Smad-INDEPENDENT TGF-β SIGNALING PATHWAY INHIBITOR, AND COMPOSITION FOR INHIBITING CANCER INVASION AND / OR METASTASIS COMPRISING THE SAME

Abstract

To develop and provide a low-toxicity anti-cancer agent capable of inhibiting cancer invasion and metastasis.SOLUTION: A Smad-independent TGF-β signaling pathway inhibitor comprising a bicyclic compound represented by the general formula (I) or a salt thereof is provided as an active ingredient for inhibiting migration and detachment of cancer cells. [In the formula, R1 and R2 each independently represent a hydrogen atom (H), or a C1-C3 straight-chain or branched alkyl group; R3 represents a hydrogen atom (H), a C1-C3 straight-chain or branched alkyl group, a sulfonic acid group, or a sulfuric acid group; and R4 represents a C1-C3 straight-chain or branched alkyl group, a sulfonic acid group, or a sulfuric acid group.]SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an inhibitor of the Smad-independent TGF-β signaling pathway, and a composition for suppressing cancer invasion and / or metastasis containing the same. [Background technology]

[0002] Cancer (malignant tumor) is a group of cells (cancer cells) that have begun to grow abnormally as a result of normal cells escaping the body's autonomous control mechanism due to genetic abnormalities, or a disease resulting from such cells.

[0003] According to the 2022 Vital Statistics of the Ministry of Health, Labor and Welfare of Japan (Non-Patent Document 1), cancer is the leading cause of all deaths in Japan, accounting for 24.6% of all mortality. Such a high mortality rate is mainly due to the formation, invasion, and metastasis of tumor masses by cancer cells.

[0004] Cancer cells that grow abnormally in the primary lesion and form a tumor mass detach from the primary lesion, invade adjacent tissues, and invade blood vessels and lymphatic vessels. They are then transported to distant tissues by the bloodstream, etc., and adhere to the vascular endothelium around the metastatic site, before detaching from the blood vessels. At the metastatic site, they induce angiogenesis and begin to grow abnormally again, forming a metastatic lesion. In this way, cancer cells are dispersed throughout the body by infiltration and metastasis, and abnormal growth continues in the various organs to which they metastasize, causing the body to consume excess nutrients and become rapidly exhausted. In addition, normal tissues are compressed by the tumor mass, causing each organ to fail to function.

[0005] If the invasion or metastasis of cancer cells could be suppressed or inhibited, the progression of cancer could be suppressed and treatment would become possible, thereby reducing the cancer mortality rate. For this reason, many studies on suppressing or inhibiting the invasion and metastasis of cancer have been reported.

[0006] For example, it has been revealed that epithelial mesenchymal transition (EMT) is closely involved in the invasion and metastasis of epithelial tumor cells (cancer cells) (Non-Patent Document 2). Epithelial mesenchymal transition refers to a phenomenon in which epithelial cells lose their epithelial properties and acquire the properties of highly motile mesenchymal cells by detachment. In epithelial cells that have undergone EMT, the expression of E-cadherin, a transmembrane glycoprotein that plays an important role in cell adhesion and maintaining normal tissue structure, is lost due to stimulation with various growth factors. On the other hand, it is also known that the expression of N-cadherin (Neural cadherin), a mesenchymal marker that contributes to the transdermal migration of cancer cells, increases.

[0007] However, drugs that effectively suppress or inhibit cancer invasion and metastasis and have fewer side effects are still in the development stage. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ministry of Health, Labor and Welfare: Overview of Vital Statistics (confirmed figures) for 2022 (Reiwa 4): Table 6 Number of deaths, mortality rate (per 100,000 population), and composition ratio by cause of death (top 10) by sex [Non-Patent Document 2] Lai X., et al., 2020, Front Cell Dev Biol, 8:760;doi: 10.3389 / fcell.2020.00760 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to develop and provide an anticancer agent with low toxicity that can inhibit cancer invasion and metastasis. [Means for solving the problem]

[0010] Based on the knowledge of epithelial-mesenchymal transition, the present inventors considered that the development of a method for inhibiting the detachment and migration of cancer cells is important for the inhibition of cancer invasion and metastasis. As a result of intensive research to solve the problem, the present inventors obtained the new knowledge that guaiazulene, which is contained in the essential oil component of chamomile and is marketed as an anti-inflammatory drug, has an inhibitory effect on cell detachment and migration. The present invention was made based on the above new knowledge and provides the following.

[0011] (1) An inhibitor of the Smad-independent TGF-β signaling pathway, comprising a bicyclic compound represented by the following general formula (I) or a salt thereof: [ka] [In the formula, R 1 and R 2 are each independently a hydrogen atom (H) or C 1 ~C 3 R represents a linear or branched alkyl group. 3 is a hydrogen atom (H), C 1 ~C 3 R represents a linear or branched alkyl group, a sulfonic acid group, or a sulfate group. 4 is C 1 ~C 3 represents a linear or branched alkyl group, a sulfonic acid group, or a sulfate group. (2) In the general formula (I), R 1 The Smad-independent TGF-β signaling pathway inhibitor described in (1), wherein represents an ethyl group or an isopropyl group. (3) The Smad-independent TGF-β signaling pathway inhibitor according to (2), which is represented by any one of the following formulas (II) to (V): [ka] [ka] [ka] [ka] (4) An inhibitor of the Smad-independent TGF-β signaling pathway according to any one of (1) to (3), wherein the Smad-independent TGF-β signaling pathway is any one of a PKC signaling pathway, a MAPK signaling pathway, a PI3K signaling pathway, a FAK signaling pathway, and a Rac / Rho signaling pathway. (5) An inhibitor of the Smad-independent TGF-β signaling pathway according to any one of (1) to (4), wherein the inhibition of the Smad-independent TGF-β signaling pathway is any one of inhibition of F-actin (Filament actin) formation, inhibition of N-cadherin expression, and inhibition of collagen synthesis. (6) A composition for inhibiting cell detachment and / or cell migration, comprising an inhibitor of the Smad-independent TGF-β signaling pathway according to any one of (1) to (5). (7) A composition for suppressing cancer invasion and / or metastasis, comprising an inhibitor of the Smad-independent TGF-β signaling pathway according to any one of (1) to (5). Effect of the Invention

[0012] According to the Smad-independent TGF-β signaling pathway inhibitor of the present invention, it is possible to inhibit the intracellular TGF-β signaling pathway that does not pass through Smad.

[0013] The cell detachment and / or cell migration inhibiting composition of the present invention can inhibit cell detachment and / or cell migration.

