KR20120088894A - A chemo-sensitizer composition comprising amurensin G showing potent SIRT1 enzyme inhibitor for treating or preventing cancer disease - Google Patents

Abstract

PURPOSE: A chemo-sensitizer composition isolated from natural products is provided to strongly suppress SIRT1 enzyme activation and to enhance cytotoxicity to cancer cells. CONSTITUTION: An anticancer aid composition for preventing and treating cancer diseases contains amurensin isolated from Vitis amurensis extract or phamacologically acceptable salt thereof as an active ingredient. The extract is isolated using water including purified water, methanol, butanol, or a mixture solvent thereof. The anticancer aid composition contains amurensin G, pharmaceutically acceptable salt, or Vitis amurensis extract. An anticancer agent contains amurensin G, adriamycin, cyclophosphamide, 5-FU, amsacrine, or taxol.

Description

A chemo-sensitizer composition comprising amurensin G showing potent SIRT1 enzyme inhibitor for treating or preventing cancer containing an Arensin (Amｕｒｅｎｓｉｎ) Ｇ compound having SIRT1 enzyme activity inhibitory activity as an active ingredient disease}

The present invention relates to an anticancer sensitizer composition for preventing and treating cancer diseases containing an amurensin G compound having SIRT1 enzyme activity inhibitory activity as an active ingredient.

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The present invention is a wild boar ( Vitis amurensis) relates to any rensin (amurensin) G compound having the SIRT1 enzyme inhibition activity isolated from the extracts on anti-cancer sensitizer composition for the prevention and treatment of cancer containing as an active ingredient.

Cancer is classified into blood cancer and solid cancer, and it occurs in almost every part of the body such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer. Among the methods used to treat these malignancies, chemotherapy agents, except surgery or radiation therapy, are collectively called anticancer agents, and most of them show anticancer activity by inhibiting the synthesis of nucleic acids. Chemotherapeutic agents are largely classified into antimetabolites, alkylating agents, antimitotic drugs, and hormones, and folates that inhibit the metabolic processes necessary for the proliferation of cancer cells. Derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, Cytarabine), etc.Introducing alkyl groups to guanine of DNA to modify the structure of DNA and chain-cutting Alkylating agents that exert the effect include nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitroso urea compounds (carmustine), triazene compounds (dacarbazine) There is. Mitosis inhibitors that inhibit mitosis by blocking mitosis as anti-mitotic drugs include plant drugs such as actinomycin D, doxorubicin, bleomycin, and anticancer drugs such as mitomycin, vincristine, and vinblastine. Alkaloids, taxoids including mitosis inhibitors including taxane rings, and the like. In addition, hormones such as corticosteroids and progesterone and platinum-containing compounds such as cisplatin are used as anticancer agents.

The biggest problem with chemotherapeutic agents is drug resistance, which, despite the initial successful response by anticancer agents, is a major factor that eventually causes treatment to fail. In connection with the problem of overcoming these side effects, efforts have recently been made to find the active ingredient in natural products that are used in the private sector.

Although chemotherapeutic agents are used to treat a variety of cancers, their use is often limited for multidrug resistant (MDR) cancer cells. MDR refers to the phenomenon of resistance to structurally different chemotherapeutic agents as well as drugs currently in use (Ozben, 2007). The most important mechanism for chemotherapeutic agents is the active efflux of anticancer drugs through increased expression of ABC (ATP-binding cassette transporters) (Bod et al., 2003; P, 2006). Three known MDR genes identified in the human body: (a) multi-drug resistance 1 (MDR1, p-glycoprotein, ABCB1), (b) multidrug resistance-associated proteins (MRPs, ABCC subfamily) and (c) breast cancer resistance protein (BCRP, ABCG2) is known (Kuo, 2007). ABC transporters are transmembrane proteins that use ATP binding to release a number of molecules across cell membranes. Despite the slight differences between them, these transporters have been known to influence the release of various anti-tumor agents (Glavinas et al., 2004). However, although the mechanism of expression of ABC transporter is clearly expressed in most tumor tissues, it is not fully understood. In addition, attempts to modulate this protein activity face limited success (Ozben, 2007).

Forkhead box-containing protein (O subfamily (FoxO) transcription factors) has a conserved DNA binding domain called a Forkhead box. Four proteins FoxO1, FoxO3, FoxO4 and FoxO6 have been identified in mammals as members of the O subfamily FoxO and the transcriptional activity of FoxO factor is regulated by the flowing system of the nuclear and cytoplasm. This system is regulated by phosphorylation-dependent ubiquitination and acetylation (Barthel et al., 2005; Vogt et al., 2005; Hoekman et al., 2006). Cell fates such as differentiation, metabolism and proliferation are regulated by FoxO factors (Accili and Arden, 2004) and are not frequently regulated in several cancer cells (Barthel et al., 2005) Arden, 2006). We found that FoxO1 is consistently upregulated in MCF-7 / ADR, adriamycin-resistant breast cancer cells, and FoxO1 plays a major role in the expression of the MDR1 gene (Han et al., 2008) .

Silent information regulator two ortholog 1 (SIRT1) is an ortholog of the yeast sir2 protein, a close family of enzymes called sirtuins (Motta et al., 2004; Borra et al., 2005). Sirtuins are known to play an important role in cellular responses to stress such as heat or starvation and to play a life-long process of calorie restriction (Borra et al., 2005 de Boer et al., 2006). Sirtuins work by removing acetyl groups from proteins in the presence of NAD ⁺ . Thus they are classified as NAD ⁺ -dependent deacetylases (de Boer et al., 2006). Several transcription factors (eg p53 and nuclear factor-kB) have been reported as substrates of SIRT1 (Yeung et al., 2003). FoxO transcription factors are also deacetylated by SIRT1 and subsequently accumulated in the nucleus (Stet al., 2007).

Compounds of the present invention are Vitis amurensis ) extract, which is a genus of Vitis belonging to the Vitaceae plant, distributed throughout the country, and as a vine, which is used for the treatment of traumatic pain, gastrointestinal pain, and headache. It has been used as a therapy (Information Shinmin Suburbs, Hyangjeom Dictionary, p300-301, Younglimsa, 1998).

