WO2014088292A1 - Composition pharmaceutique comprenant un inhibiteur de l'expression ou de l'activation de la protéine carbonyl réductase 1 pour la prévention ou le traitement du cancer colorectal ou l'inhibition d'une métastase de celui-ci - Google Patents

Composition pharmaceutique comprenant un inhibiteur de l'expression ou de l'activation de la protéine carbonyl réductase 1 pour la prévention ou le traitement du cancer colorectal ou l'inhibition d'une métastase de celui-ci Download PDF

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WO2014088292A1
WO2014088292A1 PCT/KR2013/011112 KR2013011112W WO2014088292A1 WO 2014088292 A1 WO2014088292 A1 WO 2014088292A1 KR 2013011112 W KR2013011112 W KR 2013011112W WO 2014088292 A1 WO2014088292 A1 WO 2014088292A1
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protein
reductase
expression
colorectal cancer
carbonyl
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김세미
이윤희
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한국생명공학연구원
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • composition for preventing, treating or inhibiting metastasis of colon cancer including an inhibitor of the expression or activity of carbonyl reductase 1 protein
  • the present invention relates to a pharmaceutical composition for preventing, treating or inhibiting metastasis of colorectal cancer, comprising a novel cancer treatment target carbonyl reductase Ucarbonyl reductase 1) protein expression or activity inhibitor.
  • Colorectal cancer is a refractory disease that causes 1 million cases worldwide and 530,000 deaths annually. The rate is rapidly increasing to 3rd place in Korea with .12.7% of all cancers due to aging and changes in dietary patterns. Doing. Anti-cancer agents that are currently effective against colorectal cancer are widely used by drugs such as 5-F-U, U-F, and capecitabine (Zeloda), poloropyrimidine-based drugs, irinotecan (Gamputo) and oxaliplatin. The market for colorectal cancer drugs is expected to grow rapidly from $ 900 million to $ 7.7 billion in 2017 in the seven major pharmaceutical markets, including the US and Europe. The constant use of these drugs has always been a problem of increased resistance and side effects, and it is time to study the development and diagnosis of new therapeutic agents.
  • drugs such as 5-F-U, U-F, and capecitabine (Zeloda), poloropyrimidine-based drugs, irinotecan (Gamput
  • Cancer Fetal Antigen currently used as a diagnostic marker for colorectal cancer, is a type of glycoprotein that is normally produced during fetal period, and is higher in fetal sex than adults in newborns because production of this material is normally stopped before birth. If levels of the antigen (CEA) are present, this suggests that there may be colorectal cancer or other cancers, but these levels may be associated with cirrhosis, liver disease, alcoholic pancreatitis, and smokers. It is also being used as a supplementary situation.
  • CBR 1 Human carbonyl reductase Kcarbonyl reductase 1, hereinafter CBR 1 belongs to the short chain dehydrogenase / reductase family (SDR) family and is a ubiquitous NADPH dependent enzyme. It is composed of 277 amino acid residues and also catalyzes the majority of biologically and pharmacologically active substrates, including a variety of endogenous and biobiotic carbonyl compounds (Hoffmann F, Maser E. Car bony 1 reductases and pluri potent). hydroxysteroid dehydrogenases of the short -chain dehydrogenase / reductase super f ami ly.
  • CBR 1 inactivates highly reactive lipid aldehydes, such as 4-oxonone-2-enal (ONE), and can regulate DNA and protein (Oppermann U. Car bony 1 reductases: the complex relationships of mamma 1 i an carbonyl- and qui none ⁇ reducing enzymes and their role in physiology.Annu Rev Pharmacol Toxicol 2007; 47: 293-322.).
  • CBR 1 carbonyl reductase 1, hereinafter CBR 1 increases cell survival of leukemia cells (Jang et al., Cancer Res. (2012) 72 (16); 1-11), The ability to inhibit the death of liver cancer cells (Tak et al., Jouornal of hepatology (2011) 54: 328-339) has been reported.
  • CBR 1 in cancer cells may be unclear or may vary according to the type of tumor.
  • the use of CBR 1 to regulate colon cancer cell metastasis is currently unknown.
  • the role of CBR 1 as a colon cancer growth and metastasis inducing factor was first identified. As a result, CBR 1 overexpressed in colorectal cancer cell lines and inhibited the expression of CBR 1 in colorectal cancer cell lines.
