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WO2009107262A1 - Anticancer composition containing 3”-methylated epigallocatechin gallate - Google Patents

Anticancer composition containing 3”-methylated epigallocatechin gallate Download PDF

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WO2009107262A1
WO2009107262A1 PCT/JP2008/065006 JP2008065006W WO2009107262A1 WO 2009107262 A1 WO2009107262 A1 WO 2009107262A1 JP 2008065006 W JP2008065006 W JP 2008065006W WO 2009107262 A1 WO2009107262 A1 WO 2009107262A1
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egcg
methylated
akt
cell
cancer
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French (fr)
Japanese (ja)
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橋本　修
隆登 上野
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学校法人 久留米大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/16—Tea extraction; Tea extracts; Treating tea extract; Making instant tea
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins

Definitions

  • the present invention relates to a novel anticancer agent composition.
  • the water extract of green tea contains a plurality of polyphenols, which are known as catechins.
  • catechins are known to have an anticancer effect
  • EGCG epigallocatechin gallate
  • Cell cycle arrest and anticancer effects by EGCG have been reported in cell lines or animal models derived from breast cancer, bladder cancer, colon cancer and prostate cancer (Non-patent Documents 2-6).
  • EGCG epigallocatechin gallate
  • EGCG is known to have an antioxidant effect
  • other foods having an antioxidant effect are also known to have an anti-cancer effect
  • This antioxidant action of EGCG is considered to be one of several mechanisms of the anticancer action of EGCG.
  • Non-patent Documents 10 and 11 Oxidative stress activates phosphatidylinositol 3-kinase (PI3K) -Akt signaling, and this PI3K-Akt signaling system regulates cell growth (Non-patent Documents 10 and 11). It is known that PI3K-Akt is constantly activated in various tumor cells (Non-patent Documents 12 and 13). It has been reported that LY294002, an inhibitor of PI3K-Akt, suppresses cell proliferation (Non-patent Document 14). In addition, it has been reported that downregulation by EGCG of the PI3K-Akt signaling system induces cell cycle arrest (Non-patent Documents 4 and 15).
  • Non-patent Document 16 The main methylation sites in the in vivo metabolism of EGCG are the 4 ′ and 4 ′′ positions (Non-patent Document 16), but 4 ′, 4 ”O-methylated EGCG loses the antioxidant activity known for EGCG. (Non-patent Document 17).
  • 3 "O-methylepigallocatechin gallate (hereinafter” 3 "methylated EGCG”) which is O-methylated at the 3 "position of epigallocatechin gallate, is generally found in teas of cultivars generally cultivated in Japan. It is not included, and is contained only in tea leaves of certain varieties of assam hybrids from China and Taiwan, such as “Benifuuki”. Recently, it has been found that 3 "methylated EGCG has an antiallergic effect (Patent Document 1), but the action of 3" methylated EGCG on tumor cells is not known at all.
  • a component of green tea (-)-epigallocatechin-3-gallate, promotes apoptosis in T24 human bladder cancer cells via modulation of the PI3K / Akt pathway and Bs-2 Biochem Biophys Res Commun 2007; 354: 852-7.
  • Chen C Shen G, Hebbar V, Hu R, Owuor E, Kong A. Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells. Carcinogenesis 2003; 24: 1369-78.
  • Adhami V, Ahmad N, Mukhtar H Molecular targets for green tea in prostate cancer prevention. J Nutr 2003; 133: 2417S-24S.
  • An object of the present invention is to provide a novel anticancer agent composition. Another object of the present invention is to provide a novel method for treating and / or preventing cancer.
  • the present invention provides the following formula (I):
  • the anticancer agent composition containing 3 "O-methyl epigallocatechin gallate (3" methylated EGCG) shown by these is provided.
  • composition of the present invention is suitably used for the treatment and prevention of cancer and the prevention of recurrence after cancer treatment.
  • the composition of the present invention may be provided as a pharmaceutical or a health food including a beverage.
  • the present invention also provides a method for treating and / or preventing cancer comprising administering a compound represented by formula (I) to a subject in need thereof.
  • treatment and / or prevention of cancer includes all management of cancer such as prevention, treatment, symptom reduction, symptom reduction, progression stop, metastasis suppression, recurrence prevention, etc. .
  • 3 "methylated EGCG represented by the formula (I) suppresses tumor cell growth at a very low concentration. This tumor cell growth suppression is not due to induction of apoptosis. It is speculated that this is achieved by stopping the cell cycle.
  • the present inventors further found that 3 "methylated EGCG suppresses phosphorylation of Akt (position 473). Phosphorylation of Akt is an indicator of the activity of the PI3K-Akt signaling system that is greatly involved in the cell cycle. Meaning that 3 ′′ methylated EGCG suppresses the activity of the PI3K-Akt signaling system.
  • the present inventors have also found that 3 "methylated EGCG suppresses the expression of cyclin E involved in the transition from G1 to S in the cell cycle.
  • 3 "methylated EGCG has an antioxidant effect. It is considered that the activation of the PI3K-Akt signal system by oxidative stress is involved in the growth of tumor cells. It is thought that 3 "methylated EGCG inactivates the PI3K-Akt signaling system by its antioxidant action and suppresses cell proliferation by stopping the cell cycle.
  • the inventors further confirmed that 3 "methylated EGCG inhibits tumor cell growth in vivo and completed the present invention.
  • composition of the present invention is suitable for cancer control such as cancer treatment, prevention and recurrence prevention, particularly for liver cancer control. Used.
  • Figure 6 shows the effect of EGCG and 3 "methylated EGCG on cell proliferation of human liver tumor line Huh7.
  • Figure 6 shows the effect of EGCG and 3 "methylated EGCG on cell proliferation of human liver tumor line HepG2.
  • the cell cycle distribution of Huh7 cells treated with 0, 0.5 and 5 ⁇ M 3 ′′ methylated EGCG and EGCG for 48 hours is shown.
  • the cell cycle distribution of Huh7 cells treated with 0, 0.5 and 5 ⁇ M 3 ′′ methylated EGCG and EGCG for 48 hours is shown.
  • the percentage of S phase cells in Huh7 cells cultured for 48 hours in the presence of 0, 0.5 and 5 ⁇ M 3 ′′ methylated EGCG is shown.
  • the results of examining the protein expression levels of phosphorylated Akt (P-Akt) and Akt after culturing Huh7 cells for 12 hours in the presence of each concentration of 3 "methylated EGCG are shown by Western blotting.
  • the results of examining the protein expression levels of phosphorylated Akt (P-Akt) and Akt in Huh7 cells cultured in the presence of 5 ⁇ M and 10 ⁇ M EGCG and 3 ′′ methylated EGCG by Western blotting are shown.
  • the relative ratio of the expression level of phosphorylated Akt and Akt of Huh7 cells cultured in the presence of 5 ⁇ M EGCG and 3 ′′ methylated EGCG is shown.
  • FIG. 5 shows the antioxidant effect of 5 ⁇ M EGCG and 3 ′′ methylated EGCG against oxidative stress on Huh7 cells by H 2 O 2 .
  • FIG. 5 shows the effect of 5 ⁇ M EGCG and 3 ′′ methylated EGCG on Akt phosphorylation induced by oxidative stress by H 2 O 2 .
  • the change in tumor volume is shown when physiological saline, 1 mg / kg 3 "methylated EGCG is administered to nude mice transplanted with tumor in the dorsal skin.
  • the state of the tumor of the 3rd week of the group which intraperitoneally administered physiological saline for 3 weeks to the nude mouse which transplanted the tumor in the back skin is shown.
  • the state of the tumor in the 3rd week of the group in which 1 mg of 3 "methylated EGCG was intraperitoneally administered to nude mice transplanted with tumor in the dorsal skin for 3 weeks is shown.
  • the 3 ′′ methylated EGCG used in the present invention may be prepared by chemical synthesis or extracted from natural products.
  • Natural products containing 3 ′′ methylated EGCG include darjeelin hybrids such as Nibori, Beni-Fuji, Yaeho, Shigarase, Yutaka Midori, Kanaya Midori, Okumusashi, Blue Heart Large Bread, Blue Heart Oolong, Big Leaf Oolong, Safflower, Benihikari, Yamakai, Yamamidori, Kara Variety of tea leaves such as bonito, sofu, sofu and okumidori are known.
  • it may be extracted with water or hot water, or any other known method.
  • 3" methylated EGCG is contained, The purity may be low, or the tea leaf itself or a dried and crushed tea leaf may be used.
  • the 3 ′′ methylated EGCG used in the present invention may be prepared using any known method such as the method described in JP-A-2007-306806.
  • the anticancer agent composition of the present invention can be suitably used for cancer control such as cancer treatment and prevention, and recurrence prevention after cancer treatment.
  • the anticancer agent composition of the present invention containing 3 ′′ methylated EGCG represented by the formula I as an active ingredient may be provided as a pharmaceutical composition or a health food for the purpose of maintaining and promoting health.
  • the dosage form of the composition of the present application includes tablets, powders, granules, capsules, injections according to the administration route and purpose of oral administration or parenteral administration (intravenous, intradermal, intraperitoneal administration, etc.). Appropriate dosage forms such as ointments and suppositories may be used.
  • composition of the present invention may contain an appropriate additive.
  • Additives are not particularly limited, excipients, diluents, extenders, solvents, lubricants, adjuvants, binders, disintegrants, coating agents, encapsulating agents, emulsifiers, dispersing agents, suspending agents, thickening agents. Agents, tonicity agents, buffering agents, soothing agents, preservatives, antioxidants, flavoring agents, fragrances, coloring agents, etc. That's fine.
  • composition of the present invention may contain other active ingredients as long as the object of the present invention is not violated.
  • composition of the present invention may also be provided as a health food, or may be provided as a preparation such as powder, lump or liquid for blending with food or drink.
