JP2014059210A - Screening method of cytostatic agent using inhibition of coupling between nptn and s100a8 as index - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a screening method of a chronic-inflammation inhibitor or cancer metastasis inhibitor using a new receptor of S100A8 as a target.SOLUTION: A screening method of the cytostatic agent evaluates a candidate substance as a cytostatic agent when the candidate substance of the cytostatic agent significantly inhibits coupling between NPTN and S100A8.

Description

  The present invention provides a method for screening a cell growth inhibitor targeting neuroplastin (NPTN), which is a novel receptor of S100A8, which is a protein belonging to the S100 family, particularly NPTNβ.

  S100A8 and S100A9 are known as proteins that are up-regulated in hyperproliferation and psoriasis. S100A8 and S100A9 belong to the S100 protein family having an EF-hand type calcium binding domain composed of more than 20 members (Non-Patent Document 1: Marenholz I et al., Biochem Biophys Res Commun (2004) 322: 1111- 1122). Both proteins are secreted by neutrophils, activated monocytes, and macrophages, function as chemotactic molecules for those cells, and participate in a positive feedback loop for the recruitment of inflammatory cells (Non-Patent Document 2: Roth J et al., Trends Immunol (2003) 24: 155-158). S100A8 and S100A9 positive bone marrow cells are the first cells that infiltrate in the inflammatory region (Non-patent Document 3: Odink K et al., Nature (1987) 330: 80-82). Rheumatoid arthritis (Non-patent document 4: Liao H et al., Arthritis Rheum (2004) 50: 3792-3803), multiple sclerosis (Non-patent document 5: Bogumil T et al., Neurosci Lett (1998) 247: 195-197), Crohn's disease (non-patent document 6: Lugering N, et al., Digestion (1995) 56: 406-414), and connective tissue disease (non-patent document 7: Kuruto R, et al., J Biochem). High S100A8 and S100A9 serum levels have been observed in a number of human inflammatory diseases, including (Tokyo) (1990) 108: 650-653). Therefore, S100A8 and S100A9 are considered to play an important role in the induction and propagation of inflammation.

  Regarding the biological functions performed by S100A8 and 100A9 in epithelial cells, the present inventor has previously categorized normal epidermis by forming a complex (S100A8 / A9) (also known as calprotectin) with exogenous S100A8 and S100A9. Reveals that cells (NHEK) are stimulated to produce inflammatory cytokines that are up-regulated in psoriatic lesions, and that S100A8 / A9-inducible cytokines stimulate the production and secretion of S100A8 and S100A9 in NHEK (Non-patent document 8: J Cell Biochem. 2007 Nov 28, Epub ahead of print). Furthermore, it was found that S100A8 / A9 itself enhances NHEK proliferation. These results revealed the existence of a positive feedback mechanism of NHEK proliferation and inflammation involving S100A8 / A9 as a major mediator. That is, S100A8 / A9 induces the production of inflammatory cytokines to cause inflammatory diseases, the inflammation induces cell proliferation, and further, cell proliferation induces inflammation, and the proliferation / inflammation is induced. It has been suggested that it causes continuous persistent skin inflammatory diseases such as atopic dermatitis and psoriasis.

  In order to prevent the negative cycle formation of chronic inflammation caused by S100A8 / A9, it is considered necessary to identify the receptors of S100A8 and A9. Until now, it is known that empurin is a receptor for S100A9, and inhibiting these bindings suppresses chronic inflammation and further suppresses metastasis of cancer (Patent Document 1). However, a protein that functions as a receptor for S100A8 has not been known so far.

JP 2011-47932 A

Biochem Biophys Res Commun (2004) 322: 1111-1122 Trends Immunol (2003) 24: 155-158 Nature (1987) 330: 80-82 Arthritis Rheum (2004) 50: 3792-3803 Neurosci Lett (1998) 247: 195-197 Digestion (1995) 56: 406-414 J Biochem (Tokyo) (1990) 108: 650-653 J Cell Biochem. (2008) 104: 453-464 Nature Cell Biol. (2006) 8 (12): 1369-1375 Hum Genet (2002) 111: 310-313

  The present invention provides a method for screening a cytostatic agent targeting a novel receptor of S100A8.

