WO2017142348A1 - Pharmaceutical composition for treatment of cancer, containing polyphenol compound as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer, containing a polyphenol compound as an active ingredient. The pharmaceutical composition according to the present study is very effective for the treatment and prognosis-improvement of cancer stem cells or cancer tissues having a magnitude of cancer stem cells such as poorly differentiated cancer. Further, the polyphenol compound and a biguanide compound, and an anticancer agent were observed to remarkably increase inhibitory effects on the growth of cancerous cells when they were administered to cancerous cells in combination rather than individually, thus the composition is expected to find great applications in the cancer therapy field.

Description

Pharmaceutical composition for cancer treatment containing polyphenol compound as an active ingredient

The present invention relates to a pharmaceutical composition for treating cancer comprising the polyphenol compound as an active ingredient.

Cancer is one of the most common causes of death worldwide. About 10 million new cases occur each year, accounting for about 12% of all deaths, causing the third largest number of deaths. Therefore, while efforts to develop effective anticancer drugs have been continued, most recently developed anticancer drugs target general cancer cells, and are not effective for killing cancer stem cells, which play an important role in treatment resistance and recurrence of cancer patients. The cancer stem cell is a cancer cell having unlimited regenerative capacity similar to a general stem cell, and slowly proliferates differently from a general cancer cell, and has self-renewal or differentiation ability, which is unique to stem cells, and known cancer cells. Although it is known to have a different mechanism (mechanism), the development of a drug for treating cancer stem cells targeting cancer stem cells is still very incomplete (domestic application patent 10-2011-0066035). If cancer stem cells remain in the body even after cancer treatment, cancer recurrence and / or metastasis occurs actively, so developing cancer stem cell therapeutics is an urgent task.

On the other hand, Gossypol as a polyphenol compound corresponds to a phenol derivative contained in a large amount in a cotton plant. In China, such Gosipol has been found to inhibit sperm function in males, and has been developed as a male oral contraceptive pill, but recently it has been known that there is a significant effect on Gosipol's cancer cell growth inhibition (US Patent US6114397). However, it is difficult to effectively inhibit cancer cell growth by administering Gosipol alone alone.

The present invention relates to a pharmaceutical composition for treating cancer and cancer stem cells comprising a polyphenol compound as an active ingredient, wherein the pharmaceutical composition according to the present study contains a large amount of cancer stem cells such as cancer stem cells or low-differentiated cancer. It is very effective in treating cancer tissues and improving the prognosis. In addition, polyphenol compounds, biguanide-based compounds, and anticancer agents are used in combination with cancer cells, and thus, the effects of inhibiting cancer cell growth are significantly increased compared to those administered alone, and are expected to be greatly utilized in the field of cancer treatment.

The present invention has been made to solve the above problems in the prior art, and relates to a pharmaceutical composition for treating cancer comprising a polyphenol compound as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details are set forth, such as specific forms, compositions, processes and the like. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of “in one embodiment” or “embodiment” expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. In addition, particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, "cancer" is characterized by uncontrolled cell growth, and by this abnormal cell growth, a cell mass called a tumor is formed, penetrating into surrounding tissues, and in severe cases, to other organs of the body. It is also said to be transferred. Academics are also called neoplasms. Cancer is a refractory chronic disease that, even if treated with surgery, radiation and chemotherapy, does not cure in many cases, suffers from the patient and ultimately leads to death, and there are many causes of cancer, but internal factors It is divided into external and external factors. The mechanism by which normal cells are transformed into cancer cells has not been accurately determined, but many cancers are known to be influenced by external factors such as environmental factors. Internal factors include genetic factors, immunological factors, and external factors include chemicals, radiation, and viruses. Genes involved in the development of cancer include oncogenes and tumor suppressor genes, which occur when the balance between them is broken down by internal or external factors described above. The cancer may be oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal Proximal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, chronic leukemia, acute leukemia, lymphocyte lymphoma, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma, It is not limited to this.

