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WO2015119373A1 - Composition destinée à inhiber la métastase cancéreuse, contenant de la mammaglobine - Google Patents

Composition destinée à inhiber la métastase cancéreuse, contenant de la mammaglobine Download PDF

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WO2015119373A1
WO2015119373A1 PCT/KR2014/012267 KR2014012267W WO2015119373A1 WO 2015119373 A1 WO2015119373 A1 WO 2015119373A1 KR 2014012267 W KR2014012267 W KR 2014012267W WO 2015119373 A1 WO2015119373 A1 WO 2015119373A1
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cancer
cancer metastasis
cells
protein
mamma globin
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강다원
고은하
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Gyeongsang National University GNU
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    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the inhibition of cancer metastasis, including mammaglobin, specifically, as a target molecule for inhibiting cancer metastasis, a mamma globin protein, a gene encoding a mamma globin protein, or a recombinant expression vector containing the gene as an active ingredient.
  • Pharmaceutical composition for inhibiting cancer metastasis containing a method for inhibiting cancer metastasis using the same; Cancer metastasis prediction kit; A method for selecting a cancer metastasis risk group using the kit; Screening methods of cancer metastasis inhibitors; And it relates to a method for inhibiting the movement and invasion of cancer cells using the pharmaceutical composition.
  • Cancer is a disease in which cells multiply indefinitely and interfere with normal cell function. Abnormal cells, whose proliferation and suppression are not controlled, proliferate uncontrolled and excessively proliferate and invade surrounding tissues or organs, leading to destruction of normal tissues.
  • the site of cancer is not limited, and the cause of cancer is still not known exactly.
  • mutations occur in genes of normal cells. Aberrant gene expression is closely related to unregulated cell growth and dedifferentiation, a common feature of all cancers. Numerous cancer studies have shown that the genomes of individuals with certain cancers are generally called tumor suppressor genes and normally reduce the expression of recessive genes that function to inhibit indiscriminate cell division and overgrowth. Oncogenes with the ability to modify are overexpressed. Therefore, searching for genes specifically overexpressed or suppressed in cancer cells compared to normal cells based on the relationship between gene expression and the onset or progression of cancer can develop a target for diagnosis or treatment of cancer.
  • cancer metastasis is considered to be the biggest obstacle in treating cancer.
  • cancer metastasis is an inefficient process in which only a small portion of the cancer cells that make up the primary tumor have successfully completed the various stages of metastasis and become metastatic cancer. Therefore, if we understand the metastasis process and develop a method that can effectively inhibit this by discovering clinical and biologically useful therapeutic targets, we can effectively control death from cancer metastasis.
  • Breast cancer is one of the most frequently diagnosed cancers in women in developed countries. The cause of breast cancer is not yet known. Breast cancer is a complex and heterogeneous disease regulated by several factors involved in carcinogenesis, progression, metastasis, recurrence, and the like. Therefore, even if the clinical prediction for breast cancer patients in the same stage, even if the actual outcome can be completely different treatment.
  • biomarkers including hormone receptors and human epidermal growth factor receptors (HER2)
  • HER2 human epidermal growth factor receptors
  • breast cancer has a high risk of cancer metastasis according to the characteristics of the tissues in which the mammary gland and the lymph gland are sharply distributed, and the survival possibility of the patient is largely determined by the cancer metastasis.
  • inhibition of cancer metastasis is a particularly important therapeutic direction in the field of breast cancer treatment, such as resection of peripheral lymph nodes in order to prevent cancer metastasis in advance.
  • Human mammaglobin is a breast-specific gene of the uteroglobin gene family on chromosome 11 (11q13) that encodes a 10 kDa glycoprotein.
  • hMAM is known to be overexpressed in breast epithelial and breast cancer tissues, attracting attention as a molecular marker for breast cancer. It is also proposed as a useful molecular marker for delivery of targeted drugs to breast cancer tissues as it exists on the surface of breast cancer cells.
  • mamma globin is a useful marker for breast cancer.
  • the role of mammagglobin in breast cancer is unknown, and there is no published information on its ability to be involved in tumor formation such as the proliferation, transport and invasion of breast cancer.
  • mammagglobin inhibits the migration and invasion of cancer cells, while its silencing increases the migration of cancer cells.
  • the present invention was completed by revealing that cancer metastasis can be suppressed through the regulation of mamma globin expression.
  • a mammaglobin (MAM) protein e.g., a mammaglobin (MAM) protein, a gene encoding a mamma globin protein, or an expression vector for gene therapy comprising the gene as an active ingredient.
