WO2018048008A1 - Composition for inhibiting cancer stem cell growth comprising primaquine - Google Patents

Abstract

The present invention relates to a composition for inhibiting cancer stem cell growth comprising primaquine or a pharmaceutically acceptable salt thereof as an active ingredient, and to a pharmaceutical composition, a food composition and so forth, comprising the composition, for cancer metastasis inhibition or cancer treatment or prevention. The primaquine according to the present invention inhibits the growth of breast cancer cells and the formation of breast cancer stem cells. Also, the primaquine inhibited the expression of self-renewal genes, such as Nanog, C-Myc, Oct4, Sox2, Snail and CD44, which are known to be expressed typically in breast cancer stem cells, and it has been confirmed that signal transducer STAT3 is involved therein. Accordingly, the primaquine inhibits breast cancer and breast cancer stem cell growth and can be used for treating cancer such as breast cancer.

Description

A composition for inhibiting cancer stem cell growth, comprising primaquine

The present invention comprises a primaquine or a pharmaceutically acceptable salt thereof as an active ingredient, a composition for inhibiting cancer stem cell growth, inhibiting cancer metastasis comprising the composition, or a pharmaceutical composition for treating or preventing cancer, a food composition It is about.

Interest in cancer stem cells has emerged as chemotherapy fails to effectively target and treat cell groups within tumors, leading to tumor recurrence and metastasis. Many cytotoxic anticancer drugs usually target fast-growing cells, and cancer stem cells with slow-growing characteristics can survive cytotoxic chemotherapy. Basal cell phenotype Breast cancer is believed to originate from early mammary progenitor cells in the early stages of differentiation and is known to have a poor prognosis and to be resistant to conventional chemotherapy. This is a good example to support the failure of targeted therapies for cancer stem cells.

Several treatment methods have been devised based on the cancer stem cell hypothesis, and many of the known methods use the self renewal pathway of cancer stem cells. The important point in this treatment is to target only cancer stem cell self-renewal while maintaining normal stem cell self-renewal. For example, Notch signaling is carried out by an enzyme called secretase, which can be used to suppress tumors by using secretase inhibitors in breast cancers that overexpress Notch1. Recently, targeting the Hedgehog signaling system has been shown to be anti-cancer effect. The tumor shrinked dramatically when Hedgehog inhibitor cyclopamine was administered to tumor xenograft animals.

Breast cancer, on the other hand, is a common cancer in women and is known to be the leading cause of death in female cancer patients (al A, Bray F, Center MM, Ferlay J, Ward E and Forman D. Globalcancer statistics. CA Cancer J Clin. 2011; 61 (2): 69-90). Extensive mammography and adjuvant therapy with polychemotherapy, tamoxifen, and early chemotherapy for early breast cancer have reduced breast cancer mortality, but breast cancer is still known to be the most dangerous disease due to recurrence and metastasis. Cancer stem cells (CSCs) were first identified in myeloid leukemia and then in various solid cancers, including breast, brain, colon, ovary, pancreatic, and prostate cancers. The cancer stem cells are also called tumor-initiating cells and cancer stem-like cells. It has also been shown that various cancer types, including breast cancer, originate from cancer stem cells (CSCs), a subpopulation of tumors. Such populations are known to cause changes in tumor volume through self-renewal and differentiation. Shh (Sonic hedgehog), Stat3, NF-κB, Wnt / β-catenin, TGF-β and Notch signaling pathways are known to be critical for self-renewal of CSCs.

Cancer stem cells exhibit drug resistance and radiation resistance to chemotherapy and radiation therapy and cause cancer to recur and metastasize. Thus, targeted therapies for cancer stem cells are essential for the treatment of cancer. Cancer stem cells are known to express certain proteins, including Oct4, C-myc, Nanog, and Aldehyde dehydrogenase-1 (ALDH). The ALDH is an enzyme that oxidizes toxic aldehydes, and its enzymatic activity is widely used as a CSC (cancer stem cells) marker of leukemia, head and neck, bladder, bone, colon, liver, lung, pancreas, prostate, thyroid and cervical cancer. have. ALDH is known as a therapeutic target for cancer stem cells. In addition, tumor formation is known to be superior in breast cancer populations expressing CD44 ^high / CD24 ^low in clinical samples (Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ and Clarke MF.Prospective identification of tumorigenic breast cancer cells.Proc Natl Acad Sci US A. 2003; 100 (7): 3983-3988).

Stat3 (Signal transducers and activators of transcription 3) and NF-κB are mainly activated in CSCs, and mammosphere formation is associated with JAK1 / 2-STAT3 and NF-κB pathways. Secreted IL-6 activates the JAK1 / 2-STAT3 pathway and increases expression of the Oct4 gene. The IL-6 / JAK1 / 2-STAT3 signaling pathway is known to be important for the conversion of NSCCs (Non-CSCs) to CSCs. Blocking the STAT3 signaling pathway is known to inhibit the growth of breast cancer cell-derived CD44 ^high / CD24 ^low stem cell-like cells. Nuclear factor-κB (NF-κB) transcription factor is structurally (constantly) activated in tumor cells, including colon, breast and liver cancer, and regulated by the IκB kinase (IKK) complex. Pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-κB, is known to inhibit breast cancer stem-like cells.

The breast cancer stem cells are known to be identified by expression of biomarkers such as CD44 ^high / CD24 ^low , ESA + (epithelial specific antigen) and ALDH. Chemotherapy is known to increase the proportion of cancer cells expressing CD44 ^high / CD24 ^low and mammoth formation. CSCs overexpress specific ABC transporters to protect CSCs from toxins. ABC pumps are used to isolate the side population (SP) and can be classified by ABCG2 transporter-specific Hoechst 33342 dyes. Because breast CSCs produce low levels of reactive oxygen species (ROS) compared to tumor cells, breast cancer stem-likes cells are radiation resistant. Because ROSs are a major mediator of ionizing radiation-induced cell death, CSCs are known to have less DNA damage than non-stem cancer cells (Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, Qian D, Lam JS, Ailles LE, Wong M, Joshua B, Kaplan MJ, Wapnir I, Dirbas FM, Somlo G, Garberoglio C, et al.Association of reactive oxygen species levels and radioresistance in cancer stem cells.Nature. 2009; 458 (7239): 780-783).

