KR20080030938A - Caffeic acid derivatives and compositions comprising the same as an active ingredient - Google Patents

Abstract

A caffeic acid derivative is provided to obtain an angiogenesis inhibitor and tumor growth inhibitor capable of inhibiting activation of signal transducer and activator of transcription 3 and transcription activity of hypoxia inducible factor-1alpha and vascular endothelial growth factor. A caffeic acid derivative is represented by the following formula 1. In formula 1, each of R1, R2, R3 and R4 independently represents H or a C1-C5 alkyl group. An angiogenesis inhibitor comprises the caffeic acid derivative as an active ingredient.

Description

Caffeic Acid Derivatives and Compositions Comprising the Same as an Active Ingredient}

The present invention relates to a novel synthetic caffeic acid derivative and a composition comprising the same as an active ingredient, more specifically, the newly synthesized caffeic acid derivative effectively inhibits the transcriptional activity of the vascular endothelial growth factor (VEGF) gene, thereby effectively It relates to a composition comprising as a component.

In previous studies, we found that signal transducer and activator of transcription 3 (STAT3) play an important role in hypoxia inducible factor-1α (HIF-1α) protein stability, Interaction with HIF-1α in human kidney cancer cells has been shown to improve HIF-1-mediated expression of vascular endothelial growth factor (VEGF) (1). This fact demonstrates that STAT3 is an important up-regulatory regulator of hypoxic-mediated VEGF expression in solid cancer cells.

VEGF is known to play an important role in angiogenesis and tumor progression (2), and VEGF inhibition has yielded promising results as a tumor anti-angiogenesis therapy in animal models and cancer patients (3). Since the proliferation of tumor cells is faster than the rate of blood vessel formation, hypoxic conditions usually occur inside solid cancers, making it difficult to supply blood to the tumor smoothly (4). In addition, hypoxia stimulates VEGF expression in tumors and promotes angiogenesis to meet metabolic needs for tumor growth (5). Thus, the factors regulating hypoxic angiogenesis are certain targets for chemotherapy. HIF-1 is one of these targets because it is an important transcription factor that regulates VEGF expression. HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β subunits (6), and the biological activity depends on the amount of HIF-1α, which is controlled by oxygen partial pressure. Under normal oxygen conditions, HIF-1α protein is unstable. Much research has been conducted to develop anticancer drugs targeting HIF-1α that can be induced under hypoxia (7).

Recently, STAT3 has been identified as a direct transcriptional activator of the VEGF gene and has been demonstrated to up-regulate VEGF expression in various human cancers (8), indicating that STAT3 activity contributes significantly to the overproduction of VEGF in tumors. it means. STAT3 is activated by phosphorylation of tyrosine 705 residue (Y705), which results in dimer formation, nuclear transfer, DNA binding, and target gene transcription (9). Phosphorylation of tyrosine 705 (Y705) of STAT3 is important for dimer formation and nucleus migration, a prerequisite for STAT3 activation (10). Thus, the inventors determined that inhibiting phosphorylation of STAT3 705 residues could reduce VEGF expression in tumor cells.

Throughout this specification many patent documents are referenced and their citations are indicated. The disclosures of the cited patent documents are incorporated by reference herein in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors tried to develop a novel synthetic anti-angiogenic substance, and as a result, the caffeic acid derivative inhibits the activation of STAT3 (signal transducer and activator of transcription 3), that is, phosphorylation, resulting in HIF-1α (hypoxia inducible factor-1α). The present invention was completed by effectively inhibiting the transcriptional activity of VEGF gene and vascular endothelial growth factor gene, and effectively inhibiting angiogenesis and tumor growth.

Accordingly, it is an object of the present invention to provide a novel synthetic caffeic acid derivative.

Another object of the present invention to provide a composition for inhibiting angiogenesis comprising a caffeic acid derivative as an active ingredient.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention provides a caffeic acid derivative represented by the following formula (1):

Formula 1

In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are each independently hydrogen or a C ₁ -C ₅ alkyl group.

According to another aspect of the present invention, there is provided a composition for inhibiting angiogenesis comprising the caffeic acid derivative represented by Formula 1 as an active ingredient.

The present inventors tried to develop a novel synthetic anti-angiogenic substance, and as a result, the caffeic acid derivative inhibits the activation of STAT3 (signal transducer and activator of transcription 3), that is, phosphorylation, resulting in HIF-1α (hypoxia inducible factor-1α). And VEGF (vascular endothelial growth factor) genes by effectively inhibiting the transcriptional activity, it was confirmed that it has an effect on inhibiting angiogenesis and tumor growth efficiently.

Caffeic acid (CA), a raw material of the caffeic acid derivative of the present invention, is a phenolic acid-based compound widely present in nature such as fruits, vegetables, wine, vegetable oils, and coffee components (20). The IUPAC name for caffeic acid is (E) -3- (3,4-dihydroxyphenyl) prop-2-enoic acid. Caffeic acid has a well-known antioxidant activity (11-13, 21-24), lipoxygenases, cyclooxygenase, glutathione S-transferase , Xanthine oxidase (Xanthine oxidase) and other activities to inhibit the activity (Chan, WS et al , Anticancer Res . 15: 703-707 (1995); Schefferlie, JG, and van Bladeren, PJ Food Chem . Toxicol . 31: 475-482 (1993); Koshihara, Y. et al , Biochim . Biophys . Acta . 792: 92-97 (1984); Mirzoeva, OK et al , FEBS Lett . 396: 266-270 (1996). Caffeic acid also has antitumor activity (Tanaka, T. et. al , Carcinogenesis . 14: 1321-1325 (1993); Frenkel, K. et al , Cancer Res ., 53: 1255-1261 (1993)), anti-inflammatory activity (14, 15, 24-26), HIV virus HIV replication inhibition [Kashiwada, Y. et. al , J. Nat . Prod . 58: 392-400 (1995); Fesen, MR et al , Proc . Natl . Acad . Sci . USA 90: 2399-2403 (1993) is reported. However, no activity has been reported that caffeic acid derivatives effectively inhibit angiogenesis and tumor growth by inhibiting the activity of hypoxic induced STAT3 and inhibiting the transcriptional activity of VEGF.

