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WO2014204024A1 - Composition destinée à l'induction d'un vieillissement cellulaire et contenant un agent de suppression du gène hs2st1 ou d'une protéine codifiée par ce gène, et procédé d'induction de vieillissement cellulaire ayant recours à cette composition - Google Patents

Composition destinée à l'induction d'un vieillissement cellulaire et contenant un agent de suppression du gène hs2st1 ou d'une protéine codifiée par ce gène, et procédé d'induction de vieillissement cellulaire ayant recours à cette composition Download PDF

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WO2014204024A1
WO2014204024A1 PCT/KR2013/005358 KR2013005358W WO2014204024A1 WO 2014204024 A1 WO2014204024 A1 WO 2014204024A1 KR 2013005358 W KR2013005358 W KR 2013005358W WO 2014204024 A1 WO2014204024 A1 WO 2014204024A1
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gene
cells
aging
composition
cell
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Korean (ko)
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이재선
정승희
김봉조
이형철
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Korea Institute of Radiological and Medical Sciences
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Korea Institute of Radiological and Medical Sciences
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Y208/00—Transferases transferring sulfur-containing groups (2.8)
    • C12Y208/02—Sulfotransferases (2.8.2)
    • 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 nucleic acids [NA]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications

Definitions

  • the present invention relates to a composition for inducing cell senescence comprising an HS2ST1 gene or an inhibitor for a protein encoded by the gene and a method for inducing cell senescence using the same.
  • Tumor cell premature aging is also referred to as stress-induced premature aging and refers to aging that occurs in tumor cells by various stimuli.
  • Tumor cells unlike normal cells that divide and age only a certain number of times, are generally cells that are infinitely dividing due to changes in the characteristics of replicative aging in the process of cancer, and thus, tumor cells are not likely to undergo cellular aging.
  • tumor cells have also been known to rapidly induce cellular senescence by various stimuli, which is called stress-induced premature senescence (Sugrue et al., Proc. Natl. Acad. Sci. USA). , 94: 9648-9653, 1997; Mason et al., Oncogene, 23; 9238-9246, 2004).
  • HS2ST1 (heparan sulfate 2-O sulfotransferase 1) is NCBI (National Center for Biotechnology Information) Access No.
  • the present inventors have found that when inhibiting intracellular sulfated process in tumor cells (for example, when inhibiting the heparan sulfate 2-O sulfotransferase 1) gene induces aging of tumor cells, the present invention is completed. It was.
  • an object of the present invention is to provide a composition for inducing cell aging.
  • Another object of the present invention is to provide a method of inducing cell senescence using a composition for inducing cell senescence.
  • the present invention provides a composition for inducing cell aging comprising an inhibitor for the heparan sulfate 2-O sulfotransferase 1 (HS2ST1) gene or a protein encoded by the gene.
  • HS2ST1 heparan sulfate 2-O sulfotransferase 1
  • the gene may be mRNA (messenger Ribonucleic acid), the inhibitor may be siRNA (small interfering RNA) capable of inhibiting the mRNA.
  • mRNA messenger Ribonucleic acid
  • siRNA small interfering RNA
  • the HS2ST1 gene includes the nucleotide sequence of SEQ ID NO: 1 (NCBI Access No. NM_012262) or the nucleotide sequence of SEQ ID NO: 2 (NCBI Access No. NM_001134492).
  • the nucleotide sequence of the base sequence of SEQ ID NO: 1 or the base sequence of SEQ ID NO: 2 is deleted, substituted or inserted.
  • the protein encoded by the HS2ST1 gene includes a polypeptide of SEQ ID NO: 3 or a polypeptide of SEQ ID NO: 4. Also included in the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 is the deletion, substitution or insertion of one or more amino acids.
