGENETM 6 Transfection Reagent (Roche Diagnistics Gmbh, Mannheim, Germany) was used.
  • the anti-human laminin receptor antibody for flow cytometry analysis was purchased from NEOMARKERS (Fremont, Calif.).
  • mouse IgM antibody used was one from Zymed Laboratories Inc. (San Francisco, Calif.).
  • a fluorescein isothiocyanate (FITC)-labeled anti-mouse IgM goat antibody was purchased from Southern Biotechnology Associates, Inc. (Birmingham, Ala.).
  • FACS Calibur Becton Dickinson
  • cDNA synthesis was carried out as follows: First, 1 ml of (dT) 20 primer (0.5 mg/ml) was added to 10 mg of the total RNA, left at 70° C. for 10 minutes, and then immediately cooled with ice for annealing.
  • DND39 cells were treated with 1 mM-ATRA at 37° C. for 24 hours, then subjected to RNA extraction and to cDNA synthesis.
  • primers were produced (H-LamininR-F; 5′-ATGTCCGGAGCCCTTGATGTCC-3′, H-LamininR-R; 5′-TTAAGACCAGTCAGTGGTTGCTC-3′). The primers were prepared at mM.
  • 1 ml of the synthesized cDNA, 1 ml of Ex Taq, 2 ml of 10 ⁇ Taq buffer, 1.6 ml of 2.5 mM dNTP, primers of 0.5 ml each, and 14.3 ml of dH 2 O were suspended and subjected to PCR.
  • the condition was as follows: The initial denaturation was at 95° C. for 5 minutes, the denaturation reaction was at 94° C. for 30 seconds, the annealing was at 58° C. for 30 seconds, and the extension reaction was at 72° C. for 30 seconds, 25 cycles of denaturation, annealing and extension were carried out.
  • the 67LR expression vector constructed (pT ARGE T-hLamininR) was introduced into A549 cells, using FuGENETM6 Transfection Reagent. Its details are described below.
  • the cells were inoculated into a 10% FBS-ERDF medium at 1 ⁇ 10 4 cells/ml. By keeping at 37° C. for 24 hours, the cells were adhered to the medium. Next, a fresh ERDF medium was put into a tube, and FuGENETM 6 (three times the amount of the gene) was directly added and gently mixed.
  • pT ARGE T-hLamininR was added and gently mixed, and this was kept at room temperature for 30 minutes. This was added to the medium, and kept at 37° C. for 48 hours. The cells were washed with a medium, and then the medium was changed with a fresh one.
  • an anti-67LR antibody having a final concentration of 10 g/ml (1% FBS-containing ERDF medium) was treated at 37° C. for 30 minutes, and then EGCG treatment was carried out so as to investigate the influence thereof on the antibody-treatment cells.
  • a negative control the same treatment was carried out with mouse IgM.
  • the binding of various component (5 ⁇ M) to the cells was determined by using a surface plasmon resonance sensor SPR670 (Nippon Laser and Electronic Lab., Nagoya, Japan).
  • SPR670 Surface plasmon resonance sensor
  • the cells were fixed on a gold membrane (Nippon Laser and Electronic Lab.) according to a standard fixation method for protein.
  • a gold membrane was dipped in a solution in ethanol of 10 mM of 4,4-dithiodibutyric acid, DDA (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) (this was prepared by dissolving 2.38 mg of DDA in 10 ml of 99% ethanol followed by diluting it with ethanol to 1/100) in such a manner that its gold surface could face upward, and gently stirred at room temperature for 30 minutes. Next, this was washed twice with ethanol with no pressure applied to the gold surface to thereby introduce a self-assembly membrane (SA membrane).
  • SA membrane self-assembly membrane
  • the cells were suspended to a density of 3 ⁇ 10 5 cells/ml (in a flow buffer, PBS), and 20 ⁇ l of the cell suspension was dropwise applied onto the gold membrane and kept at room temperature for 30 minutes so as to fix the cells onto the membrane.
  • a green tea catechin diluted with PBS to have a concentration of 1, 10, 25 or 50 MN was applied thereto, and the change in the surface plasmon resonance angle was determined so as to monitor its binding to the cells.
  • the binding competition test of EGCG and an anti-67LR antibody was carried out as follows: An anti-67LR antibody having a final concentration of 10 ⁇ g/ml (1% FBS-containing ERDF medium) was treated at 37° C. for 30 minutes, and then the cells were fixed on the gold membrane and tested in the same manner as above using a surface plasmon resonance sensor. In this case, the same treatment was also carried out with mouse IgM as a negative control.
  • 67LR laminin receptor
  • a secondary antibody, anti-mouse IgM FITC-labeled antibody capable of recognizing an isotype of, LR antibody was added to 25 ⁇ l, as a whole, of 1% FBS-PBS so as to have a final concentration of 12.5 ⁇ g/ml. This was incubated at 4° C. for 30 minutes, then washed twice with PBS, and re-suspended in PBS, and analyzed with a flow cytometer. A negative control IgM antibody of the same concentration (10 ⁇ g/ml) was reacted in the same manner as herein. The 67LR expression amount on the cell surfaces was indicated by the center value of the fluorescent intensity of LR.
  • the cells were treated with an EGCG-added 1% FBS-PBS having a final EGCG concentration of 50 NM, and then kept at 37° C. for 30 minutes. This was washed once with PBS. Then, this was treated in the same manner as above with the primary antibody and others, and analyzed. As a control, the cells were treated with an EGCG-free 1% FBS-PBS.
  • test data were subjected to statistical analysis according to Student's t-test.
  • pTAT-hLamininR a 67LR expression vector (pTAT-hLamininR) was introduced into A549 cells according to the method mentioned below.
  • the cells were inoculated at 1 ⁇ 10 4 cells/ml (10% FBS-ERDF medium). After 24 hours, a fresh ERDF medium was put into a tube, and FuGENETM 6 (three times the amount of the gene) was directly added and gently mixed.
  • pT ARGE T-hLamininR of varying concentrations were added and gently mixed, and this was kept at room temperature for 30 minutes. This was added to the medium, and was continuously cultured at 37° C. for 48 hours.
  • EGCG of various concentrations were added to the 67LR transient expression cells, and treated with a 5% FBS-containing ERDF medium at 37° C. for 48 hours. After the treatment, the number of the cells was counted and the survival rate thereof was determined according to a Tripanblue staining method.
  • 67LR is a membrane protein existing in a cell membrane, and the fact that the EGCG effect was enhanced in the cells with the gene expression vector introduced therein would be due to the increase in the EGCG binding. Accordingly, the EGCG binding to the cells was determined by the sue of a surface plasmon resonance sensor. EGCG was used at 5 ⁇ M.
  • Example 1 The results of Example 1 are shown in FIG. 1 , FIG. 2 and FIG. 3 .
  • the 67LR expression amount on the cell surfaces was determined. A little expression was found in the control, A549 cells. An empty vector (0.5 ⁇ g) was introduced into the cell showed almost no influence on the expression amount. However, when the 67LR expression vector (0.5 ⁇ g) was introduced into the cells, then the expression amount greatly increased. This confirms the expression of 67LR on the cell surfaces.
  • Example 2 The results in Example 2 are shown in FIG. 4 .
  • EGCG binds to cells via 67LR and exhibits its growth inhibiting activity.