  The present inventors recently focused on neuroplastin (NPTN) as a candidate protein for a novel receptor of S100A8 by database search based on homology with Emprin. It is known that NPTN has NPTNα having two immunoglobulin-like domains and NPTNβ isoform having three immunoglobulin-like domains. When NPTN was examined for expression in melanoma and keratinocytes, It was also found that NPTNβ was mainly expressed. In addition, in the binding test between RAGE, empurin or NPTNβ and S100A8, it was revealed that only NPTNβ binds to S100A8. It was shown that NPTN is involved in the promotion of keratinocyte proliferation by S100A8. Furthermore, the results of immunostaining revealed that S100A8 and NPTN were strongly expressed in the lesion skin of melanoma and atopic dermatitis, and these showed similar localization.

  Thus, the present inventors have found that NPTN (particularly NPTNβ) is a receptor for S100A8, and inhibiting the binding between them suppresses cell proliferation, thereby completing the present invention. It came to.

Accordingly, this application includes the following inventions:
(1) A method for screening a cell growth inhibitor, comprising incubating neuroplastin (NPTN) and S100A8 in the presence of a candidate substance for cell growth inhibitor to inhibit the binding between NPTN and S100A8 Selecting, as a cytostatic agent.
(2) The method according to (1), wherein the S100A8 forms a complex with S100A9.
(3) The method according to (1) or (2), wherein the NPTN is NPTNβ.
(4) A cytostatic agent comprising a drug that inhibits the binding between neuroplastin β (NPTNβ) and S100A8 or 100A8 / A9.
(5) The cell growth inhibitor according to claim 4, wherein the drug comprises one or more plants selected from the group consisting of mugwort, licorice, carrot, tea, hornon and rosemary. .
(6) A pharmaceutical composition for preventing and / or treating a disease selected from atopic dermatitis, cancer and psoriasis, which is formulated with the cell growth inhibitor according to (4) or (5).
(7) A cosmetic and / or therapeutic method for ameliorating a symptom or disease associated with abnormal cell proliferation, and requiring prevention and / or treatment of a symptom or disease associated with abnormal cell proliferation On the other hand, the method including applying the cell abnormal growth inhibitor as described in (4) or (5).
(8) A method for preventing and / or treating a disease selected from atopic dermatitis, cancer and psoriasis, the subject of (4) or (5) A method comprising applying the abnormal cell growth inhibitor of any one of the above.

  According to the present invention, a cell growth inhibitor can be searched for using inhibition of binding between NPTN (particularly NPTNβ) and S100A8 as an index. Moreover, the cell growth inhibitor obtained by the screening method of the present invention is useful for the prevention or treatment of diseases associated with abnormal cell growth, such as atopic dermatitis, cancer or psoriasis.

A structural comparison between Emprin and NPTNβ is shown. Figure 2 shows the relative expression of NPTN in melanoma and keratinocytes. The keratinocyte proliferation inhibitory effect by NPTNsiRNA is shown. The band by a Western blot which detects the coupling | bonding of RAGE and an empurin, and each S100 protein is shown. The band by a Western blot which detects the coupling | bonding of NPTN (beta) and each S100 protein is shown. The immuno-staining figure which shows the localization of S100A8 and NPTN in a melanoma. The immuno-staining figure which shows localization of S100A8 and NPTN in the epidermis of atopic dermatitis affected part. The interaction between S100A8 and NPTN in the epidermis of atopic dermatitis affected by PLA (Proximity Ligation Assay) method is shown. The NPTNβ-S100A8 binding inhibitory effect of Artemisia extract is shown. The comparison of the NPTN (beta) -S100A8 binding inhibitory effect of each plant extract is shown.

Neuroplastin (NPTN)
NPTN is also referred to as stromal cell-derived factor receptor 1 (SDFR1) or stromal cell-derived receptor 1 (SDR1), and is a single-transmembrane cell surface protein that is thought to be involved in neuronal differentiation and cell adhesion. However, little is known about its function. NPTN is known to exist in the form of NPTNα having two immunoglobulin-like domains and NPTNβ having three immunoglobulin-like domains. The structure of NPTNβ compared to Emprin is shown in FIG.