According to the present invention, various chemical residues are combined with caffeic acid of Formula 2 by an organic chemical process to provide a novel material having improved physical properties. As a result of various physiological and biochemical efficacy assays of the novel compounds of the present invention, the efficacy is increased compared to the raw material caffeic acid.

Formula 2

As used herein, the term "alkyl" refers to a straight or crushed saturated hydrocarbon group. Examples include, but are not limited to, methyl, ethyl, n -propyl, isopropyl, isobutyl, n -butyl and t -butyl.

According to a preferred embodiment of the present invention, R ₁ , R ₂ , R ₃ and R ₄ of the derivative of Formula 1 are each independently hydrogen or a C ₁ -C ₃ alkyl group, more preferably hydrogen or methyl group to be. Most preferably, R ₁ , R ₂ , R ₃ and R ₄ of the derivative of Formula 1 are each hydrogen, and a derivative having this formula is selected from CADPE ((3- (3,4-dihydroxy-phenyl)- Acrylic acid 2- (3,4-dihydroxy-phenyl) -ethyl ester)).

According to a preferred embodiment of the present invention, the active ingredient used in the present invention inhibits angiogenesis by inhibiting the transcriptional activity of the VEGF gene. Inhibition of transcriptional activity of the VEGF gene exerts various pharmacological activities by inhibiting the activity of STAT3, that is, phosphorylation of STAT3 Tyr-705. More specifically, the caffeic acid derivative, the active ingredient of the present invention, inhibits the phosphorylation of STAT3 Tyr-705, the STAT3 signaling pathway, nuclear migration, and the formation of transcription units between STAT3 / HIF-1a / p300 on the VEGF promoter, Inhibits proliferation, migration, angiogenesis and tumor growth induced by vascular endothelial cells.

Diseases, diseases or conditions that can be prevented or treated by the compositions of the present invention include various diseases associated with angiogenesis. Preferably, the disease that can be prevented or treated by the composition of the present invention is diabetic retinopathy, prematurity retinopathy, corneal transplant rejection, neovascular glaucoma, melanoma, proliferative retinopathy, cancer, psoriasis, hemophiliac joints, atherosclerosis Capillary proliferation in atherosclerotic plaques, keloids, wound granulation, vascular adhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases, atopic, allergy, Crohn's disease, restenosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcers, Cirrhosis, glomerulonephritis, diabetic nephropathy, malignant neurosis, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, diabetes, inflammatory or neurodegenerative diseases.

Cancers that can be prevented or treated by the compositions of the present invention include gastric cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, Bone cancer, skin cancer, thyroid cancer, leukemia, lymphoma, adrenal cortex cancer, parathyroid cancer, ureter cancer and kidney cancer.

Autoimmune diseases that can be prevented or treated by the composition of the present invention include alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune Autoimmune hepatitis, autoimmune ovary and testicles, autoimmune thrombocytopenia, Behcet's disease, bullous swelling, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunodeficiency syndrome, chronic inflammatory demyelinating polyneuropathy , Churg-Strauss syndrome, scar pulmonary swelling, CREST syndrome, cold-cold aggregate disease, Crohn's disease, discus lupus, congenital combined cold globulinemia, fibromyalgia-fibromyalitis, glomerulonephritis, Graves disease, Mullein Barre syndrome, Hashimoto thyroiditis, idiopathic Pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA neuritis, juvenile arthritis, lichen planus, lupus erythematosus, menis Le disease, mixed connective tissue disease, multiple sclerosis, type I or immune-mediated diabetes, myasthenia gravis, vulgaris ulcer, pernicious anemia, crystalline polyarteritis, polychondritis, autoimmune polyline syndrome, rheumatoid polymyalgia, multiple myositis and skin Myositis, Primary agammaglobulinemia, Primary biliary cirrhosis, Psoriasis, Psoriatic arthritis, Raynaud's phenomenon, Reiter syndrome, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Ankylosing syndrome, Systemic lupus erythematosus, Lupus erythematosus, Dagayasu arteritis, transient arteritis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

Examples of inflammatory diseases that can be prevented or treated by the compositions of the present invention include asthma, encephilitis, inflammatory enteritis, chronic obstructive pulmonary disease, allergies, atopy, psoriasis, pulmonary pulmonary shock, pulmonary fibrosis, Undifferentiated spondyloarthropathy, undifferentiated arthrosis, arthritis, inflammatory osteolysis, and chronic inflammation caused by chronic viral or bacterial infections.

According to a more preferred embodiment, the disease, condition or condition which can be prevented or treated by the composition of the present invention is diabetic retinopathy, prematurity retinopathy, corneal transplant rejection, neovascular glaucoma, erythematosis, proliferative retinopathy, cancer, psoriasis , Capillary hyperplasia in atherosclerotic plaques, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, allergy, atopy, Crohn's disease, restenosis, atherosclerosis, diabetes, inflammatory or neurodegenerative diseases. Most preferably, the cancer (particularly solid cancer) is kidney cancer or skin cancer.

The composition of the present invention may be prepared from a pharmaceutical composition, a functional cosmetic composition, a cosmetic composition, a neutraceutical composition or a food composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the caffeic acid derivative of formula 1 as described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the caffeic acid derivative of Formula 1 described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration, and eyedrop administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Preferably, the dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg body weight.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a cosmetically effective amount of the caffeic acid derivative of formula (1); And (b) a cosmetic composition (or functional cosmetic composition) comprising a cosmetically acceptable carrier.