[0014] The agent for suppressing cancer invasion and / or metastasis of the present invention makes it possible to suppress the progression of cancer by suppressing the invasive and / or metastatic activity of cancer cells. [Brief description of the drawings]

[0015] [Figure 1]FIG. 1 shows the effect of guaiazulene in inhibiting colony formation in cancer cells. A shows colonies of alveolar basal epithelial adenocarcinoma cell line A549 cells formed in soft agar medium on plates of each treatment group. B shows a comparison of the number of colonies on plates (a) and (b) in A. [Diagram 2] Figure 1 shows the time course results of scratch assay of A549 cells. (a) shows a negative control plate containing only DMSO, (b) shows a plate containing TGF-β, and (c) shows a plate containing guaiazulene in addition to TGF-β. In each figure, the area between the dashed lines indicates the scratch area where the cells were detached. [Diagram 3] This is a diagram showing the effect of guaiazulene in inhibiting the detachment action of human lung-derived normal fibroblast TIG-1 cells by TGF-β. (a) is a plate diagram showing the addition of DMSO as a control, (b) TGF-β alone, (c) TGF-β and a receptor inhibitor SB525334, (d) guaiazulene (Gz) alone, (e) TGF-β and guaiazulene, and (f) TGF-β and staurosporine (STS), a PKC inhibitor. [Figure 4] This figure shows the change in shape of A549 cells induced by PMA, a PKC activator. (a) shows cells treated with DMSO (control), (b) with PMA only, and (c) with PMA and guaiazulene. In the figure, the arrows indicate pseudopodia of A549 cells induced by PMA. [Diagram 5] A shows Western blotting results of each treatment group using anti-COL1 and anti-COL3 antibodies. (a) shows a sample derived from cells treated with DMSO (control), (b) with TGF-β, and (c) with TGF-β and guaiazulene. B shows the ratio of phosphorylated Smad2 to Smad2 in each treatment group, expressed as a relative value to the control.

[0016] [Figure 6-1]1 shows the inhibitory effect of guaiazulene on the induction of EMT marker expression by TGF-β, where A is the gene expression ratio of the epithelial marker E-cadherin (CDH1) and B is the gene expression ratio of the mesenchymal marker N-cadherin (CDH2) relative to the control. [Figure 6-2] 1 shows the inhibitory effect of guaiazulene on the induction of EMT marker expression by PMA, where A shows the gene expression ratio of the epithelial marker E-cadherin (CDH1) and B shows the gene expression ratio of the mesenchymal marker N-cadherin (CDH2) relative to the control. [Figure 7] FIG. 1 shows the F-actin formation inhibitory effect of guaiazulene. A shows the inhibitory effect of guaiazulene on the phosphorylation of FAK by TGF-β. B shows the inhibitory effect of guaiazulene on the F-actin formation induced by the activation of the FAK signaling pathway. In addition, in both A and B, (a) DMSO was added as a control, (b) TGF-β alone was added, (c) TGF-β and guaiazulene (Gz), and (d) TGF-β and staurosporine (STS) were added. [Figure 8] Figure 1 shows the time course results of circular wound closure assay of SAS cells. (a) shows a negative control plate containing only DMSO, (b) shows a plate containing the PKC activator PMA, and (c) shows a plate containing guaiazulene (Gz) in addition to PMA. In each figure, the area enclosed by the dashed line indicates the wound area where cells were detached. [Figure 9] This figure shows the inhibitory effect of guaiazulene on the formation of filopodia induced by TGF-β in SAS cells. (a) DMSO was added as a control, (b) TGF-β alone was added, and (c) TGF-β and guaiazulene were added. In the figure, the thorn-like extensions indicated by the arrows are filopodia. [Figure 10]This is a diagram showing the inhibitory effect of guaiazulene on the expression induction of N-cadherin (CDH2) by TGF-β or PMA in SAS cells. Fig. 10 shows the protein amount ratio of N-cadherin (CDH2), a mesenchymal marker, to the control in each sample. (a) is the case where DMSO was added as a control, (b) is the case where only TGF-β was added, (c) is the case where TGF-β and guaiazulene were added, (d) is the case where only PMA was added, and (e) is the case where PMA and guaiazulene were added. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. Smad-independent TGF-β signaling pathway inhibitors 1-1. Overview The first aspect of the present invention is an inhibitor of the Smad-independent TGF-β signaling pathway. The inhibitor of the present invention comprises a compound represented by the general formula (I) described below or a salt thereof. The inhibitor of the present invention can inhibit the Smad-independent TGF-β signaling pathway in cells.

[0018] 1-2.Definition of Terms As used herein, the following terms are defined. "TGF-β" (Transforming growth factor-β) is a cytokine that acts as a growth inhibitor on many cells. TGF-β constitutes the TGF-β superfamily along with activin, which has a similar basic structure, and BMP (bone morphogenetic protein), and functions as a ligand protein that activates the TGF-β signaling pathway.

[0019] The "TGF-β signaling pathway" is an intracellular signaling pathway that controls cell differentiation, proliferation inhibition, extracellular matrix production, and apoptosis. When the ligand TGF-β superfamily binds to two types of TGF-β receptors, type I and type II, which are transmembrane serine / threonine kinases on the cell membrane, the type II receptor phosphorylates the type I receptor. The type I receptor, activated by this phosphorylation, further phosphorylates the intracellular substrate, which is an intracellular signaling factor, thereby transmitting the signal into the cell. There are two known TGF-β signaling pathways: the Smad-dependent TGF-β signaling pathway and the Smad-independent TGF-β signaling pathway.

[0020] The "Smad-dependent TGF-β signaling pathway" (Smad / TGF-β signaling pathway) is a pathway mediated by Smad proteins as intracellular substrates of the type I receptor. It is the main pathway in the TGF-β signaling pathway and is also called the canonical-TGF-β signaling pathway or the Smad / TGF-β signaling pathway.

[0021] The "Smad-independent TGF-β signaling pathway" (non-Smad / TGF-β signaling pathway) is a pathway mediated by proteins other than Smad proteins as intracellular substrates of type I receptors. The Smad-dependent TGF-β signaling pathway branches off from the TGF-β receptor and transmits signals downstream via different intracellular signaling factors. There are several types of Smad-independent TGF-β signaling pathways. The Smad-independent TGF-β signaling pathway in the present invention may be any type, and is not particularly limited, but is preferably a pathway that controls the activity and expression of cytoskeleton and adhesion molecules, and is involved in cell elongation and contraction, invasion, and adhesion to extracellular matrix. Specific examples include the PKC (Protein kinase C) signaling pathway, the MAPK (Mitogen-activated Protein Kinase) signaling pathway, the PI3K (Phosphoinositide 3-kinase) signaling pathway, the FAK (Focal adhesion kinase) signaling pathway, and the Rac / Rho signaling pathway.

