Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• the present invention relates to a composition for suppressing cancer metastasis, and more particularly to a composition containing a VIPR2 antagonist peptide for suppressing cancer metastasis.
• Cell migration is an evolutionarily conserved mechanism that plays a role in normal processes such as embryonic development, immunity, angiogenesis and wound healing, as well as pathogenic processes such as cancer metastasis.
• Cell migration is controlled by the localization of actin filaments within the cell, resulting in the formation of protruding structures (projections) called lamellipodia at the leading edge of the cell.
• Extracellular stimulation by growth factors, cytokines, chemokines, etc. induces the formation of lamellipodia.
• GPCRs G protein-coupled receptors
• PI3K phosphatidylinositol 3-kinase
• Activated PI3K converts phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] to phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] Convert to PI(3,4,5) P3 promotes the phosphorylation of AKT and the translocation of the WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane, leading to GEF-Rac-mediated remodeling of actin filaments.
• WAVE2 verprolin homologous protein 2
• PI3K gain-of-function mutations in PI3K (ie, mutations in the p110 catalytic subunit of PI3K) enhance the PI(3,4,5) P3 signaling pathway and are frequently found in breast cancer and others.
• the activated PI(3,4,5) P3 pathway induces cancer cell growth and motility, resulting in enhanced cancer cell migration and invasion.
• Vasoactive intestinal peptide a neuropeptide closely related to pituitary adenylate cyclase-activating polypeptide (PACAP), is widely expressed in the central and peripheral nervous system. Its receptors, G protein-coupled receptors (GPCRs): VIPR1 and VIPR2 (also called VPAC1 and VPAC2), are widely expressed in the brain, but are also found in the cardiovascular, renal, digestive, immune, It is also expressed in numerous peripheral target organs such as the endocrine system and reproductive system. There are many reports on the relationship between VIP and tumor growth.
• GPCRs G protein-coupled receptors
• VIPR2 mRNA expression and/or VIPR2 gene copy number has been reported in several types of cancer, including ovarian epithelial tumor, glioblastoma, and invasive breast cancer. However, little is known yet about the pathophysiological role of VIPR2 in cancer.
• Non-Patent Document 1 and Patent Document 1 disclose novel VIPR2 antagonist peptide groups.
• KS-133 disclosed in Non-Patent Document 2, referred to as Seq-10 (SEQ ID NO: 2) in the examples described later
• Seq-10 SEQ ID NO: 2
• the decrease is suppressed (see Non-Patent Document 2).
• the problem to be solved by the present invention is, for example, to provide means for suppressing cancer metastasis by examining the role and mechanism of VIPR2 signals that affect migration of cancer cells.
• the present invention was made to solve the above problems, and VIP-VIPR2 signals localize actin for lamellipodia formation by WAVE2 via PI(3,4,5) P3 synthesis. It was created based on the discovery that it regulates cancer cell migration in vitro and in vivo by regulating growth and elongation. That is, the present invention provides means for suppressing cancer metastasis by inhibiting the function of VIPR2, and specifically includes the following embodiments.
• X 1 represents cysteine, Mpa (3-mercaptopropionic acid) or D-cysteine
• X 3 is N-methylated glycine, N-methylated alanine, 2-azetidine-2-carboxylic acid, proline, hydroxyproline, 3,4-dehydroproline, pipecolic acid, serine or lysine
• X 8 represents tyrosine, proline or arginine
• X 7 and X 11 represent lysine and aspartic acid, ornithine and glutamic acid, asparagine represents any combination of acid and lysine, glutamic acid and ornithine, lysine and glutamic acid, or glutamic acid and
• N-terminal amino group and C-terminal carboxy group may be modified or deleted.
• a cyclic peptide consisting of an amino acid sequence represented by, a derivative or modified product thereof, or a pharmacologically acceptable A composition for suppressing cancer metastasis, comprising a salt thereof.
• X 1 represents cysteine
• X 3 represents proline or serine
• X 7 represents lysine
• X 8 represents tyrosine
• X 11 represents aspartic acid
• X 12 and X 13 each independently represent leucine, isoleucine or norleucine, preferably the N-terminal amino group is acetylated and the C-terminal carboxy group is amidated.