As used herein, the term “cosmetic effective amount” means an amount sufficient to achieve the efficacy of the invention described above.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The components included in the cosmetic composition of the present invention include components commonly used in cosmetic compositions, in addition to the caffeic acid derivative of Formula 1 and the carrier component as active ingredients, and include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and Conventional adjuvants such as flavorings.

When the composition of the present invention is made of a food composition (or functional food composition), as an active ingredient, as well as the caffeic acid derivative of formula (1), it contains components that are commonly added during food production, for example, protein, carbohydrate Contains fats, nutrients, seasonings and flavorings. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

 For example, when the food composition of the present invention is prepared with a drink, in addition to the caffeic acid derivative of Formula 1 of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract and the like are added. It can be included as.

The features and advantages of the present invention are summarized as follows:

(Iii) The novel caffeic acid derivatives of the present invention have an excellent angiogenesis inhibitory effect.

(Ii) The novel caffeic acid derivative of the present invention effectively inhibits angiogenesis by inhibiting the transcriptional activity of the VEGF gene as compared to the original caffeic acid. Inhibition of transcriptional activity of this VEGF gene exerts various pharmacological activities by inhibiting the activity of STAT3, that is, phosphorylation of STAT3 Tyr-705. More specifically, the caffeic acid derivative, the active ingredient of the present invention, inhibits the phosphorylation of STAT3 Tyr-705, the STAT3 signaling pathway, nuclear migration, and the formation of transcription units between STAT3 / HIF-1a / p300 on the VEGF promoter, Inhibits proliferation, migration, angiogenesis and tumor growth of vascular endothelial cells.

(Iii) A pharmaceutical, functional cosmetic (cosmeceutical), cosmetics, functional food (neutraceutical) or food compositions can be prepared using the caffeic acid derivative of the present invention as an active ingredient.

(Iii) The composition may also prevent or treat various diseases, disorders or conditions associated with angiogenesis.

As described in detail above, the present invention provides a novel caffeic acid derivative and a composition comprising the same. The present invention is a novel caffeic acid derivative of the present invention has an excellent angiogenesis inhibitory effect. The novel caffeic acid derivatives of the present invention effectively inhibit angiogenesis by inhibiting the transcriptional activity of the VEGF gene as compared to the original caffeic acid. Inhibition of transcriptional activity of the VEGF gene exerts various pharmacological activities by inhibiting the activity of STAT3, that is, phosphorylation of STAT3 Tyr-705. More specifically, the caffeic acid derivative, the active ingredient of the present invention, inhibits the phosphorylation of STAT3 Tyr-705, the STAT3 signaling pathway, nuclear migration, and the formation of transcription units between STAT3 / HIF-1a / p300 on the VEGF promoter, Inhibits proliferation, migration, angiogenesis and tumor growth induced by vascular endothelial cells. In addition, a pharmaceutical, functional cosmetics (cosmeceutical), cosmetics, functional food (neutraceutical) or a food composition can be prepared using the caffeic acid derivative of the present invention as an active ingredient. In addition, the composition may prevent or treat various diseases, diseases or conditions associated with angiogenesis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Example

Experimental Materials and Methods

Experimental Materials and Cells

Caffeic acid (CA) was supplied from CH Kim (Sungkyunkwan University, Suwon, Korea) and resuspended in dimethylsulfoxide (DMSO) at 500 mg / ml stock concentration and stored at -20 ° C. Caffeic acid derivatives (CADPE) were synthesized by Imagene (Korea), resuspended in dimethyl sulfoxide (DMSO) at a concentration of 500 mg / ml, and stored at -20 ° C. The Caki-I human kidney cancer and COS7 monkey kidney cell lines used were cultured as previously described (1).

Western blot and fraction analysis

Western blot analysis was performed as previously described (1). The cells were washed twice followed by stop buffer (10 mM Tris, pH 7.4, 10 mM EDTA, 5 mM EGTA, 0.1 M NaF, 0.2 M sucrose, 100 μM sodium orthovanadate and 5 mM sodium pyrophosphate). , Supplemented with protease inhibitors). 400 g, after centrifugation at 4 ° C for 10 minutes, contains Diagram's Buffer A (10 mM HEPES (pH 7.9), 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, and 1% NP-40), 1 cells were lysed) supplemented with mM DTT, 100 μM sodium orthovanadate and protease inhibitors. Fractions of lysate were removed and 4 × SDS electrophoresis buffer was mixed and used to detect total protein in lysate. The remaining residue was incubated for 10 minutes at 4 ° C, then centrifuged for 2 minutes at 13,000 g, 4 ° C to pellet the nuclei. Supernatants were collected and cytoplasmic proteins were analyzed and the pellet was washed with Buffer A and centrifuged again. The obtained nuclear pellet was resuspended in Dignam's buffer C (20 mM HEPES, pH 7.9, 0.42 M NaCl, 1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, 100 μM sodium orthovanadate and protease inhibitor) Vortex and shake at 4 ° C for 15 minutes. After centrifugation at 13,000 g and 4 ° C. for 5 minutes, supernatant nuclear proteins were recovered and Dignam's buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 1 mM EDTA, 0.1 mM EGTA, 1% NP-40, 1 mM DTT, 100 μM sodium orthovanadate and protease inhibitors).

