WO2014204024A1 - Composition for inducing cellular senescence containing agent for suppressing hs2st1 gene or protein codified by the gene, and method for inducing cellular senescence using same - Google Patents

Abstract

The present invention relates to a composition for inducing cellular senescence and a method for inducing cellular senescence using same, wherein the composition can induce cellular senescence by suppressing an HS2ST1 gene in tumor cells and thus can exhibit an anticancer effect in the tumor cells.

Description

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 aging using the same

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. However, recently, 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). Genetic toxic chemicals (e.g. etoposide, cyclophophamide), radiation, and ultraviolet rays have been reported as stressors that can induce aging of tumor cells (Hemann and Narita, Genes & Dev., 21: 1-5, 2007 Chang et al., Proc. Natl. Acad. Sci. USA, 99: 389-394, 2002).

Recently, suppression of tumor cell proliferation through senescence of tumor cells using stress-induced premature aging has been suggested as an efficient mechanism for the treatment of cancer patients (Roninson et al., Drug Resist Updates, 4: 303-313, 2001; Campisi, Science, 309: 886-887, 2005), it has been suggested that increasing the effectiveness of cancer therapy through the study of cellular senescence induction mechanisms is essential (Narita and Lowe, Nature Medicine, 11: 920-922, 2005). In addition, histological analysis of cancer patients whose tumor malignancy was arrested reported that tumor cell senescence was efficiently induced (Collado et al., Nature, 436: 642, 2005), and the tumor suppressor protein p53 was applied to the cell aging. It has been demonstrated in tumor model animal experiments that tumor tissues are efficiently removed (Wue et al., Nature, 445: 656-660, 2007). This suggests that cell aging may be usefully used in the treatment of tumors. Substantially, activating the senescence mechanism of tumor cells can improve the side effects of cancer treatment by enabling the administration of a lower dose of anticancer agents or radiation than activation of the apoptosis mechanism, and overcome the cancer treatment resistance of tumor cells that have acquired resistance to cell death. (Rebbaa, Cencer Lett, 219: 1-13, 2005).

Meanwhile, HS2ST1 (heparan sulfate 2-O sulfotransferase 1) is NCBI (National Center for Biotechnology Information) Access No. The gene, NM_012262 (SEQ ID NO: 1) or NM_001134492 (SEQ ID NO: 2), is not known for early aging of tumor cells.

Therefore, 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.

Accordingly, 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.

In order to achieve the above object, 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.

In the present invention, the gene may be mRNA (messenger Ribonucleic acid), the inhibitor may be siRNA (small interfering RNA) capable of inhibiting the mRNA.

In the present invention, 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). In addition, 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.

In the present invention, 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.

It is apparent to those skilled in the art that the 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. As used herein, the term "substantial sequence identity" or "substantial 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.

In the present invention, 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.

In the present invention, 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. Preferably, 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.

The 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. In addition, diluents, dispersants, surfactants, 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. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the art.

In particular, the 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, 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.

In order to achieve the above another object, 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.

In the present invention, the cell may be a cell of a mammal except a human. Preferably 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. Preferably, the cells may be breast cancer cells, but are not limited thereto.

In order to achieve the above another object, it provides a use of an inhibitor for the 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.

The 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.

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.

4 is a diagram showing the inhibitory effect of heparan sulfate proteoglycan (HSPG), one of the intracellular sulfated process by sodium hypochlorite (NaClO ₃ ), an inhibitor of the sulfated process.

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).

8 is a result of Western blotting confirming a decrease in the expression of heparan sulphate with replication aging of the human fibroblast cell line HDF.

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.

11 is After treatment with siControl, siHS3ST1 or siHS2ST1 in human breast cancer cell line MCF-7 and incubated for 2 days, Western blotting result was confirmed the degree of phosphorylation and p21 expression of Rb.

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.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, which will be construed as to aid the understanding of the present invention.