Breast cancer cell lines MCF-7 and MDA-MB-231 are known to have a subset of cells that have similar capabilities to stem cells that can be grown in oval form without cell apoptosis without attachment in vitro. Artificially creating a non-basement condition by floating culture, the cells with stem cell properties are attached to each other to form a spherical cell mass, which is called a neurosphere. Applying this concept to human breast stem cells is the "mammosphere". Mammoth Fair contains eight times more progenitor cells than normal human breast cells, and can be passaged continuously. After several passages, 100% of the cells grow into bi-potent precursors. Mammoth is capable of differentiating into mammary gland epitherlial cells, ductal epithelial cells, and alveolar epitherlial cells, which are adult breast cells. It is observed to form a complex functional breast structure while forming a three-dimensional structure. Mammoth fair is one of the most characteristic characteristics of stem cells is capable of self-proliferation, so that a large number of mammo pairs or breast stem cells can be obtained from a single mammo pair. In addition, compared with hematopoietic stem cells, neural stem cells, embryonic stem cells, etc., many expression genes were confirmed to overlap, and mammospheres were reported to be actual breast stem cells. The standard method of analyzing the self-renewal ability of cancer stem cells is to analyze the implantation in vivo and the mammosphere formation in vitro.

In addition, cells having stem cell properties may be attached to each other to form spherical cell masses in various cancer cell lines including breast cancer cells as well as lung cancer, which is called a tumorsphere. The two pairs refers to tumor cells developed by the proliferation of one cancer stem cell or cancer progenitor cell.

To date, studies on cancer stem cells have many limitations, and the role of cancer stem cells in the formation and maintenance of tumors is not clear. In order to efficiently perform treatments targeting cancer stem cells without damaging normal stem cells, knowledge and understanding of molecular biological characteristics and its regulatory pathways that are important for the maintenance and regulation of cancer stem cells are required.

To date, there is little research on anticancer drugs or natural extracts that directly target cancer stem cells. In the prior art, experiments have been conducted to inhibit cancer stem cells by inhibiting cancer stem cells or inhibiting higher signaling proteins of cancer stem cells. However, in many tumor patients, this targeting experiment was difficult due to oncogene mutation or protein mutation.

To date, there is little research on anticancer drugs or natural extracts that directly target cancer stem cells. In the prior art, experiments have been conducted to inhibit cancer stem cells by inhibiting cancer stem cells or inhibiting higher signaling proteins of cancer stem cells. However, in many tumor patients, this targeting experiment was difficult due to oncogene mutation or protein mutation.

An object of the present invention is to provide a composition for inhibiting cancer stem cell growth, comprising a primaquin or a pharmaceutically acceptable salt thereof represented by the formula (1) as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer, comprising the composition for inhibiting cancer stem cell growth.

Another object of the present invention, to provide a pharmaceutical composition for inhibiting cancer metastasis, comprising the composition for inhibiting cancer stem cell growth.

Another object of the present invention is to provide a food composition for improving or preventing cancer, comprising the composition for inhibiting cancer stem cell growth.

Another object of the present invention is to provide a food composition for improving or preventing cancer metastasis, comprising the composition for inhibiting cancer stem cell growth.

Another object of the present invention is to provide a method for inhibiting the growth of cancer stem cells, comprising administering a primaquin or a pharmaceutically acceptable salt thereof represented by Formula 1 to a subject.

Another object of the present invention is to provide a method for inhibiting cancer metastasis, comprising administering to a subject a primaquine or a pharmaceutically acceptable salt thereof represented by the formula (1).

Another object of the present invention is to provide a method for preventing or treating cancer, comprising administering primaquine represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject.

 Another object of the present invention is to provide a use for the preparation of a medicament for inhibiting the growth of cancer stem cells, the primaquin or a pharmaceutically acceptable salt thereof represented by the formula (1).

Another object of the present invention is to provide a use for use in the manufacture of a medicament for inhibiting cancer metastasis, Primaquin or a pharmaceutically acceptable salt thereof represented by the formula (1).

Still another object of the present invention is to provide a use for the preparation of a medicament for preventing or treating cancer of primaquin or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1.

Primaquine of the present invention inhibited the growth of breast cancer cells and inhibited the formation of breast cancer stem cells. In addition, it inhibited the expression of one or more self-renewal genes selected from Nanog, C-myc, Oct4, Sox2, Snail and CD44, which are known to be characteristically expressed in breast cancer stem cells. It has been shown to inhibit the STAT3 signaling pathway known to be involved in formation. Accordingly, the compound inhibits the growth of cancer stem cells such as breast cancer and the growth of these cancers, and thus can be used for the treatment of cancers such as breast cancer.

1 shows that primaquine inhibits various cancer features in breast cancer cell lines. Specifically, (A and B) shows the survival rate of MCF-7 and MDA-MB-231 cells following the treatment with primaquine phosphate structure and primaquine. MCF-7 and MDA-MB-231 cells were treated with increasing concentrations of primaquine for 48 hours. The anti-proliferative effect of primaquine was measured by MTS analysis.

 (C, D, E) The effect of primaquine on cell death of breast cancer cells is shown. MDA-MB-231 cells were treated with primaquine for 24 hours and killed cells were analyzed by FACS using the Annexin V-PI staining kit. Caspase3 / 7 activity in MDA-MB-231 cells was analyzed by Caspase-Gloss 3/7 kit. Apoptotic cells were analyzed by fluorescence staining, and nuclei were stained with Hoechst 33258 (enlarged, x200) in breast cancer.

(F) Effects of primaquine on the migration potential of human breast cancer cells. Wound healing of MDA-MB-231 cells was taken at 0 and 18 hours, depending on whether or not primaquine treatment.

(G) shows the effect of primaquine on colony formation in human breast cancer cells. The dissociated 1000 MDA-MB-231 cells were seeded in 6-well plates and treated with the indicated concentrations of primaquine and DMSO for 7 days. Representative images of colonies were recorded. Data shown represent mean ± SD of three independent experiments. * p <0.05 vs. DMSO-treated control.

2 shows the effect of primaquine on tumor growth in xenograft models. Three million cells were injected into the mammary fat pad of immunodeficient NOD-SCID female nude mice.