Transient transformation and luciferase analysis

Luciferase assays were performed as previously described (1). Various combinations of the following constructs were co-transformed into cells; STAT3 specific siRNAs comprising 24 bp double stranded oligoribonucleotides, including sense strand 5'-CAGCAAAGAAUCACAUGCCACUUU-3 '(named siRNA 1) and antisense strand 5'-CCUGCAAGAGUCGAAUGUUCUCUAU-3' (named siRNA 2); Dominant negative mutation-STAT3YF (substituted tyrosine 705 residues of STAT3 with phenylalanine), wild-type STAT3 (250 ng of each plasmid) associated with 250 ng of human VEGF promoter reporter plasmid m67, the sequence being the 5'-adjacent region of the human VEGF gene Was inserted at about 2.7 kb.

RT-PCR

RNA isolation and RT-PCR were performed as previously described (1). CDNA fragments were amplified by PCR using primers specific for the following VEGF; Sense 5'-AGGAGGGCAGAATCATCACG-3 'and antisense 5'-CAAGGCCCACAGGGATTTTCT-3'.

Chromatin Antibody Precipitation (ChIP) Analysis

Chromatin antibody precipitation was performed as previously described (16). Promoter-specific primers include human VEGF, 5'-AGACTCCACAGTGCATACGTG-3 'and 5'-AGTGTGTCCCTCTGACAATG-3', which amplify the 235 bp fragment adjacent to the STAT3-binding element, the HIF-1α-binding element.

ELISA

VEGF levels were determined in conditioned media using Quantikine Human VEGF Immunoassay Kit (R & D Systems, Minneapolis, MN) and normalized compared to total protein content as determined using Bradford assay.

Angiogenesis Analysis

Caki-I cells were exposed to hypoxia for 24 hours with or without pretreatment with CA or CADPE for 1 hour. To determine VEGF biological activity in supernatants, HUVECs were incubated for 14 hours in supernatants of Caki-I cells cultured in the presence / absence of CA or CADPE under hypoxic or normal oxygen conditions for 24 hours. Biological activity was assessed by observing the formation of capillary-like network structures produced by endothelial cells. Growth factor-deficient matrigel was dissolved at 4 ° C. overnight. Then, the metrigel was placed in a 24-well well cell culture dish (200 μl / well) and solidified at 37 ° C. for at least 1 hour. HUVECs were incubated without serum in RPMI 1640 for 1.5 hours and then resuspended in RPMI 1640. Resuspended HUVECs were placed in 24-well plates (150,000 HUVECs / well). In addition, all conditions were supplemented with fresh RPMI 1640 to bring the total volume of each well to 2 ml. The plates were incubated at 37 ° C. for 14 hours. Angiogenesis was observed and digital photography was taken using an Olympus digital camera (New Hyde Park, NY).

Human tumor Xenograft

Male nude (BALB / cAnNCrj-nu / nu) mice were obtained from Charles River Japan Inc. (Shin-Yokohama, Japan). Animals were bred in a sterile room under controlled temperature and humidity. All animal breeding procedures were conducted according to the methods described in the Seoul National University Laboratory Animal Maintenance Manual. Caki-I cells (5 × 10 ⁶ , subcutaneous) were injected into the flanks of 25, 7-week old mice. The mice were then randomly assigned to one of five groups. The first group ( n = 5 ; control) was treated with vehicle (DMSO). The second, third, and fourth groups ( n = 5 ) had CA (5 mg every 3 days for 4 weeks after tumor size reached 100-150 mm ³ (about 15 days after Caki-I injection). / kg, intraperitoneal) or CADPE (5 mg / kg, intraperitoneal). Calculated by using the using the caliper of the tumor every two or three days was measured in two dimensions, tumor volume is axb ^2/2 (where a is the width of the widest point of the tumor and b is a width perpendicular to a) It was.

Tumor histology and CD31 , p- STAT3 , And HIF Immunostaining of -1α

After injection of the final CA, CADPE, or vehicle, mice were sacrificed and tumors removed. Tumors were fixed in formalin and embedded in paraffin. Continuous sections (6 μm thick) were cut from each paraffin block. One section was stained with hematoxylin-eosin for histological evaluation. Other sections were stained for p-STAT3, HIF-1α and endothelial marker CD31. Paraffins of the sections were removed, rehydrated in increasing alcohol concentration, and then heated in 10 mM sodium citrate (pH 6.0) for 5 minutes with microwave to restore antigen. Block nonspecific portions for 1 hour with a solution containing 2.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) and 2% normal goat serum (Life Technologies) in PBS pH 7.4 Reaction with cloned anti-CD31 antibody (diluted 1: 100 in blocking solution; Santa Cruz Biotechnology) overnight at 4 ° C. Negative control sections were reacted with diluent (blocking solution) in the absence of primary antibody. The sections were then washed and reacted with secondary antibodies labeled with the appropriate biotin, and the position of bound antibody was determined using the avidin-biotin-horseradish peroxidase complex. Diamitobenzidine was used as the final chromogen. Different sections were analyzed for each xenograft tumor. For histological evaluation, p-STAT3-positive, HIF-1α-positive cells, and CD31 positive microvascular were identified at 400x, 400x, and 600x magnifications respectively, Sony XC-77 CCD camera and microcomputer image. Analysis was performed using a device model 4 image analysis system. Expression of HIF-1α and p-STAT3, and vascular density were measured by counting the number of immunopositive cell and vascular profiles (identified by CD31 staining) per area (mm ² ) of each image.

Statistical analysis

All data were analyzed using Microsoft Excel 2000 software (Microsoft Corp., Remond, WA). Man-Whitney U test (SPSS, version 10.0; Statistical Package for the Social Sciences, Chicago, IL) to compare VEGF levels in culture medium, the number of p-STAT3 and HIF-1α-positive cells, and the number of blood vessels Was used. Tumor volume was compared between variance analysis (ANOVA) followed by untreated controls with Duncan's multi-range assay, and groups treated with CA or CADPE. If the difference is P <.05, it is considered statistically significant. All statistical tests are bilateral tests.