• composition can also be used, for example, to treat a subject containing cancer cells in which signaling pathways downstream of PI3K, such as AKT and WAVE2, have been activated via VIPR2. It can also be used to inhibit the phosphorylation of AKT in .
• a composition containing a cyclic peptide according to the present invention can suppress cancer metastasis, for example, by inhibiting the function of VIPR2.
• FIG. 1 shows the results of examining the effect of VIP on AKT phosphorylation in MCF-7 and MDA-MB-231 breast cancer cells.
• FIG. 2 shows the results of evaluation of cell motility in MCF-7 breast cancer cells overexpressing MCF-7 and VIPR2-EGFP.
• FIG. 3 shows the results of measuring the migration behavior of MDA-MB-231 breast cancer cells overexpressing EGFP or VIPR2-EGFP using a transwell migration assay.
• FIG. 4 shows the results of quantifying the area of lamellipodia.
• FIG. 5 shows the results of in vivo evaluation of migration activity of MDA-MB-231 breast cancer cells overexpressing VIPR2-EGFP.
• FIG. 6 shows the results of adding a VIPR2-selective antagonist peptide to MDA-MB-231 breast cancer cells overexpressing VIPR2-EGFP and evaluating AKT phosphorylation.
• FIG. 7 is a schematic diagram showing the structure of a transwell used for evaluation of cell motility in MDA-MB-231 breast cancer cells overexpressing VIPR2-EGFP.
• FIG. 8 shows the results of evaluation of inhibitory effects of VIPR2-selective antagonist peptides on the migration of MDA-MB-231 breast cancer cells overexpressing VIPR2-EGFP.
• Figure 9 shows the cell proliferation effect in MCF-7 cells overexpressing VIPR2 when the VIPR2 antagonist KS-133 was added (KS-133 group) or when KS-133 was not added (Control group).
• a peptide refers to a substance in which two or more amino acids are bound by an amide bond (peptide bond), and for example, 2 to 20 amino acids can be amide bonds.
• the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxy terminus).
• the first carbon atom adjacent to the carbonyl group that forms the peptide bond is called the C ⁇ carbon.
• amino acid or derivative thereof is used in its broadest sense, including naturally occurring amino acids, as well as artificial amino acids having non-natural structures, having properties known in the art that are characteristic of amino acids. Also includes chemically synthesized compounds and carboxylic acids with functional groups.
• unnatural amino acids include D-form amino acids, ⁇ / ⁇ -disubstituted amino acids whose main chain structure differs from that of the natural type (such as ⁇ -methylated amino acids such as 2-aminoisobutyric acid), N-alkyl-amino acids (N-methyl amino acids, etc.), N-substituted glycines (peptoids), amino acids with extended main chains ( ⁇ -homoamino acids and ⁇ -homoamino acids), amino acids with side chains different from the natural type (cyclohexylalanine, allylglycine, 2- (2-pyridyl)-glycine, 3-(1H-benzimidazol-2-yl)-alanine, etc.), amino acids with partial substitution in the side chain (norleucine, diaminopropanoic acid, 3-(2-pyridyl) -alanine, etc.), amino acids with extra functional groups in the side chains; , Cl, Br, I) (such as
• VIP2 means a protein of mammals such as mice, rats, dogs, monkeys and humans, "vasoactive intestinal peptide receptor 2".
• cancer metastasis suppression refers to, for example, suppression of tumor metastasis when the primary tumor has not yet metastasized, or suppression of further tumor metastasis when the tumor has already metastasized. means either or both of Metastasis of cancer cells means that cancer cells migrate from the place of origin (primary focus) and form a tumor again in a distant site.
• cyclic peptide A cyclic peptide, which is one active ingredient of the present invention, is disclosed in Patent Document 1, the entire content of which is incorporated herein by reference.
• the peptide disclosed in Patent Document 1 is stabilized by bicyclization while maintaining or enhancing the characteristics (pharmacophore) involved in the VIPR2 binding activity of VIPep-3 disclosed in Non-Patent Document 1. be. Any of the peptides disclosed in these documents can be used for the novel uses of the present invention.