  • An object of the present invention is to provide a pharmaceutical composition for preventing, treating or inhibiting colon cancer, comprising carbonyl reductase Kcarbonyl reductase 1, hereinafter CBR 1) protein expression or activity inhibitor as an active ingredient.
  • CBR 1 carbonyl reductase Kcarbonyl reductase 1, hereinafter CBR 1
  • Another object of the present invention is to provide a method for screening a colorectal cancer therapeutic agent or metastasis inhibitor candidate using the expression or activity level of CBR 1 protein.
  • the present invention provides a pharmaceutical composition for preventing, treating or inhibiting metastasis of colorectal cancer containing carbonyl reductase Kcarbonyl reductase 1) protein as an active ingredient.
  • the present invention also provides a method for screening a candidate substance for preventing, treating or inhibiting metastasis of colorectal cancer, comprising the following steps:
  • the present invention also provides a method for measuring the expression or activity level of a protein for providing information on monitoring or diagnosing colorectal cancer development or metastasis, comprising the following steps:
  • the present invention provides a method for preventing colorectal cancer, comprising administering a pharmaceutically effective amount of a carbonyl group reductase Kcarbonyl reductase 1) protein to a subject suffering from colorectal cancer.
  • the present invention provides a method for treating or inhibiting metastasis of colorectal cancer comprising administering a pharmaceutically effective amount of a carbonyl reductase Kcarbonyl reductase 1) protein to an individual suffering from colorectal cancer.
  • the present invention also provides a carbonyl reductase Kcarbonyl reductase 1) protein expression or activity inhibitor for use as a pharmaceutical composition for the prevention, treatment or metastasis of colorectal cancer.
  • the carbonyl group reductase 1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
  • Gene ID of the amino acid sequence of SEQ ID NO: 1 is 873, NCBI Reference seqence is # _001757, NP_001748.
  • the expression inhibitor of the carbonyl group reductase 1 is a group consisting of antisense nucleotides, short hairpin RNA (sh NA) and small interfering RNA (siRNA) complementary to the mRNA of the carbonyl group reductase 1 gene. It may be any one selected from, but is not limited thereto.
  • the activity inhibitor of the carbonyl group reductase 1 protein protein may be any one selected from the group consisting of peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that bind to the carbonyl group reductase 1 protein. Not determined
  • CBR 1 protein was confirmed in HEK293E cells transiently transformed with cancer cell lines and CBR1 expression vectors. It was confirmed that CBR1 is expressed in g minutes of colorectal cancer cell line (see FIG. 1).
  • CBR 1 is interpreted to function to induce colon cancer cell invasion and growth by regulating the c-Src signaling pathway.
  • the expression of CBR 1 was induced as a result of suppressing the expression of C12orf59, which confirmed that the present inventor was a colon cancer metastasis suppressor by siRNA on SW480 cells (see FIG. 5B).
  • the efficacy of C12orf59 to inhibit the colorectal cancer cell metastasis means that it may be mediated by a decrease in the expression of CBR 1.
  • CBR 1 overexpressed in colorectal cancer cell line inhibited the expression of CBR 1 in colorectal cancer cell line, and resulted in decreased cell growth, cancer cell invasion and cell mobility, matrix adhesion, FAK / c—Src related cell signaling. Since it is confirmed that this decrease, a substance that inhibits the expression or activity of CBR 1 can be used as an active ingredient of a pharmaceutical composition for preventing, treating or inhibiting metastasis of colorectal cancer.
  • the pharmaceutical composition containing an inhibitor of the expression or activity of the carbonyl group reductase 1 protein of the present invention as an active ingredient preferably contains 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition, but is not limited thereto.
  • the pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, textose solution maltodextrin solution, glycerol, ethanol, liposomes and one or more of these components.
  • Agents, buffers, bactericides and other conventional additives can be added.
  • diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsion suspensions, pills, capsules, granules or tablets.
  • Target organ specific antibodies or other ligands can be used in combination with the carrier so that they can act. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or by a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Com any, East on PA. .
  • Nucleotide or nucleic acid used in the present invention can be prepared for oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal administration.
  • the nucleic acid or vector is used in injectable form.
  • the area to be treated may be combined with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion.
