WO2019049612A1 - Antitumor agent, food and drink for preventing cancer, and tumor-suppressive micro-rna expression promoter - Google Patents

Antitumor agent, food and drink for preventing cancer, and tumor-suppressive micro-rna expression promoter Download PDF

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WO2019049612A1
WO2019049612A1 PCT/JP2018/030232 JP2018030232W WO2019049612A1 WO 2019049612 A1 WO2019049612 A1 WO 2019049612A1 JP 2018030232 W JP2018030232 W JP 2018030232W WO 2019049612 A1 WO2019049612 A1 WO 2019049612A1
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pterostilbene
mir
antitumor agent
cancer
tumor
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PCT/JP2018/030232
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French (fr)
Japanese (ja)
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孝広 落谷
蔵嵩 大塚
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キユーピー株式会社
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Priority to JP2019501742A priority Critical patent/JP6573740B2/en
Publication of WO2019049612A1 publication Critical patent/WO2019049612A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to an antitumor agent, a food and drink for cancer prevention, and an agent for enhancing the expression of a tumor suppressor microRNA.
  • the microRNA is RNA having a base pair length of about 22 bases.
  • resveratrol and pterostilbene which are stilbene derivatives are known to promote or suppress the expression of a predetermined microRNA (Non-patent Documents 1 and 2).
  • miRNA microRNA
  • cancer cancer Useful as a preventive agent
  • the compound capable of exerting an antitumor effect by oral administration can be easily applied to foods, supplements and the like.
  • this invention aims at provision of the novel antitumor agent for oral administration.
  • the present inventors have found that pterostilbene has an excellent antitumor effect by oral administration.
  • the present invention is based on this novel finding.
  • the present invention relates to, for example, the following inventions.
  • An antitumor agent for oral administration comprising pterostilbene.
  • the antitumor agent according to (1) which further contains a fat and oil.
  • the antitumor agent according to (2) wherein the oil and fat is a vegetable oil.
  • a food and drink for cancer prevention which comprises the antitumor agent according to any one of (1) to (3).
  • a tumor suppressor microRNA expression enhancer containing pterostilbene (6) The expression enhancer according to (5), which is for oral administration.
  • Tumor suppressor microRNAs from let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a and miR-193b The expression enhancer according to (5) or (6), comprising at least one selected from the group consisting of (8) The expression enhancer according to any one of (5) to (7), which is used for the treatment or prevention of cancer.
  • a method for treating or preventing cancer which comprises orally administering pterostilbene to a subject in need thereof.
  • Pterostilbene for use in the treatment or prevention of cancer by oral administration.
  • a novel antitumor agent for oral administration is provided.
  • a cancer preventive food and drink containing the antitumor agent.
  • Fig. 1 (A) shows the measurement results of cell viability when breast cancer cells are used
  • Fig. 1 (B) shows the measurement results of cell viability when gastric cancer cells are used
  • Fig. 1 (C) shows the large intestine
  • FIG. 4 (A) is a photograph showing the result of tumor luminescence 21 days after the start of administration.
  • FIG. 4 (B) is a graph showing the results of observation of the amount of luminescence over time. It is a photograph or a graph which shows the influence which the intraperitoneal administration of pterostilbene gives to the size of a tumor.
  • FIG. 5 (A) is a photograph showing the excised tumor piece.
  • FIG. 5 (B) is a graph showing the weight of the excised tumor.
  • FIG. 7 (A) is a photograph showing a tumor piece from which RNA was extracted.
  • FIG. 7 (B) is a graph showing the expression levels of tumor suppressor microRNAs (miR-200c and miR-141) in tumors.
  • FIG. 8 (A) is a photograph showing the result of tumor luminescence 21 days after the start of administration.
  • FIG. 8 (B) is a graph showing the observation result of the luminescence amount with time.
  • the present invention is characterized by providing an antineoplastic agent for oral administration, which contains pterostilbene.
  • the present invention is also characterized by providing a food and drink for cancer prevention, which comprises the above-mentioned antitumor agent.
  • the present invention is further characterized by providing a pterostilbene-containing agent for enhancing the expression of a tumor suppressor microRNA.
  • the "anti-tumor agent” in the present specification is a therapeutic or preventive agent for cancer, a cancer cell growth inhibitor, a cancer progression inhibitor, a cancer recurrence preventive (suppression) agent, a cancer metastasis suppressor, and a cancer invasion suppressor And inhibitors of secretion of extracellular vesicles such as exosomes released from cancer cells.
  • Pterostilbene is a compound represented by the following formula (1).
  • the content of pterostilbene is 0.01% by mass or more, 0.10% by mass or more, 10% by mass or less, or 5% by mass or less based on the total mass of the antitumor agent It may be.
  • pterostilbene a commercial product may be used, or one obtained by chemical synthesis may be used.
  • the antitumor agent of the present embodiment may further contain a fat and oil.
  • the antitumor agent is more suitable for oral administration.
  • the fats and oils that may be contained in the antitumor agent of the present embodiment may be animal oils or vegetable oils.
  • fats and oils for example, vegetable oils such as corn oil, soybean oil, sesame oil, rapeseed oil, safflower oil, olive oil, castor oil, castor oil, cottonseed oil, rice oil, sunflower oil, grape seed oil and wheat germ oil, and egg yolk oil
  • animal oils such as fish oil, soy sauce and liver oil, or their refined oils (salad oil), and edible oils obtained by chemical or enzymatic treatment such as MCT (medium chain fatty acid triglyceride), diglycerides, etc. may be mentioned.
  • the fats and oils may be used alone or in combination of two or more.
  • the content of the oil and fat may be 0.1% by mass or more, or 1% by mass or more, or 10% by mass or less, or 8% by mass or less, based on the total amount of the antitumor agent.
  • the antitumor agent of the present embodiment may further contain additives (except pterostilbene and oils and fats).
  • the additives include, for example, excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, and suspending agents.
  • the antitumor agent of the present embodiment may be in any form such as a solid (for example, a powder), a liquid (water-soluble or oil-soluble solution or suspension), a paste or the like.
  • a dosage form such as powder, granule, tablet, capsule, solution, suspension, syrup and the like may be used.
  • ⁇ Method of preparing antitumor agent> The various formulations described above can be prepared by mixing pterostilbene and, if necessary, the above-mentioned fats and oils and additives, and shaping as necessary. Preferred methods for preparing anti-tumor agents are described below. First, a fat or oil heated to a product temperature of 50 to 90 ° C. is prepared. Pterostilbene is added to the heated fat and dissolved. Thereafter, a mixture of pterostilbene and fats and oils, and other components are mixed, and if necessary, sterilization treatment etc. may be performed to prepare a formulation containing pterostilbene.
  • the antitumor agent of the present embodiment is used for oral administration.
  • a preparation which exerts a desired effect by parenteral administration such as intraperitoneal administration does not always exhibit a desired effect even by oral administration.
  • the antitumor agent of the present embodiment exerts an antitumor effect also by oral administration.
  • the dose, timing and period of administration of the antitumor agent can be appropriately set.
  • the living body to which the anti-tumor agent of the present embodiment is to be administered is preferably a mammal, more preferably a human.
  • the dose of the antitumor agent per day may be 0.01 mg to 5000 mg / day / person, 0. It is from 05 mg to 1000 mg / day / person, more preferably from 0.1 mg to 500 mg / day / person, even more preferably from 0.5 mg to 250 mg / day / person.
  • the antitumor agent may be administered once a day, as long as the daily dose falls within the above range, and may be divided and administered several times a day (for example, twice or three times).
  • the dose is a value based on the amount of the active ingredient (ie, pterostilbene) in the antitumor agent.
  • the antitumor agent of the present embodiment can be used as a component of products such as pharmaceuticals, quasi drugs, food and drink (drinks, food), feed, feed additives and the like.
  • examples of the beverage include water, soft drinks, fruit juice drinks, milk drinks, alcoholic drinks, sports drinks, nutritional drinks and the like.
  • examples of the food include breads, noodles, rice, tofu, dairy products, soy sauce, miso, confectionery, cream, sauces, mayonnaise, dressing and the like.
  • the antitumor agent of the present embodiment can also be used as a component of a health food, a functional display food, a special use food, a nutritional supplement, a supplement (for example, doctor's supplement) or a food for specified health.
  • the antitumor agent of the present embodiment can be used as a component of food and drink. Therefore, as one embodiment of the present invention, there is provided a food and drink for cancer prevention (food composition for cancer prevention) containing the above-mentioned antitumor agent.
  • the antitumor agent of the present embodiment may exert an antitumor effect as a result of regulating the expression of microRNA. That is, the antitumor agent of the present embodiment can also be regarded as a modulator of microRNA.
  • the microRNA expression regulator may modulate microRNA expression, for example, by suppressing the expression of a tumor-promoting microRNA or enhancing the expression of a tumor-suppressing microRNA.
  • the antitumor agent of the present embodiment exerts an antitumor effect as a result of enhancing the expression of the tumor suppressor microRNA. That is, one embodiment of the present invention provides a pterostilbene-containing agent for enhancing the expression of a tumor suppressor microRNA.
  • the tumor suppressor microRNA expression enhancer may be for oral administration.
  • the formulation form, dosage, use and the like may be the same as those in the above-mentioned antitumor agent.
  • the agent for promoting the expression of a tumor suppressor microRNA of the present embodiment may be used for the treatment or prevention of cancer.
  • tumor suppressor microRNA expression enhancer increases the microRNA from the state where the expression level of the tumor suppressor microRNA is reduced to the expression level similar to that of a healthy person (healthy person)
  • the expression level of the tumor suppressor microRNA may be maintained at the same expression level as that of a healthy individual. That is, the tumor suppressor microRNA expression enhancer can be rephrased as a tumor suppressor microRNA expression regulator, expression regulator, or expression promoter.
  • tumor suppressor microRNA is a microRNA in which the growth of cancer cells is suppressed by enhancing expression.
  • tumor suppressor microRNAs include let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a, and miR-193b. It can be mentioned.
  • the tumor suppressor microRNAs from let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141 miR-34a and miR-193b And at least one selected from the group consisting of
  • ⁇ Type of tumor suppressor microRNA> The above-mentioned tumor suppressor microRNAs are mature single-stranded microRNAs, each having a sequence as shown below.
  • let-7a UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 1)
  • let-7b UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO: 2)
  • miR-26a UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO: 3)
  • miR-107 AGCAGCAUUGUACAGGGCUAUCA (SEQ ID NO: 4)
  • miR-126 UCGUACCGUGAGUAAUAAUGCG (SEQ ID NO: 5)
  • miR-185 UGGAGAGAAGGCAGUUCCUGA (SEQ ID NO: 6)
  • miR-200c UAAUACUGCCGGGUAAUGAUGGA (SEQ ID NO: 7)
  • miR-141 UAACACUGUCUGGUAAAGAUGG (SEQ ID NO: 8)
  • One embodiment of the present invention can also be viewed as a method of treating or preventing cancer comprising orally administering pterostilbene to a subject in need thereof.
  • the administration method, administration subject, dose and the like may be the same as those in the above-mentioned antitumor agent.
  • the subject is preferably a mammal, more preferably a human.
  • One embodiment of the present invention can also be viewed as a method of enhancing the expression of a tumor suppressor microRNA, comprising orally administering pterostilbene to a subject in need thereof.