Example 1 Confirmation of Correlation between Inhibition of Intracellular Sulfation Process and Induction of Cell Aging

1.1. Confirmation of Induction of Aging by Inhibition of Sulfation Process of Tumor Cell Line Using Aging-related Betagalactosidase Activity Staining

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). Human breast cancer cell line MCF7 treated with sodium hypochlorite 0 mM, 15 mM or 30 mM, an inhibitor of intracellular sulphation, was treated with 10% fetal bovine serum for 3 days in a 37 ° C incubator at 5% CO ₂ concentration: 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: 9363-9367, 1995).

Cells were washed twice with PBS and fixed for 3-5 minutes at 3% formaldehyde at room temperature. The immobilized cells were washed once again with PBS and β-galactosidase active staining solution (1 mg / ml X-Gal, 40 mM citrate / sodium phosphate, pH 6.0), 5 mM potassium ferrocyanide, 5 5 ml of mM potassium ferricyanide, 150 mM chloride, 2 mM magnesium chloride) was added and reacted in a 37 ° C. incubator for 12-16 hours. The plate was incubated with silver foil so that light did not enter during the reaction.

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. As a result, unlike the control group, 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. In particular, the higher the concentration of sodium hypochlorite treatment, the greater the specific gravity of the cells showing the characteristics of aging cells.

In addition, 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. In particular, the higher the concentration of sodium hypochlorite treatment, the greater the percentage of cells showing positive aging-related beta-galactosidase.

Therefore, it can be seen from the results that inhibiting the intracellular sulfated process in tumor cells can induce aging of tumor cells.

1.2. Confirmation of Induction of Aging by Inhibition of Sulfation Process of Tumor Cell Line by Western Analysis

Human breast cancer cell line MCF-7 treated with sodium hypochlorite 0 mM, 15 mM or 30 mM and incubated in culture medium at 37 ° C. incubator at 5% CO ₂ concentration for 24 hours was washed with PBS (phosphate buffered saline) and then cell sample. 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 μg / ml aprotinin, 2 μg / ml) Peptin), and 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). Add 20 ug of protein 2X SDS loading buffer (60 mm Tris-Cl (pH6.8), 25% glycerol, 2% SDS, 14.4 mM mercaptoethanol, 0.1% bromophenol blue) and heat at 95 ° C. for 5 minutes. Electrophoresis was performed for 2 hours at 80V in% to 10% SDS polyacrylamide gel.

After electrophoresis, 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. In Figure 3 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.

As a result of Western blotting analysis, it was confirmed that sodium hypochlorite treatment increased the expression of aging-specific p53 and p21 in human breast cancer cell line MCF-7 and induced aging by sodium hypochlorite. In particular, as the concentration of sodium hypochlorite treatment increases, the expression of aging-specific p53 and p21 increases, indicating that the rate of induction of aging caused by sodium hypochlorite increases.

Therefore, it can be seen from the results that inhibiting the intracellular sulfated process in tumor cells can induce aging of tumor cells.

Example 2 Confirmation of Inhibition of Heparan Sulfate Proteoglycan HSPG, One of Intracellular Sulfation Processes of Sodium Hypochlorite

Sodium hypochlorite treated with 0 mM, 15 mM or 30 mM and in a 37 ° C. incubator at 5% CO ₂ concentration for 24 hours 10% fetal bovine serum (FBS, Weljin) and the antibiotic streptomycin 100 ug / Incubated in DMEM (Dulbeco's Modified Eagle's Medium) culture medium containing ml and penicillin 100 units / ml (Gibco BRL). Cell culture medium of the human breast cancer cell line MCF-7 was removed, washed twice with PBS, and allowed to stand at room temperature for 20 minutes in 3.7% formaldehyde to fix cells. After washing twice with PBS, it was placed in a PBS solution containing 3% bovine serum albumin (BSA), blocked for 30 minutes at room temperature, diluted 1: 100 anti-heparan sulfated antibody (10E4, US biological company) Was added and reacted at 4 ° C. for 16 hours. After washing three times with PBS for 10 minutes, it was reacted for 30 minutes at room temperature in PBS solution diluted 1: 200 Alexa 555 complex anti-mouse antibody (Molecular probe) as a secondary antibody. After washing twice with PBS for 10 minutes, it was mounted with 50% glycerol, and then observed at 630 × magnification by confocal microscope (LSM-710, Carl Zeiss). The results are shown in FIG.

