JP2019077657A - SIMULTANEOUS INHIBITOR OF mTOR SIGNAL TRANSDUCTION PATHWAY AND MAPK SIGNAL TRANSDUCTION PATHWAY WHICH COMPRISE RESVERATROL DIMER - Google Patents

Abstract

To provide simultaneous inhibitor of mTOR signal transduction pathway and MAPK signal transduction pathway, and to provide prophylactic or therapeutic agents for cancer which comprise the inhibitor.SOLUTION: The present invention provides a simultaneous inhibitor of mTOR signal transduction pathway and MAPK signal transduction pathway which contain a resveratrol dimer as an active ingredient having a structure represented by the following general formula (I): [in the formula (I), Rand Rare each independently selected from the group consisting of H, OH, and OCH; Ris selected from the group consisting of H, OH, OCH, and OGlc (Glc is a glucose); and Ris selected from the group consisting of H, CH, and Glc].SELECTED DRAWING: None

Description

  The present invention relates to a preventive or therapeutic agent for cancer, and more particularly to a preventive and therapeutic agent for acute myeloid leukemia (AML).

  Acute myelogenous leukemia (AML) is a rapidly progressive clonal disorder of hematopoietic progenitor cells. It is characterized by clonal aberrant proliferation of hematopoietic progenitor cells and the accumulation of immature blasts in the bone marrow, which ultimately prevents the production of normal cells. AML is the more common acute leukemia in adults, and globally, among the acute leukemias, the type with the highest annual mortality. Under 40 years of age, cure is achieved in about 40%, but the prognosis for patients older than 60 years is poor. Many AML patients reach complete remission with combination chemotherapy, but about half of them relapse within 3 years. Important factors that influence the prognosis of AML patients include age and general condition, but the most important prognostic factor is chromosome karyotype. Chromosomal karyotypes in AML patients are currently classified into three groups: good, middle and poor. AML having multiple chromosomal abnormalities is generally referred to as a complex karyotype and is included in the poor prognosis group. Among them, monosomal karyotypes (AML-MK), defined as “two autosomal monosomy or one monosomy with at least one additional chromosomal structural abnormality”, are 4 of AML patients with other complex chromosome karyotypes. It is treated as a special group in AML because its overall survival rate is 4% and its 4-year overall survival rate is disastrous, with an overall survival rate of 21% a year.

  The pathogenesis of AML is complex. It may develop as a result of accumulation of genetic abnormalities in hematopoietic stem cells with aging. Important molecular abnormalities involved in leukemogenesis include a decrease in transcription factor activity that regulates stem cell differentiation, and abnormal activation of signal transduction pathways that control cell proliferation. For example, aberrant activation of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) / AKT pathways in mammals is observed in the majority of AML patient-derived leukemia cells. In addition, constitutive activation and high expression of extracellular signal-regulated kinase 1/2 (ERK 1/2) are also detected in most AML cells. Therefore, PI3K / Akt / mTOR pathway and Ras / Raf / MEK / ERK pathway are considered as good therapeutic targets for AML (see Non-patent Document 1).

  However, it is not easy to suppress these pathways, and all past approaches have been ineffective. This is because multiple signaling pathways are activated in AML cells, and inhibition of one signaling pathway often results in activation of the other signaling pathway (see Non-Patent Documents 1 and 2). Therefore, in order to effectively kill AML cells, development of an agent that simultaneously inhibits multiple signal transduction pathways is required (see Non-patent Document 2).

  Gnetin C (resveratrol dimer) is a stilbene isolated from the seeds of Melinjo, an edible fruit native to Southeast Asia. In addition to Gnetin C, ethanol extract of Merinjo seeds containing Gnetin L, Gnemonoside A, Gnemonoside C and Gnemonoside D has free radical scavenger activity (see non-patent document 3), immunomodulatory effect (see non-patent document 4), It has been reported to have antibacterial activity and anti-angiogenic properties (see Non-patent Document 5). In the southwestern countries, Gnetin C is considered to be a highly safe natural organic substance because melinjo leaves rich in Gnetin C, fruits, seeds, etc. have been ingested for a long time (see Non-Patent Document 3) .

Ricciardi MR. J Mol Med (Berl) 2012; 90: 1133-44 McCubrey JA. Et al., Oncotarget 2012; 3: 1068-111 Kato E. et al., J Agric Food Chem 2009; 57: 2544-9 Kato H. et al., Planta Med 2011; 77: 1027-34 Kunimasa K. et al., MoI Nutr Food Res 2011; 55: 1730-4

  An object of the present invention is to provide a simultaneous inhibitor of mTOR signaling pathway and MAPK signaling pathway, and a preventive or therapeutic agent for cancer containing the inhibitor.

  We screen for antitumor compounds that induce apoptosis in leukemia cells of MK-positive patients, and resveratrol dimers such as Gnetin C, a natural stilbene, strongly injure MK-positive AML cells I found out. Examination of its mechanism of action revealed that Gnetin C simultaneously inhibits both the ERK 1/2 MAPK signaling pathway and the AKT / mTOR signaling pathway, two signals essential for leukemia cell survival. Became. Furthermore, the combination of Gnetin C and low concentrations of chemotherapeutic drugs showed a synergistic anti-tumor effect on AML cells. In addition, in an AML model of immunodeficient mice using a MK-positive AML cell line, Gnetin C was found to not only inhibit the onset of leukemia but also kill the leukemia cells and improve the survival rate. As described above, it became clear that Gnetin C exerts an anti-leukemic action by the unique action of blocking two different signal pathways, and so we came to apply for this patent.

[式（I)中、R₁およびR₃は、それぞれ独立にH、OHおよびOCH₃からなる群から選択され、R₂はH、OH、OCH₃およびOGlc（Glcはグルコース）からなる群から選択され、R₄はH、CH₃およびGlcからなる群から選択される]。
[２] レスベラトロール二量体がGnetin C、Gnetin L、Gnemonoside A、Gnemonoside CおよびGnemonoside Dからなる群から選択される[１]のmTORシグナル伝達経路およびMAPKシグナル伝達経路の同時阻害剤。
[３] レスベラトロール二量体がGnetin Cである、[２]のmTORシグナル伝達経路およびMAPKシグナル伝達経路の同時阻害剤。
[４] [１]〜[３]のいずれかの阻害剤を含む、がんの予防または治療剤。
[５] がんがAMLである、[４]のがんの予防または治療剤。
[６] 経口投与用である、[４]または[５]のがんの予防または治療剤。
[７] [４]〜[６]のいずれかのがんの予防または治療剤と、該治療剤以外のがんの化学療法剤との組合せ製剤。
[８] レスベラトロール二量体を含む、レスベラトロール二量体以外の化学療法剤と併用される医薬組成物。
[９] [１]〜[３]のいずれかの阻害剤を含む、がんの予防または治療用の飲食物。
[１０] がんがAMLである、[９]のがんの予防または治療用の飲食物。
[１１] 核型が、47,X-Y,del（1）（q32q42）,del（11）（q13q21）,+mar[27]monosomal Karyotypeを有するヒトAML細胞株。 The present invention is as follows.
[1] Simultaneous inhibitors of mTOR signaling pathway and MAPK signaling pathway comprising resveratrol dimer having a structure represented by the following general formula (I) as an active ingredient:

[In formula (I), R ₁ and R ₃ are each independently selected from the group consisting of H, OH and OCH ₃ , and R ₂ is from the group consisting of H, OH, OCH ₃ and OGlc (Glc is glucose) Selected, R ₄ is selected from the group consisting of H, CH ₃ and Glc].
[2] A simultaneous inhibitor of mTOR and MAPK signaling pathways of [1], wherein the resveratrol dimer is selected from the group consisting of Gnetin C, Gnetin L, Gnemonoside A, Gnemonoside C and Gnemonoside D.
[3] A co-inhibitor of the mTOR and MAPK signaling pathways of [2], wherein the resveratrol dimer is Gnetin C.
[4] An agent for the prophylaxis or treatment of cancer, which comprises the inhibitor of any one of [1] to [3].
[5] The agent for the prophylaxis or treatment of cancer of [4], wherein the cancer is AML.
[6] The agent for preventing or treating cancer according to [4] or [5], which is for oral administration.
[7] A combined preparation of the cancer preventive or therapeutic agent of any of [4] to [6] and a cancer chemotherapeutic agent other than the therapeutic agent.
[8] A pharmaceutical composition for use in combination with a chemotherapeutic agent other than resveratrol dimer, which comprises resveratrol dimer.
[9] A food or drink for preventing or treating cancer, which comprises the inhibitor of any one of [1] to [3].
[10] Food or drink for the prevention or treatment of the cancer of [9], wherein the cancer is AML.
[11] A human AML cell line having a karyotype of 47, XY, del (1) (q32q42), del (11) (q13q21), + mar [27] monosomal Karyotype.