In one embodiment of the present invention "poorly differentiated cancer (poorly differentiated cancer) or a poorly differentiated tumor (poorly differentiated tumor)" means a state in which the arrangement of the structure inside the cancer tissue is indistinctly clear. According to the National Cancer Institute of the National Institutes of Health, the cancer classification system is based on the nature of the cancer, and generally 1, 2, 3 or 3 depending on the teratogenicity (abnormal) of the cancer. It is divided into four stages. Stage 1 is a cancer in which the arrangement of cancer cells is almost similar to that of normal tissues. Stage 1 cancers are slow to grow and invade into surrounding tissues. In contrast, stage 3 or 4 cancers look completely different from normal cells or normal tissues, grow faster than stage 1 or 2 cancers and rapidly invade surrounding tissues. This is summarized as follows.

Precancerous stage of cancer (GX): stage in which precancerous-cancer stage cannot be determined

Stage 1 (G1): highly differentiated cancer-well differentiated

Stage 2 (G2): Moderately differentiated cancer-differentiating stage

Stage 3 (G3): Poorly differentiated stage of low differentiation cancer

Stage 4 (G4): Undifferentiated Cancer-Undifferentiated Stage

It is known that low-differentiated cancers contain a large amount of cancer stem cells, high tumor boundaries, fast metastasis, poor therapeutic effect, and poor prognosis after treatment, compared to highly differentiated or moderately differentiated cancers.

In one embodiment of the present invention, "cancer stem cell" refers to a cancer cell of a comprehensive meaning having self-renewal or differentiation ability that is unique to stem cells, for example, sphere form. Cancer cell populations and cancer tissues of poorly differentiated and poor prognosis, such as low-differentiation cancer. Normal tumor growth conditions of the cancer stem cells (the "normal tumor growth conditions" refers to a state in which there is sufficient cell nutrients (glucose) necessary for cell growth and growth conditions of the tumor microenvironment, so that there is no cellular stress.) Unlike cancer cells, they may have a slower rate of proliferation or maintain a dormant state and may have resistance to anticancer drugs. For example, expression of transcriptional regulators such as PGC-1a is controlled unlike normal tumor cells. The function of key metabolic regulators may be different compared to normal cancer cells. Through these different metabolic control mechanisms and mechanisms of mechanisms linked to these signaling pathways, we acquire resistance to apoptosis in nutrient deprivation and refer to cells that are invasive and / or metastatic. . However, any cell that can differentiate into a general cancer cell is not limited thereto.

In one embodiment of the present invention, "inhibition of growth of cancer stem cells" means inhibiting cancer stem cell maintenance, inhibiting cancer stem cell malignance and inhibiting cancer stem cell invasive activity.

In one embodiment of the present invention, "polyphenol" is a kind of chemicals found in plants and is characterized by one or more phenol groups per molecule. Polyphenols are generally classified into tannins, phenylpropanoids (flavonoids, liglines, etc.). Phenol is one hydrogen atom of benzene substituted with a hydroxy group, and polyphenol is substituted with two or more hydroxy groups. There are thousands of polyphenols, including catechins in green tea, resveratrol in wine, apple and quercetin in onions. Flavonoids in fruits and isoflavones in beans are also polyphenols.

Polyphenols have an antioxidant effect that converts free radicals (harmful oxygen) in our body into harmless substances, preventing aging. In addition, it has been reported to lower the risk of various diseases because of its ability to protect DNA damage, cellular proteins and enzymes that are damaged by exposure to free radicals. However, certain polyphenols can be toxic to cells by the action of phenolic substituents. Typical types of polyphenolic toxins include, but are not limited to, safrole, Gossypol, and coumarins.

