WO2006121219A1 - Agent for controlling epithelial-mesenchymal transition - Google Patents

Abstract

It is intended to provide an agent for controlling epithelial-mesenchymal transition, an agent for controlling Snail activity, an agent useful for the prevention of cancer metastasis, wound treatment, the induction of organogenesis or the like and a method for screening a compound having an activity of controlling epithelial-mesenchymal transition and/or controlling Snail activity. More specifically, it is intended to provide an agent for controlling epithelial-mesenchymal transition, an agent for controlling Snail activity, an agent for preventing cancer metastasis, an agent for treating wound and the agent for inducing organogenesis containing, as an active ingredient, a substance capable of controlling the intracellular zinc level, more specifically a substance regulating a zinc ion, a zinc chelator, the expression and/or function of a zinc transporter or the like and a method for screening a compound having an activity of controlling epithelial-mesenchymal transition and/or controlling Snail activity comprising measuring intracellular zinc level.

Description

 Specification

 Epithelial and mesenchymal transition regulators

 The present invention relates to an agent capable of controlling Snai 1 activity and / or epithelial-mesenchymal transition. In particular, the present invention relates to a drug capable of preventing cancer metastasis, wound healing and promoting organ formation by controlling epithelial-mesenchymal transition in cancer metastasis, wound healing, and organ regeneration. Background art

A phenomenon in which cells that normally do not move in close contact with each other, but start moving only in the case of cancer metastasis or wound healing-ontogeny, etc .: Epithelial mesenchymal transition (E pithe 1 ial— me sench yma ltransition: EMT On the other hand, the zinc (Zn) finger type transcription factor Snai1 regulates the expression of epithelial genes such as intercellular adhesion molecules and induces the expression of mesenchymal genes. In fact, many cancer cells with high invasion ability have been reported to have increased expression of Snai 1 and correlate with a decrease in the expression of intercellular adhesion molecules such as cadherin (Poser I, D orainguez D, de Herreros AG, Varna i A, Buettner R, Bosserhoff AK. Loss of E-cadherin expression in melanoma cells involves up-regulation of the transcriptional repressor Snail. J Biol Chem. 276 (27): 24661-6 ( 2001); Bla nco MJ, Moreno-Bueno G, Sarrio D, Locascio A, Cano A, Palacios J, Nieto MA. Correlation of Snail expression with histological grade and lymph no de status in breast carcinomas. Oncogene. 21 (20): 3241-6 (2002); Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R, Hofler H, Becker KF. Differential expression of the epithelial-mesenchymal transition regula tors snail, SIP1, and twist in gastric cancer Am J Pathol. 161 (5): 1881-91 (2002); Sugimachi K, Tanaka S, Karaeyama T, Taguchi K, Aishima S, Shim ada M, Sugim acni K, Tsuneyoshi M. Transcriptional repressor snail and pr ogression of human hepatocellular carcinoma. See in Cancer Res. 9 (7): 2657-64 (2003)). Although it is an established fact that Snai 1 is a master regulator in EMT today, the expression of force doherin persists in some cells despite the expression of Snai 1. The regulation mechanism for the localization and activity of Snai 1 has not been elucidated. For example, the transcription factor S nai 1 that should function in the nucleus is localized in the cytoplasm. The present inventors previously described zinc transporter L

IV 1 is a master regulator of EMT. Z n finger transcription factor S nai 1 induces localization in the nucleus and activates it to give cells mobility by suppressing the expression of intercellular adhesion molecules. (Yamashita, .S., Iyagi, C., Fukada, T., Kagara, Ν., Che, Υ.ichi S. & Hirano, T. Zinc transporter LIVI co ntrols epithelial-mesenchymal transition in zebrafish gastrula organizer.

Nature 429 (6989): 298-302 (2004)). This is the first report to elucidate the regulation of zinc auxotrophic factors by zinc transporters, but the molecular mechanism, especially the factors that can regulate EMT by regulating the intracellular localization and activity of Snai 1, are still unexplained. It has not been revealed. Disclosure of the invention

 An object of the present invention is to elucidate the EMT control mechanism in cancer, wound healing, and organ regeneration. Specifically, the present invention aims to provide an EMT regulator and an activity regulator of Snai 1 which is an EMT master regulator. To do. Another object of the present invention is to apply the EMT regulator and the Sna i 1 activity regulator to cancer metastasis prevention, wound healing and organogenesis promotion.

As a result of intensive studies in view of the above problems, the present inventors have been able to regulate the intracellular localization and activity of Snai 1 which is a master regulator of EMT by controlling the amount of intracellular zinc. We found that EMT can be controlled in cancer metastasis, wound healing, and organ formation by controlling the amount of zinc in the body, and the present invention was completed. That is, the present invention is as follows. [1] An epithelial-mesenchymal transition control agent comprising a substance capable of controlling the amount of intracellular zinc as an active ingredient;

 [2] The agent according to [1] above, wherein the substance capable of controlling the amount of intracellular zinc is zinc ion; [3] the agent according to [2] above, wherein the zinc ion is introduced into the cell by a zinc ionophore;

 [4] The agent according to [1] above, wherein the substance capable of controlling the amount of intracellular zinc is a zinc chelator;

 [5] The zinc chelator is 2,3-dimercapto 1-propanesulfonic acid (DM

PS), N, N, N, N, monotetrakis (2-pyridylmethyl) ethylenediamine (TPEN), ethylenediamine (EDTA) and N— (6-methoxy-8-quinolyl) monop-toluenesulfonamide (4) the agent described in [4] above, which is at least one selected from the group consisting of (TSQ);

 [6] The agent according to [1] above, wherein the substance capable of controlling the amount of zinc in the cell is a substance that controls the expression and Z or function of a zinc transporter;

 [7] Human ZIP, zinc transporter containing L I V family, human CD

[6] the agent described in [6], which is at least one selected from the group consisting of F; [8] the agent described in [6] above, wherein the zinc transporter is L I V I;

 [9] The agent according to [6] above, wherein the zinc transporter is ZIP 10; [10] an epithelium containing a substance capable of controlling the binding of the third Zn finger domain in the Snai1 molecule to zinc. One-mesenchymal transition control agent;

 [1 1] The agent according to the above [1] to [10], which is a S nai 1 activity control agent; [12] at least 1 selected from the group consisting of a cancer metastasis preventive agent, a wound healing agent, and an organogenesis promoter. The agent according to any one of [1] to [10] above, which is a seed medicine;

 [1 3] The agent according to [1 1] above, which is at least one drug selected from the group consisting of a cancer metastasis preventive agent, a wound healing agent and an organogenesis-promoting agent;

[14] Contains as an active ingredient a substance that can reduce the amount of intracellular zinc or a substance that can suppress the binding of zinc to the third Z_n finger domain in one Snai molecule Cancer metastasis preventive drugs;

 [15] The cancer metastasis preventive agent according to the above [14], wherein the substance capable of reducing the amount of intracellular zinc is a zinc chelator;

 [16] The zinc chelator is 2,3-dimercapto-1-propanesulfonic acid (DMP S), N, N, N, N, -tetrakis (2-pyridylmethyl) ethylene diamine (TPEN), ethylene diamine (EDTA) and N- (6-methoxy-1-8-quinolyl) 1-p-toluenesulfonamide (TSQ), which is at least one selected from the group consisting of the above-mentioned [15];

 [17] The cancer metastasis preventive drug according to [14] above, wherein the substance capable of reducing the amount of intracellular zinc is a substance that controls the expression and / or function of a zinc transporter;

 [18] The cancer metastasis preventive agent according to [17] above, wherein the zinc transporter is at least one selected from the group consisting of human Z IPs and human CDFs containing LI V family;

 [19] The cancer metastasis preventive agent according to [17] above, wherein the zinc transporter is LI VI;

 [20] The cancer metastasis preventive agent according to [17] above, wherein the zinc transporter is Z IP10;

 [21] A wound healing agent and / or a substance that can increase the amount of intracellular zinc or a substance that can promote the binding of zinc to the third Zn finger domain in Snai 1 molecule, and / or Or organogenesis-promoting agent;

 [22] The wound healing agent and / or organogenesis-promoting agent according to [21] above, wherein the substance capable of increasing the amount of intracellular zinc is zinc ion;

 [23] The wound healing agent and Z or organogenesis promoting agent according to [2 2] above, wherein zinc ions are introduced into cells by a zinc ionophore;

[24] The wound healing agent and / or organ formation promoting agent according to [21] above, wherein the substance capable of increasing the amount of intracellular zinc is a substance that controls the expression and / or function of a zinc transporter; [25] The wound healing agent and / or organ formation promoter according to [24] above, wherein the zinc transporter is at least one selected from the group consisting of human ZIPs containing LIV family and human CDFs. medicine;

 [26] The wound healing agent and / or organogenesis-promoting agent according to [24], wherein the zinc transporter is L I V I;

 [27] The wound healing agent and / or organogenesis-promoting agent according to [24] above, wherein the zinc transporter is Z I P 10;

 [28] A method for controlling epithelial-mesenchymal transition comprising controlling intracellular zinc content in vitro;

 [29] The method described in [28] above, wherein the amount of intracellular zinc is controlled by zinc ions;

 [30] The method according to [29] above, wherein the zinc ion is introduced into the cell by a zinc ionophore;

 [31] The method described in [28] above, wherein the amount of intracellular zinc is controlled by a zinc chelator;

 [32] Zinc chelator is 2,3-dimercapto-1-monopropane sulfonic acid (DMP S), N, N, N, N, monotetrakis (2-pyridylmethyl) ethylene diamine (TPEN), ethylene diamine (EDTA) And N- (6-methoxy-1-8-quinolyl) 1-p-toluenesulfonamide (TSQ), and at least one selected from the group described in [3 1] above;

 [33] The method according to [28] above, wherein the intracellular zinc content is controlled by controlling the expression and / or function of a zinc transporter;

[34] The method according to [33] above, wherein the zinc transporter is LIV 1; [35] the method according to [33] above, wherein the zinc transporter is ZIP 10; [36] A method of controlling epithelial-to-leaf transition comprising controlling the binding of the third Zn finger domain to zinc;. [3 7] comprising controlling in vitro intracellular zinc content, S nai 1 activity How to control;

 [38] The method described in [37] above, wherein the amount of intracellular zinc is controlled by zinc ions;

 [39] The method according to [3 8] above, wherein the zinc ion is introduced into the cell by a zinc ionophore;

 [40] The method described in [37] above, wherein the amount of intracellular zinc is controlled by a zinc chelator;

 [41] Zinc chelator is 2,3-dimercapto-1, 1propane sulfonic acid (D MP S), N, N, N, N, monotetrakis (2-pyridylmethyl) ethylene diamine (TPEN), ethylene diamine (EDTA) And at least one selected from the group consisting of N- (6-methoxy-1-8-quinolyl) -p-toluenesulfonamide (TSQ);

 [42] The method according to [37] above, wherein the intracellular zinc content is controlled by controlling the expression of the zinc transporter and the function of X;

 [43] The method according to [42] above, wherein the zinc transporter is L I V 1;