• a particularly suitable embodiment of the cyclic peptide for suppressing cancer metastasis has the following formula (1): c[X 1 -Pro 2 -X 3 -Tyr 4 -Leu 5 -Pro 6 -c(X 7 -X 8 -Leu 9 -Cys 10 ]-X 11 )-X 12 -X 13
• X 1 represents cysteine, Mpa (3-mercaptopropionic acid) or D-cysteine
• X 3 is N-methylated glycine, N-methylated alanine, 2-azetidine-2-carvone acid, proline, hydroxyproline, 3,4-dehydroproline, pipecolic acid, serine or lysine
• X 8 represents tyrosine, proline or arginine
• X 7 and X 11 represent lysine and aspartic acid, ornithine and glutamic acid , as
• a further preferred embodiment of the cyclic peptide is the above formula (1), wherein X 1 represents cysteine, X 3 represents proline or serine, X 7 represents lysine, X 8 represents tyrosine, X 11 represents aspartic acid, and X 12 and X 13 each independently represent leucine, isoleucine or norleucine.
• the N-terminal amino group is acetylated and the C-terminal carboxy group is amidated.
• Examples of the individual cyclic peptides included in the above formula (1) include the following examples.
• c(Mpa-Pro-Pro-Tyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH 2 (SEQ ID NO: 1) Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leu 5 -Pro 6 -c(Lys 7 -Tyr 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Ile 13 -NH 2 ( SEQ ID NO: 2) and the peptide described in paragraphs 0056 to 0059 of Patent Document 1.
• the peptide according to the present invention may be a peptide having homology in which one to several amino acids are deleted, added, and/or substituted in the amino acid sequence represented by the above formula (1), Those having binding activity are included.
• the number of those amino acids is not particularly limited as long as the peptide has VIPR2 binding activity. is preferably 1 to 5, more preferably 1 or 2.
• the sites of deletion, addition, and/or substitution may be at the terminal or in the middle of the peptide, and may be at one site or at two or more sites.
• the above amino acid sequence and BLAST Basic Local Alignment Search Tool at the National Center for Biological Information (USA) National Center for Biological Information Basic Local Alignment Search Tool
• BLAST Basic Local Alignment Search Tool at the National Center for Biological Information (USA) National Center for Biological Information Basic Local Alignment Search Tool
• the peptides according to the present invention also include various derivatives and/or modifications thereof as long as they solve the problems of the present invention.
• Such derivatives include those in which the saturated fatty chain of the peptide is replaced by an unsaturated fatty chain, those in which some of the atoms of the peptide are replaced by other atoms including radioactive or non-radioactive isotope atoms, peptides
• biotin-labeled peptides include, but are not limited to, peptides fused to membrane-permeable peptides, low-molecular-weight compounds, or molecules that induce protein ubiquitination.
• the peptide according to the present invention also includes a salt of the peptide.
• salts of peptides salts with physiologically acceptable bases or acids are used. Addition salts of acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic bases (ammonium hydroxide, or alkali , or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), addition salts of amino acids, and the like.
• the peptide according to the present invention may be a prodrug.
• a prodrug is a compound that is converted into a peptide according to the present invention by reactions with enzymes, gastric acid, etc. under physiological conditions in vivo, i.e., it undergoes enzymatic oxidation, reduction, hydrolysis, etc. to transform into the peptide according to the present invention.
• Prodrugs of the peptide of the present invention include compounds in which the amino group of the peptide of the present invention is acylated, alkylated or phosphorylated (for example, the amino group of the peptide of the present invention is eicosanoylated, alanylated, pentyl aminocarbonylated, (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonylated, tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated, or tert-butylated compound), a compound in which the hydroxy group of the peptide of the present invention is acylated, alkylated, phosphorylated, or borated (for example, the hydroxy group of the peptide of the present invention is acetylated, palmitoylated, propanoylated, pivaloylated , succinylated, fumarylated, al
• the prodrug of the peptide according to the present invention is one that changes into the peptide according to the present invention under physiological conditions as described in Hirokawa Shoten, 1990, "Drug Development", Vol. 7, Molecular Design, pp. 163-198. may
• the prodrug may form a salt, and examples of such salts include those exemplified as the salts of the peptide according to the present invention.
• the peptide according to the present invention may be a crystal, and the peptide according to the present invention includes a single crystal form or a mixture of crystal forms. Crystals can be produced by applying a crystallization method known per se to crystallize.
• the peptide according to the present invention may be a pharmaceutically acceptable co-crystal or co-crystal salt.