  • 67LR forced expression cells were treated in advance with an anti-67LR antibody, and then the influence on the binding and the growth inhibiting activity of EGCG was investigated.
  • EGCG a concentration of only 0.1 ⁇ M showed its growth inhibiting activity in 67LR forced expression cells.
  • the growth inhibiting activity was lost. In this test, no influence on the survival rate was found. It was shown that not only the EGCG binding to cells but also the growth inhibiting activity of EGCG is exerted via the binding of 67LR to EGCG.
  • Example 3 The results in Example 3 are shown in FIG. 5 and FIG. 6 .
  • EGCG binds to cell membranes via 67LR and it exhibits a cancer cell growth inhibiting activity. It was further investigated whether these effects via 67LR may be intrinsic to EGCG.
  • the other tea components tested herein are principal green tea catechins, ECG, EGC, EC, C and EGCG3, Me which is reported to have a strong antiallergic effect and to stably exist in vivo.
  • caffeine that has various physiological functions like catechins, and quercetin, one kid of flavanols that is reported also to have many physiological functions were also tested.
  • pT ARGE T-hLamininR was introduced into A549 cells by the lipofection method, followed by cultivation at 37° C. for 48 hours. Then, the cells were treated with various tea components each having a final concentration of 5 ⁇ M. The cells were further cultivated at 37° C. for 48 hours, and then the number of the cells and the survival rate thereof were determined. As a result, the gene introduction has no influence on the survival rate and the number of the cells treated with any of C, EC, EGC, caffeine and quercetin.
  • Example 4 The results in Example 4 are shown in FIG. 7 and FIG. 8 .
  • the invention can provide a novel screening method for drugs, using a 67 kDa laminin receptor as a target.

Abstract

It is intended to provide a novel method of screening a drug with the use of a 67 kDa laminin receptor and a drug obtained thereby. A method of screening a drug having an effect on inhibiting cell proliferation, an angiogenesis inhibitory effect, an effect of inhibiting cancer cell metastasis, a neuroprotective effect, and anti-allergic effect, and anti-arteriosclerotic effect and/or an effect of inhibiting infection with Creutzfeldt-Jakob disease which involves the step, of qualitatively or quantitatively measuring the degree of the binding of a test compound to a 67 kDa laminin receptor, and judging that the test compound is a drug having an effect of inhibiting cell proliferation, and angiogenesis inhibitory effect, an effect of inhibiting cancer cell metastasis, a neuroprotective effect, and anti-allergic-effect, and anti-arteriosclerotic effect and/or an effect of inhibiting infection with Creutzfeldt-Jakob disease in the case where it is found out by the results of the measurement that the test compound binds to the 67 kDa laminin receptor, and a drug obtained thereby.

Description

    TECHNICAL FIELD
  • The present invention relates to a screening-method for drugs using a 67 kDa laminin receptor, and to a drug obtained by it.
    • BACKGROUND ART
  • 67 kDa laminin receptor (hereinafter it may be referred to as “67LR”) is a protein of 67 kDa, which is derived from a 37 kDa precursor protein translated from mRNA that codes for 295 amino acids, through intracellular acylation polymerization of the precursor protein by fatty acid for homo-dimerization or hetero-dimerization thereof, and only when it moves onto the surface of a cell membrane together with integlins, it functions as a laminin receptor (Biochemistry, 1995, 34: 11276-11287, T. H. Landowski et al.; J. Cell. Biochem, 1998, 69: 244-251, S. Buto et al.). It has been clarified that the 37 kDa precursor protein participates in protein synthesis as a ribosome-related protein p40 and it is the same as that reported as a multidrug resistance-related protein (MGr1-Ag) (Cell. Mal. Life Sci., 2002, 59: 1577-1583, Y. Shi at al.). From the data of its high expression in many types of cancer cells, the laminin receptor is considered to be an oncofetal antigen as an immunogen for T cells, or that is, as a general tumor-specific transplantation antigen (Anticancer Research, 1999, 19: 5535-5542, J. H. Coggin, Jr., et al.). In addition to 67LR, a dozen or so types of laminin receptors have been already reported, and of those, the relationship between 67LR, and cancer is strongly suggested.
  • Based on detection or non-detection thereof in cancer cells, 67LR is known as an important prognostic factor in many types of cancers that indicates the degree of malignancy of human cancer patients (Breast Cancer Research and Treatment, 1998, 52: 137-145, S. Menard, et al.; Clinical Cancer Research, 1997, 3: 227-231, G. Fontanini, et al.; Clinical Cancer Research, 1996, 2: 1777-1780, F. Basolo, et al.; J. Natl. Cancer Inst., 1991, 83: 29-36, V. Coice, et al.). In animal models, it is suggested that 67LR participates in proliferation, movement, invasion and metastasis of cancer cells. For example, it is reported that the survival rate of 67LR-positive breast cancer patients is significantly lower than that of 67LR-negative ones. It is shown that the expression, of laminin, the ligand of 67LR, has no influence on prognosis but the expression of the receptor 67LR brings about a negative result in prognosis (Breast Cancer Research and Treatment, 1998, 52: 137-145, S. Menard et al.).
  • Some experiments have been reported, on the basis of this information and with expectation of an antitumor effect by inhibiting 67LR expression. It is reported that a 67LR low-expression cell line constructed by introduction of an antisense RNA of 67LR into a cancer cell line shows in-vivo significant depression of tumor proliferation capability and depression of metastasis capability in mice, as compared with the original parent cell strain, and, as a result, it increases the survival rate of individual mice (British Journal of Cancer, 1999, 80 1115-1122, K. Satoh, at al.). Further, it is reported that the 67LR low-expression cell line retards tumor neovascularization and decrease, the production of a neovascularization promotion factor, VEGF itself, as compared with the parent strain (Cancer Letters, 2000, 153: 161-168, M. Tanaka, at al.). Similarly, also in tumor metastasis experiments using an antibody for 67LR, the same effect as in the antisense experiments has been recognized (Jpn. J. Cancer Res., 1999, 90: 425-431, R. Narumi, at al.).
  • In the fields not relating to tumor, some reports have been made regarding the function of 67LR. It is reported that the growth of neovascularized blood, vessels that are induced in ischemic animal models is inhibited by a 67LR-binding laminin-derived peptide (cysteine-aspartic acid-proline-glycine-tyrosine-isoleucine-glycine-serine-arginine) or EGF-derived peptide (cysteine-valine-isoleucine-glycine-tyrosine-serine-glycine-aspartic acid-arginine-cysteine) (Am. J. Pathol., 2002, 160: 307-313, D. Gebarowska, et al.). It is reported, that eNOS expression and NO production, which are said to participate in arteriosclerosis induced by the shear force to angioendothelial cells, are inhibited by a 67LR-binding laminin-derived pentapeptide (tyrosine-isoleucine-glycine-serine-arginine) (J. Biol. Chem., 1999, 274: 15996-16002, T. Gloe, et al.).
  • A recent report says that 67LR acts as a receptor for a prion protein, which is considered as a cause for Creutzfelds-Jakob disease, and prion binding for internalization and further the binding and the internalization are inhibited by secretion of a membrane domain-deficient mutant 67LR (EMBO J., 2001, 20: 5863-5875, S. Gauczynski, at al.).