  • the administration method, administration subject, dose and the like may be the same as those in the above-mentioned antitumor agent.
  • the subject is preferably a mammal, more preferably a human.
  • ⁇ Type of cancer> examples of the cancer include breast cancer, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, lung cancer, oral cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, prostate cancer, bladder cancer, sarcoma, glioma and the like. It may be
  • pterostilbene for the preparation of an antitumor agent for oral administration.
  • the administration method, administration target, dose and the like may be the same as those in the above-mentioned antitumor agent.
  • pterostilbene for use in the treatment or prevention of cancer
  • the administration method, administration target, dose and the like may be the same as those in the above-mentioned antitumor agent.
  • reagent The following reagents were used in cell culture.
  • RPMI 1640 manufactured by Invitrogen
  • McCoy 'medium 5A modified medium manufactured by Invitrogen
  • Heat-inactivated Fetal Bovine Serum (FBS) Invitrogen
  • FBS Heat-inactivated Fetal Bovine Serum
  • Invitrogen 100X Antibiotic-Antimycotic
  • Trypsin-EDTA Invitrogen
  • Cell culture method In breast cancer cell lines and gastric cancer cell lines, RPMI 1640 supplemented with FBS and 100X Antibiotic-Antimycotic to a final concentration of 10% (v / v) and 1% (v / v), respectively, was used as a culture medium. . The culture was performed in the dark at 37 ° C., 5% CO 2 . Cell detachment at passage was performed using 0.05% Trypsin-EDTA. In colon cancer cell lines (HCT116), as the culture medium, McCoy 'medium 5A modified medium with FBS and 100X Antibiotic-Antimycotic to a final concentration of 10% (v / v) and 1% (v / v) respectively The one added was used. The culture was performed in the dark at 37 ° C., 5% CO 2 . Cell detachment at passage was performed using 0.05% Trypsin-EDTA.
  • FIG. 1 (A) to 1 (C) show the cell viability measurement results when using breast cancer cells, gastric cancer cells, or colon cancer cells, respectively. Error bars in FIG. 1 indicate standard errors.
  • Let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a and miR-193b which are known as tumor suppressor microRNAs It quantified by the following method.
  • resveratrol or pterostilbene was added to the above breast cancer cells to a concentration of 25 ⁇ M.
  • RNA was extracted according to the manufacturer's recommended protocol.
  • the extracted RNA was converted into complementary DNA (cDNA) using TaqMan® MicroRNA assay and TaqMan® MicroRNA Reverse Transcription Kit according to the protocol recommended by the manufacturer.
  • the cDNA as a template using the TaqMan (TM) Universal PCR Master Mix, NoAmpErase (TM) UNG, by StepOnePlus TM Real-Time PCR System ( Applied Biosystems), was quantitative analysis of miRNA. The same as in the above except that resveratrol or pterostilbene was not added, the one subjected to quantitative analysis of miRNA was used as a control (Mock). The results are shown in FIGS. Error bars in FIGS. 2 and 3 indicate standard errors. Moreover, the data which attached * in FIG. 2 and 3 are p ⁇ 0.05.
  • MM-231-luc-D3H2LN was used as a cancer cell transplanted to a mouse.
  • the cancer cells are suspended in phosphate buffered saline (PBS), and the obtained suspension and Matrigel (ECM Gel from Engelbreth-Holm-Swammurine sarcoma) are mixed 1: 1 and suspended for transplantation.
  • a suspension (cell number: 1 ⁇ 10 7 cells / ml) was prepared.
  • Control group (solvent administration group, dosage: 0 mg / kg / day)
  • Pterostilbene group A (dose: 12.5 mg / kg / day)
  • Pterostilbene group B (dose: 25 mg / kg / day)
  • Intraperitoneal administration was performed on the mice of each group of (i) to (iii). Intraperitoneal administration was continuously performed for 21 days starting from 2 days after the day when the cancer cells were transplanted into the above-mentioned mice (the administration start day).
  • a PBS solution containing DMSO and propylene glycol DMSO content: 10% (v / v), propylene glycol content: 10% (v / v)
  • pterostilbene pterostilbene dissolved in the above-mentioned solvent was used. The dose is a value based on the amount of pterostilbene.
  • FIG. 4 (A) shows the results of tumor luminescence at 21 days after the start of administration.
  • FIG. 4 (B) shows the observation result of the light emission amount over time.
  • the measurement result of a body weight is shown in FIG.
  • FIG. 5 shows the effect of intraperitoneal administration of pterostilbene on tumor size.
  • the removed tumor pieces are shown in FIG. 5 (A), and the weight of the removed tumor is shown in FIG. 5 (B).
  • the tumor removed after the completion of administration was used for miRNA expression analysis described later.
  • Tumor-suppressing miRNA expression analysis in human-derived tumors The expression analysis of tumor-suppressing miRNA was carried out using the tumor piece tissue formed by xenotransplantation in the above-mentioned nude mice. The expression levels of miR-200c and miR-141, which are tumor suppressor miRNAs, were measured.
  • cDNA as a template, TaqMan (R) Universal PCR Master Mix, using a No AmpErase (TM) UNG, by StepOnePlus TM Real-Time PCR System ( Applied Biosystems , Inc.), was quantified analysis of miRNA.
  • FIG. 7 (A) shows the results of tumor suppressor miRNAs.
  • the test feed (pterostilbene-containing powder feed) was prepared by the following method. That is, first, 50 g of corn oil was weighed and heated to 70 ° C. Pterostilbene was added to dissolve in heated corn oil. Reagents other than corn oil and pterostilbene (other reagents) were ground and mixed in a mortar. To the mixture of the other reagents, corn oil in which pterostilbene was dissolved was added, thoroughly ground in a mortar and mixed. Thereafter, it was further mixed for about 30 minutes with a stirrer to prepare a test feed. The compositions of various components contained in the test feed are as shown in Table 1.
  • the feed for the test was placed in a plastic bag, and after gamma sterilization with Koga Isotope Co., Ltd., it was used for animal experiments described later. Moreover, the acclimatized food (content of pterostilbene: 0 mass%) of the composition shown in Table 1 was prepared.
  • mice Five-week-old female nude mice (BALB / c-nu / nu, CAnN.Cg-Foxn1 ⁇ nu> / CrlCjlj) (Charles River Japan, Inc.) were used as experimental animals.
  • mice for each group were subjected to a cancer cell transplantation experiment.
  • a cancer cell transplantation experiment first, the cancer cells to be transplanted (MM-231-luc-D3H2LN) are suspended in phosphate buffered saline (PBS), and Matrigel (ECM Gel from Engelbreth-Holm-Swammurine sarcoma) and 1 are suspended. The mixture was mixed at a ratio of 1: 1 to prepare a suspension for transplantation (number of cells: 1 ⁇ 10 5 cells / ml).
  • transplant cancer cells of 1 ⁇ 10 4 cells to the mammary gland of a mouse (suspension for transplantation (cell number: 1 ⁇ 10 5) 100 ⁇ l of cells / ml) was introduced).
  • tissue for transplantation cell number: 1 ⁇ 10 5
  • 15 mg / ml luciferin solution Beetle Luciferin, Potassium Salt, Promega
  • the condition of the test feed and mice was properly managed daily.
  • the weight of the mice was measured at appropriate times.
  • the administration start day (day 0) was the day on which cancer cells (tumor) were transplanted. Then, the luminescence was observed with IVIS spectrum every other week from the administration start date.
  • the measurement results of tumor luminescence are shown in FIG.
  • FIG. 8 (A) shows the results of tumor luminescence 21 days after the start of administration.
  • FIG. 8 (B) shows the observation result of the light emission amount over time.
  • the measurement results of the weight of the mice in each group are shown in FIG.

Abstract

The present invention pertains to an oral antitumor agent that contains pterostilbene.

Description

抗腫瘍剤、癌予防用飲食品及び癌抑制的マイクロＲＮＡの発現亢進剤Antitumor agent, food and drink for cancer prevention and expression enhancer for tumor suppressor microRNA
　本発明は、抗腫瘍剤、癌予防用飲食品及び癌抑制的マイクロＲＮＡの発現亢進剤に関する。 TECHNICAL FIELD The present invention relates to an antitumor agent, a food and drink for cancer prevention, and an agent for enhancing the expression of a tumor suppressor microRNA.
　マイクロＲＮＡは、塩基対の長さが２２塩基程度のＲＮＡである。スチルベン誘導体であるレスベラトロール及びプテロスチルベンが、このようなマイクロＲＮＡのうち、所定のマイクロＲＮＡの発現を促進又は抑制することが知られている（非特許文献１及び２）。 The microRNA is RNA having a base pair length of about 22 bases. Among such microRNAs, resveratrol and pterostilbene which are stilbene derivatives are known to promote or suppress the expression of a predetermined microRNA (Non-patent Documents 1 and 2).
　近年、マイクロＲＮＡ（ｍｉＲＮＡ）の発現量の異常と癌発生の関係が分かってきており、ｍｉＲＮＡ（例えば、癌抑制的ｍｉＲＮＡ）の発現量を制御（亢進）し得る化合物は、抗腫瘍剤（癌予防剤）として有用である。さらに、経口投与により、抗腫瘍効果を発揮できる化合物は、食品、サプリメント等への用途展開が容易である。 In recent years, the relationship between abnormalities in microRNA (miRNA) expression levels and cancer development has been known, and compounds that can control (enhance the expression levels of miRNAs (eg, tumor suppressor miRNAs) are anti-tumor agents (cancer Useful as a preventive agent). Furthermore, the compound capable of exerting an antitumor effect by oral administration can be easily applied to foods, supplements and the like.
　そこで、本発明は、新規な経口投与用抗腫瘍剤の提供を目的とする。 Then, this invention aims at provision of the novel antitumor agent for oral administration.
　本発明者らは、プテロスチルベンが、経口投与によって、優れた抗腫瘍効果を有することを見出した。本発明は、この新規な知見に基づくものである。 The present inventors have found that pterostilbene has an excellent antitumor effect by oral administration. The present invention is based on this novel finding.
　すなわち、本発明は、例えば、以下の各発明に関する。
（１）プテロスチルベンを含有する、経口投与用抗腫瘍剤。
（２）油脂を更に含有する、（１）に記載の抗腫瘍剤。
（３）油脂が、植物油である、（２）に記載の抗腫瘍剤。
（４）（１）～（３）のいずれかに記載の抗腫瘍剤を含む、癌予防用飲食品。
（５）プテロスチルベンを含有する、癌抑制的マイクロＲＮＡの発現亢進剤。
（６）経口投与用である、（５）に記載の発現亢進剤。
（７）癌抑制的マイクロＲＮＡが、ｌｅｔ－７ａ、ｌｅｔ－７ｂ、ｍｉＲ－２６ａ、ｍｉＲ－１０７、ｍｉＲ－１２６、ｍｉＲ－１８５、ｍｉＲ－２００ｃ、ｍｉＲ－１４１、ｍｉＲ－３４ａ及びｍｉＲ－１９３ｂからなる群より選択される少なくとも１種を含む、（５）又は（６）に記載の発現亢進剤。
（８）癌の治療又は予防のために用いられる、（５）～（７）のいずれかに記載の発現亢進剤。
（９）プテロスチルベンを、それを必要とする対象に経口投与することを含む、癌を治療又は予防する方法。
（１０）経口投与用抗腫瘍剤の製造のための、プテロスチルベンの使用。
（１１）経口投与による癌の治療又は予防に使用するための、プテロスチルベン。 That is, the present invention relates to, for example, the following inventions.