However, the literature neither Vitis amurensis (Vitis There is no teaching or description of the effect of amurensin G compounds having SIRT1 enzyme activity inhibitory activity isolated from amurensis ) extracts.

Therefore, based on the assumption that the SIRT1-dependent FoxO1 activity is important for the expression and regulation of ABC transporters, the present inventors suggest that the potential of SIRT1 activation in up-regulation of MDR1 in MCF-7 / ADR cells. The role of phosphorus was investigated. In addition, the in vitro SIRT1 activity was examined and 1,820 plant extracts were confirmed that the methanol extract of Vitis amurensis showed a relatively strong SIRT1 inhibitory activity. Eight stilbene derivatives were isolated by activity-tracking fractionation of these extracts and Arensin G showed the strongest SIRT1 inhibitory activity. The inhibitory activity of Amurene G on FoxO1-mediated MDR1 expression in MCF-7 / ADR cells was measured. The present invention was also completed by testing whether Amurine G inhibits doxorubicin resistance in xenograft models of MCF-7 / ADR cells to mice.

In order to achieve the above object, the present invention has a wild worm having a strong SIRT1 inhibitory activity ( Vitis amurensis) provides an anticancer agent for secondary compositions for the prevention and treatment of cancer, including any rensin G or a pharmacologically acceptable salt thereof is separated from the extract as an active ingredient.

(I) the structure of Amurensin G

The present invention is an active wild bovine ( Vitis) having strong SIRT1 inhibitory activity Amurensis ) provides an anticancer adjuvant composition for the prevention and treatment of cancer diseases containing the extract as an active ingredient.

Vitis as defined herein amurensis ) extract is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably a mixed solvent of water and methanol, more preferably 60 to 100% methanol Contains extracts available for use.

The cancer diseases include general cancer diseases, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer , Anal leiomyoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma Cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, One or more diseases selected from brain stem glioma, pituitary adenoma, and the like, and particularly all cancer diseases caused by cancer cells that are multidrug resistant to existing anticancer agents.

Hereinafter, the present invention will be described in more detail by way of example, but is not limited thereto.

The compound of the present invention is Vitis amurensis ) can be obtained from the non-polar solvent extract of the process as follows:

For example, first of all, Vitis of the present invention ( Vitis) amurensis ), the roots are chopped , and then about 1 to 20 times, preferably about 3 to 10 times water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, of the sample weight (g). , Preferably with methanol, cold extraction, hot water extraction, ultrasonic extraction, for about 1 hour to 10 days, preferably about 2 hours to 5 hours at an extraction temperature of 20 ℃ to 100 ℃, preferably 50 ℃ to 70 ℃, After recovering the supernatant by reflux cooling extraction, more preferably using a cold extraction method, the above procedure is repeated 2 to 7 times to collect the supernatant and concentrated under reduced pressure to obtain a crude extract.

This crude extract is suspended in distilled water, and then the suspension is added 1 to 10 times by adding a nonpolar solvent such as hexane, ethyl acetate, methylene chloride and chloroform in a volume of about 1 to 100 times, preferably about 1 to 5 times, Preferably, silica gel column chromatography is performed using ethyl acetate two to five times, and the fractions obtained therefrom are subjected to recrystallization or Sephadex column chromatography and HPLC which are conventional purification methods in the art. The combination may be carried out to obtain a compound of the present invention or may additionally be subjected to conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis., 3rd Ed., Pp 6-7, 1998 ).

The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.

In addition, bases can be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the above compounds include salts of acidic or basic groups which may be present in the compound, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

The shift from chemotherapy-responsive cancer to chemotherapeutic-resistant cancers is accompanied by increased expression of multi-drug resistance 1 (MDR1, p-glycoprotein), which is the goal of multi-cancer drugs. Plays an important role in efflux from cells.

We have identified that the forkhead box-containing protein of FoxO1 (O subfamily 1) is a major regulator of MDR1 genetic transcription. Since the localization of FoxO1 is regulated by the silent information regulator of two ortholog 1 (SIRT1) deacetylases, we have found that SIRT1 may be expressed in breast cancer cells. Dominates were tested to govern MDR1 gene expression. Overexpression of SIRT1 increased both eFoxO reporter activity and nuclear levels of FoxO1. MDR1 protein expression and gene transcriptional activity were also up-regulated by SIRT1 overexpression. In addition, SIRT1 inhibition reduced both nuclear FoxO1 levels and MDR1 expression in adriamycin-resistant breast cancer cells (MCF-7 / ADR). Amurensin G, a potent SIRT1 inhibitor, was identified by searching for plant extracts and activity-tracking fractions. The compound inhibited FoxO1 activity and MDR1 expression in MCF-7 / ADR cells. In addition, 24-hour pretreatment of MCF-7 / ADR cells at 1 mg / ml of amurensin G increased the cellular uptake of doxorubicin and restored the reactivity of MCF-7 / ADR cells to doxorubicin. In xenograft studies, intraperitoneal injection of 10 mg / kg Amurensin G substantially restored the effect of doxorubicin, which inhibits MCF-7 / ADR induced tumor growth. These results confirmed that SIRT1 is a potential therapeutic target of MDR1-mediated chemoresistance, which is a useful drug for the recovery of chemotherapy resistance.

Accordingly, the present invention provides amenensin G, a pharmacologically acceptable salt thereof, or Vitis Amurensis ) provides an anticancer agent due to the inhibitory activity of SIRT1 containing the extract.

In addition, the present invention is an alensine G or a pharmacologically acceptable salt thereof or Wangeru ( Vitis) amurensis ) to provide an SIRT1 inhibitor.

The anticancer adjuvant composition for preventing and treating cancer diseases of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the present invention may also be used in the form of their pharmaceutically acceptable salts, and may also be used alone or in pharmacologically active compounds, such as anticancer agents well known in the art, namely adriamycin ( Multidrug-resistant to existing anticancer drugs in a suitable combination as well as binding with existing anticancer agents such as adriamycin, cyclophosphamide, 5-FU, amsacrine, danoomycin, and taxol It can be used as an anticancer adjuvant for inhibiting the growth of cancer cells with (Multi-drug resistance; MDR).