  • Another object of the present invention is to provide a method for inhibiting cancer cell migration and invasion, comprising administering the pharmaceutical composition for inhibiting cancer metastasis to a subject in need thereof.
  • Still another object of the present invention is to provide a method for screening a cancer metastasis inhibitor, comprising analyzing a change in mamma globin expression level by treatment of a test substance.
  • the present invention confirms that cancer metastasis can be effectively inhibited by inhibiting the migration and invasion of cancer cells through an increase in the mamma globin expression level. Therefore, the mamma globin of the present invention can be usefully used as a target molecule for inhibiting cancer metastasis.
  • FIG. 1 is a diagram confirming hMAM mRNA expression levels in normal cell lines MCF-10A, non-invasive cell lines MCF-7 and ZR75-1, invasive cell lines MDA-MB-231 and Hs-578T without tumorigenic capacity.
  • the hMAM mRNA expression levels of each cell line corrected for GAPDH expression levels were compared (p ⁇ 0.05).
  • FIG. 2 is a representative diagram measuring the expression level of hMAM in the serum of breast cancer patients. As can be seen in the figure, MAM expression was not measured in the positive patient sample, but MAM expression was confirmed in the breast cancer patient sample. GAPDH was found to be expressed in all samples, including positive patient samples. Samples were randomly selected from all samples and measured for 40 hMAM out of a total of 82 patient samples.
  • FIG. 3 is a diagram confirming the expression of hMAM protein in the ZR75-1 cell line. Specifically, hMAM protein expression was measured in ZR75-1, MCF-7 and MDA-MB-231 cells. The same amount of total protein (30 ⁇ g) was measured for each lane and the protein molecular weight was indicated on the right side of the blot.
  • Figure 4 shows the immunohistochemical analysis of hMAM protein in ZR75-1 cell line. Fluorescence images were labeled with MAM-specific antibodies and FITC-conjugated anti-rabbit IgG antibodies and nuclei stained with DAPI. As a result of confirming whether the primary antibody was luminescent by the negative control (NC), only DAPI staining was observed in the negative control (blue), and no green fluorescence signal was found.
  • the scale bar shows 20 ⁇ m, and the MCF-10A and non-invasive cells compared with the invasive cells showed a significant difference (p ⁇ 0.05).
  • MDA-MB-231 cells are transformed with pcDNA, MAM / pcDNA, scrambled siRNA and hMAM siRNA, and then the whole RNA is extracted and analyzed by semi-quantitative PCR.
  • FIG. 6 is a diagram confirming the effect of hMAM expression level on the cell viability of MDA-MB-231. Specifically, MDA-MB-231 cells were transformed with pcDNA, MAM / pcDNA, scrambled siRNA, and hMAM siRNA, and MTT analysis was performed 2 days later (p ⁇ 0.05).
  • FIG. 7 is a diagram confirming cell mobility as a result of over-expression and knock-down of hMAM in MDA-MB-231 cells. Specifically, representative fluorescence images and measurement results of mobile cells by 3-D migration analysis. Cells were seeded in culture inserts and incubated at 37 ° C. for 6 hours. Mobile cells at the bottom surface were stained with 1% methylene blue (p ⁇ 0.05).
  • FIG. 8 is a diagram confirming the invasiveness as a result of over-expression and knock-down of hMAM in MDA-MB-231 cells. Specifically, cells (1.5 ⁇ 10 4 cells / well) were seeded in a transwell culture insert and incubated at 37 ° C. for 36 hours. Invasive cells were stained at the bottom of the membrane. Total migration or invasive cells in this experiment were calculated and estimated by comparing with the number when the first cells were planted (mobile or invasive cell number / first seeded cell number). The estimated values were corrected by the corresponding controls to express the results in% shift or invasion. Each bar represents the mean ⁇ SD of 5 independent experiments. Scale bar means 100 ⁇ m. Each shows a significant difference from the corresponding control (pcDNA or scrambled siRNA) (p ⁇ 0.05).
  • FIG. 9 is a diagram confirming cell mobility as a result of over-expression and knock-down of hMAM in Hs-578T cells (E). Specifically, representative fluorescence images and measurement results of mobile cells by 3-D migration analysis. Cells were seeded in culture inserts and incubated at 37 ° C. for 6 hours. Mobile cells at the bottom surface were stained with 1% methylene blue (p ⁇ 0.05).