(1) An antitumor agent for oral administration comprising pterostilbene.
(2) The antitumor agent according to (1), which further contains a fat and oil.
(3) The antitumor agent according to (2), wherein the oil and fat is a vegetable oil.
(4) A food and drink for cancer prevention, which comprises the antitumor agent according to any one of (1) to (3).
(5) A tumor suppressor microRNA expression enhancer containing pterostilbene.
(6) The expression enhancer according to (5), which is for oral administration.
(7) Tumor suppressor microRNAs from let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a and miR-193b The expression enhancer according to (5) or (6), comprising at least one selected from the group consisting of
(8) The expression enhancer according to any one of (5) to (7), which is used for the treatment or prevention of cancer.
(9) A method for treating or preventing cancer, which comprises orally administering pterostilbene to a subject in need thereof.
(10) Use of pterostilbene for the production of an antitumor agent for oral administration.
(11) Pterostilbene for use in the treatment or prevention of cancer by oral administration.
　本発明によれば、新規な経口投与用抗腫瘍剤が提供される。本発明によればまた、当該抗腫瘍剤を含有する癌予防用飲食品が提供される。 According to the present invention, a novel antitumor agent for oral administration is provided. According to the present invention, there is also provided a cancer preventive food and drink containing the antitumor agent.
プテロスチルベン又はレスベラトロールと癌細胞の細胞生存率との関係を示すグラフである。図１（Ａ）は、乳癌細胞を用いた場合の細胞生存率の測定結果、図１（Ｂ）は、胃癌細胞を用いた場合の細胞生存率の測定結果、図１（Ｃ）は、大腸癌細胞を用いた場合の細胞生存率の測定結果を示すグラフである。It is a graph which shows the relationship between pterostilbene or resveratrol and the cell survival rate of a cancer cell. Fig. 1 (A) shows the measurement results of cell viability when breast cancer cells are used, Fig. 1 (B) shows the measurement results of cell viability when gastric cancer cells are used, and Fig. 1 (C) shows the large intestine It is a graph which shows the measurement result of the cell survival rate at the time of using a cancer cell. 癌抑制的ｍｉＲＮＡ（ｌｅｔ－７ａ、ｌｅｔ－７ｂ、ｍｉＲ－２６ａ、ｍｉＲ－１０７、ｍｉＲ－１２６、ｍｉＲ－１８５）の発現量の測定結果を示すグラフである。It is a graph which shows the measurement result of the expression level of tumor suppressor miRNA (let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185). 癌抑制的ｍｉＲＮＡ（ｍｉＲ－２００ｃ、ｍｉＲ－１４１、ｍｉＲ－３４ａ、ｍｉＲ－１９３ｂ）の発現量の測定結果を示すグラフである。It is a graph which shows the measurement result of the expression level of tumor suppressor miRNA (miR-200c, miR-141, miR-34a, miR-193b). 図４（Ａ）は、投与開始から２１日経過時点の腫瘍発光の結果を示す写真である。図４（Ｂ）は、発光量の経時観察結果を示すグラフである。FIG. 4 (A) is a photograph showing the result of tumor luminescence 21 days after the start of administration. FIG. 4 (B) is a graph showing the results of observation of the amount of luminescence over time. プテロスチルベンの腹腔内投与が腫瘍のサイズに与える影響を示す写真又はグラフである。図５（Ａ）は、摘出した腫瘍片を示す写真である。図５（Ｂ）は、摘出した腫瘍の重量を示すグラフである。It is a photograph or a graph which shows the influence which the intraperitoneal administration of pterostilbene gives to the size of a tumor. FIG. 5 (A) is a photograph showing the excised tumor piece. FIG. 5 (B) is a graph showing the weight of the excised tumor. プテロスチルベン（腹腔内投与）がマウスの体重に与える影響を示すグラフである。It is a graph which shows the influence which pterostilbene (intraperitoneal administration) gives to the weight of a mouse. 図７（Ａ）は、ＲＮＡを抽出した腫瘍片を示す写真である。図７（Ｂ）は、腫瘍中の癌抑制的マイクロＲＮＡ（ｍｉＲ－２００ｃ及びｍｉＲ－１４１）の発現量を示すグラフである。FIG. 7 (A) is a photograph showing a tumor piece from which RNA was extracted. FIG. 7 (B) is a graph showing the expression levels of tumor suppressor microRNAs (miR-200c and miR-141) in tumors. 図８（Ａ）は、投与開始から２１日経過時点の腫瘍発光の結果を示す写真である。図８（Ｂ）は、発光量の経時観察結果を示すグラフである。FIG. 8 (A) is a photograph showing the result of tumor luminescence 21 days after the start of administration. FIG. 8 (B) is a graph showing the observation result of the luminescence amount with time. プテロスチルベン（経口投与）がマウスの体重に与える影響を示すグラフである。It is a graph which shows the influence which pterostilbene (oral administration) gives to the weight of a mouse. プテロスチルベンの経口投与が腫瘍のサイズ（重量）に与える影響を示すグラフである。It is a graph which shows the influence which oral administration of pterostilbene gives to the size (weight) of a tumor.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
＜本発明の特徴＞
（抗腫瘍剤）
　本発明は、プテロスチルベンを含有する、経口投与用抗腫瘍剤を提供することに特徴を有する。 <Features of the present invention>
(Antitumor agent)
The present invention is characterized by providing an antineoplastic agent for oral administration, which contains pterostilbene.
（癌予防用飲食品）
　本発明はまた、上記抗腫瘍剤を含む、癌予防用飲食品を提供することに特徴を有する。 (Food and drink for cancer prevention)
The present invention is also characterized by providing a food and drink for cancer prevention, which comprises the above-mentioned antitumor agent.
（癌抑制的マイクロＲＮＡ発現亢進剤）
　本発明はさらに、プテロスチルベンを含有する、癌抑制的マイクロＲＮＡの発現亢進剤を提供することに特徴を有する。 (Tumor suppressor microRNA expression enhancer)
The present invention is further characterized by providing a pterostilbene-containing agent for enhancing the expression of a tumor suppressor microRNA.
＜抗腫瘍剤＞
　本明細書における「抗腫瘍剤」は、癌の治療又は予防剤、癌細胞増殖抑制剤、癌の進行抑制剤、癌の再発予防（抑制）剤、癌の転移抑制剤、癌の浸潤抑制剤、癌細胞から放出されるエクソソーム等の細胞外小胞の分泌抑制剤等を含む。 <Antitumor agent>
The "anti-tumor agent" in the present specification is a therapeutic or preventive agent for cancer, a cancer cell growth inhibitor, a cancer progression inhibitor, a cancer recurrence preventive (suppression) agent, a cancer metastasis suppressor, and a cancer invasion suppressor And inhibitors of secretion of extracellular vesicles such as exosomes released from cancer cells.
＜プテロスチルベン＞
　プテロスチルベン（Ｐｔｅｒｏｓｔｉｌｂｅｎｅ）は、下記式（１）により表される化合物である。
Figure JPOXMLDOC01-appb-C000001
 <Pterostilbene>
Pterostilbene (Pterostilbene) is a compound represented by the following formula (1).
Figure JPOXMLDOC01-appb-C000001
＜プテロスチルベンの含有量＞
　本実施形態の抗腫瘍剤において、プテロスチルベンの含有量は、抗腫瘍剤全量基準で、０．０１質量％以上、若しくは０．１０質量％以上、又は、１０質量％以下、若しくは５質量％以下であってよい。 <Content of Pterostilbene>
In the antitumor agent of the present embodiment, the content of pterostilbene is 0.01% by mass or more, 0.10% by mass or more, 10% by mass or less, or 5% by mass or less based on the total mass of the antitumor agent It may be.
＜プテロスチルベンの入手方法＞
　本実施形態において、プテロスチルベンは、市販品を用いてもよく、化学合成により得られたものを用いてもよい。 <How to obtain Pterostilbene>
In the present embodiment, as the pterostilbene, a commercial product may be used, or one obtained by chemical synthesis may be used.
＜油脂＞
　本実施形態の抗腫瘍剤は、油脂を更に含有していてもよい。この場合、抗腫瘍剤が経口投与用として、より一層好適なものとなる。 <Fat and oil>
The antitumor agent of the present embodiment may further contain a fat and oil. In this case, the antitumor agent is more suitable for oral administration.
＜油脂の種類＞
　本実施形態の抗腫瘍剤に含有していてもよい油脂は、動物油であってもよく、植物油であってもよい。油脂としては、例えば、コーン油、大豆油、ゴマ油、ナタネ油、サフラワー油、オリーブ油、ヒマシ油、綿実油、米油、ヒマワリ油、グレープシード油及び小麦胚芽油等の植物油、及び、卵黄油、魚油、鯨油、肝油等の動物油、又はこれらの精製油（サラダ油）、及びＭＣＴ（中鎖脂肪酸トリグリセリド）、ジグリセリドのように化学的あるいは酵素的処理を施して得られる食用油等が挙げられる。油脂は、１種単独で用いてもよく、２種以上を組み合わせて用いてもよい。 <Types of fats and oils>
The fats and oils that may be contained in the antitumor agent of the present embodiment may be animal oils or vegetable oils. As fats and oils, for example, vegetable oils such as corn oil, soybean oil, sesame oil, rapeseed oil, safflower oil, olive oil, castor oil, castor oil, cottonseed oil, rice oil, sunflower oil, grape seed oil and wheat germ oil, and egg yolk oil Animal oils such as fish oil, soy sauce and liver oil, or their refined oils (salad oil), and edible oils obtained by chemical or enzymatic treatment such as MCT (medium chain fatty acid triglyceride), diglycerides, etc. may be mentioned. The fats and oils may be used alone or in combination of two or more.
＜油脂の含有量＞
　油脂の含有量は、抗腫瘍剤全量基準で、０．１質量％以上、若しくは１質量％以上、又は、１０質量％以下、若しくは８質量％以下であってよい。 <Contents of fats and oils>
The content of the oil and fat may be 0.1% by mass or more, or 1% by mass or more, or 10% by mass or less, or 8% by mass or less, based on the total amount of the antitumor agent.
＜添加剤＞
　本実施形態の抗腫瘍剤は、添加剤（プテロスチルベン及び油脂を除く）を更に含有していてよい。添加剤としては、例えば、賦形剤、結合剤、滑沢剤、崩壊剤、乳化剤、界面活性剤、基剤、溶解補助剤、懸濁化剤が挙げられる。 <Additives>
The antitumor agent of the present embodiment may further contain additives (except pterostilbene and oils and fats). The additives include, for example, excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, and suspending agents.
＜製剤形態＞
　本実施形態の抗腫瘍剤は、固体（例えば、粉末）、液体（水溶性又は脂溶性の溶液又は懸濁液）、ペースト等のいずれの形状であってもよい。また、散剤、顆粒剤、錠剤、カプセル剤、液剤、懸濁剤、シロップ剤等のいずれの剤形であってもよい。 <Formulation form>
The antitumor agent of the present embodiment may be in any form such as a solid (for example, a powder), a liquid (water-soluble or oil-soluble solution or suspension), a paste or the like. In addition, any dosage form such as powder, granule, tablet, capsule, solution, suspension, syrup and the like may be used.