  The screening method of the present invention is not particularly limited, but NPTN (particularly NPTNβ) and S100A8 are incubated in the presence of the candidate substance, and the candidate drug that significantly inhibits the binding between NPTN and S100A8 protein is subjected to cell proliferation. It consists of selecting as an inhibitor. As the evaluation criteria, for example, 10% or more, 20% or more, or 30% or more, or 50% or more, or 70% or more, or 100% inhibition as compared with the case where the binding between NPTN and S100A8 protein acts as a control. If so, it may be determined to “significantly inhibit” chronic inflammation or cancer metastasis.

  As described above, S100A8 may form a complex with S100A9, and this complex may bind to the empurin. Therefore, the S100A8 / A9 complex may be used for the screening of the present invention.

  In addition, the present inventors have now found that empurin, NPTNα, and NPTNβ form a homodimer and a heterodimer, respectively. Therefore, the NPTN used in the screening of the present invention may form a homodimer, or may form a heterodimer with either empurin or NPTNα or NPTNβ.

  A means for detecting inhibition of binding between NPTN (particularly NPTNβ) and S100A8 is not particularly limited, but a calibration curve for binding between NPTN and S100A8 (or S100A8 / A9) is prepared based on the ELISA method. A molecule that inhibits binding, that is, a molecule that decreases in absorbance can be detected as a candidate drug for a cytostatic agent. From the viewpoint of ensuring good detection sensitivity, the molecule adsorbed on the solid support is preferably NPTN having a large molecular weight.

S100A8 and A9
The amino acid sequences of S100A8 and A9 and the DNA sequence encoding them are disclosed in, for example, Hum Genet (2002) 111: 310-313 (Non-patent Document 10). S100A8 and A9 that can be used in the present invention are usually human-derived natural types or recombinant proteins, but modified, heterogeneous, or non-purified products can be used as long as they have activity. The recombinant protein of S100A8 and A9 is prepared according to a method well known in the art, for example, by inserting the isolated or PCR-synthesized S100A8 or A9 gene (cDNA) into a plasmid, virus, etc. Is introduced into a host cell, for example, a cultured cell such as a microorganism, an animal cell, or a plant cell, and expressed in large quantities.

  S100A8 is dissolved in water or a medium, for example, a medium suitable for culturing epidermal keratinocytes, such as the EpiLife (registered trademark) medium, and added to the screening system of the present invention. Although the amount added cannot be generally specified, the concentration is about 1 ng / ml to 1 mg / ml, preferably about 10 ng / ml to about 100 μg / ml, more preferably about 100 ng / ml to about 10 μg / ml. The addition of S100A8 or S100A8 / A9 is preferably performed in the presence of calcium chloride. The culture conditions such as incubation time and incubation temperature in the presence of S100A8 or S100A8 / A9 are not particularly limited, preferably 30 to 37 ° C for 1 to 14 hours, more preferably 34 to 37 ° C for 2 to 7 Incubation is carried out for a time, preferably under 5% CO2.

  The cell growth inhibitor of the present invention is used as a pharmaceutical or cosmetic effective for improvement such as prevention or treatment of abnormal cell proliferation-related diseases caused by S100A8 or S100A8 / A9, such as atopic dermatitis, cancer, or psoriasis. it can.

  Examples of the cell growth inhibitor obtained by the screening method of the present invention include a plant selected from the group consisting of mugwort, licorice, carrot, tea, hornon and rosemary, or an extract thereof.

Mugwort (scientific name: Artemisia indica var. Maximowiczii) is a perennial plant belonging to the family Asteraceae distributed in Japan and the Korean peninsula, and the leaves of mugwort are known as herbal medicines with hemostatic action.
Licorice (scientific name: Glycyrrhiza) is a leguminous perennial plant that grows naturally in the Mediterranean region, Asia Minor, South Russia, Central Asia, North China, North America, etc. The dried roots are herbal medicines that have anti-inflammatory effects. Are known.
The carrot is preferably rapeseed carrot (scientific name: Panax ginseng CAMeyer), which is a perennial plant belonging to the family Araceae that originates in China and the Korean Peninsula. It is known as a herbal medicine having effects such as fatigue recovery, nerve cell activation, and enhancement of immunity.
Cha (scientific name: Camellia sinensis) is an evergreen tree of the camellia family that originates in Japan and China, and tea extract is known to have antioxidant and antibacterial effects.
Ogon is the root of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi), which is native to Russia and China, and is known as a herbal medicine with antipruritic activity.
Rosemary (scientific name: Rosmarinus officinalis) is an evergreen shrub that originates in the Mediterranean coastal region and belongs to the Labiatae family. Rosemary extract is known to have a blood circulation promoting action and an antioxidant action.