[0022] As used herein, the term "cell detachment" refers to the release of cells from surrounding tissues and the like due to a decrease or loss of cell adhesive ability caused by, for example, a decrease in the expression of E-cadherin.

[0023] As used herein, the term "cell migration" refers to a state in which a cell released from an adherent state by detachment acquires motility and enters a migratory state.

[0024] As used herein, the term "subject" refers to a living body to which a drug or composition is applied. Examples of such a body include humans, livestock, racehorses, pets, and laboratory animals. Preferably, the body is a human (in this case, the body is specifically referred to as a "subject").

[0025] As used herein, "information on a subject" refers to various individual information on the living body to which the invention is applied, and in the case of a subject, includes the overall health condition, the progression and severity of a disease or injury if present, age, weight, sex, diet, drug sensitivity, the presence or absence of concomitant medication, and resistance to treatment.

[0026] 1-3.Configuration The Smad-independent TGF-β signaling pathway inhibitor of the present invention comprises a bicyclic compound represented by the following general formula (I) and a salt thereof.

[0027] [ka]

[0028] In the above formula, R 1 and R 2 are each independently a hydrogen atom (H) or C 1 ~C 3 R represents a linear or branched alkyl group. 3 is a hydrogen atom (H), C 1 ~C 3 R represents a linear or branched alkyl group, a sulfonic acid group, or a sulfate group. 4 is C 1 ~C 3 represents a linear or branched alkyl group, a sulfonic acid group, or a sulfate group.

[0029] Said “C 1 ~C 3 Specific examples of the linear or branched alkyl group of R 1 is preferably an ethyl group or an isopropyl group.

[0030] Among the bicyclic compounds represented by the general formula (I), guaiazulene represented by the following formula (II), chamazulene represented by the formula (III), egualene represented by the formula (IV), and guaiazulene sulfonic acid represented by the formula (V) are particularly preferred as Smad-independent TGF-β signaling pathway inhibitors of the present invention, without being limited thereto.

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] [ka]

[0035] "Guaiazulene" is C 15 H 18 It is a bicyclic sesquiterpene having the molecular formula: and is a deep blue crystalline hydrocarbon represented by the above formula (II). It is contained in the resin of Guaiacum officinale and the essential oil of German chamomile (Matricaria recutita), and has anti-inflammatory, anti-allergic, and ultraviolet absorbing properties, and is almost free of skin irritation, skin sensitization, and cytotoxicity, and is therefore used as an active ingredient in anti-inflammatory drugs for stomatitis, dermatitis, ulcers, etc., and as an ingredient in cosmetics.

[0036] "Camazulene" is C 14 H 16It is a bicyclic sesquiterpene having the molecular formula: and is a blue-purple crystalline hydrocarbon represented by the formula (III). It is contained in the essential oils of German chamomile and Artemisia absinthium, and has an anti-inflammatory effect similar to guaiazulene.

[0037] "Egualen" is C 15 H 17 O 3 It is a bicyclic compound with the molecular formula S.

[0038] "Guaiazulene sulfonic acid" is a C 15 H 18 O 3 It is a bicyclic compound with the molecular formula S.

[0039] In the present specification, the term "salt thereof" refers to a salt of a compound represented by general formula (I) or formulas (II) to (V), preferably a pharma-ceutically acceptable non-toxic salt. For example, inorganic acid salts such as sulfonate, sulfate, hydrochloride, nitrate, phosphate, carbonate, hydrogencarbonate or perchlorate, organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate or ascorbate, etc. For example, but not limited to, egualen sodium represented by the following formula (VI) and azulenesulfonic acid sodium represented by formula (VII) are included.

[0040] [ka]

[0041] [ka]

[0042] "Egualen sodium" is the sodium salt of egualen, and its hydrate is known as the pharmaceutical drug Azuloxa, which is an anti-gastric ulcer drug.

[0043] "Sodium azulene sulfonate" is the sodium salt of guaiazulene sulfonic acid, the hydrate of which is known as the pharmaceutical azulene. Azulene is used as a treatment for oral inflammation and wounds, such as pharyngitis, tonsillitis, stomatitis, acute gingivitis, and glossitis.

[0044] 2. Cell detachment and migration inhibitor composition 2-1. Overview A second aspect of the present invention is a cell detachment / migration inhibitory composition. The composition of the present invention is characterized in that it contains the Smad-independent TGF-β signaling pathway inhibitor described in the first aspect as an active ingredient. The cell detachment / migration inhibitory composition of the present invention can inhibit the detachment and / or migration of cells, particularly cancer cells.

[0045] 2-2.Configuration 2-2-1. Constituent factors The composition of this embodiment contains an active ingredient as an essential component and a solvent and / or a carrier as optional components. Each of the components will be specifically described below.

[0046] (1) Active ingredient The composition contains the Smad-independent TGF-β signaling pathway inhibitor described in the first embodiment as an essential active ingredient. Here, the composition may contain one or two or more different Smad-independent TGF-β signaling pathway inhibitors. For example, the composition may contain two types of inhibitors, guaiazulene and chamazulene, or three types, sodium guaiazulene sulfonate, sodium egualene, and chamazulene. The composition may also contain other active ingredients that have medicinal properties that are effective in inhibiting cell detachment and / or cell migration, or in suppressing or treating other diseases.

[0047] The content of the Smad-independent TGF-β signaling pathway inhibitor contained in the present composition varies depending on the type and / or effective amount thereof, the dosage form of the composition, the type of carrier or additive described below, and the type of disease to which the composition is administered, and therefore may be determined appropriately taking into account each of the conditions.

[0048] As used herein, the term "effective amount" refers to an amount necessary for the Smad-independent TGF-β signaling pathway inhibitor to function as an active ingredient in the composition, and an amount that causes little or no harmful side effects in the living body to which it is applied. This effective amount may vary depending on various conditions such as subject information, administration route, and number of administrations. In addition, the effective amount is sufficient if the total amount of the active ingredient administered per day (daily dosage unit) reaches that amount. Therefore, the composition can be administered multiple times per day as long as the daily dosage unit of the active ingredient is within a specified range.

[0049] The effective amount of the Smad-independent TGF-β signaling pathway inhibitor contained in the composition is not limited. For example, in the case of guaiazulene sulfonate such as azulene, the LD50 in intraperitoneal administration is known to be very safe, with 105 mg / kg in mice and 180 mg / kg in rats (https: / / medical.nihon-generic.co.jp / uploadfiles / medicine / AZUGG IF 1002.pdf). Therefore, for humans, the daily dosage unit may be 5 mg or more but less than 30 mg, 6 mg or more but less than 25 mg, 7 mg or more but less than 24 mg, 8 mg or more but less than 23 mg, 9 mg or more but less than 22 mg, 10 mg or more but less than 21 mg, 11 mg or more but less than 20 mg, 12 mg or more but less than 19 mg, 13 mg or more but less than 18 mg, 14 mg or more but less than 17 mg, 15 mg or more but less than 16 mg, or 10 mg or more but less than 20 mg.