＜抗腫瘍剤の調製方法＞
　上述の各種製剤は、プテロスチルベンと、必要に応じて、上記油脂及び添加剤と、を混和し、必要に応じて成形することによって調製することができる。
　抗腫瘍剤の好適な調製方法を以下に説明する。まず、品温５０～９０℃に加温した油脂を準備する。加温した油脂に、プテロスチルベンを添加して、溶解させる。その後、プテロスチルベン及び油脂の混合物と、その他の成分と、を混合し、必要に応じて、滅菌処理等を行い、プテロスチルベンを含有する製剤を調製することができる。 <Method of preparing antitumor agent>
The various formulations described above can be prepared by mixing pterostilbene and, if necessary, the above-mentioned fats and oils and additives, and shaping as necessary.
Preferred methods for preparing anti-tumor agents are described below. First, a fat or oil heated to a product temperature of 50 to 90 ° C. is prepared. Pterostilbene is added to the heated fat and dissolved. Thereafter, a mixture of pterostilbene and fats and oils, and other components are mixed, and if necessary, sterilization treatment etc. may be performed to prepare a formulation containing pterostilbene.
＜投与方法＞
　本実施形態の抗腫瘍剤は、経口投与用として用いられる。通常、腹腔内投与等の非経口投与により、所望の効果を奏する製剤は、必ずしも経口投与によっても、所望の効果を奏するとは限らない。一方で、本実施形態の抗腫瘍剤は、非経口投与によって、抗腫瘍効果が奏されることに加えて、経口投与によっても抗腫瘍効果が奏される。抗腫瘍剤の投与量、投与タイミング、投与期間については、適宜、設定することができる。 <Method of administration>
The antitumor agent of the present embodiment is used for oral administration. In general, a preparation which exerts a desired effect by parenteral administration such as intraperitoneal administration does not always exhibit a desired effect even by oral administration. On the other hand, in addition to the antitumor effect being exerted by parenteral administration, the antitumor agent of the present embodiment exerts an antitumor effect also by oral administration. The dose, timing and period of administration of the antitumor agent can be appropriately set.
＜投与対象＞
　本実施形態の抗腫瘍剤の投与対象となる生体は、好ましくは哺乳動物であり、より好ましくはヒトである。 <Subject of administration>
The living body to which the anti-tumor agent of the present embodiment is to be administered is preferably a mammal, more preferably a human.
＜投与量＞
　具体的な投与量の一例として、例えば、ヒト成人男子（体重６０ｋｇ）に投与する場合、一日当たりの抗腫瘍剤の投与量は、０．０１ｍｇ～５０００ｍｇ／日／人であってよく、０．０５ｍｇ～１０００ｍｇ／日／人、より好ましくは０．１ｍｇ～５００ｍｇ／日／人、さらに好ましくは０．５ｍｇ～２５０ｍｇ／日／人である。抗腫瘍剤は、１日当たりの投与量が上記範囲内であれば、１日１回投与されてもよく、１日複数回（例えば、２回又は３回）に分けて投与されてもよい。なお、投与量は、抗腫瘍剤中の有効成分（すなわち、プテロスチルベン）の量を基準とした値である。 <Dose amount>
As an example of a specific dose, for example, when administered to adult human male (body weight 60 kg), the dose of the antitumor agent per day may be 0.01 mg to 5000 mg / day / person, 0. It is from 05 mg to 1000 mg / day / person, more preferably from 0.1 mg to 500 mg / day / person, even more preferably from 0.5 mg to 250 mg / day / person. The antitumor agent may be administered once a day, as long as the daily dose falls within the above range, and may be divided and administered several times a day (for example, twice or three times). The dose is a value based on the amount of the active ingredient (ie, pterostilbene) in the antitumor agent.
＜抗腫瘍剤の用途＞
　本実施形態の抗腫瘍剤は、医薬品、医薬部外品、飲食品（飲料、食品）、飼料、飼料添加物等の製品の成分として使用することができる。飲料としては、例えば、水、清涼飲料水、果汁飲料、乳飲料、アルコール飲料、スポーツドリンク、栄養ドリンク等が挙げられる。食品としては、例えば、パン類、麺類、米類、豆腐、乳製品、醤油、味噌、菓子類、クリーム、ソース類、マヨネーズ、ドレッシング等が挙げられる。また、本実施形態の抗腫瘍剤は、健康食品、機能性表示食品、特別用途食品、栄養補助食品、サプリメント（例えば、ドクターズサプリメント）又は特定保健用食品等の成分として使用することもできる。 <Use of antineoplastic agent>
The antitumor agent of the present embodiment can be used as a component of products such as pharmaceuticals, quasi drugs, food and drink (drinks, food), feed, feed additives and the like. Examples of the beverage include water, soft drinks, fruit juice drinks, milk drinks, alcoholic drinks, sports drinks, nutritional drinks and the like. Examples of the food include breads, noodles, rice, tofu, dairy products, soy sauce, miso, confectionery, cream, sauces, mayonnaise, dressing and the like. In addition, the antitumor agent of the present embodiment can also be used as a component of a health food, a functional display food, a special use food, a nutritional supplement, a supplement (for example, doctor's supplement) or a food for specified health.
＜癌予防用飲食品＞
　本実施形態の抗腫瘍剤は、飲食品の成分として使用することが可能である。よって、本発明の一実施形態として、上記抗腫瘍剤を含有する、癌予防用飲食品（癌予防用食品組成物）が提供される。 <Food and drink for cancer prevention>
The antitumor agent of the present embodiment can be used as a component of food and drink. Therefore, as one embodiment of the present invention, there is provided a food and drink for cancer prevention (food composition for cancer prevention) containing the above-mentioned antitumor agent.
＜マイクロＲＮＡの発現調節剤＞
　本実施形態の抗腫瘍剤は、マイクロＲＮＡの発現を調節する結果として、抗腫瘍効果が奏されるものであってよい。すなわち、本実施形態の抗腫瘍剤は、マイクロＲＮＡの調節剤と捉えることもできる。マイクロＲＮＡの発現調節剤は、例えば、癌促進的マイクロＲＮＡの発現抑制、又は、癌抑制的マイクロＲＮＡの発現亢進によって、マイクロＲＮＡの発現を調節するものであってよい。 <MicroRNA expression regulator>
The antitumor agent of the present embodiment may exert an antitumor effect as a result of regulating the expression of microRNA. That is, the antitumor agent of the present embodiment can also be regarded as a modulator of microRNA. The microRNA expression regulator may modulate microRNA expression, for example, by suppressing the expression of a tumor-promoting microRNA or enhancing the expression of a tumor-suppressing microRNA.
＜癌抑制的マイクロＲＮＡの発現亢進剤＞
　本実施形態の抗腫瘍剤は、癌抑制的マイクロＲＮＡの発現を亢進する結果として、抗腫瘍効果が奏される。すなわち、本発明の一実施形態は、プテロスチルベンを含有する、癌抑制的マイクロＲＮＡの発現亢進剤を提供する。癌抑制的マイクロＲＮＡの発現亢進剤は、経口投与用であってよい。なお、本実施形態の癌抑制的マイクロＲＮＡの発現亢進剤において、製剤形態、投与量、用途等については、上記の抗腫瘍剤におけるものと同様であってよい。本実施形態の癌抑制的マイクロＲＮＡの発現亢進剤は、癌の治療又は予防のために用いられるものであってよい。ここで、「癌抑制的マイクロＲＮＡの発現亢進剤」とは、癌抑制的マイクロＲＮＡの発現量が低下している状態から健常人（健常者）と同様の発現量まで当該マイクロＲＮＡを増加させるものであってもよく、癌抑制的マイクロＲＮＡの発現量を健常人と同様の発現量に維持するものであってもよい。つまり、癌抑制的マイクロＲＮＡの発現亢進剤は、癌抑制的マイクロＲＮＡの、発現調節剤、発現制御剤、又は発現促進剤と言い換えることもできる。 <Expression Promoter for Tumor Suppressive MicroRNA>
The antitumor agent of the present embodiment exerts an antitumor effect as a result of enhancing the expression of the tumor suppressor microRNA. That is, one embodiment of the present invention provides a pterostilbene-containing agent for enhancing the expression of a tumor suppressor microRNA. The tumor suppressor microRNA expression enhancer may be for oral administration. In the tumor suppressor microRNA expression enhancer of the present embodiment, the formulation form, dosage, use and the like may be the same as those in the above-mentioned antitumor agent. The agent for promoting the expression of a tumor suppressor microRNA of the present embodiment may be used for the treatment or prevention of cancer. Here, the term "tumor suppressor microRNA expression enhancer" increases the microRNA from the state where the expression level of the tumor suppressor microRNA is reduced to the expression level similar to that of a healthy person (healthy person) The expression level of the tumor suppressor microRNA may be maintained at the same expression level as that of a healthy individual. That is, the tumor suppressor microRNA expression enhancer can be rephrased as a tumor suppressor microRNA expression regulator, expression regulator, or expression promoter.
＜癌抑制的マイクロＲＮＡ＞
　本明細書において、「癌抑制的マイクロＲＮＡ」とは、発現が亢進することにより、癌細胞の増殖が抑制されるマイクロＲＮＡである。癌抑制的マイクロＲＮＡとしては、例えば、ｌｅｔ－７ａ、ｌｅｔ－７ｂ、ｍｉＲ－２６ａ、ｍｉＲ－１０７、ｍｉＲ－１２６、ｍｉＲ－１８５、ｍｉＲ－２００ｃ、ｍｉＲ－１４１、ｍｉＲ－３４ａ、ｍｉＲ－１９３ｂが挙げられる。本実施形態において、癌抑制的マイクロＲＮＡは、ｌｅｔ－７ａ、ｌｅｔ－７ｂ、ｍｉＲ－２６ａ、ｍｉＲ－１０７、ｍｉＲ－１２６、ｍｉＲ－１８５、ｍｉＲ－２００ｃ、ｍｉＲ－１４１ｍｉＲ－３４ａ及びｍｉＲ－１９３ｂからなる群より選択される少なくとも１種を含んでいてよい。 <Tumor suppressor microRNA>
As used herein, a "tumor suppressor microRNA" is a microRNA in which the growth of cancer cells is suppressed by enhancing expression. Examples of tumor suppressor microRNAs include let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a, and miR-193b. It can be mentioned. In this embodiment, the tumor suppressor microRNAs from let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141 miR-34a and miR-193b And at least one selected from the group consisting of
＜癌抑制的マイクロＲＮＡの種類＞
　上記の癌抑制的マイクロＲＮＡは、成熟した一本鎖のマイクロＲＮＡであり、それぞれ以下に示すとおりの配列を有する。
ｌｅｔ－７ａ：　ＵＧＡＧＧＵＡＧＵＡＧＧＵＵＧＵＡＵＡＧＵＵ　（配列番号１）
ｌｅｔ－７ｂ：　ＵＧＡＧＧＵＡＧＵＡＧＧＵＵＧＵＧＵＧＧＵＵ　（配列番号２）
ｍｉＲ－２６ａ：　ＵＵＣＡＡＧＵＡＡＵＣＣＡＧＧＡＵＡＧＧＣＵ　（配列番号３）
ｍｉＲ－１０７：　ＡＧＣＡＧＣＡＵＵＧＵＡＣＡＧＧＧＣＵＡＵＣＡ　（配列番号４）
ｍｉＲ－１２６：　ＵＣＧＵＡＣＣＧＵＧＡＧＵＡＡＵＡＡＵＧＣＧ　（配列番号５）
ｍｉＲ－１８５：　ＵＧＧＡＧＡＧＡＡＡＧＧＣＡＧＵＵＣＣＵＧＡ　（配列番号６）
ｍｉＲ－２００ｃ：　ＵＡＡＵＡＣＵＧＣＣＧＧＧＵＡＡＵＧＡＵＧＧＡ　（配列番号７）
ｍｉＲ－１４１：　ＵＡＡＣＡＣＵＧＵＣＵＧＧＵＡＡＡＧＡＵＧＧ　（配列番号８）
ｍｉＲ－３４ａ：　ＵＧＧＣＡＧＵＧＵＣＵＵＡＧＣＵＧＧＵＵＧＵ　（配列番号９）
ｍｉＲ－１９３ｂ：　ＡＡＣＵＧＧＣＣＣＵＣＡＡＡＧＵＣＣＣＧＣＵ　（配列番号１０） <Type of tumor suppressor microRNA>
The above-mentioned tumor suppressor microRNAs are mature single-stranded microRNAs, each having a sequence as shown below.