  • examples of the food and drink include beverages containing the compound of formula I, such as tea beverages.
  • the dosage of the anticancer agent composition of the present invention may be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the subject and is not particularly limited.
  • 3 "methylated EGCG may be administered 50 to 5000 mg per day, preferably 100 to 3000 mg, for example 500 mg once per day, 3 times per day.
  • the 3 "methylated EGCG used in the present invention has no apparent toxicity, and it is considered that no health problems will occur even if a large amount is consumed.
  • Naturally derived 3 "O-methylepigallocatechin gallate (3" methylated EGCG) was obtained from Nagara Science Co., Ltd. (Gifu City, Japan) (purity 98%).
  • EGCG and 3 ′′ methylated EGCG were dissolved in PBS to prepare a 50 mM concentration stock solution and stored at 4 ° C. The stock solution was used within 48 hours of preparation.
  • Effect on Cell Proliferation The effect of 3 "methylated EGCG and EGCG on cell proliferation of human hepatoma derived cell lines Huh7 and HepG2 was examined. Each cell line was seeded in a 96-well plate at 1250 cells / well.
  • Western blot analysis Western blot analysis was performed as follows: Whole cell lysate was added to 1 mM phenylmethylsulfonyl fluoride, protein inhibitor cocktail (Sigma-Aldrich Japan), lysis buffer (0.1% Nodidet p-40, 50 mM HEPES; pH 7.5; 10 mM NaF and 1 mM Na3VO4), pH 7.5 , 250 mM NaCl; 20 mM EDTA). Cell lysates were incubated for 15 minutes at 4 ° C. and clarified by centrifugation (14000 ⁇ g) for 15 minutes at 4 ° C.
  • the protein in the supernatant was measured using a DC protein assay kit (Bio-RAD Laboratories, Hercules, CA) according to the method indicated in the kit.
  • the entire cell lysate was added to 2 ⁇ sample loading buffer containing equal volumes of 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol and boiled for 5 minutes.
  • Akt phosphorylation positive and negative proteins from cell total protein (50 ⁇ g) and Phosph-Akt detection kit (Cell Signaling Technologh, Inc., Bvely, MA, USA) were loaded onto SDS-polyacrylamide gel, respectively, for high sensitivity chemiluminescence (ECL) transferred to nitrocellulose membrane (Paul Life Science, Ann Arbor, MI, USA).
  • the membrane was blocked with regular shaking for 1 hour at room temperature with TBS (TBST) containing 0.1% Twin 20 and 5% (w / v) non-fat dry milk for 1 hour. After washing with the same buffer at least 3 times, the membrane was incubated overnight at 4 ° C. with the following primary antibody appropriately diluted with regular shaking: Antibodies: Akt, phosphorylated Akt (Ser473) (Cell Signaling Technology, Inc.), cyclin A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), cyclin E (Santa Cruz Biotechnology, Inc), cyclin D1 (Oncogene Research Producta, Cambridge, MA), p21 (BD Bioscience, San Jose, CA), actin antibody (Sigma-Aldrich).
  • the membrane was washed with TBST buffer at least three times and incubated with a horseradish peroxidase (HRP) -conjugated second antibody at room temperature for 1 hour.
  • HRP horseradish peroxidase
  • the protein-antibody complex was detected by the method described in the manual using ECL (Amersham Biosciences Biotech, Buckinghamshire, United Kingdom).
  • a positive signal from the target protein was visualized with an image analyzer LAS-1000 plus (Fuji Film, Tokyo, Japan). Each band was scanned and the optical density of the band was analyzed with the NIH Image J software program.
  • Huh7 cells were cultured in non-added medium (control), 5 ⁇ M 3 ′′ methylated EGCG or 5 ⁇ MEGCG for 3 hours. Huh7 cells were then cultured in 50 mM 2,7-dichlorodihydrofluorescein diacetate (DCF).
  • DCF 2,7-dichlorodihydrofluorescein diacetate
  • DCF-DA Sigma-Aldrich
  • the cells were treated with 0.1 mM or 1 mM hydrogen peroxide for 30 minutes, then collected by trypsin treatment, and washed twice with cold PBS.
  • the change in fluorescence intensity of Huh7 cells was analyzed by flow cytometry using FacsCalibur at an excitation wavelength of 488 nm. Peak fluorescence intensity and cell number were measured.
  • FIG. 2A shows the results of flow cytometry analysis
  • Figure 2B shows the G1, S and G2 phases of the cell cycle in cells treated with 3 "methylated EGCG or EGCG at various concentrations for 48 hours. It is a graph which shows the ratio of the cell which exists in.
  • FIG. 2C shows the fraction of cell fraction in S phase relative to total cells treated with 3 ′′ methylated EGCG at various concentrations for 48 hours. * P ⁇ 0.05 vs. control.
  • 3 ”methylated EGCG had a function of arresting the cell cycle of tumor cells in the G1 phase in a concentration-dependent manner, but the cell cycle distribution even when treated with EGCG up to 5 ⁇ M.
  • the ratio of S phase of Huh7 cells was untreated, but 43.3% was reduced to 30.5% by 5 ⁇ M 3 "methylated EGCG treatment (FIG. 2C).
  • Huh7 cells were cultured for 12 hours in the presence of 3 ′′ methylated EGCG at concentrations of 0, 1, 5, 10, 20 ⁇ M. After 12 hours, activated Akt (phosphorylated Akt (Ser473), p ⁇ ) in each cell. The protein expression levels of Akt) and total Akt (Akt) were examined by Western blotting to obtain the relative ratio, and the results are shown in FIG. After treating Huh7 cells with 5 and 10 ⁇ M EGCG or 3 ′′ methylated EGCG for 12 hours, p-Akt / Akt was measured as described above. The results are shown in FIGS. 4A and 4B.
  • FIG. 3 shows the protein expression levels of phosphorylated Akt and total Akt, and the graph shows the relative ratio of phosphorylated Akt to Akt.
  • This p-Akt / Akt ratio is an indicator of the activity of the PI3K-Akt signaling system.
  • 3 methylated EGCG inhibited Akt phosphorylation at concentrations of 5 ⁇ M or more.
  • FIG. 4B after treatment with 5 ⁇ M EGCG and 3” methylated EGCG, The p-Akt / Akt relative ratio was 0.97 and 0.53, respectively.
  • Huh7 cells were treated with 5 ⁇ M3" methylated EGCG or 5 ⁇ M EGCG for 3 hours, and then the cells 0.1 mM or 1 mM hydrogen peroxide was allowed to act for 30 minutes.
  • the intracellular oxidation level was examined by incorporating it into DCF-DA cells, which serve as an index of oxidation, using flow cytometry. After treatment with 0.1 mM and 1 mM hydrogen peroxide, the oxidation levels of Huh7 cells were 2.3 times and 3.5 times that of the untreated control, respectively.
  • Huh7 cells were recovered after treatment for 30 minutes in the presence or absence of 1 mM hydrogen peroxide.
  • the Huh7 lysate was subjected to Western blot analysis for pAkt and Akt, and the optical density of the observed band was measured.
  • the relative ratio of p-Akt / Akt increased 2.2-fold over the untreated control.
  • Huh7 cells were treated with 5 ⁇ M methylated EGEG or 5 ⁇ M EGCG for 3 hours and then treated with 1 mM hydrogen peroxide for 30 minutes and then harvested.
  • Western blot analysis was performed for p-Akt and Akt (FIG.
  • In vivo anti-tumor activity The in vivo anti-tumor activity of 3 "methylated EGCG was investigated. 5x10 6 Huh7 cell lines were inoculated into the dorsal skin of nude mice. 1 mg / kg of 3 ′′ methylated EGCG or 1 mg / kg of EGCG was administered intraperitoneally once a day for 3 weeks. Tumor volume was measured every week. Physiological saline was similarly administered to the control group. The test was conducted with 7 animals in each group.
  • FIG. 9 shows a graph of tumor volume in the 1 mg / kg 3" methylated EGCG-treated and control groups. * P ⁇ 0.05, ** p ⁇ 0.01 (vs. control)
  • FIG. 10A shows a photograph of the third week mouse.
  • FIG. 10A shows a control mouse (administered with physiological saline), and
  • FIG. 10B shows a mouse administered with 1 mg / kg of methyl EGCG.
  • FIG. 10 also shows that administration of 1 mg / kg of 3 ′′ methylated EGCG significantly suppressed tumor growth.

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Abstract

It is intended to provide an anticancer composition which contains 3”-O-methylepigallocatechin gallate represented by the following formula (I).

Description

３”メチル化エピガロカテキンガレート含有抗癌剤組成物Anticancer agent composition containing 3 "methylated epigallocatechin gallate
　本発明は、新規な抗癌剤組成物に関する。 The present invention relates to a novel anticancer agent composition.
　緑茶の水抽出物には複数のポリフェノール類が含まれ、これらはカテキン類として知られている。一般にカテキン類には抗癌作用があることが知られており、そのうちエピガロカテキンガレート（以下「ＥＧＣＧ」）についてもっともよく研究されている（非特許文献１）。ＥＧＣＧによる細胞周期停止並びに抗癌作用が、乳癌、膀胱癌、大腸癌および前立腺癌由来のセルラインあるいは動物モデルにおいて報告されている（非特許文献２－６）。Ｋｈａｎらの総説によれば、ＥＧＣＧの抗癌作用の作用機序としては、幾つかの機序が考えられており細胞内シグナルの不活性化により誘導される細胞増殖抑制、細胞形質転換、アポトーシス、並びに血管新生、腫瘍浸潤および転移の抑制などが挙げられる（非特許文献７）。 The water extract of green tea contains a plurality of polyphenols, which are known as catechins. In general, catechins are known to have an anticancer effect, and epigallocatechin gallate (hereinafter “EGCG”) is most frequently studied (Non-patent Document 1). Cell cycle arrest and anticancer effects by EGCG have been reported in cell lines or animal models derived from breast cancer, bladder cancer, colon cancer and prostate cancer (Non-patent Documents 2-6). According to a review by Khan et al., Several mechanisms are considered as the mechanism of action of anticancer activity of EGCG. Cell growth suppression, cell transformation, apoptosis induced by inactivation of intracellular signals And suppression of angiogenesis, tumor invasion and metastasis (Non-patent Document 7).