Experiment result

CA  And CADPE Is Hypoxia -Judo STAT3 It inhibits the activity of and movement of position into the nucleus.

Since phosphorylation of STAT3 Tyr-705 is required for nuclear transfer and binding of specific promoter sequences on target genes, phosphorylation blocking of Tyr-705 provides an effective method of inhibiting the STAT3 signaling pathway in human cancers. Therefore, we investigated the activity of CA and CADPE on in vitro STAT3 phosphorylation using Caki-I cells. As can be seen in FIGS. 1A and 1B, phosphorylation of STAT3 on tyrosine 705 (Y705) increased with hypoxic state and decreased by CA or CADPE concentration-dependently. In addition, such inactivation of STAT3 was not caused by a decrease in protein levels. In addition, to rule out the possibility that the cytotoxic effect of CA or CADPE may affect the dephosphorylation of STAT3, we assessed cell viability after treatment of 10-300 μM of CA or CADPE using MTT assay. It was.

We then investigated whether CA or CADPE reduced the shift of position of STAT3 into the nucleus. To confirm the inhibitory effect on hypoxic-induced STAT3 position shift of CA and CADPE, we transformed wild type STAT3 with COS7 (STAT3 deficient cell line) and then fractionated the total cell extract to obtain nuclear and cytoplasmic proteins. Western blotting was then performed using anti-STAT3 and p-STAT3 (Y705) antibodies. As can be seen in FIG. 1C, phosphorylated STAT3 is shifted from the cytoplasm to the nucleus according to the hypoxic state, but STAT3 remains in the cytoplasm in cells pretreated with 100 μM CA or 30 μM CADPE despite hypoxic stimulation. In addition, we confirmed the expression of Lamin B in the nuclear fraction.

Taken together, CA or CADPE inhibits STAT3 (Y705) phosphorylation necessary for nucleotide position shift of STAT3 and suppresses hypoxia-induced position shift of STAT3 to the nucleus.

CA  or CADPE On by STAT3 Active destruction of the Src  Tyrosine Kinase  By suppression.

To identify the STAT3 inhibition mechanism, we investigated some tyrosine kinase upstream activity of STAT3. Src tyrosine kinases are known to activate the Jak and STAT3 pathways (17), and it has been found that Src tyrosine kinase-induced VEGF expression is required in mouse 3T3 fibroblasts (8). In addition, STAT3 has been reported to regulate Src-dependent hypoxic-induced VEGF expression in pancreatic and prostate cancers (18). To clarify whether Src tyrosine kinase is involved in STAT3 signal transduction that upregulates HIF-1α induction under hypoxic conditions, we examined the STAT3 signaling system under hypoxic conditions by western blotting. 1D shows that the hypoxic states increase Src (Y416), STAT3 (Y705), and HIF-1α induction in order. Interestingly, 100 μM CA or 30 μM CADPE significantly inhibited the Src / STAT3 / HIF-1α pathway.

To determine whether Src is the upstream regulator of hypoxic-induced STAT3 activity and the first target of CA or CADPE, we have identified Caki-I kidney cancer cells as either Src structurally active (Y527F) or Src dominant negative ( Y416F), followed by confirmation of STAT3 inhibition and HIF-1α induction by CA or CADPE under hypoxic conditions. We have confirmed STAT3 phosphorylation and HIF-1α induction in the overexpressed active form of Src (Y527F) even though CA or CADPE was pretreated in cells. However, we were unable to detect STAT3 phosphorylation and HIF-1α induction in the overexpressed dominant negative form of Src (Y416F) (FIG. 2E).

These results indicate that Src tyrosine kinase is required for STAT3-mediated HIF-1α induction under hypoxic conditions, and disruption of STAT3 activity by CA or CADPE is by Src tyrosine kinase inhibition.

CA  or CADPE Is Hypoxia  According to the condition STAT3 Suppress the VEGF  By inhibiting the regulator of the promoter VEGF Inhibits transcriptional activity of

In previous studies, we found that STAT3 up-regulates HIF-1-mediated VEGF expression under hypoxic conditions of kidney cancer cells (1). We therefore investigated whether CA or CADPE inhibits VEGF expression by inhibiting STAT3. First, we performed luciferase analysis on Caki-I cells using m67 (VEGF promoter reporter plasmid) in which a 5 ′ flanking region of about 2.7 kb of the human VEGF gene was inserted. Hypoxia increased reporter activity (approximately 4 fold) in Caki-I cells (FIG. 2A, the first two columns) but not in cells pretreated with 100 μM CA or 30 μM CADPE (FIGS. 2A, 3 and Fourth column). Next, we transformed cells with STAT3-specific siRNA and evaluated the hypoxic-induced reporter activity of the VEGF promoter, which was found to be greatly reduced by siRNA-mediated inhibition of STAT3 (FIG. 2A, fifth). And sixth column). In addition, the reporter activity of the VEGF promoter was significantly lower in cells transformed with STAT3YF (dominant negative STAT3).

In order to confirm whether the inhibitory effect on VEGF transcriptional activity of CA or CADPE was due to STAT3 inhibition, we performed luciferase analysis using COS7 cells (STAT3-deficient cell line). As shown in FIG. 2B, the hypoxic state induced VEGF promoter (about 5 fold) reporter activity compared to normal oxygen state (first two columns), due to HIF-1. This is because both HIF-1 and STAT3 are involved in regulating VEGF expression (1). However, after treatment with 100 μM CA or 30 μM CADPE, it was found that the hypoxic-induced reporter activity of the VEGF promoter was not reduced in COS7 cells (FIG. 2B, third and fourth columns), which was Caki-I cells. In contrast to the observations in (STAT3-containing cell line).