let-7a: UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 1)
let-7b: UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO: 2)
miR-26a: UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO: 3)
miR-107: AGCAGCAUUGUACAGGGCUAUCA (SEQ ID NO: 4)
miR-126: UCGUACCGUGAGUAAUAAUGCG (SEQ ID NO: 5)
miR-185: UGGAGAGAAGGCAGUUCCUGA (SEQ ID NO: 6)
miR-200c: UAAUACUGCCGGGUAAUGAUGGA (SEQ ID NO: 7)
miR-141: UAACACUGUCUGGUAAAGAUGG (SEQ ID NO: 8)
miR-34a: UGGCAGUGUCUAUGCUGGUUGU (SEQ ID NO: 9)
miR-193b: AACUGCCCCUCAAAGUCCCGCU (SEQ ID NO: 10)
＜癌を治療又は予防する方法＞
　本発明の一実施形態は、プテロスチルベンを、それを必要とする対象に経口投与することを含む、癌を治療又は予防する方法と捉えることもできる。当該方法において、投与方法、投与対象、投与量等については、上記の抗腫瘍剤におけるものと同様であってよい。対象は哺乳動物であることが好ましく、ヒトであることがより好ましい。 <Method for treating or preventing cancer>
One embodiment of the present invention can also be viewed as a method of treating or preventing cancer comprising orally administering pterostilbene to a subject in need thereof. In the method, the administration method, administration subject, dose and the like may be the same as those in the above-mentioned antitumor agent. The subject is preferably a mammal, more preferably a human.
＜癌抑制的マイクロＲＮＡの発現を亢進する方法＞
　本発明の一実施形態は、プテロスチルベンを、それを必要とする対象に経口投与することを含む、癌抑制的マイクロＲＮＡの発現を亢進する方法と捉えることもできる。当該方法において、投与方法、投与対象、投与量等については、上記の抗腫瘍剤におけるものと同様であってよい。対象は哺乳動物であることが好ましく、ヒトであることがより好ましい。 <Method for enhancing expression of tumor suppressor microRNA>
One embodiment of the present invention can also be viewed as a method of enhancing the expression of a tumor suppressor microRNA, comprising orally administering pterostilbene to a subject in need thereof. In the method, the administration method, administration subject, dose and the like may be the same as those in the above-mentioned antitumor agent. The subject is preferably a mammal, more preferably a human.
＜癌の種類＞
　上記癌は、例えば、乳癌、胃癌、大腸癌、結腸直腸癌、卵巣癌、肺癌、口腔癌、食道癌、肝細胞癌、膵臓癌、胆管癌、前立腺癌、膀胱癌、肉腫、神経膠腫等であってもよい。 <Type of cancer>
Examples of the cancer include breast cancer, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, lung cancer, oral cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, prostate cancer, bladder cancer, sarcoma, glioma and the like. It may be
＜経口投与用抗腫瘍剤の製造のための、プテロスチルベンの使用＞
　本発明の一実施形態として、経口投与用抗腫瘍剤の製造のための、プテロスチルベンの使用が提供される。なお、当該実施形態における、投与方法、投与対象、投与量等については、上記の抗腫瘍剤におけるものと同様であってよい。 <Use of Pterostilbene for Production of Antitumor Agent for Oral Administration>
In one embodiment of the present invention, there is provided use of pterostilbene for the preparation of an antitumor agent for oral administration. In the embodiment, the administration method, administration target, dose and the like may be the same as those in the above-mentioned antitumor agent.
＜癌の治療又は予防に使用するための、プテロスチルベン＞
　本発明の一実施形態として、経口投与による癌の治療又は予防に使用するための、プテロスチルベンが提供される。なお、当該実施形態における、投与方法、投与対象、投与量等については、上記の抗腫瘍剤におけるものと同様であってよい。 <Pterostilbene for use in the treatment or prevention of cancer>
As an embodiment of the present invention, pterostilbene is provided for use in the treatment or prevention of cancer by oral administration. In the embodiment, the administration method, administration target, dose and the like may be the same as those in the above-mentioned antitumor agent.
　以下、実施例等に基づいて、本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be more specifically described based on examples and the like. However, the present invention is not limited to the following examples.
（統計解析）
　以下の実施例において、統計解析はKaleida Graph4.5（HULINKS社）を用いて、分散分析（One-Way ANOVA）を行った後、多重比較（Tukey’s HSD Test）で検定した。 (Statistical analysis)
In the following examples, statistical analysis was performed by analysis of variance (One-Way ANOVA) using Kaleida Graph 4.5 (HULINKS) and then tested by multiple comparison (Tukey's HSD Test).
〔試験例１：細胞を用いた抗腫瘍効果の評価〕
１．細胞培養の条件
（細胞）
　乳癌細胞株(MDA-MB231-luc-D3H2LN)、胃癌細胞株(MKN45)、及び大腸癌細胞株(HCT116)を使用した。 [Test Example 1: Evaluation of antitumor effect using cells]
1. Cell culture conditions (cells)
Breast cancer cell lines (MDA-MB231-luc-D3H2LN), gastric cancer cell lines (MKN45), and colon cancer cell lines (HCT116) were used.
（試薬）
　細胞培養においては、以下の試薬を用いた。
RPMI1640(Invitrogen社製)
McCoy’ medium 5A modified medium(Invitrogen社製)
Heat-inactivated Fetal Bovine Serum(FBS)(Invitrogen社製)
100X Antibiotic-Antimycotic(Invitrogen社製)
0.5% Trypsin-EDTA(Invitrogen社製) (reagent)
The following reagents were used in cell culture.
RPMI 1640 (manufactured by Invitrogen)
McCoy 'medium 5A modified medium (manufactured by Invitrogen)
Heat-inactivated Fetal Bovine Serum (FBS) (Invitrogen)
100X Antibiotic-Antimycotic (Invitrogen)
0.5% Trypsin-EDTA (Invitrogen)
（細胞培養方法）
　乳癌細胞株及び胃癌細胞株では、培養培地として、RPMI1640にFBS及び100XAntibiotic-Antimycoticを終濃度がそれぞれ１０％（ｖ／ｖ）及び１％（ｖ／ｖ）になるように添加したものを用いた。培養は暗所にて、３７℃、５％ＣＯ２の条件で行った。継代の際の細胞剥離は0.05% Trypsin-EDTAを用いて行った。
　大腸癌細胞株（ＨＣＴ１１６）では、培養培地として、McCoy’ medium 5A modified mediumにFBS及び100X Antibiotic-Antimycoticを終濃度がそれぞれ１０％（ｖ／ｖ）及び１％（ｖ／ｖ）になるように添加したものを用いた。培養は暗所にて、３７℃、５％ＣＯ２の条件で行った。継代の際の細胞剥離は0.05% Trypsin-EDTAを用いて行った。 (Cell culture method)
In breast cancer cell lines and gastric cancer cell lines, RPMI 1640 supplemented with FBS and 100X Antibiotic-Antimycotic to a final concentration of 10% (v / v) and 1% (v / v), respectively, was used as a culture medium. . The culture was performed in the dark at 37 ° C., 5% CO 2 . Cell detachment at passage was performed using 0.05% Trypsin-EDTA.
In colon cancer cell lines (HCT116), as the culture medium, McCoy 'medium 5A modified medium with FBS and 100X Antibiotic-Antimycotic to a final concentration of 10% (v / v) and 1% (v / v) respectively The one added was used. The culture was performed in the dark at 37 ° C., 5% CO 2 . Cell detachment at passage was performed using 0.05% Trypsin-EDTA.
２．細胞生存率の測定
（試薬及びキット）
　以下に示す試薬及びキットを使用した。
レスベラトロール(trans-Resveratrol、Cayman Chemical社製)
プテロスチルベン(Pterostilbene、Cayman Chemical社製)
Cell Counting Kit-8(株式会社 同仁化学研究所製) 2. Measurement of cell viability (reagent and kit)
The following reagents and kits were used.
Resveratrol (trans-Resveratrol, manufactured by Cayman Chemical)
Pterostilbene (Pterostilbene, manufactured by Cayman Chemical)
Cell Counting Kit-8 (Dojin Chemical Research Institute, Inc.)
（評価方法）
　上記の乳癌細胞、胃癌細胞、及び大腸癌細胞を９６ウェルプレートに各ウェル当たり細胞数が２，５００個となるように播種した。播種した翌日に、レスベラトロール又はプテロスチルベンを、それぞれ１２．５μＭ、２５μＭ、５０μＭ又は１００μＭとなるように添加した。３日間培養後に、Cell Counting Kit-8を用い、製造元のプロトコールの条件に従って、４５０ｎｍの吸光度を測定することにより、生細胞数を測定した。コントロール（Ｍｏｃｋ）としては、レスベラトロール又はプテロスチルベンを添加しなかったこと以外は、上記同様にして、生細胞数を測定したものを用いた。４５０ｎｍの吸光度測定にはSynergyTM H4 Hybrid Microplate Reader(BioTek社製)を使用した。各群（レスベラトロール群又はプテロスチルベン群）の細胞生存率は、コントロールにおける生細胞数を１００％としたときの値とした。結果を図１に示す。図１（Ａ）～（Ｃ）は、それぞれ乳癌細胞、胃癌細胞、又は大腸癌細胞を用いたときの細胞生存率測定結果を示す。図１中、エラーバーは、標準誤差を示す。 (Evaluation method)
The above-mentioned breast cancer cells, gastric cancer cells and colon cancer cells were seeded in 96-well plates so as to have 2,500 cells per well. The day after seeding, resveratrol or pterostilbene was added to be 12.5 μM, 25 μM, 50 μM or 100 μM, respectively. After 3 days of culture, the number of viable cells was determined by measuring absorbance at 450 nm using Cell Counting Kit-8 according to the conditions of the manufacturer's protocol. As a control (Mock), the one in which the number of viable cells was measured was used in the same manner as described above except that resveratrol or pterostilbene was not added. The absorbance measurement of 450nm using the Synergy TM H4 Hybrid Microplate Reader (BioTek Co., Ltd.). The cell viability of each group (resveratrol group or pterostilbene group) was a value when the number of living cells in the control was 100%. The results are shown in FIG. FIGS. 1 (A) to 1 (C) show the cell viability measurement results when using breast cancer cells, gastric cancer cells, or colon cancer cells, respectively. Error bars in FIG. 1 indicate standard errors.