In one embodiment of the present invention "Gossypol" is a poly, which is contained in the detachable pigment glands of seeds, leaves, stems, and roots of Gossypium plants and Malvaceae plants. A kind of phenolic compound, also called polyphenolic gossypol or cotton thread pigment. Provide plants with resistance to pests. When Gosipol is added to poultry feed, feed availability, decreased egg productivity and egg yolk discoloration have been reported. The ruminant axis, on the other hand, inactivates Gosipol by fermentation. Free gopopol is physiologically toxic while bound gopopol is inactive. Non-protein components in cotton yarn also combine with Gosipol to form insoluble and / or non-digestible complexes. This binding detoxifies Gosipol in cotton but decreases protein and biological value. The addition of iron in a ratio of 2: 1 or 3: 1 to free Gosipol can effectively reduce the toxicity of Gosipol in the liver. In China, Gosipol has been found to inhibit sperm function in men and has been studied as a male oral contraceptive. Gosipol in the present invention is preferably a compound represented by the following formula (1) or derivatives thereof, but is not limited thereto.

[Formula 1]

In one embodiment of the present invention, "biguanide-based compound" is preferably a biguanide-based diabetes treatment agent, more preferably metformin, phenformin, buformine But it is not limited to any biguanide-based compounds that interfere with the production of energy in the cell to induce a nutritional deficiency-like state.

In one embodiment of the present invention, "anticancer agent" is a generic term for chemotherapeutic agents used for the treatment of malignant tumors. Most anticancer drugs intervene in various metabolic pathways of cancer cells and mainly inhibit the synthesis of nucleic acids or exhibit anticancer activity. Anticancer drugs currently used for cancer treatment are classified into six categories according to biochemical mechanisms of action.

(1) Alkylating agents: A highly reactive substance with the ability to introduce an alkyl group R-CH2 to a compound, and when reacted to a cell, most of it reacts with the N7 of guanine in the DNA to modify its DNA structure, and It causes cleavage and shows anticancer effect and cytotoxic effect. Examples of drugs belonging to the above include: 1) nitrogen mustard system: nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, etc. 2) ethyleneimine type: thiotepa ③ alkylsulfonate system: sulphate ④ tri Azine, hydrazine: DTIC (daccarbine), procarbazine ⑤ nitrosourea: BCNU, CCNU, methyl-CCNU and the like.

(2) metabolic antagonists (代謝 拮抗劑: antimetabolites): drugs belonging to this group have the effect of inhibiting metabolic processes necessary for the proliferation of cancer cells ① folic acid derivatives: mesotrexate (MTX) ② purine derivatives: 6-mercaptopurine (6-MP), 6-thioguanine ③ pyrimidine derivatives: 5-fluorouracil, cytarabine and the like.

(3) Antibiotics (antibiotics): Among antibiotics produced by bacteria, anti-cancer activity includes adriamycin, daunorubicin, bleomycin, mitomycin-C, and actinomycin-D.

(4) vinca alkaloids: These drugs are specific to the mitotic phase and stop cell division in the metaphase during mitotic phase. Vincristine, vinblastine, VP-16-213 and VM-26.

(5) Hormone: Some types of cancer can be treated with hormones. Male hormones are used to treat breast cancer, female hormones to prostate cancer, progesterone to endometrial cancer, and corticosteroids. Is used for the treatment of acute lymphocytic leukemia and lymphoma, and tamoxifen, an anti-female hormone, is used for breast cancer.

(6) Others: cisplatin, L-asparagine, o, p-DDD, and the like. As described above, there are about 40 kinds of anticancer drugs currently used for cancer treatment, and each drug has a big difference in its anticancer range.

In one embodiment of the present invention, "Irinotecan" is a kind of anticancer agent used in recurrent, metastatic gastric cancer, rectal cancer or colon cancer, and preferably, irinotecan hydrochloride, but is not limited thereto.