  • It is also reported that 67LR is expressed in a subset group of CD4+CD8− or CD4−CD8+ of CD45RO+/CD45RA− memory cells that are a subset of T cells, and an effect of 67LR to the immune system is suggested (J. Immunol., 1999, 163: 3430-3440, S. M. Canfield, et al.).
  • There are some reports relating to the expression of mRNA of 67LR. It is reported that the expression is inhibited by a cancer-inhibiting factor p53 or anticancer factors TNF-alpha and IFN-gamma (Biochem. Biophys. Res. Commun., 1998, 251: 564-569, N. Clauses, at al.). However, there is no report relating to low-molecular compounds for 67LR which is presumed to participate in such diverse functions.
  • On the other hand, among catechins, epigallocatechin gallate (hereinafter this may be referred to as “EGCG”) is an main ingredient: of tea catechins, accounting for about 50% thereof. In addition, tea catechins include epigallocatechin, epicatechin gallate and epicatechin (hereinafter these may be referred to as “EGC”, “ECG” and “EC”, respectively).
  • There is a long history that tea has been used as a drug from the past, and recently, tea has been analyzed for the relation between its efficacy and its components. Of the components, EGCG is a component discovered by A. Bradfield, et al. in 1947 (J. Chem. Soc., 1947, 32: 2249, A. E. Bradfield, et al.).
  • Various physiological effects of tea catechins including EGCG have been reported, for example, antioxidation, anticancer, suppression of plasma cholesterol increase, suppression of blood pressure increase, platelet aggregation inhibition, suppression of blood sugar increase, dementia prevention, antiulcer, antiinflammation, antiallergy, antibacterial, anticaries, antiviral, detoxication, enteroflora improvement, deodorization (Functions of Tea, edited by Keiichiro Muramatsu, Society Publishing Center, 2002).
  • Of those, there are many reports relating to anticancer effect that includes anti-mutation effect, anti-carcinogenic promotion effect, antitumor proliferation-inhibiting effect, anti-invasion/metastasis inhibiting effect, anti-neovascularization inhibiting effect. Recent reports say that EGCG inhibits DNA synthesis in leukemia cells, thereby inducing apoptosis (Int. J Mol. Med., 2001, 7: 645-652, D. M. Smith, et al.), and that GCG inhibits the growth of breast cancer cells (J. Cell. Biochem., 2001, 82: 387-398, K. T. Kavanagh, et al.). Further, there is a report saying that EGCG inhibits the proliferation of cancer cells more strongly than that of normal cells (Arch. Biochem. Biophys., 2000, 376: 338-346, N. Ahmad, et al.).
  • Regarding invasion and metastasis, it is reported that catechin inhibits the invasion of high-metastatic cells in an invasion test using a matrigel, and that EGCG inhibits adhesion of cancer cells to fibronectin and laminin (Cancer Lett., 1995, 98: 27-31, M. Susuka, et al.; Cell Biol. Int., 1993, 17: 559-564, M. Isemura, et al.; Cancer Lett., 2001, 173: 15-20, Y. Suzuki, et al.).
  • Further, molecular-level analysis of these catechin effects has been reported recently. For example, EGCG concentration-dependently inhibits the proliferation of Her-2 antigen high-expression cells which, as suggested, may have relation to cancer. As reported, its functional mechanism would be inhibition of the downstream signal transmission through Her-2 phosphorylation inhibition (Cancer Res., 2002, 62: 652-655, S. Pianetti, et al.).
  • It has been reported that catechins including EGCG inhibit neovascularization which has close relationship with tumor growth. As indicated, the mechanism is that catechins inhibit the phosphorylation of VEGFR-1, a receptor for VEGF, which is a growth factor for angioendothelial cells. It is reported that this does not depend on the antioxidation and anti-radical activity of catechins (Cancer Res., 2002, 62: 381-385, S. Lamy, et al.).
  • Similarly, it is reported that catechins inhibit the phosphorylation of PDGF-R-beta by another growth factor, PDGF-BB in vascular smooth muscle cells, thereby inhibiting the hypertrophy of blood vessels (FASEB J., 2002, 16: 893-895, A. Sachinidis, et al.).
  • Further, it is reported that EGCG inhibits in-vivo neovascularization and growth of endothelial cells by EGF-2 (Nature, 1999, 389: 381, Y. Cao, et al.). There is a report saying that EGCG binds to an apoptosis-inducing Fas protein (Biochem. Biophys. Res. Commun., 2001, 285: 1102-1106, S. Hayakawa, et al.). However, it is not clarified as to whether the above-mentioned EGCG's effect could have relation to Pas, but suggesting the presence of any other factor that may interact with EGCG.
  • It has become clear on the molecular level that catechins have various physiological effects in addition to the antitumor effect thereof. It is reported that EGCG inhibits glucose production in hepatic cells and promotes tyrosine phosphorylation of the insulin receptor and IRS-1, and is therefore effective against diabetes (J. Biol. Chem., 2002, 277: 34933-34940, M. E. Waltner-Law, et al.).
  • From a report indicating that, in Parkinson model mice, EGCG shows a strong neuroprotective effect (J. Biol. Chem., 2002, 277: 30574-30580, Y. Levites, et al.), EGCG is expected to inhibit many types of neuropathy. There are a report saying that the expression of Fc-epsilon RI in basophils, which is a cause for allergy, is inhibited by EGCG and its methylated derivative (J. Agric. Food Chem., 2002, 50: 5729-5734, Y. Fujimura, et al.); and a report saying that the expression of COX-2 and NO synthase-2, as induced by IL-1-beta in cartilage, is inhibited by EGCG (Free Radical Biology & Medicine, 2002, 33: 1097-2002, S. Ahmed, et al.).
  • However, so long as the present inventors know, there is no report at all up to the present, relating to the fact that EGCG functions as a cell growth-inhibiting factor via 67LR and to the fact that 67LR may be used as a target in drug screening for low-molecular compounds having a cell growth-inhibiting effect.
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  • Breast Cancer Research and Treatment, 1998, 52: 137-145
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    • DISCLOSURE OF THE INVENTION
  • The present inventors conducted extensive studies in consideration of the above-mentioned problems, and, as a result, have found that 67LR can be used as a target for drugs having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect, and have completed the present invention.
  • Specifically, the invention is as follows:
  • [1] A method of screening a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect, which comprises a step of qualitatively or quantitatively determining the degree of binding of a test compound to a 67 kDa laminin receptor, and, when the test compound binds to the 67 kDa laminin receptor from the test data, then judging that the test compound is a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • [2] The screening method of [1], wherein the drug has a cell growth-inhibiting effect, a neovascularization-inhibiting effect and/or a cancer cell metastasis activity-inhibiting effect.
  • [3] A drug obtainable according to the screening method of [1] or [2].
  • [4] The drug of any of [1] to [3], wherein the active ingredient is a compound having a galloyl group.
  • [5] The drug of [4], wherein the compound is a catechin.
  • [6] The drug of [5], wherein the catechin is epigallocatechin gallate.
  • [7] The drug of any of [3] to [6], which is used for a disease capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect, the cancer cell metastasis activity-inhibiting effect, the neuroprotective effect, the anti-allergic effect, the anti-arteriosclerotic effect and/or the Creutzfelds-Jakob disease infection-inhibiting effect thereof.
  • [8] The drug of any of [3] to [6], which is used for a disease capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect and/or the cancer cell metastasis activity-inhibiting effect thereof.