  Resveratrol dimers such as Gnetin C can simultaneously inhibit both mTOR and MAPK signaling pathways activated in cancer cells. As a result, it induces apoptosis in cancer cells and damages cancer cells. Resveratrol dimers such as Gnetin C can be used as agents for preventing or treating cancer.

FIG. 5 shows the antitumor effect of Gnetin C and various drugs on AML cells. FIG. 2 shows the antitumor effect of Gnetin C on AML cells. It is a figure which shows the induction | stimulation effect of the apoptosis of the leukemia cell strain by Gnetin C. FIG. FIG. 2 shows annexin V staining, Giemsa staining and Mitocapture reagent staining of leukemia cell lines treated with Gnetin C. It is a figure which shows the induction | stimulation effect of the apoptosis of the primary leukemia cell by GnetinC. It is a figure which shows the induction | guidance | derivation effect of apoptosis of THP-1, MV4 and HL-60 cell by GnetinC. It is a figure which shows the induction | stimulation effect of the apoptosis of OUN1, KH88, and K562 cell by Gnetin C. FIG. 2 shows the effect of Gnetin C on the cell cycle of AML-MT cells. FIG. 2 shows the effect of Gnetin C on the cell cycle of AML cells. FIG. 6 shows the effect of Gnetin C on the expression of cell cycle proteins in AML-MT cells. FIG. 6 shows the effect of Gnetin C on the expression of cell cycle proteins in AML cells. FIG. 6 shows the cytotoxic effect of Gnetin C on AML cells. FIG. 6 shows the effect of Gnetin C on the colony forming ability of bone marrow mononuclear cells. FIG. 4 shows the synergy between Gnetin C and chemotherapeutic agents. FIG. 5 shows the results of identification of molecular targets of Gnetin C in AML cells. FIG. 7 shows the results of identification of molecular targets of Gnetin C in AML cells (densitometry data). FIG. 6 shows the pathways disrupted by Gnetin C based on the identified target proteins. FIG. 7 shows blocking of the ERK 1/2 MAPK pathway by Gnetin C. FIG. 6 shows blocking of mTOR signaling activity by Gnetin C by Western blotting. FIG. 4 shows blockade of mTOR signaling activity by Gnetin C by flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an outline of in vivo experiments using a mouse AML xenograft model. FIG. 1 shows the incidence of leukemia in a mouse AML xenograft model. FIG. 7 shows Giemsa stained images of peripheral blood samples collected from a mouse AML xenograft model. FIG. 7 shows weight change over time in a mouse AML xenograft model. It is a figure which shows the outline | summary of the experiment which confirms the anti-tumor effect with respect to AML cell of Gnetin C in vivo. It is a figure which shows the antitumor effect by Gnetin C in the mouse | mouth which administered the leukemia cell. FIG. 5 shows the number of AML cells in peripheral blood, bone marrow and spleen in Gnetin C-treated mice. FIG. 5 shows the percentage of dead cells in peripheral blood, bone marrow and spleen in Gnetin C-treated mice. FIG. 4 is a flow cytometry showing the percentage of leukemia cells (hCD45 +) dead cells (7AAD +) in peripheral blood, bone marrow and spleen in Gnetin C-treated mice. It is a figure which shows the detection result of the leukemia cell in the tissue derived from Gnetin C process mouse | mouth.

  Hereinafter, the present invention will be described in detail.

  The present invention is a simultaneous inhibitor which simultaneously inhibits the mTOR and MAPK signaling pathways. The inhibitor can be used as a preventive or therapeutic agent for cancer.

  The inhibitor contains resveratrol dimer as an active ingredient. Resveratrol dimer has a structure represented by the following general formula (I).

式（I)中、R₁およびR₃は、それぞれ独立にH、OHおよびOCH₃からなる群から選択され、R₂はH、OH、OCH₃およびOGlc（Glcはグルコース）からなる群から選択され、R₄はH、CH₃およびGlcからなる群から選択される。

In formula (I), R ₁ and R ₃ are each independently selected from the group consisting of H, OH and OCH ₃ , and R ₂ is selected from the group consisting of H, OH, OCH ₃ and OGlc (Glc is glucose) R ₄ is selected from the group consisting of H, CH ₃ and Glc.

Among these, Gnetin C, Gnetin L, Gnemoside A, Gnemoside C or Gnemoside D described below are preferable, and Gnetin C is more preferable.
Gnetin CR ₁ : H, R ₂ : OH, R ₃ : H, R ₄ : H
C ₂₈ H ₂₂ O ₈
Gnetin LR ₁ : OCH ₃ , R ₂ : H, R ₃ : OH, R ₄ : H
C ₂₉ H ₂₄ O ₇
Gnemonoside AR ₁ : H, R ₂ : OGlc, R ₃ : H, R ₄ : Glc
C ₄₀ H ₄₂ O ₁₆
Gnemonoside CR ₁ : H, R ₂ : OGlc, R ₃ : H, R ₄ : H
C ₃₄ H ₃₂ O ₁₁ ,
Gnemonoside DR ₁ : H, R ₂ : OH, R ₃ : H, R ₄ : Glc
C ₃₄ H ₃₂ O ₁₁

  Resveratrol dimer is a stilbene which can be obtained by separating and purifying from extract of seeds, leaves, fruits, etc. which are edible fruits of melinjo (Gnetum gnemon L.). In particular, it is preferable to use a seed extract. For example, purification can be performed using a general fractionation technique such as chromatography. For example, extract the seeds of Melinjo with an organic solvent such as ethanol, methanol, acetone, hexane, ether, ethyl acetate, chloroform, butanol, propanol and the like, and further use resveratrol by chromatography using hydrophobic interaction. The body can be separated and purified.

Melinjo is a tree of the Gunnum family dimorphism distributed from Southeast Asia to the West Pacific Islands.
Also, the resveratrol dimer may be synthetically produced. Those skilled in the art can easily synthesize them based on the above chemical formulas.

  In the southwestern countries, Gnetin C is considered safe for humans because resveratrol dimer rich melinjo leaves such as Gnetin C, fruits and seeds have been consumed for a long time. The absence of serious adverse events in mice receiving Gnetin C also demonstrates the safety of this compound.

  The mTOR (mammalian target of rapamycin) signaling pathway is also called AKT / mTOR signaling pathway, and mTOR, which is a serine / threonine kinase, is involved. AKT activated by phosphorylation is a pathway that activates mTOR (phosphorylation), and is involved in pathological conditions such as cancer. The mTOR signaling pathway is shown on the left of Figure 6-3.

  MAPK (mitogen-activated protein kinase) signaling pathway is also called ERK (extracellular signal-regulated kinase) 1/2 MAPK signaling pathway, and it is a serine / threonine kinase including ERK 1/2 (ERK1 · ERK2 subfamily) etc. It is a pathway that controls cell proliferation etc. by activation of certain MAPKs by growth factors etc. and activation of activated MAPKs to phosphorylate substrates such as other transcription factors and transmit amplified signals to the nucleus. . The putative MAPK signaling pathway is shown on the right of Figure 6-3.

  In many cancers, including AML, both mTOR and MAPK signaling pathways are activated. Inhibition of either the mTOR signaling pathway or the MAPK signaling pathway results in activation of the other, usually by a feedback mechanism. Gnetin C induces apoptosis of cancer cells by simultaneously inhibiting both mTOR and MAPK signal transduction pathways and damages cancer cells.

  Therefore, Gnetin C can be used as a preventive or therapeutic agent for cancer. The present invention seeks to use Gnetin C, which is a simultaneous inhibitor of the mTOR signaling pathway and the MAPK signaling pathway, as a cancer preventive or therapeutic agent.

  Furthermore, Gnetin C exerts a synergistic therapeutic effect by being used in combination with a cancer chemotherapeutic agent (anticancer agent). The metabolism of cytarabine (Ara-C), hydroxycarbamide (hydroxyurea (HU)), methotrexate, aminopterin, 6-mercaptopurine, 5-fluorouracil, carmofur, gemcitabine etc. as a chemotherapeutic agent whose combination may be useful Antagonists; Enzyme preparations such as L-asparaginase (L-Asp); Anticancer antibiotics such as adriamycin (doxorubicin), dactinomycin, actinomycin D, chromomycin, daunomycin, bleomycin, pepomycin, donorubicin, epirubicin, mitomycin C, etc. Nitrogen mustard (cyclophosphamide etc.), dacarbazine, carmustine (BCNU), busulfan, ifosfamide, nimustine hydrochloride, lomustine (CCNU) (nitrosourea agent), lanimustine (MCNU) (nitrosourea agent), proca Alkylated preparations such as rubazin; plant alkaloid agents such as vinblastine, vincristine, paclitaxel, docetaxel, CPT-11 (irinotecan), etoposide; hormone preparations such as prednisolone, dexamethasone, hydrocortisone; biological function modifiers such as lentinan, picibanil, bestatin And platinum preparations such as cisplatin and carboplatin.