(A) The effect of primaquine on tumor growth in immunodeficient nude mice producing MCF-7 cells. The drug dosage used is 10 mg / kg / day. After 9 weeks, the images were captured as Odyssey® images (LICOR, pearl image system, USA). High grade of tumor using IRDye 800 CW optical probe (2DG) was used to detect breast tumors in 800 nm channels and marked in pseudo-color.

(B) Tumor volume was measured twice a week using a caliper and calculated as (width ² × length) / 2. Tumor growth curves were monitored during the experiment.

(C) shows the effect of primaquine on the weight of the tumor.

Tumor volume was measured after the start of treatment. * P <0.05 compared to the control. Representative images were captured at the end of 9 weeks post-treatment, and the results were shown in vehicle treated control, primaquine treated mice.

3 shows the effect of primaquine on mammoth pair formation. MCF-7 and MDA-MB-231 cells were incubated under mammoth pairing conditions for 7 days.

(A) shows the effect of primaquine on MCF-7 cell-derived mammoth pair formation. Primary mammoth pairs were incubated with primaquine (2.5 and 5 μΜ) or DMSO.

(B) shows the effect of primaquine on the formation of mammoth pair derived from MDA-MB-231 cells. The mammo pairs were incubated with primaquine (10 and 20 μM) or DMSO. MCF-7 and MDA-MBB-231 cells were treated with primaquine and DMSO for 7 days of culture. Images were obtained with a 10x magnification microscope and show representative mammoth pairs (scale bar = 100 μm).

 Data shown represent mean ± SD of three independent experiments. * p <0.05 vs. DMSO-treated control.

4 shows the effect of primaquine on the expression of cancer stem cell markers in breast cancer cell lines.

(A) Shows CD44high / CD24low cell population analyzed by flow cytometer analysis in Primaquin (5 μM) or DMSO treated MDA-MB-231 cells. For FACS analysis, 50,000 cells were obtained. Gating was based on control antibodies.

(B) shows the effect of primaquine on ALDH positive cell population. MDA-MB-231 cells were treated with primaquin (10 μM) or DMSO for 2 days, followed by ALDEFLUOR analysis and FACS analysis. The right panel shows ALDH positive cells treated with DEAB, an ALDH inhibitor as negative control, and the left panel shows ALDH positive cells untreated with DEAB. ALDH positive populations are marked in boxes.

5 shows the effect of primaquine on the STAT3 signaling pathway in breast cancer mammospheres.

(A) shows the effect of primaquine on the STAT3 signaling pathway in mammoth pairs. Nuclear protein expression and activation of STAT3 and NF-kB were measured in mammoth pairs with antibodies to pSTAT3, STAT3, P65 and Lamin B. Primaquine reduced levels of nuclear p-STAT3 protein in mammoths.

(B) EMSA (electrophoretic mobility shift) analysis of MDA-MB-231 cell-derived mammoth nuclear lysates treated with primaquine. Nuclear lysates were incubated with a biotin-labeled Stat3 probe and isolated by 6% PAGE.

Lane 1: probe alone; Lane 2: probe + nuclear extract; Lane 3: probe + primaquine treated nuclear extract; Lane 4: self competition; Lane 5: nuclear extract incubated with mutant STAT3 probe. The primaquine reduced DNA / STAT3 interaction in mammoth nuclear lysate.

(C) Transcriptional expression levels of the CSC markers Nanog, C-myc, Oct4, Sox2, snail and CD44 genes were determined by real-time PCR (RT-PCR) using CSC marker specific primers in primaquine and DMSO-treated mammoth pairs. It was analyzed using. β-actin was used as an internal control.

(D) shows the effect of primaquine on mammosphere growth. The primaquine inhibits mammoth growth. The primaquine and DMSO treated mammoth pairs were dissociated into single cells for 2 days and plated in 6 cm dishes with the same cell number. After 24 hours of plating, cells were counted. On days 2 and 3, cells were counted and plotted to mean value. The data represent the mean ± SD of three independent experiments. * p <0.05 vs. DMSO-treated control.

The present inventors confirmed that cancer stem cells inhibit growth of cancer stem cells using various compounds, and among them, primaquine selectively inhibits breast cancer stem cells. Primaquine is known to control malaria with antimalarial drugs, but for the first time we inhibit breast cancer stem cell growth and selectively inhibit STAT3 signaling pathways in breast cancer-derived mammoths as compared to MCF-7 bulk cells. It was confirmed that. In addition, mouse xenograft models were used to effectively inhibit tumor growth. Accordingly, Primaquin has confirmed that the targeting of CSCs can inhibit the growth of cancer stem cells, including breast cancer, and can be used to treat cancers including breast cancer.

In order to achieve the above object, the present invention provides a composition for inhibiting cancer stem cell growth comprising a primaquin or a pharmaceutically acceptable salt thereof represented by Formula 1 as an active ingredient.

[Formula 1]

In the present invention, the primaquin is known as an antimalarial drug, but it was confirmed by the present inventors to inhibit the growth of breast cancer stem cells for the first time. In the present invention, the pharmaceutically acceptable salt of primaquin may be primaquin phosphate, and may be represented by Formula 2, but is not limited thereto.

[Formula 2]

In the present invention, the term "cancer" generally refers to or describes the physiological state of a mammal that is characterized by unregulated cell growth. "Cancer" refers to a condition in which a problem occurs in the regulation of normal division, differentiation and death of cells, abnormally proliferating and invading surrounding tissues and organs to form agglomerates and destroy or modify existing structures.

In the present invention, the term "cancer stem cell" is an undifferentiated cell having the ability to differentiate into various cancer cells, the cancer including colorectal cancer and colorectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer , Brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer, small intestine cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer , Kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Penile cancer, central nervous system (CNS) tumors, primary CNS lymphoma, spinal cord tumors, brainstem glioma or pituitary adenoma. Although not limited thereto, the cancer stem cells may be stem cells of breast cancer.

As used herein, the term "breast cancer stem cell" refers to an undifferentiated cell having the ability to differentiate into breast cancer cells.

In the present invention, the term "breast cancer stem cell growth inhibition" is meant to include breast cancer stem cell maintenance (maintenance) inhibition, breast cancer stem cell malignance (inhibition), breast cancer stem cell migration and breast cancer stem cell invasive activity (invasive) inhibition.