  Among these plants or extracts, mugwort extract, in particular, has been confirmed to significantly inhibit the binding between NPTNβ and S100A8 (FIG. 8A), and therefore is a preferred active ingredient of a cell growth inhibitor. is expected. Here, the plant body of each plant used in the present invention or an extract thereof is the various parts of each plant body (flowers, flower spikes, fruit skin, fruit, stem, leaves, branches, branch leaves, stem, bark, rhizome, roots). Skin, roots, seeds, whole grass, etc.) as they are or dried and pulverized to give a dry powder, or as it is or after drying and pulverizing and then extracted with a solvent.

  In the case of an extract, the extraction solvent used for extraction may be any solvent that is usually used for extraction, in particular alcohols such as methanol, ethanol or 1,3-butylene glycol, hydrous alcohols, acetone, ethyl acetate, etc. These organic solvents can be used alone or in combination. Of these, alcohols and hydrous alcohols are particularly preferred, and methanol, ethanol, 1,3-butylene glycol, hydrous ethanol or hydrous 1,3-butylene glycol are particularly preferred. . The solvent is preferably used at a temperature between room temperature and the boiling point of the solvent.

  There are no particular limitations on the extraction method, but usually it may be in the range of the boiling point of the solvent at room temperature to normal pressure. After extraction, the solution is filtered, ion exchanged, adsorbed, decolored and purified. , Paste, gel, and powder. In many cases, it can be used as it is, but if necessary, further purification treatment such as deodorization and decoloration may be added as long as the effect is not affected. An activated carbon column or the like may be used, and usual means generally applied depending on the extracted substance may be arbitrarily selected.

  For the extraction of plant bodies, leaves are considered for mugwort and tea, roots and stems for licorice, roots for carrots and dragons, and leaves and flowers for rosemary. The extraction site is not limited to these.

  The extract obtained by extraction with the above solvent can be used as it is or, for example, an extract concentrated by lyophilization or the like, and if necessary, an adsorbent method, for example, an ion exchange resin removed impurities, A polymer (for example, Amberlite XAD-2) adsorbed on a column, eluted with a desired solvent, and further concentrated can be used.

  The cell growth inhibitor of the present invention is preferably composed of one or two or more of the above-mentioned plants or extracts thereof, but contains other various components as long as the effects of the present invention are not impaired. be able to. In addition, the dose, usage, and dosage form of the cell growth inhibitor of the present invention can be appropriately determined according to the purpose of use. For example, the dosage form of the cell growth inhibitor of the present invention may be oral, parenteral, external use and the like. Examples of the dosage form include oral preparations such as tablets, powders, capsules, granules, extracts, and syrups, or parenteral preparations such as injections, drops, and suppositories, ointments, creams, emulsions, and lotions. And external preparations such as packs and bath preparations.

  The blending amount of the extract component of the cell growth inhibitor of the present invention can be appropriately determined according to the use, but is generally 0.0001 to 20.0% by mass, preferably 0.0001 to 10.0% by mass as a dry product in the total amount of the inhibitor. . Artemisia extract, licorice extract, carrot extract, tea extract, ougon extract and rosemary extract are considered to inhibit cell abnormal growth in a concentration-dependent manner.

  In addition to the above-mentioned drugs, for example, excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants, sweeteners other than the above-mentioned drugs are included in cell growth inhibitors. Whitening agent, moisturizer, oily component, UV absorber, surfactant, thickener, alcohols, powder component, colorant, ordinarily used in cosmetics, acidulants, seasonings, colorants, fragrances, etc. An aqueous component, water, various skin nutrients, etc. can be suitably mix | blended as needed.