In addition, hypoxic-induced reporter activity in COS7 cells was further increased (about 2.5-fold) by overexpressing wild-type STAT3 compared to the mock control (FIG. 2B, second and fourth columns). Interestingly, 100 μM CA or 30 μM CADPE significantly inhibited the hypoxic-induced reporter activity of the VEGF promoter in COS7 cells overexpressing wild type STAT3 (FIG. 2B, fourth, sixth and seventh columns), while CA or A decrease in reporter activity by CADPE could not be observed in the absence of STAT3.

Western blot was performed on STAT3 protein in Caki-I cells to examine the transformation efficiency of STAT3 siRNA (FIG. 2C). Interestingly, disruption of STAT3 with siRNA reduced HIF-1α protein levels in the hypoxic state. RT-PCR was used to further investigate the inhibitory effect on VEGF gene expression of CA or CADPE. Caki-I cells were pretreated with CA or CADPE or without CA or CADPE and then placed in hypoxic state. As can be seen in FIG. 2D, CA or CADPE reduced hypoxia-induced VEGF mRNA expression concentration-dependently. Summarizing these results, the inhibitory effect on VEGF expression of CA and CADPE is due to STAT3 inhibition.

The results show that either CA or CADPE decreases the hypoxic-induced transcriptional activity of VEGF by inhibiting STAT3. Therefore, in order to clarify whether CA or CADPE inhibits the action of STAT3 on the VEGF promoter according to the hypoxic state, we conducted a chromatin immunoprecipitation assay. As can be seen in FIG. 2E, increased binding of STAT3 on the human VEGF promoter was observed in the hypoxic state. In addition, the binding of HIF-1α and p300 on the human VEGF promoter was increased in the hypoxic state. However, their binding on human VEGF promoters was reduced in hypoxic cells pretreated with 100 μM CA or 30 μM CADPE. This result is consistent with previous findings (FIG. 1C), that is, CA or CADPE inhibit the positional shift of hypoxic-mediated STAT3 to the nucleus.

Taken together these results, both CA and CADPE inhibit the transcription of the VEGF gene by blocking the action of STAT3 and the formation of transcriptional units between STAT3, HIF-1a, and p300 on the VEGF promoter.

CA  And CADPE Is Matrigel  On a substrate VEGF  Secretion and HUVECs  Inhibits blood vessel formation

To analyze the inhibitory effect of CA or CADPE on angiogenesis, we measured the amount of VEGF secreted by Caki-I cells pretreated with CA or CADPE under hypoxic conditions or without CA or CADPE. As can be seen in FIG. 3A, VEGF secretion was significantly reduced in cells pretreated with 100 μM CA or 30 μM CADPE compared to the case of only hypoxia (FIG. 3A, second, third and fourth columns). In addition, VEGF secretion was significantly reduced by siRNA-mediated STAT3 inhibition (FIG. 3A, fifth and sixth column) and significantly reduced in cells transformed with STAT3YF (dominant negative of STAT3) (FIG. 4A, seventh and Eighth column).

Next, we used a micro-dimensional (3D) HUVECs angiogenesis assay for the angiogenesis process to confirm the anti-angiogenic activity of CA or CADPE. Caki-I cells were stimulated with hypoxia for 24 hours without treatment with 100 μM CA or 30 μM CADPE for 1 hour or without 100 μM CA or 30 μM CADPE, and then the supernatants were collected. In order to analyze the biological activity of VEGF in the supernatant, HUVECs were cultured in the collected supernatant and evaluated for capillary-like network structure formation by endothelial cells. Angiogenesis by HUVECs was found to be reduced in the presence of 100 μM CA or 30 μM CADPE (FIG. 3B). These results demonstrate that both CA and CADPE inhibit hypoxic-induced VEGF secretion and that they have anti-angiogenic activity.

CA  or CADPE of Genographed  Effect on the development of human tumors.

Because of the observed in vitro effects of CA and CADPE, we investigated whether CA or CADPE inhibited angiogenesis and tumor growth by inhibiting STAT3 in vivo . Living Caki-I cells (5 × 10 ⁶ ) were injected subcutaneously into the flanks of mice and then randomly assigned to one of three groups. The first group ( n = 5 ) was the control group and injected only vehicle (DMSO). The second and third groups ( n = 5 ) measured tumors of 100-150 mm ³ (about 15 days), CA (5 mg / kg) or CADPE (5 mg / kg) was intraperitoneally injected every three days for four weeks. Tumors of CADPE-treated mice were visually smaller than tumors of CA or vehicle-treated mice (FIG. 4A). Changes in tumor size were measured and plotted against time versus average tumor size (FIG. 4B). After tumors stabilized, tumor growth was stopped in mice treated with CA or CADPE ( P <.05 compared to vehicle-treated group). However, the body weight of the CA- or CADPE-treated group did not decrease as compared to the vehicle-treated group. These results indicate that CA or CADPE significantly inhibits tumor growth in mice with Caki-I tumors.

Nude mouse Caki Histopathology and immunohistochemistry of -I.

In order to determine whether the inhibitory effect of CA or CADPE on tumor growth is associated with angiogenesis inhibition, we analyzed the distribution of endothelial marker CD31 in tumor tissue. Very few CD31-immunopositive vessels were observed in tumor sections of CA or CADPE treated mice, but many vessels were observed in tumor sections of vehicle-treated mice (FIG. 5A, top panel). As can be seen from the in vitro data of the present invention, STAT3 is important in angiogenesis, so we evaluated the activity of STAT3 in tumor sections of vehicle- and CA- or CADPE-treated mice. Caki-I tumors in vehicle-treated mice showed many p-STAT3 immune-responsive cells (FIG. 5A, middle panel), whereas tumor sections of CA or CADPE treated mice showed little p-STAT3 immune-responsive cells. .