[44] The method according to [42] above, wherein the zinc transporter is ZIP 10; [45] comprising controlling the binding of zinc to the third Zn finger domain in the Snai1 molecule, A method for controlling Snail activity;

 [46] A screening method for a compound having an epithelial-mesenchymal transition-controlling action and a compound having a Snai-1 activity-controlling action, which comprises measuring intracellular zinc levels;

 [47] A screening method for a compound having an epithelial-mesenchymal transition-controlling action and / or a compound having a Snai-1 activity-controlling action, comprising the following steps:

 (1) contacting the third Zn finger domain present in the Snai1 molecule with zinc in the presence or absence of the test compound;

 (2) measuring the degree of binding between the zinc finger domain and zinc; and

(3) Substances that significantly affect the binding between zinc finger domains and zinc Qualifying as a compound having a skin-mesenchymal transition control action and / or a compound having a Snai 1 activity control action;

 [48] a method for controlling epithelial-mesenchymal transition in the subject, comprising administering to the subject an effective amount of a substance capable of controlling the amount of intracellular zinc;

 [4 9] An epidermis mesenchyme in the subject, comprising administering to the subject an effective amount of a substance capable of controlling the binding of zinc to the third Zn finger domain in the Snai 1 molecule. Conversion control method;

 [50] a method of controlling Snai l activity in a subject, comprising administering to the subject an effective amount of a substance capable of controlling the amount of intracellular zinc;

 [5 1] Including administration of an effective amount of a substance capable of reducing the amount of intracellular zinc or a substance capable of suppressing the binding of zinc to the third Zn finger domain in the Snai 1 molecule to the subject. A method for preventing cancer metastasis in the subject;

 [52] This includes administering to a subject an effective amount of a substance capable of increasing the amount of intracellular zinc or a substance capable of promoting the binding of zinc to the third Zn finger domain in Snai 1 molecule. A method of promoting wound healing and / or organ formation in the subject;

[5 3] Use of a substance capable of controlling the amount of intracellular zinc in the production of an epithelial-mesenchymal transition regulator;

 [5 4] Use of a substance capable of controlling the binding of zinc to the third Zn finger domain in the Snai1 molecule in the production of an epithelial-mesenchymal transition regulator;

 [5 5] Use of a substance capable of controlling the amount of intracellular zinc in the manufacture of Snai 1 activity regulator;

 [5 6] Use of a substance that can reduce the amount of intracellular zinc or a substance that can suppress the binding of the third Zn finger domain in the Snai1 molecule to zinc in the production of a cancer metastasis preventive agent;

[5 7] A substance capable of increasing the amount of intracellular zinc in the manufacture of wound healing drugs and organogenesis promoters, or the binding of the third Zn finger domain in Snai 1 molecule to lead. Use of substances that can be accelerated; [58] A drug containing a substance that can control the amount of intracellular zinc as an active ingredient, and a statement describing that the drug can or should be used to control epithelial-mesenchymal transition Including commercial packages;

 [59] A drug containing, as an active ingredient, a substance capable of controlling the binding between the third Zn finger domain in one molecule of Snai and zinc, and the drug is used for controlling epithelial-mesenchymal transition. A commercial package containing a statement stating that it should be obtained or used;

 [60] A drug containing a substance capable of controlling the amount of zinc in the cell as an active ingredient, and a statement describing that the drug can or should be used to control Snai 1 activity. Including commercial package;

 [6 1] A drug containing a substance capable of reducing the amount of intracellular zinc or a substance capable of inhibiting the binding of the third Zn finger domain in the Snai 1 molecule and zinc as an active ingredient, and the drug A commercial package containing a statement stating that can be or should be used for the prevention of cancer metastasis;

 [62] A drug containing as an active ingredient a substance capable of increasing the amount of intracellular zinc or a substance capable of promoting the binding of zinc to the third Zn finger domain in Snai 1 molecule, and the drug A commercial package containing a statement that states that can or should be used for wound healing and organogenesis promotion. Brief Description of Drawings

 FIG. 1 is a graph showing changes in intracellular localization of Snai 1 by regulating intracellular zinc levels in human eclampsia cancer cells HeLa.

 FIG. 2 is a diagram showing the relationship between the zinc binding ability of the Sn ai 1 intramolecular Zn finger domain and changes in subcellular localization.

Figure 3 shows EGF-induced nuclear accumulation of SNAIL 1 and zinc during EMT in human keratinocytes H a C a T. (A) S NA IL 1, E-cadherin, and vimentin in Ha C a T cells during EGF-induced E MT Expression. GAPDH was used as a loading control. (B) EGF-induced Ha Ca T cell morphology, SNA IL subcellular localization, and nuclear staining during EMT.

(c) Phylogenetic relevance and expression pattern of ZI moths and moths in Ha C a T cells in the presence or absence of EGF. (D) — (f) ZGF-specific uptake and its nuclear accumulation in EGF-stimulated Ha C a T cells. Zinc uptake (d) and its substrate specificity (e) were assayed using ⁶⁵ Zn. The intracellular localization of zinc (f) was visualized using ZnAF-2 DM. Each point is the average of data from a representative experiment; each experiment was performed in triplicate, 3 experiments were performed for each condition, and the error bar indicates soil 1 SD.

FIG. 4 shows the dependence of SNA IL 1 nuclear accumulation via ZIP 10 on zinc during EGF-induced wound healing by Ha C a T cells. (A) ― (b) Control siRNA introduction (Control), ZIP 6 knockdown (ZIP 6), ZIP 10 knockdown (ZIP 1 0), and ZIP 6; ZIP 10 double knockdown (ZIP 6 ; ZIP 10) Expression of Ζ I Ρ 6 and Ζ IPI 0 (a) and EGF-induced ⁶⁵ Z n uptake activity (b) of Ha C a T cells. Each point is the average of representative experimental data. Each experiment was performed in triplicate, 3 experiments were performed for each condition, and the error bar indicates ± 1 SD. (C) Kontoronore _s i RN A introduced (C ontrol), ZIP 6 knockdown (ZIP 6), ZIP 10 knockdown (ZIP 10), ZIP 6; ZIP 10 double knockdown (ZIP 6; ZIP 1 0) and TP EN treatment (TP EN) Haca C T cell motility and migratory behavior were angularly analyzed with an in vitro wound healing assay (WHA). SNA IL 1 was labeled by immunostaining and zinc was labeled with the fluorescent marker Zn AF-2 DM. The arrow indicates the tip of the migratory cell. Each experiment was performed at least three times and shows one experiment with typical results.

FIG. 5 shows that zinc coordination in the Zn finger domain of mouse S nai 11 is essential for its nuclear accumulation. (A) — (c) LGF present (b). In the absence (a) EGF-stimulated GFP-fused mouse S in H a C a T cells Intracellular localization of various mutants of nail 1 (c). The C 2H2 motif (C 2H2) was mutated into the G 2 G 2 motif (G 2G2) to eliminate the zinc coordination activity in each finger domain. WT, wild type. (D)-(e) With or without LMB (e) or without (d) EG F-stimulated The percentage of cells that markedly show nuclear GFP of various mutants of mouse Snai 1 1 in H a C a T cells It was measured. Each point is the average of representative experimental data; each experiment was performed in triplicate, 3 experiments were performed for each condition, and the error bar indicates 1 SD.

 FIG. 6 shows the zinc-dependent regulation of EMT in cancer progression. (A) Endogenous expression of ZIP 10 and E—forced doherin was observed in various invasive / metastatic cancer cell lines (He La, MDA—MB—231, and MD A—MB—435 S), and non- It was inversely correlated in metastatic cell lines (MCF 7, T47D, ZR 75-1 and ZR 75-30). The expression levels of Z IP10, ZIP6, E-force doherin, and vimentin were analyzed by RT-PCR. GAPDH was used as a loading control. (B) Control si RNA introduction (Control), ZIP 6 knockdown (ZIP 6), ZIP 10 knockdown (ZIP 10), and ZIP 6; ZIP 10 double knockdown (ZIP 6; ZIP 1 0) He La Invasive properties of HeLa cells analyzed by transwell migration assay of cells. Each point is the average of representative experimental data; each experiment was performed in triplicate, 3 experiments were performed for each condition, and error bar indicates soil 1 S. D. BEST MODE FOR CARRYING OUT THE INVENTION

Epithelial-to-mesenchymal transition (EMT) is an invasive cell that acts as a mesenchymal cell during the process of embryogenesis, organ and tissue regeneration, and cancer metastasis. (Thiery JP. Epith elial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2 (6): 442-54. (2002)) ₀ On the other hand, the regulation of EMT is to suppress or promote the above-described changes in cell phenotype. That is, in the present invention, “control” By both positive and negative adjustment is meant. In the present invention, the control of Snai 1 activity means controlling the function of EMT as a master regulator. Specifically, Snai protein is transferred to the nucleus or cytoplasm (intracellular site of Snai 1). Control), intercellular adhesion control and associated EMT control. In the present invention, “control of intracellular zinc *” means in addition to the literal meaning of increasing or decreasing the amount of intracellular zinc, particularly zinc in the zinc finger domain present in the cell. In other words, it also means an effect that can have an effect similar to the effect on EMT and Z or Snai 1 activity given by increasing or decreasing the amount of zinc in the cell. It is not restricted by the amount of intracellular zinc. In the present invention, the cell to be controlled for the amount of cytoplasmic zinc is not particularly limited as long as it expresses Snai 1 family and undergoes epithelial-mesenchymal transition, and specifically, kidney cells (for example, MD CK, etc.), epithelial cells such as keratinocytes, breast cancer cells and the like. The expression of one molecule of Snai may be naturally expressed or forcedly expressed using genetic engineering techniques. In the present specification, a cell to be controlled is sometimes referred to as “target cell” for convenience.

Examples of “substances that can control the amount of intracellular zinc” include “zinc ions”, “zinc chelators”, “zinc ionophores”, “substances that regulate the expression and / or function of zinc transporters”, etc. “Ion” and “Zinc ionophore” can directly increase the amount of zinc in the target cell by adding it, while “Zinc killer” increases the amount of zinc in the target cell by adding it. In particular, it is possible to directly reduce the amount of zinc in the Zn fin guard main body. “A substance that regulates the expression and / or function of a zinc transporter” refers to a cell that regulates the expression and / or function of a zinc transporter in controlling EMT or Snai 1 activity of a target cell. A phenomenon similar to that caused by an increase or decrease in the amount of zinc can be induced. That is, the action of zinc can be indirectly controlled. In “Substances that regulate the expression and / or function of zinc transporters”, any substance that regulates the expression or function in a direction that promotes its expression or function can be analyzed. A substance that increases the responsiveness of the sporter to zinc and regulates the expression or function in a direction that suppresses the expression can further suppress the responsiveness. In addition, the “substance that regulates the expression and Z or function of zinc-requiring protein” also affects the balance of the amount of intracellular zinc, and is therefore used as the “substance that can control the amount of zinc in the cell” of the present invention. be able to.

 “Zinc ions” are introduced into target cells by zinc ionophores. In other words, zinc ions are introduced into target cells in the form of a complex with a zinc ionophore. Zinc ionophores include various compounds that are commonly used in the art, and preferably are commercially available. Examples include zinc pyrithione, heterocyclic amine, dithiocarbamate, and vitamins.

