JP2006249015A - Cellular senescence inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cellular senescence inhibitor and a health food or a cosmetic having cellular senescence-inhibiting action. <P>SOLUTION: The present invention provides a cellular senescence inhibitor containing a substance inhibiting unbalanced growth of cell, a health food or a cosmetic containing the cellular senescence inhibitor, a method for screening a compound having cellular senescence-inhibiting effect and a screening kit, etc., of the compound having cellular senescence-inhibiting effect. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell aging inhibitor, a health food or cosmetic containing the cell aging inhibitor, a method for screening a compound having a cell aging inhibitory effect, a compound screening kit having a cell aging inhibitory effect, and the like.

Adherent somatic cells collected from normal mammalian tissues stop growing after a certain number of cell divisions when they are subcultured, and become hypertrophic, flattened, and indefinite. This phenomenon is called passaged aging (Hayflick, L., Moorehead PS, Exp cell Res, 1961. 25, pp 585-621:. Non-Patent Document 1). On the other hand, oxidative stress, radiation, topoisomerase inhibitors, histone modifying enzymes, demethylation factors, cell cycle regulators, signal transduction mediators, etc. also induce cell senescence (Young, J. and JR Smith, Exp Gerontol, 2000. 35 (1), pp. 23-32: Non-Patent Document 2). Many of these stresses induce apoptosis when applied in excess. In addition, apoptosis is generally induced in cancer cell lines. Currently, cell aging is caused by metabolic inhibition by cellular metabolites, changes in cell surface, increased cross-linking between cytoskeletal molecules, accumulation of degraded molecules in cells due to free radicals, transcription and translation of genetic information Accumulation of errors, programming of genetic cell life span, damage of intracellular DNA and reduction of its repair ability, etc. are considered, and various cell aging hypotheses based on these have been proposed. However, the molecular mechanism is still unknown.

When examining the various cellular senescence systems reported so far, delays in DNA replication and morphological changes (hypertrophy, flattening, and irregularity) are observed without exception. This phenomenon is nothing but the long-established unbalanced growth. On the other hand, 5-bromo-2-deoxyuridine (abbreviated as BrdU) is known to rapidly age any cell line (Michishita, E., et al., J Biochem (Tokyo), 1999. 126 (6), pp. 1052-9: Non-Patent Document 3). When the mechanism was examined in detail, it was observed that the morphological change occurred early. Although senescent cells commonly show a delay in DNA replication in S phase, the various senescence induction means described above directly or indirectly inhibit DNA replication. If this disproportionate growth continues for a long time (about 1 week), charged RNA and proteins accumulate excessively in the cell (Hoffmann, EK and PB Dunham, Int Rev Cytol, 1995. 161 , pp. 173 -262: Non-patent document 4). In order to compensate for the osmotic pressure, water flows in and the cells expand (Hamill, OP and B. Martinac, Physiol Rev, 2001. 81 (2), pp. 685-740: Non-Patent Document 5). The membrane is tensioned and induced or activated by mechanical non-selective substance channels, volume sensitive ion channels, phosphorylated inositol metabolism, cytoskeletal rearrangement, Rho GTPase family proteins, membrane-bound receptors, MAPK signaling pathways, etc. Is done. These reactions are intended to release osmotic pressure and reduce cell damage (Jakab, M., et al., Cell Physiol Biochem, 2002. 12 (5-6), pp. 235-58: Non Patent Document 6). However, cells that exceed the limit capacity deviate from the homeostatic mechanism and lose the ability to divide.
Hayflick, L., Moorehead PS, Exp cell Res, 1961. 25, pp. 585-621. Young, J. and JR Smith, Exp Gerontol, 2000. 35 (1), pp. 23-32. Michishita, E., et al., J Biochem (Tokyo), 1999. 126 (6), pp. 1052-9. Hoffmann, EK and PB Dunham, Int Rev Cytol, 1995. 161, pp. 173-262. Hamill, OP and B. Martinac, Physiol Rev, 2001. 81 (2), pp. 685-740. Jakab, M., et al., Cell Physiol Biochem, 2002. 12 (5-6), pp. 235-58.