  Further, when the cell growth inhibitor of the present invention is used as an external preparation for skin, auxiliary agents commonly used for external preparations for skin, such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, Metal sequestrants such as gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof Drugs such as vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, etc., sugars such as glucose, fructose, mannose, sucrose, trehalose, retinoic acid, retinol, retinol acetate, palmitic acid Bitin such as retinol Min A such like can also be appropriately blended.

  Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this.

Example 1. Relative expression of NPTN in melanoma and keratinocytes Quantitative PCR was performed using the following primer set.
NPTN-panF: gtaagaatgccagcaacatggagt (SEQ ID NO: 1)
NPTN-panR: gccaactgacttgcaatacatagtgg (SEQ ID NO: 2)
NPTN-betaR: aggatgataatttcagccagaattccc (SEQ ID NO: 3)
NPTN-panF and NPTN-panR were used for quantification of NPTNα, and NPTN-panF and NPTN-betaR were used for quantification of NPTNβ. The obtained values were corrected and compared based on the expression level of G3PDH (G3-F: GGTGAAGGTCGGAGTCAACGGATTTGGTCG (SEQ ID NO: 4), G3-R: TATTGGAACATGTAAACCATGTAGTTGAGG (SEQ ID NO: 5)).
The cDNAs used were two melanoma cell lines (SK-MEL-2, SK-MEL-5), and keratinocytes with different differentiation stages (KC80%: growth phase, KC100%: confluent, KC-Ca: after confluence, 1.2 mM calcium was added, and further cultured for 2 days. KC-Air: after confluence, air exposure for 15 minutes, and then further cultured for 2 days). As shown in FIG. 2, NPTNβ was mainly expressed in both melanoma and keratinocytes.

Example 2. Suppression of keratinocyte growth by NPTN siRNA To suppress NPTN expression, RNAiMax was used to culture NPTN siRNA (Santaq Cruz Biotechnology, Inc, Neuroplastin siRNA (h): sc-90193) in the growth phase to a final concentration of 20 nM. Keratinocytes were transfected. As a control, control siRNA (Santa Cruz Biotechnology, Inc., sc-3707) having no homology with any part of the human gene was used. Transfection was performed after the culture medium was replaced with a basal medium containing no growth factor. Proliferated keratinocytes were stimulated with S100A8, S100A9, S100A8 + A9 (10 μg / ml) 24 hours after transfection, and RNA was collected after another 24 hours. As a result, as shown in FIG. 3, when NPTN siRNA was transfected, keratinocyte proliferation was significantly suppressed as compared with the case of using the control.

Example 3 Binding of S100 protein and receptor Insert each S100 protein with Myc tag (S100A4, S100A7, S100A8, S100A9, S100A11, S100B) cDNA, RAGE cDNA with HA tag, NPTN cDNA and Emprin cDNA into a vector with CMV promoter Thus, an expression construct was prepared. HEK293 cells were transfected as described above and 48 hours later, cell extracts were prepared. These samples were subjected to an immunoprecipitation reaction using Myc antibody. Western blotting was performed by a conventional method, and detection was performed using Myc antibody and HA antibody. The results are shown in FIGS. 4A and 4B.

Example 4 Localization of S100A8 and NPTN in the epidermis 1) Immunostaining The melanoma patient tissue was fixed with 4% paraformaldehyde and then embedded in paraffin to prepare 4 μm sections. After deparaffinization with xylene, the primary antibody (anti-S100A8 antibody: SC8112, Santaq Cruz Biotechnology) was blocked using immunoblock (DS Pharma Biomedical Co., Ltd., Cat. No. KN001A) to block nonspecific antibody binding. , Inc, and Anti-Neuroplastin / SDFR1 (N-term), ACRIS Antibodies [Manufacturer Model] AP31309PU-N) for 1 hour at 37 ° C., washed, and secondary antibody (Molecular Probe, Alexa Fluor 594 anti -rabbit, A-21207, Alexa Fluor 488, A-21467) and observed with a fluorescence microscope (Olympus, BX51).
Similarly, biopsy was also performed from a lesion site of atopic dermatitis, staining was performed in the same manner as described above, and the localization of S100A8 and NPTN was observed.
As a result, S100A8 and NPTN showed the same localization on the melanoma tissue and the atopic dermatitis lesion tissue (FIGS. 5A and 5B).