Therefore, the present invention is metabolism of the compound and other anticancer agents, preferably folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, Cytarabine) and the like used in conventional chemotherapy. Antagonists (antimetabolites); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer anticancer agents such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine and vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Hormones such as corticosteroids and progesterone; Platinum-containing anticancer agents such as cisplatin; more preferably at least one anticancer agent selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, taxol and anticancer activity It provides an anticancer composition that can be maximized in combination ratio, for example, preferably in a ratio of 1: 0.1 to 10 times the ratio of the existing anticancer agent and the alensin compound.

Accordingly, the present invention is an antimetabolites such as the alensine G and folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine) and pyrimidine derivatives (5-fluorouracil, Cytarabine); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer anticancer agents such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine and vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Hormones such as corticosteroids and progesterone; A platinum-containing anticancer agent such as cisplatin; more preferably a combination with at least one anticancer agent selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, taxol An anticancer agent for inhibiting the growth of cancer cells having multi-drug resistance (MDR) as an active ingredient is provided.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the compound. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dose is not intended to limit the scope of the invention in any way.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

As described above, the alensine G of the present invention is useful as an anticancer adjuvant composition and anticancer drug for preventing and treating cancer diseases in combination with a conventional anticancer agent by strongly inhibiting the activity of SIRT1.

1 is a diagram showing the role of SIRT1 in the MDR1 gene transcription process, (A) Transactivation of the MDR1 gene by SIRT1 overexpression, p195-MDR1 (left panel) and MDR1 protein expression (right panel) in MCF-7 cells ) Reporter activity was transiently infected with SIRT1-CA (30-300 ng / well) or pCMV5 (mock transfection, 300 ng) plasmid and data indicated mean SD of four different samples (significant versus the control, * p <0.05; ** p <0.01). (B) shows FoxO1 activation by SIRT1 overexpression and reporter activity of the FHRE reporter (left panel) in MCF-7 cells was combined with SIRT1-CA (30-300 ng / well) or pCMV5 (mock transfection, 300 ng) plasmid Transiently infected with plasmid (FHRE minimal reporter plasmid) and data indicate mean SD of four different samples (significant versus the control, * p <0.05; ** p <0.01). Intranuclear contents of FoxO1 and FoxO3a (right panel) in MCF-7 cells were infected with SIRT1-CA (30-300 ng / well) or pCMV5 (mock transfection, 300 ng) plasmid and Western blot analysis was performed 24 hours after infection. Performed using nuclear extracts from serum-free cells and FoxOl and Fox3a levels in nuclear fractions were immunochemically analyzed using specific antibodies. (C) is a diagram showing the effect and analysis results (Immunoblot analyses; left panel) of SIRT1 inhibitors on FoxO1-mediated MDR1 expression, Representative immunoblots according to the presence of 1 mM nicotinamide (NAM) for 24 hours Protein expression of MDR1, nuclear FoxO1 and FoxO3 in cultured MCF-7 and MCF-7 / ADR cells is shown. Equivalent loading of proteins is evidenced by actin or lamin B1 immunooblot and Rhodamine-123 retention assay (right panel). After incubating MCF-7 and MCF-7 / ADR cells for 90 minutes with 20 mM R-123, R-123 fluorescence of cell lysates was measured at excitation and emission wavelengths (480 and 540 nm). 1 mM NAM was added 24 hours before rhodamine-123 loading. The figures were divided by the total protein amount of the individual samples. Data represent mean SD values of four different sample groups (significant versus the control MCF-7 cells, ** p <0.01 significant versus the vehicle-treated MCF-7 / ADR cells, ^# p <0.05);
Figure 2 is a diagram showing the complementary effect of FoxO1 and SIRT1, (A) showing the upregulation of MDR1 transactivation by FoxO1 in the presence or absence of SIRT1 overexpression MCF-7 cells SIRT1 overexpression plasmid (SIRT1-CA, 300ng / The luciferase activities after co-infection of p195-MDR1 reporter and pCMV5-FoxO1 (30 ng / well) with or without the presence of the wells were measured 18 hours after infection (left panel). Mean versus SD of different samples (significant versus the pCMV5-transfected MCF-7 cells, **

0.01; significant versus the FoxO1-trasnfected MCF-7 cells, ^## p

0.01). Immunoblot analysis of MDR1 (right penel) and MCF-7 cells were infected with pCMV5-FoxO1 (100ng / well) according to the presence or absence of SIRT1 overexpressing plasmid (300ng / well), and total cell lysates were infected for 24 hours after infection. (B) is a diagram showing the conversion of SIRT1-induced MDR1 gene transcription by FoxO1 inhibition, 300ng / well pCMV5 or SIRT1- in combination with control siRNA or FoxO1 siRNA (20 p mole / well, left panel). P195-MDR1 reporter activity was measured in MCF-7 cells after co-administration with CA, and data represent mean (SD) of three different samples (significant versus the control siRNA and pCMV5-transfected MCF-7 cells , **

<0.01; significant versus the control siRNA and SIRT1-transfected MCF-7 cells, ^##

<0.01). Results of immunoblot analysis of MDR1 (right penel), total cell lysates were administered 24 hours after infection in combination with 300ng / well SIRT1-CA in combination with 60 p g / well control siRNA or FoxO1 siRNA in MCF-7 cells. Obtained.
Figure 3 is a diagram showing the MDR1 inhibition of amurensin G, a natural SIRT1 inhibitor, (A) the chemical structure of amurensin G, (B) alensine G and nicotinamide (left panel) By In In vitro SIRT1 inhibition and FHRE reporter activity (right panel) is shown, MCF-7 cells co-administered with pCMV5 or SIRT1 (300ng) with FHRE reporter plasmid and cells for an additional 18 hours after infection ( incubated with vehicle dimethylsulfoxide) or amurensin G (0.1-1 μg / ml), and the data were prepared as mean (SD) of three different samples (significant versus the pCMV5-transfected MCF-7 cells, **