  Preventing cell aging is a long-standing hope of mankind to prolong life and promote beauty. However, as described above, various studies have been conducted on cell aging, but no effective cell aging inhibitor has been developed so far. Therefore, development of an effective cell aging inhibitor capable of suppressing cell aging is desired.

  The present inventor considered that a gene specific to cell aging is a gene that responds to changes in membrane structure, and assumed that swelling due to chronic imbalanced growth is the principle of cell aging. Therefore, as a result of examining in detail whether cell senescence occurs by inducing unbalanced growth with a DNA replication inhibitor in cells that do not cause cell damage, the present invention has been completed.

That is, the present invention
[1] Cell aging inhibitor containing a substance that suppresses unbalanced growth of cells;
[2] The cell aging inhibitor according to [1] above, which contains a MAP kinase inhibitor and / or a protein synthesis inhibitor as a substance that suppresses unbalanced growth of cells;
[3] The cell aging inhibitor according to [2] above, wherein the MAP kinase inhibitor is an ERK inhibitor;
[4] The cell aging inhibitor according to [3] above, wherein the MAP kinase inhibitor is U0126;
[5] The cell aging inhibitor according to [2] above, wherein the protein synthesis inhibitor is selected from the group consisting of cycloheximide, emetine, puromycin, and rapamycin;
[6] A health food containing the cell aging inhibitor according to any one of [1] to [5] above;
[7] A cosmetic containing the cell aging inhibitor according to any one of [1] to [5] above;
[8] A screening method for a compound having a cell aging inhibitory effect,
(1) inducing unbalanced growth in cells,
(2) continuing the unbalanced growth for a certain period, and (3) administering the test compound and observing the cells,
A screening method comprising:
[9] The screening method according to [8] above, wherein the step of inducing unbalanced growth in the cell comprises a step of treating the cell with a DNA replication inhibitor or a DNA damaging agent;
[10] The screening method according to [9] above, wherein the DNA replication inhibitor or DNA damaging agent is aphidicolin.
[11] A screening kit for a compound having a cell aging inhibitory effect, comprising a cell, a DNA replication inhibitor or a DNA damaging agent, and a MAP kinase inhibitor;
Etc.

  ADVANTAGE OF THE INVENTION According to this invention, the cell aging inhibitor which has the effect of suppressing cell aging, the cosmetics containing this, and health food can be provided. Further, according to the screening method of the present invention, a substance having an effect of inducing cell aging or an effect of suppressing cell aging can be efficiently searched.

1. Cell aging inhibitor The 1st aspect of this invention is related with the cell aging inhibitor containing the substance which suppresses the imbalanced proliferation of a cell. In the present specification, “cell aging” is a phenomenon in which proliferation is stopped after a certain number of cell divisions when subculture is continued, and enlargement, flattening, indefinite state (passaging aging), or It refers to cell enlargement, flattening, and indetermination induced by oxidative stress, radiation, topoisomerase inhibitors, histone modifying enzymes, demethylation factors, cell cycle regulators, signal transduction mediators, and the like. In addition, “unbalanced growth of cells” means a phenomenon in which synthesis of RNA, protein, metabolites, etc. proceeds almost normally under conditions where DNA replication is delayed, and the cell expands. Another aspect of the “cell aging” phenotype. In the present invention, the screening method for a cell aging inhibitor and a compound having a cell aging inhibitory effect has been completed by paying attention to this “cell imbalanced proliferation”. Therefore, in the present specification, “cell senescence suppression” refers to suppression of one or more of the above “cell senescence” phenotype and cell imbalanced proliferation. Examples of “substances that suppress imbalanced growth” include MAP kinase inhibitors, protein synthesis inhibitors, and the like. These may be used alone or in combination of two or more.