  • FIG. 10 is a diagram confirming the invasiveness as a result of over-expression and knock-down of hMAM in Hs-578T cells. Specifically, cells (1.5 ⁇ 10 4 cells / well) were seeded in a transwell culture insert and incubated at 37 ° C. for 36 hours. Invasive cells were stained at the bottom of the membrane. Total migration or invasive cells in this experiment were calculated and estimated by comparing with the number when the first cells were planted (mobile or invasive cell number / first seeded cell number). The estimated values were corrected by the corresponding controls to express the results in% shift or invasion. Each bar represents the mean ⁇ SD of 5 independent experiments. Scale bar means 100 ⁇ m. Each shows a significant difference from the corresponding control (pcDNA or scrambled siRNA) (p ⁇ 0.05).
  • FIG. 11 shows that, through RT-PCR experiments, E-cadherin levels are increased and non-mentin levels are lowered in hMAM overexpressing cells.
  • RT-PCR was performed 36 hours after transformation, and mRNA expression levels of cells transformed with MAM / pcDNA and controls transformed with the empty vector (pcDNA) were compared.
  • the E-cadherin and non-mentin were used as markers of epithelial and mesenchymal cells, respectively.
  • FIG. 12 is a diagram illustrating the results of densitometric analysis of the results of FIG. 11.
  • the expression of E-cadherin and non-mentin is altered by hMAM.
  • Bar graphs show mRNA expression levels of E-cadherin and non-mentin corrected for GAPDH expression levels. Each bar represents the mean ⁇ SD of 3 independent experiments. Significant differences from control (pcDNA) are shown (p ⁇ 0.05).
  • One embodiment of the present invention provides a pharmaceutical composition for inhibiting cancer metastasis, which contains a mammaglobin (MAM) protein, a gene encoding a mamma globin protein, or an expression vector for gene therapy comprising the gene as an active ingredient.
  • the composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • cancer metastasis refers to the proliferation of cancer cells by leaving the primary organs and moving to other organs.
  • the spread of cancer to other parts of the body is largely divided into primary cancers in which cancerous tissues grow and directly invade surrounding organs, and distant metastases along blood vessels or lymphatic vessels to other organs in the distance.
  • mamma globin of the present invention is a 10 kDa glycoprotein, which is characterized in particular in breast-specific expression in humans. Mamma globin increases expression in breast cancer and is therefore known as a breast cancer marker (Mark A. Watson and Timothy P. Fleming, 'Mammaglobin, a Mammary-specific Member of the Uteroglobin Gene Family, Is Overexpressed in Human Breast Cancer', Cancer Res 1996; 56: 860-865). That is, mamma globin is only recognized as a cancer marker or carcinogen in cancer.
  • Carcinoma in which the movement and invasion of cancer cells can be suppressed by the expression control of mamma globin in the present invention is not particularly limited as long as it can cause metastasis, for example, breast cancer, lung cancer, colon cancer, prostate cancer, etc., In particular breast cancer.
  • Mamma globin protein of the present invention may be a human-derived mamma globin (hMAM), in particular may have an amino acid sequence of SEQ ID NO: 1.
  • mamma globin expression / protein levels of mamma globin were identified in invasive cancer cells or non-invasive cancer cells.
  • the mamma globin expression level in non-invasive MCF-7 breast cancer cells and invasive MDA-MB-231 breast cancer cells were compared by RT-RCR, Western blot (Fig. 1 to 4) .
  • mRNA expression levels of human mammaglobin in ZR75-1 and MCF-7 cells were MDA-MB231 and Hs-578T (estrogen receptor (ER) -negative invasive cells). It was confirmed that the high compared to the protein was also measured only in non-invasive cell line ZR75-1.
  • the effects of mammaglobin expression on the migration of breast cancer cells by 3-D mobility analysis showed that overexpression of MAM reduced cell migration and invasion, while silencing of MAM increased migration and invasion of these cells. It was confirmed (see FIGS. 5 to 10).
  • the present invention first identified that mamma globin expression levels are associated with migration invasion of human cancer cells. To date, the role of mamma globin has been studied as a breast cancer trigger gene or breast cancer marker. The mamma globin gene has been reported to be overexpressed in human carcinomas, including breast cancer, but the association between its expression level and cancer cell metastasis has not been reported. The present invention reveals that mammaglobin expression levels are associated with cell migration and invasion, which are crucial processes of cancer metastasis, and propose the use of mamma globin as a new target molecule for cancer treatment through cancer metastasis inhibition.
  • the present invention provides a pharmaceutical composition for inhibiting cancer metastasis, which comprises a mamma globin protein, a gene encoding the protein or an expression vector for gene therapy comprising the gene as an active ingredient.
  • Mamma globin protein in the present invention refers to a mamma globin protein of SEQ ID NO: 1 or a protein having a substantially equivalent physiological activity. Proteins having substantially equivalent physiological activity include the mamma globin protein of SEQ ID NO: 1 and its functional equivalents and functional derivatives.