<0.01; significant versus the SIRT1-trasnfected and vehicle-treated MFC-7 cells, ^#

<0.05;

^# p <0.01), (C) is a diagram showing the inhibitory activity of Arensin G on FoxO1-induced MDR1 expression, MCF-7 / ADR cells presence of amurensin G (0.3-3μg / ml) Total cell lysates and nuclear fractions were subjected to immunoblot for MDR1, FoxO1 and FoxO3 after incubation for 24 hours, and (D) changes in cell viability after treatment of MCF-7 / ADR cells with amurensin G. Cell viability was measured by MTT assay after culturing MCF-7 / ADR cells with or without amurensin G (0.1-3 μg / ml) for 24 hours. Mean of samples (expressed as means ± SD);
4 is a diagram overcoming doxorubicin resistance in MCF-7 / ADR cells by amurensin G, (A) is a diagram showing the cellular doxorubicin uptake, and for 24 hours, alensine G (0.1 After culturing MCF-7 and MCF-7 / ADR cells with or without -3 μg / ml), doxorubicin (30 μM) was treated for 60 minutes and doxorubicin fluorescence present in the cell lysates of MCF-7 and MCF-7 / ADR Intensity was measured at excitation (470 nm) and emission wavelength (570 nm) and the value was divided by the total protein amount of the individual samples and the data expressed as the mean (means ± SD) of three different samples (significant versus the untreated MCF- 7 / ADR cells, *

<0.05; **

<0.01; control level = 1). (B) is a diagram showing the intercellular concentration of doxorubicin. After incubation of MCF-7 and MCF-7 / ADR cells according to the presence or absence of amurensin G for 24 hours, doxorubicin (30 μM) was treated for 60 minutes and cells of doxorubicin. Liver concentration was measured as described in this method, this value was divided by the total protein amount of the individual samples, and the data expressed as the mean (means ± SD) of four different samples (significant versus the untreated MCF-7 / ADR cells, *

<0.05; **

<0.01). (C) is a diagram showing synergistic cytotoxicity by Arensin G and doxorubicin in MCF-7 / ADR cells, pre-cultured Arensin G (0.1-1 μg / ml) for 24 hours and MCF-7 and MCF-7 / ADR cells were exposed to doxorubicin (30 μM) for an additional 24 hours, cell viability was performed by crystal violet assay and data expressed as mean (means ± SD) of 14 different samples (significant versus the doxorubicin alone-treated MCF-7 / ADR cells, **

<0.01). (D) is a diagram showing the augmentation of doxorubicin-mediated DNA synthesis inhibition by Amurensin G. MCF-7 / ADR cells were treated as described in panel C and BrdU assay was performed. Shows the mean (amean ± SD) of eight different samples (significant versus the doxorubicin alone-treated MCF-7 / ADR cells, **

<0.01). (E) is a representative picture of TUNEL assay in cells cultured with or without 30 μM doxorubicin for 24 hours, MCF-7 / ADR cells were pretreated with CPP343 (0.3-1 μg / ml) 24 hours before doxorubicin exposure, (F) is a diagram showing a synergistic inhibitory effect of cell growth by Arensin G and paclitaxel (Taxol) in MCF-7 / ADR cells, Alensine G (0.1-1 μg / ml) is pre-cultured for 24 hours and MCF- 7 / ADR cells were exposed to doxorubicin (30 μM) for an additional 48 hours in 10% FBS-containing medium, relative cell numbers were determined by MTT assay, and data were the mean (means ± SD) of eight different samples. (Significant versus the paclitaxel alone-treated MCF-7 / ADR cells, **

<0.01).
Figure 5 is a diagram showing the recovery of doxorubicin reactivity by Arensin G in xenograft test, (A) is a diagram showing the inhibition of MCF-7 / ADR tumor growth by Arensin G, doxorubicin (4mg / kg, ip Representative pictures of tumor-induced non-thymus mice with or without Arencin G (10 mg / kg, ip twice a week for 4 weeks) (upper panel), tumor size every 3 days Measured with a caliper measurement over and the size of the standard formula (width ²⁾ x length 0.52), and data are expressed as mean (SD) of four different mice (significant versus the untreated control, * p <0.05, ** p <0.01; significant versus the doxorubicin alone-treated group, ^# p <0.05, ^## p <0.01). (B) is a diagram showing the histology and immunohistochemistry of the tumor tissue, H & E staining with immunohistochemical staining for PCNA, Ki-67 and TUNEL staining on paraffin fragments from solid tumor tissue and the individual picture Values are expressed as mean (SD) of four different samples (significant versus the doxorubicin alone-treated group, * p <0.05, ** p <0.01). (C) is a diagram showing MDR1 expression in tissue lysate, tumor tissue was homogenized in PBS, centrifuged at 10,000 g, the supernatant was subjected to immunoblot, individual lanes were loaded with 20 g of tissue lysate and the same protein Loading was verified using actin as the internal standard and the results were confirmed in two separate experiments.

Hereinafter, the present invention will be described in detail. However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example 1 Amurensin G Isolation

1-1. Preparation of Crude Extracts

V. amurensis (J., Maysan, Jinan-gun, Jeollabuk-do, Korea) Jul. 2008, Professor Young-Hee Moon, Chosun University; Jeong-Won Cho, College of Herbal Medicine Sample Number: CU-0235 Extract 2 kg of stem with 100% methanol (10L × 72h × 2) at room temperature. And concentrated to give crude extract (250 g).

1-2. By solvent Fraction  Produce

The crude extract was suspended with water and successively partitioned into n- hexane (1.5 L x 3), EtOAc (1.5 L x 3), and BuOH (1.5 L x 3). The EtOAc fraction (87 g) was subjected to silica gel column chromatography (10 × 35 cm; 63200 m particle size) using a developing solvent ( n- hexane / acetone gradient; 5: 1 to 0: 1) to obtain 7 fractions (F1F7). The sixth fraction (Fraction 6; 20.5 g) was partitioned into three subfractions (F6.1-F6.3) on a Sephadex column (Sephadex LH-20) using MeOH as the mobile phase.