@0011 Examples of the MAP kinase inhibitor that can be used in the present invention include neutralizing antibodies against MAP kinase, compounds that inhibit the activity of MAP kinase, compounds that inhibit transcription of a gene encoding MAP kinase (for example, antisense nucleic acid). , RNAi, ribozyme) and the like. MAP kinase inhibitors preferably used in the present invention include U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene), PD98059 (2- (2′-amino- 3'-methoxyphenyl) -oxanaphthalen-4-one), SB203580 (4- (4-Fluorophenyl) -2- (4-methylsulfonylphenyl) -5 (4-pyridyl) imidazole), SP600125 (anthra [1,9-cd] pyrazole-6 (2H) -one) SL327), peptides, and plant components (eg, polyphenols, flavonoids, glycosides) and the like. In the present invention, E0 inhibitor U0126 is particularly preferable. U0126 [Chemical name 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) -butadiene (1,4-Diamino-2,3-dicyano-1,4-bis (2 -aminophenylthio) -butadiene)] has the following structure:

@ 0011

  Examples of the protein synthesis inhibitor that can be used in the present invention include a compound that inhibits the enzyme activity of a protein synthesis enzyme, a compound that inhibits transcription of a gene encoding the protein synthesis enzyme (for example, an antisense nucleic acid, RNAi , Ribozyme) and the like. Specific examples of the protein synthesis inhibitor include cycloheximide, emetine, puromycin, and rapamycin. In the present invention, cycloheximide is preferred.

  In the present invention, a neutralizing antibody against a protein synthesis enzyme or MAP kinase can be used as a “substance that suppresses unbalanced growth”. The neutralizing antibody that can be used in the present invention may be either a monoclonal antibody or a polyclonal antibody as long as it has an effect of inhibiting cell aging. In the present invention, the “antibody” means the whole antibody molecule or a fragment thereof (for example, Fab or F (ab ′) 2 fragment) capable of binding to an antigen protein synthesis enzyme or MAP kinase. The antibody used in the present invention can be produced by any of various methods. Methods for producing such antibodies are well known in the art [see, eg, Harlow E. & Lane D., Antibody, Cold Spring Harbor Laboratory Press (1988)].

  Examples of the “substance that suppresses unbalanced growth” include compounds that inhibit transcription of a gene encoding a protein synthesizing enzyme such as antisense nucleic acid, RNAi, ribozyme or MAP kinase. An antisense nucleic acid having a base sequence complementary to or substantially complementary to the base sequence of a gene DNA encoding a protein synthesis enzyme or MAP kinase, or a part thereof, encodes a protein synthesis enzyme or MAP kinase. Any antisense nucleic acid can be used as long as it has a base sequence complementary to or substantially complementary to the base sequence of the gene DNA to be processed and has a function capable of suppressing the expression of the DNA. Although it may be present, antisense DNA is preferred. “Substantially complementary nucleotide sequence” means, for example, about 70% or more, preferably about 80% or more of the entire or partial nucleotide sequence of the nucleotide sequence complementary to the gene DNA (ie, complementary strand) More preferred is a base sequence having a homology of about 90% or more, most preferably about 95% or more. In particular, of the total base sequence of the complementary strand, about 70% or more of the complementary strand of the base sequence encoding the N-terminal site of the protein synthesizing enzyme or MAP kinase (eg, the base sequence near the start codon, etc.), preferably Antisense nucleic acids having a homology of about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable.

  In the present specification, the term “homology” refers to the amount (number of numbers) that can be determined to be the same in the matching relationship between the bases constituting the chain between the two chains in the base sequence. ) And the degree of sequence correlation between two base sequences. Homology can be easily calculated. Many methods for measuring homology between nucleotide sequences are known, and the term “homology” (also referred to as “identity”) is well known to those skilled in the art. Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman, Examples disclosed in D., SIAM J. Applied Math., 48: 1073 (1988) are not limited thereto. Preferred methods for measuring homology include those designed to obtain the largest match between the two sequences being tested. An example of such a method is one assembled as a computer program. Preferred computer program methods for measuring the homology between two sequences include the GCG program package (Devereux, J. etal., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol., 215: 403 (1990)) and the like, but are not limited thereto, and methods known in the art can be used.

  Antisense nucleic acids are usually composed of about 10-40 bases, preferably about 15-30 bases. In order to prevent degradation by a hydrolase such as nuclease, phosphate residues (phosphates) of each nucleotide constituting an antisense nucleic acid are converted to chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. May be substituted. These antisense nucleic acids can be produced using a known DNA synthesizer.