  Among them, when combined with Gnetin C, cytarabine (Ara-C), hydroxyurea (HU) or L-asparaginase (L-Asp), which are known as chemotherapeutic agents for acute leukemia, show synergistic effects on AML cells Be The present invention includes combination preparations or combination kits combining resveratrol dimers such as Gnetin C and other chemotherapeutic agents. Furthermore, the present invention encompasses pharmaceutical compositions in combination with chemotherapeutic agents other than resveratrol dimers such as Gnetin C. Resveratrol dimers such as Gnetin C and other chemotherapeutic agents may be administered simultaneously or separately. Alternatively, they may be administered sequentially, and the administration order may be resveratrol dimer such as Gnetin C first, or other chemotherapy agents first.

  The cancer targeted by the prophylactic or therapeutic agent of the present invention includes AML, chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), malignant lymphoma (Hodgkin's lymphoma; Lymphoma, MALT lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma (Parkitt's lymphoma, Lymphoblastic lymphoma, etc.), blood cancer such as multiple myeloma, as well as liver cancer, lung cancer, gastric cancer , Pancreatic cancer, colon cancer, colon cancer, anal and rectal cancer, esophagus cancer, uterine cancer, uterine cancer, breast cancer, renal cancer, bladder cancer, prostate cancer, brain and nerve tumor, skin cancer, adrenal cancer, renal pelvic and ureteral cancer, urethral cancer, The cancer includes any cancer including solid cancer (epithelial cell cancer, non-epithelial cell cancer) such as penile cancer, testicular cancer, bone and osteosarcoma, leiomyoma, rhabdomyosarcoma and the like. Among them, monosomal karyotype AML, which is effective for the prevention or treatment of leukemia, particularly AML, among which the presence of two autosomal monosomy or one monosomy with at least one additional chromosomal structural abnormality is defined. It is effective for

  The pharmaceutical composition which is an agent for the prophylaxis or treatment of cancer containing the simultaneous inhibitor of mTOR signaling pathway and MAPK signaling pathway of the present invention can be administered orally or intravenously, intramuscularly, subcutaneously, rectally It may be administered parenterally such as administration and transdermal administration. The pharmaceutical composition may contain a pharmaceutically acceptable excipient, a lubricant, a binder, a disintegrant, a coating agent and the like. In addition, coloring agents, flavoring agents, preservatives and the like may be contained. As an excipient, for example, lactose, glucose, corn starch, sorbite, crystalline cellulose etc., as lubricant, for example, talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil, etc., as binder, for example, dimethylcellulose, polyvinyl alcohol, Examples of the disintegrant include polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, polyvinyl pyrrolidone and the like. Starch, sodium alginate, gelatin powder, calcium carbonate, calcium citrate, dextrin and the like. The form in use is not particularly limited, and may be in the form of capsule, powder, granular, tablet, liquid or the like.

  The simultaneous inhibitor of the mTOR signaling pathway and the MAPK signaling pathway of the present invention can be used as a supplement, or can be mixed with food and used as a food and drink composition.

  When used as a supplement, it can be used in the form of tablets or granules, and may be mixed with other useful ingredients.

  When used as a composition for eating and drinking, it can be provided as a health food and drink, food and drink for specified health, functional indication food, nutritional function food and drink, health supplement food and drink, and the like. These food and drink can be displayed with functions such as preventing or ameliorating cancer and preventing or ameliorating a tumor.

  The composition for food and drink is in various forms such as solid, liquid, powder, granule, paste, etc. Specifically, for example, soft drink, lactic acid drink, taste drink, coffee, green tea, black tea, oolong tea, etc. Beverages; Candy, chocolate, biscuits, sweets, cakes, sweets, sweets such as rice cakes; Fruit drinks, vegetable drinks, jams, pastes and other processed vegetables and fruits; sake, shochu, wine Alcoholic beverages such as Chinese liquor, whiskey, vodka, brandy, gin, lamb, liquor, beer, refreshing alcoholic beverages, fruit liquor, liqueurs; Yogurt, ice cream, butter, cheese, condensed milk, dairy products such as milk powder Dressings, mayonnaise, processed oils and fats such as tempura oil, salad oil; soy sauce, sauce, vinegar, mirin, dressing type seasoning, etc. Seasonings; Powdered juice, powdered soup, instant coffee, instant noodles, instant curry, instant miso soup, cooked foods, cooked beverages, dried foods and beverages such as cooked soups, processed cereal products such as processed flour products, processed starch products Goods etc. For example, it may be used as a solid such as candy, cookie, chewing gum or biscuit, or may be liquid such as soft drink, milk, yogurt or syrup. In the case of food and drink, additives commonly used for food and drink such as citric acid, lactic acid and casein can be blended.

  The resveratrol dimer content in a pharmaceutical composition, a supplement, a food or drink composition such as a food or drink containing a simultaneous inhibitor of mTOR signaling pathway and MAPK signaling pathway, which comprises resveratrol dimer, Although different depending on the dosage form, usually, resveratrol dimer may be contained in an amount of about 0.1 to 50% by weight, preferably about 0.1 to 20% by weight, based on the total composition. The intake in the case of resveratrol dimer can be appropriately increased or decreased in consideration of the user's age, sex, weight, type of symptoms, degree of symptoms, etc., but the daily intake is 0.01 to 250 mg / kg. It is preferable to set according to the form of food and drink so that It may be taken by administration once or several times a day, by feeding, etc.

  The pharmaceutical composition containing the resveratrol dimer of the present invention and the composition for eating and drinking such as supplements and foods are intended for mammals including human. Examples of mammals other than human include primates such as monkeys, rodents such as rats and mice, sheep, pigs, cows, cats, dogs and the like.

  The invention also encompasses human AML-MK cell lines. The cell line was established from leukemia patients of human AML-MK patients. Leukemic cells collected from the peripheral blood of patients are passaged in a medium containing recombinant human SCF (eg, 100 ng / ml), IL-3 (eg, 100 ng / ml) and FLT-3L (eg, 50 ng / ml) It can be established by culturing. In the cell line, the expression of CD38 is attenuated and the expression of CD34 is enhanced. The karyotype of the cell line is represented by 47, XY, del (1) (q32q42), del (11) (q13q21), + mar [27]. The cell line is useful for research on pathogenesis of leukemia, etc., or for research on treatment methods.

  The present invention is specifically described by the following examples, but the present invention is not limited by these examples.

Materials and Methods in the Examples Protease inhibitor cocktail, phosphatase inhibitor cocktail, anti-alpha tubulin and anti-beta actin antibody were purchased from Sigma. Anti-CDK3 antibodies were purchased from Genetex, and anti-CDK4 and anti-pAMPK1 / 2 (172/183) antibodies were purchased from Santa Cruz Biotechnology. Anti-phospho MSK 1/2 (S376 / S360) and anti-RSK1 (S380) were purchased from R & D Systems, Inc. All other antibodies were purchased from Cell Signaling Technology.

Cell culture
AML cell line MV4 was purchased from ATCC (Rockville, MD, USA). AML cell lines THP1, U937 and HL60, and chronic myelogenous leukemia (CML) cell line K562 cells were purchased from Health Science Research Resource Bank. BCR-ABL1 positive CML strain OUN1 and KH88 cells were provided by Professor Yasukawa of Ehime University. The cells were cultured in RPMI-1640 medium (37 ° C., 5% CO ₂ atmosphere) supplemented with 10% FBS and 1% penicillin and streptomycin and tested periodically to confirm negative for mycoplasma contamination. The primary leukemia cells used in this study were obtained from bone marrow or peripheral blood of various newly diagnosed or relapsed AML patients (Table 1). Samples were obtained during routine diagnostic procedures after informed consent was obtained from all patients according to the Helsinki Declaration and in accordance with institutional guidelines and studies approved by the Kanazawa University Ethics Committee. Bone marrow mononuclear cells and peripheral blood mononuclear cells containing 90% or more of leukemia cells were isolated using Ficoll-Hypaque gradient centrifugation (Pharmacia Biotech), aliquoted until use and cryopreserved. Cryopreserved blasts for use in in vitro experiments were over 90% viable as demonstrated by trypan blue staining. 20 ng / ml RPMI supplemented with 100 ng / ml 20% fetal bovine serum (FBS) stem cell factor (SCF), 100 ng / ml Fms related tyrosine kinase 3 ligand (FLT3-L), and GM-CSF. It culture | cultivated by culture medium. Gnetin C prepared from Melinjo extract (Kato E. et al., J Agric Food Chem 2009; 57: 2544-9) was obtained from Hosoda SHC and solubilized in DMSO. Leukemia cells were treated with various concentrations of Gnetin C or excipients. In some experiments, peripheral blood mononuclear cells (n = 3) and bone marrow samples (n = 3) isolated from heparinized peripheral blood samples of healthy individuals were used as controls.