1-3. Preparation of compounds

The double fraction (Fraction F6.2; 5.5 g) was applied to a column (C-18 silica gel column chromatography) and eluted with a stepwise solvent system (MeOH / H ₂ O; 1: 1 to 10: 1). F6.2.1-F6.2.6). A small fraction (F6.2.3; 300 mg) was purified by HPLC (ODS-H80 column [(20 × 150 mm, 4 m particle size); solvent system; MeOH in H ₂ O with 0.1% formic acid, 0-25 min: 35% MeOH, 35 min: 100% MeOH); Flow rate 3 mL / min; UV detection at 280 and 320 nm] was performed to obtain amurensin G ( t _R = 20 min, 28 mg) having the following characteristics.

Appearance: brown amorphous powder;

mp 263-264 ° C;

[] 25D +28 ( c 0.1, MeOH);

UV (MeOH) _max (log): 213 (3.80), 217 (3.95), 224 (4.00), 282 (4.32) nm; IR (film) ν _max 3320, 1610, 1510, 1180, 1040, 840 cm ⁻¹ ;

¹ H- and ¹³ C-NMR are consistent with existing data (He et al., 2009; He S, Lu Y, Jiang L, Wu B, Zhang F, Pan Y. Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high-speed counter-current chromatography in stepwise elution mode.J Sep Sci. 2009 Jul; 32 (14): 2339-45.)

EI-MS m / z 681 [M + H] ⁺ (Calc. For C ₄₂ H ₃₂ O ₉ ).

Reference Example  1. Preparation for experiment

1-1. Experimental material

Anti-MDR1 antibodies were purchased from Calbiochem (Darmastadt, Germany) and FoxOl and FoxO3a specific antibodies, SIRT1 antibodies, and horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs (Cell Signaling Technology). Beverly, Mass.). Also IgG (Alkaline phosphatase-conjugated donkey anti-mouse IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG) were purchased from the company (Jackson Immunoresearch Laboratories; West Grove, PA). Most of the reagents used in the experiment were purchased from the company (Sigma; St. Louis, Mo.). siRNA targeting human FoxO1 was purchased from Ambion, Austin, TX. Buffers used for human recombinant SIRT1, Fluor de Lys SIRT1 deacetyalse substrate, Fluor de Lys Developer II NAD + and assays were purchased from Plymouth Meeting (PA). 1,820 plant samples were obtained from the 21C Frontier R & D Program Plant Diversity Research Center in the Republic of Korea.

1-2. Cell culture

MCF-7 cells and adriamycin-resistant MCF-7 (MCF-7 / ADR) cells were cultured with Dulbecco's modified Eagle's medium (DMEM); 10% fetal bovine serum (FBS), 100 units / ml penicillin, And 100 mg / ml streptomycin) at 37 ° C. under a humid 5% CO ₂ atmosphere.

1-3. Plasmid Preparation

Plasmids (p195-MDR1-Lucreporter plasmids) were prepared by conjugation of the PCR-amplified MDR1 promoter regions with a vector (pGL3-enhancer vector; Promega, Madison, Wis.) (Han et al., 2008). Plasmid (FHRE-Luc, FoxO response element-containing a reporter plasmid) was purchased from the company (Addgene Inco .; Cambridge, Mass.). The active plasmid (SIRT1 constitutive active plasmid) was provided by Chonnam National University (Professor Gwang-yeol Lee, Gwangju Metropolitan City, Korea).

1-4. Nuclear extract  Ready

Nuclear extracts were prepared as disclosed in the literature (Schreiber et al., 1990). In summary, cells in dishes are washed with ice-cold PBS, scraped, transferred to microtubes, and 100 ml lysis buffer [10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES, pH 7.9), containing 0.5% Nonidet P-40, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonylfluoride (PMSF). Cell membranes were disrupted by vortexing and the lysates were incubated for 10 minutes on ice and centrifuged at 7,200 g for 5 minutes. Pellets containing crude nuclei were resuspended in 60 ml extraction buffer [20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM DTT and 1 mM PMSF] and incubated on ice for 30 minutes. The sample was centrifuged at 15,800 g for 10 minutes to obtain a supernatant containing the nuclear extract and stored at -80 ° C until needed.

1-5. Immunoblot ( Immunoblot analysis ) analysis

After washing with sterile PBS (phosphate buffered saline), MCF-7 and MCF-7 / ADR cells were washed with EBC lysis buffer (20 mM Tris®Cl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM -glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, and 1 mg / ml leupeptin). Cell lysates were centrifuged at 10,000 g for 10 minutes to remove impurities and proteins were fractionated using a gel (10% separating gel). Fractionated proteins were electrophoretically transferred to nitrocellulose paper and immunoblotted with specific antibodies. Antibodies (Horseradish peroxidase- or alkaline phosphatase-conjugated anti-IgG antibodies) were used as secondary antibodies. The nitrocellulose paper was developed using 5-bromo-4-chloro-3-indolylphosphate (BCIP) / 4-nitroblue tetrazolium (NBT) or the system (ECL chemiluminescence system). The instrument (LAS3000-mini; Fujifilm, Tokyo, Japan) was used for fluorescence detection.

Experimental Example 1. SIRT1 inhibition experiment

1-1. Gene Assays Reporter gene assay )

Promoter activity was measured using a dual-luciferase reporter assay system (Promega, Madison, Wis.).

In summary, cells (3 × 10 ⁵ cells / well) are incubated overnight on plates (12-well plates) and temporarily infected with reagents (Hilymax®reagent; Dojindo Molecular Technologies, Gaithersburg, MD) (THRE). -Luc or p195-MDR1 reporter plasmid / phRL-SV plasmid; hRenilla luciferase expression for normalization-Promega, Madison, WI). Cells were then incubated in serum-free medium for 18 hours and firefly and enzyme (hRenilla luciferase) activity in the cell lysate was measured using a luminometer (LB941, Berthold Technologies, Bad Wildbad, Germany). Relative enzyme (luciferase) activity was calculated by normalizing promoter-driven firefly luciferase to hRenilla luciferase.