[0050] (2) Solvent The composition may contain a pharma- ceutically acceptable solvent. The solvent is an optional component of the composition and may be added as necessary. "Pharmaceutically acceptable" means that the solvent is harmless or has low toxicity to the living body, can be generally used in the technical field of formulations, and can preferably be used in pharmaceutical compositions.

[0051] Examples of the solvent include water or an aqueous solution, or an organic solvent. Examples of the pharma- ceutically acceptable aqueous solution include physiological saline, an isotonic solution containing glucose or other auxiliary agents, a phosphate buffer, and a sodium acetate buffer. Examples of the auxiliary agent include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Examples of the pharma-ceutically acceptable organic solvent include ethanol, butanol, etc.

[0052] (3) Carrier The composition may include a pharma- ceutically acceptable carrier, which is an optional component of the composition and may be added if desired.

[0053] Examples of the carrier include suspending agents, diluents, solubilizing agents, dispersing agents, surfactants, emulsifying agents, soothing agents, stabilizers, preservatives, antiseptics, antioxidants, buffers, and isotonic agents.

[0054] In addition to the above, if necessary, the composition may appropriately contain excipients, fillers, binders, disintegrants, absorption enhancers, bulking agents, moisturizing agents, humectants, adsorbents, disintegration inhibitors, coating agents, colorants, and the like that are commonly used in pharmaceuticals.

[0055] Such carriers are primarily used to facilitate the formation of dosage forms and maintain the dosage form and pharmaceutical effects, as well as to make the active ingredient, the fermented product, less susceptible to decomposition in the body, and may be used appropriately as needed.

[0056] 2-2-2. Dosage form The dosage form of the composition is not particularly limited as long as it does not inactivate the active ingredient, the Smad-independent TGF-β signaling pathway inhibitor, and can exert the pharmacological effect of the active ingredient in vivo after administration. The specific dosage form of the composition may be appropriately selected depending on the administration method and / or prescription conditions. Generally, the administration method can be broadly divided into parenteral administration and oral administration, and the composition may be in a dosage form suitable for each administration method.

[0057] (1) Dosage forms for parenteral administration Parenteral administration can be divided into systemic administration and local administration, and local administration can be further divided into intratissue administration, transdermal administration, transmucosal administration, and transrectal administration. The dosage form for parenteral administration may be administered by an appropriate administration method known in the art according to each administration method. For example, a dosage form suitable for intratissue administration in systemic administration or local administration can be an injection, which is a liquid. Dosage forms suitable for transdermal or transmucosal administration can be liquids (including liniments, eye drops, nasal drops, and inhalants), suspensions (including emulsions and creams), powders (including nasal drops and inhalants), pastes, gels, ointments, plasters, etc. Dosage forms suitable for rectal administration can be suppositories, etc.

[0058] (2) Oral dosage form In the case of oral administration, the dosage form includes solid agents (including tablets, pills, sublingual agents, capsules, and drops), granules, powders, powders, liquid agents (including oral water agents, suspensions, emulsions, and syrups), etc. The solid agents or granules can be made into dosage forms with coatings known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, if necessary.

[0059] 2-3. Method of administration The method of administration of the composition can be appropriately selected depending on the type of disease to be administered, its symptoms, or the progress of the disease. The administration method includes parenteral administration and oral administration as described above, and the composition may be administered by either method. Parenteral administration can be further divided into local administration and systemic administration. Oral administration is generally systemic administration since it is absorbed through the oral mucosa or intestinal mucosa and then passes through the circulatory system. In the case of the composition, it may be either local administration, systemic administration, or a combination thereof. Considering that the action effect of the composition is inhibition of cell detachment and / or migration, and that mesenchymal cells that have been detached from a tumor mass by epithelial-mesenchymal transition and have acquired motility mainly move through the circulatory system, systemic administration is preferred, although not limited thereto, and parenteral administration in which the composition is administered directly into the circulatory system, such as intravascular injection, is particularly preferred.

[0060] 2-4. Use and application area The use and application site of the composition of this embodiment are not particularly limited. Since the effect of the composition is to inhibit cell detachment and / or migration, it may be applied to a use or site according to the purpose. For example, as in the third embodiment described below, it can be used as a pharmaceutical composition for suppressing cancer invasion and / or metastasis.

[0061] 3. Cancer invasion and metastasis inhibitors 3-1. Overview The third aspect of the present invention is a composition for suppressing cancer invasion and metastasis. The composition of the present invention is characterized in that it contains the Smad-independent TGF-β signaling pathway inhibitor described in the first aspect as an active ingredient. The composition for suppressing cancer invasion and metastasis of the present invention can be understood as a composition in which the target cells to which the cell detachment and cell migration inhibiting composition of the second aspect is applied are specified as cancer cells. Therefore, the composition of this aspect can suppress cancer invasion and metastasis by inhibiting the detachment and migration of cancer cells.

[0062] 3-2.Configuration The composition of this embodiment has the same basic structure as the cell detachment / cell migration inhibition composition of the second embodiment, except that the target of application is cancer and the use is different, that is, to inhibit the invasion and metastasis of the cancer. Therefore, the description thereof will be omitted here.

[0063] 3-3. Method of administration The composition of this embodiment may be administered basically in the same manner as described in "2-3. Administration Method" for the cell detachment / cell migration inhibition composition of the second embodiment. That is, although not limited thereto, systemic administration is preferred, and parenteral administration in which the composition is administered directly into the circulatory system, such as by intravascular injection, is particularly preferred. However, since the composition of this embodiment is intended to inhibit cancer invasion and / or metastasis, direct local administration by injection into the primary and / or metastatic lesions is also preferred, either together with or independently of the above administration.

[0064] 3-4. Use and application area The composition of this embodiment is used to inhibit cancer invasion and metastasis, and therefore is intended to be used essentially against cancer (malignant tumors).

[0065] The type of cancer to which the composition of this embodiment is applied is not particularly limited. For example, it may be any of epithelial tumors (cancer), non-epithelial tumors (sarcoma), and hematopoietic tumors. Preferably, it is an epithelial tumor (cancer).

[0066] There is no limitation on the type of epithelial tumor to which the composition of this embodiment is applied, and it may be, for example, any of skin cancer, brain cancer, tongue cancer, pharyngeal cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, thyroid cancer, breast cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, cervical cancer, and ovarian cancer.