  • sequence of the HS2ST1 gene in the present invention is exemplary and not limited thereto. Sequences having substantial sequence identity or substantial sequence homology to those sequences are also within the scope of the present invention.
  • sequence identity or substantial sequence homology
  • sequence homology is used to express that the sequence represents substantial structural or functional identity with another sequence. This difference is due, for example, to inherent variations in codon usage between different species. If there is a significant amount of sequence overlap or similarity between two or more different sequences, the structural differences are negligible if they have similar physical properties, even if their lengths or structures differ.
  • the inhibitor may be an siRNA capable of inhibiting HS2ST1 gene expression. That is, the inhibitor may be siRNA capable of inhibiting the expression of mRNA for the HS2ST1 gene.
  • the siRNA is a sense sequence of SEQ ID NO: 5 (5'-UGU AGU CUC UCC CAC AGA U-3 ') and an antisense sequence of SEQ ID NO: 6 (5'-AUC UGU GGG AGA GAC UAC A-3') It may be a double stranded siRNA.
  • the siRNA may be a thymine 2 base (dTdT) is coupled to the 3 'end of the sense sequence and / or antisense sequence.
  • Inhibitors to the protein in the present invention may be an antibody.
  • the antibody may be a monoclonal antibody, polyclonal antibody, and / or recombinant antibody that can specifically bind to a protein encoded by the HS2ST1 gene, and may be purchased commercially or prepared directly by a known method.
  • the cell may be a cancer cell.
  • the cancer is liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer Species, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, osteosarcoma and central nervous system tumors.
  • the cells may be breast cancer cells, but are not limited thereto.
  • the composition for inducing cellular senescence of the present invention may be a pharmaceutical composition, and aging may be induced in tumor cells by an inhibitor of the HS2ST1 gene or a protein encoding the gene in the composition for inducing cellular senescence of the present invention to exhibit anticancer activity. have.
  • composition for inducing cell aging of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological preparations.
  • Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for in vivo delivery of the composition, and may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. It can be used, and other conventional additives such as antioxidant, buffer, bacteriostatic agent can be added as needed.
  • diluents may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • diluents such as aqueous solutions, suspensions, emulsions and the like.
  • dispersants such as aqueous solutions, suspensions, emulsions and the like.
  • surfactants such as aqueous solutions, suspensions, emulsions and the like.
  • binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • it can be preferably formulated according to each disease or component using appropriate methods in the art.
  • composition for inducing cell senescence of the present invention may be administered in a dose of 0.01ng / kg ⁇ 100mg / kg once a day on the basis of a gene inhibitor, for example, siRNA in order to induce aging of tumor cells
  • a gene inhibitor for example, siRNA
  • Protein inhibitors for example, can be administered in a dose of 2 ⁇ 10mg / kg once daily on an adult basis
  • intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, Administration can be in conventional manner via the nasal, inhaled, topical, rectal, oral, intraocular or intradermal route.
  • the therapeutically effective amount of the composition for inducing cell aging of the present invention may vary depending on various factors, for example, the method of administration, the target site, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing.
  • the present invention provides a method for inducing senescence of a cell comprising the step of treating the cell composition for inducing cellular senescence comprising an inhibitor for the HS2ST1 gene or a protein encoded by the gene.
  • the cell may be a cell of a mammal except a human.
  • the cell may be a cancer cell.