  Similar to the above-described antisense nucleic acid, double-stranded RNA (siRNA or RNAi; RNA interference method), ribozyme, and the like can also suppress the expression of a gene encoding a protein synthesis system enzyme or MAP kinase. Here, double-stranded RNA is designed based on the base sequence of a gene encoding a protein synthesizing enzyme or MAP kinase according to a known method, for example, the method described in Nature, 411, 494, 2001. Can be manufactured. The ribozyme is designed based on the base sequence of a gene encoding a protein synthesizing enzyme or MAP kinase according to a known method, for example, the method described in TRENDS in Molecular Medicine, 7, 221, 2001. Can be manufactured. For example, a desired ribozyme can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. Examples of the RNA encoding the protein of the present invention include a portion (RNA fragment) close to the cleavage site on the RNA of the present invention that can be cleaved by a known ribozyme.

  “Substances that suppress imbalanced growth” further include artificial peptides (eg, anti-MAP kinase antibodies) that inhibit protein synthesizing enzymes or MAP kinases. Since peptides are rich in diversity, the design of peptides that bind to the reaction sites of various enzymes has become a common technique. In addition, as described above, highly safe plant-derived compounds can be mentioned. For example, aromatic compounds such as polyphenols and flavonoids having an antioxidant action, glycosides, and compounds such as alkaloids having various biological activities are also included.

  A substance that suppresses unbalanced growth as described above is effective as an active ingredient of a cell aging inhibitor. Specifically, the cell aging inhibitor of the present invention is effective in preventing the appearance of senescent cells, returning the aging cells to the growth cycle, and selectively removing senescent cells. A major cause of cell aging in the living body is cell damage due to oxidative stress. Therefore, it is considered that the above-mentioned compounds having an antioxidative action suppress not only the protein synthesizing enzyme or MAP kinase inhibitory action but also suppress unbalanced growth by reducing oxidative stress, and consequently suppress cell aging. .

  When the anti-aging inhibitor of the present invention is used as a pharmaceutical, the above-mentioned substance (active ingredient) that suppresses unbalanced growth alone or in combination with a pharmaceutically acceptable carrier, oral, parenteral or topical It can be administered by any of these routes. Such administration is performed in single or multiple doses. In general, the active ingredient is combined with various pharmaceutically acceptable carriers to produce tablets, powders, capsules, lozenges, troches, hardened candy, powders, sprays, creams, waxes, suppositories, jelly, gels, pastes, lotions , Ointments, suspensions, solutions, elixirs, syrups, etc. Such preparations include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, emulsifiers, stabilizers, buffers, preservatives, flavoring agents, fragrances. Coloring agents and the like can be added.

  For example, tablets can be used for excipients such as starch, lactose, glucose, microcrystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, coating agents such as gelatin, hydroxypropylcellulose, gelatin, gum arabic, methylcellulose, etc. Binders and disintegrants such as starch, agar, gelatin, sodium bicarbonate may be included. In addition, lubricants such as magnesium stearate and talc are also useful for preparing tablets. Similar types of solid compositions may also be employed as fillers for gelatin capsules; suitable materials in this regard include lactose as well as high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredient is combined with various sweetening or flavoring agents, coloring agents or dyes, and, optionally, diluents such as water, ethanol, propylene Emulsifiers and / or suspending agents can be combined with glycols, glycerin and various combinations thereof.

  In general, the active ingredient is contained in the oral dosage form in an amount of 5% to 70%, preferably 10% to 50% by weight. The compound of the present invention in the form of a solution can be injected parenterally, for example, intradermally, subcutaneously, intravenously or intramuscularly. Such solutions are, for example, sterile aqueous solutions, aqueous suspensions and edible oil solutions. The aqueous solution is suitably buffered (preferably pH> 8) and contains a sufficient amount of salt or glucose to make the solution isotonic with blood. Aqueous solutions are suitable for intravenous injection purposes. Aqueous suspensions may contain suitable dispersing or suspending agents, for example, sodium carboxymethyl cellulose, methyl cellulose, polyvinyl pyrrolidone or gelatin. Aqueous suspensions can be used for subcutaneous or intramuscular injection. Edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil can be employed for the edible oil solution.