 A “zinc chelator” is a substance capable of removing zinc from the cell by forming a complex with zinc in the target cell. For example, 2,3-dimercapto 1-propanesulfonic acid ( DM PS), N, N, N, N, -tetrakis (2 monopyridylmethyl) ethylenediamine (TPEN), ethylenediamine (EDTA), N— (6-methoxy-1-8-quinolyl) 1 ρ-toluenesulfonamide ( TSQ) and the like. Both are commercially available. In addition, it is included as an active ingredient in the EMT, Snail activity regulator of the present invention, and the cancer metastasis preventive agent, wound healing agent and organ formation promoter (hereinafter also simply referred to as the medicament of the present invention). The amount of zinc chelator as a substance capable of controlling the amount of intracellular zinc is appropriately determined within the range in which the amount of zinc in the cell can be controlled. Further, the zinc chelator to be included may be one type or two or more types. When two or more types are included, the blending amount is appropriately set according to the type.

“A substance that regulates the expression and / or function of a zinc transporter” means that the mechanism of action is not particularly limited as long as the expression and / or function of the zinc transporter can be regulated as a result. Regulation at the gene level Even the regulation at the protein level may be used. Examples of regulation at the gene level include transcription regulation and gene expression regulation. Protein level regulation includes metabolic regulation, phosphorylation, Examples include dephosphorylation, sugar addition, lipid addition, coordinate bond with zinc, degradation, ubiquitination, acetylation, and the like. “Zinc transporters” include, for example, human ZIPs containing LIV family (BAB 70848, h Z ip 4, BI GM 103, KI AAO 06 2, KIAA 1 265, h L iv— 1, AAHO 88 53, h K E4, XP_208649, h ZIP l, h ZIP 2, h ZIP 3, BAA 9 2 100, B AC 04504, h ZIPIO, etc., human CDFs (hZ nt—5, h Z nt—7 H Z nt— 1, h Z nt— 6, h Z nt— 3, h Z nt— 2, h Z nt— 8, h Z nt— 4, h Z nt— 3, h Z nt— 9, etc.) Etc. LIV 1 was originally identified as an estrogen-regulated breast cancer protein, and more recently the ZIP zinc transporter subfamily (Z rt-, I) called LZT (LI V-1 subfamily of ZIP zinc transporters). rt-like protein) (Taylor, KM & Nicholson, RI The LZT proteins; the LIV-1 subfamily of zinc transporters. Biochim. Biophys. Acta 1611 (1-2): 1 6-30 (2003)) It has been shown to function as a zinc transporter (Taylor, K. Μ., Morgan, HE, Johnson, A., Hadiey, LJ & Nicholson, RI Structure-function analysis of LIV-1, the breast cancer- associated prot ein that belongs to a new subfamily of zinc transporters. Biochera. J. 37 5 (pt 1): 51—9 (2003)). Other examples include CD F (cationdiffusion factor), ZIP 10 and the like.

For example, the zinc transporter LIV 1 is regulated by STAT 3 (Yamashita, S., Miyagi, C., Fukada, Τ., Kagara, N., Che, Y.—S. & Hirano, T Zinc transporter LIVI controls epithelial-mesenchymal trans ition in zebrafish gastrula organizer. Nature 429 (6989): 298-302 (2004)) ₀ For example, zinc transporter ZIP 10 is subject to expression regulation by EGF.

The “substance that modulates the Z or function of zinc transporter” may be a known compound or a new compound that will be developed in the future. In addition, low molecular compound It may be a product or a polymer compound. Here, the low molecular weight compound is a compound having a molecular weight of less than about 3000, and includes, for example, organic compounds that can be normally used as pharmaceuticals, derivatives thereof, and inorganic compounds. Derivatives, naturally-occurring compounds and derivatives thereof, small nucleic acid molecules such as promo 1 and various metals, and preferably organic compounds that can be used as pharmaceuticals, derivatives thereof, and nucleic acid molecules. The high molecular compound is a compound having a molecular weight of about 3000 or more, and includes proteins, polynucleic acids, polysaccharides, and combinations thereof, and is preferably a protein. These low-molecular compounds or high-molecular compounds are commercially available if they are known, or can be obtained through steps such as collection, production, and purification according to each report literature. These may be of natural origin, prepared by genetic engineering, or obtained by semisynthesis.

 The zinc transporter can control the amount of intracellular zinc by its zinc transport function. For example, a substance that regulates the expression or function of LIV 1 that controls the transport of zinc from outside the cell and induces the transfer of the Snai 1 molecule into the nucleus controls the amount of zinc in the cell. Specific examples of substances that can be obtained include the following.

 (1) DN A as described in any of a) to d) below

 a) DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 2, 4 or 6 (L I V1 protein)

b) DNA consisting of the sequence described in SEQ ID NO: 1, 3 or 5 (LIV 1 gene) c) In the amino acid sequence described in SEQ ID NO: 2, 4 or 6, one or more amino acid sequences are substituted, deleted, inserted and DNA that encodes a protein having an added amino acid sequence (protein similar to LIV 1)

d) DNA _r that hybridizes with the sequence of SEQ ID NO: 1, 3 or 5 under stringent conditions

(2) A vector in which the DNA according to any one of a) to d) below is inserted a) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6

b) DN A consisting of the sequence described in SEQ ID NO: 1, 3 or 5

c) DNA encoding a protein having an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4 or 6

d) DNA that hybridizes with the sequence described in SEQ ID NO: 1, 3 or 5 under stringent conditions

 (3) A protein encoded by the DNA according to any one of a) to d) below

a) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6

b) DN A consisting of the sequence described in SEQ ID NO: 1, 3 or 5

c) DNA encoding a protein having an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted and Z or added in the amino acid sequence of SEQ ID NO: 2, 4 or 6

d) DNA that hybridizes with the sequence described in SEQ ID NO: 1, 3 or 5 under stringent conditions

 (4) An antisense oligonucleotide having a DNA sequence consisting of the sequence of SEQ ID NO: 1, 3 or 5 as a target sequence

 (5) Double-stranded RNA having a sequence identical or similar to a part of DNA consisting of the sequence described in SEQ ID NO: 1, 3 or 5

 (6) An antibody against a protein having the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6

 (7) Substances (including natural and non-natural products) that associate with the protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6 and regulate its function.

(1) to (3) positively regulate the expression and function of zinc transporter (4) and (5) negatively regulate the expression and function or function of the zinc transporter (decrease expression, and Substance that suppresses the function or function).

 L I V 1 protein can be prepared by various methods well known to those skilled in the art. For example, it can be prepared by producing and purifying a protein in a transformant holding a vector inserted with a DNA (LIV1 gene) consisting of the nucleotide sequence set forth in SEQ ID NO: 1, 3 or 5. The vector to be used can be appropriately selected depending on the translation system used for protein production. LIV 1 is known to be expressed in hormonal tissues such as breast, prostate, pituitary gland, and brain (Taylor, KM & Nicholson, RI The LZT proteins; the LIV-1 suofamily of zinc transp orters. Biochim. Biophys. Acta 1611 (1-2): 16-30 (2003)). When an anti-LIVI protein antibody is prepared by a well-known method and a affinity column is prepared using the antibody, the LIVI protein can be purified from a cell extract expressing LIV1. To the extent that it can function as a zinc transporter, a protein similar to LIV 1 is also a substance capable of controlling the amount of intracellular zinc, and as an example, in the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6 Hybridizes under stringent conditions with a protein consisting of an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted and / or added, or a DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 Mention may be made of proteins encoded by DNA.

For the preparation of a protein similar to LIV 1, for example, by performing hybridization using the base sequence encoding LIV 1, a transformant is prepared from the obtained highly homologous DNA, and the transformation is performed. The body can produce a desired protein. As an example of a method for obtaining highly homologous DNA, a part of the base sequence described in SEQ ID NO: 1, 3 or 5 is used as a probe, and stringent hyperplasia is obtained from cells of vertebrate animals other than humans. Consider the method of high-prioritization under the condition of dialysis. _ The above stringent high pre-crystallization conditions are suitable for those skilled in the art. It can be selected as appropriate. One example is 25% formamide, and under more severe conditions 50% honremamide, 4 XS SC, 5 OmM Hepesp H 7.0, 10 X Denhardt solution, 20 // g / niL denatured salmon sperm DNA. After pre-hybridization at 42 ° C in dialysis solution, add labeled probe and incubate at 42 ° C to incubate. In the subsequent cleaning, the cleaning solution and temperature conditions are about “1 XSSC, 0.1% SDS, 37 ° C”, and the more severe conditions are about “0.5 XS SC, 0.1% SDS, 42 ° C”. More severe conditions can be implemented at “0.2 XS SC, 0.1% SDS, 65 ° C”. Thus, the isolation of DNA having high homology with the probe sequence can be expected as the conditions for washing high-precipitation become more severe. However, combinations of SSC, SDS and temperature conditions are examples, and those skilled in the art will recognize the above or other factors that determine the stringency of the hybridization (eg, probe concentration, probe length). In addition, a stringency similar to that described above can be realized by appropriately combining the hybridization reaction time and the like.

Polypeptides encoded by DNA isolated using such hybridization techniques usually have high homology in amino acid sequence with LIVI. High homology means at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and further preferably 97% or more (for example, 98-99%) sequence homology. The identity of the amino acid sequence is, for example, the algorithm by Karlinand Altschul B LAST (Proc. Natl. Ac ad. Sci. USA 8 7: 2264-2268, 1 990, Proc. Natl. Ac a d. S ci. USA 90: 5873-58 77, 1 9 93). Based on this algorithm, a program called B LASTX has been developed (Altschuleta 1. J. Mo 1. B iol. 21 5: 40. 3— 41 0, 1 990). When analyzing amino acid sequences by B LASTX The norm shall be ί arrangement score = 50 and wo rdlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known (http: Zz www.ncbi.n1m.nih.go v.)

 Preparation of a protein similar to L I V 1 can also be performed by other known means. For example, LIVI DNA can be artificially modified by site-directed mu tagenesis such as deletion mu tant production method using exonuclease or cassette mutation method, and desired protein can be prepared using the modified LIV 1 DNA. .

 Another preferred example of a substance that can be used as a substance that regulates the expression and function of a zinc transporter is DNA comprising the sequence described in SEQ ID NO: 1, 3, or 5. The DNA encodes the protein L I V 1.

 The above DNA should be prepared from cDNA of cells expressing LIV 1 by a hybridization technique well known to those skilled in the art using a part of the sequence shown in SEQ ID NO: 1, 3 or 5 as a probe. Can do. It can also be obtained by carrying out RT-PCR from mRNA using a part of the sequence described in SEQ ID NO: 1, 3 or 5 as a primer. Alternatively, it may be artificially synthesized using a commercially available DNA synthesizer.