  • the cancer is liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer Species, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, osteosarcoma and central nervous system tumors.
  • the cells may be breast cancer cells, but are not limited thereto.
  • HS2ST1 heparan sulfate 2-O sulfotransferase 1 gene or a protein encoded by the gene for the production of a composition for inducing tumor cell aging.
  • composition for inducing cell senescence according to the present invention may induce senescence of the cells by inhibiting the HS2ST1 gene in tumor cells, and may exhibit anticancer effects in tumor cells.
  • FIG. 1 is a phase contrast microscopy of a human breast cancer cell line MCF7 after treatment with or without concentration of sodium hypochlorite, an inhibitor of intracellular sulphation (control), and staining using a staining sample specific for aging-related beta galactosidase.
  • Figure shows the results observed using ECLIPSE TE300, Nikon).
  • FIG. 2 shows age-related staining using a staining sample specific to aging-related beta galactosidase after treatment or control of sodium hypochlorite, an inhibitor of intracellular sulfation, by concentration, in human breast cancer cell line MCF7. It is a graph showing the proportion of cells showing beta-galactosidase positive.
  • Figure 3 is a Western blotting result confirming the expression level of p53 and p21 after treatment with sodium hypochlorite concentration in human breast cancer cell line MCF-7 by concentration and cultured for 24 hours.
  • HSPG heparan sulfate proteoglycan
  • NaClO 3 sodium hypochlorite
  • FIG. 5 is a diagram illustrating a photograph observed with a phase contrast microscope after staining using a staining sample specific to activated aging-related beta-galactosidase for each passage of the human fibroblast cell line HDF.
  • Figure 6 shows the percentage of cells showing aging-related beta-galactosidase positive after staining using a specific staining method for activated aging-related beta-galactosidase activated by passage of human fibroblast cell line HDF. The graph shown.
  • Figure 7 is a diagram showing the reduction of the expression of heparan sulfate with replication aging of the human fibroblast cell line HDF observed at 630 X magnification by confocal microscopy (LSM-710, Carl Zeiss).
  • FIG. 9 is a diagram showing the inhibitory effect of heparan sulfate by the HS2ST1 inhibitor or the HS3ST1 inhibitor.
  • Figure 10 shows the cell proliferation rate by treatment with HS2ST1 inhibitor or HS3ST1 inhibitor.
  • Figure 12 is treated with siControl, siHS2ST1 siHS3ST1 in human breast cancer cell line MCF7, respectively, and subjected to staining using a staining sample specific for aging-related beta galactosidase, and then observed using a phase contrast microscope (ECLIPSE TE300, Nikon) It is a diagram showing.
  • Figure 13 shows the percentage of cells showing aging-related beta-galactosidase positive after treatment with siControl, siHS2ST1 siHS3ST1 to human breast cancer cell line MCF7 and staining with staining samples specific for aging-related beta galactosidase. Is a graph.
  • Aging-related beta galactosidase activity staining was performed to investigate the correlation between the inhibition of intracellular sulphation and cell aging of human breast cancer cell line MCF-7 (ATCC, USA).
  • Aging-related beta galactosidase activity after incubation in DMEM (Dulbeco's Modified Eagle's Medium) culture medium containing 100 ug / ml of antibiotics streptomycin and 100 units / ml of penicillin (Gibco BRL) Staining was performed. The staining was performed according to the method of Dimri et al. (Dimri et al., Proc. Natl. Acad. Sci. USA, 92: 93
  • the human breast cancer cell line MCF7 was treated with or without sodium hypochlorite, an inhibitor of intracellular sulfated process (control), and the degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon). 1 is shown.
  • the human breast cancer cell line MCF-7 three days after treatment with sodium hypochlorite, which is an inhibitor of the sulfated process can be confirmed that the cell size, which is characteristic of senescent cells, becomes flat and flat.