　図１に示すとおり、レスベラトロール又はプテロスチルベンを添加することにより、いずれの癌細胞においても細胞生存率が低減した（コントロールとの対比）。プテロスチルベンを添加した場合、レスベラトロールを添加した場合と比較して、細胞生存率がより低減された。 As shown in FIG. 1, addition of resveratrol or pterostilbene reduced the cell survival rate in any cancer cells (contrast to control). When pterostilbene was added, cell viability was further reduced compared to when resveratrol was added.
３．癌抑制的マイクロＲＮＡ（ｍｉＲＮＡ）の定量
（細胞）
　癌抑制的マイクロＲＮＡ（ｍｉＲＮＡ）の定量においては、上記の乳癌細胞株(MDA-MB231-luc-D3H2LN)を使用した。 3. Determination of tumor suppressor microRNA (miRNA) (cell)
The above breast cancer cell line (MDA-MB231-luc-D3H2LN) was used for quantification of tumor suppressor microRNA (miRNA).
（試薬）
　試薬及びキットとしては、以下のものを用いた。
レスベラトロール(trans-Resveratrol、Cayman Chemical社製)
プテロスチルベン(Cayman Chemical社製)
miRNeasy（登録商標） Mini Kit（QIAGEN社製）
TaqMan（登録商標） MicroRNA assay（Applied Biosystems社製）
TaqMan（登録商標） MicroRNA Reverse TranscriptionKit（Applied Biosystems社製）
TaqMan（登録商標） Universal PCR Master Mix, NoAmpErase（登録商標） UNG（AppliedBiosystems社製） (reagent)
The following were used as reagents and kits.
Resveratrol (trans-Resveratrol, manufactured by Cayman Chemical)
Pterostilbene (manufactured by Cayman Chemical)
miRNeasy (registered trademark) Mini Kit (manufactured by QIAGEN)
TaqMan (registered trademark) MicroRNA assay (manufactured by Applied Biosystems)
TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems)
TaqMan (registered trademark) Universal PCR Master Mix, NoAmpErase (registered trademark) UNG (manufactured by Applied Biosystems)
（癌抑制的マイクロＲＮＡの定量解析）
　癌抑制的マイクロＲＮＡとして知られているｌｅｔ－７ａ、ｌｅｔ－７ｂ、ｍｉＲ－２６ａ、ｍｉＲ－１０７、ｍｉＲ－１２６、ｍｉＲ－１８５、ｍｉＲ－２００ｃ、ｍｉＲ－１４１、ｍｉＲ－３４ａ及びｍｉＲ－１９３ｂを以下の方法により定量した。
　まず、上記の乳癌細胞に、レスベラトロール又はプテロスチルベンを、それぞれ濃度２５μＭになるように添加した。２日後にmiRNeasy（登録商標） Mini Kit を用いて、製造元推奨のプロトコールに従い、ＲＮＡを抽出した。抽出したＲＮＡは、TaqMan（登録商標） MicroRNA assay とTaqMan（登録商標） MicroRNA Reverse TranscriptionKitとを用いて、それぞれ製造元推奨のプロトコールに従い、相補的ＤＮＡ（ｃＤＮＡ）に変換した。そのｃＤＮＡを鋳型にして、TaqMan（登録商標） Universal PCR Master Mix, NoAmpErase（登録商標） UNG を用いて、StepOnePlusTMReal-Time PCR System（Applied Biosystems）により、ｍｉＲＮＡの定量解析を行った。レスベラトロール又はプテロスチルベンを添加しなかったこと以外は上記同様にして、ｍｉＲＮＡの定量解析を行ったものをコントロール（Ｍｏｃｋ）とした。結果を図２及び３に示す。図２及び３中、エラーバーは、標準誤差を示す。また、図２及び３中、＊を付したデータはｐ＜０．０５である。 (Quantitative analysis of tumor suppressor microRNA)
Let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a and miR-193b, which are known as tumor suppressor microRNAs It quantified by the following method.
First, resveratrol or pterostilbene was added to the above breast cancer cells to a concentration of 25 μM. Two days later, using the miRNeasy® Mini Kit, RNA was extracted according to the manufacturer's recommended protocol. The extracted RNA was converted into complementary DNA (cDNA) using TaqMan® MicroRNA assay and TaqMan® MicroRNA Reverse Transcription Kit according to the protocol recommended by the manufacturer. The cDNA as a template, using the TaqMan (TM) Universal PCR Master Mix, NoAmpErase (TM) UNG, by StepOnePlus TM Real-Time PCR System ( Applied Biosystems), was quantitative analysis of miRNA. The same as in the above except that resveratrol or pterostilbene was not added, the one subjected to quantitative analysis of miRNA was used as a control (Mock). The results are shown in FIGS. Error bars in FIGS. 2 and 3 indicate standard errors. Moreover, the data which attached * in FIG. 2 and 3 are p <0.05.
（評価結果）
　図２及び３に示すとおり、レスベラトロール又はプテロスチルベンを添加することにより、コントロールと比較して、癌抑制的ｍｉＲＮＡの発現が亢進していることが示された。 (Evaluation results)
As shown in FIGS. 2 and 3, it was shown that the addition of resveratrol or pterostilbene enhances the expression of tumor suppressor miRNA as compared to the control.
〔試験例２：腹腔内投与による抗腫瘍効果の評価〕
４－１．プテロスチルベンの腹腔内投与実験
（実験動物）
　実験動物として、５週齢のメスのヌードマウス（BALB/cAJc1-nu/nu）（日本クレア社）を用いた。 [Test Example 2: Evaluation of antitumor effect by intraperitoneal administration]
4-1. Intraperitoneal administration experiment of pterostilbene (experimental animals)
Five-week-old female nude mice (BALB / cAJc1-nu / nu) (CLEA Japan, Inc.) were used as experimental animals.
（試薬）
　試薬としては、以下のものを用いた。
プテロスチルベン（Cayman Chemical社製）
ジメチルスルホキシド（DMSO）（Merck社製）
ECM Gel from Engelbreth-Holm-Swam murine sarcoma（Invitrogen社製）
エスカイン(登録商標)吸入麻酔液（Pfizer社製）
Beetle Luciferin, Potassium Salt（Promega社製）
プロピレングリコール（Sigma-Aldrich社製） (reagent)
The following were used as reagents.
Pterostilbene (manufactured by Cayman Chemical)
Dimethyl sulfoxide (DMSO) (Merck)
ECM Gel from Engelbreth-Holm-Swam murine sarcoma (Invitrogen)
Escaine (R) Inhalation Anesthesia (Pfizer)
Beetle Luciferin, Potassium Salt (Promega)
Propylene glycol (Sigma-Aldrich)
（飼育方法）
　温度２０～２５℃、湿度４０～６０％の条件下に保持された環境下で、上記の実験動物を飼育した。 (How to breed)
The experimental animals described above were reared under an environment maintained at a temperature of 20-25 ° C. and a humidity of 40-60%.
（試験方法）
　５週齢のメスのヌードマウス（BALB/cAJc1-nu/nu）を、実験開始前に１週間ほど環境に順応させた。マウスに移植する癌細胞としては、MM-231-luc-D3H2LNを用いた。この癌細胞をリン酸緩衝生理食塩水（PBS）に懸濁し、得られた懸濁液とマトリゲル（ECM Gel from Engelbreth-Holm-Swammurine sarcoma）とを１：１で混ぜ合わせて、移植用の懸濁液（細胞数：１×１０７ｃｅｌｌ／ｍｌ）を準備した。 (Test method)
Five-week-old female nude mice (BALB / cAJc1-nu / nu) were acclimated to the environment for about one week before the start of the experiment. MM-231-luc-D3H2LN was used as a cancer cell transplanted to a mouse. The cancer cells are suspended in phosphate buffered saline (PBS), and the obtained suspension and Matrigel (ECM Gel from Engelbreth-Holm-Swammurine sarcoma) are mixed 1: 1 and suspended for transplantation. A suspension (cell number: 1 × 10 7 cells / ml) was prepared.
　本試験例では、イソフルラン（エスカイン（登録商標）吸入麻酔液）による麻酔下にて、マウスの乳腺に１×１０６ｃｅｌｌｓの癌細胞を移植（上記の移植用の懸濁液（細胞数：１×１０７ｃｅｌｌ／ｍｌ）を１００μｌ導入）した。癌細胞が移植されていることの確認は、上記方法で移植した後にしばらく放置してから、１５０ｍｇ／ｋｇの量で、１５ｍｇ／ｍｌルシフェリン溶液（Beetle Luciferin, Potassium Salt、Promega 社）を腹腔内に投与し、約１０分後にIVIS Spectrum（Perkin Elmer社）で移植された細胞由来の発光（ｌｕｃｉｆｅｒａｓｅ活性）を測定することにより行った。移植後２日間飼育した後に、再度細胞由来の発光を測定した。当該測定結果に基づいて、試験用のマウスを１群当たり５匹として、以下の（ｉ）～（ｉｉｉ）の３群に群分けした。
（ｉ）コントロール群（溶媒投与群、投与量：０ｍｇ／ｋｇ／ｄａｙ）
（ｉｉ）プテロスチルベン群Ａ（投与量：１２．５ｍｇ／ｋｇ／ｄａｙ）
（ｉｉｉ）プテロスチルベン群Ｂ（投与量：２５ｍｇ／ｋｇ／ｄａｙ） In this test example, 1 × 10 6 cells of cancer cells were transplanted into the mammary gland of a mouse under anesthesia with isoflurane (Escaine® Inhalation Anesthesia) (suspension for transplantation (cell count: 1 above) 100 μl of × 10 7 cells / ml) was introduced). After confirming that cancer cells have been transplanted, leave for a while after transplantation according to the above-mentioned method, and then at a dose of 150 mg / kg, 15 mg / ml luciferin solution (Beetle Luciferin, Potassium Salt, Promega) in the abdominal cavity The measurement was carried out by measuring luminescence (luciferase activity) derived from cells implanted with IVIS Spectrum (Perkin Elmer) after about 10 minutes. After breeding for 2 days after transplantation, the luminescence from cells was again measured. Based on the measurement results, the test mice were divided into three groups of (i) to (iii), with five mice per group.