　一方、ＥＧＣＧが抗酸化作用を有することが知られており、また抗酸化作用を有する他の食物が抗癌作用を有することも知られている（非特許文献８、９）。このＥＧＣＧの抗酸化作用はＥＧＣＧの抗癌作用の幾つかのメカニズムのうちの一つであると考えられている。 On the other hand, EGCG is known to have an antioxidant effect, and other foods having an antioxidant effect are also known to have an anti-cancer effect (Non-Patent Documents 8 and 9). This antioxidant action of EGCG is considered to be one of several mechanisms of the anticancer action of EGCG.
　酸化ストレスはホスファチジルイノシトール３－キナーゼ（ＰＩ３Ｋ）－Ａｋｔシグナル伝達を活性化させ、このＰＩ３Ｋ－Ａｋｔシグナル伝達系は細胞の増殖を調節している（非特許文献１０、１１）。種々の腫瘍細胞においてＰＩ３Ｋ－Ａｋｔが恒常的に活性化していることが知られている（非特許文献１２，１３）。ＰＩ３Ｋ―Ａｋｔの抑制剤であるＬＹ２９４００２が細胞増殖を抑制すること（非特許文献１４）が報告されている。またＰＩ３Ｋ－Ａｋｔシグナル伝達系のＥＧＣＧによるダウンレギュレートが細胞の周期停止を誘導することが報告されている（非特許文献４、１５）。 Oxidative stress activates phosphatidylinositol 3-kinase (PI3K) -Akt signaling, and this PI3K-Akt signaling system regulates cell growth (Non-patent Documents 10 and 11). It is known that PI3K-Akt is constantly activated in various tumor cells (Non-patent Documents 12 and 13). It has been reported that LY294002, an inhibitor of PI3K-Akt, suppresses cell proliferation (Non-patent Document 14). In addition, it has been reported that downregulation by EGCG of the PI3K-Akt signaling system induces cell cycle arrest (Non-patent Documents 4 and 15).
　ＥＧＣＧについての臨床上の報告はまだあまりないが、疫学的および実験室データからは緑茶の飲用により複数の癌を抑制できることが示唆されている（非特許文献２）。
　ＥＧＣＧのインビボ代謝物はＯ－メチル化物であることが知られている。Ｏ－メチル化ＥＧＣＧを下式に示す：
Figure JPOXMLDOC01-appb-C000002
 ＥＧＣＧ：Ｒ１＝Ｒ２＝Ｒ３＝Ｒ４＝Ｈ
４”Ｏ－メチルＥＧＣＧ：Ｒ１＝Ｒ２＝Ｒ３＝Ｈ、Ｒ４＝ＣＨ３
３”Ｏ－メチルＥＧＣＧ：Ｒ１＝Ｒ２＝Ｈ、Ｒ３＝ＣＨ３、Ｒ４＝Ｈ
３’Ｏ－メチルＥＧＣＧ：Ｒ１＝ＣＨ３，Ｒ２＝Ｒ３＝Ｒ４＝Ｈ
４’Ｏ－メチルＥＧＣＧ：Ｒ１＝Ｈ、Ｒ２＝ＣＨ３，Ｒ３＝Ｒ４＝Ｈ
４’，４”ジ－Ｏ－メチルＥＧＣＧ：Ｒ１＝Ｈ、Ｒ”＝ＣＨ３、Ｒ３＝Ｈ、Ｒ４＝ＣＨ３ Although there are not many clinical reports on EGCG yet, epidemiological and laboratory data suggest that drinking green tea can suppress multiple cancers (Non-patent Document 2).
In vivo metabolites of EGCG are known to be O-methylates. O-methylated EGCG is shown below:
Figure JPOXMLDOC01-appb-C000002
 EGCG: R1 = R2 = R3 = R4 = H
4 ″ O-methyl EGCG: R1 = R2 = R3 = H, R4 = CH 3
3 ″ O-methyl EGCG: R1 = R2 = H, R3 = CH 3 , R4 = H
3′O-methyl EGCG: R1 = CH 3 , R2 = R3 = R4 = H
4′O-methyl EGCG: R1 = H, R2 = CH 3 , R3 = R4 = H
4 ′, 4 ″ di-O-methyl EGCG: R1 = H, R ″ = CH 3 , R3 = H, R4 = CH 3
　ＥＧＣＧの生体内代謝において主なメチル化部位は４'および４"位であるが（非特許文献１６）、４'，４"Ｏ－メチル化ＥＧＣＧはＥＧＣＧについて知られた抗酸化作用が消失することが報告されている（非特許文献１７）。 The main methylation sites in the in vivo metabolism of EGCG are the 4 ′ and 4 ″ positions (Non-patent Document 16), but 4 ′, 4 ”O-methylated EGCG loses the antioxidant activity known for EGCG. (Non-patent Document 17).
　エピガロカテキンガレートの３”位置がＯ－メチル化された３”Ｏ－メチルエピガロカテキンガレート（以下「３”メチル化ＥＧＣＧ」）は一般的に日本で栽培されている品種の茶にはほとんど含まれておらず、「べにふうき」などの中国、台湾系統のアッサム雑種の特定の品種の茶葉にのみ含有されている。最近になって３”メチル化ＥＧＣＧに抗アレルギー作用があることが見出されたが（特許文献１）、３”メチル化ＥＧＣＧの腫瘍細胞に対する作用については全く知られていない。 3 "O-methylepigallocatechin gallate (hereinafter" 3 "methylated EGCG"), which is O-methylated at the 3 "position of epigallocatechin gallate, is generally found in teas of cultivars generally cultivated in Japan. It is not included, and is contained only in tea leaves of certain varieties of assam hybrids from China and Taiwan, such as “Benifuuki”. Recently, it has been found that 3 "methylated EGCG has an antiallergic effect (Patent Document 1), but the action of 3" methylated EGCG on tumor cells is not known at all.
特開２００７－１２６３７３号公報JP 2007-126373 A
　本発明は新規抗癌剤組成物を提供することを目的とする。本発明はまた、新規な癌の治療および／または予防法を提供することを目的とする。 An object of the present invention is to provide a novel anticancer agent composition. Another object of the present invention is to provide a novel method for treating and / or preventing cancer.
　本発明は下式（Ｉ）：
Figure JPOXMLDOC01-appb-C000003
 で示される３”Ｏ－メチルエピガロカテキンガレート（３”メチル化ＥＧＣＧ）を含有する、抗癌剤組成物を提供する。 The present invention provides the following formula (I):
Figure JPOXMLDOC01-appb-C000003
 The anticancer agent composition containing 3 "O-methyl epigallocatechin gallate (3" methylated EGCG) shown by these is provided.
　本発明の組成物は癌の治療、予防、並びに癌治療後の再発予防に好適に用いられる。本発明の組成物は、医薬品として提供されてもよいし、または飲料を含む健康食品として提供されてもよい。 The composition of the present invention is suitably used for the treatment and prevention of cancer and the prevention of recurrence after cancer treatment. The composition of the present invention may be provided as a pharmaceutical or a health food including a beverage.
　本発明はまた、式（Ｉ）で示される化合物を必要な対象に投与することを含む、癌の治療および／または予防方法を提供する。本明細書および請求の範囲において、「癌の治療および／または予防」とは、予防、治療、症状の軽減、症状の減退、進行停止、転移抑制、再発防止等、癌のあらゆる管理が含まれる。 The present invention also provides a method for treating and / or preventing cancer comprising administering a compound represented by formula (I) to a subject in need thereof. In the present specification and claims, “treatment and / or prevention of cancer” includes all management of cancer such as prevention, treatment, symptom reduction, symptom reduction, progression stop, metastasis suppression, recurrence prevention, etc. .
　本発明者らは、式（Ｉ）で示される３”メチル化ＥＧＣＧが非常に低濃度で腫瘍細胞の増殖を抑制することを見出した。この腫瘍細胞の増殖抑制は、アポトーシスの誘導によるものではなく細胞周期を止めることによって達成されているものと推測される。 The present inventors have found that 3 "methylated EGCG represented by the formula (I) suppresses tumor cell growth at a very low concentration. This tumor cell growth suppression is not due to induction of apoptosis. It is speculated that this is achieved by stopping the cell cycle.
　本発明者らはさらに、３”メチル化ＥＧＣＧがＡｋｔのリン酸化（４７３位）を抑制することを見出した。Ａｋｔのリン酸化は細胞周期に大きく関与するＰＩ３Ｋ－Ａｋｔシグナル伝達系の活性の指標であり、３”メチル化ＥＧＣＧがＰＩ３Ｋ－Ａｋｔシグナル伝達系の活性を抑制していることを意味する。また、本発明者らは細胞周期のＧ１からＳへの移行に関与するサイクリンＥの発現を３”メチル化ＥＧＣＧが抑制することを見出した。 The present inventors further found that 3 "methylated EGCG suppresses phosphorylation of Akt (position 473). Phosphorylation of Akt is an indicator of the activity of the PI3K-Akt signaling system that is greatly involved in the cell cycle. Meaning that 3 ″ methylated EGCG suppresses the activity of the PI3K-Akt signaling system. The present inventors have also found that 3 "methylated EGCG suppresses the expression of cyclin E involved in the transition from G1 to S in the cell cycle.