As a result, unlike the control group, 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. In particular, the higher the concentration of sodium hypochlorite treatment, the higher the inhibition of heparan sulfate, which is one of the sulfated protein intracellular polysaccharides.

Example 3 Linkage of Replication Aging to Heparan Sulfation of Protein Polysaccharides

3.1. Replicating aging according to passage number

To study replication aging, 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 The cells were cultured in a cell culture in which the concentration and the temperature of 37 ° C. were maintained at all times. 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. If you go through this process once, it counts as the number of passages 1, and if you pass 10 times, p10, if you pass 20, p20, if you pass 30, p30 Aging-related beta galactosidase activity staining was performed to determine whether cell aging occurred properly by dividing into young cells, medium-old cells, and old cells, respectively. The staining was performed according to the method of Dimri et al. (Dimri et al., Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995).

Cells were washed twice with PBS and fixed for 3-5 minutes at 3% formaldehyde at room temperature. The immobilized cells were washed once again with PBS and β-galactosidase active staining solution (1 mg / ml X-Gal, 40 mM citrate / sodium phosphate, pH 6.0), 5 mM potassium ferrocyanide, 5 1 ml of mM potassium ferricyanide, 150 mM chloride, 2 mM magnesium chloride) was added and reacted in a 37 ° C. incubator for 12-16 hours. The plate was incubated with silver foil so that light did not enter during the reaction.

The degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon), and the results are shown in FIG. 5. In addition, 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. In the case of p10 and p20 in Figure 6 it was not within the detectable range. In the case of p10 and p20 in Figure 6 it was not within the detectable range.

As shown in Figures 5 and 6, it can be seen that as the number of passages increases the proportion of cells showing age-related beta-galactosidase positive.

Therefore, it can be seen that replicating aging progresses as the number of passages increases.

3.2. Changes in the Heparan Sulfation Level of Protein Polysaccharides with Replication Aging

In order to confirm the association between replicative aging and the heparan sulfate process of protein polysaccharide, staining was performed using an anti-heparan antibody. Specifically, human fibroblast cell line HDF (ATCC, USA) on 100 mm culture dish (SPL) and 10% fetal bovine serum (FBS, Weljin) and 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 The cells were cultured in a cell culture in which the concentration and the temperature of 37 ° C. were maintained at all times. 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. Wash the cells twice with PBS and leave the cells in 3.7% formaldehyde for 20 minutes at room temperature to fix the cells. Thereafter, washed twice with PBS, then placed in a PBS solution containing 3% bovine serum albumin (BSA), blocked for 30 minutes at room temperature, and diluted 1: 100 anti-heparan sulfated antibody (10E4, US biological) The reaction was carried out at 4 ℃ for 16 hours. After washing three times with PBS for 10 minutes, the secondary antibody was reacted with PBS solution diluted with Alexa 555 conjugated anti-mouse antibody (Alexa 555 conjugated goat anti-mouse IgG, Molecular probe) 1: 200 at room temperature for 30 minutes. After washing twice with PBS for 10 minutes, it was mounted with 50% glycerol, and then observed at 630 × magnification with a confocal microscope (LSM-710, Carl Zeiss).

After washing the cells of the same experimental group as in Fig. 7 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 μg / ml aprotinin, 2 μg / 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). 20 μg of protein 2 × SDS loading buffer (60 mm Tris-Cl (pH6.8), 25% glycerol, 2% SDS, 14.4 mM mercaptoethanol, 0.1% bromophenol blue) was added and heated at 95 ° C. for 5 minutes. Electrophoresis was performed for 2 hours at 80V on 8% to 10% SDS polyacrylamide gel. After electrophoresis, 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. 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.

As shown in Figures 7 and 8, it was confirmed that as the replication aging proceeds, the degree of sulfated intracellular heparan decreased.