1-2. SIRT1 deacetylase enzyme assay

Fluor de Lys fluorescence assay was used to determine the SIRT1 activity of photochemicals using the literature (BioMol product sheets-Borra et al., 2005).

In summary, the assay uses Fluor de Lys-SIRT1, NAD +, SIRT1 enzymes in SIRT1 assay buffer (50 mM Tris-Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl ₂ , 1 mg / ml bovine serum albumin). It was performed using. After 10 minutes preincubation of the SIRT1 enzyme with assay buffer for 10 minutes, the reaction was carried out with Fluor de Lys peptide and NAD ⁺ . Initiation was added.

After 45 minutes of incubation, 2 mM nicotinamide was added to the developer (Developer II) in a buffer (histone deacetylase assay buffer; 25 mM Tris / Cl pH 8.0, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl ₂ ).

At each measurement point, 50 μL reaction was taken and mixed with 50 μL developer solution. In conclusion, fluorescence was measured using a microplate reader (Varioskan, Thermo Electron Co.) at excitation wavelength (λ = 360 nm) and emission wavelength (λ = 460 nm).

Relationship between FoxO1-dependent MDR1 expression and SIRT1; In a previous study we found that FoxO1 bound to the MDR1 promoter plays a major role in MDR1 expression in MCF-7 / ADR cells (Han et al., 2008). FoxO factors include two different mechanisms; That is, it is regulated by phosphorylation and acetylation. Multiple kinase pathways, including phosphatidylinositol 3-kinase (PI3-kinase) / Akt, extracellular signal regulated kinase and p38 kinase, have been shown to regulate FoxO through phosphorylation (Vogt et al., 2005; Asada et al. al., 2007). SIRT1 has also been shown to cause nuclear translocation of FoxO1 and increase its transcriptional activity through deacetylation (Frescas et al., 2005). Since SIRT1 is considered a promising goal of anticancer drug development (Saunders et al., 2007), we were interested in the potential association of SIRT1 with MDR1 gene transcription.

We first investigated whether SIRT1 activity is essential for FoxO1-dependent MDR1 expression. Reporter activity and MDR1 protein levels of p195-MDR1-Luc including the FoxO1 binding site (Han et al., 2008) were significantly elevated by overexpression of SIRT1 constitutive active plasmids. Confirmation (see FIG. 1A). SIRT1 overexpression also increased the nuclear level of FoxO1 and transcriptional activity of the forkhead-response element (FHRE) minimal reporter selectively (see FIG. 1B). In addition, 1 mM nicotinamide, a representative SIRT inhibitor, was found to inhibit nuclear accumulation of FoxO1 and reduced MDR1 protein levels in MCF-7 / ADR cells (see Figure 1C left).

These results demonstrate that SIRT1 activity is closely linked to FoxO1-mediated MDR1 expression. We also measured the transport activity of MDR1 using an assay (Rhodamine-123 (MDR1 substrate) retention assay).

Reduced accumulation of R-123 in MCF-7 / ADR cells was significantly recovered by 1 mM nicotinamide (see FIG. 1C right). This suggests that MDR1 activity as well as its expression is regulated by SIRT1 deacetylase.

FoxO1 Dependence MDR1  About manifestation SIRT1 Synergistic effect.

To determine whether SIRT1 regulates the FoxO1-mediated MDR1 gene transcription pathway, MCF-7 cells were infected with structurally active SIRT1 plasmids in the presence or absence of FoxO1 overexpression. SIRT1 overexpression in the presence of FoxO1 synergistically enhanced both poromotor activity and protein expression of MDR1 as was only FoxO1 overexpression (see FIG. 2A). In addition, SIRT1-induced MDR1 protein expression or MDR1 transcription was inhibited by FoxOl siRNA (see FIG. 2B). These data demonstrate that SIRT1 is a major regulator of MDR1 expression through FoxO1-dependent mechanisms and FoxO1 and SIRT1 cooperate to induce MDR1 expression.

Powerful from natural products SIRT1  Inhibitor search and Amurensin  Of G MDR1  Inhibitory effect.

Given that SIRT1 regulates cell cycle and apoptosis during tumorigenesis (Saunders et al., 2007) and considers a major role of SIRT1 in regulating MDR1 expression, the deacetylase may be an attractive anticancer target. Several SIRT inhibitory chemicals have been identified, but potential toxicity or low efficacy has been an obstacle to the development of new anticancer drugs. Thus, the present inventors have sought to identify the result of the powerful inhibitors SIRT1 from 1820 plant sample, Vitis amurensis (Vitis amurensi ) methanol extract showed a significant SIRT1 inhibitory activity (35% inhibition; 30g / mL).

An active compound having SIRT1 inhibitory activity was traced from the extract by an activity-tracking fraction to isolate amurensin G (see FIG. 3A).

Amurine G showed stronger in vitro inhibitory activity than 1 mM nicotinamide for SIRT1 enzyme activity (see Figure 3B left). The present inventors tested the inhibitory effect on SIRT1 / FoxO1-dependent transcriptional activity using the reporter plasmid of alensine G. After introducing activating SIRT1 plasmid to increase FHRE reporter activity, MCF-7 cells were exposed to Amurine G. Amurensin G reduced SIRT1-induced FHRE transcriptional activity in a concentration-dependent manner, and 1 μg / ml Amurensin G was sufficient to completely inhibit SIRT1-dependent FHRE reporter activity (see FIG. 3B right). In order to determine whether Arencin G inhibits MDR1 expression, a western blot analysis was performed. Amurensin G strongly inhibited the expression of MDR1 in MCF-7 / ADR cells. In addition, while FoxO3 remained unchanged, nuclear FoxO1 levels were also reduced by Arencin G treatment (see FIG. 3C).

Experimental Example  2. MTT  Cell survival Assay ( cell viability assay )

To measure cell viability, cells were placed at constant concentration (10 ⁴ cells / well) in 96-well plates. In order to measure the cytotoxicity of Amurine G, MCF-7 / ADR cells were incubated in a medium (FBS-free medium) for 24 hours in the presence or absence of Arensin G (0.1-3μg / ml). Viable fixed cells were stained with reagent (MTT; 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide 2 mg / ml) for 4 hours. The medium was removed and the resulting formazan crystals were dissolved by adding 200 μl dimethylsulfoxide / well. Absorbance was measured at 540 nm. Cell viability is expressed as a ratio relative to untreated control cells.