• Co-crystals or co-crystal salts as used herein refer to two or more compounds at room temperature that each have different physical properties (e.g., structure, melting point, heat of fusion, hygroscopicity, solubility, stability, etc.). It means a crystalline substance composed of distinct solids.
• a co-crystal or a co-crystal salt can be produced according to a co-crystallization method known per se.
• composition containing cyclic peptide A cyclic peptide, derivative or modification thereof as one active ingredient of the present invention can be used in the form of a composition as a pharmaceutical, diagnostic agent, or research reagent.
• the dosage form of the composition is not particularly limited, and may be oral administration or parenteral administration.
• Parenteral administration includes, for example, transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, or rectal administration), injection administration (intravenous injection, subcutaneous injection, intramuscular injection, etc.), transmucosal administration Skin administration and the like can be mentioned.
• transmucosal administration nonasal, oral, ocular, pulmonary, vaginal, or rectal administration
• injection administration injection administration
• subcutaneous injection subcutaneous injection, intramuscular injection, etc.
• transmucosal administration Skin administration and the like can be mentioned.
• Various modifications can be made to the peptides in the composition in view of their tendency to be metabolized
• biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. are used as sustained-release bases.
• PLGA polylactic acid glycol
• porous hydroxyapatite hydroxyapatite
• liposomes surface-modified liposomes
• emulsions prepared with unsaturated fatty acids nanoparticles, nanospheres, etc.
• the peptide may be encapsulated therein.
• a weak electric current can be applied to the skin surface to permeate the stratum corneum (iontophoresis method).
• compositions may use the active ingredient as it is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, and the like.
• Dosage forms include, for example, liquids (injections, etc.), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, Examples include inhalants, ointments, eye drops, nose drops, ear drops, poultices, and the like.
• These formulations may be controlled release formulations such as immediate release formulations or sustained release formulations (sustained release microcapsules, etc.).
• excipients for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjustment Agents, preservatives, antioxidants and the like can be used as appropriate and can be carried out in a conventional manner.
• ingredients used for formulation include purified water, saline, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, and crystals.
• surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, saponin, etc.
• Bile salts such as taurocholic acid
• Chelating agents such as EDTA and salicylic acids
• Fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, mixed micelles
• Pills or tablets can also be coated with sugar-coated, gastric-soluble, or enteric-coated substances.
• Injections can include distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oils, alcohols and the like.
• wetting agents, emulsifiers, dispersing agents, stabilizers, solubilizers, solubilizers, preservatives and the like can be added. If necessary, conventional additives such as preservatives, antioxidants, coloring agents, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.
• the composition of the present invention has a cancer metastasis inhibitory effect, it can be administered to patients with tumor metastasis and patients at high risk of tumor metastasis.
• the composition may be administered to a subject before metastasis of a tumor has occurred or may be administered to a subject after metastasis of a tumor has occurred.
• the subject to be applied is not particularly limited as long as it is a cell, but tumor cells are preferred.
• the type of cancer is not limited, and examples include head and neck cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, liver cancer, gallbladder/bile duct cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, and uterine cancer. , oral cancer, pharyngeal cancer, throat cancer, tongue cancer, esophageal cancer, renal cancer, ovarian cancer, and the like.
• composition of the present invention may be used in combination with other medicaments and therapeutic methods such as various chemotherapy, surgical treatments, and radiotherapy useful for the above diseases.
• the dosage may vary depending on the symptoms, age, sex, body weight of the patient. , susceptibility difference, administration method, administration interval, type of active ingredient, depending on type of formulation, but not particularly limited. can do.
• the coupling reagent Oxima Pure and diisopropylcarbodiimide (DIC) were added and reacted at 50°C for 3 hours.
• TFA was added to deprotect the protecting group of the thiol group, and at the same time the monocyclic peptide was cleaved from the resin.
• the monocyclic peptide was purified by RT-HPLC using a SunFire C18 column (10 ⁇ 150 mm) (manufactured by Waters) and then lyophilized.
• Bicyclic peptides Seq-1 and Seq-10 were purified by RT-HPLC using a SunFire C18 column (10 ⁇ 150 mm) (manufactured by Waters), and then lyophilized. The molecular weight of the finally obtained peptide was measured using microflex (Bruker) to identify the desired product.