[0067] The specific application site varies depending on the type of cancer. For example, when used for gastric cancer, the stomach, particularly the tumor site, is the main application site. EXAMPLES

[0068] Example 1: Inhibition of colony formation of cancer cells by guaiazulene (the purpose) The cancer cell proliferation inhibitory effect of the Smad-independent TGF-β signaling pathway inhibitor of the present invention will be verified. Epithelial cells grow in an anchorage-dependent manner, but epithelial cancer cells can grow in an anchorage-independent manner. Since soft agar does not serve as a scaffold, normal epithelial cells cannot grow when cells are seeded in soft agar medium, but epithelial cancer cells can grow and form colonies. By utilizing this property, we confirmed whether the growth of cancer cells is inhibited in soft agar medium to which the Smad-independent TGF-β signaling pathway inhibitor of the present invention has been added.

[0069] (material) The cells used were A549 cells, an alveolar basal epithelial adenocarcinoma cell line, and 100 μM guaiazulene (Gz) (Tokyo Chemical Industry Co., Ltd.) dissolved in DMSO was used as an inhibitor of the Smad-independent TGF-β signaling pathway.

[0070] (method) (1) Preparation of base agar 1% agar was dissolved in pure water, cooled to 40℃, and mixed with an equal amount of 2xDMEM (Nacalai Tesque) containing 20% ​​FBS (Nichirei Biosciences) and antibiotics (penicillin-streptomycin mixed solution (PC+SM) (Nacalai Tesque)) kept at 40℃. 1.5mL of the solution was added to each of four 35mm plates, cooled and solidified to prepare 10% FBS / PC+SM / 1xDMSO / 0.5% Agar medium.

[0071] (2) Preparation of top agarose A549 cells were cultured in 10% FBS / PC+SM / 1×DMEM medium at a cell count of 2×10 5 The cell suspension was adjusted to a cell density of 100 cells / mL. 0.1 mL of the cell suspension was gently mixed with 3 mL of 20% FBS / PC+SM / 2×DMEM and 3 mL of 0.7% agarose, which had been kept at 40°C, and then 1.5 mL of each mixture was placed on each of the base agar plates prepared in (1).

[0072] (3) Cell culture Cells were cultured in a humidified incubator at 37 °C for 4 weeks, with 10% FBS / 1×DMEM medium added to each plate once or twice a week. For the added medium, DMSO was added as a control in (a), guiazulene was added at a final concentration of 100 μM in (b) and (d), and after standing for 1 hour, TGF-β was added at a final concentration of 5 ng / mL in (c) and (d).

[0073] (4) Crystal violet staining After culturing, the culture medium was discarded, and a 0.1% crystal violet solution was added to each plate, and the gel was rotated for staining for about 20 minutes. After staining, each plate was washed 4 - 5 times with sterile water and then photographed using a gel imager with a white light filter.

[0074] (Results) The results are shown in Figure 1. A shows the images of each plate after crystal violet staining. A549 cells in soft agar medium formed colonies with only DMSO in (a) and only TGF-β in (c), but when guiazulene was added, the number of colonies decreased at a visually detectable level in both plates (b) and (d). B shows a graph comparing the number of colonies on plates (a) and (b) of A. In (b) with added guiazulene, the number of colonies decreased to less than 15% of that in (a). This result suggests that guiazulene has an inhibitory effect on the growth of cancer cells.

[0075] In addition, cell migration in soft agar medium induced by TGF-β was also confirmed in plate (c) (arrow), but was inhibited in (d).

[0076] (Example 2: Inhibitory effect of guiazulene on cell migration in A549 cells) (Objective) Verify the inhibitory effect of guiazulene on cell migration by scratch assay. The scratch assay is an experiment to examine the effect of surrounding cells migrating to fill (close) the gap created when part of cells cultured on a plate is scratched. It is known that cells that have undergone epithelial-mesenchymal transition due to TGF-β induction have increased cell migration activity, and therefore fill the gap more quickly. Using these properties, we will confirm that guaiazulene has an inhibitory effect on cell migration.

[0077] (method) 2×10 5 A549 cells were seeded in a 6-well plate and incubated at 5% CO 2 After culturing in an incubator at 37°C for 24 hours, DMSO was added to (a) and guaiazulene was added to (c) at a final concentration of 100 μM, and then TGF-β was added to (b) and (c) at a final concentration of 5 ng / mL. After physically peeling off the cells proliferating on the bottom surface of the plate, the cells on the plate were photographed (0 hours) and cultured again. Photographs were taken in the same way 24 and 48 hours after culture. The area of ​​the detached region (scratch area) was measured from the photographed image data, and relative values ​​were calculated for each time point, with the time point at which culture was 0 being set as 100%.

[0078] (result) The results are shown in FIG.

[0079] [Table 1]

[0080] Figure 2 shows the cells on the plate at each incubation time after scratch assay, and Table 1 shows the scratch area on each plate at each incubation time as a relative value to the zero-hour incubation time.

[0081] When TGF-β was added (b), cell migration was activated and the area of ​​the scratch area decreased over time, but when guaiazulene was added to TGF-β (c), the area of ​​the scratch area became similar to that of the control, demonstrating that guaiazulene completely inhibited TGF-β-induced cell migration.

[0082] These results suggest that guaiazulene has the activity of inhibiting the migration of cancer cells.

[0083] Example 3: Inhibitory effect of guaiazulene on cell detachment caused by TGF-β (the purpose) TGF-β weakens the adhesion between normal cells and plates, making the cells more likely to detach from the plates. Since guaiazulene inhibited cell migration caused by TGF-β in Example 2, we will confirm whether it can also inhibit the cell detachment action caused by TGF-β.

[0084] (method) 2x10 cells cultured in 10% FBS / PC+SM / DMEM 5 TIG-1 cells (human lung-derived normal fibroblasts) were seeded on a 35 mm Cell+ Cell Culture Plate (Sarstedt) with a special surface treatment and incubated at 37 °C for 1 h in 5% CO. 2 After culturing in an incubator at 37°C for 24 hours, DMSO was added as a control to (a), the TGF-β receptor inhibitor SB525334 was added to (c) at a final concentration of 10 μM, guaiazulene was added to (d) and (e) at a final concentration of 100 μM, and the PKC inhibitor staurosporine (STS) was added to (f) at a final concentration of 5 nM. After leaving the cells to stand for 1 hour, TGF-β was added to (b), (c), (e), and (f) at a final concentration of 5 ng / mL. After culturing for another 16 hours, the state of the cells on each plate was photographed.

[0085] (result) The results are shown in Figure 3. As is clear from (b), TIG-1 cells cultured on the Cell+ Cell Culture Plate were detached from the plate by the addition of TGF-β. This cell detachment action caused by TGF-β was completely inhibited by the addition of the TGF-β receptor inhibitor SB525334 (c) or the PKC inhibitor staurosporine (f). On the other hand, the result in (e) revealed that guaiazulene can also inhibit the cell detachment action caused by TGF-β, similar to SB525334 (c) and staurosporine (f).