Cell Viability, Apoptosis Test and Cell Cycle Analysis Assessment of cell viability using a colorimetric MTT assay was performed according to the manufacturer's instructions (Roche). Absorbance intensity was measured at 490 nm using a MultiSkan microplate reader (Thermo Scientific) with a reference wavelength of 620 nm. All experiments were performed three times and cell viability was calculated (Sugimori N. et al., PloS One 2015; 10: e0120709). Screening of candidate compounds capable of inhibiting the growth of leukemia cells with MK karyotype was performed by culturing AML-MK blasts for 48 hours in the presence of various agents with reported anti-tumor properties Then, cell viability was evaluated by MTT assay, and cell suppression rate was calculated by the method described in Quoc Trung L. et al. PLoS One 2013: 8; e55183.

  Quoc Trung L. et al. PLoS One 2013: 8; based on annexin V staining (Invitrogen) in cells cultured in the presence or absence of Gnetin C or other drugs as described in e55183 , Or mitochondrial apoptosis detection kit Mitocapture BioVision Inc. (Milpitas) was used to evaluate. Giemsa staining in Gnetin C-treated cells and untreated cells was performed by a known method.

Human phosphokinase membrane array
Screening of potential targets of Gnetin C was performed using two proteome profiler membrane arrays, human phospho-MAPK array kit (cat # ARY002B) and human phosphokinase array kit (cat # ARY003B, R & D Systems). Spot intensity, which correlates with the expression level of the target protein, was assessed using Quickspot imaging software (R & D Systems).

Flow cytometry Immunostaining of various cell surface receptors in leukemia cells was performed by co-culturing the cells with a fluorescently labeled monoclonal antibody (BD-Biosciences). Intracellular staining was performed using BD's Cytofix / Cytoperm kit (BD Biosciences). KI-67 is detected using PerCP Cy5.5 anti-human KI-67 monoclonal antibody (BD Biosciences), and activated mTOR is detected using anti-phospho mTOR antibody (Ser2448), clone D9C2 (cell signaling) Followed by staining with PE labeled anti-mouse IgG antibody (BD Biosciences). Data were acquired on a BD FACSCANTO II instrument (BD Biosciences) and analyzed using FlowJo software package, version 10.0.7.

Western Blot Analysis Cells were treated with Gnetin C at the indicated dose, time. After treatment, whole cell protein samples were prepared by lysing cells in M-PER® Mammalian Protein Extraction Reagent (Pierce) and proteins were detected using antibodies.

Animal Experiment Immunodeficient mice, 57BL / 6.Rag2 ^null / Il2rg ^null / NOD-Sirpa (Yamauchi T. et al., Blood 2013; 121: 1316-25) were used. Animals were housed in a pressurized ventilated cage in a pathogen-free room at Kanazawa University. Six or eight week old animals were used. This study was formally approved by the Kanazawa University Animal Care and Use Review Board. Leukemia was induced with some modifications according to the method described by Agliano A. et al., Int J Cancer 2008; 123: 2222-7. Briefly, non-irradiated mice were injected intraperitoneally with 2 × 10 ⁶ primary leukemia AML-MT cells or AML cell lines (THP-1 and HL-60 cells). Leukemia was diagnosed by detecting human leukemia cells (HLA-ABC + or hCD45 + cells) in bone marrow and peripheral blood samples using flow cytometry. As previously reported, leukemia was detected at an early stage by PCR using a primer specific to the alpha-satellite region of human chromosome 17 (Agliano A. et al., Int J Cancer 2008; 123: 2222-7 ). By optimizing the experimental conditions, it was found that THP-1 and AML-MT cells cause mice to develop leukemia-like more reliably. Therefore, they were used in experiments to investigate the anti-leukemic effect of Gnetin C in vivo. To expose mice to Gnetin C, Gnetin C was dissolved in corn oil (Sigma) and orally administered using disposable feeding needles (Fuchigami Co). In some experiments, plasma samples were isolated by centrifugation from mouse peripheral blood collected 24 hours after vehicle or Gnetin C administration. Plasma levels of Gnetin C and its metabolite Gnetin C monoglucuronide (GC-MNG) were measured at Japan Clinical Laboratories Inc.

Pathological examination of mouse tissues Mice were euthanized under ether anesthesia and the organs were fixed in 3.7% formalin (Sigma) for 48 hours. The tibia and femurs were decalcified in phosphate buffered formalin (3.7% formalin, pH 7.4) containing 20% EDTA (Sigma) for 48 hours prior to paraffin embedding. Tissues were processed using ethanol and xylene series according to standard methods. Sections (4 μm) were mounted on slides and processed according to the standard method using a xylene and ethanol series. Deparaffinized sections were stained with hematoxylin-eosin (H & E). Deparaffinized sections were incubated in Target Retrieval Solution (Dako Japan) for 20 minutes at 95 ° C. for pretreatment prior to human CD45 and MHC class I immunostaining. Block the endogenous peroxidase activity in methanol-hydrogen peroxide for 20 minutes and incubate in normal goat serum (1:10; Vector Lab, Burlingame, CA, USA) for 20 minutes, and then prime the sections against human CD45. Incubate overnight at 4 ° C. with monoclonal antibodies (clone 2B11 + PD7 / 26, mouse IgG1 kappa, Dako Japan) or human MHC class IA, B, C (clone EMR8-4, mouse IgG1, 1 μg / ml, North Star), Then, after benzidine reaction using Dako Envision-HRP (Dako Japan), the sections were additionally stained with hematoxylin.

Screening of various drugs for anti-leukemia effect
AML blasts with Monosomal karyotype (MK) were cultured in RPMI medium supplemented with 20% FBS, SCF (100 ng / ml), GM-CSF (20 ng / ml) and FLT3 ligand (100 ng / ml). Cells were aliquoted at 10,000 cells / well seeded at n = 3 in 96 well round bottom plates. Two hours after dispensing, various drugs (5 μL / well) or excipients (0.05% DMSO) were added, cells were cultured for 48 hours, and cell viability was evaluated by MTT assay. Compounds selected for this screening were used at doses reported to induce cell death of leukemia cells and other malignant cells in vivo. The following reagents were used:
Panobinostat (50 nM), resveratrol (50 μM), cytarabine (500 nM), doxorubicin (500 nM), bortezomib (50 nM), apigenin (100 μM), L-asparaginase (0.1 IU), methotrexate (250 nM), ascorbic acid (5 μM) , Hydroxyurea (100 μM), etoposide (15 μM), benftochiamine (15 μM), valproic acid (4 mM), dasatinib (5 μM), AG 490 (100 μM), Gnetin C (25 μM), sorafenib (5 μM), Thymoquinone (2 μg / ml) Retinoic acid (50 nM), Albendazol (0.5 μM) (19 μg), gefitinib (5 μM), mitomycin (5 μM), lactoferrin (100 μg / ml), tunicamycin (50 ng / ml), menadione (5 μM), pyocyanin (0.5 μg / ml), SB202190 (25 μM)
Other than dasatinib (Cayman), gefinitib (Tocris Bioscience), pyocyanin (Enzo Life Sciences) and Gnetin C (Hosoda Nutritional) were obtained from Sigma Aldrich.

Examination of synergistic effects of various drugs
The IC50 was evaluated using a pharmacological analysis tool ED50 v1.0 (Yanada M. et al., J Clin Med 2016; 5). The synergistic effects of Gnetin C and other drugs are cooperative indexes based on the Chou-Talalay method (Yanada M. et al., J Clin Med 2016; 5, Lechman ER. Et al., Cancer Cells 2016; 29: 214-23). Determined according to (CI). Here, CI is the sum of growth inhibition of single agent treatment / growth inhibition combined treatment. When CI <1, CI = 1, and CI> 1, the effects can be judged to be synergistic, additive, and antagonistic, respectively. Cell cycle analysis was performed using a FACS Canto II flow cytometer (BD Biosciences).