  • the higher the concentration of sodium hypochlorite treatment the greater the specific gravity of the cells showing the characteristics of aging cells.
  • FIG. 1 a graph of the percentage of cells showing age-related beta-galactosidase positive by measuring the number of stained cells using a microscope (ECLIPSE TE300, Nikon) is shown in FIG.
  • the higher the concentration of sodium hypochlorite treatment the greater the percentage of cells showing positive aging-related beta-galactosidase.
  • PBS phosphate buffered saline
  • Cell lysis buffer 50 mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 50 mM NaF, 0.2 mM Na 3 VO 4 , 10 ⁇ g / ml aprotinin, 2 ⁇ g / ml) Peptin
  • the extracted protein was centrifuged at 11,000 rpm for 10 minutes, and then only the supernatant was taken.
  • Bradford method (bradford, M., Anal. Biochem. 72: 248-254 (1976).
  • the separated proteins were transferred to nitrocellulose membrane (Whatman).
  • the membrane to which the protein has been transferred is placed in a PBS solution containing 5% skim milk powder, left to stand at room temperature for 1 hour, blocked, and put in primary antibodies diluted 1: 500 to 1: 1000, and then 4 hours at 4 ° C. Reacted.
  • Primary antibody is polyclonal Anti-p21 antibody (polyclonal anti-p21 Ab, Santa Cruz), polyclonal anti-actin antibody (polyclonal anti-actin Ab, Santa Cruz), polyclonal anti-p53 antibody (polyclonal anti-p53 Ab, Leica)
  • the secondary antibody was an enhanced chemiluminescence (ECL) reagent using a horse radish peroxidase conjugated anti-rabbit or anti-rat antibody (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz). (Amersham) confirmed the cell aging. The results are shown in FIG.
  • Figure 3 is a Western blotting result confirming the expression level of p53 and p21 after treatment with sodium hypochlorite concentration in human breast cancer cell line MCF-7 by concentration and cultured for 24 hours.
  • Actin Actin was used as a reference to show whether the same amount of protein was analyzed as a gene (housekeeping gene) expressed at the same level in all cells.
  • heparan sulfation one of the intracellular protein polysulfation processes, was inhibited in the human breast cancer cell line MCF-7 after 24 hours after treatment with sodium hypochlorite, an inhibitor of the sulfidation process.
  • the higher the concentration of sodium hypochlorite treatment the higher the inhibition of heparan sulfate, which is one of the sulfated protein intracellular polysaccharides.
  • human fibroblast cell line HDF (ATCC, USA) was treated with 10% fetal bovine serum (FBS, Weljin) and 100 ug of antibiotic streptomycin on a 100 mm culture dish (SPL). 5% CO in DMEM (Dulbeco's Modified Eagle's Medium) culture medium containing / ml and 100 units / ml of penicillin (Gibco BRL) 2
  • FBS fetal bovine serum
  • SPL 100 mm culture dish
  • DMEM Dulbeco's Modified Eagle's Medium
  • penicillin Gibco BRL
  • HDF cells After 3-4 days, when HDF cells are more than 90% full in the culture dish, remove the culture medium, wash the remaining culture solution with 10 ml of PBS (phosphate buffered saline) and trypsin (Welljin Co., Ltd.). Treatment removes the HDF attached to the culture dish and transfers only one quarter of the cells to the new culture dish in 5% CO in the above culture medium. 2 The cells were cultured in a cell culture in which the concentration and the temperature of 37 ° C. were maintained at all times.
  • PBS phosphate buffered saline
  • trypsin Welljin Co., Ltd.
  • the degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon), and the results are shown in FIG. 5.
  • a graph of the ratio of cells showing age-related beta-galactosidase-positive to total cells by measuring the number of stained cells using a microscope (ECLIPSE TE300, Nikon) is shown in FIG. 6.
  • p10 and p20 in Figure 6 it was not within the detectable range.
  • p10 and p20 in Figure 6 it was not within the detectable range.
  • human fibroblast cell line HDF ATCC, USA
  • FBS fetal bovine serum
  • antibiotics 100 ug / ml of streptomycin and penicillin 100 5% CO in DMEM (Dulbeco's Modified Eagle's Medium) culture medium containing units / ml (Gibco BRL) 2