Furthermore, DNA similar to the above DNA is also an example of “a substance that regulates the expression or function of a zinc transporter”. Examples of the similar DNA include DNA that hybridizes with the sequence described in SEQ ID NO: 1, 3 or 5 under stringent conditions. The method for preparing this DNA has already been described above. When each of the above-mentioned DNAs is used, it can be inserted into an appropriate vector. A product inserted into a vector is also one embodiment of the present invention. The vector to be used can select an appropriate vector according to the purpose. Specifically, vectors derived from mammals (for example, p cDNA3 (manufactured by Invitrogen) or EF-BO S (Nucleic Acids. Re s., 18 (17), p. 5322, 1 990) PEF, pCDM8, pCXN), vectors derived from insect cells (eg “B ac-to- BAC baculovirus expression syst em” (Invitrogen), B ac P AK8), plant-derived expression vectors (eg, ρ べ 1, pMH2), animal virus-derived vectors (eg, pH SV, pMV, p A dex L cw), retrovirus-derived vectors (for example, p ZIP neo), yeast-derived vectors (for example, “Pichia Expression Kit J (manufactured by Invitrogen), NV 1 1, SP—Q 01). And vectors derived from Bacillus subtilis (for example, pPL608, pKTH50), E. coli vectors (M13 vector, pUC vector, pBR322, pBluescript ^ p CR — S cript), etc. In the present invention, it is preferable to use a vector that can be expressed in mammalian cells, and it is also preferable to use an expression vector In order to introduce a vector into cells, for example, the calcium phosphate method '(Virology, VoI 52, p. 456, 1 973), D EAE dextran method, method using Cationic Liposome DOTAP (manufactured by Kuchishu Diagnostics), electroporation method (Nucleic Acids Res., Vol. 1 5, p. 1 3 1 1, 1 98 7), lipofection method (J. C 1 in. B ioch em. Nutr., Vo l. 7, p. 1 75, 1 989), virus introduction method (pMX, MS CV, etc .; S c Am., Ρ. 34, 1 994), particle gun etc.

Examples of “substances that regulate the expression and function or function of zinc transporter” include antisense oligonucleotides that target DNIV or mRNRN encoding LIV 1. An antisense oligonucleotide against DNA encoding LIV 1 prevents the expression of the endogenous LIV 1 gene and suppresses zinc transport from the outside of the cell into the cell. Examples of such an antisense oligonucleotide include an antisense oligonucleotide having a DNA having the sequence described in SEQ ID NO: 1, 3 or 5 or an mRNA generated from the DNA as a target sequence. As an example, the oligonucleotide described in SEQ ID NO: 7 or 8 can be used. In addition, from the sequence described in SEQ ID NO: 1, 3 or 5 above DNA that is included in the antisense oligonucleotide of the present invention and comprises the sequence described in SEQ ID NO: 1, 3, or 5 or It does not have to be completely complementary to the corresponding mRNA.

 The antisense oligonucleotide can be used by inserting it into an appropriate vector according to the purpose. For example, for the purpose of application to gene therapy, virus vectors such as retro-inenores vector, adeno-winores vector, vaccinia wino-less vector, non-viral vectors such as cationic ribosome, ligand DNA complex, etc. It can be selected as appropriate. It is also conceivable to administer a large amount of aqueous solution as naked plasmid DNA (naked DNA) without using a carrier.

Another “substance that regulates the expression and / or function of a zinc transporter” includes a double-stranded RNA having the same or similar sequence as a part of DNA encoding LIV 1. Double-stranded RNA having the same or similar sequence as the target gene sequence can cause RNA interference (RNA i) that prevents expression of the target gene. RNA i is a phenomenon in which when mRNA is introduced into a cell, the mRNA corresponding to the RNA sequence is specifically decomposed and is not expressed as a protein. It prevents the expression of LIVI gene and suppresses zinc transport from the outside of the cell into the cell. The region that forms a double strand may form a double strand in all regions, or a part of the region (for example, both ends or one end) may be a single strand or the like. . Therefore, the double-stranded RNA of the present invention may also contain a region that is not double-stranded. Oligo RNA used for RNA i is often 10 to 100 bp RNA, and usually 19 to 23 bp RNA. RNA i method is Na ture, Vo l. 39 1,. 806, 1 998, Pro c. Na tl. Ac a d. S ci. US A Vo l. 95,. 1 5502, 1 998, Na ture , Vo l .395, .p. 854, 1 998, Proc. Na tl. Ac a d. S ci. USA V o 1. 96,. 5049, 1 99 99, Cell, Vo l. 95, p. 1 01 7, 1 998, Proc. Na tl. Ac a d. S ci. USA Vo l. 96, p. 145 1, 1 999, Proc. Na tl. Ac a d. S ci. USA Vo l. 95, p. 1 3959, 1 9 98, Nature Cell Biol., Vo l. 2, p. 70 , 2000, etc.

 Even when “Zinc transporter” is other than LIV 1 (for example, ZIP 10 etc.), various methods are performed in the same manner as in LIV 1 based on the known amino acid sequence or base sequence of the zinc transporter. DNA, vectors, proteins, antisense oligonucleotides, double-stranded RNA, antibodies, etc. can be prepared. Information on various amino acid sequences and nucleotide sequences can be obtained from various databases that can be browsed by N C B I or the like. For example, information on the amino acid sequence and base sequence of ZIP 10 can be obtained from NCB I (accession number; NM—020342 (SEQ ID NO: 69), NP—065075 (SEQ ID NO: 70), NM — 1 7265 3, NP—766241, NM—2006 71, NP—9569 65).

The substance that regulates the expression and Z or function of zinc-requiring protein is not particularly limited as long as the expression and z or function of zinc-requiring protein can be regulated as a result. It may be a regulation at the protein level or a protein level. Examples of regulation at the gene level include transcription regulation and gene expression regulation. Examples of the regulation at the protein level include metabolic regulation, phosphorylation, dephosphorylation, sugar addition, lipid addition, coordinate bond with zinc, degradation, ubiquitination, acetylation, and the like. The term “zinc-requiring protein” as used in the present invention affects the amount of zinc in the target cell by regulating its expression and Z or function, resulting in EMT regulation and Snai 1 activity regulation. Examples of proteins that have an influence include a protein having a Zn finger, a protein having a RING finger, a protein having a LIM domain, and a protein having a PHD zinc finger. Z n fingers can only have a higher order structure when cysteine or histidine and amino acids are coordinated with zinc. A protein having a unique nucleic acid binding ability, and a protein with a Zn finger includes P KC a (Korichneva I, Hoyos B, Chua R, Levi E, Hammer 1 in g U. Zinc release from protein kinase C as the common event during activation by lipid second messenger or reactive oxygen.J. Biol. Chera.277 (4 6): 44327-31 (2002)), GEF— HI (Krendel M, Zenke FT, Bokoch GM. Nucle otide exchange factor GEF-HI mediates cross-talk between microtubules an d the actin cytoskeleton.Nature Cell Biology 4 (4): 294-301 (2002)), HD AC (Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A , Yao TP. The deacetylase HDAC6 regulates aggresome formation and ceil viability in res ponse to misfolded protein stress.Cell 115 (6): 727-738 (2003)), SQSTM 1 (Joung I, Strorainger JL, Shin J. Molecular cloning of a phosphotyrosine-independent ligand of the p561ck SH2 domain. Proc Natl Acad Sci USA, 93 (2): 5991-5995 (1996)). The RI NG finger is a variant of the zinc finger and is suggested to be involved in protein-protein interactions. A protein with the RI NG finger includes ubiquitin-protein ligase E 3 (ubiquitin-proteinligase E 3) active, ubiquitin-coupled protein E 2 (ubiquitin-conjugating enzymes E 2), TRAF 6 (signal molecule of TNF CK; Kobayashi N, Kadono Y, Naito A, Matsumoto K, Yamamoto T , Tan aka S, Inoue J. Segregation of TRAF6-mediated signaling pathways clarif i es its role in osteoclastogenesis.The EMBO J. 20 (6): 1271-1280 (2001)) has been reported. . The LIM domain consists of 60 amino acids in which the positions of 6 cysteines and 1 histidine are conserved. Although it has a structure similar to a Zn finger, its DNA binding ability is unknown. It also functions as a domain that mediates binding to PKC. P a X i 1 1 in (Brown MC, Perrotta JA, Turner CE .. Identification of LIM3 as the principal determinant of paxillin focal a dhesion localization and characterization of a novel motif on paxillin d irecting vinculin and focal adhesion kinase binding.J. Cell Biol. '135 (4): 1109-1123 (1996)), Z yxin (Sadler I, Crawford AW, Michelsen JW, Examples include Beckerle MC. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J. Cell Biol. 119 (6): 1573-1587 (1992)). The PHDZ n finger is a Zn finger-like domain that is a nuclear protein suggested to be involved in chromatin-mediated transcriptional regulation and is predicted to have DNA-binding ability. Proteins with PHDZ n fingers include Jade— 1 (Panchenko MV, Zhu ou MI, Cohen HT.von Hippel-Lindau partner Jade-1 is a transcriptional co-activator associated with histone acetyltransf erase activity. J. Biol. Chem. 279 (53): 56032-56041 (2004)).

 R af-1 is a protein key that leads extracellular signals from R as _ GTP to ERK _ cascade, and R af — 1 is one of the CDF families Z n T 1 1 Zinc It is activated by exporters, and inactivated by zinc, that is, Raf — 1 activity, which is responsible for R as-ERK pathway, is activated by Z I T from Z よ り ίΗko via Z n T-1 (Jirakulaporn T, Muslin AJ. Cation diffusion faci litator proteins modulate Raf-1 activity.J Biol Chem. 279 (26): 27807-15 (2004), Bruinsma JJ, Jirakulaporn T, Muslin AJ, Kornfeld K. Zinc ions an d cation diffusion facilitator proteins regulate Ras-mediated signaling. Dev Cell. 2 (5): 567-78 (2002)).

The “substance that regulates the expression and / or function of a zinc-requiring protein” may be a known compound or a novel compound that will be developed in the future. Further, it may be a low molecular compound or a high molecular compound. Here, the low molecular weight compound is a compound having a molecular weight of less than about 300, for example, an organic compound that can be normally used as a pharmaceutical, a derivative or an inorganic compound thereof, and is manufactured using an organic synthesis method or the like. Compounds and their derivatives, naturally derived compounds and their derivatives, promoters, etc. These are small nucleic acid molecules, various metals, etc., preferably organic compounds that can be used as pharmaceuticals, their derivatives, and nucleic acid molecules. In addition, the high molecular compound is a compound having a molecular weight of about 300 or more; and includes proteins, polynucleic acids, polysaccharides, and combinations thereof, and is preferably a protein. These low-molecular compounds or high-molecular compounds are commercially available as long as they are known, or can be obtained through steps such as collection, production, and purification according to each report literature. These may be of natural origin, prepared by genetic engineering, or obtained by semisynthesis.

 Examples of “substances that regulate the expression and Z or function of zinc-requiring proteins” include various DNAs obtained in the same manner as LIV 1 described above, based on the known amino acid sequence and base sequence of each protein, Examples include vectors, proteins, antisense oligonucleotides, double-stranded RNA, and antibodies.