  • [9] The drug of [8]., wherein the disease is cancer.
  • [10] A method for producing a pharmaceutical composition, which comprises a step of producing the drug of any of [3] to [9] by chemical synthesis, and a step of adding a pharmaceutically acceptable carrier thereto.
  • [11] A pharmaceutical composition obtainable according to the production method of [10].
  • [12] A screening method for a drug, which comprises a step of qualitatively or quantitatively determining the degree of binding of a compound having a galloyl group and a test compound to a 67 kDa laminin receptor, and, when the degree of binding of the test compound with the 67 kDa laminin receptor is higher than that of binding of the compound having a galloyl group to the 67 kDa laminin receptor from the test data, then judging that the test compound is a drug having the same pharmacological effect as that of catechins.
  • [13] A screening method for a drug, which comprises a step of making competition between the binding of a compound having a galloyl group to a 67 kDa laminin receptor and the binding of a test compound to the 67 kDa laminin receptor, and as a result of the competition, when the site at which the test compound has bound with the 67 kDa laminin receptor is the same as the site at which the compound having a galloyl group has bound with the 67 kDa laminin receptor, then judging that the test compound is a drug having the same pharmacological effect as that of the compound having a galloyl group.
  • [14] The screening method of [12] or [13], wherein the pharmacological effect of the compound having a galloyl group is a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • [15] The screening method of [12] or [13], wherein the pharmacological effect of the compound having a galloyl group is a cell growth-inhibiting effect, a neovascularization-inhibiting effect and/or a cancer cell metastasis activity-inhibiting effect.
  • [16] The screening method of any of [12] to [15], wherein the compound is a catechin having a galloyl group.
  • [17] The screening method of any of [12] to [15], wherein the catechin is epigallocatechin gallate.
  • [18] A drug obtainable according to the screening method of any of [12.] to [17].
  • [19] The drug of [18], which is used for a disease capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect, the cancer cell metastasis activity-inhibiting effect, the neuroprotective effect, the antiallergic effect, the antiarteriosclerotic effect and/or the Creutzfelds-Jakob disease infection-inhibiting effect thereof.
  • [20] The drug of [18] or [19], which is used for a disease capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect and/or the cancer cell metastasis activity-inhibiting effect thereof.
  • [21] The drug of [20], wherein the disease is cancer.
  • [22] A method for producing a pharmaceutical composition, which comprises a step of producing the drug of any of [18] to [21] by chemical synthesis, and a step of adding a pharmaceutically-acceptable carrier thereto.
  • [23] A pharmaceutical composition obtainable according to the production method of [22].
  • [24] A compound capable of binding to a 67 kDa laminin receptor at a site thereof that is the same as the site at which a compound having a galloyl group binds to the 67 kDa, laminin receptor.
  • [25] The compound of [24], which is a catechin.
  • [26] The compound of [26], wherein the catechin is epigallocatechin gallate.
  • [27] A cell growth inhibitor containing the compound of any of [24] to [26].
  • [28] A neovascularization inhibitor containing the compound of any of [24] to [26].
  • [29] A cancer cell metastasis activity inhibitor containing the compound of any of [24] to [26].
  • [30] An anticancer agent inhibitor containing the compound of any of [24] to [26].
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1:
  • It is a drawing showing the test data with a surface plasmon resonance sensor in Example 1.
  • FIG. 2:
  • It is a drawing showing a result of the cell growth test in Example 1.
  • FIG. 3:
  • It is a drawing showing a result of the cell growth test in Example 1.
  • FIG. 4:
  • It is a drawing showing a result of analysis through flow cytometry in Example 2.
  • FIG. 5:
  • It is a drawing showing a result of the cell growth test in Example 3.
  • FIG. 6:
  • It is a drawing showing the test data with a surface plasmon resonance sensor in Example 3.
  • FIG. 7:
  • It is a drawing showing the test data with a surface plasmon resonance sensor in Example 4.
  • FIG. 8:
  • It is a drawing showing a result of the cell growth test in Example 4.
    •  BEST MODE FOR CARRYING OUT THE INVENTION
  • The invention is described in detail hereinunder.
  • The invention provides a novel screening methods for drugs, using 67LR as a target.
  • One embodiment of the invention is a screening method for drugs having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect, which comprises a step of qualitatively or quantitatively determining the degree of binding of a test compound to a 67 kDa laminin receptor, and, when the test compound binds to the 67 kDa laminin receptor from the test data, then judging that the test compound is a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • 67LR used in the present invention itself is a known protein, and, for example, based on GenBank Accession No. NM-002295 registered cDNA sequence, cDNA of 67LR can be readily obtained according to an ordinary process by using the PCR with a template of various libraries to sandwich the sequence which encoding the present protein therebetween. The cDNA thus obtained may be inserted into various commercially-available vectors in the form that enables protein expression, whereby it is easy to construct a cell line capable of expressing the present protein and to obtain the present protein itself. Apart from it, there are some reports relating to cDNA production and protein expression (Proc. Natl. Acad. Sci. U.S.A., 1988, 85: 6394, H. You, et al.; British Journal of Cancer, 1999, 80: 1115-1122, K. Satoh, et al.; Biochemistry, 1995, 34: 11276-11287, T. H. Landowski, et al.).
  • As a gene thereof, 67LR is a 40 S ribosome-binding protein of 37 kDa, but it is known that when the protein is expressed in membranes, it has 67 kDa. In the invention, any and every protein that has 67 kDa when expressed in membranes and has the ability of adhesion to bind to laminin is defined as 67LR for use in the present invention. Not only an intact protein, but also its partial peptide can be used herein. Depending on the means for screening employed herein, 67LR may be used in any form of, for example, a purified protein, or a soluble protein, or a protein bonded to a carrier, or a protein fused with any other protein.
  • In the invention, a test compound is bound with 67LR whereby drugs are screened for those having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect. The test compound in this step is not particularly limited and may have any form of cell extracts, cell culture supernatants, fermented microorganism products, organism extracts, plant extracts, purified or crude proteins, peptides, non-peptidic compounds, synthetic low-molecular compounds, natural compounds, gene libraries and others that are used in ordinary drug screening.
  • For the binding of a test compound to 67LR, any suitable mode may be selected in accordance with the form of the test compound used. For example, a method of adding a test compound to a culture of 67LR expressing cells may be used.
  • In the manner as above, the degree of binding of a test compound to 67LR is determined. The method for the determination to be employed herein may be any of a qualitative method or a quantitative method. One example of the method of determining the binding degree comprises using a surface plasmon resonance sensor as demonstrated in Examples given hereinunder.
  • As a result of the measurement of the binding degree thereof, when the test compound has substantially bound to 67LR, then the test compound is judged to be a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect. Above all, the screening method of the invention is favorable for drugs having a cell growth-inhibiting effect, a neovascularization-inhibiting effect and/or a cancer cell metastasis activity-inhibiting effect.
  • As demonstrated in Examples given hereinunder, a compound having a galloyl group which is suggested to have a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect has bound with 67LR and has extremely inhibited the cell growth.
  • This suggests that a compound having a galloyl group may function as a cell growth factor via 67LR, further indicating that, like the compound having a galloyl group, a substance capable of binding to 67LR may have the same effects as those of the compound having a galloyl group, which are a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and/or a Creutzfelds-Jakob disease infection-inhibiting effect.