  • the “functional equivalent” refers to an amino acid sequence variant in which some or all of the protein amino acids of SEQ ID NO: 1 are substituted, or a part of the amino acids are deleted or added, and have substantially the same biological activity as the mamma globin protein of SEQ ID NO: 1. .
  • the “functional derivative” is a protein that has been modified to increase or decrease the physicochemical properties of the mamma globin protein, which means that it has a physiological activity substantially equivalent to that of the mamma globin protein of SEQ ID NO: 1.
  • Mamma globin gene in the present invention is characterized in that it comprises a nucleotide sequence encoding the mamma globin protein or a functional equivalent thereof, the nucleotide sequence includes all DNA, cDNA and RNA sequences.
  • the mamma globin gene is represented by the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, most preferably represented by the nucleotide sequence of SEQ ID NO: 2.
  • Mamma globin protein in the present invention can be prepared using DNA recombination techniques for this purpose can be used methods known in the art. Specifically, the mamma globin protein can be prepared by genetic engineering methods using recombinant mamma globin gene in a bacterial, yeast, plant cell line and animal cell line.
  • the expression vector for gene therapy included in the pharmaceutical composition of the present invention refers to a recombinant expression vector into which the mamma globin gene is inserted, but is not limited thereto, and preferably refers to plasmids, viruses or other media known in the art. do.
  • the mamma globin gene was inserted into the pcDNA3.1 vector to express the mamma globin gene and a recombinant expression vector MAM / pcDNA3.1 was prepared.
  • the term "gene therapy” is intended to treat various genetic diseases and cancers caused by gene abnormalities, by directly injecting a gene associated with the disease into the patient's body and injecting the gene into cells.
  • the expression vector for gene therapy in the present invention refers to an expression vector capable of inhibiting the movement and invasion of cancer cells by directly expressing and expressing the mamma globin gene in the body.
  • the expression vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells It can be introduced into cells by the method of.
  • the expression vector was prepared by inserting the mamma globin gene of SEQ ID NO: 2 into the pcDNA3.1 vector, and named it 'MAM / pcDNA3.1'.
  • the prepared expression vector was transformed into ZR75-1 cells to confirm that the mamma globin protein was successfully expressed from the expression vector (FIG. 3, left blot).
  • Mamma globin protein produced by the expression vector according to the present invention is characterized by inhibiting the movement and invasion of cancer cells can effectively inhibit cancer metastasis. Therefore, the expression vector or mamma globin protein can be used as an active ingredient of the pharmaceutical composition for inhibiting cancer metastasis.
  • one aspect of the present invention provides a method for inhibiting cancer cell migration and invasion, comprising administering the pharmaceutical composition for inhibiting cancer metastasis to a subject in need thereof.
  • the term "subject” refers to a mammalian animal having a disease in which cancer metastasis can be inhibited by administering the pharmaceutical composition of the present invention, preferably human, monkey, dog, goat, pig or rat.
  • Cancer metastasis inhibiting pharmaceutical composition according to the present invention and a method of inhibiting the movement and invasion of cancer cells using the same can be effectively inhibited cancer metastasis, but not limited to these brain tumor (Brain Tumor) Low-grade astrocytoma, High-grade astrocytoma, Pituitary adenoma, Meningioma, CNS lymphoma, Oligodendrogliryn, Craniophaloma ), Ependymoma, Brain stem tumor, Head & Neck Tumor, Larygeal cancer, Oropharyngeal cancer, Nasal cavity / PNS tumor, Nasopharyngeal tumor, Salivary gland tumor, Hypopharyngeal cancer, Thyroid cancer, Oral cavity tumor, Chest Tumor, Small cell lung cancer Non-small cell Lung cancer (NSCLC), thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, breast cancer, male breast cancer, abdomen-pelvis tumor, stomach cancer cancer, liver cancer,
  • the pharmaceutical composition of the present invention specifically inhibits the migration and invasion of cancer cells, effectively inhibits cancer metastasis, when used in combination with other anticancer agents known to inhibit the growth and proliferation of cancer cells, both anti-cancer and cancer metastasis inhibitory effects are observed. Achievements can lead to significant progress in chemotherapy.
  • Anticancer agents that can be administered in combination with the pharmaceutical composition of the present invention include DNA alkylating agent (DNA alkylating agent), methyllothamine (mechloethamine), chlorambucil, phenylalanine (phenylalanine), mustard (mustard), cyclo Phosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin (streptozotocin), busulfan, thiotepa, Cisplatin and carboplatin;
  • Anti-cancer antibiotics include dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin (idaunorubicin), idarubicin, mitoxantrone, and pyrimamycin (plicamycin), mitomycin and C Bleomycin; And plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel,
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient described above for administration.