• Table 1 shows the theoretical molecular weight, measured molecular weight, purity, cyclization type, and sequence of the peptides synthesized in this example. Among them, the amino acid sequences of Seq-1 and Seq-10 are shown below. In addition, in Table 1 and the following amino acid sequences, an amino acid without D-form represents the L-form.
• Seq-1 c(Mpa-Pro-Pro-Tyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile- NH2 (SEQ ID NO: 1)
• Seq-10 Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leu 5 -Pro 6 -c(Lys 7 -Tyr 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Ile 13 -NH2 (SEQ ID NO: 2)
• Seq-1 and Seq-10 remove the C-terminal 3 residues (Leu-Arg-Ser) of VIPep-3 (see Non-Patent Document 1), and the S-S bond between positions 1-10 and 7-position It is a peptide bicyclized by an amide bond between the -11 positions.
• pCMV6-AN-Myc-DDK vector PS100016
• S10C-0600 negative control siRNA
• the pEGFP-N2 vector was purchased from Takara Clontech.
• the pCMV6-VIPR2-Myc-DDK plasmid was constructed from the pCMV6-AN-Myc-DDK vector and VIPR2 cDNA.
• the VIPR2-Myc region was cloned from the resulting plasmid into the pEGFP-N2 vector.
• Human VIPR2-siRNA (si1, 3024813653-000080 and -000090; si2, 3024813653-000020 and -000030; si3, 3024813653-000050 and -000060) were purchased from Sigma-Aldrich.
• Anti-GAPDH antibody (#2118), anti-WAVE2 antibody (#3659), anti-pan AKT antibody (#4691), anti-phosphorylated AKT antibody (Ser473; #4060), anti-phosphorylated AKT antibody (Thr308; #2965) It was purchased from Cell Signaling Technology.
• MCF-7 and MDA-MB-231 cell lines were purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Health and Nutrition). Control siRNA and VIPR2 siRNA were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's recommendations. MCF-7 and MDA-MB-231 cells were transfected with an expression vector encoding VIPR2-EGFP or a control EGFP expression vector and cultured for 14 days in the presence of 1 mg/mL G418 (Nacalai Tesque). , established cell lines that stably express VIPR2-EGFP or EGFP.
• MCF-7 cells (1.5 ⁇ 10 4 cells) were seeded in a 35 mm culture dish in serum-free medium, cultured until adherence, and then stimulated with 100 nM VIP (Cayman Chemical Company).
• VIP Cosmetic Chemical Company
• cells were monitored by live cell imaging (BZ-X800; KEYENCE CORPORATION) at 37° C. every hour for 12 hours using a thermoplate (manufactured by Tokai Hit Co., Ltd.). Cell tracking analysis was performed using Image-Pro premiere ver. 9.4 (Media Cybernetics, Inc.).
• NightOWL NightOWL
• FIG. 2A In the presence of 100 nM VIP, MCF-7 cells migrate with the passage of culture time, and this migration was strongly inhibited by siRNA against VIPR2. On the other hand, in MCF-7 cells stably expressing VIPR2-EGFP, in addition to cell migration, cell morphology changed, and pseudopodia were significantly observed.
• FIG. 2B is the results of analysis and graphing of track plots of MCF-7 cells transfected with control siRNA and VIPR2 siRNA.
• FIG. 2C shows a comparison of travel speeds in a bar graph. The migration speed of MCF-7 cells was strongly suppressed by siRNA against VIPR2, whereas it was significantly accelerated in MCF-7 cells stably expressing VIPR2-EGFP. Data are shown as mean ⁇ SD. *** between indicated groups: p ⁇ 0.001 (Kruskal-Wallis test followed by Dunn's multiple comparison test).
• the transwell chamber assay shown in Figure 7 was used to assess the migration ability of MDA-MB-231 cells.
• Cells were suspended in serum-free medium (1 ⁇ 10 4 cells/100 mL). 600 ⁇ L of serum-free medium 6 containing 200 nM VIP was added to each well, insert 3 having 3 ⁇ m pore 5 was submerged in the well, and then suspended cells 1 were added to insert 3 and incubated at 37° C., 5% CO. Cultured under two conditions. After 30 hours, the cells were fixed and the cells on the surface of the insert 3 were removed, and the EGFP fluorescence of the cell 2 that had migrated to the back surface induced by VIP was detected with a fluorescence microscope.