In one embodiment of the present invention, to determine whether the primaquine can inhibit the growth of breast cancer stem cells, primaquine to the primary mammosphere (mammosphere) derived from MCF-7 and MDA-MB-231 cells As a result, it was confirmed that primaquine inhibited the formation of primary mammoths derived from breast cancer cell lines. Specifically, the number of mammoths derived from MCF-7 and MDA-MB-231 cells, which are breast cancer cells, was confirmed. Not only was confirmed to decrease by 70-90%, it was also confirmed that the size of the mammoth pair is also reduced (FIGS. 3A and 3B). Accordingly, it was confirmed that the primaquin of the present invention can inhibit the formation of mammospheres or inhibit the proliferation of mammospheres.

Accordingly, the compound of the present invention can inhibit the formation of breast cancer-derived mammoth (mammosphere), or inhibit the proliferation of breast cancer-derived mammoth.

In one embodiment of the present invention, the breast cancer stem cells may express one or more self-renewal genes selected from Nanog, C-myc, Oct4, Sox2, Snail and CD44. In one embodiment of the present invention, primaquin inhibited the expression of self-renewing genes such as Nanog, C-myc, Oct4, Sox2, Snail and CD44, which are known to be characteristically expressed in breast cancer stem cells (FIG. 5C). It was confirmed that the STAT3 signaling pathway was inhibited by being involved in mammosphere formation of cells (FIGS. 5A and 5B). Accordingly, it was confirmed that the compound can inhibit the growth of breast cancer stem cells.

The composition of the present invention can be used as a pharmaceutical composition or food composition.

When the composition of the present invention is utilized as a pharmaceutical composition, it may include the primaquine or a pharmaceutically acceptable salt thereof.

 As used herein, the term "pharmaceutically acceptable salts" refers to all salts that retain the desired biological and / or physiological activity of the compound and exhibit minimal unwanted toxicological effects. Salts prepared according to conventional methods in the art, which methods are known to those skilled in the art. Specifically, the pharmaceutically acceptable salts include, but are not limited to, salts derived from pharmacologically or physiologically acceptable inorganic acids and organic acids and bases.

For example, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases may include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines; Substituted amines, including naturally occurring substituted amines; And isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, Hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamine, theobromine, purine, piperazine, piperidine, and / or N-ethylpiperi Salts of cyclic amines, including dean. Also included are other carboxylic acid derivatives, such as carboxamides, lower alkyl carboxamides, di (lower alkyl) carboxamides, and the like.

For example, pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids are acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid , But may include, but is not limited to, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and / or salicylic acid.

In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier or additive. In the present invention, the "pharmaceutically acceptable" means that the subject of application (prescription) is not toxic as long as it is adaptable without inhibiting the activity of the active ingredient. The term "carrier" is defined as a compound that facilitates the addition of the compound into cells or tissues.

Primaquines of the present invention may be administered alone or in admixture with any convenient carrier and the like, and such dosage forms may be single dose or repeated dose formulations. The pharmaceutical composition may be a solid formulation or a liquid formulation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like. Liquid formulations include, but are not limited to, solutions such as water, propylene glycol solutions, suspensions, emulsions, and the like, and may be prepared by adding suitable colorants, flavors, stabilizers, viscosity agents, and the like. For example, powders may be prepared by simple mixing of the active ingredient of the present invention primaquine with a suitable pharmaceutically acceptable carrier such as lactose, starch, microcrystalline cellulose. Granules are the primaquin of the present invention; Suitable pharmaceutically acceptable carriers; And a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone, hydroxypropyl cellulose, and the like, and then prepared by using a wet granulation method using a solvent such as water, ethanol, isopropanol, or a dry granulation method using a compressive force. Can be. Tablets may also be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting using a tableting machine.

Primaquine of the present invention can be administered orally, by injection (eg, intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalation, nasal administration, depending on the condition and condition of the subject to be treated. It may be administered as a vaginal agent, rectal administration agent, sublingual agent, transdermal agent, topical agent, etc., but is not limited thereto. It may be formulated into a suitable dosage unit dosage form comprising a pharmaceutically acceptable carrier, excipient, vehicle, conventionally used and nontoxic, depending on the route of administration.

The pharmaceutical composition of the present invention may be administered daily from about 0.0001 mg / kg to about 10 g / kg, and may be administered in a daily dosage of about 0.001 mg / kg to about 1 g / kg. However, the dosage may vary depending on the degree of purification of the mixture, the condition of the patient (age, sex, weight, etc.), the severity of the condition being treated, and the like. If desired, the total daily dose may be divided several times a day for convenience.

When the composition of the present invention is used as a pharmaceutical composition, the content of primaquine in the composition can be appropriately adjusted to an effective amount capable of exhibiting anti-inflammatory activity according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, and the like, for example, The amount of queen may be 0.0001 wt% or more, specifically 0.001 wt% or more, 80 wt% or less, specifically 50 wt% or less, based on the total weight of the total composition, but is not limited thereto.

In addition, the primaquin of the present invention was confirmed that inhibits the growth (proliferation) of breast cancer cell-derived mammoth, can be used as a food composition for inhibiting breast cancer stem cell growth.

When the composition of the present invention is used as a food composition, it may include an acceptable food supplement additive, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of food.

In the present invention, the food means a natural product or a processed product containing one or more nutrients, and specifically, means a state in which it can be directly eaten through a certain degree of processing. Used to include all functional foods, beverages, food additives and beverage additives. Examples of the foods include various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, the food of the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other dietary supplements. The functional food, beverages, food additives or beverage additives may be prepared by a conventional manufacturing method.

The term "functional food" refers to the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body control function related to the living body, specifically, it may be a health functional food.

As used herein, the term "health functional food" refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, the term "function" means obtaining a useful effect for health purposes such as nutrient control or physiological action on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food. Food composition of the present invention can be prepared in various forms of formulation, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material, and excellent portability, the present invention Dietary supplements are available as supplements to enhance the effects of breast cancer stem cell growth inhibition.

In addition, the functional food may include a food-acceptable food supplement additive, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition, in the food composition, the amount of primaquine may be at least 0.00001% by weight, specifically 0.1% by weight or more, 80% by weight or less, specifically 50% by weight or less, more specifically 40% by weight or less of the total weight of the food composition. When the food is a beverage, based on 100 ml of the total volume of the food, 0.001 g or more, specifically 0.01 g or more, 50 g or less, specifically 10 g or less, more specifically 2 g or less May be, but is not limited thereto.