  • a pharmaceutically acceptable carrier, excipient or diluent refers to a carrier, excipient or diluent which does not irritate the organism and does not inhibit the biological activity and properties of the active ingredient administered together.
  • the carrier, excipient and diluent may be an unnatural substance, but are not limited thereto.
  • the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • compositions of the present invention can be prepared in a variety of mixtures with pharmaceutically acceptable carriers, as described above, and in particular for formulations for inhalation or injection.
  • pharmaceutically acceptable carriers for example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampules or in multiple dosage forms. It may be formulated into other solutions, suspensions, tablets, pills, capsules, sustained release preparations and the like.
  • Drug delivery via inhalation is one of the non-invasive methods, in particular therapeutic nucleic acid delivery via inhalation dosage forms (eg, aerosols) may be advantageously used for a wide range of treatment of lung diseases. . This is because the anatomical structure and location of the lung allows for an immediate and non-invasive approach and can be subject to topical application of the gene delivery system without affecting other organs.
  • therapeutic nucleic acid delivery via inhalation dosage forms eg, aerosols
  • suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, preservatives and the like may be further included.
  • Liquid preparations for oral administration include, but are not limited to, suspensions, solvents, emulsions, syrups, and the like, and various excipients, such as wetting agents, sweeteners, and fragrances, in addition to commonly used simple diluents such as water and liquid paraffin. , Preservatives and the like can be added.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.
  • base of the suppository utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • compositions of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically), depending on the method of delivering the gene therapy agent, and the dosage may be determined by the condition and body weight of the patient. Depending on the severity of the disease, the form of the drug, the route of administration and the duration, it may be appropriately selected by those skilled in the art.
  • compositions of the present invention may also be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modulators for effective cancer metastasis inhibition.
  • one aspect of the invention provides a kit for predicting cancer metastasis comprising an antibody specific for mamma globin or a primer pair specific for mamma globin mRNA encoding said protein.
  • an antibody refers to a specific protein molecule directed against an antigenic site.
  • an antibody refers to an antibody that specifically binds to the mamma globin protein of the present invention, which is a protein encoded by the marker gene by cloning each gene into an expression vector according to a conventional method. Can be obtained and prepared from conventional proteins by conventional methods.
  • the antibody suitable for the present invention is preferably a monoclonal antibody, more preferably a humanized monoclonal antibody.
  • primer refers to a nucleic acid sequence having a short free 3′-terminal hydroxyl group, which may form a base pair that interacts with a complementary template, Short nucleic acid sequence as a starting point for copying a template.
  • Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffers and temperatures.
  • reagents for polymerization ie, DNA polymerase or reverse transcriptase
  • four different nucleoside triphosphates at appropriate buffers and temperatures.
  • PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.
  • Primer pairs suitable for the present invention can be used without limitation, as long as it can specifically recognize and amplify the mamma globin-encoding mRNA, but is not particularly limited to bases described in SEQ ID NO: 3 and SEQ ID NO: 4 It is preferred to have a sequence.
  • kits refers to a tool capable of predicting cancer metastasis by confirming mRNA expression level or protein expression level of a mammaglobin gene as a target molecule.
  • the cancer metastasis predictive kit of the present invention includes at least one other component composition suitable for analytical methods as well as primers, probes or optionally to recognize a marker for measuring mamma globin expression level as a target molecule for cancer metastasis diagnosis, Solutions, or devices may be included.
  • one aspect of the present invention provides a method of selecting a cancer metastasis risk group by measuring the expression level of a mamma globin gene or protein.
  • step 2) When the level of the mamma globin protein detected in step 1) or the gene encoding the same is lower than the level measured in metastatic cancer cells, it is determined that cancer metastasis is likely, and when the level is higher than the level measured in metastatic cancer cells, cancer metastasis is determined. Determining that the probability is low.
  • the sample is not limited thereto, but may be a peeled sample, a crushed solution, or total RNA obtained from a cancer tissue sample.
  • Step 1) detects a mamma globin protein or a gene encoding the same from a cancer tissue sample by using a mamma globin specific antibody or a primer pair specific to a mamma globin mRNA encoding the protein included in a cancer metastasis predictive kit. It's a step.
  • detection of the mamma globin protein or gene encoding it can be performed by Northern blot, Western blot, immunohistochemical staining, in situ hybridization, reverse transcriptase polymerization.
  • Reaction Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), real-time quantitative RT-PCR (real-time quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay) can be performed using.