  When applied to the skin (regeneration of skin, wrinkle removal, stain removal, etc.), the compound of the present invention can also be administered locally. In this case, it is preferably prepared in the form of a cream, jelly, gel, paste, ointment or the like according to standard techniques.

  When the cell aging inhibitor of the present invention is administered to a patient by any of the above routes, the daily dose per patient body weight is about 0.01 mg / kg to about 3000 mg / kg, preferably about 0.01 mg / kg to About 1000 mg / kg, more preferably about 0.1 mg / kg to about 100 mg / kg is administered in a single or divided dose. However, it can be varied as appropriate depending on the weight and condition of the patient being treated, the type of active ingredient employed, the condition being treated, and the particular route of administration selected.

2. Health food containing cell aging inhibitor of the present invention The second aspect of the present invention relates to a health food containing the cell aging inhibitor. That is, the health food of the present invention contains a substance (active ingredient) that suppresses the above-mentioned cell imbalanced growth. In this specification, the term “health food” is used as a broad concept term including beverages, supplements, dietary supplements, foods for specified insurance, and the like.
The health food of the present invention comprises the above active ingredient in a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula, Add to cake mixes, powdered or liquid dairy products, bread, cookies, etc., and if necessary, process into pellets, tablets, granules, etc. together with excipients such as dextrin, lactose, starch, flavorings, pigments, etc. Alternatively, it can be prepared by coating with gelatin or the like and molding into a capsule. In the production of these health foods, flavoring agents, coloring agents, flavoring agents, stabilizers, extenders and the like can be used in combination as appropriate.
In addition, content of the active ingredient in health food is not specifically limited, For example, it can contain 1 to 99%.

3. Cosmetics containing the cell aging inhibitor of the present invention A third aspect of the present invention relates to a cosmetic containing the cell aging inhibitor. The cosmetic of the present invention has a cell aging inhibitory action, and is particularly suitable as an anti-wrinkle agent, a wrinkle remover, and a stain remover. The cosmetics of the present invention are cosmetics such as lotions, creams, emulsions, foundations, oily cosmetics, packs, skin cleansers, sprays, aerosols, etc., and medicinal cosmetics (quasi-drugs), ointments according to conventional methods. It can be prepared and used as a pharmaceutical external preparation such as an agent, a liniment, a lotion or an emulsion.
Examples of the base suitable for the cosmetic of the present invention include various basic cosmetic bases that have been conventionally used, such as various alcohols, hydrocarbons, fatty acids, phospholipids, fats and oils, and waxes. These can be used alone or in combination. Regardless of which base is used, the external preparation of the present invention preferably contains water and alcohol.

In the cosmetic of the present invention, in order to enhance absorption into the skin, for example, surfactants, terpenes, 1-alkylpyrrolidones, tesylmethyl sulfoxide, isopropyl myristate, phospholipid microspheres or the like can be used as absorption promoters. A mixture of
For the production of the cosmetic of the present invention, a method usually used in the technical field of cosmetics, medicinal cosmetics or external preparations can be used. In the production, preservatives, fragrances, humectants, stabilizers, anti-aging agents, pigments, hormones and vitamins can be appropriately selected and added as desired. In addition, content of the active ingredient in cosmetics is not specifically limited, For example, it can be made to contain 1 to 99%.

4). Screening method and screening kit for compound having cell aging inhibitory effect A fourth aspect of the present invention relates to a method for screening a compound having a cell aging inhibitory effect. Specifically, the screening method of the present invention is a screening method for a compound having a cell aging inhibitory effect, which comprises (1) a step of inducing unbalanced growth in cells, and (2) continuing unbalanced growth for a certain period. A screening method comprising the steps of: (3) administering a test compound and observing cells; As such a screening method, more specifically, (i) morphology of cells proliferating imbalanced, and (ii) morphology of cells after contacting the cells proliferating imbalanced with the test compound Or (iii) the amount of protein contained in the cells proliferating imbalanced, and (iv) the cells proliferating imbalanced and the test compound. Examples thereof include a screening method for a compound having a cell aging inhibitory effect, which is characterized by comparing the amount of protein contained in a cell after contact.