The food composition of the present invention may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients. Sweeteners may be used in amounts that give the food a suitable sweet taste, and may be natural or synthetic. Specifically, a natural sweetener is used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose. Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing specifically ,. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. Moreover, what was obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like. As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used. As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.

In addition, the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetening agent.

Such preservatives, emulsifiers and the like are preferably added and used in very small amounts as long as the use to which they are added can be achieved. By trace amounts it is meant numerically in the range of 0.0005% to about 0.5% by weight based on the total weight of the food composition. Examples of preservatives that can be used include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like. Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like. Examples of acidulants that may be used include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste. Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling agents and the like.

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing cancer, comprising the composition for inhibiting cancer stem cell growth.

In one embodiment of the present invention, when treated with primaquine in the MCF-7 cell line and MDA-MB-231 cell line, it was confirmed that the growth of breast cancer cell lines is inhibited (Fig. In addition, in one embodiment of the present invention, the primaquine inhibitor reduced the population expressing CD44 ^high / CD24 ^low in breast cancer cells (FIG. 4A), and confirmed that the ratio of ALDH positive breast cancer cells was reduced (FIG. 4B). . Accordingly, in the present invention, the cancer may be, but is not limited to, breast cancer, the composition may inhibit the growth of breast cancer cells expressing CD44 ^high / CD24 ^low , aldehyde dehydrogenase (ALDH) positive breast cancer Inhibit the growth of cells.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis, comprising the composition for inhibiting cancer stem cell growth.

The cancer is classified into primary cancer existing at the site of occurrence and metastatic cancer that has spread from the site of development to other parts of the body. The "cancer metastasis" refers to a state in which a malignant tumor has propagated to another tissue away from the organ. Cancer cells are formed by spreading through the blood circulation or lymph circulation, usually by blood circulation to other organs and then growing into new tumors. Cancer cells, on the other hand, are formed by moving directly to neighboring tissues. In the present invention, cancer metastasis is a metastasis of cancer cells by invasion in which cancer cells move and penetrate directly into neighboring tissues, and cancer cells move through the bloodstream to form new tumors in organs that are not physically adjacent to primary cancer. Include all metastasis. On the other hand, in the cancer metastasis, the movement of cells is essential. Therefore, it is obvious that inhibiting the migration of cancer cells is the primary method of preventing cancer metastasis.

In the present invention, the cancer is not limited thereto, but may be breast cancer. In the present invention, the terms "cancer", "cancer stem cells", "cancer stem cell growth inhibition", "pharmaceutical composition" is as described above.

In one embodiment of the present invention, it was confirmed that primaquine inhibits the migration and colony formation of MDA-MB-231 cells in a concentration-dependent manner (FIGS. 1F and 1G). Accordingly, the composition of the present invention can inhibit cancer metastasis by inhibiting the movement of cancer cells, it can be utilized as a pharmaceutical composition for inhibiting cancer metastasis.

In another aspect, the present invention provides a food composition for improving or preventing cancer, comprising the composition for inhibiting cancer stem cell growth.

In the present invention, the terms "cancer", "cancer stem cells", "cancer stem cell growth inhibition", "food composition" is as described above. In one embodiment of the present invention, when treated with primaquine in the MCF-7 cell line and MDA-MB-231 cell line, it was confirmed that the growth of breast cancer cell lines is inhibited, can be used as a food composition for improving or preventing cancer Do. In the present invention, the cancer may be breast cancer, but is not limited thereto.

In another aspect, the present invention provides a food composition for improving or preventing cancer metastasis, comprising the composition for inhibiting cancer stem cell growth.

In the present invention, the terms "cancer metastasis", "cancer stem cells", "cancer stem cell growth inhibition", "food composition" is as described above. In one embodiment of the present invention, it was confirmed that primaquine inhibits the migration and colony formation of MDA-MB-231 cells in a concentration-dependent manner, and thus inhibits cancer metastasis. As a food composition for improving or preventing cancer metastasis, Available. In the present invention, the cancer may be breast cancer, but is not limited thereto.

In another aspect, the present invention provides a method of inhibiting the growth of cancer stem cells, comprising administering to a subject a primaquine or a pharmaceutically acceptable salt thereof represented by Formula 1 below.

[Formula 1]

In the present invention, the description of the terms "primaquine", "cancer", "cancer stem cell", "cancer stem cell growth inhibition" is as described above.

In the present invention, the term "individual" means all animals including humans having cancer metastases or having cancer. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, wherein the growth of cancer stem cells is inhibited by administration of the composition of the present invention, thereby limiting the individual to whom the cancer is treated. Includes without.

Another object of the present invention is to provide a method for inhibiting cancer metastasis, comprising administering a primaquin or a pharmaceutically acceptable salt thereof represented by Formula 1 to a subject.

In the present invention, the description of the terms "primaquine", "cancer", "individual", "cancer metastasis" is as described above.

Still another object of the present invention is to provide a method for treating or preventing cancer, comprising administering to a subject a primaquine or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1.

In the present invention, the description of the terms "primaquine", "cancer", "individual", "treatment", "prevention" is as described above.

 Another object of the present invention is to provide a use for the preparation of a medicament for inhibiting the growth of cancer stem cells, the primaquin or a pharmaceutically acceptable salt thereof represented by the formula (1).

In the present invention, the description of the terms "primaquine", "cancer", "cancer stem cell", "cancer stem cell growth inhibition" is as described above.

Another object of the present invention is to provide a use for the preparation of a medicament for inhibiting cancer metastasis of primaquin or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1.

In the present invention, the description of the terms "primaquine", "cancer", "cancer metastasis" is as described above.

Still another object of the present invention is to provide a use for the preparation of a medicament for preventing or treating cancer of primaquin or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1.

In the present invention, the description of the terms "primaquine", "cancer", "treatment", "prevention" is as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<A: Experimental Materials and Methods>

Example  1: test material

Six-well culture plates containing very low adherent cluster plates were obtained from Corning (Tewksbury, MA, USA). Primaquine was purchased from Sigma-Aldrich Co. (St. Louis, Mo., USA). Cell viability was measured using CellTiter 96® aqueous one solution cell proliferation assay kit (Promega, Madison, Wis., USA). The ALDEFLUOR ™ Kit was purchased from STEMCELL Technologies Inc (Vancouver, BC, Canada).