  • Step 2) is a step of determining the possibility of cancer metastasis. If the level of the mamma globin protein detected in step 1) or the gene encoding the same is lower than the level measured in metastatic cancer cells, it is determined that cancer metastasis is likely, and metastatic It is determined that cancer metastasis is unlikely if it is higher than the level measured in cancer cells.
  • the present invention provides an information providing method for selecting a cancer metastasis risk group using the screening method.
  • one aspect of the present invention provides a method for screening cancer metastasis inhibitors by targeting mamma globin protein.
  • test substance having increased by analyzing the expression level of the mamma globin protein or the gene encoding the intracellular before and after treatment of the test substance.
  • the cell including the mamma globin protein or the gene encoding the same may be a cell transformed to express the mamma globin protein or cancer cells detached from body tissues such as tumor tissue or blood or lymph fluid of a cancer patient.
  • the cell is treated with a test substance to determine whether it is a cancer metastasis inhibitor.
  • test substance in the present invention is a substance capable of increasing the expression level of mamma globin, and includes without limitation oligonucleotides, proteins, compounds, and the like.
  • step 2) the expression level of the mamma globin protein or the gene encoding it is expressed by Northern blot, Western blot, immunohistochemical staining, in situ hybridization method, reverse transcriptase polymerization (RT-PCR), real-time quantitative RT-PCR, ELISA, etc. It is detected using the method of.
  • a test substance that significantly increases the expression level of a mammaglobin protein or a gene encoding it is selected as a cancer metastasis inhibitor by comparing the detected expression level with that before the test substance treatment.
  • RT-PCR Peripheral blood transcriptase-polymerase chain reaction
  • CDNA was synthesized by extracting total RNA from peripheral blood samples obtained from the above patient groups.
  • EMT epithelial-mesenchymal transition
  • hMAM human mammaglobin
  • RT-PCR was performed using the primers of Table 2 and cDNA obtained from the patient group. RT-PCR conditions were: 40 seconds of amplification of 30 seconds at 95 °C, 30 seconds at 60 °C, 30 seconds at 72 °C was repeated, and finally extended to 72 minutes at 72 °C. It was electrophoresed on 2.5% agarose gel to measure the expression level of each gene.
  • Human breast cancer cell lines Hs-578T, MCF-7, MDA-MB231 and ZR75-1 were used by the Korea Cell Line Bank. Normal breast epithelial cells MCF-10A were distributed from Prof. Mun Aeri of Duksung Women's University. MCF-10A, MDA-MB231 and Hs-578T were incubated in DMEM containing 5% fetal bovine serum (FBS) and antibiotics (100 U / ml penicillin / 100 ⁇ g / ml streptomycin: Pen-Strep). . MCF-7 and ZR75-1 were incubated in RPMI 1640 with 5% FBS and Pen-Step. Cells were incubated in 95% air 5% CO 2 conditions. The culture was replaced every two days.
  • FBS fetal bovine serum
  • Pen-Strep Pen-Strep
  • Recombinant expression vector hMAM / pcDNA3.1 was prepared by cloning the human mamma globin gene (secretoglobin family 2A, member 2, SCGB2A2; GenBank accession number NM_002411) and subcloning into pcDNA 3.1 vector. Cells were inoculated at a cell concentration of 5 ⁇ 10 4 in a 24-well dish 48 hours prior to transformation and cultured in culture containing 5% FBS. The magnetofection (Magnetofection TM system) was used to perform transformation to inject hMAM / pcDNA3.1 into the cells according to the manufacturer's instructions.
  • Oligonucleotide using a dT (TM SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen Carlsbad, CA, USA) first from the total RNA of cancer cell-strand cDNA was synthesized. The first-stranded cDNA was used as a template for PCR amplification, and PCR was performed using primers specific to each gene and Taq polymerase (Takara, Otsu, Japan). PCR conditions were: after the initial denaturation at 94 ° C for 4 minutes, repeating the amplification process for 30 seconds at 94 ° C, 30 seconds at 58 ° C, and 60 seconds at 72 ° C for 27 to 30 cycles, and finally at 10 ° C for 10 minutes. The stretching process was performed.
  • dT TM SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen Carlsbad, CA, USA
  • the DNA fragments obtained by RT-PCR were analyzed using ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, CA, USA). Expression levels were quantified based on GAPDH expression levels. PCR products were electrophoresed in 1.2% (w / v) agarose gel to confirm product size.
  • the supernatant protein thus obtained was separated using 12% SDS-polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane through a semi-dry transfer (Bio-Rad, CA, SUA).