In colony assays, healthy human (3) bone marrow mononuclear cells remaining after filtration of bone marrow fluid and 4 AML patients (AML 1, AML 2, AML 3) remained after bone marrow collection for hematopoietic stem cell transplantation. , And AML 9) bone marrow cells were used. A methylcellulose solution (Metho Cult GF M 3434, StemCell Technologies, Minato-Ku) containing 1 × 10 ⁴ patient-derived AML cells or bone marrow mononuclear cells was cultured in a 35 mm Petri dish. Gnetin C was added at the start of the culture at various concentrations. At the same time, those to which only the excipient (DMSO) was added as a control were simultaneously cultured. On day 14 of culture, clusters of 40 or more cells were counted as colonies of AML blasts under an inverted microscope. Similarly, granulocyte-macrophage precursor cells (CFU-GM) and BFU-E derived from bone marrow mononuclear cells of healthy subjects: burst-forming-unit-erythroid (BFU-E) were inverted on day 14 of culture I counted below.

AML with Monosomal karyotype: Case presentation
A 22-year-old man was referred to our hospital for evaluation of an anterior mediastinal mass with left pleural effusion. The patient suffered from fever with weakness, fatigue and dry cough and sweating at night. Hematological examinations performed at other hospitals were suspected of malignant lymphoma because LDH (394 IU / L) and sIL-2R (2,508 U / mL) were elevated. There was no history of hematologic disease. The patient's pulse rate was 100 / min, SpO ₂ 93% (room air), and the respiration rate was 26 / min. Elastic hard lymph nodes were incised in the bilateral clavicle fossa. The breathing sound was attenuated in the lower left back. Blood tests (Table 2) confirmed leukocytosis (25, 680 / μl) with 66% blasts and elevated LDH (1047 IU / L). FDG / PET-CT examination showed increased FDG uptake in the mediastinum (SUVmax 10), pleura, clavicle and axillary nodes, and bone marrow. The bone marrow smear is a hyperplastic bone marrow containing a small number of erythroblasts and 74% blasts. The blasts are large, monocyte-like, bluish gray cytoplasm, and an increase in nuclear: cytoplasmic ratio, etc., for acute leukemia. Was suggested. Leukemic cells were positive for CD4, CD33, and CD56, and myeloperoxidase (MPO) staining was also positive. Leukemia cells isolated from pleural fluid were also MPO positive. Immunophenotypes of the surface of leukemia cells are: CD33 +/- (78%), CD38 +/- (90%), CD4 +/- (74.5%), HLA-DR +/- (39.9%), CD56 + /-(28%), CD13 +/- (15.9%), CD45 +/- (10%), CD14 +/- (3%), CD22 +/- (42.8%), CD10 +/- (5%) ), CD19- (5.8%), CD34 +/- (3%), CD20- (0.9%), CD3-, CD5-, CD7-, CD8-, CD20-, CD21-, CD22-, CD25- and CD41a -Met. Chromosome examination revealed the following karyotype. 46 XY, add (11) (q 13), add (16) (p 11.2) [3] 45, idem, -Y [4] 44, idem, -Y, -15 [3] 45, idem, -15, -add (16), + add (16) (p11.2) [1] 46, XY [3].

  FISH analysis of MLL (11 q23) was negative and PCR of BCR / ABL was also negative. From these results, it was diagnosed as AML having monosomal karyotype. The patient received a induction regimen with idarubicin (IDR) and cytarabine (Ara-C). The patient received a second chemotherapy with IDR + Ara-C because she did not reach a complete remission, but a decrease in the percentage of blasts in the bone marrow (3%) was observed, but mediastinum and lymphadenopathy The survival remained and a complete remission was not obtained. She also developed a skin infiltration of leukemia, so she received a high-dose Ara-C-containing regimen (HD-AC) as salvage therapy. Cutaneous leukemia improved temporarily, and FDG / PET-CT examination also showed improvement of mediastinal and lymph node lesions. However, during the hematopoietic recovery period after the second HD-AC therapy, blasts in the bone marrow rapidly increased to 80%, and blasts also appeared in the peripheral blood. After the blast count was temporarily reduced by the third HD-AC therapy, peripheral blood stem cell transplantation (R-PBSCT) from the siblings was performed after pretreatment with fludarabine (Flu) and total body irradiation (TBI). Cyclophosphamide, tacrolimus (FK506) and MTX were used for the purpose of preventing GVHD. Neutrophil engraftment was confirmed by day 13 after transplantation, but as a result of bone marrow examination performed on day 26 after transplantation, numerous blasts were confirmed. Chemotherapy with azacitidine (AZA) was discontinued, but was discontinued due to a further increase in the number of blasts, and chemotherapy with mitoxantrone, etoposide, moderate Ara-C (MEC regimen) for the second hematopoietic stem cell transplantation Did. However, the percentage of blasts in bone marrow was also 90% in MEC therapy. Therefore, after pretreatment with Flu / Mel / TBI (4 Gy), peripheral blood stem cell transplantation (PBSCT) was performed from the mother. The patient developed hemorrhagic cystitis, but progressed without any other complications, and on day 15, engraftment of donor cells and remission of leukemia were confirmed. However, at the second month of transplantation, 40% blasts appeared in the blood test, and 70% blasts were also found in the bone marrow. Although the third MEC therapy was performed, the number of blasts gradually increased and died 10 days later due to sepsis.

Establishment and Maintenance of AML-MT Cells Peripheral blood was collected from this AML-MK patient who relapsed after the second refractory hematopoietic stem cell transplantation on December 23, 2014, from refractory and very active. The white blood cell count was 90,800 / L and 95% were blasts. LymphoprepTM gradient-separated leukemia cells containing 20% inactivated fetal bovine serum (FBS) 100 ng / ml SCF, 100 ng / ml IL-3, 50 ng / ml FLT-3L The cells were suspended in RPMI-1640 medium (Gibco / Thermo Scientific) and cultured in an incubator at 37 ° C., 5% CO ₂ , 100% humidity. The concentration of cells was adjusted to 1 × 10 ⁶ cells / ml in a T10 culture flask. The culture medium was replaced every 4 days. After 33 days of culture initiation, recombinant SCF and IL-3 in the medium were reduced to 50 ng / ml and 20 ng / ml, respectively. FLT-3L was maintained at 50 ng / ml. When some of the cells were transferred to a cytokine-free medium, the cells were killed within 48 hours. Subsequently, the cells were observed to survive without IL-3, but since cell growth was extremely poor, IL-3 was added again, and culture medium containing SCF, IL-3, FLT-3L was used. After 67 days of culture, cell growth was stable. Since cell lines were established, morphological and immunophenotyping studies were performed as follows.

  Cells adsorbed to slide glass in Cytospin were stained with Giemsa (MG), myeloperoxidase (MPO). Patient peripheral blood and AML-MK cells were stained with fluorochrome-conjugated antibody and cell surface antigens were analyzed by flow cytometry. The AML-MK cell line showed an immunophenotype similar to that of the original leukemia cells, but in AML-MK cells there was a decrease in CD38 expression and an increase in CD34 expression (Table 3). MPO staining was also positive in the AML-MK cell line. Granules were present in about 35% of the cells and vacuoles were observed in the cytoplasm of some cells. The nuclei were often separated, having 1 to 3 nucleoli, and having a basophil cytoplasm containing fine azurophilic granules.

To confirm that the AML-MK cells were indeed from the original leukemic cells, cytogenetic analysis was performed using trypsin-Gimsa banding method (GTG) and spectral karyotyping (SKY). For analysis, those maintained in a test tube for 17 months were used. Twenty-one metaphase cells were analyzed and karyotypes were defined according to International Cytogenetic Nomenclature 2016 (Burgess MR. Et al., Blood 2014; 124: 3947-55). Fluorescence in situ hybridization (FISH) and SKY were performed by known methods. The karyotype of AML-MK is 47, XY, del (1) (q32q42), del (11) (q13q21), +, combined with cytogenetic findings from GTG, DAPI banding, FISH, SKY. It turned out to be mar [27]. Cytogenetic analysis revealed that del (1) (q32q42) and del (11) (q13q21) detected in AML-MK were identical to those of the original leukemic blast.
AML-MK cells remained floating cells and continuously proliferated for more than 22 months with a doubling time of 60-72 hours.

Data analysis All data were reported as the mean of three experiments ± standard deviation. The results were analyzed using Student-t test, and survival rates of mice were estimated using Kaplan-Meier method. All analyzes were performed using the GraphPad Prism software package, version 5.02 (San Diego). P value <0.05 was determined to be significant.