Example  2: human breast cancer cell culture and Mammoth Fair ( mammospheres ) formation

Human breast cancer cells, MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were treated with Dulbecco's Modified Essential Medium (DMEM; Hyclone, 10% fetal bovine serum (FBS; Hyclone), 100 U / ml penicillin, and 100 μg / ml streptomycin (Hyclone). In Logan, UT, USA). The MCF-7 and MDA-MB-231 cells were maintained at 37 ° C. in a humidified incubator containing 5% CO ₂ . Cells were plated at a density of 1 × 10 ⁶ cells in a 10 cm culture dish. To establish the primary mammoth, single cell suspended MCF-7 and MDA-MB-231 cells were ultra-low attachment 6-well containing 2 ml of complete MammoCult ^™ medium (StemCell Technologies, Vancouver, BC, Canada). Plates were inoculated with cell numbers of 3.5-4 × 10 ⁴ and 0.5-1 × 10 ^{4 per} well. The complete MammoCult ^™ medium was supplemented with 4 μg / ml heparin, 0.48 μg / ml hydrocortisone, 100 U / ml penicillin and 100 μg / ml streptomycin. The cells were incubated for 7 days at 37 ° C., 5% CO ₂ incubator.

Example  3: breast cancer Mammoth  Automatic calculation

On day 7 of culturing, the cell culture plates were placed in a scanner (Epson Perfection V700 PHOTO, Epson Korea, Co, Seoul, Korea) to obtain 8-bit gray scale images of mammo pairs. At low resolution (300 dpi) images were obtained using a NICE software program and downloaded from ftp://ftp.nist.gov/pub/physics/mlclarke/NICE. For counting, the desired number of rows and columns (e.g., 2 x 3 for 6-well plates) were selected to generate ROIs of interest, and after selecting the elliptical setting of the NICE program, the individual ROIs were The provided ROI shape was moved and scaled to define. The background signal of the image was negated using a threshold algorithm, and the selected image was automatically counted. The mammosphere formation assay determined the formation efficiency (MFE,%) of mammo pairs corresponding to the number of mammo pairs per well / total number of plated cells per well x100.

Example  4: Breast Cancer Cell Proliferation Assay

Proliferation rates of MCF-7 and MDA-MB-231 cells were measured using CellTiter 96® aqueous one solution cell proliferation kit. MCF-7 and MDA-MB-231 cells were incubated in 96-well plates in the presence of primaquin 0, 5, 10, 20, 40, and 80 μM for 48 hours. Absorbance was determined at 490 nm using a 96-well plate reader (Dynex Revelation, Dynex Ltd., Billingshurst, UK) according to the manufacturer's protocol. Each data was determined by measuring three sets.

Example  5: Caspase -3/7 ( Caspase -3/7) analysis

Cancer cells were treated with different concentrations of primaquine for 24 hours. Caspase-3 / 7 activity was according to the manufacturer's instructions for the Capase-Glo 3/7 kit (Promega, Wisconsin, USA). 100 μl of Caspase-Glo3 / 7 reagent was added to 96-well cultured cancer cells. Plates were covered with a plate sealer and incubated at room temperature for 6 hours and measured using a plate-reading luminometer, GloMax® Explorer (Promega, Wisconsin, USA).

Example  6: Annexin ( Annexin A) V / PI staining analysis

Cancer cells were cultured in 6-well plates and incubated with primaquine (20 μΜ) or DMSO for 24 hours. Bistained with PI and FITC-Annexin V according to manufacturer's instructions. The samples were analyzed by flow cytometry (Accuri C6, BD, San Diego, CA, USA).

Example  7: Cell death assay by fluorescence staining

MDA-MB-231 cells were treated with primaquin 20 μM for 24 hours, and the cells were incubated for 10 minutes at 37 ° C. with Hoechst 33258 solution (10 mg / ml). The cells were then observed under a fluorescence microscope.

Example  8: Clonogenicity  analysis

MDA-MB-231 cells were seeded at low density in 6-well plates and treated with different concentrations of primaquine in DMEM medium. After 24 hours the medium was replaced with fresh medium and incubated for 7 days of growth. Grown colonies were counted.

Example  9: scratch  Moving analysis

MDA-MB-231 cells were seeded in 6-well plates and grown to 90% confluency. Using a sterile white micro pipette tip, the cell layer was scratched. After washing with DMEM medium, breast cancer was treated with primaquine or DMSO. At 18 hours, wounded areas were photographed with a 10x optical microscope.

Example  10: CD44 and CD24 expression Flow cell  Analysis (Flow cytometric  analysis)

Expression of CD44 and CD24 in MDA-MB-231 cells was measured by FACS analysis. After isolation and harvesting cells using 1 × Trypsin / EDTA, one million cells were suspended and FITC-bound anti-human CD44 and PE-bound anti-human CD24 antibodies (BD Pharmingen, San Diego, CA, USA) ) And incubated at 4 ° C. for 30 minutes. The cells were then washed three times with 1 × PBS and analyzed by flow cytometry (Accu C6, BD, San Diego, Calif., USA).

Example  11: real time PCR  (RT- PCR )

According to the manufacturer's instructions, levels of transcripts were measured with a One Step SYBR PrimeScript RT-PCR kit (Takara, Tokyo, Japan) using SYBR Green as a double stranded DNA specific dye. One-step RT-PCR reactions were performed on 1 μg total RNA, 10 μl 2X One Step SYBR RT-PCR Buffer IV, 1 μl PrimeScript 1 step Enzyme Mix II, CD44, NANOG, OCT4, SOX2, C-myc, Snail and β-actin. A final volume of 20 μl per reaction was performed, including 10 μM PCR forward primer, and PCR reverse primer.

The forward and reverse primers are as follows.