(I) Control group (solvent administration group, dosage: 0 mg / kg / day)
(Ii) Pterostilbene group A (dose: 12.5 mg / kg / day)
(Iii) Pterostilbene group B (dose: 25 mg / kg / day)
　（ｉ）～（ｉｉｉ）の各群のマウスに対し、腹腔内投与を行った。腹腔内投与は、上記マウスに癌細胞を移植した日の２日後（投与開始日）から起算して、２１日間継続して行った。なお、溶媒としては、ＤＭＳＯ及びプロピレングリコールを含有するＰＢＳ溶液（ＤＭＳＯの含有量：１０％（ｖ／ｖ）、プロピレングリコールの含有量：１０％（ｖ／ｖ））を用いた。プテロスチルベンの投与には、上記溶媒にプテロスチルベンを溶解したものを用いた。投与量は、プテロスチルベンの量を基準とする値である。 Intraperitoneal administration was performed on the mice of each group of (i) to (iii). Intraperitoneal administration was continuously performed for 21 days starting from 2 days after the day when the cancer cells were transplanted into the above-mentioned mice (the administration start day). As a solvent, a PBS solution containing DMSO and propylene glycol (DMSO content: 10% (v / v), propylene glycol content: 10% (v / v)) was used. For administration of pterostilbene, pterostilbene dissolved in the above-mentioned solvent was used. The dose is a value based on the amount of pterostilbene.
　各群のマウスにおいて、体重は適時計測し、一週間おきにＩＶＩＳ　ｓｐｅｃｔｒｕｍを用いて、発光を計時観察した。移植した細胞由来の発光の測定結果を図４に示す。図４（Ａ）に、投与開始から２１日経過時点の腫瘍発光の結果を示す。図４（Ｂ）に発光量の経時観察結果を示す。図６に体重の測定結果を示す。 In each group of mice, body weight was measured appropriately, and luminescence was clocked and observed using IVIS spectrum every other week. The measurement results of luminescence from the transplanted cells are shown in FIG. FIG. 4 (A) shows the results of tumor luminescence at 21 days after the start of administration. FIG. 4 (B) shows the observation result of the light emission amount over time. The measurement result of a body weight is shown in FIG.
　投与終了後（投与開始日から２１日経過後）には、イソフルラン麻酔下にて腫瘍を摘出し、安楽死を行った。図５に、プテロスチルベンの腹腔内投与が腫瘍のサイズに与える影響を示す。図５（Ａ）に摘出した腫瘍片を示し、図５（Ｂ）に、摘出した腫瘍の重量を示す。投与終了後に摘出した腫瘍は、後述するｍｉＲＮＡ発現解析に用いた。 After the administration was completed (21 days after the administration start date), the tumor was excised under isoflurane anesthesia and euthanasia was performed. Figure 5 shows the effect of intraperitoneal administration of pterostilbene on tumor size. The removed tumor pieces are shown in FIG. 5 (A), and the weight of the removed tumor is shown in FIG. 5 (B). The tumor removed after the completion of administration was used for miRNA expression analysis described later.
（評価結果）
　図４及び５に示すとおり、プテロスチルベンを腹腔内投与した群では、コントロール群と比べて、腫瘍の増大が抑制された。プテロスチルベンの用量依存的に腫瘍の増大が抑制されることが示された。なお、プテロスチルベンの腹腔内投与は、マウスの体重に大きな影響を与えないことが示された（図６参照）。 (Evaluation results)
As shown in FIGS. 4 and 5, in the group administered intraperitoneally with pterostilbene, the increase in tumor was suppressed as compared to the control group. It was shown that the tumor growth was suppressed in a dose-dependent manner of pterostilbene. Incidentally, it was shown that intraperitoneal administration of pterostilbene does not significantly affect the weight of mice (see FIG. 6).
４－２．ヒト由来の腫瘍中の癌抑制的ｍｉＲＮＡ発現解析
　上記ヌードマウスに異種移植して形成された腫瘍片組織を用いて、癌抑制的ｍｉＲＮＡの発現解析を行った。癌抑制的ｍｉＲＮＡであるｍｉＲ－２００ｃ及びｍｉＲ－１４１の発現量を測定した。 4-2. Tumor-suppressing miRNA expression analysis in human-derived tumors The expression analysis of tumor-suppressing miRNA was carried out using the tumor piece tissue formed by xenotransplantation in the above-mentioned nude mice. The expression levels of miR-200c and miR-141, which are tumor suppressor miRNAs, were measured.
（試薬、キット及び実験器具）
　試薬、キット及び実験器具としては、以下のものを用いた。
３ｍｌ破砕チューブ（安井器械株式会社製）
メタルコーン（安井器械株式会社製）
miRNeasy（登録商標） Mini Kit（QIAGEN社製）
TaqMan（登録商標） MicroRNA assay（Applied Biosystems社製）
TaqMan（登録商標） MicroRNA Reverse TranscriptionKit（Applied Biosystems社製）
TaqMan（登録商標） Universal PCR Master Mix, NoAmpErase（登録商標） UNG（AppliedBiosystems社製） (Reagent, kit and laboratory equipment)
The following were used as reagents, kits and experimental instruments.
3 ml crushing tube (made by Yasui Instruments Co., Ltd.)
Metal cone (made by Yasui Kikai Co., Ltd.)
miRNeasy (registered trademark) Mini Kit (manufactured by QIAGEN)
TaqMan (registered trademark) MicroRNA assay (manufactured by Applied Biosystems)
TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems)
TaqMan (registered trademark) Universal PCR Master Mix, NoAmpErase (registered trademark) UNG (manufactured by Applied Biosystems)
（試験方法）
　上記腫瘍片組織を約３ｍｍ角の大きさに切り取り、３ｍｌ破砕チューブにメタルコーンとともに入れた。液体窒素で冷却しながら、マルチビーズショッカー（登録商標）（安井器械株式会社）で破砕した。その後はmiRNeasy（登録商標） Mini Kitを用いて、製造元推奨のプロトコールでＲＮＡを抽出した。得られたＲＮＡは、TaqMan（登録商標） MicroRNA assay とTaqMan（登録商標） MicroRNA Reverse TranscriptionKitとを用いて、製造元推奨のプロトコールに従い相補的ＤＮＡ（ｃＤＮＡ）に変換した。ｃＤＮＡを鋳型にして、TaqMan（登録商標） Universal PCR Master Mix, No AmpErase（登録商標）UNGを用いて、StepOnePlusTM Real-Time PCR System（Applied Biosystems社）により、ｍｉＲＮＡの定量解析を行った。 (Test method)
The tumor piece tissue was cut into a size of about 3 mm and placed in a 3 ml crushing tube with a metal cone. While cooling with liquid nitrogen, it was crushed with Multibeads Shocker (registered trademark) (Yasui Kikai Co., Ltd.). After that, RNA was extracted using the miRNeasy® Mini Kit according to the manufacturer's recommended protocol. The obtained RNA was converted to a complementary DNA (cDNA) using TaqMan® MicroRNA assay and TaqMan® MicroRNA Reverse Transcription Kit according to the protocol recommended by the manufacturer. cDNA as a template, TaqMan (R) Universal PCR Master Mix, using a No AmpErase (TM) UNG, by StepOnePlus TM Real-Time PCR System ( Applied Biosystems , Inc.), was quantified analysis of miRNA.
　癌抑制的ｍｉＲＮＡの結果を図７に示す。ＲＮＡの抽出は、図７（Ａ）中の丸で示したものを用いた。図７（Ｂ）は、それぞれｍｉＲ－２００ｃ及びｍｉＲ－１４１の発現量の測定結果を示す。 The results of tumor suppressor miRNAs are shown in FIG. Extraction of RNA used what was shown by the circle in FIG. 7 (A). FIG. 7 (B) shows the measurement results of the expression levels of miR-200c and miR-141, respectively.
（評価結果）
　図７（Ｂ）に示すとおり、プテロスチルベン群（腹腔内投与）では、コントロール群と比べて、癌抑制的ｍｉＲＮＡ（ｍｉＲ－２００ｃ及びｍｉＲ－１４１）の発現が亢進した。 (Evaluation results)
As shown in FIG. 7 (B), in the pterostilbene group (intraperitoneal administration), the expression of tumor suppressor miRNAs (miR-200c and miR-141) was enhanced as compared to the control group.
〔試験例３：経口投与による抗腫瘍効果の評価〕
５．プテロスチルベンの経口投与実験
（試験用粉末飼料）
　試験用の粉末飼料の調製には、以下の試薬を用いた。
カゼイン（オリエンタル酵母株式会社）
ＤＬ－メチオニン（和光純薬工業株式会社）
β－コーン澱粉（オリエンタル酵母株式会社）
スクロース（ＫＨ１　精上白糖）（和田製糖株式会社）
セルロースパウダー（オリエンタル酵母株式会社）
コーン油（胚芽の恵みコーン油）（株式会社Ｊ－オイルミルズ）
ミネラルミックス（ＡＩＮ－９３Ｇ　ミネラル混合）（オリエンタル酵母株式会社）
ビタミンミックス（ＡＩＮ－９３　ビタミン混合）（オリエンタル酵母株式会社）
重酒石酸コリン（和光純薬工業株式会社）（Ｌｏｔ．Ｎｏ．：ＰＤＱ０５７５、ＣＴＫ０２４１）
９０％プテロスチルベン（シルビトール）（サビンサジャパンコーポレーション製） [Test Example 3: Evaluation of antitumor effect by oral administration]
5. Oral administration experiment of pterostilbene (powder feed for test)
The following reagents were used for the preparation of the powder feed for the test.
Casein (Oriental Yeast Co., Ltd.)
DL-Methionine (Wako Pure Chemical Industries, Ltd.)
β-corn starch (Oriental Yeast Co., Ltd.)
Sucrose (KH1 refined white sugar) (Wada Sugar Co., Ltd.)
Cellulose powder (Oriental Yeast Co., Ltd.)
Corn oil (Gemme corn oil) (J-Oil Mills Co., Ltd.)
Mineral mix (AIN-93G mineral mix) (Oriental Yeast Co., Ltd.)
Vitamin Mix (AIN-93 Vitamin Blend) (Oriental Yeast Co., Ltd.)
Choline bitartrate (Wako Pure Chemical Industries, Ltd.) (Lot. No .: PDQ0575, CTK0241)
90% pterostilbene (silbitol) (manufactured by Sabinsa Japan Corporation)
（プテロスチルベン含有粉末飼料の調製）
　試験用の飼料（プテロスチルベン含有粉末飼料）は、以下の方法により調製した。すなわち、まず、コーン油を５０ｇ秤量し、加温して７０℃とした。加温したコーン油にプテロスチルベンを添加して溶解させた。コーン油及びプテロスチルベン以外の試薬（その他の試薬）は乳鉢ですり潰し、混合した。その他の試薬の混合物に、プテロスチルベンを溶解させたコーン油を添加し、乳鉢で十分にすり潰して混合した。その後、攪拌機で約３０分間更に混合して、試験用の飼料を調製した。試験用の飼料に含まれる各種成分の組成は、表１に示すとおりである。試験用の飼料はビニール袋に入れ、株式会社コーガアイソトープでガンマ線滅菌を行った後、後述する動物実験に用いた。また、表１に示す組成の馴化食（プテロスチルベンの含有量：０質量％）を用意した。 (Preparation of Pterostilbene-Containing Powdered Feed)
The test feed (pterostilbene-containing powder feed) was prepared by the following method. That is, first, 50 g of corn oil was weighed and heated to 70 ° C. Pterostilbene was added to dissolve in heated corn oil. Reagents other than corn oil and pterostilbene (other reagents) were ground and mixed in a mortar. To the mixture of the other reagents, corn oil in which pterostilbene was dissolved was added, thoroughly ground in a mortar and mixed. Thereafter, it was further mixed for about 30 minutes with a stirrer to prepare a test feed. The compositions of various components contained in the test feed are as shown in Table 1. The feed for the test was placed in a plastic bag, and after gamma sterilization with Koga Isotope Co., Ltd., it was used for animal experiments described later. Moreover, the acclimatized food (content of pterostilbene: 0 mass%) of the composition shown in Table 1 was prepared.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（実験動物）
　実験動物としては、５週齢のメスのヌードマウス（BALB/c-nu/nu, CAnN.Cg-Foxn1<nu>/CrlCjlj）（日本チャールズリバー社）を用いた。 (Experimental animals)
Five-week-old female nude mice (BALB / c-nu / nu, CAnN.Cg-Foxn1 <nu> / CrlCjlj) (Charles River Japan, Inc.) were used as experimental animals.