Interestingly, tumor sections of CA or CADPE treated mice showed little HIF-1α protein despite the tumor being hypoxic and solid cancer (FIG. 5A, lower panel). These results are consistent with previous results that STAT3 up-regulates the stability of HIF-1α protein under hypoxic conditions. We quantified the number of p-STAT3-positive, HIF-1α-positive cells, and CD31-positive blood vessels in tumor sections of vehicle- and CA or CADPE-treated mice (FIG. 5B). Levels of HIF-1α, p-STAT3 immunoreactive cells, and angiogenesis were significantly lower in mice treated with CA or CADPE than vehicle-treated mice.

In order to reconfirm the inhibitory effect of CA and CADPE on STAT3 in Caki-I tumors, we used phosphorylation of STAT3 in Caki-I tumor lysates by western blotting using anti-phospho-STAT3 (Y705) antibodies. Investigate. It was found that STAT3 (Y705) phosphorylation levels were significantly lower in tumor lysates from CA- or CADPE-treated mice than in vehicle-treated tumors. In addition, HIF-1α expression was lower in CA- or CADPE-treated tumors than in vehicle-treated tumors (FIG. 5C). In addition, VEGF mRNA levels were lower in CA- or CADPE-treated tumors than in vehicle-treated tumors (FIG. 5D).

In summary, the inhibition of STAT3 by CA or CADPE reduces HIF-1α and VEGF expression and blocks tumor growth and angiogenesis in Caki-I tumors.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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1A-1E show that both CA and CADPE inhibit the hypoxic-induced phosphorylation of STAT3 and position shift to the nucleus. CA is caffeic acid, CADPE is a synthetic derivative thereof (3- (3,4-dihydroxy-phenyl) -acrylic acid 2- (3,4-dihydroxy-phenyl) -ethyl ester), and STAT3 stands for signal transducer and activator of transcription 3. 1A and 2A, before incubating Caki-I cells under normal oxygen (20% O ₂ vol / vol) or hypoxic (1% O ₂ vol / vol) conditions for 3 hours, the indicated concentrations of CA (FIG. 1A) ) Or CADPE (FIG. 1B) for 1 hour. Phosphorylation of STAT3 and expression of STAT3 were analyzed by immunoblotting using anti-pSTAT3 (Y705) and anti-STAT3. In FIG. 1C, COS7 cells were plated in cell culture dishes at a density of 1 × 10 ⁶ cells per ml. Cells are overexpressed with STAT3 for 24 hours, then treated with 100 μM CA or 30 μM CADPE for 1 hour and normal oxygen (20% O ₂ vol / vol) or hypoxic (1% O ₂ vol / vol) conditions for 3 hours. Incubated at. The cells were then separated into cytoplasm and nuclear proteins. STAT3 phosphorylation and expression of STAT3 and lamin B were analyzed by immunoblotting using anti-pSTAT3 (Y705), anti-STAT3 and anti-Lamin B. In FIG. 1D, 100 μM CA or 30 μM CADPE for 1 hour prior to incubation of Caki-I cells under normal oxygen (20% O ₂ vol / vol) or hypoxic (1% O ₂ vol / vol) conditions for the indicated times. Treated with. Phosphorylation of Src and STAT3, and expression of STAT3 were analyzed by immunoblotting with anti-Src (Y416), anti-STAT3 (Y705) and anti-STAT3. In FIG. 1E, Caki-I cells were plated in cell culture dishes at 1 × 10 ⁶ per ml. Src active (Y527F) or Src dominant negative (Y416F) plasmid DNA was transfected into cells using Lipofectamine ™ and cells were treated with 100 μM CA or 30 μM CADPE for 1 hour. Cells were then incubated for 3 hours under normal or hypoxic conditions. Phosphorylation of Src and STAT3, and expression of STAT3 were analyzed by immunoblotting using anti-Src (Y416), anti-STAT3 (Y705) and anti-STAT3.

2A-2E show that CA and CADPE inhibit transcriptional activity and the action of modulators on the VEGF promoter depending on the hypoxic state. VEGF represents vascular endothelial growth factor and HIF-1α represents hypoxia inducible factor-1α. In FIGS. 3A and 3B, Caki-I (FIG. 3A) or COS7 (FIG. 3B) cells were seeded on 24-well plates at a density of 4 × 10 ⁴ cells per well. The VEGF reporter plasmid DNA was simultaneously transformed with Lipofectamine ™ with a combination of STAT3 specific siRNA or dominant negative STAT3 (STAT3YF) or wild type STAT3 (STAT3WT). Cells were then treated with 100 μM CA or 30 μM CADPE for 1 hour before incubating for 24 hours under normal or hypoxic conditions. Cells were then lysed and subjected to luciferase assay. Data is mean ± SD for four independent experimental results. In FIG. 2C, Caki-I cells were transformed with STAT3-specific siRNA and incubated for 24 hours under normal or hypoxic conditions for 24 hours. Expression of STAT3, HIF-1α and β-actin was analyzed by immunoblotting using anti-STAT3, anti-HIF-1α and anti-β actin antibodies. In FIG. 2D, Caki-I cells were pretreated with 50 or 100 μM CA, or 15 or 30 μM CADPE for 1 hour and then exposed to hypoxic conditions for 24 hours. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using specific primers for VEGF and β actin. In FIG. 2E, Caki-I cells were treated with 100 μM CA or 30 μM CADPE for 1 hour and then incubated for 3 hours under normal or hypoxic conditions. The cells were then fixed with formaldehyde. Soluble chromatin samples were immunoprecipitated with anti-STAT3, anti-HIF-1α or anti-p300 antibodies at 4 ° C. overnight. DNA isolated from the immunoprecipitated material was then amplified using PCR. Promoter-specific primers include human VEGF, 5'-AGACTCCACAGTGCATACGTG-3 'and 5'-AGTGTGTCCCTCTGACAATG-3', which amplify the 235 bp fragment adjacent to the STAT3-binding element, the HIF-1α-binding element.