 “Zinc ion”, “Zinc ionophore”, “Zinc chelator”, “Substance that regulates the expression and / or function of zinc transporter” and “Substance that regulates the expression and Z or function of zinc-requiring protein” All regulate the amount of zinc in humans, mammals such as horses, horses, dogs, mice, rats, etc., specifically in cells showing EMT and Z or Snai 1 activity. And similar effects on cells. Substances that can reduce intracellular sub-amounts suppress EMT through the suppression and activation of S nai 1 nuclei, and substances that increase the amount of intracellular zinc transfer to S nai 1 nuclei EMT can be enhanced through the promotion of activation. EMT is observed during cancer metastasis, wound healing, and organ formation. Therefore, the EMT regulator of the present invention that suppresses EMT is useful as a cancer metastasis preventive agent. In addition, the EMT control agent of the present invention that enhances EMT is useful as a wound healing agent and an agent for promoting organ formation.

The EMT regulator, the Snai 1 activity regulator, the cancer metastasis preventive agent, the wound healing agent, and the organogenesis promoter of the present invention include the above-described series of active ingredients (zinc ion, zinc ionophore, zinc chelator, zinc transporter). That regulate the expression and / or function of Substances that regulate the quality and expression of zinc-requiring protein and Z or function), and optionally include pharmaceutically acceptable excipients and additives. Pharmaceutically acceptable excipients and additives include carriers, binders, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. Can be mentioned. In addition, different types of “substances that control the amount of intracellular zinc” can be used in combination as active ingredients.

 Pharmaceutically acceptable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, ratatoses, pectin, dextrin, starch, gelatin, tragacanth, methinoresenololose, sodium canoleboxoxymethinoresenorero , Low melting wax, cocoa butter, etc. -Furthermore, tablets can be made into tablets with ordinary coatings as required, for example, sugar-coated tablets, enteric-coated tablets, film-coated tablets, double-layer tablets, and multilayer tablets. Powders are formulated with a pharmaceutically acceptable powder base. Examples of bases include tanolec, ratatoose, and starch. Drops can be formulated with an aqueous or non-aqueous base and one or more pharmaceutically acceptable diffusing agents, suspending agents, solubilizing agents, and the like. Capsules can be produced by filling a compound as an active ingredient together with a pharmaceutically acceptable carrier. The compounds can be mixed with pharmaceutically acceptable excipients or filled into force capsules without excipients. A cachet agent can be produced by a similar method. When the present invention is prepared as a suppository, it is usually used together with a base such as vegetable oil (castor oil, olive oil, peanut oil, etc.), mineral oil (petrol, white petrolatum, etc.), waxes, partially synthetic or fully synthetic glycerin fatty acid ester. It is formulated by the technique.

 Examples of injection solutions include solutions, suspensions, and emulsions. For example, an aqueous solution, a water-propylene glycol solution and the like can be mentioned. The solution can also be produced in the form of a solution of polyethylene glycol and polyethylene or propylene dallicol, which may contain water.

A solution suitable for oral administration can be produced by adding a compound as an ingredient to water and adding a colorant, a fragrance, a stabilizer, a sweetener, a solubilizer, a thickener and the like as necessary. A solution suitable for oral administration can also be produced by adding the compound together with a dispersant to water to make it viscous. Examples of the thickener include pharmaceutically acceptable natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or known suspending agents.

 Examples of the topical administration agent include the above-mentioned solutions, creams, aerosols, sprays, powders, lotions, ointments and the like. The above-mentioned topical preparation can be produced by mixing a compound as an active ingredient with a pharmaceutically acceptable diluent and carrier. Ointment opium cream is formulated, for example, by adding a thickener and Z or gelling agent to an aqueous or oily base. Examples of the base include water, liquid paraffin, and vegetable oil. Examples of the thickening agent include soft paraffin, stearic acid anoleminium, cetostearyl alcohol, propylene glycolol, polyethylene glycol, lanolin, hydrogenated lanolin, and honey bees. If necessary, antiseptics such as methyl hydroxybenzoate, oral pills of hydroxybenzoic acid, chlorocresol, benzalkonium chloride, and bacterial growth inhibitors can be added to the topical preparation. For lotions, add one or more pharmaceutically acceptable stabilizers, suspending agents, emulsifiers, diffusing agents, thickeners, coloring agents, fragrances, etc. to an aqueous or oily base. Can do.

 The thus obtained EMT regulator, Snai 1 activity regulator, cancer metastasis preventive agent, wound healing agent and organogenesis promoter of the present invention are administered orally or parenterally. When administered orally, it can be administered in a dosage form commonly used in the art. When administered parenterally, it can be administered in a dosage form such as a topical administration agent (transdermal agent, etc.), a rectal administration agent, an injection, or a nasal agent.

Examples of the oral preparation or rectal preparation include capsules, tablets, pills, powders, drops, cachets, suppositories, and liquids. Examples of injections include sterile solutions or suspensions. Examples of topical agents include creams, ointments, lotions, transdermal agents (ordinary patches / matrixes, matrix agents) and the like. Dosage, number of doses is the type of substance that can control the amount of intracellular zinc used, It can be set appropriately depending on the shape, age, weight, dosage form, and the like.

 Examples of the cancer that can be the subject of the “cancer metastasis preventive agent” of the present invention include cancers such as breast cancer, cervical cancer, and gastric cancer. Particularly useful are metastatic cancers such as breast cancer. Examples of the wound that can be the subject of the “wound healing agent” of the present invention include wounds such as burns and gastrointestinal ulcers, and ulcer healing is particularly useful. EMT Master regulator S nai 1 is translocated to the nucleus, and induces activation. LIV 1 expression is regulated by STAT 3, which is essential for lumen formation in the kidney and liver. The substance possessed is useful as an organogenesis promoter.

 Furthermore, based on the knowledge obtained by the present inventors that EMT control and / or Snai 1 activity can be controlled by controlling the amount of intracellular zinc, E It becomes possible to screen a compound having an MT control action and a Z or S nai 1 activity control action. For example, this screening method is performed by the following steps.

 (1) A process in which cells that desire EMT control and Z or Snai 1 activity control are divided into two groups, and one is treated with the test compound (the remaining untreated is the control)

 (2) A step of measuring the amount of intracellular zinc in each of the treated cells and untreated control cells.

 (3) A process for selecting a compound having a significant variation in zinc amount compared with the amount of zinc in control cells and certifying it as a compound having an EMT control action or a Snai 1 activity control action.

 Cells that desire EMT control and Z or S nai 1 activity control are not particularly limited as long as S nai 1 is activated and EMT occurs, and specific examples include normal kidney cells and keratinocytes. Examples thereof include epithelial cells and breast cancer cells. Appropriately treat one of the two groups with the test compound. The other is not treated and used as a control cell. In addition, compounds known to affect the amount of zinc, such as zinc chelator, TPEN, may be used as a positive control compound.

Intracellular zinc levels can be measured using methods commonly used in this field. It can be performed according to this. For example, it can be measured directly by an atomic absorption method (flame method) or by a fluorescence spectrophotometer using a specific probe (for example, a fluorescent reagent).

 In addition, based on the knowledge obtained by the present inventors that the third Zn finger domain in the Snai1 molecule is involved in the intracellular localization of Snai1, in the presence or absence of the test compound. By measuring the degree of binding between the zinc finger domain and zinc in the presence, it becomes possible to screen a compound having an EMT regulating action and / or a Snai 1 activity regulating action. For example, the screening method is implemented by the following steps.

 (1) contacting the third Zn finger domain present in the Snai1 molecule with zinc in the presence or absence of the test compound;

 (2) a step of measuring the degree of binding between the zinc finger domain and zinc, and

(3) A step of recognizing a substance having a significant effect on the binding between a zinc finger domain and zinc as a compound having an EMT regulating action and / or a compound having a Snai 1 activity regulating action.

 The third Zn finger domain present in Snai 1 molecule is specifically a polypeptide composed of amino acids from 2 10th cysteine to 2 30th histidine of SEQ ID NO: 8 It is. Even if this polypeptide is obtained by a known method such as separation, extraction, purification, etc. from nature, genetic engineering technology is applied based on the sequence of the gene encoding the polypeptide (SEQ ID NO: 7). It may be obtained by the synthesis used, chemical synthesis or a combination thereof. Moreover, even if it is isolated as a polypeptide, it may be used in a state expressed in cells. In addition, the third zinc finger domain present in one molecule of Snai as long as the binding to zinc is not lost is substitution, deletion or insertion of one to several amino acids in its amino acid sequence. And / or additions may be made. ,

S nai The third Z n finger domain present in one molecule (hereinafter referred to as Z n The degree of binding between the zinc finger domain and zinc can be measured by utilizing the property that, when zinc binds to zinc, the intranuclear migration of a single Snai molecule is induced. That is, using a labeled S nai molecule containing a Zn finger domain or a fragment thereof, in the presence or absence of a test compound, in the presence of zinc, the labeled S nai 1 molecule or a fragment thereof in the nucleus Z n finger domain is considered to be bound to zinc if transition to is observed, and Z n finger domain is not bound to zinc if transfer to the nucleus is not observed. available. Those that significantly affect Snai 1 molecule translocation into the nucleus when compared with controls are identified as compounds having EMT-controlling activity and compounds having Snai-1 activity-controlling activity of the present invention. be able to.

 For the labeling of a Snai 1 molecule containing a Zn finger domain or a fragment thereof, a method commonly used in the art, for example, a radiolabel or a fluorescent label is used. However, it is particularly useful when expressed in a cell. In this case, a fluorescent label that can be observed in real time in the nucleus is preferable.