Example 1 Screening of agents showing cytotoxicity against monosomal karyotype AML cells In order to identify candidate compounds showing cytotoxic activity against complex karyotype AML cells, a series of published reports having anti-cancer properties The compounds were screened in AML blasts from AML-MK patients who were resistant to various anti-leukemic drugs and were not fully remitted.

  Leukemia cells from patients with AML-MK were aliquoted three times in each condition and treated with Gnetin C or various cytotoxic agents for 48 hours. Cell suppression rates were then assessed by MTT assay. The results are shown in Figure 1-1. The only compound that showed potent antiproliferative effects in AML-MK cells was Lnet-asparaginase (L-Asp), bortezomib, methotrexate and Panobinostat, as well as the naturally occurring stilbene Gnetin C ( Figure 1-1).

  To explore the potential of Gnetin C as an anti-leukemic agent, a panel of nine patient-derived AML samples was screened. Gnetin C was able to inhibit cell growth with an IC 50 in the range of 2.5 μM to 17 μM in 8 out of 9 samples (Table 4). Remarkably, Gnetin C is resistant to currently available chemotherapy regimens because the growth inhibitory ability of this compound is also observed in patient specimens with cytogenetic characteristics with poor prognosis and relapsed leukemia cells. May have an effect on sexual patients.

  AML cells from three different patients were cultured with Gnetin C at the concentrations shown in FIG. 1-2A for 48 hours and cell viability was assessed by MTT assay. The results are shown in Figure 1-2A. In addition, cell viability of AML 1 and AML 9 cells treated with Gnetin C (17 μM) was evaluated by MTT assay at 24, 48 and 72 hours. The results are shown in Figure 1-2B. Gnetin C showed a growth inhibitory effect in a dose-dependent manner (FIG. 1-2A) and a time-dependent manner (FIG. 1-2B). The growth inhibitory effect was pronounced within 24 hours and diminished within 72 hours after treatment.

  Furthermore, the antitumor effect of Gnetin C was also examined on seven myeloid cell lines including CML cell lines K562, KH88 and OUN1, and AML cell lines U937, HL-60, THP-1 and MV4. . These cell lines were treated with various concentrations of Gnetin C for 48 hours and cell viability was assessed by MTT assay. The results are shown in FIGS. 1-2D and 1-2E. A dose-dependent growth inhibitory effect was observed in all AML cell lines examined (FIG. 1-2D). On the other hand, Gnetin C showed no inhibitory activity against the three CML cell lines examined (FIG. 1-2E).

  Finally, in order to confirm the safety of the present compound, the concentration of Gnetin C is increased (up to 100 μM), peripheral blood mononuclear cells from healthy individuals and bone marrow-derived CD34 positive hematopoietic progenitor cells of 3 healthy individuals Were cultured for 48 hours and cell viability was assessed by MTT assay. Gnetin C at a concentration that effectively inhibits the growth of AML cells did not impair the survival of peripheral blood mononuclear cells. However, as expected, at very high concentrations (100 μM), the viability of normal cells was significantly reduced (FIGS. 1-2C and 1-2F).

Example 2 Gnetin C Induces Apoptosis and Cell Cycle Arrest in AML Cells An AML cell line (AML-MT) was established that maintains the characteristics of the patient's leukemia cells (AML-MK) used in screening experiments. To the best of the inventors' knowledge, AML-MT cells are the first cell line to have MK karyotype dependent on Flt-3L, SCF and IL-3 growth factors. AML-MT cells, which retain a phenotype and karyotype similar to those of the original AML cells, engraft in immunodeficient mice and develop leukemia. AML-MT cells and AML cells as a source of AML-MT cells were cultured for 48 hours with Gnetin C at the concentration shown in FIG. 2-1, and cell viability was evaluated by MTT assay. As with the original leukemia cells, AML-MT cells were sensitive to the cytotoxic effects of Gnetin C (Figure 2-1).

  AML-MT cells were cultured for 24 hours with Gnetin C at the concentration shown in FIG. 2-2, and then after staining with annexin FITC, they were analyzed by flow cytometry (upper panel). Alternatively, cells adsorbed to slide glass by cytospin were stained with Giemsa, and cell morphology was evaluated with a light microscope (middle panel). AML-MT cells were cultured for 12 hours with the indicated concentrations of Gnetin C, and cells stained with Mitocapture reagent were observed under a fluorescent microscope (lower panel). The results are shown in Figure 2-2. The cell growth inhibitory ability of Gnetin C in AML cells was determined by cell membrane protrusion, aggregated chromatin, nuclear fragments in the annexin V staining assay (FIG. 2-2, upper panel) and Giemsa staining (FIG. 2-2, central panel) It showed typical morphological signs of apoptosis, including traces of transformed and condensed basophil cytoplasm. These were similar to the induction of apoptosis seen in patient-derived AML cells. Interestingly, a significant reduction in membrane potential was observed in Gnetin C-treated cells (FIG. 2-2, lower panel). This suggests that cell apoptosis induced by Gnetin C is due to mitochondrial membrane dysfunction in AML cells.

  Three patient-derived AML cells were treated with the indicated concentrations of Gnetin C for 36 hours and they were analyzed by flow cytometry after staining with annexin FITC. As a result, the ability of Gnetin C to induce apoptosis was also shown in patient-derived AML cells derived from three different patients (FIG. 2-3).

  Furthermore, THP-1 cells were treated with Gnetin C at the concentrations indicated on FIG. 2-4 for 48 hours and then they were analyzed by flow cytometry after staining with annexin FITC. In addition, THP-1, HL-60, MV4, KH88 and K562 cells were treated with various concentrations of Gnetin C for 48 hours, similarly stained with annexin FITC, and analyzed by flow cytometry. Furthermore, OUN1 cells were treated with Gnetin C at the concentration shown on FIGS. 2-5 for 48 hours, and stained with annexin FITC, and analyzed by flow cytometry. The results are shown in FIGS. 2-4 and 2-5. Gnetin C consistently induced apoptosis in cell lines of the AML lineage of MV4, HL-60 and THP-1 (Figure 2-4), but against the CML lines OUN1, KH88 and K562 It did not induce apoptosis (Fig. 2-5).

  Next, AML-MT cells were treated with 17 μM Gnetin C for 12 hours, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Also, AML 2, AML 3 and AML 9 cells were treated with 17 μM Gnetin C for 24 hours, stained with propidium iodide, and analyzed for DNA content by flow cytometry. These cell cycle analyzes revealed that Gnetin C confined the cell cycle of leukemia cells to the G1 phase (FIG. 3-1 (AML-MT) and FIG. 3-2 (A: AML2, B: AML3, C: AML 9) (* P <0.05 vs UNT)).

  In addition, AML-MT cells were treated with Gnetin C at the concentrations shown in FIG. 3-3 for 12 hours (FIG. 3-3A) or with 17 μM Gnetin C for the indicated times (FIG. 3-3B) . Total protein extracts were prepared and subjected to Western blotting using antibodies specific for CDK1, CDK3, CDK4 and CDC42. In addition, AML blasts were treated similarly and whole protein extracts were prepared and subjected to Western blotting using antibodies specific for CDK3, CDK4, p27 and CDC42. The results are shown in FIGS. 3-3 and 3-4 (A: AML2, B: AML3, C: AML9). Cell cycle regulatory proteins such as cyclin dependent kinase 3 (CDK3) and cell division regulatory protein 42 homolog (CDC 42) were constitutively highly expressed in AML-MT cells, but their expression was significantly reduced by Gnetin C treatment did. On the other hand, no significant changes were observed in the expression of other cell cycle regulatory proteins such as CDK1, CDK2 and CDK4 (FIGS. 3-3 and 3-4).

Next, colony assays were performed to further clarify the cytotoxic effects of Gnetin C on AML cells. Leukemia cells (5 × 10 ⁴ cells / plate) obtained from 4 AML patients are treated with Gnetin C or vehicle at the concentration shown in FIG. 4-1, cultured in MethoCult H04034, and more than 40 The colonies consisting of cells were counted 14 days later. Treatment with Gnetin C reduced the colony number of patient-derived AML cells in a dose-dependent manner (FIG. 4-1 (A: AML1, B: AML2, C: AML3, D: AML9)). Remarkably, higher doses of Gnetin C were required to inhibit the colony forming ability of AML9 cells. This was consistent with the low sensitivity of this patient sample to the cytotoxic effects of Gnetin C. When bone marrow mononuclear cells of 3 healthy subjects were cultured using the same methyl cellulose medium, no significant phenomenon was observed in the number of BFU-E and CFU-GM colonies in the presence of 20 and 40 μM Gnetin C. . However, at high concentrations of 80 μM or more, both BFU-E and CFU-GM decreased significantly (FIG. 4-2 (A: BFU-E, B: BFU-GM)).