  • Accordingly, in the screening method of the invention, it may be judged that a test compound capable of binding to 67LR is a drug having a cell growth-inhibiting effect, a neovascularization-inhibiting effect, a cancer cell metastasis activity-inhibiting effect, a neuroprotective effect, an anti-allergic effect, an anti-arteriosclerotic effect and a Creutzfelds-Jakob disease infection-inhibiting effect.
  • The compound having a galloyl group mentioned above includes galloyl group-having catechins such as epigallocatechin gallate and its 3-methyl-substituted derivative, and epicatechin gallate; galloyl group-having polyphenols; and trigalloylglucose, pentagalloylglucose, strictinin, pyrogallol. Preferred is epigallocatechin gallate.
  • Other embodiments of the screening method of the invention are described.
  • In other embodiments thereof, the invention provides a screening method for drugs, using a compound having a galloyl group in addition to 67LR. Specifically, the screening method for drugs comprises a step of qualitatively or quantitatively determining the degree of binding of a compound having a galloyl group and a test compound to 67LR, and, when the degree of binding of the test compound to 67LR is higher than that of binding of the compound having a galloyl group to 67LR from the test data, then judging that the test compound is a drug having the same pharmacological effect as that of the compound having a galloyl group.
  • The invention also provides a screening method for drugs, which comprises a step of making competition between the binding of a compound having a galloyl group to a 67 kDa laminin receptor and the binding of a test compound to the 67 kDa laminin receptor, and as a result of the competition, when the site at which the test compound has bound to the 67 kDa laminin receptor is the same as the site at which the compound having a galloyl group has bound to the 67 kDa laminin receptor, then judging that the test compound is a drug having the same pharmacological effect as that of the compound having a, galloyl group.
  • As demonstrated in Examples given hereinunder, a monoclonal antibody to 67LR interferes with the binding of a compound having a galloyl group to 67LR, therefore interfering with the cell growth-inhibiting effect of a compound having a galloyl group. Contrary to this, a compound having a galloyl group interferes with the binding of an anti-67LR antibody to 67LR. Specifically, the binding site of a compound having a galloyl group overlaps with the antibody-recognition site thereof. This indicates that, in competitive reaction of a compound having a galloyl group and a test compound with 67LR, when the test compound has bound at the same site as that at which the compound having a galloyl group has bound, then the test compound may be considered to have the same effect as that of the compound having a galloyl group.
  • The screening method of the other embodiment of the invention not using a compound having a galloyl group has been described in detail hereinabove, and its description shall apply to the embodiments of the invention using a compound having a galloyl group. The compound having a galloyl group as referred to herein for use in the screening method of these embodiments of the invention may be suitably diluted with a buffer such as PBS before its use, as in Examples given hereinunder. In case where a test compound and a compound having a galloyl group are added to a 67LR expression cell culture to induce the intended competitive reaction therein, the order of adding the test compound and the compound having a galloyl group thereto is not specifically limited.
  • The drugs screened as above are usable as those for diseases capable of being prevented and/or treated owing to the cell growth-inhibiting effect, the neovascularization-inhibiting effect, the cancer cell metastasis activity-inhibiting effect, the neuroprotective effect, the anti-allergic effect, the anti-arteriosclerotic effect and/or the Creutzfelds-Jakob disease infection-inhibiting effect thereof. Of those diseases, the invention is the most suitable for cancer.
  • In the invention, once a drug is selected through the screening, the drug may then be produced through ordinary chemical synthesis. In addition, a pharmaceutically-acceptable carrier may be added thereto. Accordingly, a method for producing a pharmaceutical composition which comprises a step of producing the drug obtainable in the above-mentioned screening method, through chemical synthesis, and a step of adding a pharmaceutically-acceptable carrier thereto; and the pharmaceutical composition obtainable according to the production method are within the scope of the invention.
  • When the drug is used as a pharmaceutical composition thereof, then the pharmaceutically-acceptable carrier therein includes, for example, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer. The drug may be suitably combined with any of these and may be formulated into a pharmaceutical composition. It may be administered to patients in any mode of, for example, intra-arterial injection, intravenous injection, subcutaneous injection or oral administration. Depending on the drug and on the body weight, the age and the condition of the patient, the administration route may be suitably selected. Similarly, the does may be suitably selected and determined depending on the drug and on the body weight, the age and the condition of the patient. When a test compound may be encoded by a DNA thereof, then the DNA can be inserted into a gene therapy vector for carrying out gene therapy with it.
  • A compound capable of binding to 67LR at a site thereof that is the same as the site at which a compound having a galloyl group binds to 67LR is also within the scope of the invention. As demonstrated in Examples given hereinunder, this is well supported by the following: A monoclonal antibody to 67LR interferes with the binding of a compound having a galloyl group to 67LR, therefore interfering with the cell growth-inhibiting effect of a compound having a galloyl group. Contrary to this, a compound having a galloyl group interferes with the binding of an anti-67LR antibody to 67LR. Specifically, the binding site of a compound having a galloyl group overlaps with the antibody-recognition site thereof. Since the compound of the type is considered to have the same effect as that of a compound having a galloyl group, it is usable as a cell growth inhibitor, a neovascularization inhibitor and a cancer cell metastasis activity inhibitor., or that is, as an anticancer agent.
    • EXAMPLES
  • The invention is described in more detail with reference to the following Examples, to which, however, the invention should not be limited.
    • Materials and Methods (1) Cells and Cell Culturing:
  • The human lung cancer cell line A549 (ATCC Number: CCL-185) used in this experiment were subcultivated and kept in an ERDF medium (Kyokutoh Pharmaceutical) supplemented with 10% fetal bovine serum (FBS) (Bio Source International, Camarillo, Calif.), at 37° C. in a water vapor-saturated 5% CO2 condition. 1.125 g of NaHCO3 (Wako Pure Chemicals, Osaka, Japan) was added to one liter of the ERDF medium. The cells were cultivated and kept in their logarithmic growth phase. Human Burkit's lymphoma cell line DND39 were subcultivated and kept in a 5% FBS-added RPMI-1640 medium (Nissui, Japan) at 37° C. in a water vapor-saturated 5% CO2 condition. To the RPMI-1640 medium, were added 100 U/ml penicillin (Meiji Pharmaceutical Company, Tokyo, Japan), 100 mg/ml streptomycin (Meiji Pharmaceutical Company), 12.5 mM NaHCO3 (Wako Pure Chemicals), and 10 mM HEPES (Wako Pure Chemicals).
    • (2) Green Tea Catechins:
  • Green tea catechins, epigallocatechin-3-O-gallate (EGCG), epicatechin-3-O-gallate (ECG), epigallocatechin (EGC), epicatechin (EC), catechin (C) and epigallocatechin-3-(3-O-methyl)-gallate (EGCG3″Me) were dissolved in a phosphate buffer (PBS) to have a concentration of 5 mM. Before use, it was suitably thawed. PBS was prepared by dissolving 8.0 g of NaCl (Nacalai Tesque, Inc.), 0.2 g of KCl (Nacalai Tesque, Inc.), 1.15 g of Na2HPO4 (Nacalai Tesque, Inc.) and 0.2 g of KH2 PO4 (Nacalai Tesque, Inc.) in 1 liter of ultra-pure water.