　本発明者らはまた、３”メチル化ＥＧＣＧが抗酸化作用を有していることを確認した。腫瘍細胞の増殖においては酸化ストレスによるＰＩ３Ｋ－Ａｋｔシグナル系の活性化が関与しているものと考えられているが、３”メチル化ＥＧＣＧはその抗酸化作用によりＰＩ３Ｋ－Ａｋｔシグナル伝達系を不活性化し、細胞周期を止めることによって細胞増殖を抑制すると推察される。 The present inventors have also confirmed that 3 "methylated EGCG has an antioxidant effect. It is considered that the activation of the PI3K-Akt signal system by oxidative stress is involved in the growth of tumor cells. It is thought that 3 "methylated EGCG inactivates the PI3K-Akt signaling system by its antioxidant action and suppresses cell proliferation by stopping the cell cycle.
　本発明者らはさらに、３”メチル化ＥＧＣＧがインビボにおいて腫瘍細胞の増殖を抑制することを確認して本願発明を完成した。 The inventors further confirmed that 3 "methylated EGCG inhibits tumor cell growth in vivo and completed the present invention.
　本発明の組成物の有効成分である３”メチル化ＥＧＣＧは、強い腫瘍増殖抑制作用を有している。細胞増殖抑制効果は抗酸化作用による細胞周期の停止による細胞増殖抑制によるものと推察され、アポトーシスによるものでないことより正常細胞への障害が少ない可能性も示唆される。従って本発明の組成物は、癌の治療、予防、再発予防など癌の制御、特に肝臓癌の制御に好適に用いられる。 3 ”methylated EGCG, which is an active ingredient of the composition of the present invention, has a strong tumor growth-inhibiting effect. It is presumed that the cell growth-inhibiting effect is due to cell growth inhibition due to cell cycle arrest due to antioxidant action. Therefore, it is also suggested that there is little possibility of damage to normal cells because it is not due to apoptosis, and therefore the composition of the present invention is suitable for cancer control such as cancer treatment, prevention and recurrence prevention, particularly for liver cancer control. Used.
ＥＧＣＧおよび３”メチル化ＥＧＣＧのヒト肝臓腫瘍株Ｈｕｈ７の細胞増殖に及ぼす作用を示す。Figure 6 shows the effect of EGCG and 3 "methylated EGCG on cell proliferation of human liver tumor line Huh7. ＥＧＣＧおよび３”メチル化ＥＧＣＧのヒト肝臓腫瘍株ＨｅｐＧ２の細胞増殖に対する作用を示す。Figure 6 shows the effect of EGCG and 3 "methylated EGCG on cell proliferation of human liver tumor line HepG2. ０、０．５および５μＭの３”メチル化ＥＧＣＧおよびＥＧＣＧで４８時間処理したＨｕｈ７細胞の細胞周期の分布を示す。The cell cycle distribution of Huh7 cells treated with 0, 0.5 and 5 μM 3 ″ methylated EGCG and EGCG for 48 hours is shown. ０、０．５および５μＭの３”メチル化ＥＧＣＧおよびＥＧＣＧで４８時間処理したＨｕｈ７細胞の細胞周期の分布を示す。The cell cycle distribution of Huh7 cells treated with 0, 0.5 and 5 μM 3 ″ methylated EGCG and EGCG for 48 hours is shown. ０，０．５および５μＭの３”メチル化ＥＧＣＧの存在下で４８時間培養したＨｕｈ７細胞中のＳ相の細胞の割合を示す。The percentage of S phase cells in Huh7 cells cultured for 48 hours in the presence of 0, 0.5 and 5 μM 3 ″ methylated EGCG is shown. Ｈｕｈ７細胞を各濃度の３”メチル化ＥＧＣＧの存在下で１２時間培養した後のリン酸化Ａｋｔ（Ｐ－Ａｋｔ）とＡｋｔのタンパク質発現量をウエスタンブロット法にて調べた結果を示す。The results of examining the protein expression levels of phosphorylated Akt (P-Akt) and Akt after culturing Huh7 cells for 12 hours in the presence of each concentration of 3 "methylated EGCG are shown by Western blotting. ５μＭおよび１０μＭのＥＧＣＧおよび３”メチル化ＥＧＣＧの存在下で培養したＨｕｈ７細胞のリン酸化Ａｋｔ（Ｐ－Ａｋｔ）とＡｋｔのタンパク質発現量をウエスタンブロット法にて調べた結果を示す。The results of examining the protein expression levels of phosphorylated Akt (P-Akt) and Akt in Huh7 cells cultured in the presence of 5 μM and 10 μM EGCG and 3 ″ methylated EGCG by Western blotting are shown. ５μＭのＥＧＣＧおよび３”メチル化ＥＧＣＧの存在下で培養したＨｕｈ７細胞のリン酸化ＡｋｔとＡｋｔの発現量の相対比を示す。The relative ratio of the expression level of phosphorylated Akt and Akt of Huh7 cells cultured in the presence of 5 μM EGCG and 3 ″ methylated EGCG is shown. ５μＭの３”メチル化ＥＧＣＧおよびＥＧＣＧの存在下で２４時間まで培養したＨｕｈ７細胞における、リン酸化Ａｋｔ、Ａｋｔ、及びアクチンのタンパク質発現量を示す。The protein expression levels of phosphorylated Akt, Akt, and actin in Huh7 cells cultured for up to 24 hours in the presence of 5 μM 3 ″ methylated EGCG and EGCG are shown. ５μＭの３”メチル化ＥＧＣＧおよびＥＧＣＧの存在下で２４時間まで培養したＨｕｈ７細胞における、ｐ２１、サイクリンＤ１、サイクリンＡ、サイクリンＥおよびアクチンのタンパク質発現量を示す。The protein expression levels of p21, cyclin D1, cyclin A, cyclin E and actin in Huh7 cells cultured for up to 24 hours in the presence of 5 μM 3 ″ methylated EGCG and EGCG are shown. Ｈ２Ｏ２によるＨｕｈ７細胞への酸化ストレスに対する５μＭのＥＧＣＧおよび３”メチル化ＥＧＣＧの抗酸化作用を示す。FIG. 5 shows the antioxidant effect of 5 μM EGCG and 3 ″ methylated EGCG against oxidative stress on Huh7 cells by H 2 O 2 . Ｈ２Ｏ２による酸化ストレスにより誘導されるＡｋｔのリン酸化に対する５μＭのＥＧＣＧおよび３”メチル化ＥＧＣＧの作用を示す。FIG. 5 shows the effect of 5 μM EGCG and 3 ″ methylated EGCG on Akt phosphorylation induced by oxidative stress by H 2 O 2 . 背部皮内に腫瘍を移植したヌードマウスへ生理的食塩水、１ｍｇ／ｋｇの３”メチル化ＥＧＣＧを投与した場合の腫瘍体積の変化を示す。The change in tumor volume is shown when physiological saline, 1 mg / kg 3 "methylated EGCG is administered to nude mice transplanted with tumor in the dorsal skin. 背部皮内に腫瘍を移植したヌードマウスへ、三週間生理的食塩水を腹腔内投与した群の３週間目の腫瘍の状態を示す。The state of the tumor of the 3rd week of the group which intraperitoneally administered physiological saline for 3 weeks to the nude mouse which transplanted the tumor in the back skin is shown. 背部皮内に腫瘍を移植したヌードマウスへ、三週間１ｍｇの３”メチル化ＥＧＣＧを腹腔内投与した群の３週間目の腫瘍の状態を示す。The state of the tumor in the 3rd week of the group in which 1 mg of 3 "methylated EGCG was intraperitoneally administered to nude mice transplanted with tumor in the dorsal skin for 3 weeks is shown.
　本発明において用いられる３”メチル化ＥＧＣＧは、化学合成によって調製しても、天然物から抽出してもよい。３”メチル化ＥＧＣＧを含有する天然物としては、ダージリン系雑種であるべにふうき、べにほまれ、べにふじ、やえほ、するがわせ、ゆたかみどり、かなやみどり、おくむさし、青心大パン、青心ウーロン、大葉ウーロン、紅花、べにひかり、やまかい、やまみどり、からべに、香駿、そうふう、および、おくみどり、などの品種の茶葉が知られている。茶葉より３”メチル化ＥＧＣＧを抽出する場合は、水または湯で抽出しても、その他公知のいずれの方法にて抽出してもよい。３“メチル化ＥＧＣＧが含有されていれば抽出物の純度は低くてもよく、または該茶葉そのもの、もしくは茶葉を乾燥、粉砕したものを用いても良い。その他本発明において用いられる３”メチル化ＥＧＣＧは特開２００７－３０６８０６記載の方法等、公知のいずれの方法を用いて調製されたものであってもよい。 The 3 ″ methylated EGCG used in the present invention may be prepared by chemical synthesis or extracted from natural products. Natural products containing 3 ″ methylated EGCG include darjeelin hybrids such as Nibori, Beni-Fuji, Yaeho, Shigarase, Yutaka Midori, Kanaya Midori, Okumusashi, Blue Heart Large Bread, Blue Heart Oolong, Big Leaf Oolong, Safflower, Benihikari, Yamakai, Yamamidori, Kara Variety of tea leaves such as bonito, sofu, sofu and okumidori are known. When extracting 3 "methylated EGCG from tea leaves, it may be extracted with water or hot water, or any other known method. If 3" methylated EGCG is contained, The purity may be low, or the tea leaf itself or a dried and crushed tea leaf may be used. In addition, the 3 ″ methylated EGCG used in the present invention may be prepared using any known method such as the method described in JP-A-2007-306806.