CD44 forward primer: AGAAGGTGTGGGCAGAAGAA (SEQ ID NO: 1),

CD44 reverse primer: AAATGCACCATTTCCTGAGA (SEQ ID NO: 2),

NANOG forward primer: ATGCCTCACACGGAGACTGT (SEQ ID NO: 3),

NANOG reverse primer: AAGTGGGTTGTTTGCCTTTG (SEQ ID NO: 4),

OCT4 forward primer: AGCAAAACCCGGAGGAGT (SEQ ID NO: 5),

OCT4 reverse primer: CCACATCGGCCTGTGTATATC (SEQ ID NO: 6),

SOX2 forward primer: TTGCTGCCTCTTTAAGACTAGGA (SEQ ID NO: 7),

SOX2 reverse primer: CTGGGGCTCAAACTTCTCTC (SEQ ID NO: 8),

C-myc forward primer: AATGAAAAGGCCCCCAAGGTAGTTATCC (SEQ ID NO: 9),

C-myc reverse primer: GTCGTTTCCGCAACAAGTCC (SEQ ID NO: 10),

Snail forward primer: ACCACTATGCCGCGCTCTT (SEQ ID NO: 11),

Snail reverse primer: GGTCGTAGGGCTGCTGGAA (SEQ ID NO: 12),

β-actin forward primer: TGTTACCAACTGGGACGACA (SEQ ID NO: 13),

β-actin reverse primer: GGGGTGTTGAAGGTCTCAAA (SEQ ID NO: 14).

The relative expression level of mRNA of the target gene was calculated using the comparative CT method. At least three independent PCR procedures were performed following statistical analysis. PCR products were normalized to the β-actin gene as an internal control.

Example  12: ALDEFLUOR  analysis

The ALDEFLUOR assay system provides a novel approach to the identification, evaluation and isolation of CSCs based on the activity of aldehyde dehydrogenase (ALDH). Active reagent BODIPY-aminoacetaldehyde was added to breast cancer cells and converted to fluorescent BODIPY-aminoacetate by aldehyde dehydrogenase (ALDH). Diethylaminobenzaldehyde (DEAB), an ALDH inhibitor, was used as a negative control. MDA-MB-231 cells were treated with 10 μM for 24 hours, and the percentage of ALDH positive cells was analyzed by ALDEFLUOR assay. ALDH positive and negative cells were sorted using flow cytometry (Accuri C6, BD, San Diego, CA, USA).

Example  13: Weston Blotting

Proteins isolated from primaquine treated MCF-7 and MDA-MB-231 mammoth pairs were separated on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass., USA). The membrane was blocked in PBS-Tween 20 (0.1%, v / v) containing 5% skim milk powder at room temperature for 30 minutes. The blots were incubated overnight at 4 ° C. with blocking solutions containing primary antibodies. Primary antibodies used were as follows: Stat3, p65, Lamin B, and phospho-Stat3 (Cell Signaling, Beverly, MA, USA). β-actin (Santa Cruz Biotechnology) was used as a loading control. After washing with PBS-Tween 20 (0.1%, v / v), the blots were incubated with horseradish peroxidase-bound secondary antibody and photosensitized with a chemiluminescence detection kit (Santa Cruz Biotechnology).

Example  14: Electrophoretic  mobility shift assays ( EMSA )

EMSA was detected using Lightshift's chemiluminscet EMSA kit (Thermoscientific, IL, USA) according to the manufacturer's instructions. The biotin-top and bottom probs of the Stat3 probe (5'-CTTCATTTCCCGGAAATCCCTA-Biotin3 ', SEQ ID NO: 15 and 5'-TAGGGATTTCCGGGAAATGAAG-Biotin3', SEQ ID NO: 16) were annealed and the double-stranded oligonucleotides were terminally labeled with biotin. Nuclear extracts from MCF-7 and MDA-MB-231 cells were prepared as shown in the references (Choi HS, Hwang CK, Kim CS, Song KY, Law PY, Wei LN and Loh HH. Transcriptional regulation of mouse mu opioid receptor gene: Sp3 isoforms (M1, M2) function as repressors in neuronal cells to regulate the mu opioid receptor gene.Mol Pharmacol. 2005; 67 (5): 1674-1683).

Biotin-labeled DNA probes were incubated with primaquine treated nuclear proteins in a final volume of 20 μL EMSA buffer containing 1 μg / μL poly [dI-dC] at room temperature for 20 minutes. The reaction mixture was electrophoresed on 6% polyacrylamide unmodified gel in 0.5 × TBE (45 mM Tris borate and 1 mM EDTA) at 4 ° C. and visualized using a chemiluminescent nucleic acid detection kit (Thermoscientific, IL, USA) It was.

Example  15: Immunodeficiency NOD- Producing Breast Cancer Cells SCID  ( BALB Of cSIc  (nu / nu)) chemotherapy in female nude mice

NOD-SCID (BALB / cSIc (nu / nu)) female nude mice producing a total of 18 breast cancer cells were divided into three groups. Six mice as negative controls did not receive chemotherapy. However, the tumor volume of control mice was measured every three days and calculated using the formula (width ² × length) / 2. The other six nude mice received primaquine using breast fat pad infusion at an optimal dose of 10-50 mg / kg / day. The last group remaining was used as non-tumor group without treatment.

Example  16: Statistical analysis

All data were expressed as mean ± standard deviation (SD). Data was analyzed using student's t-test. P values lower than 0.05 were considered statistically significant (GraphPad Prism 5 Software, San Diego, CA, USA).

<B: Experimental Example > Effect on breast cancer stem cells and breast cancer

Experimental Example  One: Primaquine  Induction of cell death and inhibition of proliferation of human breast cancer cells

In order to investigate the antiproliferative effect in the human breast cancer cell lines MCF-7 and MDA-MB-231, which are shown in FIG. 1A, primaquin was treated by concentration, and then MTS analysis was performed. As a result, 48 hours after primaquin treatment, growth of breast cancer cell lines was inhibited in a concentration-dependent manner at a concentration of 80 μM or more of primaquin in MCF-7 cell line and 40 μM or more of primaquin in MDA-MB-231 cell line (FIG. 1B).

Next, it was confirmed that the number of mammary cancer cells (annexin V +) killed in MDA-MB-231 cells increased by treatment with Primaquin 20 μM (FIG. 1C).

Next, caspase 3/7 fluorescence was performed in MDA-MB-231 cells, and as a result, it was confirmed that the caspase 3/7 activity was induced by the treatment of primaquin 30 μM (FIG. 1D). . In addition, it was confirmed that apoptotic body formation was induced in the breast cancer cell line MDA-MB-231 by the treatment with primaquine (FIG. 1E). Primaquine also inhibited the migration and colony formation of MDA-MB-231 cells in a concentration-dependent manner (FIGS. 1F and 1G). These results indicate that primaquines effectively inhibit various cancer features (proliferation, migration, cell death and colony formation).