  Here, it is necessary to include a step of treating the cells with a DNA replication inhibitor or a DNA damaging agent as the step of inducing unbalanced growth in the cells. As the DNA replication inhibitor or DNA damaging agent to be used, a compound selected from the group consisting of aphidicolin, excess thymidine and hydroxyurea, particularly aphidicolin, is preferable. In addition, various DNA replication inhibiting factors such as hydrogen peroxide solution, high concentration calcium, high oxygen partial pressure of the incubator, and osmotic pressure change of the culture solution can be used. The step of inducing unbalanced growth in cells can be performed as follows. That is, a DNA replication inhibitor or a DNA damaging agent is administered to cells under mild conditions to induce unbalanced growth. Subsequently, the cells can be aged by continuing unbalanced growth for a period of time, for example 5 to 15 days, preferably 7 to 10 days, more preferably 7 days, and most preferably 14 days. Here, cell senescence can be confirmed by morphological changes peculiar to senescent cells, or induction of cell senescence markers (for example, cell senescence specific β-galactosidase activity). Further, loss of cell division ability may be detected.

  After detecting cell aging, a test compound can be administered to the cells to screen for compounds having a cell aging inhibitory effect. The test compound may be any compound such as a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, plant extract, or animal tissue extract. In addition, the compound may be a new substance or a known substance.

  For example, the number of cells having a normal size in the case of (ii) is measured, and the number is about 10% or more, preferably about 20% or more, compared to the case of (i). A test compound which is preferably about 30% or more, more preferably about 50% or more can be selected as a compound having a cell aging inhibitory effect.

  A fifth aspect of the present invention relates to a screening kit for a compound having a cell aging inhibitory effect. The screening kit includes cells, DNA replication inhibitors or DNA damaging agents, MAP kinase inhibitors, experimental reagents, and the like. The DNA replication inhibitor or DNA damaging agent includes a compound selected from the group consisting of aphidicolin, excess thymidine, hydroxyurea, hydrogen peroxide, and high-concentration calcium, particularly aphidicolin, and MAP kinase The inhibitor preferably contains a compound such as U0126, PD98059, SB202190, SB203580, SL327, and particularly U0126. The cells may be any cells, but normal cells are highly sensitive to various stresses and easily undergo cellular senescence, and thus have high proliferation ability and are relatively insensitive to stress such as HeLa cells. Is preferred.

  The compound obtained using the screening method or screening kit of the present invention is the above-mentioned test compound, for example, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract. It is a compound selected from liquid, plasma and the like, and is a compound having a cell aging inhibitory effect. Moreover, the said compound can be utilized as cosmetics or health food which has a cell aging inhibitory effect by adding and mix | blending with cosmetics or food-drinks.

Examples and Experimental Examples The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.

First, a general experimental method will be described, and then examples of the present invention will be described.
General Experimental Procedure (1) Cell Culture In this experiment, a human cervical cancer-derived HeLa cell line clone was used. The cells were cultured in a plastic petri dish containing ES medium supplemented with 5% FCS under conditions of 37 ° C., 5% CO ₂ , humidity 95%, 5% CO ₂ . The number of cells was measured with a hemocytometer, and the survival rate was measured by staining the generated colonies with crystal violet.

(2) Senescence-specific β-galactosidase assay Cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde for 5 minutes. Next, the staining solution (1 mg / mll 5-bromo-4-chloro-3-indolyl β-D-galactoside, 29.48 mM citric acid (pH 6.0), 58.96 mM disodium hydrogen phosphate, 5 mM potassium ferricyanide, 5 mM ferro Potassium cyanide, 150 mM sodium chloride, 2 mM magnesium chloride) was added and incubated at 37 ° C. for 16 hours. After washing with PBS, it was observed and photographed with an optical microscope.

(3) Analysis of the cell cycle by flow cytometry After treating cells with 0.1% Triton X-2 mg / ml RNase A solution to degrade double-stranded RNA, DNA was added with 0.1 mg / ml propidium iodide (PI) solution. Were analyzed with a flow cytometer (Coulter EPICS XL). The amount of DNA was analyzed by a fluorescent signal, and the cell size was analyzed by a scattered signal.