  • Caffeine and quercetin were purchased from Nacalai Tesque, Inc., and the former was suspended in PBS while the latter was in dimethylsulfoxide (DMSO) (Nacalai Tesque, Inc.), each having a concentration of 5 mM.
    • (3) Reagents and Instruments:
  • Tripanblue (Wako Pure Chemicals) was suspended in PBS to have a concentration of 1%, and sterilized in an autoclave at 121° C. for 20 minutes. All-trans-retionic acid (ATRA) was purchased from Sigma (St. Louis, Mo.), and dissolved in ethanol.
  • TRIzol used for RNA extraction was purchased from Invitrogen (Carlsbad, Calif.). Aqueous 0.1% diethyl pyrocarbonate (DEPC) was purchased from Sigma (St. Louis, Mo.). A DEPC solution used for RNA dissolution was prepared by adding DEPC to distilled water to have a final concentration of 0.1%, then stirring it for 2 hours and autoclaving it. Oligotex-dT30 and human placenta-derived RNase inhibitor were purchased from Takara (Kyoto, Japan); and Molony murine leukemia virus (MMLV)-reverse transferase was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). We asked BIOSYNTHESIS (Japan) to synthesize oligonucleotides such as primers. Tag DNA polymerase was purchased from Fermentas (Vilnius, Lithuania), and Ex Tag was purchased from Takara. For polymerase chain reaction (PCR), used was GeneAmp PCR System 2400 (Parkin-Elmer, Tokyo, Japan). As an agarose, used was ultra-pure agarose (Sawaday Technology, Tokyo, Japan).
  • As a cloning vector, used was pTARGET™ Mammalian Expression Vector System (Promega, Madison, Wis.). For purification, used was QIAGEN Plasmid Midi Kit or EndoFree Plasmid Maxi Kit (both QIAGEN).
  • An LB medium was prepared by dissolving 10 g of Bacto Tryptone (Becton Dickinson, Sunnyvale, Calif.), 5 g of Bacto Yeast Extract (DIFCO LABORATORIES, Detroit, Mich.) and 5 g of NaCl in 1 liter of ultra-pure water, and autoclaving it. After cooled to 60° C., 1000 ml of ampicillin (150 mg/ml) (prepared by dissolving ampicillin sodium (Wako Pure Chemicals) in ultra-pure water to have a concentration of 150 mg/ml and filtering it for sterilization) was added to it. For an LB plate, 2 g of Bacto Tryptone, 1 g of Bacto Yeast Extract, 2 g of NaCl and 5 g of Bacto Agar (DIFCO) were dissolved in 200 ml of ultra-pure water and autoclaved. After cooled to 60° C., 200 ml of ampicillin (150 mg/ml) was added to it, and this was put into 10-ml dishes (Falcon), as divided into portions of 10 ml each. Isopropyl-b-D(-)-thiogalactopyranoside (IPTG) was purchased from Wako Pure Chemicals, and it was formulated to have a concentration of 0.1 M. 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) (Wako Pure Chemicals) was dissolved in N,N-dimethylformamide (Wako Pure Chemicals) to have a concentration of 20 mg/ml. SOC was prepared as follows: H2O was added to 3 g of Bacto Tryptone, 0.75 g of Bacto Yeast Extract, 0.078 g of NaCl and 0.017 g of KCl so as to make 148.5 ml in total. The solution was autoclaved. 1.5 ml of 2 M Mg2+ solution (12.324 g of MgSO4.7H2O and 10.165 g of MgCl2.6H2O were mixed with ultra-pure water to make 50 ml) that had been autoclaved separately from it was added to it. 1 ml of 2 M glucose was added to 100 ml of this solution.
  • For a DNA sequencer, used was ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan), in which was used ABI PRISM BigDye™ Terminator Cycle Sequencing Ready Reaction Kits Version 2.0 (Applied Biosystems). Template Suppression Reagent (TSR) was purchased from Applied Biosystems.
  • For gene introduction, FuGENE™ 6 Transfection Reagent (Roche Diagnistics Gmbh, Mannheim, Germany) was used.
  • The anti-human laminin receptor antibody for flow cytometry analysis was purchased from NEOMARKERS (Fremont, Calif.). As a negative control antibody, mouse IgM antibody, used was one from Zymed Laboratories Inc. (San Francisco, Calif.). A fluorescein isothiocyanate (FITC)-labeled anti-mouse IgM goat antibody was purchased from Southern Biotechnology Associates, Inc. (Birmingham, Ala.). In a flow cytometer, used was FACS Calibur (Becton Dickinson).
  • (4) RNA Extraction and cDNA Synthesis:
  • Cells that had been previously treated with 1 mM ATRA at 37° C. for 24 hours were washed with PBS, and Trizol was added thereto in an amount of 1 ml per 1×107 cells, and immediately suspended and completely dissolved. After left statically at room temperature for 5 minutes, 0.2 ml of chloroform was added to it and vigorously stirred by turning it upside down. Then, after statically kept at room temperature for 3 minutes, it was centrifuged at 12000×g and at 4° C. for 15 minutes. 0.5 ml of 2-propanol was added to the centrifuged supernatant, and vigorously stirred by turning it upside down. Then, after statically kept at room temperature for 10 minutes, it was centrifuged at 12000×g and at 4° C. for 10 minutes. The supernatant was removed, and the precipitate was rinsed with 1 ml of 75% ethanol. This was centrifuged at 12000×g and at 4° C. for 5 minutes, and the supernatant ethanol was removed as much as possible. The precipitated total RNA was dissolved in 20 ml of aqueous DEPC. cDNA synthesis was carried out as follows: First, 1 ml of (dT)20 primer (0.5 mg/ml) was added to 10 mg of the total RNA, left at 70° C. for 10 minutes, and then immediately cooled with ice for annealing. Next, 2 ml of 10 mM dNTP, 0.1 ml of RNase inhibitor, and 4 ml of 5× buffer attached to MMLV-reverse transferase were added to it, and aqueous DEPC was added to it to make 20 ml in total. The resulting mixture was kept at 37° C. for 1 hour for cDNA synthesis, and it was kept at 97° C. for 5 minutes to inactivate the enzyme.
    • (5) Construction of 67 kDa Laminin Receptor (67LR Expression Vector):
  • DND39 cells were treated with 1 mM-ATRA at 37° C. for 24 hours, then subjected to RNA extraction and to cDNA synthesis. With reference to the full-length cDNA (Yow, et al., Proc. Natl. Acad. Sci. USA., 85: 6394-6398 (1988)), primers were produced (H-LamininR-F; 5′-ATGTCCGGAGCCCTTGATGTCC-3′, H-LamininR-R; 5′-TTAAGACCAGTCAGTGGTTGCTC-3′). The primers were prepared at mM. 1 ml of the synthesized cDNA, 1 ml of Ex Taq, 2 ml of 10× Taq buffer, 1.6 ml of 2.5 mM dNTP, primers of 0.5 ml each, and 14.3 ml of dH2O were suspended and subjected to PCR. The condition was as follows: The initial denaturation was at 95° C. for 5 minutes, the denaturation reaction was at 94° C. for 30 seconds, the annealing was at 58° C. for 30 seconds, and the extension reaction was at 72° C. for 30 seconds, 25 cycles of denaturation, annealing and extension were carried out. This was subjected to electrophoresis with 1.2% agarose gel, and the intended band was purified by the use of Wizard SV Gel and PCR Clean-Up System (Promega). Its sequencing confirmed that this is the intended product. 4.18 ml of dH2O was added to 1 ml of T4 DNA Ligase 10× buffer, 1 ml of pTARGE™ Vector, 1 ml of T4 DNA Ligase and 2.80 ml of the PCR product, and kept at 4° C. overnight for ligation. The following operation is the same as that for subcloning. After reciprocal judgment through colony PCR, the colonies were gathered with a platinum loop and transferred into an LB medium, and cultured by shaking therein overnight at 37° C. and at 150 rpm. The cells were collected and purified by the use of EndoFree Plasmid Maxi Kit. Its sequencing confirmed that this is 67LR.