In one embodiment of the present invention, "diagnosis" refers to confirming the presence or characteristics of a pathological condition, and for the purpose of the present invention, the diagnosis is to confirm the presence or absence of cancer, proliferation, and metastasis. Cancer ”means“ cancer stem cells ”. Cancer can be diagnosed by visual or cytological confirmation of tissue from a patient suspected of developing cancer or metastasis, and a sample of tissue suspected of developing cancer or metastasis (clinically, cells, blood, sap, pleural fluid, ascites, joint fluid, pus) (Iv), secretion, bile, pharyngeal mucus, urine, bile, stool, etc.), using a cancer-compatible antibody, directly detecting a cancer-related protein in the sample, or encoding a cancer-related protein Cancer can be diagnosed by direct detection of nucleic acids. Diagnostic means using antigen-antibody binding or direct detection of cancer-related proteins include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and ouktero. Ouchterlony immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip ( protein chip), but the present invention is not limited thereto, and methods for directly detecting nucleic acids encoding cancer-related proteins include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, and real-time. Reverse Transcription Polymerase Reaction (Real-time RT-PCR), RNase Protection Assay (RPA), Northern Blotting or DNA Chips. It doesn't happen.

In one embodiment of the invention “treatment” refers to a series of activities performed to alleviate or / or ameliorate a desired disease. Treatment for the purposes of the present invention inhibits the numerical or quantitative proliferation of cancer cells, including cancer stem cells, kills cancer cells, inhibits the growth of cancer tissues, reduces the size of cancer tissues, cancer tissues Activity that inhibits the development of neovascularization within.

In one embodiment of the present invention, "metastasis" means that cancer cells leave the primary organs and go to other organs, and the term "cancer" means "cancer stem cells". The spread of cancer to other parts of the body is largely divided into primary cancers in which the cancerous tissue grows and directly invades the surrounding organs and distantly metastasizes along the blood vessels or lymphatic vessels to other organs in the distance. Metastasis can be regulated through inhibition of expression of a gene associated with cancer development or inhibition of protein activity of the gene.

In one embodiment of the present invention, "pharmaceutical composition" means a composition to be administered for a specific purpose. For the purposes of the present invention, a pharmaceutical composition of the present invention comprises a polyphenol compound and is used to treat or inhibit the metastasis of cancer, including cancer stem cells, and the compounds and pharmaceutically acceptable carriers, excipients or Diluents. In addition, the pharmaceutical composition according to the present invention may be provided further comprising a biguanide compound and an anticancer agent in the polyphenol compound, the biguanide compound is phenformin, and the anticancer agent is irinotecan (Irinotecan). Is preferably, but is not limited thereto. In addition, the pharmaceutical composition according to the present invention contains 0.1 to 50% by weight of the active ingredient of the present invention based on the total weight of the composition. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In one embodiment of the invention “administration” means introducing the composition of the invention to the patient in any suitable way, the route of administration of the composition of the invention via any general route as long as it can reach the target tissue. May be administered. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but are not limited thereto. Do not. In the present invention, the effective amount is defined as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently. In adults, the therapeutic pharmaceutical composition can be administered once in the body in an amount of 50ml ~ 500ml, 0.1ng / kg-10mg / kg for a compound, 0.1ng / kg-10mg for a monoclonal antibody. It may be administered at a dose of / kg. Dosing interval may be 1 to 12 times a day, if 12 times a day may be administered once every two hours. In addition, the pharmaceutical composition of the present invention may be administered alone or in combination with other therapies known in the art for the treatment of cancer stem cells of interest, such as chemotherapeutic agents, radiation and surgery. The pharmaceutical compositions of the present invention may also be administered in admixture with other therapies designed to enhance immune responses, for example adjuvant or cytokines (or nucleic acids encoding cytokines), as is well known in the art. Other standard delivery methods may be used, such as biolistic delivery or ex vivo treatment. In vitro treatment, for example, antigen presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells can be obtained from a patient or appropriate donor and then activated in vitro with the pharmaceutical composition and then administered to the patient. have.