(4) Blot analysis In Northern blot analysis, total RNA extracted from cells was transferred to a nylon membrane after agarose gel electrophoresis under denaturing conditions. Hybridization was performed using a probe labeled with ³² P. Autoradiography or an image analyzer (FUJI BAS2000) was used for signal detection.
In Western blot analysis, proteins extracted from cells were transferred to PVDF membrane after SDS-polyacrylamide gel electrophoresis. After the primary antibody was bound to the target protein, chemiluminescence generated by the secondary antibody was detected with an X-ray film.

Proof of the basic principle A DNA replication inhibitor was administered to HeLa cells derived from cervical cancer under mild conditions to induce unbalanced growth. Excess thymidine (1.5 mM), a natural allosteric inhibitor of ribonucleoductase, or aphidicolin (2 mM), an inhibitor of DNA polymerase α, were selected as inhibitors. Here, HeLa cells were used as a cell line because normal cells easily enter cellular senescence due to various stresses. As shown in FIG. 1a, addition of an appropriate concentration of excess thymidine or aphidicolin slowed cell growth. Excess thymidine was found to be ideal for inducing disproportionate growth, since it was not cytotoxic at the concentrations used. On the other hand, aphidicolin caused cell lysis or apoptosis at the high concentrations normally observed with chemical inhibitors. As shown in FIG. 2a, the cells with delayed growth were enlarged and flattened. As shown in FIG. 1b, such cells gradually lost their ability to divide and almost completely lost within 10 days. After 2 weeks in culture, almost all cells (90%) were clearly stained with senescence-specific β-galactosidase, as shown in FIG. 2b. The morphological changes peculiar to senescent cells and induction of other cell senescence markers were also observed by this imbalanced growth. In addition, 3-4 days after addition of excess thymidine or aphidicolin, the actin filaments became thick and clear and then disappeared. Actin filaments were also lost in normal human fibroblasts (TIG-7) that were passaged. When the cell cycle was analyzed with a flow cytometer for cells cultured for 1 week under unbalanced growth conditions, as shown in FIG. 3a, excess thymidine accumulated cells from the early stage to the middle stage of S phase. Aphidicolin, on the other hand, accumulated cells from the middle to late S phase. The cell diameter expanded about 4-5 times and the volume increased to about 110 times. Cells that had been in disproportionate growth for 1 week were examined for activation of the MAP kinase family (ERK, p38, JNK) by Western blot analysis. As a result, only ERK was remarkably activated (all were activated in normal human fibroblasts aged by culture). When the induction of representative cell aging marker genes was examined by Northern blot analysis, all genes were remarkably induced. Therefore, the phenomenon induced by sustained imbalanced growth of cells was completely consistent with the phenomenon that was made specific for cell aging.

Suppression of cell aging First, we examined whether imbalanced growth could be corrected by inhibiting protein synthesis. When cycloheximide (3μM) is used as a protein synthesis inhibitor, as shown in Fig. 1b and Fig. 2a, morphological changes, senescence-specific β-galactosidase activity, loss of division ability, ERK activation, cell aging marker gene All inductions were suppressed. Next, when the activation of ERK, a member of the MAP kinase family, was inhibited by U0126 (10 μM), the induction of all aging markers was remarkably suppressed as compared with the case of cycloheximide. Inhibitors of stress kinases (p38 and JNK) belonging to the same family did not show a suppressive effect. The results are shown in FIGS. 1a, 1b and 2a. Mechanical stimulation to cells is known to activate small molecule Rho GTPase family proteins (RhoA, Rac and Cdc42) and PI3 kinase. However, these inhibitors also showed an improvement effect on the cell morphology, but did not show suppression of the division ability. Therefore, the cell size and protein amount when cycloheximide or U0126 was added simultaneously were measured, and the results are shown in FIGS. 3b and 3c. As a result, cycloheximide simultaneously reduced cell enlargement and the amount of protein per cell. On the other hand, U0126 inhibited cell hypertrophy similar to cycloheximide, but did not affect the amount of protein per cell. These results indicate that cycloheximide and U0126 suppress unbalanced growth by different mechanisms. These results suggest that ERK is an important factor involved in the late reaction of cellular senescence.