Therefore, it can be seen from the results that inhibiting the intracellular sulfated process in tumor cells can induce aging of tumor cells.

Example 4 Identification of Inhibition of Heparan Sulfate Proteoglycan (HSPG) by HS2ST1 Inhibitors and HS3ST1 Inhibitors

Enzymes that mediate the process of heparan sulfate of protein polysaccharide 1) HS3ST1 {(heparan sulfate 3-O sulfotransferase; NCBI Access No. NM_005114 (SEQ ID NO: 7)}, 2) HS2ST1 (heparan sulfate 2-O sulfotransferase; Access No Tumor cell line MCF-7 (ATCC, USA), 10% fetal bovine serum (fetal bovine), was tested to determine the effect of decreased gene expression of NM_012262 (SEQ ID NO: 1) or NM_001134492 (SEQ ID NO: 2). serum: FBS, Weljin) and 2 μl of RNAiMAX (invitrogen, Cat # 13778) incubated in DMEM culture medium containing antibiotics (Gibco BRL) and passaged in 60 mm culture plates one day before injection of small interfering RNA. -075) and OptiMEM® I culture medium (Invitrogen, Cat # 31985) were mixed and small interfering 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 (dTdT) bound to the cells were treated at a concentration of 50 nM. After 6 hours, 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.

In addition, 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.

All siRNA used what was produced by requesting Bioneer (Korea) was used. Specifically, siRNAs were synthesized using β-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. Subsequently, the 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. After washing twice with PBS, it was placed in a PBS solution containing 3% bovine serum albumin (BSA), blocked for 30 minutes at room temperature, and diluted 1: 100 anti-heparan sulfated antibody (10E4, US biological) Was added and reacted at 4 ° C. for 16 hours. After washing three times with PBS for 10 minutes, it was reacted for 30 minutes at room temperature in PBS solution diluted 1: 200 Alexa 555 complex anti-mouse antibody (Molecular probe) as a secondary antibody. After washing twice with PBS for 10 minutes, the cells were mounted with 50% glycerol and observed at 630 × magnification by confocal microscopy (LSM-710, Carl Zeiss). The results are shown in FIG.

As a result, unlike the control group (siCON), 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.

Example 5 Confirmation of Change of Cell Proliferation Rate by Treatment of HS2ST1 Inhibitor or HS3ST1 Inhibitor

5% after injecting human breast cancer cell line MCF-7 with specific small interfering RNA (siHS3ST1) for HS3ST1, specific small interfering RNA (siHS2ST1) for HS2ST1 or nonspecific small interfering RNA (siControl) prepared as in Example 4 10% fetal bovine serum (FBS, Weljin) and the antibiotics streptomycin 100 ug / ml and penicillin 100 units / ml (Gibco BRL) for 1 to 3 days at 37 ℃ incubator with CO ₂ concentration After culturing in the included DMEM (Dulbeco's Modified Eagle's Medium) culture medium by harvesting the cell supernatant by date (0 days, 1 day, 2 days, 3 days) shown in Figure 10 and after the cells are removed using trypsin After washing twice with PBS, mixed 1: 1 with 0.4% trypan blue (NUNK), and left at room temperature for 5 minutes. Cell proliferation as a percentage of the number of cells is shown in FIG. Served. In FIG. 10, the x-axis represents the days of cell culture after treatment with siHS3ST1, siHS2ST1, or siControl, and the y-axis represents the cell proliferation rate based on the number of cells on day 0.

As shown in FIG. 10, 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.