In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising a polyphenol compound as an active ingredient, wherein the polyphenol compound is safrole, Gossypol, and coumarins. It provides a pharmaceutical composition which is at least one selected from the group consisting of, wherein the polyphenol compound provides a pharmaceutical composition contained in an amount of 0.5 to 500 mM, the pharmaceutical composition further comprises a biguanide compound To provide a composition, the biguanide-based compound provides a pharmaceutical composition of any one or more selected from the group consisting of metformin, buformin, and phenformin, wherein the pharmaceutical composition is It provides a pharmaceutical composition further comprising an anticancer agent, wherein the anticancer agent provides a pharmaceutical composition which is Irinotecan (Irinotecan), the cancer Providing a pharmaceutical composition comprising cancer stem cells, wherein the cancer provides a pharmaceutical composition that is gastric cancer, wherein the treatment inhibits the increase in the number of cancer cells, including cancer stem cells, inhibits quantitative proliferation, Provided are pharmaceutical compositions comprising killing cells, reducing or maintaining the size of cancerous tissue, including cancer stem cells, or inhibiting neovascular development in cancerous tissue, including cancer stem cells.

In another embodiment of the present invention, there is provided a pharmaceutical composition for inhibiting cancer metastasis, comprising a polyphenol compound as an active ingredient, wherein the polyphenol compound is safrole, Gossypol, and coumarins. Provided is a pharmaceutical composition which is at least one selected from the group consisting of, wherein the polyphenol compound provides a pharmaceutical composition comprising an amount of 0.5 to 500 mM, the pharmaceutical composition further comprises a biguanide-based compound To provide, the biguanide-based compound provides a pharmaceutical composition of any one or more selected from the group consisting of metformin, buformin, and phenformin, wherein the pharmaceutical composition is an anticancer agent It provides a pharmaceutical composition comprising a further, wherein the anticancer agent provides a pharmaceutical composition is Irinotecan (Irinotecan), the cancer It provides that the pharmaceutical composition comprising the stem cells, the cancer is gastric cancer provides a pharmaceutical composition.

Hereinafter, the present invention will be described in detail step by step.

Cancer stem cells are cancer cells with unlimited regenerative capacity similar to those of general stem cells, and slowly proliferate differently from general cancer cells, and have a mechanism of self-renewal or differentiation, which is unique to stem cells, and different mechanisms from known cancer cells. It is known to have mechanism. If cancer stem cells remain in the body even after cancer treatment, cancer recurrence and / or metastasis occurs actively, so developing cancer stem cell therapeutics is an urgent task.

The present invention relates to a pharmaceutical composition for treating cancer comprising a polyphenol compound as an active ingredient, wherein the pharmaceutical composition according to the present study is for treating cancer tissue including cancer stem cells or cancer stem cells in large amounts such as low-differentiated cancer. And very effective in improving prognosis. In addition, polyphenol compounds, biguanide-based compounds, and anticancer agents are used in combination with cancer cells, and thus, the effects of inhibiting cancer cell growth are significantly increased compared to those administered alone, and are expected to be greatly utilized in the field of cancer treatment.

1 is a result confirming the effect of inhibiting the growth of gosipole on gastric cancer cell line according to an embodiment of the present invention.

Figure 2 is in accordance with an embodiment of the present invention, the result of confirming the ATP synthesis inhibitory effect of Gosipol on gastric cancer cell line.

Figure 3 is a diagram confirming the cell death promoting effect of Gosipol on gastric cancer cell line according to an embodiment of the present invention.

Figure 4 is a graph confirming the cell death promoting effect of Gosipol on gastric cancer cell line according to an embodiment of the present invention.

5 is a result of analyzing the cell cycle of gosipole treated gastric cancer cell line according to an embodiment of the present invention.

6 is a result of administering Gosipol to general gastric cancer cells and gastric cancer stem cells according to an embodiment of the present invention, confirming the IC50 concentration.