Example 3 Gnetin C showed synergy in combination with a chemotherapeutic agent
To evaluate the utility of combining Gnetin C with a therapeutic drug for AML, including hydroxyurea (HU) and cytarabine (Ara-C), AML-MT cells in the presence or absence of Gnetin C The cells were cocultured with low concentrations of HU (20 μM), Ara-C (50 μM) or L-Asp (0.01 IU / ml) for 48 hours. Cell proliferation was assessed by MTT assay and cooperative index (CI) analysis was performed to determine the interaction between Gnetin C and chemotherapeutic agents. The effects of CI <1, CI = 1 and CI> 1 were defined as synergistic, additive and antagonistic, respectively. The combination of Gnetin C with Ara-C (FIG. 5A) or HU (FIG. 5B) significantly increases the anti-cancer effect of these compounds, and cooperative index (CI) is less than 1 at most concentrations used Met. This indicates that there is a synergistic effect. Intriguingly, similar synergy is also observed when Gnetin C is used in the treatment of lymphoid malignancies, and in combination with L-Asp (Fig. 5C), which has recently also been shown to have therapeutic effects in AML. An effect was observed.

Example 4 Identification of Molecular Target of Gnetin C in AML Cells
To identify the anti-tumor mechanism of Gnetin C in AML, total protein extracts from Gnetin C-treated AML cells were evaluated for expression of active forms of multiple protein kinases and MAPK pathway members using membrane arrays. That is, after AML-MT cells are left untreated or treated with 17 μM Gnetin C for 4 hours, a total protein extract is prepared and a human phosphorylated MAPK array (FIG. 6-1) or a human phosphorylated kinase membrane It evaluated by the array (FIG. 6-2). Representative results are shown in FIGS. 6-1A and 6-2A. Each two spots represent an activated protein target. The targets highlighted by squares are listed at the bottom of the figure. FIGS. 6-1B and 6-2B show data of densitometrically measured dot intensities of representative active proteins detectable in AML-MT cells. As shown in FIGS. 6-1 and 6-2, Gnetin C treatment inhibited constitutive activation of p70SK and AKT in AML-MT cells, consistent with an inhibitory effect on the AKT / mTOR pathway. Furthermore, constitutive activation of activated p90 ribosomal S6 kinase (RSK1) and mitogen and stress activated protein kinase (MSK 1/2), which are downstream members of ERK 1/2 MAPK, was also demonstrated in Gnetin C of AML-MT cells. It was inhibited by the treatment. From the above, it was concluded that Gnetin C targets both ERK 1/2 and AKT / mTOR signaling in AML cells.

  Figure 6-3 shows the pathways inhibited by Gnetin C based on the identified target proteins. Inhibition by Gnetin C induces cell cycle arrest, induces apoptosis (Apoptosis cell death), and regulates cell growth (Growth inhibition).

Example 5 Gnetin C Blocks the ERK 1/2 MAPK Pathway Constitutively Activated in AML Cells Western Blot with specific antibodies of active MAPK signaling members to validate the results of array screening Did. That is, AML-MT cells are left untreated (NC) or treated with 17 μM Gnetin C for 30 minutes, 1, 3, 4 or 6 hours (GC), and then a protein extract is prepared and phosphorylated. Western blot was performed using antibodies specific for ERK and ERK. Furthermore, AML-MT cells were cultured for 4 hours in the presence or absence of Gnetin C, and protein extracts were subjected to Western blot using antibodies specific for phosphorylated ERK, ERK and β-actin. As a result, ERK 1/2 kinase, which is constitutively activated in AML-MT cells, is always inhibited at 3 hours after Gnetin C treatment, and ERK 1/2 inhibition peaks at 6 hours after ERK treatment. (FIG. 7A), and Gnetin C treatment was shown to be involved in concurrent p38 MAPK and pJNK 1/2 signaling (FIG. 7A). In addition to activation of JNK 1/2 and p38 MAPK signaling, inhibition of ERK 1/2 has also been reported by other agents that induce apoptosis in AML cells (Sugimori N. et al., Plos One 2015; 10: e0120709, Konopleva M. et al., Leukemia 2005; 19: 1350-4). The AML cytotoxicity of Gnetin C was dose dependent (FIG. 7B), and was confirmed in various patient-derived AML cells in addition to AML-MT (FIG. 7C). On the other hand, since constitutive activation of ERK1 / 2 signaling and its inhibition by Gnetin C were hardly detected in AML9 cells that were relatively resistant to Gnetin C, constitutive activation of ERK1 / 2 signaling was Activation appeared to correlate with the sensitivity of Gnetin C to the cytotoxic effects. Interestingly, p38 MAPK activation, normally induced by Gnetin C treatment, was not observed in any of the AML cells examined here (FIG. 7C). In AML 2 and AML 3 cells, p38 MAPK was rather suppressed, so activation of p38 MAPK was considered to be unnecessary for the cytotoxic effect of Gnetin-C.

Example 6 Gnetin C Blocks mTOR Signaling Activity Activated Constitutively in AML Cells To validate findings from membrane arrays, we next examined Gnetin C treatment for mTOR signaling. The impact was assessed. Briefly, AML-MT cells were either untreated (NC) or cultured with 17 μM Gnetin C for 30 minutes, 1, 3, 4 or 6 hours (GC). In addition, the cells were cultured for 4 hours in various concentrations of Gnetin C. Protein extracts were prepared and western blotted with antibodies specific for p-AKT, AKT and p-P70SK6. Western blot showed time-dependent and dose-dependent inhibition of p70 SK6 and AKT mTOR phosphorylation (FIGS. 8-1A and 8-1B). In addition, total protein extracts are prepared from various patient-derived AML cells treated with no treatment (NC) or 17 μM Gnetin C (GC) for 4 hours, and antibodies specific for p-AKT, AKT and p-P70SK6 When Western blotting was performed, time-dependent and dose-dependent inhibition of p70 SK6 and AKT mTOR phosphorylation was also observed in various patient-derived AML cells (FIG. 8-1C). The effect of Gnetin C on mTOR was further evaluated by flow cytometry using specific antibodies for active mTOR on er 967. Thus, AML-MT cells were left untreated (control) or cultured for 4 hours with 17 μM Gnetin C. Cultured cells were then membrane processed, incubated with anti-mTOR or anti-Ki67 antibody followed by fluorescently labeled anti-mouse antibody and analyzed by flow cytometry. Similar treatment was performed on cells from 3 AML patients. Gnetin C inhibited the expression of active mTOR in AML-MT cells (FIGS. 8-2A and 8-2B). Also, Ki-67 protein expression, a cell marker closely related to cell proliferation, was reduced by Gnetin C treatment (Fig. 8-2A, lower panel). On the other hand, mTOR and p70SK6 were not constitutively activated in AML9 cells, and no inhibition of AKT by Gnetin C treatment was observed. This was consistent with the lower sensitivity of Gnetin C to the cytotoxic effects (Figures 1-2B and 8-1C). These findings indicate that the anti-tumor effects of Gnetin C involve blockade of the AKT / mTOR pathway.

Example 7 Gnetin C suppresses leukemia onset in mice
To investigate whether Gnetin C is also effective in vivo, a mouse AML xenograft model was created by injecting AML-MT or NB4 cells intraperitoneally into immunodeficient mice. In this model, all mice develop leukemia within 3 weeks after injection of leukemia cells and die within 4 to 5 weeks. In order to examine whether Gnetin C prevents the onset of leukemia in vivo, Gnetin C (5 mg / kg) starting one week prior to immunodeficient mice (n = 7 for each group) receiving leukemia cells Alternatively, the vehicle was orally administered every two days and continued for the entire test period (4 weeks) (Figure 9-1). The percentage of mice that developed leukemia was determined by detecting human leukemia cells in peripheral blood, bone marrow and spleen using flow cytometry. The experiment was repeated three times. In contrast to vehicle-treated mice that developed leukemia and died within 5 weeks, the onset of leukemia was significantly delayed in Gnetin C-treated mice, and 40% of mice did not develop leukemia at all (Figure 9-2 and Fig. 9-3). FIG. 9-3 shows representative Giemsa staining results of peripheral blood samples collected from vehicle-treated and Gnetin C-treated mice. Peripheral blood samples were subjected to erythrocyte lysis prior to staining. It was found that normal granulocytes were present in the Gnetin C-treated mice, and leukemia cells were not found, whereas granulocytes were very few in the excipient-treated mice and leukemia cells were numerous. When the body weight of Gnetin C-treated mice and vehicle-treated mice was measured every 5 days, Gnetin C administration did not cause significant weight loss compared to the control (FIG. 9-4). This allowed us to accurately measure plasma levels of Gnetin C and its major metabolites. The mean ± SEM plasma levels of Gnetin C and Gnetin C monoglucuronide were 175 ± 156 ng / mL and 49 ± 30.15 ng / mL, respectively.