　本発明の抗癌剤組成物は、癌の治療、予防および癌の治療後の再発予防等、癌の制御に好適に用いることができる。式Ｉで示される３”メチル化ＥＧＣＧを有効成分とする本発明の抗癌剤組成物は、医薬組成物として提供されても、健康の維持及び増進を目的とする健康食品として提供されてもよい。 The anticancer agent composition of the present invention can be suitably used for cancer control such as cancer treatment and prevention, and recurrence prevention after cancer treatment. The anticancer agent composition of the present invention containing 3 ″ methylated EGCG represented by the formula I as an active ingredient may be provided as a pharmaceutical composition or a health food for the purpose of maintaining and promoting health.
　本願の組成物の剤形としては、経口投与あるいは非経口投与（静脈内、皮内、腹腔内投与等）等の投与経路や投与目的に応じて錠剤、粉剤、顆粒剤、カプセル剤、注射剤、軟膏、坐剤など適当な剤形とすればよい。 The dosage form of the composition of the present application includes tablets, powders, granules, capsules, injections according to the administration route and purpose of oral administration or parenteral administration (intravenous, intradermal, intraperitoneal administration, etc.). Appropriate dosage forms such as ointments and suppositories may be used.
　本発明の組成物には適当な添加剤を含有させてもよい。添加剤としては特に限定されず賦形剤、希釈剤、増量剤、溶剤、潤滑剤、補助剤、結合剤、崩壊剤、被覆剤、カプセル化剤、乳化剤、分散剤、懸濁剤、増粘剤、等張化剤、緩衝剤、無痛化剤、保存剤、抗酸化剤、矯味剤、芳香剤、着色剤など、製剤学に関する一般書籍に記載されているものから必要に応じて適宜選択すればよい。 The composition of the present invention may contain an appropriate additive. Additives are not particularly limited, excipients, diluents, extenders, solvents, lubricants, adjuvants, binders, disintegrants, coating agents, encapsulating agents, emulsifiers, dispersing agents, suspending agents, thickening agents. Agents, tonicity agents, buffering agents, soothing agents, preservatives, antioxidants, flavoring agents, fragrances, coloring agents, etc. That's fine.
　本発明の組成物には、本発明の目的に反しない限り他の有効成分を含有させてもよい。 The composition of the present invention may contain other active ingredients as long as the object of the present invention is not violated.
　本発明の組成物はまた、健康食品として提供されてもよいし、飲食物に配合するための粉、塊、液状等の製剤として提供されてもよい。飲食物としては、式Ｉの化合物を含有する飲料、例えば茶飲料が例示される。 The composition of the present invention may also be provided as a health food, or may be provided as a preparation such as powder, lump or liquid for blending with food or drink. Examples of the food and drink include beverages containing the compound of formula I, such as tea beverages.
　本発明の抗癌剤組成物の投与量は対象者の年齢、性別、体重、症状、投与経路等によって適宜決定すればよく、特に限定されない。例えば、成人男性の癌の予防または治療のために経口投与する場合、３”メチル化ＥＧＣＧを１日５０～５０００ｍｇ、好ましくは１００～３０００ｍｇ、例えば１回５００ｍｇを１日３回投与すればよい。本発明に用いられる３”メチル化ＥＧＣＧには明らかな毒性は認められておらず、多量に摂取しても健康上の問題が生じることはないと考えられる。 The dosage of the anticancer agent composition of the present invention may be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the subject and is not particularly limited. For example, when administered orally for the prevention or treatment of cancer in adult males, 3 "methylated EGCG may be administered 50 to 5000 mg per day, preferably 100 to 3000 mg, for example 500 mg once per day, 3 times per day. The 3 "methylated EGCG used in the present invention has no apparent toxicity, and it is considered that no health problems will occur even if a large amount is consumed.
以下実施例により本願を更に詳細に説明する。下記実施例は説明のためのものであって、本願を限定するものではない。
材料および方法
細胞および試薬
　特に断らない限り、下記実施例において細胞の培養には１０％牛胎児血清並びに１００Ｕ／ｍｌのペニシリンとストレプトマイシンを添加したＤＭＥＭ培地を用い、３７℃、５％ＣＯ２インキュベーター内で行った。
　ヒト肝細胞由来細胞株Ｈｕｈ７およびＨｅｐＧ２は東北大学附属癌細胞保存施設（日本国仙台市）より入手した。
　天然由来の（－）－エピガロカテキンガレート（ＥＧＣＧ）は和光純薬工業株式会社（日本国大阪市）より入手した（純度９０％）。
　天然由来の３”Ｏ－メチルエピガロカテキンガレート（３”メチル化ＥＧＣＧ）は長良サイエンス株式会社（日本国岐阜市）より入手した（純度９８％）。
ＥＧＣＧおよび３”メチル化ＥＧＣＧはＰＢＳに溶解して５０ｍＭ濃度の保存溶液を調製し、４℃にて保存した。保存溶液は調製から４８時間以内に使用した。
細胞増殖に対する作用
　ヒト肝癌由来細胞株Ｈｕｈ７およびＨｅｐＧ２の細胞増殖に対する３”メチル化ＥＧＣＧおよびＥＧＣＧの作用を調べた。各細胞株を、１２５０個／ウエルとなるよう９６穴プレートへ播種した。ここへ各種濃度のＥＧＣＧまたは３”メチル化ＥＧＣＧを添加し、４８時間培養した。コントロールとしては何も添加しないで同様に培養した。４８時間処理後の各細胞の増殖能を、ブロモデオキシウリジン（ＢｒｄＵ）取り込み（ＥＬＩＳＡ法）（Roche Applied Science, Penzberg, Germany）にて、キットの手順にしたがって測定した。 Hereinafter, the present application will be described in more detail by way of examples. The following examples are for illustrative purposes and do not limit the present application.
Materials and methods
Cells and Reagents Unless otherwise specified, in the following examples, cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum and 100 U / ml penicillin and streptomycin in a 37 ° C., 5% CO 2 incubator.
Human hepatocyte-derived cell lines Huh7 and HepG2 were obtained from Tohoku University cancer cell storage facility (Sendai City, Japan).
Naturally derived (−)-epigallocatechin gallate (EGCG) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) (purity 90%).
Naturally derived 3 "O-methylepigallocatechin gallate (3" methylated EGCG) was obtained from Nagara Science Co., Ltd. (Gifu City, Japan) (purity 98%).
EGCG and 3 ″ methylated EGCG were dissolved in PBS to prepare a 50 mM concentration stock solution and stored at 4 ° C. The stock solution was used within 48 hours of preparation.
Effect on Cell Proliferation The effect of 3 "methylated EGCG and EGCG on cell proliferation of human hepatoma derived cell lines Huh7 and HepG2 was examined. Each cell line was seeded in a 96-well plate at 1250 cells / well. Various concentrations of EGCG or 3 ″ methylated EGCG were added and incubated for 48 hours. As a control, the cells were cultured in the same manner without adding anything. The proliferation ability of each cell after 48 hours treatment was measured by bromodeoxyuridine (BrdU) incorporation (ELISA method) (Roche Applied Science, Penzberg, Germany) according to the procedure of the kit.
細胞周期に対する作用
　Ｈｕｈ７細胞を３”メチル化ＥＧＣＧまたはＥＧＣＧの存在下で４８時間培養した後の細胞周期をフローサイトメトリーにて観察した。４８時間培養後の細胞を２回ＰＢＳで洗浄し、０．１％トリトンＸ－１００および５０μｇ／ｍｌのヨウ化プロピジウム（ＰＩ）を含有するバッファーに分散させた。フローサイトメトリー分析はＦＡＣＳｃａｌｉｂｕｒ（Becton Dickinson Franklin Lakes, NJ, USA)を用いて行い、結果をQuad Statistics of CellQuest software program (Becton Dickinson)を用いて解析した。試験は３連で行った。 Effect on Cell Cycle Huh7 cells were observed by flow cytometry after culturing for 48 hours in the presence of 3 "methylated EGCG or EGCG. The cells after 48 hours were washed twice with PBS, 0 Dispersed in a buffer containing 1% Triton X-100 and 50 μg / ml propidium iodide (PI) Flow cytometric analysis was performed using FACSCalibur (Becton Dickinson Franklin Lakes, NJ, USA) The analysis was performed using the Quad Statistics of CellQuest software program (Becton Dickinson).