[0086] These results suggest that guaiazulene interferes somewhere in the PKC / TGF-β signaling pathway.

[0087] Example 4: Inhibitory effect of guaiazulene on the PKC / TGF-β signaling pathway (the purpose) Determine where in the PKC / TGF-β signaling pathway guaiazulene exerts its inhibitory effect.

[0088] (method) 2x10 cells cultured in 10% FBS / PC+SM / DMEM 5 A549 cells were seeded onto a 35 mm plate and incubated at 4 °C for 30 min at 5% CO. 2 After 24 hours of incubation at 37°C in an incubator, DMSO was added to (a) and guaiazulene was added to (c) at a final concentration of 100 μM. After standing for 1 hour, PMA (phorbol 12-myristate 13-acetate) was added to (b) and (c) at 10 ng / mL. PMA is a PKC activator and is known to enhance cell migration (Sumagin R., et al., 2013, PLOS ONE, https: / / doi.org / 10.1371 / journal.pone.0055775). After culturing for another 5 hours, the state of the cells on each plate was photographed using an inverted microscope.

[0089] (result) The results are shown in Figure 4. In (b), the activation of PKC by PMA caused the formation of pseudopodia (arrows) in A549 cells, while in (c), the addition of guaiazulene almost completely suppressed the formation of pseudopodia. These results suggest that guaiazulene can interfere with the pathway after PKC in the PKC / TGF-β signaling pathway and inhibit cell migration activated by PKC.

[0090] <Example 5: Inhibitory effect of Smad-independent TGF-β signaling pathway by guaiazulene> (the purpose) In the TGF-β signaling pathway, various signaling pathways can be activated in addition to the Smad-dependent TGF-β signaling pathway (Smad / TGF-β signaling pathway) mediated by Smad proteins downstream of the receptor. In Example 4, since guaiazulene inhibited the PKC / TGF-β signaling pathway not mediated by Smad, it was confirmed whether or not the Smad-dependent TGF-β signaling pathway can be inhibited, and whether or not other Smad-independent TGF-β signaling pathways can be inhibited in the same manner as the PKC / TGF-β signaling pathway.

[0091] (method) 2x10 cells cultured in 10% FBS / PC+SM / DMEM 5 A549 cells were seeded onto a 35 mm plate and incubated at 4 °C for 30 min at 5% CO. 2 After culturing in an incubator at 37°C for 24 hours, DMSO was added as a control to (a) and guaiazulene was added to (c) at a final concentration of 100 μM. After leaving the cells to stand for 1 hour, TGF-β was added to (b) and (c) at a final concentration of 5 ng / mL.

[0092] After culturing for another 24 hours, the cells were washed with PBS and dissolved in sample buffer (0.125M Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 10% 2-mercaptoethanol, bromophenol blue) to prepare a sample solution. Next, the cells were heat-treated at 90°C and applied to an 8% polyacrylamide gel for electrophoresis. After electrophoresis, the separated proteins were transferred to a PVDF membrane using a semi-dry transfer device.

[0093] After blocking with EzBlock Chemi (ATTO) blocking agent for 1 hour at room temperature, primary antibodies were added and incubated overnight at 4°C to carry out the primary antibody reaction. The dilution rates of each antibody added were as follows: Mouse anti-COL1 (type I collagen) monoclonal antibody (R&D); 500:1 Rabbit anti-COL3 (type III collagen) polyclonal antibody (Proteintech); 500:1 Rabbit anti-tubulin polyclonal antibody (Proteintech); 1000:1 Rabbit anti-phosphorylated Smad2 rabbit polyclonal antibody (Affinity); 500:1 Goat anti-Smad2 polyclonal antibody (R&D); 1000:1

[0094] After the primary antibody reaction, the membrane was washed three times with TBS-T, and then the secondary antibody reaction was carried out at room temperature for 1 hour using an HRP-conjugated secondary antibody for each antigen animal.

[0095] After chemiluminescence using Immobilon HRP substrate (Merck) and imaging with an imager (Thermo Fisher Scientific), the ratio of phosphorylated Smad2 protein / Smad2 protein was calculated relatively by measuring the intensity of each band using ImageJ.

[0096] (result) The results are shown in Figure 5. A is a Western blotting diagram using anti-COL1 antibody and anti-COL3 antibody for each treatment group. (b) Type I collagen (COL1) and type III collagen (COL3) induced in A549 cells by the addition of TGF-β were suppressed in expression by the addition of guaiazulene (Gz) as shown in (c). It is known that the expression of type I and type III collagen by TGF-β is induced by activation of the MAPK signaling pathway or the PI3K signaling pathway that does not involve Smad in the TGF-β signaling pathway (Kimoto K., et al., 2004, Invest Ophthalmol Vis Sci, 4:2431-7; Hu X., et al.,2020, Laboratory Investigation, 100:801-811).

[0097] B shows the ratio of phosphorylated Smad2 to Smad2 in each treatment group, expressed as a relative value to the control. Smad2 was phosphorylated by the addition of TGF-β, but the phosphorylation was suppressed by SB525334 (SB), a TGF-β receptor inhibitor. On the other hand, guaiazulene could not suppress this phosphorylation.

[0098] These results suggest that guaiazulene does not inhibit the Smad-dependent TGF-β signaling pathway, but inhibits various Smad-independent TGF-β signaling pathways that are not mediated by Smads.

[0099] Example 6: Inhibitory effect of guaiazulene on transcription induction of EMT markers (the purpose) We confirm that guaiazulene can suppress the transcriptional induction of EMT markers induced by TGF-β and PMA.

[0100] (method) For induction by TGF-β, 2x10 cells were cultured in 10% FBS / PC+SM / DMEM. 5 A549 cells were seeded onto three 35 mm plates and incubated in 5% CO 2After culturing in an incubator at 37°C for 24 hours, DMSO was added as a control to (a) and guaiazulene was added to (c) at a final concentration of 100 μM. After leaving the cells to stand for 1 hour, TGF-β was added to (b) and (c) at a final concentration of 5 ng / mL.

[0101] In addition, for PMA induction, 2x10 cells cultured in 10% FBS / PC+SM / DMEM were used. 5 A549 cells were seeded onto five 35 mm plates and incubated in 5% CO 2 After culturing in an incubator at 37°C for 24 hours, DMSO was added as a control to (a), guaiazulene was added to (c) at a final concentration of 100 μM, U0126, a highly selective inhibitor of MAPK / ERK kinases MEK1 and MEK2, was added to (d) at a final concentration of 10 μM, and staurosporine, a PKC inhibitor, was added to (e) at a final concentration of 5 nM. After leaving the cells to stand for 1 hour, PMA was added to (b) to (e) at a final concentration of 10 ng / mL.