  • the pharmaceutical compositions of the present invention may in particular comprise isotonic sterile solutions or lyophilized compositions which allow the composition of injectable solutions upon the addition of dry, in particular sterile water or appropriate physiological saline. Direct injection of nucleic acid into a patient's tumor is advantageous because it allows the treatment efficiency to be focused on the infected tissue.
  • the dosage of nucleic acid used can be adjusted by various parameters, in particular genes, vectors, mode of administration used, disease in question or alternatively desired duration of treatment.
  • the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and the severity of the disease.
  • Daily dose g is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times daily.
  • the pharmaceutical composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or thoracic injection during parenteral administration. It can be administered by internal injection and can be used in the form of general pharmaceutical preparations.
  • the present invention also provides a method for screening a candidate substance for preventing, treating or inhibiting metastasis of colorectal cancer, comprising the following steps:
  • the present invention also provides a method for screening a candidate substance for preventing, treating or inhibiting metastasis of colorectal cancer, comprising the following steps:
  • the cell line of step 1) is colon cell or colon cancer cell line g can be.
  • the expression level of the protein of step 2) is determined by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blotting, and It is preferred to measure with any one selected from the group consisting of flow cytometry (FACS), but any method of measuring the amount of transcript or protein encoded therefrom known to those skilled in the art can be used.
  • the present invention also provides a method for screening a candidate substance for preventing, treating or inhibiting metastasis of colorectal cancer, comprising the following steps:
  • the cell line of step 1) may be a colorectal cell or a colorectal cancer cell line.
  • the activity of the protein of step 2) is preferably measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry and protein chip, but not limited thereto. It doesn't work.
  • the present invention provides a method for measuring the level of expression or activity of a protein for providing information on monitoring or diagnosing colorectal cancer development or metastasis, comprising the following steps:
  • the present invention also provides a method for diagnosing colon cancer by measuring the expression or activity level of carbonyl reductase 1 protein from cells or tissues isolated from a subject.
  • the subject is a vertebrate and preferably a mammal, more preferably a viable animal such as a rat, a rabbit, a guinea pig, a hamster, a dog, a cat, and most preferably a chimpanzee, a gorilla, It is the same anthropoid animal.
  • the cell line of step 1) may be a colorectal cell or a colorectal cancer cell line.
  • CBR 1 overexpressed in colorectal cancer cell lines inhibited the expression of CBR 1 in colorectal cancer cell lines, reduced cell growth, cancer cell invasion and cell mobility, substrate adhesion, FAK / c-Src-related cell signaling Since it was confirmed that this decrease, the expression or activity level of CBR 1 can be used in the screening method for candidates for preventing, treating or inhibiting metastasis of colorectal cancer.
  • the expression or activity level of CBR 1 may be used in a method for measuring the expression or activity level of the protein to provide information on the development or monitoring or diagnosis of colorectal cancer.
  • the present invention provides a kit for diagnosing colorectal cancer comprising an antibody that specifically binds to a carbonyl group reductase 1 protein.
  • the carbonyl group reductase 1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
  • Gene ID of the amino acid sequence of SEQ ID NO: 1 is 873, NCBI Reference seqence is ⁇ _001757, ⁇ _00 ⁇ 748.
  • Antibodies that can be used in the kit include polyclonal antibodies, monoclonal antibodies, fragments capable of binding to epitopes, and the like.
  • the polyclonal antibody may be produced by a conventional method of injecting any one of the peptide markers into an animal and collecting blood from the animal to obtain serum containing the antibody.
  • Such polyclonal antibodies can be purified by any method known in the art and can be made from a host of any animal, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog.
  • the monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et al., Nature 256: 495-497, 1975; Kozbor D et al., J Immunol Methods 81: 31-42, 1985; Cote RJ et al., Proc Natl Acad Sci 80: 2026-2030, 1983; and Cole SP et al., Mol Cell Biol 62: 109-120; 1984).
  • antibody fragments containing specific binding sites for any of the peptide markers can be prepared (Huse WD et al., Science 254: 1275-1281, 1989). As described above, a method for preparing an antibody against a peptide having a specific sequence is obvious to those skilled in the art.
  • Antibodies that can be used in the kit can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex.
  • the kit may additionally include a detection antibody that specifically binds to the peptide marker.
  • the detection antibody may be a conjugate labeled with a detector such as a chromophore, a fluorescent substance, a radioisotope or a colloid, and is preferably a secondary antibody capable of specifically binding to the marker. It doesn't work.