  • PVDF polyvinylidene fluoride
  • the membrane was blocked with 5% skim milk powder and then incubated with hMAM monoclonal antibody (1: 1000 dilution) and ⁇ -tubulin monoclonal antibody (1: 10000 dilution). It was then incubated with peroxidase-linked anti-rabbit or anti-mouse secondary antibody (1: 10000 dilution). Immune-positive bands were observed using an ECL kit (ECL Plus kit, ELPIS, Daejeon, Korea).
  • Anti-hMAM antibodies obtained from the mouse IgG used above and mamma globin from human, monkey, mouse and rabbit were purchased from ZETA company.
  • ZR75-1 cells were cultured in a round cover slide coated with poly-L-lysine and fixed for 30 minutes with 0.1 M phosphate buffer saline (PBS) containing 4% paraformaldehyde. The samples were then washed and incubated for 2 hours with gentle shaking in a blocking solution containing 1% normal goat serum and 0.1% Triton X-100. Cells were incubated overnight at 4 ° C. in PBS diluted 1: 400 of polyclonal antibodies purified by a binding affinity to hMAM protein. After washing three times with PBS, cells were incubated with FITC-conjugated anti-rabbit IgG secondary antibody (1: 400) diluted 1: 400 in PBS for 1 hour and 30 minutes in the dark. The cells were treated with 4 ', 6'-diamidino-2-phenylindole (DAPI) to stain nuclei. Stained cells were observed mounted in the wet state using a confocal laser scanning microscope.
  • PBS phosphate
  • Cells migrated to the bottom surface were stained with cell-staining solution (1% methylene blue) for 10 minutes. Five microscopic photographs were obtained using the microscope and the average number of cells moved was counted. The stained inserts were washed, dissolved in extract solution (Triton X-100) and incubated for 10 minutes. Each sample was transferred to a 96 well microtiter plate and absorbance was measured using a plate reader (Infinite F200, Tecan, Switzerland) at 560 nm absorbance.
  • cell-staining solution 1% methylene blue
  • the invasive assay used an invasive chamber consisting of cell culture inserts placed in tissue culture wells. This insert has an 8 ⁇ m pore size polycarbonateite membrane coated with an ECMatrix TM layer. Cells suspended in a serum-free medium at a density of 2.3 ⁇ 10 5 cells / ml were inoculated into the culture insert. The cells were allowed to invade the basement membrane layer for 36 hours. Cells invaded through the membrane were stained with cell-staining solution (1% methylene blue), and non-invasive cells were removed by wiping off the top surface of the membrane and observed under a microscope.
  • QCM TM 24-well cell invasion assay used an invasive chamber consisting of cell culture inserts placed in tissue culture wells. This insert has an 8 ⁇ m pore size polycarbonateite membrane coated with an ECMatrix TM layer. Cells suspended in a serum-free medium at a density of 2.3 ⁇ 10 5 cells / ml were inoculated into the culture insert. The cells were allowed to invade
  • hMAM ON-TARGET Non-targeting Pool
  • the target sequences of hMAM are GACAGCAGUCUUUGUGAUU (SEQ ID NO: 15), CUACAAAUGCCAUAGAUGA (SEQ ID NO: 16), GACAAUCAAUCCACAAGUG (SEQ ID NO: 17), and GAAACUCUGAGCAAUGUUG (SEQ ID NO: 18).
  • hMAM mRNA was reduced by about 80% after 2 days of transfection.
  • Relative levels of mRNA and protein were measured by calibrating GAPDH and ⁇ -tubulin, respectively. Images of agarose gels and Western blots were captured with a luminescence image analyzer LAS-4000 (Fujifilm corp, Tokyo, Japan). Bands obtained from RT-PCR were quantified using Sigma Gel image analysis software (version 1.0, Jandel Scientific, San Rafael, Calif., USA).
  • MRNA levels of mamma globin in normal breast epithelial MCF-7 cells and human breast cancer cells were compared.
  • expression levels of mamma globin were compared by semi-quantitative RT-PCR in breast cancer cell lines ZR75-1, MCF-7, MDA-MB231 and Hs-578T.
  • HMAM protein was found only in the ZR75-1 cell line, and not measured at all in MCF-7 and MDA-MB231 (FIG. 3, right blot).
  • MDA-MB-231 cells were inserted into the empty vector (pcDNA3.1, pcDNA), hMAM / pcDNA vector with hMAM inserted into pcDNA3.1, scrambled siRNA (scrambled siRNA). And MAM siRNA. Two days after the transformation, the expression level of mRNA was analyzed by semi-quantitative PCR.