Experimental Example  2: Primaquine  Inhibitory effect of tumor growth in xenograft models

Primaquine was confirmed in FIG. 1 to inhibit the proliferation of breast cancer cells in vitro. Next, it was examined whether primaquine inhibited tumor induction in a xenograft tumor model. As a result, the tumor volume in the primaquin administration group was smaller than the control group not treated with primaquin (FIGS. 2A and 2B). In addition, the tumor weight in the primaquin treatment group was smaller than the control group not treated with primaquine (FIG. 2C). However, the body weights of the mice in the primaquine treated group were similar to the control (FIG. 2A). These results indicate that primaquines effectively inhibit tumor development in xenograft models.

Experimental Example  3: Primaquine  Inhibitory effect on breast cancer stem cells

To assess whether primaquine can inhibit the formation of tumorspheres, primary mammospheres derived from MCF-7 and MDA-MB-231 cells were treated with different concentrations of primaquine. . As shown in FIG. 3, primaquine inhibited the formation of primary mammoths derived from breast cancer cell line MCF-7. The number of mammo pairs was reduced by 70-90%, and the size of mammo pairs was also reduced (FIG. 3A). In addition, the treatment with pramaquin also reduced the number and size of primary mammospheres derived from MDA-MB-231 cells (FIG. 3B).

Experimental Example  4: Primaquine CD44high Of CD24low  Expressing populations ALDH  Reduces the proportion of benign breast cancer cells

MDA-MB-231 cells were treated with primaquine for 24 hours and the effects of the primaquine inhibitors were examined in subpopulations expressing CD44 ^high / CD24 ^low in breast cancer cells. As a result, the primaquine inhibitor reduced the population expressing CD44 ^high / CD24 ^low in breast cancer cells (FIG. 4A). MDA-MB-231 cells were treated with primaquine for 24 hours and ALDEFLUOR assay was performed to investigate the effect of primaquine inhibitors on the proportion of ALDH positive breast cancer cells. As a result, it was confirmed that primaquin reduced the ratio of ALDH positive breast cancer cells from 0.9% to 0.3% (FIG. 4B).

Experimental Example  5: Primaquine At Mammoth Fair STAT3  Effect of inhibiting signal pathways

To investigate the cellular function of primaquine, under STATquine treatment, STAT3 and NF-kB signaling pathways were investigated in mammoth pairs derived from MCF-7 cells. As a result, primaquine reduced phosphorylation of nuclear STAT3 protein as compared to the control. However, primaquine did not reduce the protein level of nuclear p65 (FIG. 5A).

In addition, the binding of the primaquine treated nuclear extract to Stat3 DNA was analyzed using a biotin-labeled Stat binding probe that binds STAT3 with high affinity. As a result, primaquine inhibited the binding of the biotin-labeled Stat probe to Stat3, as shown in FIG. 5B (FIG. 5B, lane 3). Specificity of the pStat3 / biotin-labeled Stat probe was determined by unlabeled excess self-competitor (Fig. 5B, lane 4) and mutated-Stat competitor (Fig. 5B, lane). 5). These data indicate that the Stat3 signaling pathway is important for regulating the growth and self-renewal of mammoth pairs, and that primaquine inhibits the self-renewal of mammoth pairs via the Stat3 signaling pathway.

Experimental Example  6: Primaquine Of cancer stem cells (CSCs)  Self-renewal gene expression Mammoth  Inhibiting proliferation

In order to confirm that primaquine inhibited the expression of self regenerative genes, self regenerative gene expression was examined by real-time PCR (RT-PCR). As a result, primaquine reduced the expression of self-renewing genes such as Nanog, Sox2, Oct4, snail, C-myc and CD44 in breast cancer cells (FIG. 5C).

Next, to confirm whether the primaquine inhibits the growth of the mammo pair, the mammoth pair was treated with the primaquine, and the number of cells of the mammo pair was counted. As a result, primaquine induced apoptosis of mammoth pairs, and the number of cells observed in primaquine treated mammoth pairs was small. Through these results, it was found that primaquin significantly reduced mammosphere proliferation (FIG. 5D).

Through the above experimental examples, the primaquine of the present invention not only inhibits the proliferation of breast cancer, but also confirms that it inhibits the growth of stem cells of breast cancer, and found that it can be used for growth inhibition of breast cancer and its stem cells. Could.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

The present invention was supported by the National Research Foundation (NRF) by the Basic Science Research Program (2016R1A6A1A03012862) and the Local University Specialization Project (CK-I).

Claims (14)

Comprising a primaquin or a pharmaceutically acceptable salt thereof represented by the formula (1) as an active ingredient, cancer stem cell growth inhibition composition. [Formula 1]

The composition of claim 1, wherein the pharmaceutically acceptable salt of primaquine is primaquine phosphate The composition of claim 1, wherein the cancer stem cells are stem cells of breast cancer. The composition of claim 1, wherein the compound inhibits the formation of mammospheres derived from breast cancer or inhibits the proliferation of mammospheres derived from breast cancer. The composition of claim 1, wherein the breast cancer stem cells express one or more self-renewal genes selected from Nanog, C-myc, Oct4, Sox2, Snail, and CD44. A pharmaceutical composition for treating or preventing cancer, comprising the composition of any one of claims 1 to 5. The pharmaceutical composition of claim 6, wherein the cancer is breast cancer. The pharmaceutical composition of claim 6, wherein the composition inhibits the growth of breast cancer cells expressing CD44 ^high / CD24 ^low . The pharmaceutical composition of claim 6, wherein the composition inhibits the growth of aldehyde dehydrogenase (ALDH) positive breast cancer cells. A pharmaceutical composition for inhibiting cancer metastasis, comprising the composition of any one of claims 1 to 5. The pharmaceutical composition of claim 10, wherein the cancer is breast cancer. A food composition for improving or preventing cancer, comprising the composition of any one of claims 1 to 5. A food composition for improving or preventing cancer metastasis, comprising the composition of any one of claims 1 to 5. A method of inhibiting the growth of cancer stem cells, comprising administering a primaquin or a pharmaceutically acceptable salt thereof represented by Formula 1 to a subject.  [Formula 1]

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