（試薬）
　試薬としては、以下のものを用いた。
ECM Gel from Engelbreth-Holm-Swam murine sarcoma（Invitrogen社）
エスカイン（登録商標）吸入麻酔液（Pfizer社）
Beetle Luciferin, Potassium Salt（Promega社）
プロピレングリコール（Sigma-Aldrich社） (reagent)
The following were used as reagents.
ECM Gel from Engelbreth-Holm-Swam murine sarcoma (Invitrogen)
Escaine (R) Inhalation Anesthetic Fluid (Pfizer)
Beetle Luciferin, Potassium Salt (Promega)
Propylene glycol (Sigma-Aldrich)
（飼育方法）
　温度２０～２５℃、湿度４０～６０％の条件下に保持された環境下で、上記の実験動物を飼育した。給餌器としては、吊り下げ型の給餌器を用いた。 (How to breed)
The experimental animals described above were reared under an environment maintained at a temperature of 20-25 ° C. and a humidity of 40-60%. A hanging feeder was used as the feeder.
（試験群）
　プテロスチルベン含有粉末飼料の経口投与による癌細胞への影響の検討として、１群当たり８匹又は１０匹として、（ｉ）コントロール群（０％（ｗ／ｗ）プテロスチルベン群、ｎ＝１８）、及び（ｉｉ）０．２５％（ｗ／ｗ）プテロスチルベン群（ｎ＝１８）の２群の試験区を設定した。 (Test group)
(I) Control group (0% (w / w) pterostilbene group, n = 18), with 8 or 10 animals per group, for examining the effect on cancer cells by oral administration of powder feed containing pterostilbene And (ii) two test zones of 0.25% (w / w) pterostilbene group (n = 18) were set up.
（試験方法及び評価結果）
　まず、５週齢のメスのヌードマウスを、実験開始前に１週間ほど環境に順応させた。
　プテロスチルベン含有粉末飼料の経口投与による癌細胞への影響の検討として、まず、コントロール食（馴化食）を吊り下げ型給餌器で約１週間与えた後に、（ｉ）コントロール群、及び（ｉｉ）０．２５％（ｗ／ｗ）プテロスチルベン群の２群に群分けした。コントロール群では、馴化食を経口投与し、プテロスチルベン群では、プテロスチルベン含有粉末飼料（プテロスチルベンの含有量：０．２５質量％）を経口投与した。 (Test method and evaluation results)
First, 5-week-old female nude mice were acclimated to the environment for about a week before the start of the experiment.
As an examination of the effect on cancer cells by orally administering pterostilbene-containing powder feed, first, after giving a control food (conditioned food) with a suspension feeder for about 1 week, (i) control group, and (ii) The 0.25% (w / w) pterostilbene group was divided into 2 groups. In the control group, the conditioned food was orally administered, and in the pterostilbene group, pterostilbene-containing powder feed (pterostilbene content: 0.25% by mass) was orally administered.
　群分けしてから１週間経過時点で、各群のマウスに対して、癌細胞の移植実験を行った。癌細胞の移植実験では、まず、移植する癌細胞（MM-231-luc-D3H2LN）をリン酸緩衝生理食塩水（PBS）に懸濁し、マトリゲル（ECM Gel from Engelbreth-Holm-Swammurine sarcoma）と１：１の比率で混ぜ合わせて、移植用の懸濁液（細胞数：１×１０５ｃｅｌｌ／ｍｌ）を準備した。次いで、イソフルラン（エスカイン（登録商標）吸入麻酔液）による麻酔下にて、マウスの乳腺に１×１０４ｃｅｌｌｓの癌細胞を移植（上記の移植用の懸濁液（細胞数：１×１０５ｃｅｌｌ／ｍｌ）を１００μｌ導入）した。癌細胞が移植されていることの確認は、移植後にしばらく放置し、１５０ｍｇ／ｋｇの量で、１５ｍｇ／ｍｌルシフェリン溶液(Beetle Luciferin, Potassium Salt、Promega社)を腹腔内に投与し、約１０分後にＩＶＩＳ　Ｓｐｅｃｔｒｕｍ（Perkin Elmer社）で細胞由来の発光（ｌｕｃｉｆｅｒａｓｅ活性）を測定することにより行った。試験用の飼料及びマウスの状態は、毎日適切に管理した。マウスの体重は適時計測した。投与開始日（０日目）は、癌細胞（腫瘍）を移植した日とした。そして、投与開始日から一週間おきにＩＶＩＳ　ｓｐｅｃｔｒｕｍにて発光を計時観察した。腫瘍発光の測定結果を図８に示す。図８（Ａ）に、投与開始から２１日経過時点の腫瘍発光の結果を示す。図８（Ｂ）に、発光量の経時観察結果を示す。各群におけるマウスの体重の計測結果を図９に示す。 One week after grouping, mice for each group were subjected to a cancer cell transplantation experiment. In a cancer cell transplantation experiment, first, the cancer cells to be transplanted (MM-231-luc-D3H2LN) are suspended in phosphate buffered saline (PBS), and Matrigel (ECM Gel from Engelbreth-Holm-Swammurine sarcoma) and 1 are suspended. The mixture was mixed at a ratio of 1: 1 to prepare a suspension for transplantation (number of cells: 1 × 10 5 cells / ml). Then, under anesthesia with isoflurane (Escaine® inhalation anesthetic solution), transplant cancer cells of 1 × 10 4 cells to the mammary gland of a mouse (suspension for transplantation (cell number: 1 × 10 5) 100 μl of cells / ml) was introduced). For confirmation that cancer cells have been transplanted, leave for a while after transplantation, and administer 15 mg / ml luciferin solution (Beetle Luciferin, Potassium Salt, Promega) intraperitoneally at a dose of 150 mg / kg for about 10 minutes. It was carried out by measuring the luminescence (luciferase activity) derived from cells later with IVIS Spectrum (Perkin Elmer). The condition of the test feed and mice was properly managed daily. The weight of the mice was measured at appropriate times. The administration start day (day 0) was the day on which cancer cells (tumor) were transplanted. Then, the luminescence was observed with IVIS spectrum every other week from the administration start date. The measurement results of tumor luminescence are shown in FIG. FIG. 8 (A) shows the results of tumor luminescence 21 days after the start of administration. FIG. 8 (B) shows the observation result of the light emission amount over time. The measurement results of the weight of the mice in each group are shown in FIG.
　投与終了後には、イソフルラン麻酔下にて腫瘍を摘出し、安楽死を行った。各群（コントロール群又はプテロスチルベン群）における摘出した腫瘍のサイズ（重量）の測定結果を図１０に示す。なお、本試験例は独立した試験を２回繰り返し行ったものである。 After completion of administration, the tumor was removed under isoflurane anesthesia and euthanasia was performed. The measurement results of the size (weight) of the excised tumor in each group (control group or pterostilbene group) are shown in FIG. This test example is an independent test repeated twice.
　図１０中、エラーバーは、標準誤差を示し、図１０中、＊を付したデータは、Ｐ＜０．０５であり、＊＊を付したデータは、Ｐ＜０．０１である。 In FIG. 10, error bars indicate standard errors, and in FIG. 10, data with * is P <0.05, and data with ** is P <0.01.
（評価結果）
　図８及び１０に示すとおり、プテロスチルベンを経口投与した群（プテロスチルベン群）では、コントロール群と比べて、腫瘍の増大が抑制された。なお、プテロスチルベンの経口投与は、マウスの体重に大きな影響を与えないことが示された（図９参照）。 (Evaluation results)
As shown in FIGS. 8 and 10, in the group orally administered with pterostilbene (pterostilbene group), the tumor growth was suppressed as compared to the control group. In addition, it was shown that oral administration of pterostilbene did not significantly affect the weight of mice (see FIG. 9).

Claims (11)

  1. 　プテロスチルベンを含有する、
    　経口投与用抗腫瘍剤。     Contains pterostilbene,
    Antitumor agent for oral administration.
  2. 　請求項１に記載の抗腫瘍剤において、
    　油脂を更に含有する、
    　抗腫瘍剤。     In the antitumor agent according to claim 1,
    Further contains fats and oils,
    Antineoplastic agent.
  3. 　請求項２に記載の抗腫瘍剤において、
    　前記油脂が、植物油である、
    　抗腫瘍剤。     In the antitumor agent according to claim 2,
    The oil and fat is a vegetable oil,
    Antineoplastic agent.
  4. 　請求項１～３のいずれか一項に記載の抗腫瘍剤を含む、
    　癌予防用飲食品。     An antitumor agent according to any one of claims 1 to 3 comprising
    Food and drink for cancer prevention.
  5. 　プテロスチルベンを含有する、
    　癌抑制的マイクロＲＮＡの発現亢進剤。     Contains pterostilbene,
    Tumor suppressor microRNA expression enhancer.
  6. 　請求項５に記載の発現亢進剤において、
    　経口投与用である、発現亢進剤。     In the expression enhancer according to claim 5,
    An expression enhancer for oral administration.
  7. 　請求項５又は６に記載の発現亢進剤において、
    　前記癌抑制的マイクロＲＮＡが、ｌｅｔ－７ａ、ｌｅｔ－７ｂ、ｍｉＲ－２６ａ、ｍｉＲ－１０７、ｍｉＲ－１２６、ｍｉＲ－１８５、ｍｉＲ－２００ｃ、ｍｉＲ－１４１、ｍｉＲ－３４ａ及びｍｉＲ－１９３ｂからなる群より選択される少なくとも１種を含む、発現亢進剤。     In the expression enhancer according to claim 5 or 6,
    A group consisting of let-7a, let-7b, miR-26a, miR-107, miR-126, miR-185, miR-200c, miR-141, miR-34a and miR-193b An expression enhancer comprising at least one selected from the group consisting of
  8. 　請求項５～７のいずれか一項に記載の発現亢進剤において、
    　癌の治療又は予防のために用いられる、発現亢進剤。     In the expression enhancer according to any one of claims 5 to 7,
    An expression enhancer used for the treatment or prevention of cancer.
  9. 　プテロスチルベンを、それを必要とする対象に経口投与することを含む、癌を治療又は予防する方法。 A method of treating or preventing cancer comprising orally administering pterostilbene to a subject in need thereof.
  10. 　経口投与用抗腫瘍剤の製造のための、プテロスチルベンの使用。 Use of pterostilbene for the preparation of an antitumor agent for oral administration.
  11. 　経口投与による癌の治療又は予防に使用するための、プテロスチルベン。 Pterostilbene for use in the treatment or prevention of cancer by oral administration.
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