ウエスタンブロット分析
　ウエスタンブロット分析は下記のごとく実施した：
　細胞全体のライセートを１ｍＭのフェニルメチルスルホニルフルオライド、タンパク質インヒビターカクテル（Sigma-Aldrich Japan)、１０ｍＭのＮａＦおよび１ｍＭのＮａ３ＶＯ４を加えたリシスバッファー（0.1% Nodidet p-40, 50 mM HEPES; pH7.5, 250 mM NaCl; 20 mM EDTA)を用いて調製した。細胞ライセートを４℃にて１５分間インキュベートし、４℃にて１５分間遠心分離（１４０００×ｇ）により清澄化した。上清中のタンパク質をＤＣタンパク質アッセイキット (Bio-RAD Laboratories, Hercules, CA)を用いて、キットに指示された方法にて測定した。細胞ライセート全体を等容量の２％ドデシル硫酸ナトリウム（ＳＤＳ）および５％２－メルカプトエタノールを含有する２×サンプルローディングバッファーへ加え、５分間煮沸した。細胞全タンパク質（５０μｇ）およびPhosph-Akt検出キット（Cell Signaling Technologh, Inc., Bvely, MA, USA）のＡｋｔリン酸化ポジティブ及びネガティブタンパク質を、それぞれＳＤＳ－ポリアクリルアミドゲルへロードし、高感度化学発光（ＥＣＬ）ニトロセルロースメンブレン（Paul Life Science, Ann Arbor, MI, USA）に移した。メンブレンを１時間０．１％のツイン２０を含有するＴＢＳ（ＴＢＳＴ）および５％（ｗ／ｖ）の無脂肪ドライミルクとともに１時間室温で規則的に振とうしながらブロックした。少なくとも３回同じバッファーにて洗浄した後、メンブレンを規則的に振とうしながら適当に希釈した下記一次抗体とともに４℃にて一晩インキュベートした：
抗体：　Ａｋｔ、リン酸化Ａｋｔ（Ｓｅｒ４７３）（Cell Signaling Technology, Inc.）、サイクリンＡ(Santa Cruz Biotechnology, Inc.,  Santa Cruz, CA), サイクリンＥ(Santa Cruz Biotechnology, Inc),サイクリンＤ１ (Oncogene Research Producta, Cambridge, MA), p21 (BD Bioscience, San Jose, CA), アクチン抗体 (Sigma-Aldrich)。 Western blot analysis Western blot analysis was performed as follows:
Whole cell lysate was added to 1 mM phenylmethylsulfonyl fluoride, protein inhibitor cocktail (Sigma-Aldrich Japan), lysis buffer (0.1% Nodidet p-40, 50 mM HEPES; pH 7.5; 10 mM NaF and 1 mM Na3VO4), pH 7.5 , 250 mM NaCl; 20 mM EDTA). Cell lysates were incubated for 15 minutes at 4 ° C. and clarified by centrifugation (14000 × g) for 15 minutes at 4 ° C. The protein in the supernatant was measured using a DC protein assay kit (Bio-RAD Laboratories, Hercules, CA) according to the method indicated in the kit. The entire cell lysate was added to 2 × sample loading buffer containing equal volumes of 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol and boiled for 5 minutes. Akt phosphorylation positive and negative proteins from cell total protein (50 μg) and Phosph-Akt detection kit (Cell Signaling Technologh, Inc., Bvely, MA, USA) were loaded onto SDS-polyacrylamide gel, respectively, for high sensitivity chemiluminescence (ECL) transferred to nitrocellulose membrane (Paul Life Science, Ann Arbor, MI, USA). The membrane was blocked with regular shaking for 1 hour at room temperature with TBS (TBST) containing 0.1 % Twin 20 and 5% (w / v) non-fat dry milk for 1 hour. After washing with the same buffer at least 3 times, the membrane was incubated overnight at 4 ° C. with the following primary antibody appropriately diluted with regular shaking:
Antibodies: Akt, phosphorylated Akt (Ser473) (Cell Signaling Technology, Inc.), cyclin A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), cyclin E (Santa Cruz Biotechnology, Inc), cyclin D1 (Oncogene Research Producta, Cambridge, MA), p21 (BD Bioscience, San Jose, CA), actin antibody (Sigma-Aldrich).
　インキュベーション終了後、メンブレンを少なくとも３回ＴＢＳＴバッファーにて洗浄し、室温でホースラディッシュパーオキシダーゼ（ＨＲＰ）複合化第２抗体とともに１時間インキュベートした。タンパク質－抗体複合体はＥＣＬ (Amersham Biosciences Biotech, Buckinghamshire, United Kingdom) にて説明書に記載の方法にて検出した。標的タンパク質からの陽性シグナルをイメージアナライザーＬＡＳ－１０００プラス（富士フイルム、日本国東京）にて可視化した。各バンドをスキャンし、バンドの光学密度をＮＩＨイメージＪソフトウエアプログラムにて解析した。 After completion of the incubation, the membrane was washed with TBST buffer at least three times and incubated with a horseradish peroxidase (HRP) -conjugated second antibody at room temperature for 1 hour. The protein-antibody complex was detected by the method described in the manual using ECL (Amersham Biosciences Biotech, Buckinghamshire, United Kingdom). A positive signal from the target protein was visualized with an image analyzer LAS-1000 plus (Fuji Film, Tokyo, Japan). Each band was scanned and the optical density of the band was analyzed with the NIH Image J software program.
細胞内酸化レベルに対する作用
　Ｈｕｈ７細胞を無添加培地（コントロール）、５μＭの３”メチル化ＥＧＣＧまたは５μＭＥＧＣＧ添加培地で３時間培養した。Ｈｕｈ７細胞を次いで５０ｍＭの２，７－ジクロロジヒドロフルオレセインジアセテート（ＤＣＦ－ＤＡ、Sigma-Aldrich)にて１５分処理した。ＤＣＦ－ＤＡはフリーラジカルの生成を検出するために用いられる。この細胞浸透性剤の蛍光強度は酸化により増強される。即ちＤＣＦ－ＤＡ蛍光強度は細胞内酸化レベルを示す指標である。 Effect on intracellular oxidation levels Huh7 cells were cultured in non-added medium (control), 5 μM 3 ″ methylated EGCG or 5 μMEGCG for 3 hours. Huh7 cells were then cultured in 50 mM 2,7-dichlorodihydrofluorescein diacetate (DCF). -DA, Sigma-Aldrich) DCF-DA is used to detect the generation of free radicals, the fluorescence intensity of this cell penetrating agent is enhanced by oxidation, ie DCF-DA fluorescence Intensity is an indicator of intracellular oxidation level.
　細胞を０．１ｍＭまたは１ｍＭの過酸化水素にて３０分間処理した後トリプシン処理にて回収し、冷ＰＢＳで２回洗浄した。Ｈｕｈ７細胞の蛍光強度の変化をＦａｃｓＣａｌｉｂｕｒを用い、励起波長４８８ｎｍとしてフローサイトメトリー分析を行った。ピークの蛍光強度及び細胞数を測定した。 The cells were treated with 0.1 mM or 1 mM hydrogen peroxide for 30 minutes, then collected by trypsin treatment, and washed twice with cold PBS. The change in fluorescence intensity of Huh7 cells was analyzed by flow cytometry using FacsCalibur at an excitation wavelength of 488 nm. Peak fluorescence intensity and cell number were measured.
結果
細胞増殖に対する作用
　ヒト肝癌由来細胞株Ｈｕｈ７およびＨｅｐＧ２の細胞増殖に対する３”メチル化ＥＧＣＧおよびＥＧＣＧの作用を調べた。
　各細胞株を、１２５０個／ウエルとなるよう９６穴プレートへ播種した。ここへ１、５、１０、２０μＭとなるようにＥＧＣＧまたは３”メチル化ＥＧＣＧを添加し、４８時間培養した。コントロールとしては何も添加しないで同様に培養した。４８時間処理後の各細胞の増殖能を、ＢｒｄＵ取り込み（ＥＬＩＳＡ法）にて測定した。結果を図１に示す。図１ＡはＨｕｈ７、図１ＢはＨｅｐＧ２細胞株の結果をそれぞれ示す。＊ｐ＜０．０５（対コントロール）。 result
Effects on cell proliferation The effects of 3 "methylated EGCG and EGCG on the cell proliferation of human hepatoma derived cell lines Huh7 and HepG2 were investigated.
Each cell line was seeded in a 96-well plate at 1250 cells / well. Here, EGCG or 3 ″ methylated EGCG was added so as to be 1, 5, 10, 20 μM, and cultured for 48 hours. As a control, the cells were cultured in the same manner without adding anything. The proliferation ability was measured by BrdU incorporation (ELISA method), and the results are shown in Fig. 1. Fig. 1A shows the results of Huh7 and Fig. 1B shows the results of HepG2 cell line, respectively * p <0.05 (vs. control).
　図１Ａから明らかなように、Ｈｕｈ７細胞においてはＢｒｄＵ取り込みは少なくとも５μＭの３”メチル化ＥＧＣＧにて４８時間処理後に減少した。５μＭの３”メチル化ＥＧＣＧ処理は、５μＭのＥＧＣＧ処理と比較してＨｕｈ７細胞において細胞増殖を４８％、ＨｅｐＧ２細胞において３５％抑制した。 As is apparent from FIG. 1A, BrdU incorporation was reduced in Huh7 cells after treatment with at least 5 μM 3 ″ methylated EGCG for 48 hours. 5 μM 3 ″ methylated EGCG treatment was compared to 5 μM EGCG treatment. Cell proliferation was suppressed by 48% in Huh7 cells and 35% in HepG2 cells.
細胞周期に対する作用
　Ｈｕｈ７細胞を０、０．５および５μＭの３”メチル化ＥＧＣＧまたはＥＧＣＧの存在下で４８時間培養した時点における細胞周期をフローサイトメトリーにて観察した。試験は３連で行った。結果を図２Ａ～Ｃに示す。図２Ａはフローサイトメトリー分析結果を、図２Ｂは各濃度の３”メチル化ＥＧＣＧまたはＥＧＣＧで４８時間処理した細胞において、細胞周期のＧ１，ＳおよびＧ２相に有る細胞の割合を示すグラフである。図２Ｃは各濃度の３”メチル化ＥＧＣＧにより４８時間処理した全細胞に対するＳ相にある細胞フラクションの割合を示す。＊ｐ＜０．０５、対コントロール Effect on cell cycle The cell cycle was observed by flow cytometry when Huh7 cells were cultured for 48 hours in the presence of 0, 0.5 and 5 μM of 3 ″ methylated EGCG or EGCG. The test was performed in triplicate. The results are shown in Figures 2A to C. Figure 2A shows the results of flow cytometry analysis, and Figure 2B shows the G1, S and G2 phases of the cell cycle in cells treated with 3 "methylated EGCG or EGCG at various concentrations for 48 hours. It is a graph which shows the ratio of the cell which exists in. FIG. 2C shows the fraction of cell fraction in S phase relative to total cells treated with 3 ″ methylated EGCG at various concentrations for 48 hours. * P <0.05 vs. control.