[0102] In both cases, the cells were cultured again for 24 hours after induction, and the cells were collected. Total RNA was extracted from the cells of each treatment group using RNA iso plus (Takara Bio Inc.), and then reverse transcription reaction was performed using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo Co., Ltd.) to prepare cDNA. The expression of E-cadherin (CDH1), an epithelial marker, and N-cadherin (CDH2), a mesenchymal marker, was measured by real-time PCR using the synthesized cDNA as a template. Real-time PCR was performed using THUNDERBIRD (registered trademark) SYBR (registered trademark) qPCR Mix (Toyobo Co., Ltd.) with an ABI 7500fast real-time PCR system (Thermo Fisher Scientific Co., Ltd.). The PCR conditions were an initial denaturation step at 95°C for 20 seconds, and a PCR step of 95°C for 3 seconds and 60°C for 30 seconds, with 40 cycles performed. The gene expression ratio of each treatment group relative to the control was calculated using the ΔΔCt method with the 18S rRNA gene as the reference.

[0103] (result) The results are shown in Figure 6-1 and Figure 6-2. In both figures, A shows the gene expression ratio of E-cadherin (CDH1) and B shows the gene expression ratio of N-cadherin (CDH2) relative to the control.

[0104] In Figure 6-1, the results in A show that guaiazulene (Gz) did not affect the expression of the epithelial marker E-cadherin, but as shown in the results in B, it significantly suppressed the expression of the mesenchymal marker N-cadherin induced by TGF-β.

[0105] In addition, in Figure 6-2, the results in A show that the expression of the epithelial marker E-cadherin, which was significantly suppressed by PMA, could not be restored by the addition of guaiazulene (Gz). On the other hand, the results in B show that the expression of the mesenchymal marker N-cadherin, which was significantly induced by PMA, was significantly suppressed by guaiazulene (Gz).

[0106] These results indicate that guaiazulene does not affect the expression of the epithelial marker E-cadherin, but significantly inhibits the expression of the mesenchymal marker N-cadherin, suggesting that guaiazulene may be an inhibitor of epithelial-mesenchymal transition (EMT).

[0107] <Example 7: Inhibitory effect of guaiazulene on F-actin formation> (the purpose) Confirm that guaiazulene can inhibit the formation of F-actin. Activation of the FAK signaling pathway by TGF-β causes actin protein polymerization and the formation of F-actin (filamentous actin) within the cell. We will confirm whether the phosphorylation of FAK by TGF-β is suppressed by the addition of guaiazulene, and whether guaiazulene can inhibit the formation of F-actin by activation of the FAK signaling pathway.

[0108] (method) 2x10 cells cultured in 10% FBS / PC+SM / DMEM 5A549 cells were seeded onto a 35 mm plate and incubated at 4 °C for 30 min at 5% CO. 2 After culturing in an incubator at 37°C for 24 hours, DMSO was added as a control to (a), guaiazulene (Gz) was added to (c) at a final concentration of 100 μM, and staurosporine (STS) was added to (d) at a final concentration of 10 ng / mL. After leaving the cells to stand for 1 hour, TGF-β was added to (b) to (d) at a final concentration of 5 ng / mL. After culturing for another 2 hours, the cells were washed with PBS.

[0109] The phosphorylation of FAK was verified by Western blotting. The basic protocol for Western blotting was similar to the method described in Example 5, so a detailed explanation will be omitted here. The dilution rates of each primary antibody added were as follows: Rat anti-phosphorylated FAK monoclonal antibody (R&D); 500:1 Rat anti-FAK monoclonal antibody (Biolegend); 1000:1 Mouse anti-GAPDH monoclonal antibody (Wako); 2000:1

[0110] For F-actin staining, the washed cells were fixed with 4% paraformaldehyde-PBS at room temperature for 15 minutes, washed with PBS, and treated with PBS-T for 5 minutes. Phalloidin (Phalloidin-iFluor 488: Cayman) was then added at a dilution rate of 1500:1 and treated in the dark at room temperature for 1 hour. After washing with PBS, the cells were mounted on a slide glass and observed under a confocal fluorescence microscope, and images were taken.

[0111] (result) The results are shown in Figure 7. A shows the ratio of phosphorylated FAK to FAK in each treatment group, expressed as a relative value to the control. FAK was phosphorylated by the addition of TGF-β, but this phosphorylation could be suppressed by the addition of guaiazulene and also by the PKC inhibitor staurosporine (STS).

[0112] B shows images of phalloidin fluorescence in cells in each treatment group. In (b), F-actin formation induced by the addition of TGF-β was confirmed by phalloidin staining, but in plates further containing guaiazulene (c) or staurosporine (d), F-actin formation was inhibited.

[0113] These results suggest that guaiazulene can also interfere with the FAK / TGF-β signaling pathway, inhibiting the phosphorylation of FAK induced by TGF-β and suppressing the formation of F-actin activated by phosphorylated FAK.

[0114] Example 8: Inhibitory effect of guaiazulene on cell migration in SAS cells (the purpose) In Examples 1 to 7, the effect of guaiazulene was examined using A549 cells, which are an alveolar basal epithelial adenocarcinoma cell line. In the following Examples 8 to 10, the effect of guaiazulene was examined using SAS (squamous carcinoma), which is a tongue cancer-derived poorly differentiated squamous cell carcinoma cell line.

[0115] Tongue squamous cell carcinoma is the most common type of oral squamous cell carcinoma. It is known to be highly malignant because it is highly progressive and involves lymph node metastasis and distant organ metastasis. It is induced by chronic chemical stimuli such as drinking and smoking, and chronic mechanical stimuli such as constant contact due to misaligned teeth, and the number of cases has been increasing in recent years.

[0116] In this example, first, the cell migration inhibitory effect of guaiazulene is verified using a circular wound closure assay.

[0117] In the circular wound closure assay, cells are cultured on a plate until they reach confluence. Then, a part of the cells on the plate is scraped in a circular shape to create a gap (circular wound), and the experiment examines whether the surrounding cells migrate to fill the gap (close the wound). This is similar to the scratch assay described in Example 2.

[0118] (method) 2×10 5 SAS cells were seeded in a 6-well plate and incubated at 5% CO 2 The cells were cultured in an incubator at 37°C until confluence was reached. Then, the tip of a 10μL tip was pressed vertically against the cells in the center of the plate to aspirate, creating a circular wound, and the cells on the plate were photographed at 0 hours. Next, DMSO for control was added to (a), PMA, a PKC activator, was added to (b) and (c) at a final concentration of 10ng / mL, and guaiazulene was added to (c) at a final concentration of 100μM, and the cells were cultured again, and photographed in the same manner after 6 and 8 hours of culture. From the photographed image data, the area of ​​the wound area was measured, and the relative value at each elapsed time was calculated, with the time of culture at 0 hours being 100%.