Basic cytotoxicity of Amurine G against MCF-7 / ADR cells was measured, demonstrating no change in cell viability up to 3 μg / ml (see FIG. 3D).

Experimental Example  3. In vitro  Exam (anticancer drug increase and decrease effect)

3-1. Crystal violet  Assay cell viability assay )

Doxorubicin-induced cell death was measured using crystal violet staining as described in the literature (Vellonen et al., 2004).

The cells were stained with 0.4% crystal violet in methanol for 30 minutes at room temperature and washed with tap water. Stained cells were extracted with 50% methanol and dye extracts were measured at 550 nmp using a microtiter plate reader (Berthold Technologies, Bad Wildbad, Germany).

3-2. Doxorubicin cell uptake assay Cellular uptake of doxorubicin )

Doxorubicin uptake was quantified in MCF-7 and MCF-7 / ADR cells. Cells (3x10 ⁶ cells) were incubated with 30 μM doxorubicin for 60 minutes, washed three times with PBS, EBC lysis buffer (20 mM Tris Cl, pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10 % glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM -glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, and 1 mg / ml leupeptin). After centrifugation of the sample at 10,000 g for 10 minutes, the change in the fluorescence of doxorubicin in the supernatant was measured at excitation (470 nm) and emission wavelength (590 nm), respectively. Absorption intensity is expressed as the ratio of fluorescence to doxorubicin-treated group.

3-3. Doxorubicin  Cell concentration measurement

To measure the cell concentration of doxorubicin, the cell lysate was recovered as described as part of the 'cellular uptake of doxorubicin'. 200 μl acetonitrile containing an internal standard (e-acetoaminocarproic acid, 1 mg / ml) was added to 50 μl cell lysate and the mixture was shaken for 15 seconds and centrifuged at 12,000 rpm for 5 minutes. The organic layer was transferred to another tube and dried under nitrogen atmosphere. The dry sample was reconstituted with 120 μl solvent (0.1% formic acid in 30% acetonitrile solution), shaken and 50 μl transferred to an autosample vial and 5 μl injected into the HPLC system. A certain amount (5 μl) of each sample extract was analyzed for analysis column (Gemini-NX C ₁₈ analytical column; 150 × 2.0 mm id). Example Compounds were eluted by pumping a mobile phase (10 mM ammonium formate in water: acetonitrile = 70: 30, v / v) at a flow rate of 300 ml / min. Under these conditions, typical standard retention times for internal standards and doxorubicin were 1.2 and 1.5 minutes. Total chromatographic run time was 3.5 minutes. Mass spectrometers (LC / MS / MS 1200L, Varian, CA) equipped with electrosprays in positive mode were subjected to transitions 544.2> 361.0 with collision energy 25 eV for doxorubicin and internal standards. Multiple reaction monitoring (MRM) was prepared, which measures the transition (174.0> 114.1) with collision energy 14eV.

3-4. TUNEL ( TdT mediated dUTP nick end labeling ) Assay

A TUNEL Assay for Measuring Apoptosis in A situ cell death detection kit (Roche Diagnostics GmbH, Germany) was used.

After 18 hours of incubation with doxorubicin (30 μΜ) or alensine G (0.3-1 μg / ml), MCF-7 or MCF-7 / ADR cells were washed with PBS. Cells on the slides were fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 1 hour and infiltrated with 0.1% Triton ^® X-100 in 0.1% sodium citrate for 2 minutes on ice. . It was washed with PBS, incubated at 37 ° C. for 60 minutes with the addition of 50 μl TdT enzyme solution, 50 μl anti-fluorescent antibody (Fab fragment from sheep conjugated with alkaline phosphatase) and incubated at 37 ° C. for 30 minutes. And further incubated for 10 minutes in the presence of BCIP / NBT solution. Slides were washed with brine (phosphate-buffered saline), covered under cover-slip and analyzed by light microscopy.

< Amurenicin  By G Increased  Doxorubicin uptake and synergistic cytotoxicity>

The inventors tested whether doxorubicin enhanced cellular uptake by amurensin G using doxorubicin itself fluorescence detection (FIG. 4A) or LC / Ms / Ms-based cell concentration assay (FIG. 4B). Doxorubicin was measured in MCF-7 / ADR cells less than in MCF-7 cells, and doxorubicin uptake in MCF-7 / ADR cells increased with increasing Arencin G concentration (FIG. 4A; see FIG. 4B).

We additionally tested whether doxorubicin responsiveness was restored by Arencin G in cell culture tests. Doxorubicin (30 μM) -mediated cell death was measured after 24 hours preculture of MCF-7 / ADR cells with excipient or Ammuresin G (0.1, 0.3, 1 μg / ml). Amurensin G treatment significantly increased the toxicity of doxorubicin, as evidenced by crystal violet (see FIG. 4C) and BrdU uptake (see FIG. 4D) assays. In addition, TUNEL staining demonstrates that 24 hours of doxorubicin (30 μM) causes severe cell death upon exposure to control MCF-7 cells, but not MCF-7 / ADR cells (see FIG. 4E). . However, TUNEL-positive cells were found when pretreatment of MCF-7 / ADR cells with Amurine G (0.3 or 1 mg / ml) for 24 hours (see FIG. 4E) and this indicated that the MDR1 sub-regulatory effect of Arensin G ( down-regulatory effect) restores the cellular sensitivity of doxorubicin.

We have determined the possible synergistic effect of Aurexin G on paclitaxel-mediated cell proliferation inhibitory activity in MCF-7 / ADR cells. As expected, amurensin G promoted paclitaxel-mediated cytotoxicity in a concentration dependent manner (see FIG. 4F).