  • the kit may further include a wash solution or an eluent which can remove enzymes, color reaction reaction substrates, unbound proteins, and the like and retain only bound peptide markers.
  • CBR 1 carbonyl reductase Kcarbonyl reductase 1, hereinafter CBR 1
  • CBR 1 is a diagram analyzing the expression of carbonyl reductase Kcarbonyl reductase 1, hereinafter CBR 1) in various colorectal cancer cell lines:
  • pcDNA-CBR 1 Lysate of cells which transiently transformed CBR 1 expression vector into HE 293E cells.
  • FIG. 2 is a diagram showing the results of invasion and cell migration assay of colorectal cancer cell line by inhibition of expression of CBR 1:
  • siGL3 control siRNA.
  • Figure 3 is a diagram showing the cell growth results of colorectal cancer cell line by inhibiting the expression of CBR 1:
  • Figure 4 is a diagram confirming the degree of cell-substrate adhesion (ceU-matrix adhesion assay) by inhibiting the expression of CBR 1.
  • FIG. 5A is a diagram confirming the reduction of FAK / c—Src related cell signaling by inhibition of CBR-1 expression.
  • Figure 5B is a diagram confirming the increased expression of CBR 1 by inhibiting the expression of C12orf59 in colorectal cancer cells. [Best form for implementation of the invention]
  • SW480, HCT116, HCT15, HT29, and Colo205 cell lines were cultured in RPMI1640 (GIBCO, USA) medium containing 10% FBS, penicillin-straptomycin L-glutamine, sodium pyruvate.
  • WiDr, Caco-2 cell line 10% FBS (fetal bovine serum: GIBCO, USA), penicillin-streptomycin (penici 11 in-streptomycin), L-glutamine (Lg hit amine), sodium pyruvate, non-essential amino acids (nonessent ial amino acids) 0] contain the minimal essential medium MEM: GIBCO, USA) and cultured in a culture medium.
  • HEK293E was incubated in DMEM medium.
  • Cell lines were cultured at 37 ° C, 5% C0 2 conditions.
  • SW480 sub is the subpopulat ion obtained by the inventors from SW80.
  • the pcDNA-CBR 1 vector was constructed to express the wild type CBR1 protein by introducing the CBR 1 gene into the pcDNA (invitrogen) vector.
  • the prepared pcDNA-CBR 1 vector was introduced into HEK293E cells for transient transfection.
  • the pcDNA-CBRl vector was added to Lipofectamine 2000 reagent in HEK293E cells. After 48 hours, HEK293E cells with pcDNA-CBRl vector were introduced into RIPA buffer (10 mM Tris, pH7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium). orthovanadate, 50 mM NaF, 1 mM PMSF, com lete protease inhibitor) and used for Western blot analysis.
  • RIPA buffer 10 mM Tris, pH7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium.
  • orthovanadate 50 mM NaF, 1 mM PMSF, com lete protease inhibitor
  • the cell lines obtained in Examples 1 and 2 were prepared using RIPA buffer (10 mM).
  • Tris pH7.2, 150 mM NaCl, 1% deoxycholate, 1 Triton X-100, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, complete protease inhibitor).
  • Cell lysates were quantified by commercially available modified Bradford assay (Bio-Rad Laboratories, Hercules, CA). Cell lysate 30 was heated in combination with SDS sample buffer and run on 8% SDS-PAGE gel. The isolated protein was transferred to nitrocellulose membrane and blocked with 5% skim milk.
  • CBR 1 was confirmed in HEK293E cells in which the pcDNA-CBR 1 vector expressing wild type CBR 1 was used as a positive control, and CBR 1 was expressed in most colorectal cancer cell lines. It was confirmed that it is expressed (Fig. 1).
  • the method for transforming cancer cell lines with CBR 1 specific siRNA is as follows.
  • SW480 cells were transformed with microporation (Neon Transfection system, Invitrogen) using the manufacturer's instructions. Briefly, cells were harvested, washed with PBS, resuspended in R buffer at a concentration of 3 ⁇ 10 5 cells / 12, 40 ⁇ siRNA 2 was added and a microporator instrument. Transformation was performed using. After 48 hours, cells were harvested for invasion / cell mobility, cell growth, cell-substrate adhesion loss, or lysis and western blot experiments were performed.
  • siRNA of CBR 1 (5'-cacagaauuacucccucua-3 ': SEQ ID NO: 2) used at this time was custom-made in estifamp.