7 is a result of confirming the cell viability after administration of Gosipol to normal gastric cancer cells and gastric cancer stem cells, 48 hours after the embodiment of the present invention.

8 is a result of administering Gosipol to PAGS and SAGS cells according to an embodiment of the present invention, and confirming the IC50 concentration.

Figure 9 is a result confirming the cell growth inhibitory effect when combined administration of Gosipol and conventional drugs to the gastric cancer cell line according to an embodiment of the present invention.

10 is a result of confirming the decrease in intracellular mitochondrial activity upon co-administration of Gosipol and existing drugs to gastric cancer cell line according to an embodiment of the present invention.

Figure 11 shows the results of confirming the cell death effect in combination administration of Gosipol and conventional drugs to the gastric cancer cell line according to an embodiment of the present invention.

12 is a graph showing the results of confirming the cell killing effect when co-administration of gosipol and the existing drug to the gastric cancer cell line according to an embodiment of the present invention.

Cancer cells that survived for 4 weeks of continuous metabolic stress from normal gastric cancer cell AGS (PAGS, parental AGS) were expressed as cancer stem cell markers, tumor spheroid forming ability, standard anticancer drug resistance, and tumor forming ability in immunosuppressive mice. The growth inhibition effect by gosipol was confirmed for AGS cells (SAGS, selected AGS, Cell Death Dis. 2015 Jul 2; 6: e1805) selected by phenotypic analysis. Cell culture and viability measurement method was performed in the same manner as in Example 2-1. The experimental results showed that the IC50 gopopol concentration of PAGS was 4.2 mM, whereas the IC50 gopopol concentration of SAGS, which has the characteristics of cancer stem cells, was 2.8 mM, depending on whether the cells of the same origin have the characteristics of stem cells. Therefore, it was found that drug sensitivity to Gosipol was significantly different.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1 Confirm the Effect of Gosipol on Gastric Cancer Cells

Example 1-1. Confirmation of Gosipol's Inhibitory Effect on Gastric Cancer Cells

Six different gastric cancer cell lines (MKN28, MKN45, AGS, SNU668, Kato3, and HS746T) were prepared to confirm the inhibitory effect of Gosipol on gastric cancer cell lines. Definitions of cell lines known to the Korean Cell Line Bank or the International Cell Line Bank (ATCC) are shown in Table 1.

Cell line name	Justice	Eruption
MKN28	Human stomach adenocarcinoma (tubular)	Moderately differentiated
MKN45	Human stomach adenocarcinoma	Poorly differentiated
AGS	Human stomach adenocarcinoma	Poorly differentiated
SNU668	Human stomach carcinoma, signet ring cell	Poorly differentiated
Kato3	Human stomach carcinoma	Poorly differentiated
HS746T	Human stomach carcinoma	Poorly differentiated

The concentration of each culture in 96-well plates for 24 hours, the notice pole of 0, 10, 20, 40 or 60μM -; the six types of cells ^{(1 X 10 4 / well 1} X 10 3 cells can) After treatment, the cells were incubated for an additional 48 hours. Cells were then fixed with 50% by volume trichloroacetic acid (TCA) for 1 hour at 4 ° C., washed with distilled water and dried at room temperature. Each well containing immobilized cells was stained with 100 μl of 0.4 wt% Sulforhodamine B dissolved in 1 vol% acetic acid solution for 10 min and washed 4 times with 1 vol% acetic acid solution. The stained plates were dried at room temperature and 100 μl of 10 mM Tris buffer (pH 7.5) was added to each well, and the eluted pigment was absorbed at 515 nm. Growth inhibition effect of the cancer cell line according to the goshipol concentration derived as the above result is described in FIG.