  • DMEM Dulbeco's Modified Eagle's Medium
  • HDF cells After 3-4 days, when HDF cells are more than 90% full in the culture dish, remove the culture medium, wash the remaining culture solution with 10 ml of PBS (phosphate buffered saline) and trypsin (Welljin Co., Ltd.). Treatment removes the HDF attached to the culture dish and transfers only one quarter of the cells to the new culture dish to 5% CO in the culture medium. 2
  • the cells were cultured in a cell culture in which the concentration and the temperature of 37 ° C. were maintained at all times. Once this process has been performed, the number of passages is calculated to be 1, which is p10 when passaged 10 times, p20 when passaged 20 times, and p30 when passaged 20 times, respectively, and divided into young cells, medium-old cells, and old cells.
  • PBS cell samples were cell lysis buffer (50mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM PMSF, 50 mM NaF, 0.2 mM Na 3 VO 4 , 10 ⁇ g / ml aprotinin, 2 ⁇ g / ml lupeptin), and the extracted protein was centrifuged at 13,200 rpm for 20 minutes, and only the supernatant was taken.
  • the Bradford method ⁇ bradford, M., Anal. Biochem. 72: 248-254 (1976).
  • the primary antibody used was a monoclonal anti-heparan (monoclonal anti-HS Ab, US biology) antibody and a polyclonal anti-actin antibody (polyclonal anti-actin Ab, Santa Cruz) and the secondary antibody was horse radish peroxidase Heparan of intracellular protein polysaccharides using ECL (enhanced chemiluminescence) reagent (Amersham) using conjugated anti-rabbit or anti-rat antibodies (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz) The degree of sulfation was measured. 8 is a result of Western blotting confirming the decreased expression of heparan sulphate with replication aging of human fibroblast cell line HDF.
  • RNAiMAX invitrogen, Cat # 13778
  • RNA for the HS3ST1 gene ⁇ siHS3ST1 5'-CUG ACU ACA CCC AAG UGU U-3 '(SEQ ID NO: 8) and 5'- AAC ACU UGG GUG UUG UAG UCA G-3 '(SEQ ID NO: 9) is a double-stranded siRNA consisting of a nucleotide sequence of 3' end of each sequence A second base (dTdT) is engaged state Im) ⁇ or small interfering RNA for the gene HS2ST1 ⁇ siHS2ST1; Double-stranded siRNA consisting of 5'-UGU AGU CUC UCC CAC AGA U-3 '(SEQ ID NO: 5) and 5'-AUC UGU GGG AGA GAC UAC A-3' (SEQ ID NO: 6), each of the 3 'ends Ethine 2 base (dTd
  • the culture medium was replaced with DMEM culture medium containing 10% fetal bovine serum (FBS, Weljin) and 100 ug / ml of antibiotic streptomycin and 100 units / ml of penicillin (Gibco BRL). Afterwards, the cells were cultured in a 37 ° C. incubator at 5% CO 2 concentration for 4 days.
  • FBS fetal bovine serum
  • Gibco BRL penicillin
  • non-specific small interfering RNA ⁇ siControl; Double-stranded siRNA consisting of 5'-CCU ACG CCA CCA AUU UCG U-3 '(SEQ ID NO: 10) and 5'-ACG AAA UUG GUG GCG UAG G-3' (SEQ ID NO: 11), each sequence
  • the cell line injected with thymine dibasic (dTdT) bound to the 3 'end was used as a control.
  • siRNAs were synthesized using ⁇ -cyanoethyl phosphoramidite by linking phosphodiester bonds that form a skeleton of DNA structure (Sinha et al., Nucleic Acids Research, 12: 4539-4557, 1984). That is, using an RNA synthesizer (Perseptive Biosystems 8909, PE Biosystems, USA), a series of deblocking, coupling, oxidation, and cappings on a nucleotide-attached solid support The procedure was repeated to obtain a reaction containing RNA of the desired length.
  • RNA synthesizer Perseptive Biosystems 8909, PE Biosystems, USA
  • reaction product was applied to HPLC LC918 (Japan Analytical Industry, Japan) using Daisogel C18 (Daiso, Japan) to separate RNA and apply it to MALDI-TOF mass absorber (Shimadzu, Japan) to synthesize it. It was confirmed that it matches the base sequence. Then, by combining the sense and antisense RNA strands, each of the desired double-stranded siRNA was prepared.
  • Human breast cancer cell lines were injected with specific small interfering RNA for HS3ST1 (siHS3ST1), specific small interfering RNA for HS2ST1 (siHS2ST1), or nonspecific small interfering RNA (siControl) for 3 days at 37 ° C. at 5% CO 2 concentration. After culturing in an incubator, the cell culture solution is removed, washed twice with PBS, and allowed to stand at room temperature for 20 minutes in 3.7% formaldehyde to fix the cells.