    • (6) Construction of Transient Expression System for 67LR:
  • The 67LR expression vector constructed (pTARGET-hLamininR) was introduced into A549 cells, using FuGENE™6 Transfection Reagent. Its details are described below. The cells were inoculated into a 10% FBS-ERDF medium at 1×104 cells/ml. By keeping at 37° C. for 24 hours, the cells were adhered to the medium. Next, a fresh ERDF medium was put into a tube, and FuGENE™ 6 (three times the amount of the gene) was directly added and gently mixed. Next, pTARGET-hLamininR was added and gently mixed, and this was kept at room temperature for 30 minutes. This was added to the medium, and kept at 37° C. for 48 hours. The cells were washed with a medium, and then the medium was changed with a fresh one.
    • (7). Influence of Various Components on 67LR Forced Expression Cells:
  • Various components of various concentrations were added to the 67LR transient expression cells, and treated in a 5% FBS-containing ERDF medium at 37° C. for 48 hours. After the treatment, the number of the cells was counted, and the survival rate of the cells was determined according to a Tripanblue staining method.
  • Regarding EGCG pretreatment, an anti-67LR antibody having a final concentration of 10 g/ml (1% FBS-containing ERDF medium) was treated at 37° C. for 30 minutes, and then EGCG treatment was carried out so as to investigate the influence thereof on the antibody-treatment cells. As a negative control, the same treatment was carried out with mouse IgM.
  • (8) Analysis of Various Components for their Binding to 67LR Forced Expression Cells:
  • The binding of various component (5 μM) to the cells was determined by using a surface plasmon resonance sensor SPR670 (Nippon Laser and Electronic Lab., Nagoya, Japan). For the measurement, the cells were fixed on a gold membrane (Nippon Laser and Electronic Lab.) according to a standard fixation method for protein. Precisely, a gold membrane was dipped in a solution in ethanol of 10 mM of 4,4-dithiodibutyric acid, DDA (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) (this was prepared by dissolving 2.38 mg of DDA in 10 ml of 99% ethanol followed by diluting it with ethanol to 1/100) in such a manner that its gold surface could face upward, and gently stirred at room temperature for 30 minutes. Next, this was washed twice with ethanol with no pressure applied to the gold surface to thereby introduce a self-assembly membrane (SA membrane). 25 mg of a water-soluble carbodiimide, EDC (Wako Pure Chemicals) was dissolved in 1 ml of ultra-pure water; and 15 mg of N-hydroxysuccinimide, NHS (Wako Pure Chemicals) was dissolved in 9 ml of 1,4-dioxane (Nacalai Tesque, Inc.). These solutions: were mixed, and the SA membrane-processed gold membrane was dipped into the mixture, and gently stirred at room temperature for 10 minutes. 10 ml of ultra-pure water was added, and further stirred at room temperature for 5 minutes. This was washed twice with ultra-pure water with no water pressure applied to the gold surface thereof, then dried (in air), and mounted on a cartridge. The cells were suspended to a density of 3×105 cells/ml (in a flow buffer, PBS), and 20 μl of the cell suspension was dropwise applied onto the gold membrane and kept at room temperature for 30 minutes so as to fix the cells onto the membrane. Next, a green tea catechin diluted with PBS to have a concentration of 1, 10, 25 or 50 MN was applied thereto, and the change in the surface plasmon resonance angle was determined so as to monitor its binding to the cells.
  • The binding competition test of EGCG and an anti-67LR antibody was carried out as follows: An anti-67LR antibody having a final concentration of 10 μg/ml (1% FBS-containing ERDF medium) was treated at 37° C. for 30 minutes, and then the cells were fixed on the gold membrane and tested in the same manner as above using a surface plasmon resonance sensor. In this case, the same treatment was also carried out with mouse IgM as a negative control.
    • (9) Flow Cytometry Analysis:
  • It is known that 67LR is expressed on cell surfaces. Therefore, 67LR expressed on the surfaces of the cells was detected through flow cytometry analysis using an anti-human laminin receptor (LR) antibody. The cells were collected, and using a 1.5-ml Eppendorf tube, they were added to 100 μl, as a whole, of 1% FBS-PBS at 1×106 cells therein. A primary antibody, anti-human LR antibody was added to it to have a final concentration of 10 μg/ml. This was incubated at 4° C. for 30 minutes, and then washed once with PBS. Next, a secondary antibody, anti-mouse IgM FITC-labeled antibody capable of recognizing an isotype of, LR antibody was added to 25 μl, as a whole, of 1% FBS-PBS so as to have a final concentration of 12.5 μg/ml. This was incubated at 4° C. for 30 minutes, then washed twice with PBS, and re-suspended in PBS, and analyzed with a flow cytometer. A negative control IgM antibody of the same concentration (10 μg/ml) was reacted in the same manner as herein. The 67LR expression amount on the cell surfaces was indicated by the center value of the fluorescent intensity of LR.
  • For the purpose of investigating the influence of EGCG treatment on the cell surface expression of 67LR, the cells were treated with an EGCG-added 1% FBS-PBS having a final EGCG concentration of 50 NM, and then kept at 37° C. for 30 minutes. This was washed once with PBS. Then, this was treated in the same manner as above with the primary antibody and others, and analyzed. As a control, the cells were treated with an EGCG-free 1% FBS-PBS.
    • (10) Statistical Analysis:
  • The test data were subjected to statistical analysis according to Student's t-test.
    • Example 1 Influence of 67LR Gene Introduction on the Cell Binding and the Cell Growth Activity of EGCG
  • Using FuGENE™ 6 Transfection Reagent, a 67LR expression vector (pTAT-hLamininR) was introduced into A549 cells according to the method mentioned below. The cells were inoculated at 1×104 cells/ml (10% FBS-ERDF medium). After 24 hours, a fresh ERDF medium was put into a tube, and FuGENE™ 6 (three times the amount of the gene) was directly added and gently mixed. Next, pTARGET-hLamininR of varying concentrations were added and gently mixed, and this was kept at room temperature for 30 minutes. This was added to the medium, and was continuously cultured at 37° C. for 48 hours. EGCG of various concentrations were added to the 67LR transient expression cells, and treated with a 5% FBS-containing ERDF medium at 37° C. for 48 hours. After the treatment, the number of the cells was counted and the survival rate thereof was determined according to a Tripanblue staining method.
  • As a result, EGCG concentration-dependent cell growth inhibition was observed in the cells where 67LR was transiently expressed. In addition, in proportion to the 67LR gene amount introduced into the cells, cell growth inhibition was also observed.