3A-3B show that CA and CADPE significantly inhibit VEGF secretion and HUVECs angiogenesis on metrigel matrix. HUVECs represent human umbilical vein endothelial cells. In FIG. 3A, Caki-I cells were transformed with siRNA or STAT3YF specific for STAT3 for 24 hours, or treated with 100 μM CA or 30 μM CADPE. The amount of VEGF protein in Caki-I cells in conditioned media was measured using an ELISA kit. The Caki-I cells were treated with CA or CADPE or transformed with STAT3YF or siRNA and incubated under normal or hypoxic conditions for 24 hours. VEGF concentrations were quantified twice compared to the series of VEGF standard samples included in the assay kit. Bars represent the mean and 95% confidence intervals of four independent experimental results. ^* Indicates significant differences compared to supernatant controls of cells cultured under normal oxygen conditions ( P <0.001 ); # Indicates significant difference compared to supernatant control of cells cultured under hypoxic conditions ( P <0.001 ). In FIG. 3B, growth factor-deficient methgel was placed in 24-well cell culture dishes using pre-cooled pipette tips and cell culture dishes. HUVECs were incubated for 14 hours in supernatants of Caki-I cells without or without hypoxic stimulation with 100 μM CA or 30 μM CADPE for 1 hour or without CA or CADPE treatment before reaching hypoxic state. Biological activity was determined by reacting the plate at 37 ° C. for 14 hours and then evaluating capillary-like network structure formation by endothelial cells. Digital photographs were taken using an Olympus digital camera (New Hyde Park, NY).

4A-4B show the effect of CA and CADPE on the growth of xenografted human tumors. In FIG. 4A, Caki-I kidney cancer cells (5 × 10 ⁶ ) were injected subcutaneously into the flanks of male nude mice. After tumors reach 100-150 mm ³ (about 15 days), mice are intraperitoneally injected every three days with CA (5 mg / kg), CADPE (5 mg / kg) or vehicle (DMSO) for 4 weeks. It was. In FIG. 4B, differences between tumor sizes in vehicle- and CA- or CADPE-treated groups were compared using a deviation analysis. Data represent mean ± SEMs; ^* Indicates P <0.05 compared to control. Solid diamonds represent the vehicle, solid triangles the CA, and solid squares the CADPE. Data points represent means (control n = 5 ; CA n = 5 ; CADPE n = 5 ).

5A-5D show histopathology and immunohistochemistry of Caki-I tumors grown in nude mice. In FIG. 5A, Caki-I kidney cancer cells (5 × 10 ⁶ ) were injected subcutaneously into the flanks of male nude mice. After tumors reach 100-150 mm ³ (15 days post-injection), mice are CA (5 mg / kg, intraperitoneal), CADPE (5 mg / kg, intraperitoneal) or vehicle every three days for four weeks. (DMSO) was injected. The mice were then euthanized and tumors removed, fixed in formalin and embedded in paraffin. Tumor sections were cut from paraffin blocks and immunohistochemically stained with anti-CD31 antibody, anti-pSTAT3 antibody or anti-HIF-1α antibody to detect endothelial cells. In FIG. 5B, STAT3 phosphorylation, HIF-1α expression and vascular density. Histological evaluation was performed on two sections per xenograft (5-10 fields per section). (5 control groups, 5 CA and 5 CADPE-treated genograft groups were examined). Bars represent mean and low or high 95% confidence intervals. Statistical significance of differences between treatment groups was compared using the Mann-Whitney U test. The numbers above the error bars represent the difference in P values relative to the control. In FIG. 5C, after the final treatment, mice were euthanized and lysates were prepared for immunoblotting by removing tumors. Tumor lysates of vehicle-treated and CA- and CADPE-treated mice were evaluated for STAT3 phosphorylation. In FIG. 5D, mRNA levels of VEGF and β-actin in tumor lysates were measured by RT-PCR.

Claims (9)

Caffeic acid derivative represented by the following formula (1): Formula 1

In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are each independently hydrogen or a C ₁ -C ₅ alkyl group. The caffeic acid derivative according to claim 1, wherein R ₁ , R ₂ , R ₃ and R ₄ of the derivative of Formula 1 are each independently hydrogen or a C ₁ -C ₃ alkyl group. The caffeic acid derivative according to claim 1, wherein R ₁ , R ₂ , R ₃ and R ₄ of the derivative of Formula 1 are each independently hydrogen or a methyl group. The caffeic acid derivative according to claim 1, wherein R ₁ , R ₂ , R ₃ and R ₄ of the derivative of Formula 1 are each hydrogen. The composition for inhibiting angiogenesis comprising the caffeic acid derivative of any one of claims 1 to 4 as an active ingredient. The composition of claim 5, wherein the active ingredient inhibits angiogenesis by inhibiting the transcriptional activity of the vascular endothelial growth factor (VEGF) gene. The composition of claim 6, wherein the active ingredient inhibits the activity of the hypoxic-induced signal transducer and activator of transcription 3 (STAT3) and inhibits the action of the regulator of the VEGF promoter, thereby inhibiting the transcriptional activity of VEGF. . 6. The composition of claim 5, wherein the composition is diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma, melanoma, proliferative retinopathy, cancer, psoriasis, hemophiliac joints, capillary proliferation in atherosclerotic plaques, Keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, atopic, allergy, Crohn's disease, restenosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, diabetic nephropathy A composition for the treatment or prevention of malignant neuropathy, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, diabetes, inflammatory or neurodegenerative diseases. 6. The composition of claim 5, wherein the composition is a pharmaceutical composition, a cosmeceutical composition, a cosmetic composition, a nutraceutical composition or a food composition.

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