The test compound may be a known compound or a new compound to be developed in the future. Further, it may be a low molecular compound or a high molecular compound. Here, a low molecular weight compound is a compound having a molecular weight of less than about 300, for example, organic compounds that can be normally used as pharmaceuticals, derivatives thereof, and inorganic compounds, and are manufactured using organic synthesis methods and the like. And organic compounds and their derivatives and nucleic acid molecules that can be used as medicines, such as small nucleic acid molecules such as promoters, various kinds of metals, and the like. The high molecular compound is a compound having a molecular weight of about 300 or more, and includes proteins, polynucleic acids, polysaccharides, and combinations thereof, and is preferably a protein. These low-molecular compounds or high-molecular compounds can be obtained commercially if they are known, or can be obtained through steps such as collection, production, and purification according to each report literature. , These are natural sources, but also genes :! May be prepared scientifically or obtained by semi-synthesis etc. The

 The treatment time of the cells with the test compound is appropriately set depending on the type of cells used and the test compound. When a positive control compound such as T PEN is used, it can be carried out with reference to the fact that fluctuations in the amount of zinc are confirmed when treated with the control compound. The concentration of the test compound for treating the cells is also appropriately determined depending on the type of cells used, the type of test compound, and the treatment time. Example

 Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the scope of the present invention in any way. All publications cited throughout this application are hereby incorporated by reference. In addition, the reagents, devices, and materials used in the present invention are commercially available unless otherwise specified. Example 1: Intracellular localization of GFP—Snai 1 in human cervical cancer cells HeLa and its change by regulation of intracellular zinc content

A plasmid that expresses a fusion protein of Snail of SEQ ID NO: 7 and GFP (Green Fluorescent Protein) is constructed according to a conventional method, and then transferred to He La cells by ribofectamine for 4 hours. I made a cission. When cultured in DMEM medium for 24 hours in the presence of serum (10% FCS), GFP-Snail expression was confirmed. GFP—Snai 1 was localized in the nucleus. When this was treated with 50 zinc chelators TP EN (Mo 1 ecu 1 ar Probe) for 2 hours, the localization of GFP-Snai 1 was transferred to the cytoplasm (Fig. 1). It was shown that the intracellular localization of Snai 1 can be controlled by reducing the amount of intracellular zinc or chelating zinc in the Snai 1 molecule. Example 2: Snai 1 intramolecular finger domain zinc binding ability and changes in cellular and subcellular localization GFP—Snai1 expressed in HeLa cells in Example 1 was localized in the nucleus and transferred to the cytoplasm by reducing the amount of cells and zinc (for example, treatment with the zinc chelator T PEN). Therefore, each of the four Zn finger domain C 2 H 2 motifs in the S nai 1 molecule was mutated to G 2G 2 using STRATGENE's Quick Change Site—D irected Mu tagenesiskit, and in the presence of zinc. Intracellular localization was examined (Fig. 2). These mutations are thought to be due to the lack of the Zn-binding ability of the Zn finger domain. As a result, only one Snai molecule in which a mutation was introduced into the third Zn finger domain was inhibited from being transferred to the nucleus by zinc (localized in the cytoplasm). That is, it was revealed that only the third Zn finger domain plays an important role in intracellular localization of Snai 1. Example 3: Zinc binding state between nuclear-localized snail and cell-localized snail

 In order to further investigate the mechanism of regulation and activity of intracellular S n a i 1 by intracellular zinc, we analyzed the binding state of zinc in the nuclear localization S n a i l and the cytoplasmic localization S n a i l. Cells expressing nuclear-localized Snail and forcibly-expressing GFP-Snai1 and cells exhibiting cytoplasmic-localized Snai1 were cultured in the presence of zinc, respectively. When the zinc content was measured, it was found that cytoplasmic localized S nai 1 had a lower zinc content than nuclear localized Snail. S n a i 1 is thought to change its localization by binding and dissociating zinc in the Zn finger domain. Example 4

 (Method)

Cell culture, RNA i, and zinc chelation

The human keratinocyte cell line HaCaT (Boukarap, P. et al., J Cell Biol, 106, 761-71 (1988)) was maintained in DMEM supplemented with 10% FBS. Cells They were starved for 24 hours in medium without pups and then stimulated for 24 hours or indicated times with medium containing 100 ng / ml human EGF. SiRNA for human ZIP 6 and ZIP 10 and the control mouth (si CONTROL Non-targeting siRNA # 1) were obtained from Dharma con. Target sequences for R NA i are: 5'-GAAGUUAUCUGUAAUCUUGUU-3 '(SEQ ID NO: 9), 5'-GAAGUGACCUCAACUGUGUUU-3' (SEQ ID NO: 1 0), 5, -UGAAGGAACUCACUUUCUAUU-for ZIP 6 3, (SEQ ID NO: 1 1), 5, -UGACUUUGCUGUUCUACUAUU-3 '(SEQ ID NO: 1 2); For ZIP 10, 5, -GAACGUCACUCAGUUAUUAUU-3, (SEQ ID NO: 1 3), 5, -GGAAGAAUAUGAUGCUGUAUU -3 ′ (SEQ ID NO: 14), 5, -CCACAAACCUGAUCGUGUAUU-3 ′ (SEQ ID NO: 15), 5, -GAAAGGACUUGUUGCUCUAUU-3, (SEQ ID NO: 16). RNA interference (RNA i) screening was performed according to the manufacturer's protocol. To chelate zinc, TPEN (Mo 1 ecular Probes) was added to the culture medium (10 / M in DMSO) 24 hours prior to observation.

Plasmid

The expression constructs for GFP-fused full-length mouse S naill (1 -264) and C-terminal deleted mouse S naill (1-1 5 1) are as described above (Yamashita, S. et al., Nature, 429). , 298-302 (2004)). C 2 H 2 / G 2 G 2 Z n finger mutations were detected in C 1 56, C 1 59, H 1 72, and in the first Z n finger domain of full-length mouse S nai 1 1 (1-264). H 1 76, C 1 82, C 1 85, HI 98, and H 202 in the second Z n finger domain, C 2 10, C 21 3, H 226, and H 202 in the third Zn n finger domain C H238, and C 238, C 241, H252, and H254 in the 4th Zn finger domain were intercalated using Quick-Changgesite-directed mu tagenesisk ι CS tratagene). All S nai 1 1 mutants are cloned into the pCS 2. expression vector and tagged with GFP at the end, in-frame. It was. Transient transfection of cells with Snai 11 1 construct was performed using Lipofect amine-P lus reagent (Invitrogen) according to the instruction manual. To inhibit nuclear export, lebutomycin B (Sigma) was added to the culture 3 hours before observation (5 ng / m 1 in ethanol). Traditional RT—P CR

 The primers used for conventional RT-PCR are as follows:

SNA I L 1; 5'-AAGGACCCCACATCCTTCTC-3 '(sense; SEQ ID NO: 17), 5'-TCGGGGCATCTCAGACTCTA-3' (antisense; SEQ ID NO: 18),

E-cadherin; 5, -GTCATTGAGCCTGGCAATTT-3, (sense; SEQ ID NO: 19), 5, -GCTTGAACTGCCGAAAAATC-3, (antisense; SEQ ID NO: 20).

Vimentin; 5, -CCTTGAACGCAAAGTGGAAT-3 '(sense; SEQ ID NO: 2 1),

5′-GCTTCAACGGCAAAGTTCTC-3 ′ (antisense; SEQ ID NO: 22).

GAPDH; 5′-TGAAGGTCGGAGTCAACGGAT-3, (sense; SEQ ID NO: 2 3), 5′-CATGTGGGCCATGAGGTCCAC-3 ′ (antisense; SEQ ID NO: 24).

Z I P 1; 5, -CCTATCTCCCTCCTCCCATC-3, (sense; SEQ ID NO: 2 5),

5,-GGGTAACAGGTCCCAAAACA-3, (antisense; SEQ ID NO: 2 6),

Z I P 2; 5, -TCCACAGCCATGGACATTTA-3, (sense; SEQ ID NO: 2 7),

5,-TAGCTAGCTCCCGTGGAAGA-3, (antisense; SEQ ID NO: 2 8),

Z ΓΡ 3; 5,-GCGACTCGGAGTATGAGAGC-3, (sense; SEQ ID NO: 29),

5′-ACCTTGAGCAGACGGTCACT-3 ′ (antisense; SEQ ID NO: 30),

 Z I P 4; 5′-CCCAGAGTTGAGGCTACTGC-3 ′ (sense; SEQ ID NO: 3 1),

5'-AGGGCAGGGTATCAGAAGGT-3 '(antisense; SEQ ID NO: 3 2),

Z I P 5; 5,-TCCTCCTGGGAGATGGTCTA-3, (sense; SEQ ID NO: 3 3),

5,-AGGGTTATGGCAAGCATGAG-3, (antisense; SEQ ID NO: 34),

Z I P 6; 5,-TCTGTCACAAATCCCCTTCA-3, (sense; SEQ ID NO: 3 5),

5, -GGAGGGCTCTTGTGAGTCTG-3 '(antisense; SEQ ID NO: 3 6),

ZIP 7; 5,-GGCTGGCAGTCTTACAGAGG-3, (sense; SEQ ID NO: 3 7), 5'-CCCCTCGATTCAAACACAGT-3 '(antisense; SEQ ID NO: 3 8), ZIP 8; 5'-AGATTCCACGAAAAGCTGGA-3'(sense; SEQ ID NO: 3 9), 5'-TTGTTGGGCTTTGTCAATCA-3 '(antisense; sequence No. 40), ZIP 9; 5, -GATCCAGAAGCAGCAAGGTC-3, (sense; SEQ ID NO: 4 1), 5, -GATGAGAGGGATGAGGCAAC-3 '(antisense; SEQ ID NO: 4 2),

ZIP 1 0 ； 5,-CCTGGTTCCTGAAGATGAGG-3, (sense; SEQ ID NO: 4 3) 5 '-CATGGCAGAGAGGAGGTTGT-3' (antisense; SEQ ID NO: 44), ZIP 1 1; 5, CACAACGTTCCAGAGGGTCT-3, (sense ; SEQ ID NO: 4 5) 5 '-CCAACGTCCAGTGACATCAT-3' (antisense; SEQ ID NO: 4 6), ZIP 1 2; 5,-CCCACATGAAATGGGAGACT-3, (sense; SEQ ID NO: 4 7) 5 '-TCATGGTGTTGGAAACCAAA- 3 '(antisense; SEQ ID NO: 4 8), ZIP 1 3; 5,-CGCTTCCTMGACCCTTTCC-3' (sense; SEQ ID NO: 4 9) 5 '-TCTGAAAACATCCACGGTGA-3' (antisense; SEQ ID NO: 50) , ZIP 14; 5, -GCGCAGAGGTCAGTCCTAGT-3, (sense; SEQ ID NO: 5 1) 5'-CGCTACCCAAACAGAGAAGC-3 '(antisense; SEQ ID NO: 5 2),

ZNT 1; 5,-GTGTGAACTTGCCTGCAGAA-3, (sense; SEQ ID NO: 5 3), 5'-GCTTTCCAAAACAGTCACAGC-3 '(antisense; SEQ ID NO: 54), ZNT 2; 5,-TGATCCTGGTGTTGATGGAA-3, (sense; SEQ ID NO: 5 5), 5'-CCCTGGCTGTAGTCAGATGG-3 '(antisense; SEQ ID NO: 5 6), ZNT 3; 5'-CATCTCCCCATGGTTCATGT-3' (sense; SEQ ID NO: 5 7), 5'-GGAGTCCTCCCTCTTTCAGG- 3 '(antisense; SEQ ID NO: 5 8), ZNT4; 5'-ACAAGATTGCTGACCCCATC-3' (sense; SEQ ID NO: 5 9), 5, CAGCTGTCAGGGATTCCATT-3, (antisense; SEQ ID NO: 60), ZNT 5 5,-AGTCTCAGTTGGAGGGCTGA-3, (sense; SEQ ID NO: 6 1), 5'-AGCAGAATGACGCCAAAAAT-3 '(antisense; SEQ ID NO: 6 2),

ZNT 6; 5, ACCTACACCAACACCCAAGC-3, (sense; SEQ ID NO: 6 3), 5'-GAAGGAGATCAGACTCCCAAAA-3 '(antisense; SEQ ID NO: 64), ZNT 7; 5,-TCCAGATGTCCACCATGAGA-3, (sense; SEQ ID NO: 6 5),

5, -TGCAACTGCTGTACCCTCTG-3 '(antisense; SEQ ID NO: 66),

ZNT 8; 5 '-GGCCACAATCACAAGGAAGT-3, (sense; SEQ ID NO: 6 7),

5,-AGCAATTTCTCTCCGAACCA-3 '(antisense; SEQ ID NO: 68)

Immunostaining

 Immunostaining was performed using Al x a 488 En h anc e kit (Mole c u lar P r o b e s). The nuclei were counterstained with DAP I (MolecularProbes).