2) Demonstration of interaction between S100A8 and NPTN in atopic skin It was confirmed by PLA (Proximity Ligation Assay) method that S100A8 and NPTN were not simply bound but actually interacted with each other. According to the PLA method, two types of antibodies labeled with DNA probes are used to hybridize complementary DNAs labeled with fluorescent dyes, thereby clarifying whether these proteins interact. Can do. The PLA method is much more sensitive than normal immunostaining.
Interaction tests were performed using the Duolink in situ PLA kit from Olink. The affected skin tissue obtained from an atopy patient was fixed with 4% paraformaldehyde and then embedded in paraffin by a usual method. After chopping 4 μm, washing with PBS after xylene treatment, ethanol treatment, blocking, and primary antibody (anti-S100A8 antibody: Calgranulin A [C-19, sc-8112] [Santa Cruz Biotechnology], anti-neuroplus Tin antibody: AP31309PU-N [Acris Antibodies, Inc.]) and reacted overnight at 4 ° C. After washing with PBS, it was reacted with PLA probe (anti-goat PLUS, anti-rabbit MINUS [OLINK Bioscience]) at 37 ° C. for 2 hours.
After washing, hybridization with the DNA probe was performed, washing with TBS-T, ligase was added and incubated at 37 ° C. for 15 minutes to fuse the probe. Polymerase was added and incubated at 37 ° C. for 90 minutes to amplify the ligated DNA probe. Detection kit 613 (Olink) was used to label the fluorescent dye and microscopic observation was performed. As a result, it was confirmed that S100A8 and NPTN have a strong interaction in the upper layer of the atopic dermatitis affected skin and in the vicinity of the granule layer (FIG. 6).

Example 5. EPTNβ-S100A8 ELISA Screening phCMV-FSRTM vector (Genlantis, San Diego, Calif.) With a His-tagged, soluble neuroplastin (sol-NPTN, 1-338AA) lacking the transmembrane domain and intracellular domain Was inserted and introduced into HEK293 cells. After 48 hours, a cell extract was prepared, and sol-NPTN was purified with a nickel column. sol-NPTN (2 μg / ml in 100 ml of PBS) was dispensed into each well of a 96-well plate and allowed to stand at 4 ° C. overnight to immobilize sol-NPTN. After blocking with 5% BSA, 250 ng / ml of 100A8 and a display substance were added (total 100 μl), incubated at 37 ° C. for 1 hour, washed 4 times with PBS-Tween, and then washed with rabbit anti-S100A8 antibody at 37 ° C., 1 ° Reacted for hours. After washing, after reacting with HRP-conjugated anti-Rabbit Fab (GE Healthcare) at 37 ° C. for 1 hour, color was developed using TMB Peroxydase EIA substrate kit (Bio-Rad) and measured at 630 nm. For binding inhibition, the absorbance when only S100A8 was added was taken as 100%, and cases below this were determined to have inhibitory activity. As a result, strong inhibitory activity was shown for mugwort extract, licorice extract, carrot extract, tea extract, ougon extract and rosemary extract (FIGS. 7A and 7B).

Claims (6)

  A method for screening a cell growth inhibitor, comprising incubating neuroplastin (NPTN) and S100A8 in the presence of a candidate substance for a cell growth inhibitor, and inhibiting the growth of a substance that inhibits the binding between NPTN and S100A8 Selecting a method as an agent.   The method of claim 1, wherein S100A8 is complexed with S100A9.   The method according to claim 1, wherein the NPTN is NPTNβ.   A cytostatic agent comprising an agent that inhibits the binding between neuroplastin β (NPTNβ) and S100A8 or 100A8 / A9.   The cell growth inhibitor according to claim 4, wherein the drug comprises one or more plants selected from the group consisting of mugwort, licorice, carrot, tea, hornon and rosemary.   A pharmaceutical composition for preventing and / or treating a disease selected from atopic dermatitis, cancer and psoriasis, comprising the cytostatic agent according to claim 4 or 5.