• FIG. 3 shows the results of measuring the migration of MDA-MB-231 cells stably expressing EGFP or VIPR2-EGFP using a transwell chamber assay.
• VIP 200 nM
• cells were added to the inserts, incubated at 37° C. for 30 hours, and cells that migrated to the back side of the inserts were quantified and shown graphically.
• the number of cells that migrated to the back side of the insert increased, and cells expressing VIPR2-EGFP further remarkably enhanced their migration.
• Data are shown as mean ⁇ SD. *: p ⁇ 0.05, ***: p ⁇ 0.001 between indicated groups (Kruskal-Wallis test followed by Dunn's multiple comparison test).
• the results are shown in Figure 4.
• the bar graph shows the area of lamellipodia. Data are shown as mean ⁇ SD. **: p ⁇ 0.01, ***: p ⁇ 0.001 between indicated groups (Kruskal-Wallis test and Dunn's multiple comparison test). n. s. indicates not statistically significant.
• VIP stimulation expanded the area of WAVE2-rich lamellipodia of MDA-MB-231. MDA-MB-231 cells stably expressing VIPR2-EGFP showed more marked expansion of the area of lamellipodia compared to control MDA-MB-231 cells expressing EGFP.
• FIG. 5A Panels are fluorescence images of MDA-MB-231 cells expressing EGFP or VIPR2-EGFP at 1, 4 and 6 weeks in vivo.
• the graph in FIG. 5B shows the distance (distance to the furthest cell population from the administration site) between the implanted area at week 1 and the next dense area (arrowhead) at the indicated weeks.
• KS-133 Inhibits Activation of the PI3K/PI(3,4,5) P3 Pathway by VIP in MDA-MB-231 Cells Stably Expressing VIPR2-EGFP> VIP-induced phosphorylation of AKT in MDA-MB-231 cells stably expressing VIPR2-EGFP was assessed in the absence and presence of KS-133. KS-133 strongly inhibited AKT phosphorylation induced by VIP in a concentration-dependent manner. KS-133 did not affect AKT expression.
• the VIPR2 selective antagonist KS-133 suppresses the migration of MDA-MB-231 breast cancer cells stably expressing VIPR2-EGFP>
• the transwell shown in FIG. 7 was used to examine the effect of KS-133 on the migration of MDA-MB-231 breast cancer cells stably expressing VIPR2-EGFP.
• MDA-MB-231 cells stably expressing VIPR2-EGFP were suspended in serum-free medium containing KS-133 at concentrations of 0 to 1000 nM (1 ⁇ 10 4 cells/100 ⁇ L).
• FIG. 8A is a fluorescence micrograph showing migrated cells.
• FIG. 8B and Table 2 show the number of migrated cells when each concentration of KS-133 was added.
• VIP-induced cell migration of MDA-MB-231 stably expressing VIPR2-EGFP was suppressed by the addition of KS-133 (Kruskal-Wallis test, Dunn's mltiple comparison test showed that KS-133 A significant difference was observed only when 100 nM or more of KS-133 was added).
• KS-133 a selective VIPR2 antagonist
• MCF-7 cells overexpressing VIPR2-EGFP were seeded in a 35 mm culture dish at a density of 1 ⁇ 10 5 cells/dish for 6 hours, and the culture medium was mixed with 2% FBS, 100 nM VIP, and 0 or 100 nM KS-133. (day 0). Cells were observed under a BZ-X800 (Keyence, Kyoto, Japan) fluorescence microscope and counted by trypan blue dye staining every 24 hours for up to 4 days using a hemacytometer (timecourse experiment).
• BZ-X800 Keyence, Kyoto, Japan
• the VIPR2 antagonist KS-133 was added to MCF-7 cells in which VIPR2-EGFP was overexpressed (Non-Patent Document 2), and the proliferation effect of the cells was confirmed. The confirmed results are shown in FIG. 9 and Table 3. In MCF-7 cells overexpressing VIPR2, addition of KS-133 markedly inhibited cell growth from day 2 onwards of culture. On the other hand, daily cell proliferation was confirmed in the control group. This confirmed result reveals that VIP-VIPR2 signaling induces proliferation of breast cancer cells.
• a composition containing a cyclic peptide according to the present invention may be used as a drug such as a cancer metastasis inhibitor.

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