　図２Ａから明らかなように、３”メチル化ＥＧＣＧは濃度依存的に腫瘍細胞の細胞周期をＧ１相で停止させる作用を有していたが、５μＭまでのＥＧＣＧで処理しても細胞周期の分布に変化は認められなかった。無処理、５μＭのＥＧＣＧ４８時間処理、および５μＭの３”メチル化ＥＧＣＧ４８時間処理後のＧ１相の細胞はそれぞれ４８％、４１％および５８％であった（図２Ｂ）。Ｈｕｈ７細胞のＳ相の割合は無処理で４３．３％のものが５μＭの３”メチル化ＥＧＣＧ処理にて３０．５％に下がった（図２Ｃ）。これらの結果は３”メチル化ＥＧＣＧが細胞周期の停止を誘導し、低濃度にて細胞増殖を抑制することを示唆するものである。 As is clear from FIG. 2A, 3 ”methylated EGCG had a function of arresting the cell cycle of tumor cells in the G1 phase in a concentration-dependent manner, but the cell cycle distribution even when treated with EGCG up to 5 μM. There was no change in the G1 phase cells after treatment with 5 μM EGCG 48 hours and 5 μM 3 ″ methylated EGCG 48 hours, respectively, (48%, 41% and 58%) (FIG. 2B). . The ratio of S phase of Huh7 cells was untreated, but 43.3% was reduced to 30.5% by 5 μM 3 "methylated EGCG treatment (FIG. 2C). These results indicate that 3" methylated EGCG It is suggested to induce cell cycle arrest and suppress cell proliferation at low concentrations.
ＰＩ３Ｋ－Ａｋｔシグナル系に対する作用Action on PI3K-Akt signal system
　Ｈｕｈ７細胞を０、１、５、１０、２０μＭの濃度の３”メチル化ＥＧＣＧの存在下で１２時間培養した。１２時間後の各細胞中の活性化Ａｋｔ（リン酸化Ａｋｔ（Ｓｅｒ４７３）、ｐ－Ａｋｔ）および全Ａｋｔ（Ａｋｔ）のタンパク質発現量をウエスタンブロット法により調べ、その相対比を得た。結果を図３に示す。
　Ｈｕｈ７細胞を５および１０μＭのＥＧＣＧまたは３”メチル化ＥＧＣＧにて１２時間処理した後のｐ－Ａｋｔ／Ａｋｔを上記と同様に測定した。結果を図４Ａおよび図４Ｂに示す。 Huh7 cells were cultured for 12 hours in the presence of 3 ″ methylated EGCG at concentrations of 0, 1, 5, 10, 20 μM. After 12 hours, activated Akt (phosphorylated Akt (Ser473), p−) in each cell. The protein expression levels of Akt) and total Akt (Akt) were examined by Western blotting to obtain the relative ratio, and the results are shown in FIG.
After treating Huh7 cells with 5 and 10 μM EGCG or 3 ″ methylated EGCG for 12 hours, p-Akt / Akt was measured as described above. The results are shown in FIGS. 4A and 4B.
　またＨｕｈ７細胞を５μＭのＥＧＣＧまたは３”メチル化ＥＧＣＧの存在下で０、３、６、１２および２４時間培養した後のＡｋｔおよびｐ－Ａｋｔタンパク質発現量をウエスタンブロット法にて調べた。結果を図５に示す。 Further, the expression levels of Akt and p-Akt proteins after culturing Huh7 cells in the presence of 5 μM EGCG or 3 ″ methylated EGCG for 0, 3, 6, 12 and 24 hours were examined by Western blotting. As shown in FIG.
　図３はリン酸化Ａｋｔと全Ａｋｔのそれぞれのタンパク質発現量を示し、グラフはリン酸化ＡｋｔのＡｋｔに対する相対比を示す。このｐ－Ａｋｔ／Ａｋｔ比はＰＩ３Ｋ－Ａｋｔシグナル伝達系の活性の指標である。図３から明らかなように、３”メチル化ＥＧＣＧは５μＭ以上の濃度でＡｋｔのリン酸化を抑制した。また図４Ｂより明らかなように５μＭのＥＧＣＧおよび３”メチル化ＥＧＣＧにて処理した後のｐ－Ａｋｔ／Ａｋｔ相対比はそれぞれ０．９７および０．５３であった。５μＭの３”メチル化ＥＧＣＧによる処理は同濃度のＥＧＣＧによる処理と比して有意に（ｐ＜０．０５）ｐ－Ａｋｔ／Ａｋｔ相対比を減少させた、即ち、３”メチル化ＥＧＣＧはＡｋｔのリン酸化を抑制した。 FIG. 3 shows the protein expression levels of phosphorylated Akt and total Akt, and the graph shows the relative ratio of phosphorylated Akt to Akt. This p-Akt / Akt ratio is an indicator of the activity of the PI3K-Akt signaling system. As is apparent from FIG. 3, 3 ”methylated EGCG inhibited Akt phosphorylation at concentrations of 5 μM or more. As is clear from FIG. 4B, after treatment with 5 μM EGCG and 3” methylated EGCG, The p-Akt / Akt relative ratio was 0.97 and 0.53, respectively. Treatment with 5 μM 3 ″ methylated EGCG significantly (p <0.05) decreased the p-Akt / Akt relative ratio compared to treatment with the same concentration of EGCG, ie 3 ″ methylated EGCG was Akt. Inhibited phosphorylation.
　また図５から明らかなように、５μＭの３”メチル化ＥＧＣＧにて６時間以上処理した後は、ｐ－Ａｋｔ／Ａｋｔ相対比が減少したが、５μＭのＥＧＣＧによる処理によってはこの比には変化なかった。
　これらの結果は、３”メチル化ＥＧＣＧがＰＩ３Ｋ－Ａｋｔシグナル伝達経路の活性化を低濃度の処理により抑制することを強く示唆するものである。 As is clear from FIG. 5, the p-Akt / Akt relative ratio decreased after treatment with 5 μM 3 ″ methylated EGCG for 6 hours or more, but this ratio changed depending on the treatment with 5 μM EGCG. There wasn't.
These results strongly suggest that 3 "methylated EGCG suppresses activation of the PI3K-Akt signaling pathway by low concentration treatment.
ＰＩ３Ｋ－Ａｋｔシグナル伝達系関連因子の発現に対する作用
　Ｈｕｈ７細胞株を５μＭの３”メチル化ＥＧＣＧまたはＥＧＣＧの存在下で２４時間培養した。０、３時間、６時間、１２時間および２４時間培養後のＰ－Ａｋｔ（リン酸化Ａｋｔ（Ｓｅｒ４７３））、Ａｋｔ、サイクリンＤ１、サイクリンＡ、サイクリンＥおよびアクチンの発現量をウエスタンブロット法により測定した。結果を図６に示す。 Effect on expression of PI3K-Akt signaling system related factor Huh7 cell line was cultured for 24 hours in the presence of 5 μM 3 "methylated EGCG or EGCG. After 0, 3, 6, 12 and 24 hours of culture The expression levels of P-Akt (phosphorylated Akt (Ser473)), Akt, cyclin D1, cyclin A, cyclin E, and actin were measured by Western blotting, and the results are shown in FIG.
　サイクリンＥのタンパク質の発現レベルが３”メチル化ＥＧＣＧ処理によって6時間以降下がった。しかしながら、ｐ２１は３”メチル化ＥＧＣＧあるいはＥＧＣＧ処理によって変化無かった。サイクリンＤ１は３”メチル化ＥＧＣＧ処理によっても減らなかった。この結果は、３”メチル化ＥＧＣＧ処理後の細胞周期のＧ１相停止は細胞周期の進行を調節するタンパク質に対する作用に起因することを示唆する。 The expression level of cyclin E protein decreased after 6 hours by 3 "methylated EGCG treatment. However, p21 was not changed by 3" methylated EGCG or EGCG treatment. Cyclin D1 was not reduced by 3 "methylated EGCG treatment. This result suggests that G1 phase arrest of the cell cycle after 3" methylated EGCG treatment is due to an action on proteins that regulate cell cycle progression. To do.
細胞酸化に関する作用
　Ｈｕｈ７細胞の過酸化水素処理後の酸化レベルに対する３”メチル化ＥＧＣＧおよびＥＧＣＧの効果を調べた。Ｈｕｈ７細胞を５μＭ３”メチル化ＥＧＣＧまたは５μＭのＥＧＣＧで３時間処理し、次いでこの細胞へ０．１ｍＭまたは１ｍＭの過酸化水素を３０分作用させた。細胞内酸化レベルをフローサイトメトリーを用い、酸化の指標となるＤＣＦ－ＤＡ細胞に取り込ませて調べた。０．１ｍＭと１ｍＭの過酸化水素処理の後、Ｈｕｈ７細胞の酸化レベルはそれぞれ無処理コントロールの２．３倍、３．５倍となった。０．１ｍＭの過酸化水素処理による酸化レベルを３”メチル化ＥＧＣＧとＥＧＣＧはそれぞれ１．５倍、０．８倍と顕著に抑制した。１ｍＭの過酸化水素による細胞内酸化は、３”メチルＥＧＣＧでは抑制したが、ＥＧＣＧは係る抑制作用を示さなかった（図７）。これらの結果は３”メチル化ＥＧＣＧが低用量で酸化を抑制することができることを示す。 Effects on Cellular Oxidation The effect of 3 "methylated EGCG and EGCG on the oxidation level after treatment with hydrogen peroxide in Huh7 cells was investigated. Huh7 cells were treated with 5 μM3" methylated EGCG or 5 μM EGCG for 3 hours, and then the cells 0.1 mM or 1 mM hydrogen peroxide was allowed to act for 30 minutes. The intracellular oxidation level was examined by incorporating it into DCF-DA cells, which serve as an index of oxidation, using flow cytometry. After treatment with 0.1 mM and 1 mM hydrogen peroxide, the oxidation levels of Huh7 cells were 2.3 times and 3.5 times that of the untreated control, respectively. The oxidation level by treatment with 0.1 mM hydrogen peroxide was markedly suppressed by 3 "methylated EGCG and EGCG, 1.5 times and 0.8 times, respectively. The intracellular oxidation by 1 mM hydrogen peroxide was 3" methyl. Although EGCG suppressed, EGCG did not show the inhibitory effect (FIG. 7). These results indicate that 3 "methylated EGCG can inhibit oxidation at low doses.