Example 8 Gnetin C has an antitumor effect on AML cells in vivo Next, experiments with a treatment model were performed in which administration of a drug was started one week after the injection of leukemia cells. That is, human leukemia cells were injected intraperitoneally into immunodeficient mice (n = 7 per group). One week after injection (until the end of the test period) Gnetin C (5 mg / kg) or vehicle was orally administered every two days (FIG. 10-1). In this model, Gnetin C showed an antitumor effect in vivo and significantly prolonged the survival of mice (FIG. 10-2). Using the same model, mouse organs are collected 10, 21 and 28 days after injection of leukemia cells, and whether the percentage of leukemia cells in the organs of Gnetin C-treated mice is different compared to the control group Was evaluated. That is, peripheral blood, spleen and bone marrow specimens are collected at 10, 21 and 28 days from mice treated with Gnetin C or vehicle 56 days after injection of leukemia cells, and single cell suspensions are prepared. The percentage of leukemia cells (hCD45 +) and photoreceptors (7AAD: 7-amino-actinomycin D) was evaluated by flow cytometry. The number of AML cells in peripheral blood and bone marrow was significantly reduced in Gnetin C-treated mice as compared to vehicle-treated mice (FIG. 10-3, A: blood, B: spleen, C: Bone marrow). Furthermore, many of the leukemia cells detected in Gnetin C-treated mice were dead cells, suggesting that Gnetin C induces leukemia cell death in vivo (FIG. 10-4 and FIG. 10-5). ). Finally, in order to confirm the in vivo anti-leukemia effect of Gnetin C, mouse tissue (spleen and bone marrow) preparations were evaluated by H & E staining and immunohistochemical staining. In contrast to bone marrow and spleen tissue samples from vehicle-treated mice that were heavily infiltrated by leukemia cells, leukemia cells were not seen in tissues from Gnetin C-treated mice. (Figure 10-6).

Discussion To the best of our knowledge, Gnetin C is the first naturally-occurring stilbene to be able to simultaneously inhibit two distinct cell survival pathways of AML: the AKT / mTOR signaling pathway and the MAPK ERK 1/2 pathway. . Gnetin C at concentrations that induced apoptosis in AML cells did not inhibit colony formation of normal bone marrow progenitor cells. Furthermore, oral administration of Gnetin C induced leukemia cell death in vivo without apparent toxicity in mouse models of human leukemia and significantly prolonged survival.

  Dysregulation of mTOR in hematopoietic cells is known to be involved in leukemogenesis. Aberrant activation of the AKT / mTOR pathway is detected in 50-80% of AML patients, and the prognosis for these patients is extremely poor. Several inhibitors of AKT, mTOR and PI3K show promising results in preclinical models and are currently in clinical development. However, most past clinical trials using these agents have failed. According to previous clinical trials, treatment of AML cells with MEK / ERK inhibitors results in feedback activation of PI3K / AKT / mTOR, and further, PI3K / AKT / mTOR inhibition in turn induces ERK1 / 2 activation It has been reported that. Therefore, the failure of clinical trials of these drugs is thought to be due to the presence of a feedback loop.

  On the other hand, recent studies have shown that PI3K / AKT / mTOR signaling enhancement is restricted to leukemic progenitors in the cell cycle. In contrast, PI3K / AKT / mTOR signaling is suppressed in quiescent leukemia stem cells that cause relapse. This suggests that, even if the leukemia is treated with a PI3K / AKT / mTOR inhibitor, the leukemic stem cells in the quiescent phase will remain. The simultaneous inhibition of PI3K / AKT / mTOR and MEK / ERK pathway showed strong anti-leukemia effect by the present inventors' preclinical tests, therefore, an anti-leukemia regimen combined with Gnetin C is considered to be useful for leukemia treatment Be Another recent study has also shown that inhibition of ERK 1/2 signaling induces apoptosis in leukemia stem cells. Thus, dual inhibition of the AKT / mTOR and ERK 1/2 pathways by Gnetin C may be more useful for eradicating leukemia stem cells.

  By using cationic dyes that fluoresce differently in the cytoplasm and mitochondria, we observed loss of membrane potential in Gnetin C-treated AML cells. This was a finding consistent with mitochondrial membrane dysfunction. The opening of the mitochondrial permeability transition pore is an indication characteristic of the early stages of programmed cell death. The resulting changes in membrane potential allow passage of ions and small molecules, resulting in uncoupling of the respiratory chain and release of cytochrome c into the cytosol, triggering various apoptotic pathways. The AKT / mTOR pathway is involved in the regulation of mitochondrial homeostasis via AKT-mediated phosphorylation of IP3R, hexokinase 2 and phosphoflic acid cluster sorting protein 2. mTOR C2 defects disrupt mitochondrial associated endoplasmic reticulum membrane (MAM) and cause mitochondrial defects including elevated mitochondrial membrane potential, ATP production, and calcium uptake.

  MAPK regulates cellular function by phosphorylating target proteins including structural proteins, enzymes, transcription factors and downstream kinases. Among the target kinases, RSK isoforms are activated by ERK1 / 2. Furthermore, ERK1 / 2 activates MSK1 kinase and its closely related isoform MSK2. Interestingly, constitutive activation of MSK 1/2 has been reported in AML patients, particularly those with the FLT3-L mutation. Several MEK / ERK 1/2 inhibitors have been developed and currently over 80 clinical trials are in progress. Some of these studies are for AML patients.

  In this example, it was the first success in establishing a human AML cell line having monosomal karyotype. Human leukemia cell lines are an abundant and unrestricted source of cells with specific molecules and chromosomal abnormalities, and so are very useful in elucidating the molecular mechanisms of leukemia and in developing new drugs. The newly established AML-MT cell line has cytogenetic characteristics similar to the patient's original leukemia cells and induced leukemia in mice. AML-MT cells are also characterized in that they are cytokine-dependent in vitro.

  Gnetin C is a clinically promising drug, and can be expected to be particularly effective for AML patients resistant to standard chemotherapy. Also, since Gnetin C acts synergistically with Ara-C, HU and L-Asp to more effectively injure AML cells, the combination of Gnetin C with other chemotherapy is an attractive treatment and Is expected to be Gnetin C may be useful for preventing recurrence in patients who have achieved complete remission, based on the finding that administration of Gnetin C to mice prior to leukemia cell administration delayed the onset of leukemia. In conclusion, Gnetin C is considered a promising therapeutic for AML and other malignancies where the AKT / mTOR signaling and ERK1 / 2 MAPK pathways are essential for survival.

  Gnetin C is useful as a preventive or therapeutic agent for cancer.

Claims (11)

Simultaneous inhibitors of mTOR signaling pathway and MAPK signaling pathway comprising resveratrol dimer having a structure represented by the following general formula (I) as an active ingredient:

[In formula (I), R ₁ and R ₃ are each independently selected from the group consisting of H, OH and OCH ₃ , and R ₂ is from the group consisting of H, OH, OCH ₃ and OGlc (Glc is glucose) Selected, R ₄ is selected from the group consisting of H, CH ₃ and Glc].   The simultaneous inhibitor of mTOR signaling pathway and MAPK signaling pathway according to claim 1, wherein the resveratrol dimer is selected from the group consisting of Gnetin C, Gnetin L, Gnemonoside A, Gnemonoside C and Gnemonoside D.   The simultaneous inhibitor of mTOR signaling pathway and MAPK signaling pathway according to claim 2, wherein the resveratrol dimer is Gnetin C.   An agent for the prophylaxis or treatment of cancer, comprising the inhibitor according to any one of claims 1 to 3.   The agent for preventing or treating cancer according to claim 4, wherein the cancer is AML.   The agent for preventing or treating cancer according to claim 4 or 5, which is for oral administration.   A combined preparation of the cancer preventive or therapeutic agent according to any one of claims 4 to 6 and a cancer chemotherapeutic agent other than the therapeutic agent.   A pharmaceutical composition for use in combination with a chemotherapeutic agent other than resveratrol dimer, comprising resveratrol dimer.   Foods and beverages for the prevention or treatment of cancer, comprising the inhibitor according to any one of claims 1 to 3.   Food and drink for the prevention or treatment of the cancer according to claim 9, wherein the cancer is AML.   A human AML cell line having a karyotype of 47, XY, del (1) (q 32 q 42), del (11) (q 13 q 21), + mar [27] monosomal Karyotype.

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