In vitro wound healing and cell migration assay ''

 H a C a T cells are cultured in the presence of F C S until they reach subconfluence,

They were starved for 24 hours and subjected to an in vivo wound healing assembly. The cell-free area was introduced by cleaving the monolayer with a yellow pipette tip. An additional 24 hours of cell migration into the cell-free area was evaluated in the presence of starvation or 100 ηg / m 1 EGF. Using cell migration assembly according to the manufacturer's protocol 5. 0 111 pore size T r a n s w e 1 1 i in s er t (C o s t a r) ¾ ■? T, this.

Zinc Atsey

^{For 65} Zn uptake, grow cells to 50% confluence,

They were cultured in D MEM containing ⁶⁵ of human EGF and a particular concentration of times 100 ng / m 1 of the display in _{37 ° C Z n C l 2} (O ak R idge N ational L aboratory), 3 in PB S Washed twice and then collected. Cell-associated radioactivity was determined on a Packard Auto-Gamma 5650 counter. Zinc Contact Yopi other metal chloride salt _{(C a C 1 2, C} d C 1 2, C o C 1 2, C u C 1 2, F e C 1 2, Hg C 1 2, Mg C Stock solutions of 1 ₂ , Mn C 1 ₂ , and Ni C 1 ₂ ) were added to the culture medium at a concentration of 5- / zM. Each CPM was normalized by cell number. To visualize intracellular zinc distribution, load cells by adding Zn AF-2 DM (Daiichi Pure Chemicals) to the medium at a final concentration of 5 μΜ 30 min before observation. did. Fluorescence image obtained using FI TC filter set It was.

 (Result)

During EMT in gastrulation, wound healing, and cancer progression, the SNA IL-1 transcription factor is involved in the transcriptional repression of the epithelial gene E-forced herrin, thus reducing the level of this cell indirect adhesion molecule Allows cells to detach and migrate from their neighbors (Batlle, E. et al., Nat Cell Biol, 2, 84-9 (2000); Cano, A. et al., Nat Cell Biol, 2, 76-83 (2000); Thiery, JP & Sleeman, JP, Nat Rev Mol Cell Biol, 7, 131-42 (2006)). In many cell lines, there is an inverse correlation between E-forced doherin and SNAIL 1 mRNA levels that should be considered, but sometimes E force doherin and SNA I LI mRNA are expressed in the same system. (Dominguez, D. et al., Mol Cell Biol, 23, 5078-89 (2003)). Here, the human keratinocyte line Ha C a T (Boukamp, P. et al. J Cell Biol 106, 761-71 (1988)) expressed SNA ILI and showed a high expression level of E-force doherin. However, we found that the mesenchymal gene vimentin is undetectable and reflects the epithelial morphology of this system (Figures 3a and 3b; DIC). In response to EGF, Ha C a T cells downregulate E-cadherin, upregulate vimentin, and develop a mesenchymal-like phenotype without increasing SNA IL 1 expression. (Figs. 3a and 3b; DIC), suggesting that EGF regulates the activity of SNA IL 1 protein post-translationally. Since the activity of Snai 11 is regulated by its intracellular arrangement (Dominguez, D. et al., Mol Cell Biol, 23, 5078-89 (2003)), SNA IL 1 activity in Ha C a T cells The effect of EGF on subcellular localization was examined (Fig. 3b). Without EGF stimulation, SNA IL 1 was localized in the cytoplasm, consistent with the epithelial-like morphology of the cell line. In contrast, EGF induced the adoption of a mesenchymal-like morphology of SNA IL 1 nuclear accumulation opal cells. These data suggested that the activity of SNA IL 1 in Ha C a T cells is controlled by its intracellular configuration, which is determined in response to extracellular stimuli. In EGF-dependent wound healing by mouse keratinocytes, S tat 3 is It transmits signals necessary for migration rather than proliferation of these cells (Sano, S. etal., Embo J, 18, 4657-68 (1999)). In addition, Stat 3 signaling regulates nuclear localization of S nai 1 1 through the zinc receptor transporter Li V 1 / Z ip 6 and actively migrates during zebrafish gastrulation Induces EMT of organizer cells (Yamashita, S. et al., Nature, 429, 298-302 (2004)). These facts imply that zinc transporter-dependent regulation of SNA IL 1 subcellular localization may play a role in EMT of wound healing by mammalian keratinocytes. To test this idea, the ZIPs (ZIP 1-14) (Eide, DJ, Plugers Arch, 447, 796-800 (2004)) and cytoplasm that are thought to function in zinc influx into the cytoplasm. In order to examine the expression of ZNTs (ZNT 1-8) (Palmiter, RD & Huang, L., Plugers Arch, 447, 744-51 (2004)) that mediate zinc efflux from C a T cells were used (Fig. 3c). The most closely related member of the ZIP family of zinc transporters (see their proximity in the phylogenetic tree analysis based on their amino acid sequence; Fig. 3 c) The expression of ZIP 6 and ZIP 10 is associated with EG F stimulation In response, it was upregulated in Ha C a T cells. These results supported the idea that ZIP 6 and ZIP 10 could function in nuclear localization of SNA IL 1 in EGF-induced EMT of wound healing by Ha C a T cells. Therefore, siRNA against human ZIP 6 and ZIP 10 was used to produce ZIP 6 and ZIP 10 knockdown H a C a T cells (Figure 4a). Using these cells, we performed an in vitro wound healing assay and analyzed the intracellular localization of SNA IL 1 under EGF stimulation (Fig. 4c :). ZIP 10 knockdown EGF-induced in vitro wound healing by Ha C a T cells is markedly impaired (Fig. 4c; ZIP 10 and ZIP 6; ZIP 10), whereas control Ha C a T cells migrate normally The wound was completely healed after stimulation with EGF (Fig. 4c; control). Using ZI-6 knockdown H a C a T cells, the wound healed after EGF stimulation, although there was some disturbance at the tip of the migratory cells (Figure 4c; ZI-6). Consistent with this, ZIP 10 in H a C a T cells Knockdown strongly reduced EGF-induced nuclear accumulation of SNA IL 1 (FIG. 4c; ZIP 10 and ZIP 6; ZIP 10). These results indicated that ZIP 10 has an essential function in the nuclear accumulation of SNA IL 1 during EGF-induced EMT of wound healing by Ha C a T cells. In addition, our results indicate that ZIP-mediated regulation of nuclear localization of SNA IL 1 is involved in growth factor-mediated wound healing by human keratinocytes as well as zebrafish gastrulation. It is suggested that.

Once the above results are obtained, the most important questions that have not yet been answered are how the membrane molecule ZIP regulates SNA IL 1 nuclei ^ Ϊ and zinc is involved in this process. It is whether it is included. To answer these questions, we first analyzed zinc metabolism in Ha C a T cells using a ⁶⁵ Zn uptake assembly. EGF-stimulated Ha C a sputum cells took up significantly more ⁶⁵ Zn than resting Ha C a T cells during a series of assemblies (FIG. 3d). EGF optic lobe induced ⁶⁵ Z n up write is inhibited by an excess of non-radioactive zinc rather than other metals (Figure 3 c :), show that EGF is specifically induce Ha C a T cell by zinc uptake ing. Importantly, only low levels of zinc uptake were detected in ZIP 10 knockdown H a C a T cells (FIG. 4 b ′ :). Considering that ZI Ρ family members mediate zinc influx into the cytoplasm (Eide, DJ, Pflugers Arch, 447, 796-800 (2004)), these results are due to H a C a T cells It clearly shows that EGF-induced zinc uptake depends on ZIP 10. Furthermore, when combined with the essential role of ZIP 10 in nuclear accumulation of SNA IL in EGF-stimulated Ha C a T cells, these results indicate that nuclear accumulation of SNA IL in response to extracellular stimulation is at least To some extent, it strongly suggested that it depends on zinc actively transported by ZIPs.

To investigate whether EGF-mediated uptake of zinc via ZIPs regulates subcellular localization of SN AIL in Ha C a T cells, we next examined whether the fluorescent zinc indicator Z n AF-2 (Hirano , T., Kikuchi, Κ., Urano, Y., T, H. & Nagano, Τ., J Am Chera Soc, 122, 12399-400 (2000)) was used to analyze the intracellular distribution of zinc in the cells (Fig. 3f and 3c :). Z nAF-2 is very specific for zinc; it is thought to form a ternary complex with zinc metalloproteins, where zinc acts as a mediator; the formation of this complex is Enables direct evaluation of intracellular distribution (Kay, AR & Toth, K., J Neurophysiol, 95, 1949-56 (2006)) ₀ Epithelial-like morphology Opause with cytoplasmic distribution of SNA IL In a C a T cells, zinc was uniformly distributed. After EGF stimulation, the amount of zinc bound to intracellular proteins increased, and zinc accumulated in the nucleus of Ha C a T cells (Fig. 3f) and correlated with nuclear localization of SNA IL-1. This redistribution was not observed in ZIP 10 knockdown H a C a T cells in which EGF-induced nuclear accumulation of SNA IL 1 disappeared (Fig. 4c; ZI Ρ 10 and ZI Ρ 6; ZI Ρ 10). ZIP 10-dependent nuclear colocalization of zinc and SNA IL 1 in £ 0 stimulated ^ 1 a C a T cells suggested that zinc could be involved in the nuclear localization of SNA IL 1. In fact, intracellular zinc depletion using membrane-permeable zinc chelators N, Ν, Ν ', Ν, -tetrakis (2-pyridylmethyl) ethylenediamine (ΤΡΕΝ) abolishes EG F-induced SNA IL 1 nuclear accumulation And impaired wound healing by them (Figures 4b and 4c; TPEN +) ₀

The C-terminal half of mouse Snai11 contains four C2H2-type Zn finger domains and is essential for Snail1 nuclear localization (Dorainguez, D. et al., Mol Cell Biol , 23, 5078-89 (2003)). All results for this point suggest that EGF-induced zinc uptake via ZIPs regulates SNA IL 1 nuclear localization through interaction with the Zn finger domain of SNA IL 1 Yes. To test this hypothesis, C 2 H 2 / G 2G 2 mutations were introduced into all four Zn finger domains of mouse S nai 1 1 (Fig. 5c; 1 234) and the zinc binding activity was abolished. (Nomura, A. & Sugiura, Tsuji, Inorg Chem, 41, 3693-8 (2002)). Next, the intracellular localization of Snail 1. in EGF-stimulated Ha C a T cells was examined (FIGS. 5a to 5d). Consistent with the requirements of S ai 1 1 zinc for nuclear accumulation (Figures 5a and 5d; TPEN). And its own C-terminal half (Figures 5a and 5d; ΔC), 1 2 34 mutants are located in the cytoplasm, not in the nucleus (Figures 5a and 5d; 1 2 34), and zinc coordination at the C-terminal half of Snail 1 This indicates that it is important for the accumulation of Snail 1 nuclei.