  ADVANTAGE OF THE INVENTION According to this invention, the cell aging inhibitor which has cell aging effect | action, health food, cosmetics, etc. containing this can be provided. These cell aging inhibitors and health foods and cosmetics containing the same are effectively used for preventing cell aging, for example, promoting beauty. Moreover, according to the screening method of the present invention, a substance having a cell senescence action can be efficiently searched.

It is a figure which shows the growth, the survival rate, and the morphological change at the time of the unbalanced proliferation continuation of a HeLa cell. It is a growth curve under various culture conditions. CHX, dT, AC, and U0126 represent growth curves when 3 μM cycloheximide, 1.5 mM thymidine, 2 μM aphidicolin, and 10 μM U0126 are added, respectively. It is a figure which shows the growth, the survival rate, and the morphological change at the time of the unbalanced proliferation continuation of HeLa cell. The survival rate under various culture conditions is shown. The survival rate of the cells after culturing for a certain time was counted by the number of colonies that appeared after culturing in a normal medium for 2 weeks. It is a figure which shows the growth, the survival rate, and the morphological change at the time of the unbalanced proliferation continuation of a HeLa cell. The morphological changes under various culture conditions are shown. The morphological changes after 1 week of culture were photographed. It is a figure which shows the growth, the survival rate, and the morphological change at the time of the unbalanced proliferation continuation of HeLa cell. The measurement result of aging-specific β-galactosidase is shown. The acid β-galactosidase activity in 2 weeks of culture was stained and photographed. It is a figure which shows the growth, the survival rate, and the morphological change at the time of the unbalanced proliferation continuation of HeLa cell. It is the figure which observed F-actin cytoskeleton. Cells stably expressing GFP-actin were cultured and photographed. It is a figure which shows the analysis of the cell cycle at the time of imbalanced growth continuation of a HeLa cell, a cell size, and the protein content per cell. The results of flow cytometry are shown. It is a figure which shows the analysis of the cell cycle at the time of imbalanced growth continuation of a HeLa cell, a cell size, and the protein content per cell. It is the result of analyzing the size of a cell. It is a figure which shows the analysis of the cell cycle at the time of imbalanced growth continuation of a HeLa cell, a cell size, and the protein content per cell. It is the result of analyzing the protein content per cell. It is a figure which shows the induction | guidance | derivation of the activation of a MAP kinase, and the aging related gene at the time of the imbalanced growth of HeLa cell. It is an analysis result of Western blotting of MAP kinase. It is a figure which shows the induction | guidance | derivation of the activation of a MAP kinase, and the aging related gene at the time of the imbalanced growth of HeLa cell. It is the result of the Northern blot analysis of an aging specific gene.

Claims (11)

  A cell aging inhibitor containing a substance that suppresses unbalanced growth of cells.   The cell aging inhibitor according to claim 1, comprising a MAP kinase inhibitor and / or a protein synthesis inhibitor as a substance that suppresses cell imbalance growth.   The cell aging inhibitor according to claim 2, wherein the MAP kinase inhibitor is an ERK inhibitor.   The cell aging inhibitor according to claim 3, wherein the MAP kinase inhibitor is U0126.   The cell aging inhibitor according to claim 2, wherein the protein synthesis inhibitor is selected from the group consisting of cycloheximide, emetine, puromycin, and rapamycin.   A health food containing the cell aging inhibitor according to any one of claims 1 to 5.   Cosmetics containing the cell aging inhibitor according to any one of claims 1 to 5. A method for screening a compound having a cell aging inhibitory effect,
(1) inducing unbalanced growth in cells,
(2) continuing the unbalanced growth for a certain period, and (3) administering the test compound and observing the cells,
A screening method comprising:   The screening method according to claim 8, wherein the step of inducing unbalanced growth in the cell comprises a step of treating the cell with a DNA replication inhibitor or a DNA damaging agent.   The screening method according to claim 9, wherein the DNA replication inhibitor or DNA damaging agent is aphidicolin.   A screening kit for a compound having a cell aging inhibitory effect, comprising a cell, a DNA replication inhibitor or a DNA damaging agent, and a MAP kinase inhibitor.

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