Experimental Example  4. Animal model test (cancer sensitizing effect)

4-1. Xenograft research Xenograft study )

Xenograft model mice were prepared from mice (6 week old female BALB / c nude mice; Central Lab. Animal Inc., Seoul, Korea) and tumor cells (5 × 10 ⁶ MCF-7 / ADR cells) were treated with 50% Matrigel ( Matrigel) was suspended in 1 ml serum-free medium and injected percutaneously into the upper flank of individual nude mice. When tumor cells reached about 200 mm ³ (day 12), mice were randomly assigned to vehicle control, doxorubicin-treated or amurensin G + doxorubicin-treated groups, respectively. Assigned. After two weeks of infusion, 0.1 ml of Alensine G solution (10 mg / kg) was injected intraperitoneally for four weeks [twice per week (Mon and Thursday) and doxorubicin was intraperitoneally for 4 weeks (once a week (Wednesday)) at 4 mg / kg. Injected. Tumor size was measured as disclosed in the literature (Ahn et al., 2010). The animal protocols used in this study were performed and approved in accordance with the terms of the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC) in accordance with the Code of Ethics and Scientific Procedures (Approval Number PNU-2009-0024). .

Mice were sacrificed and the excised tumors were fixed with 10% buffered formalin and impregnated with paraffin. For pathological examination, 4 mm thick tissue sections were stained with H & E. Immunohistochemical staining was performed by the antibody (avidin-biotin complex method) using an antibody (anti-proliferating cell nuclear antigen (PCNA).) Immune responses were visualized with reagents (3,3-diaminobenzidine) and stained. Back staining with (Mayer's hematoxylin) Tissue TUNEL assays were performed using ApopTag Plus Peroxidase In Situ Apoptosis Detection kits (Intergen, Purchase, NY) according to the manufacturer's instructions.

In summary, paraffin was removed from the slides and placed in 3% hydrogen peroxide to inhibit endogenous peroxidase. Slides were incubated for 1 hour at 37 ° C. in reaction buffer containing enzyme (terminal deoxynucleotidyl transferase). The slides were incubated with an antibody (peroxidase-conjugated anti-digoxigenin antibody) for 30 minutes and the reaction product visualized with a solution containing 2 mmol hydrogen peroxide (0.03% 3,3-diaminobenzidine solution). Counterstaining was performed with 0.5% methyl green. PCNA and TUNEL-positive cells were counted and labeled as the average of the five highest areas within a single × 200 field. Some of the tumor tissue was homogenized and immunoblotting was performed for MDR1.

Statistical analysis

Scanning densitometry was performed using an instrument (LAS-3000mini; Fujifilm, Japan). One way analysis of variance (ANOVA) was used to determine the significant deviation from the treatment group, where the Newman-Keuls test was used to determine the multiple group mean. statistical significance was expressed as p <0.05 or p <0.01.

In xenotransplantation research Amurensin  G restores doxorubicin reactivity.

We measured tumor growth in non-thymus nude mice with MCF-7 / ADR cells. Only the 4 mg / kg doxorubicin treated group (once a week for 4 weeks) showed slight inhibition of tumor growth, but the co-treated group with amurensin G (10 mg / kg, twice weekly) significantly inhibited tumor doxorubicin-mediated inhibition. (See FIG. 5A). On histological examination, the tumor showed solid growth of undifferentiated carcinoma without glandular or ductal differentiation. However, Amurine-G co-administered tumors resulted in a wider cell death than control or doxorubicin-only tumor groups (see FIG. 5B). PCNA is a representative marker of cancer cell differentiation. Histologic analysis revealed that all tumor cells in the group of vehicle-treated control cells and doxorubicin alone were PCNA-positive, but the numbers were co-administered with Arensin G. Hour showed a significant decrease (see FIG. 5B). PCNA is not expressed in the entire cell cycle, but Ki-67 tumor-specific antigen is expressed in proliferative cells throughout the G1, S, G2 and M phases. Thus, immunohistochemical analytical Ki-67 staining can provide a reliable indicator of tumor cell proliferation (Brown et al., 1990). Ki-67 staining intensity was also reduced due to co-administration of doxorubicin with Amurene G (see FIG. 5B). In addition, co-administration with amurensin G increased the number of TUNEL-positive cells (see FIG. 5B). We measured MDR1 protein expression in tumor tissue lysate. MDR1 protein expression was clearly reduced in the Arensin-G treatment test group (see FIG. 5C). These results demonstrate that the native SIRT1 inhibitor Arensin G strongly inhibits MCF-7 / ADR-derived tumor growth as a MDR1 down-regulation mechanism.

Claims (8)

An anticancer adjuvant composition for the prevention and treatment of cancer diseases, comprising as an active ingredient Arencin G or its pharmacologically acceptable salts isolated from Vitis amurensis extract having potent SIRT1 inhibitory activity. An anticancer adjuvant composition for cancer disease prevention and treatment containing Vitis amurensis extract having potent SIRT1 inhibitory activity as an active ingredient. The method of claim 2,
The extract is a composition that is an extract available in a solvent selected from a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, such as water, methanol, ethanol, butanol, including purified water. 3. The method according to claim 1 or 2,
The cancer may be breast cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer, chronic or acute One or more selected from leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma A composition that is a disease. Arencin G, its pharmacologically acceptable salts, or Vitis anti-cancer sensitizer due to the inhibitory activity of SIRT1 activity containing amurensis ) extract. Inhibitors of SIRT1 containing Amurensin G or its pharmacologically acceptable salt or Vitis amurensis extract. Metabolic antagonists (antimetabolites) such as amurine G and folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine) and pyrimidine derivatives (5-fluorouracil, Cytarabine); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer anticancer agents such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine and vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Hormones such as corticosteroids and progesterone; An anticancer agent for inhibiting the growth of cancer cells having a multi-drug resistance (MDR) as an active ingredient in combination with at least one anticancer agent selected from a platinum-containing anticancer agent such as cisplatin. 8. The method of claim 7,
Multidrug resistance as an active ingredient comprising a combination of at least one anticancer agent selected from amurene G and adriamycin, cyclophosphamide, 5-FU, amsacrine, and taxol Anticancer agent for inhibiting the growth of cancer cells with Multi-drug resistance (MDR).

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