To further limit the sites involved in Sn nail 1 nuclear localization, a series of constitutives with C 2 H 2 / G 2 G 2 mutations in 1, 2 or 3 Zn fingers were prepared ( Figure 5 c), the intracellular arrangement of mutants in EGF-stimulated HaCa T cells. A single C 2 H 2 / in the third Zn finger domain, not the first, second, or fourth Zn finger domain (Figure 5c; 1, 2, or 4) G 2 G 2 mutation (Fig. 5c; 3) Force EGF-stimulated Inhibition of Snai 11 nuclear localization in Ha C a T cells (Figs. 5a and 5d). In addition, any combination of C 2H2 / G 2 G 2 mutations containing a mutated third Zn finger domain (Figures 5a, 5c and 5d; 1 3, 2 3, 3 4, 1 2 3, 1 34, or 2 34) abolished nuclear accumulation of mutants in EGF-stimulated Ha C a T cells, and the ability of the third Zn finger domain of S nai 1 1 to nai 1 1 is essential for nuclear accumulation. Reciprocally, any combination of C 2 H2 / G 2 G 2 mutations in any Zn finger domain except the third (Figures 5a, 5c and 5d; l, 2, 4, 1 2, 1 4 , 24, or 1 24) It did not disrupt the nuclear localization of the mutant. Above all, the nuclear localization of mutant 1 24 (Figs. 5a, 5c and 5d; 1 24) shows that the coordination of zinc by Snaill's third Zn binger domain is induced by EGF-stimulated H We clearly demonstrated that it is necessary and sufficient for nuclear localization of S nai 11 in a C a T cells. Finally, nuclear export receptor CRM 1 (Fukuda, M. et al., Nature, 390, 308-11 (1997); Xu, L. & Massague, J., Nat Rev Mol Cell Biol, 5, 209- 19 (2004)) in the presence of leptomycin B, a specific inhibitor (Figures 5b and 5e; LMB), a construct carrying the third Zn finger domain of the mutant. (Fig. 5b, 5c and 5e; 3, 1 3, 2 3, 34, 1 2 3, 1 34, 2 34, or 1 2 3 4) even localized in the nucleus. The third Z n finger is essential for S nai 1 1's nuclear mooring, This indicates that it is not included in the translocation of Snail 1 to the nucleus.

 To examine whether EMT zinc-dependent regulation of wound healing by human keratinocytes also occurs in cancer progression, HeLa, MDA—MB—231, and MDA— ZIP in a variety of invasive and metastatic cancer cell lines, including MB—43 5 S cells, especially non-metastatic cell lines such as MCF 7, T47D, ZR 75—1, and ZR 75-30 cells The expression of 10 and ZIP 6 was analyzed. Consistent with observations in keratinocytes, ZIP 10 up-regulation correlated with E-force doherin down-regulation and vimentin up-regulation, but ZIP 6 expression was associated with a mesenchymal phenotype It was not done (Fig. 6a). These results are consistent with previous findings that the expression level of ZIP 6 is not associated with the malignant cell phenotype in breast cancer (Kasper, G. et al., Int J Cancer, 117, 961-73 (2005)). Thus, ZIP 10 suggests that it is the primary ZIP in the zinc-dependent regulation of EMT in cancer progression.

 Therefore, we next examined the role of 2 1 to 10 2 2 6 in cell migration in He La cells (Fig. 6b). The control HeLa cells showed high migration activity, and Z I P 10 but not Z IP 6 inhibited it. These results indicate that ZIP10 regulates EMT not only in physiological but also pathological conditions, suggesting that zinc is essential for EMT in certain human cancers. In fact, the depletion of intracellular zinc by TPEN inhibits migration of He La cells (Fig. 6b; TPEN), and zinc is clearly involved in cancer progression.

The results establish that zinc is a regulator of EMT during wound healing in Invitro and cancer progression in Invitro and in vivo, and zinc stimulates extracellular stimuli to zinc metalloproteins via zinc transporters. It was suggested that it could act as a second messenger to convert. Zinc is actively transported by ZIPs and coordinated in the Zn finger domain of SNA IL 1, impeding its nuclear export, and leading to increased levels of SNA IL 1 in the nucleus, hence the EMT It enables guidance. These results are based on the zinc transporter ZIP A molecular explanation of susceptibility sideling To understand EGF-stimulated Zn finger protein SNA IL's nuclear accumulation and its intracellular zinc signaling used by other zinc metalloproteins Provides a conceptual basis for Industrial applicability.

 Intracellular localization and activity of S nai 1, the master regulator of EMT, by controlling the amount of zinc in the cell by using zincions, zincone chelator, or regulating the expression and function of zinc transporters This makes it possible to control the EMT. The drug of the present invention capable of controlling EMT is useful for preventing cancer metastasis, promoting wound healing and organ formation.

 This application is based on Japanese Patent Application No. 2005-141684 (filing date: May 13, 2005) filed in Japan, the contents of which are incorporated in full herein.

Claims

The scope of the claims

I. An epithelial-mesenchymal transition control agent containing a substance capable of controlling the amount of intracellular zinc as an active ingredient.

2. The agent according to claim 1, wherein the substance capable of controlling the amount of intracellular zinc is zinc ion.

3. The agent according to claim 2, wherein the zinc ion is introduced into the cell by a zinc ionophore.

 4. The agent according to claim 1, wherein the substance capable of controlling the amount of intracellular zinc is a zinc chelator.

5. The zinc chelator is 2,3-dimercapto 1-propanesulfonic acid (DMP

S), Ν, Ν, Ν ', Ν, -tetrakis (2-pyridylmethyl) ethylene diamine (ΤΡΕΝ), ethylene diamine (EDTA) and Ν— (6-methoxy-1-8-quinolyl) one; ρ-toluenesulfonamide 5. The agent according to claim 4, which is at least one selected from the group consisting of (TSQ).

6. The agent according to claim 1, wherein the substance that can control the amount of intracellular zinc is a substance that controls the expression and function or function of a zinc transporter.

 7. The agent according to claim 6, wherein the zinc transporter is at least one selected from the group consisting of human Z I moths and human CDFs including the L IV family.

8. The agent according to claim 6, wherein the zinc transporter is L I V 1.

 9. The agent according to claim 6, wherein the zinc transporter is Z I Ρ10.

 10. An epithelial-mesenchymal transition regulator comprising a substance capable of controlling the binding of zinc to the third Zn finger domain in the Snai1 molecule.

 The agent according to any one of claims 1 to 10, which is an I I. Snai l activity control agent.

 1 2. The agent according to any one of claims 1 to 10, which is at least one pharmaceutical selected from the group consisting of a cancer metastasis preventive, a wound healing agent and an organogenesis-promoting agent. .

1 3. A small group selected from the group consisting of cancer metastasis preventives, wound healing agents, and organogenesis promoters. The agent according to claim 11, which is at least one pharmaceutical.

 14. A cancer metastasis-preventing agent comprising, as an active ingredient, a substance that can reduce the amount of intracellular zinc or a substance that can inhibit the binding of the third Zn finger domain in the Snai1 molecule to zinc.

 1 5. Substance X that can increase the amount of intracellular zinc contains a wound healing agent and a nodule containing a substance capable of promoting the binding of zinc to the third Zn finger domain in Snai 1 molecule as an active ingredient. Or organogenesis promoter.

 1 6. A method for controlling epithelial-to-mesenchymal transition comprising controlling intracellular zinc levels in vitro.

 1 7. A method of controlling Snai 1 activity, comprising controlling intracellular zinc levels in vitro.

 18. A screening method for a compound having an epithelial-mesenchymal transition-controlling action and a compound having an action-controlling action of Sn ai1 activity, comprising measuring intracellular zinc content.

1 9. A screening method for a compound having an epithelial-mesenchymal transition-controlling action and / or a compound having a SnaiL activity-controlling action comprising the following steps

 (1) contacting the third Zn finger domain present in the Snai1 molecule with zinc in the presence or absence of the test compound;

 (2) measuring the degree of binding between the zinc finger domain and zinc; and

(3) A step of identifying a substance that significantly affects the binding between the zinc finger domain and zinc as a compound having an epidermal-mesenchymal transition control action or a compound having a Snai 1 activity control action .

 20. A method for controlling epithelial-mesenchymal transition in a subject, comprising administering to the subject an effective amount of a substance capable of controlling the amount of intracellular zinc.

 21. Control of epithelial-mesenchymal transition in a subject comprising administering to the subject an effective amount of a substance capable of controlling the binding of zinc to the third Zn finger domain in the Snai 1 molecule Method. ,.

22. administering to the subject an effective amount of a substance capable of controlling the amount of intracellular zinc; A method for controlling Snail activity in the subject.

 2 3. It includes administering to the subject an effective amount of a substance capable of reducing the amount of intracellular zinc or a substance capable of suppressing the binding of zinc to the third Zn finger domain in the Snai 1 molecule. A method for preventing cancer metastasis in the subject.

24. Administration of an effective amount of a substance capable of increasing the amount of intracellular zinc or a substance capable of promoting the binding of zinc to the third Zn finger domain in Snai 1 molecule. A method for promoting wound healing and / or organ formation in the subject.

2 5. Use of a substance capable of controlling the amount of intracellular zinc in the production of an epithelial-mesenchymal transition regulator.

2 6. Use of a substance capable of controlling the binding of zinc to the third Zn finger domain in the Snai1 molecule in the production of an epithelial-mesenchymal transition regulator.

2 7. Use of substances capable of controlling the amount of intracellular zinc in the production of Snai 1 activity regulators.

 28. Use of a substance that can reduce the amount of intracellular zinc or a substance that can suppress the binding of the third Zn finger domain in the Snai1 molecule to zinc in the production of a cancer metastasis preventive agent.

 29. Substances that can increase the amount of intracellular zinc in the manufacture of wound healing and / or organogenesis-promoting drugs, or substances that can promote the binding of zinc to the third Zn winger domain in Snai 1 molecule Use of.

 30. A commercial containing a drug containing a substance capable of controlling the amount of zinc in the cell as an active ingredient, and a statement describing that the drug can or should be used to control epithelial-mesenchymal transition Package.

 3 1. A drug containing as an active ingredient a substance that can control the binding of the third zinc finger domain in the S nai 1 molecule to zinc, and the drug can be used to control epithelial-mesenchymal transition Or a commercial knockout containing a statement stating that it should be used. ,.

3 2. A drug containing a substance capable of controlling the amount of zinc in the cell as an active ingredient, and A commercial package containing a statement stating that the drug can or should be used to control Snai 1 activity.

 3 3. A drug containing as an active ingredient a substance capable of reducing the amount of intracellular zinc or a substance capable of inhibiting the binding of zinc to the third Zn finger domain in Snai 1 molecule, and said drug as a cancer A commercial package containing a statement stating that it can or should be used to prevent metastasis.

 3 4. A drug containing a substance capable of increasing the amount of intracellular zinc or a substance capable of promoting the binding of zinc to the third Zn finger domain in Snai 1 molecule as an active ingredient, and the drug as a wound A commercial package containing a statement stating that it can or should be used for healing and promoting Z or organogenesis.