  • MAM siRNA The expression level of MAM mRNA was increased in MAM overexpressed cells and decreased in cells transformed with MAM siRNA (FIG. 5).
  • knock-down of hMAM in cells did not result in a significant change in silver cell viability (FIG. 6).
  • hMAM expression was confirmed by 3-D migration and invasive analysis.
  • Scrambled siRNA and MAM siRNA were transiently transformed into MDA-MB-231 cells.
  • cell migration was reduced by MAM overexpression in MDA-MB-231 cells, whereas cell migration was increased in MAM siRNA-transfected cells.
  • MAM overexpression reduced the migration of MDA-MB-231 cells by 41.6 ⁇ 14.5%
  • MDA-MB-231 and Hs-578T cells are invasive cancer cells and have been characterized by the conversion of epithelial cells into mesenchymal cells (EMT). EMT is involved in aggressive, invasive and potentially metastatic in epithelial cancer cells. To determine whether hMAM overexpression reduces migration and invasion through inhibition of EMT, semi-quantitative PCR was performed on MAM overexpressed MDA-MB-231 cells and analyzed for changes in expression of EMT markers.
  • the RT-PCR experiment confirmed that the decrease of non-mentin (EMT marker) and the increase of E-cadherin (characteristic of epithelial marker) (FIGS. 11 and 12). Specifically, in the case of overexpressing MAM, E-cadherin expression was increased by about 2.1-fold compared to the control group (pcDNA empty vector transformed cells), but it was confirmed that non-mentin expression was reduced by about 60 ⁇ 7%. Keratin was very high due to overexpression of MAM in Hs-578 cells. In MDA-MB-231 cells, keratin mRNA was not seen and the expression of E-cadherin was lower than that of non-mentin.
  • EMT marker EMT marker
  • E-cadherin characteristic of epithelial marker

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Abstract

La présente invention concerne une composition destinée à inhiber la métastase cancéreuse, contenant de la mammaglobine. L'invention concerne en particulier : une composition pharmaceutique destinée à inhiber la métastase cancéreuse, contenant, en tant que principe actif, la protéine mammaglobine comme molécule cible pour l'inhibition de la métastase cancéreuse, un gène codant pour la protéine mammaglobine ou un vecteur d'expression recombinant contenant le gène, et un procédé destiné à inhiber la métastase cancéreuse à l'aide son utilisation ; un kit permettant de prédire la possibilité d'une métastase cancéreuse ; un procédé permetant d'identifier, au moyen de ce kit, un groupe à risque pour la métastase cancéreuse ; et un procédé destiné au criblage d'inhibiteurs de la métastase cancéreuse. Comme décrit ci-dessus, la présente invention démontre que la métastase cancéreuse peut être inhibée de façon efficace en inhibant le mouvement et l'invasion de cellules cancéreuses via une augmentation du niveau d'expression de la mammaglobine. En conséquence et ainsi que décrit dans la présente invention, la mammaglobine peut être utilisée en tant que molécule cible pour l'inhibition de la métastase cancéreuse.
PCT/KR2014/012267 2014-02-04 2014-12-12 Composition destinée à inhiber la métastase cancéreuse, contenant de la mammaglobine Ceased WO2015119373A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024187968A1 (fr) * 2023-03-15 2024-09-19 侯明宏 Composition pharmaceutique pour traitement du cancer du sein triple négatif

Citations (2)

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Publication number Priority date Publication date Assignee Title
JP2001516569A (ja) * 1997-09-18 2001-10-02 ワシントン ユニバーシティ ママグロビン、分泌された乳腺‐特異乳癌タンパク質
KR20070031925A (ko) * 2004-06-04 2007-03-20 베리덱스, 엘엘씨 유방암 경과의 진단 또는 예측

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JP2001516569A (ja) * 1997-09-18 2001-10-02 ワシントン ユニバーシティ ママグロビン、分泌された乳腺‐特異乳癌タンパク質
KR20070031925A (ko) * 2004-06-04 2007-03-20 베리덱스, 엘엘씨 유방암 경과의 진단 또는 예측

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DATABASE NCBI [O] 30 November 2013 (2013-11-30), "Homo sapiens secretoglobin, family 2A, member 2(SCGB2A2), mRNA", XP055219737, Database accession no. NM_002411.3 *
KOH, E.H. ET AL.: "Upregulation of human mammaglobin reduces migration and invasion of breast cancer cells", CANCER INVESTIGATION, vol. 32, pages 22 - 29 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024187968A1 (fr) * 2023-03-15 2024-09-19 侯明宏 Composition pharmaceutique pour traitement du cancer du sein triple négatif

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