  • specific small interfering RNA for HS3ST1 siHS3ST1
  • siHS2ST1 specific small interfering RNA for HS2ST1
  • siControl nonspecific small interfering RNA
  • the specific small interfering RNA treatment group for HS3ST1 (siHS3ST1) and the specific small interfering RNA treatment group for si2ST1 (siHS2ST1) were sulfated of intracellular protein polysaccharide in human breast cancer cell line MCF-7. It can be seen that one of the processes, heparan sulfate is inhibited.
  • FIG. 10 Cell proliferation as a percentage of the number of cells is shown in FIG. Served.
  • the x-axis represents the days of cell culture after treatment with siHS3ST1, siHS2ST1, or siControl
  • the y-axis represents the cell proliferation rate based on the number of cells on day 0.
  • the siHS2ST1 treatment group showed a lower cell proliferation rate of tumor cells (human breast cancer cell line MCF7) than the control group (siControl) group and siHS3ST1 treatment group over time. Therefore, when the HS2ST1 gene expression is suppressed in tumor cells (for example, human breast cancer cell line MCF7), it can be confirmed that aging of tumor cells is induced and cell proliferation rate is lowered.
  • tumor cells for example, human breast cancer cell line MCF7
  • DMEM Dulbeco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • Streptomycin 100 ug / ml and penicillin 100 units / ml Gibco BRL
  • Human breast cancer cell line MCF-7 cultured in culture medium was washed with PBS (phosphate buffered saline), and then the cell samples were treated with cell lysis buffer (50 mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, Protein was extracted using 50 mM NaF, 0.2 mM Na 3 VO 4 , 10 ⁇ g
  • nitrocellulose membrane (Whatman).
  • the membrane to which the protein has been transferred is placed in a PBS solution containing 5% skim milk powder, left to stand at room temperature for 1 hour, blocked, and put in primary antibodies diluted 1: 500 to 1: 1000, and then 4 hours at 4 ° C. Reacted.
  • Primary antibodies are polyclonal anti-p21 antibody (polyclonal anti-p21 Ab, Santa Cruz), polyclonal anti-actin Ab (Santa Cruz), phosphorylated amino acids 807 / in serine residues of pRb
  • An anti-p-pRb antibody anti-phospho-pRb Ab, cell signaling
  • the secondary antibody was a horse radish peroxidase conjugated anti-rabbit antibody (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG (Santa Cruz) was used to confirm cell aging with ECL (enhanced chemiluminescence) reagent (Amersham). The results are shown in FIG.
  • FIG. 11 is a Western blotting result confirming the degree of phosphorylation and p21 expression of Rb after treatment with siControl, siHS3ST1 or siHS2ST1 in human breast cancer cell line MCF-7 and cultured for 2 days.
  • actin was used as a standard showing whether the same amount of protein was analyzed as a gene (housekeeping gene) expressed at the same level in all cells.
  • the degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon), and the results are shown in FIG. 12.
  • a graph of the percentage of cells showing age-related beta-galactosidase positive by measuring the number of cells stained using a microscope (ECLIPSE TE300, Nikon) is shown in FIG. 13.
  • siHS2ST1 treatment group treated with the specific small interfering RNA (siHS2ST1) for HS2ST1 can be confirmed that the aging phenomenon is significantly higher than the control group (siCON or siControl) and siHS3ST1 treatment group treated with siControl.
  • siHS2ST1 treatment group treated with the specific small interfering RNA (siHS2ST1) for HS2ST1 can be confirmed that the aging phenomenon is significantly higher than the control group (siCON or siControl) and siHS3ST1 treatment group treated with siControl.
  • tumor cells eg, human breast cancer cell line MCF-7
  • the senescence of tumor cells can be induced to show anticancer effects.
  • the composition for inducing cellular senescence may induce senescence of the cells by inhibiting the HS2ST1 gene in tumor cells, and thus may be used to exhibit anticancer effects in tumor cells.