　次いで１ｍＭの過酸化水素処理により誘導されるＡｋｔのリン酸化を観察した。１ｍＭ過酸化水素の存在、非存在下で３０分間処理した後のＨｕｈ７細胞を回収した。Ｈｕｈ７のライセートにつき、ｐＡｋｔおよびＡｋｔについてのウエスタンブロット解析を行い、観察されたバンドの光学濃度を測定した。１ｍＭの過酸化水素処理群では、ｐ－Ａｋｔ／Ａｋｔの相対比は無処理コントロールに対して２．２倍に増えた。Ｈｕｈ７細胞を５μＭのメチル化ＥＧＥＧまたは５μＭのＥＧＣＧにて３時間処理し、次いで１ｍＭの過酸化水素にて３０分処理した後に回収した。ｐ－ＡｋｔとＡｋｔについてウエスタンブロット分析を行った（図８Ａ）。ｐ－Ａｋｔ／Ａｋｔ強度の相対比は５μＭの３”メチル化ＥＧＣＧと５μＭのＥＧＣＧでそれぞれ０．９倍、１．６倍となった（図８Ｂ）。本データは５μＭの３”メチル化ＥＧＣＧによる前処理により酸化レベルを抑え、１ｍＭの過酸化水素により誘導されるＡｋｔのリン酸化が抑制されることを示す。 Next, phosphorylation of Akt induced by treatment with 1 mM hydrogen peroxide was observed. Huh7 cells were recovered after treatment for 30 minutes in the presence or absence of 1 mM hydrogen peroxide. The Huh7 lysate was subjected to Western blot analysis for pAkt and Akt, and the optical density of the observed band was measured. In the 1 mM hydrogen peroxide treatment group, the relative ratio of p-Akt / Akt increased 2.2-fold over the untreated control. Huh7 cells were treated with 5 μM methylated EGEG or 5 μM EGCG for 3 hours and then treated with 1 mM hydrogen peroxide for 30 minutes and then harvested. Western blot analysis was performed for p-Akt and Akt (FIG. 8A). The relative ratio of p-Akt / Akt intensities was 0.9 and 1.6 times for 5 μM 3 ″ methylated EGCG and 5 μM EGCG, respectively (FIG. 8B). This data is 5 μM 3 ″ methylated EGCG. This shows that the oxidation level is suppressed by pretreatment with, and phosphorylation of Akt induced by 1 mM hydrogen peroxide is suppressed.
インビボ抗腫瘍作用
　３”メチル化ＥＧＣＧのインビボでの抗腫瘍作用を検討した。ヌードマウスの背部皮内へＨｕｈ７細胞株を一匹当たり５×１０６個接種した。このマウスへ０．１ｍｇ／ｋｇ、１ｍｇ／ｋｇの３”メチル化ＥＧＣＧ、または１ｍｇ／ｋｇのＥＧＣＧを腹腔内へ１日１回、３週間投与した。１週間毎に腫瘍の体積を計測した。コントロール群には生理的食塩水を同様に投与した。試験は各群７匹で行った。 In vivo anti-tumor activity The in vivo anti-tumor activity of 3 "methylated EGCG was investigated. 5x10 6 Huh7 cell lines were inoculated into the dorsal skin of nude mice. 1 mg / kg of 3 ″ methylated EGCG or 1 mg / kg of EGCG was administered intraperitoneally once a day for 3 weeks. Tumor volume was measured every week. Physiological saline was similarly administered to the control group. The test was conducted with 7 animals in each group.
　０．１ｍｇ／ｋｇの３”メチル化ＥＧＣＧ（ｎ＝７）、１ｍｇ／ｋｇの３”メチル化ＥＧＣＧ（ｎ＝１２）、１ｍｇ／ｋｇのＥＧＣＧ（ｎ＝７）および生理的食塩水コントロール（ｎ＝１２）を投与した各群の腫瘍体積はそれぞれ１週間後は３５３±１８４ｍｍ３、１４９±３３ｍｍ３、２５６±１２３ｍｍ３、および２０２±９２ｍｍ３、２週間後はそれぞれ９１０±３４３ｍｍ３、４１８±２１８ｍｍ３、８３１±３８７ｍｍ３および８３４±３６５ｍｍ３、および３週間後はそれぞれ１６３４±５５１ｍｍ３、８３１±２４５ｍｍ３、１６３１±７５９ｍｍ３，および１６０１±５８７ｍｍ３であった。 0.1 mg / kg 3 "methylated EGCG (n = 7), 1 mg / kg 3" methylated EGCG (n = 12), 1 mg / kg EGCG (n = 7) and saline control (n = 12), respectively after 1 week the tumor volume of each group were administered with 353 ± 184mm 3, 149 ± 33mm 3, 256 ± 123mm 3, and 202 ± 92 mm 3, after 2 weeks respectively 910 ± 343mm 3, 418 ± 218mm 3, 831 ± 387mm 3 and 834 ± 365 mm 3, and after 3 weeks, respectively 1634 ± 551mm 3, 831 ± 245mm 3, 1631 ± 759mm 3, and 1601 was ± 587mm 3.
　１ｍｇ／ｋｇの３”メチル化ＥＧＣＧによる処理は腫瘍の増殖をコントロールの５０％迄（２週間、ｐ＜０．０５，３週間、ｐ＜０．０１）抑制したが、同濃度のＥＧＣＧ処理（ｉｐ投与、１ｍｇ／ｋｇ／日）はコントール群と相違がなかった。０．１ｍｇの３”メチル化ＥＧＣＧもまた、コントロール群と相違がなかった。処置期間中コントロール、ＥＧＣＧ処理または３”メチル化ＥＧＣＧ処理群間で体重には相違なかった。図９に、１ｍｇ／ｋｇの３”メチル化ＥＧＣＧ処理群とコントロール群の腫瘍体積のグラフを示す。＊ｐ＜０．０５、＊＊ｐ＜０．０１（対コントロール） Treatment with 1 mg / kg 3 "methylated EGCG suppressed tumor growth to 50% of control (2 weeks, p <0.05, 3 weeks, p <0.01), but treatment with EGCG at the same concentration ( ip administration, 1 mg / kg / day) was not different from the control group. 0.1 mg of 3 "methylated EGCG was also not different from the control group. There was no difference in body weight between the control, EGCG-treated or 3 "methylated EGCG-treated groups during the treatment period. FIG. 9 shows a graph of tumor volume in the 1 mg / kg 3" methylated EGCG-treated and control groups. * P <0.05, ** p <0.01 (vs. control)
　また、図１０Ａに３週間目のマウスの写真を示す。図１０Ａがコントロールマウス（生理的食塩水投与）、図１０ＢがメチルＥＧＣＧ１ｍｇ／ｋｇを投与したマウスである。図１０からも、１ｍｇ／ｋｇの３”メチル化ＥＧＣＧの投与により有意に腫瘍の増殖が抑制されたことがわかる。 FIG. 10A shows a photograph of the third week mouse. FIG. 10A shows a control mouse (administered with physiological saline), and FIG. 10B shows a mouse administered with 1 mg / kg of methyl EGCG. FIG. 10 also shows that administration of 1 mg / kg of 3 ″ methylated EGCG significantly suppressed tumor growth.

Claims (6)

  1. 下式（Ｉ）：
    Figure JPOXMLDOC01-appb-C000001
     で示される３”－（Ｏ）メチルエピガロカテキンガレートを含有する、抗癌剤組成物。     Formula (I):
    Figure JPOXMLDOC01-appb-C000001
     The anticancer agent composition containing 3 "-(O) methyl epigallocatechin gallate shown by these.
  2. 健康食品である、請求項１記載の組成物。 The composition according to claim 1, which is a health food.
  3. 飲料である、請求項１記載の組成物。 The composition of claim 1, which is a beverage.
  4. 医薬品である、請求項１記載の組成物。 The composition according to claim 1, which is a pharmaceutical product.
  5. 癌予防、治療または癌治療後の再発防止のためのものである、請求項１～４何れかに記載の組成物。 The composition according to any one of claims 1 to 4, which is used for cancer prevention, treatment or prevention of recurrence after cancer treatment.
  6. 癌が肝臓癌である、請求項１～５何れかに記載の組成物。 The composition according to any one of claims 1 to 5, wherein the cancer is liver cancer.
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JP2012031101A (en) * 2010-07-30 2012-02-16 Kurume Univ Composition for amelioration of non-alcoholic steatohepatitis
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JP2019506147A (en) * 2016-01-26 2019-03-07 ネステク ソシエテ アノニム Compositions comprising 3'-O-methyl-5-O-sulfate epicatechin and therapeutic uses of such compositions
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Cited By (5)

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WO2011162320A1 (en) 2010-06-23 2011-12-29 国立大学法人九州大学 Combination of egcg or methylated egcg and a pde inhibitor
JP2012031101A (en) * 2010-07-30 2012-02-16 Kurume Univ Composition for amelioration of non-alcoholic steatohepatitis
CN107438423A (en) * 2014-12-09 2017-12-05 株式会社爱茉莉太平洋 Activate the composition of longevity gene
JP2019506147A (en) * 2016-01-26 2019-03-07 ネステク ソシエテ アノニム Compositions comprising 3'-O-methyl-5-O-sulfate epicatechin and therapeutic uses of such compositions
JP2022092966A (en) * 2020-12-11 2022-06-23 トヨタ自動車株式会社 Dipeptidyl peptidase IV inhibitor, food with functional claims

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