[0119] (result) The results are shown in FIG.

[0120] [Table 2]

[0121] Figure 8 shows the cells on the plate at each incubation time after the circular wound closure assay, and Table 2 shows the wound area on each plate at each incubation time relative to the area at 0 hours of incubation.

[0122] When PMA was added (b), cell migration was activated and the area of ​​the wound area decreased over time compared to the control (a). However, when guaiazulene was added to PMA (c), no decrease in the area of ​​the wound area was observed, demonstrating that cell migration was completely inhibited.

[0123] The results of the scratch assay using A549 cells in Example 2 and the results of the present application suggest that guaiazulene has activity to inhibit cell migration regardless of the type of cancer cells.

[0124] <Example 9: Inhibitory effect of guaiazulene on filopodia formation> (the purpose) Since cell migration was inhibited by guaiazulene in Example 8, it was confirmed whether or not guaiazulene inhibits the formation of filopodia, which are extended from lamellipodia by actin polymerization in the leading edge of migrating cells in response to TGF-β.

[0125] (method) 2×10 5 SAS cells were seeded in a 6-well plate containing 10% FBS / PC+SM / DMEM and incubated at 5% CO 2 The cells were cultured in an incubator at 37°C for 24 hours. Then, DMSO was added as a control to (a) and guaiazulene was added to (c) at a final concentration of 100 μM and allowed to stand for 1 hour. TGF-β was added to (b) and (c) at a final concentration of 5 ng / mL, and the cells were fixed with 4% paraformaldehyde / phosphate buffer (Wako) after 2 hours. F-actin staining was performed according to the method described in Example 7.

[0126] (result) The results are shown in Figure 9. In the figure, some of the filopodia are indicated by arrows. In the control (a), some filopodia were formed. On the other hand, in (b), the addition of TGF-β activated the FAK signaling pathway, which polymerized actin protein and formed F-actin within the cells, resulting in a significant increase in filopodia. However, the results in (c) show that guaiazulene almost completely inhibited the formation of filopodia enhanced by TGF-β.

[0127] These results suggest that guaiazulene inhibited the formation of filopodia in SAS cells as well as in A549 cells, by suppressing the formation of F-actin induced by TGF-β, as in the results of Example 7.

[0128] Example 10: Inhibitory effect of guaiazulene on the induction of EMT marker expression (the purpose) We will confirm that guaiazulene can suppress the expression of EMT markers induced by TGF-β and PMA at the protein level in SAS cells.

[0129] (method) For induction by TGF-β, 2x10 cells were cultured in 10% FBS / PC+SM / DMEM. 5 SAS cells were seeded onto five 35 mm plates and incubated in 5% CO 2 After culturing in an incubator at 37°C for 24 hours, DMSO was added as a control to (a) and guaiazulene was added to (c) at a final concentration of 100 μM and allowed to stand for 1 hour, after which TGF-β was added to (b) and (c) at a final concentration of 5 ng / mL.

[0130] For PMA induction, cells were seeded onto two 35 mm plates and incubated in 5% CO 2 After culturing in an incubator at 37° C. for 24 hours, guaiazulene was added to (e) at a final concentration of 100 μM and allowed to stand for 1 hour, after which PMA was added to (d) and (e) at a final concentration of 10 ng / mL.

[0131] After induction, the cells were cultured for 24 hours again and then harvested. Next, the cells from each test group were dissolved in a detergent-containing sample buffer (2% SDS, 5% β-mercaptoethanol, 10% glycerol, 50 mM Tris-HCl (pH 6.8), bromophenol blue) and then subjected to Western blotting. N-cadherin was detected using a 1000-fold diluted anti-mouse N-cadherin monoclonal antibody (Biolegend), and as an internal control, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), a housekeeping protein, was detected using a 2000-fold diluted anti-mouse GAPDH monoclonal antibody (Wako). Each antibody was chemiluminesced using Immobilon Forte Western HRP substrate (Millipore) and photographed with an imager (Thermo Fisher Scientific). Next, the intensity of each band was measured by image analysis using ImageJ, and the protein amount in each test group was normalized based on the amount of GAPDH, and the relative protein amount to the protein amount in the control test group was calculated.

[0132] (result) The results are shown in Figure 10. Figure 10 shows the protein amount ratio of N-cadherin (CDH2) to the control. Guaiazulene (Gz) significantly suppressed the expression of N-cadherin, which was significantly induced by TGF-β and PMA, also at the protein level. These results indicate that guaiazulene can suppress the expression of mesenchymal marker N-cadherin at the protein level in SAS cells, and suggest that guaiazulene can be an inhibitor of epithelial-mesenchymal transition (EMT) regardless of the type of cancer cells.

Claims

1. An inhibitor of the Smad-independent TGF-β signaling pathway, comprising a bicyclic compound represented by the following general formula (I) or a salt thereof. 【Chemistry 1】 [In the formula, R 1 and R 2 are each independently a hydrogen atom (H) or C 1 ~C 3 R represents a linear or branched alkyl group. 3 is a hydrogen atom (H), C 1 ~C 3 R represents a linear or branched alkyl group, a sulfonic acid group, or a sulfate group. 4 is C 1 ~C 3 represents a linear or branched alkyl group, a sulfonic acid group, or a sulfate group.

2. In the general formula (I), R 1 The Smad-independent TGF-β signaling pathway inhibitor of claim 1, wherein represents an ethyl group or an isopropyl group.

3. The Smad-independent TGF-β signaling pathway inhibitor of claim 2, represented by any one of the following formulas (II) to (V): 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】

4. The Smad-independent TGF-β signaling pathway inhibitor of claim 1 or 2, wherein the Smad-independent TGF-β signaling pathway is any one of the PKC signaling pathway, the MAPK signaling pathway, the PI3K signaling pathway, the FAK signaling pathway, and the Rac / Rho signaling pathway.

5. The Smad-independent TGF-β signaling pathway inhibitor of claim 1 or 2, wherein the inhibition of the Smad-independent TGF-β signaling pathway is any one of inhibition of F-actin formation, inhibition of N-cadherin expression, and inhibition of collagen synthesis.

6. A composition for inhibiting cell detachment and / or cell migration, comprising an inhibitor of the Smad-independent TGF-β signaling pathway according to claim 1 or 2.

7. A composition for suppressing cancer cell invasion and / or metastasis, comprising an inhibitor of the Smad-independent TGF-β signaling pathway according to claim 1 or 2.

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