  • C12orf59-siRNA (Dharmacon) used the one described in patent application No. 2011-00896.
  • the control siRNA (siGL3; sense strand 5'-CUU ACG CUG AGU ACU UCG ATT-3 ': SEQ ID NO: 3, anti-sense strand 5' -UCG AAG UAC UCA GCG UAA GTT- 3 ': SEQ ID NO: 4, estifam) is also the transformation method described in ⁇ Example 4>
  • the siRNA used was a siRNA consisting of SEQ ID NOs: 3 and 4, not CBR-1 specific siRNA. 48 hrs post-transfection of S 80 year old pouch and invasion assay and cells Cell migration assays were performed.
  • Invasion assays were performed specifically for 100 ⁇ Matrigel (BD Biosciences, USA) with 250 fig / dilution of porous membranes in 24-well transwell plates (8 pore size; Costar, USA) diluted with serum free medium. ) it was coated with was performed to solidify and left at room temperature for 1 hour.
  • the lower layer of the transwell plate is a chemical handout
  • a chemoat tract ant As a chemoat tract ant, it was coated using collagen type I (Sigma) 100 ⁇ at a concentration of 5 ⁇ g / ⁇ i. 3 ⁇ 10 4 cells resuspended in serum-free medium were dispensed into the upper chamber and allowed to migrate from the upper chamber to the lower chamber while incubating for 48 hours at 37 ° C., 5% CO 2 conditions. Unmoved cells were removed from the surface of the upper chamber. Cells transferred to the lower layer chamber were fixed with 3.7% paraformaldehyde dissolved in PBS and stained with 2% crystal violet solution. The excess crystal vial solution was washed with distilled water and photographed of the selected area (X100), and the number of cells moved was counted at five selected areas. The average value of the counted numbers was calculated and the relative% was obtained. Cell mobility experiments were performed by dispensing 1.5 ⁇ 10 4 cells into the upper chamber without Matrigel coating.
  • SW480 cells transformed with siRNA were harvested and dispensed at 1 ⁇ 10 4 cells / well into coated 24-well plates. After incubation for 20 minutes at 37 ° C, 5% C0 2 conditions, it was fixed with 3.7% paraformaldehyde dissolved in PBS, and stained with 2 >> crystal vial solution. The excess crystal violet solution was washed with distilled water and photographed of the selected area (X100) and the number of cells adhered to the substrate (matrixXcollagen I or fibronectin) was counted.
  • CBR 1 is interpreted as a function of inducing colon cancer cell invasion and growth by regulating the c—Src signaling pathway.
  • C12orf59 is a colorectal cancer metastasis suppressor whose function has been verified by the present inventors (Patent Application 2011—0089601).
  • Patent Application 2011—0089601 the expression of CBR 1 was confirmed by inhibiting the expression of C12ori59 by siRNA in SW480 cells.

Abstract

La présente invention valide en premier lieu le rôle de CR 1 comme facteur d'induction de la croissance et de la métastase du cancer colorectal. On a découvert que CBR 1 était surexprimé dans une lignée cellulaire de cancer colorectal. On a également découvert que la croissance cellulaire, l'invasion par les cellules cancéreuses, la mobilité cellulaire, l'attachement au substrat et la signalisation cellulaire associés à FAK/c-Src sont réduits par inhibition de CR 1 dans une lignée cellulaire de cancer colorectal. Par conséquent, une substance qui inhibe l'expression ou l'activation de CBR 1 peut être utilisée pour prévenir et traiter le cancer colorectal.
PCT/KR2013/011112 2012-12-05 2013-12-03 Composition pharmaceutique comprenant un inhibiteur de l'expression ou de l'activation de la protéine carbonyl réductase 1 pour la prévention ou le traitement du cancer colorectal ou l'inhibition d'une métastase de celui-ci WO2014088292A1 (fr)

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KR102120927B1 (ko) * 2018-04-20 2020-06-11 경희대학교 산학협력단 Cbr 1의 유전자 발현 억제를 통한 두경부암의 방사선 민감도 증진용 조성물
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KR20230115797A (ko) 2022-01-27 2023-08-03 중앙대학교 산학협력단 βPix의 SH3 도메인에 특이적으로 결합하는 항체를 포함하는 암 치료 또는 암 전이 억제용 약학적 조성물

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