Example 6 Confirmation of Induction of Cellular Aging by Treatment of HS2ST1 Inhibitor or HS3ST1 Inhibitor

6.1. Western analysis of HS2ST1 inhibitors or HS3ST1 inhibitors to induce aging

37 ° C. constant temperature of 5% CO ₂ concentration after injection of specific small interfering RNA (siHS3ST1) for HS3ST1, specific small interfering RNA (siHS2ST1) or nonspecific small interfering RNA (siControl) prepared as in Example 4 DMEM (Dulbeco's Modified Eagle's Medium) containing 10% fetal bovine serum (FBS, Weljin) and the antibiotic Streptomycin 100 ug / ml and penicillin 100 units / ml (Gibco BRL) for 2 days in the incubator 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 μg / ml aprotinin, 2 μg / ml lupetin), and the extracted protein was centrifuged at 11,000 rpm for 10 minutes, and then Bradford was prepared by taking only the supernatant. Law {bradford, M., Anal. Biochem. 72: 248-254 (1976). Add 20 ug of protein 2X SDS loading buffer (60 mm Tris-Cl (pH6.8), 25% glycerol, 2% SDS, 14.4 mM mercaptoethanol, 0.1% bromophenol blue) and heat at 95 ° C. for 5 minutes. Electrophoresis was performed for 2 hours at 80V in% to 10% SDS polyacrylamide gel.

After electrophoresis, 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 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) that specifically recognizes 811 was used. 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. In FIG. 11, 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.

Western blotting analysis showed that siHS2ST1 treatment group showed decreased phosphorylation of aging-specific Rb and increased expression of aging-specific p21 in human breast cancer cell line MCF-7 compared to control group (siControl) and siHS3ST1 treatment group. It can be seen that more is induced.

Therefore, when the HS2ST1 gene expression is suppressed in tumor cells, it can be confirmed that aging of tumor cells can be induced.

6.2. Confirmation of Induction of Aging by HS2ST1 Inhibitors or HS3ST1 Inhibitors in Tumor Cell Lines Using Aging-related Betagalactosidase Activity Staining

5% CO ₂ concentration of human breast cancer cells MCF7 injected with specific small interfering RNA (siHS3ST1) for HS3ST1, specific small interfering RNA (siHS2ST1) for HS2ST1, or nonspecific small interfering RNA (siControl) prepared as in Example 4 Aging-related beta galactosidase activity staining was performed after 3 days of incubation in a 37 ° C. incubator. The staining was performed according to the method of Dimri et al. (Dimri et al., Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995).

Cells were washed twice with PBS and fixed for 3-5 minutes at 3% formaldehyde at room temperature. The immobilized cells were washed once again with PBS and β-galactosidase active staining solution (1 mg / ml X-Gal, 40 mM citrate / sodium phosphate, pH 6.0), 5 mM potassium ferrocyanide, 5 5 ml of mM potassium ferricyanide, 150 mM chloride, 2 mM magnesium chloride) was added and reacted in a 37 ° C. incubator for 12-16 hours. The plate was incubated with silver foil so that light did not enter during the reaction.

The degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon), and the results are shown in FIG. 12. In addition, 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.

Compared to the siControl-treated control group (siCON or siControl) and siHS3ST1 treatment group, it can be seen that the number of cells stained in the siHS2ST1 treatment group, that is, aging-related beta-galactosidase positive cell number. In particular, the 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.

Therefore, on the basis of the above results, it can be confirmed that when the HS2ST1 gene expression is suppressed in tumor cells (eg, human breast cancer cell line MCF-7), the senescence of tumor cells can be induced to show anticancer effects.

According to the present invention, 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.

Claims (8)

1. A composition for inducing cell aging comprising an HS2ST1 (heparan sulfate 2-O sulfotransferase 1) gene or an inhibitor for a protein encoded by the gene.
2. The composition of claim 1, wherein the inhibitor is siRNA.
3. The composition of claim 1, wherein the siRNA has a sense sequence of SEQ ID NO: 5 and an antisense sequence of 6.
4. The composition of claim 1, wherein the inhibitor for the protein is an antibody.
5. The composition of claim 1, wherein the cells are cancer cells.
6. The composition of claim 5, wherein the cancer cells are breast cancer cells.
7. Mammalian cells, except humans, comprising treating cells of mammals other than humans with a composition for inducing cellular senescence comprising a heparan sulfate 2-O sulfotransferase 1 (HS2ST1) gene or an inhibitor for a protein encoded by the gene Method of inducing aging.
8. The method of claim 7, wherein the cell is a cancer cell.

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