  • 67LR is a membrane protein existing in a cell membrane, and the fact that the EGCG effect was enhanced in the cells with the gene expression vector introduced therein would be due to the increase in the EGCG binding. Accordingly, the EGCG binding to the cells was determined by the sue of a surface plasmon resonance sensor. EGCG was used at 5 μM.
  • Only a little angle change (indicating binding amount) was found in the A549 cells with an empty vector introduced therein. In the cells with 0.25 μg of the 67LR expression vector introduced therein, a great increase in the angle was found; and in those with 0.5 μg thereof introduced therein, a further greater increase in the angle was found. This indicates that the 67LR expression vector introduction increases the binding of EGCG to cell surfaces.
  • The results of Example 1 are shown in FIG. 1, FIG. 2 and FIG. 3.
    • Example 2 Determination of 67LR Expression Amount on Cell Surfaces
  • The above indicates that the 67LR expression vector introduction increases the binding of EGCG to cell surfaces. Then, it was investigated as to whether the increase in the binding was caused by the increase in the 67LR expression on the cell membranes, using the flow cytometry.
  • The 67LR expression amount on the cell surfaces was determined. A little expression was found in the control, A549 cells. An empty vector (0.5 μg) was introduced into the cell showed almost no influence on the expression amount. However, when the 67LR expression vector (0.5 μg) was introduced into the cells, then the expression amount greatly increased. This confirms the expression of 67LR on the cell surfaces.
  • Further, in order to clarify as to whether EGCG binds to the cells via 67LR, EGCG was allowed to act on the cells before the anti-67LR antibody was allowed to act on them. As a result, the 67LR expression seen in the control cells apparently disappeared. The same phenomenon was found in cells with the empty vector introduced thereinto. In addition, it was the same with the 67LR-introduced cells. From this, it is understood that EGCG previously allowed to act on the cells in advance immediately bound to 67LR on the cell surfaces and therefore the anti-67LR antibody could not bind to 67LR on the cell surfaces and, as a result, the apparent 67LR expression could not be detected.
  • These results clearly show that the 67LR vector introduction increases the 67LR expression on cell surfaces, also indicating that the increase in the EGCG binding is the increase in the 67LR expression in cell membranes. In addition, it was suggested that EGCG binds to cells via 67LR. Specifically, it was shown that 67LR is a receptor of EGCG.
  • The results in Example 2 are shown in FIG. 4.
    • Example 3 Influence of Anti-67LR Antibody on the Binding and the Growth Inhibiting Activity of EGCG
  • It was shown that EGCG binds to cells via 67LR and exhibits its growth inhibiting activity. To further clarify this, 67LR forced expression cells were treated in advance with an anti-67LR antibody, and then the influence on the binding and the growth inhibiting activity of EGCG was investigated.
  • First, the influence of the antibody on the binding was investigated by using a surface plasmon resonance sensor. When an anti-67LR antibody was allowed to act on the cells, then the reduction in the EGCG binding speed and also in the EGCG binding amount itself was formed the angle change. This result was not found in the cells treated with a negative control antibody.
  • In addition, the influence of the antibody on the cancer cell growth inhibition by EGCG was also investigated. EGCG of a concentration of only 0.1 μM showed its growth inhibiting activity in 67LR forced expression cells. When the cells were subjected to antibody treatment before treating with EGCG, the growth inhibiting activity was lost. In this test, no influence on the survival rate was found. It was shown that not only the EGCG binding to cells but also the growth inhibiting activity of EGCG is exerted via the binding of 67LR to EGCG.
  • The results in Example 3 are shown in FIG. 5 and FIG. 6.
    • Example 4 Influence of 67LR Expression on the Binding and The Growth Inhibiting Activity of Other Tea Components
  • It was shown that EGCG binds to cell membranes via 67LR and it exhibits a cancer cell growth inhibiting activity. It was further investigated whether these effects via 67LR may be intrinsic to EGCG. The other tea components tested herein are principal green tea catechins, ECG, EGC, EC, C and EGCG3, Me which is reported to have a strong antiallergic effect and to stably exist in vivo. In addition, caffeine that has various physiological functions like catechins, and quercetin, one kid of flavanols that is reported also to have many physiological functions were also tested.
  • First, the influence of the substances on the growth of 67LR forced expression cells was investigated. Similarly to the above, 0.5 μg of pTARGET-hLamininR was introduced into A549 cells by the lipofection method, followed by cultivation at 37° C. for 48 hours. Then, the cells were treated with various tea components each having a final concentration of 5 μM. The cells were further cultivated at 37° C. for 48 hours, and then the number of the cells and the survival rate thereof were determined. As a result, the gene introduction has no influence on the survival rate and the number of the cells treated with any of C, EC, EGC, caffeine and quercetin. On the other hand, however, the cells treated with any of ECG and EGCG3″Me both having a galloyl group like EGCG showed the increase in the growth inhibiting effect thereof, like those treated with EGCG. These results suggest that the galloyl group-having components exhibit a growth inhibiting activity in 67LR forced expression cells.
  • In addition, the binding of various tea components to 67LR forced expression cells was determined by using a surface plasmon resonance biosensor. C, EC and EGC showed no binding to A549 cells, and there was no change also in 67LR forced expression cells. In addition, caffeine and quercetin also did not show cell binding, showing no change also in the forced expression cells. ECG and EGCG″3Me both showed cell binding though not comparable to that of EGCG, and it was clarified that their cell binding increased in 67LR forced expression cells.
  • The results in Example 4 are shown in FIG. 7 and FIG. 8.
    • INDUSTRIAL APPLICABILITY
  • The invention can provide a novel screening method for drugs, using a 67 kDa laminin receptor as a target.
  • The present application was filed, claiming a priority to Japanese Patent Application No. 2003-097652.

Claims (10)

1-30. (canceled)
31. A method of treating cancer, inhibiting cancer cell growth, or inhibiting cancer cell metastasis activity, comprising the step of providing to a subject a catechin which binds to a 67 kDa laminin receptor, wherein binding of said 67 kDa laminin receptor inhibits cancer cell growth or inhibits cancer metastasis activity of a cancer.
32. The method of claim 31 wherein said step of providing is performed orally.
33. The method of claim 31 wherein said catechin includes a galloyl group.
34. The method of claim 33 wherein said catechin is selected from (-)-epicatechin-3-O-gallate (ECG) and (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG″3Me).
35. The method of claim 31 wherein said catechin is (-)-epigallocatechin-3-O-gallate (EGCG).
36. The method of claim 31 wherein said catechin binds to said 67 kDa laminin receptor at a different locating from a laminin binding site where laminin binds to said 67 kDa laminin receptor.
37. The method of claim 31 wherein said cancer is a laminin receptor expression positive cancer.
38. A method of inhibiting cancer cell growth, comprising the step of exposing cancer cells to a catechin which binds to a 67 kDa laminin receptor, wherein binding of said 67 kDa laminin receptor inhibits cell growth of said cancer cells, wherein said step of exposing is performed in vitro.
39. A method of inhibiting cancer cell growth, comprising the step of exposing cancer cells to a catechin which binds to a 67 kDa laminin receptor, wherein binding of said 67 kDa laminin receptor inhibits cell growth of said cancer cells, wherein said step of exposing is performed in vivo.
US12/760,772 2003-04-01 2010-04-15 Method of Screening Drug with the Use of 67 kDa Laminin Receptor and Drug Obtained Thereby Abandoned US20100222424A1 (en)

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