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  • Microbiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une composition destinée à l'induction de vieillissement cellulaire et un procédé d'induction de vieillissement cellulaire ayant recours à cette composition, ladite composition permettant d'induire un vieillissement cellulaire par la suppression d'un gène HS2ST1 dans des cellules tumorales, et donc de présenter un effet anticancer dans les cellules tumorales.
PCT/KR2013/005358 2013-06-18 2013-06-18 Composition destinée à l'induction d'un vieillissement cellulaire et contenant un agent de suppression du gène hs2st1 ou d'une protéine codifiée par ce gène, et procédé d'induction de vieillissement cellulaire ayant recours à cette composition Ceased WO2014204024A1 (fr)

Priority Applications (1)

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PCT/KR2013/005358 WO2014204024A1 (fr) 2013-06-18 2013-06-18 Composition destinée à l'induction d'un vieillissement cellulaire et contenant un agent de suppression du gène hs2st1 ou d'une protéine codifiée par ce gène, et procédé d'induction de vieillissement cellulaire ayant recours à cette composition

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2013/005358 WO2014204024A1 (fr) 2013-06-18 2013-06-18 Composition destinée à l'induction d'un vieillissement cellulaire et contenant un agent de suppression du gène hs2st1 ou d'une protéine codifiée par ce gène, et procédé d'induction de vieillissement cellulaire ayant recours à cette composition

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WO2014204024A1 true WO2014204024A1 (fr) 2014-12-24

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PCT/KR2013/005358 Ceased WO2014204024A1 (fr) 2013-06-18 2013-06-18 Composition destinée à l'induction d'un vieillissement cellulaire et contenant un agent de suppression du gène hs2st1 ou d'une protéine codifiée par ce gène, et procédé d'induction de vieillissement cellulaire ayant recours à cette composition

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070066564A1 (en) * 2003-05-19 2007-03-22 Koji Kimata Sulfotransferase inhibitor
US20080038189A1 (en) * 2006-06-21 2008-02-14 Reliance Life Sciences Pvt. Ltd. RNA interference mediated inhibition of aurorakinase B and its combinations as anticancer therapy
WO2010003023A2 (fr) * 2008-07-01 2010-01-07 Zacharon Pharmaceuticals, Inc. Inhibiteurs d'héparane sulfate
KR20110130627A (ko) * 2010-05-28 2011-12-06 한국원자력의학원 항암용 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070066564A1 (en) * 2003-05-19 2007-03-22 Koji Kimata Sulfotransferase inhibitor
US20080038189A1 (en) * 2006-06-21 2008-02-14 Reliance Life Sciences Pvt. Ltd. RNA interference mediated inhibition of aurorakinase B and its combinations as anticancer therapy
WO2010003023A2 (fr) * 2008-07-01 2010-01-07 Zacharon Pharmaceuticals, Inc. Inhibiteurs d'héparane sulfate
KR20110130627A (ko) * 2010-05-28 2011-12-06 한국원자력의학원 항암용 조성물

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 17 April 2013 (2013-04-17), accession no. M_012262 *
LEE, JAE SEON: "Aging Cancer Cell ? Development on Aging Marker for New Cancer Treatment", SCIENCE AND TECHNOLOGY, vol. 483, 2009, pages 67 - 69 *

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