Experimental results showed that most low-differentiated cancer cells (MKN45, AGS, SNU668 and Kato3) inhibited cell proliferation in comparison with the neutralized cancer cells (HS746T) at all concentration conditions. In addition, when treated with 60 μM of gosipol, all kinds of low-differentiated cancer cells (MKN45, AGS, SNU668, Kato3 and HS746T) used in the experiments inhibited cell proliferation more than neutral-differentiated cancer cells (HS746T). GI50 values of each cell line derived from the above experimental results are shown in Table 2.

Cell line name	GI50 (μM)
MKN28	5.32
MKN45	4.65
AGS	6.43
SNU668	4.11
Kato3	3.15
HS746T	9.89

Example 1-2. Inhibition of Gosipol's ATP Synthesis on Gastric Cancer Cells

ATP (adenosine triphosphate) is an energy source for living organisms. When the intracellular ATP synthesis is inhibited, energy metabolic activity decreases. The gastric cancer cell lines MKN28, MKN45 and SNU668 of Example 1-1 were confirmed to inhibit the ATP synthesis of Gosipol.

MKN28, MKN45, and each cell of the SNU668 (cell ^{number; 1 X 10 3 - 1 X} 10 4 / plate) were incubated for 24 hours in 60mm culture dishes and treated for examination pole in each 0, 10, or the concentration of 20μM Then, incubated for an additional 48 hours. Cells were then harvested and counted and diluted in 100 μl of RPMI culture with 10 volume% FBS, which was transferred to each well of a 96-well plate. 100 μl of an assay buffer (rL / L reagent + reconstitution buffer) of the Promega ATP assay kit (G7572, Promega, Durham, NC, USA) was added to the wells containing the cells, and the fluorescence emission was measured at 560 nm. . The results are shown in FIG.

Experimental results showed that ATP synthesis was inhibited in proportion to gosifol concentration in all gastric cancer cell lines used in the experiment. This suggests that Gosipol effectively reduces energy levels in cancer cells.

Example 1-3. Confirmation of Gosifol's Cell Death Promoting Effect on Gastric Cancer Cells

From Examples 1-1 to 1-2, Gosipol was expected to have an effect of promoting cell death against cancer cells, and the following experiment was conducted.

Examples 1-1 of MKN28 (jungbun Mars cells) with each cell of MKN45 (low molecular chemical conversion cells) (cell ^{count; 1 X 10 3 - 1 X} 10 4 / plate) for 24 hours in a 100mm Petri dish, and the notice Poles were treated at concentrations of 0, 5, 10, or 20 μM, respectively, and then diluted in 400 μl of binding buffer (0.1 M Hepes, pH 7.4, 1.4 M NaCl, and 25 mM CaCl ₂ ). The Gosipol 0 μM treated control cells were divided into 4 groups, each non-stained, Annexin V stained, propidium iodide (PI) stained and Annexin V + PI stained, and treated with Gosipol 5, 10, or 20 μM, respectively. One cell was stained with Annexin V + PI. Staining of cells was performed for 10 minutes in the dark. The stained cells were subjected to flow cytometry (FACS) to derive cell death rate to total cells. The results are shown in FIGS. 3 to 4 (BD Biosciendes, # 556547).

As a result, MKN28 (differentiated cells) showed 9.59%, 17.72%, 28.85% and 55.62% cell death rate according to gosypol concentrations of 0, 5, 10 and 20 μM, respectively. Cases were 11.77 &, 45.75%, 65.8% and 82.22%. The results showed that the cell death rate of MKN45 (low-differentiating cells) was higher than that of MKN28 (low-differentiating cells) at all concentrations of Gosipol-treated cells. . In addition, considering that low-differentiation cancer contains a lot of cancer stem cells, Gosipol is expected to have a significant effect on the growth inhibition of cancer stem cells.

Example 1-4. Cell Cycle Analysis of Gosifol-treated Gastric Cancer Cells

Six different gastric cancer cell lines (MKN28, MKN45, AGS, SNU668, Kato3 and HS746T) of Example 1-1 were incubated for 24 hours in a 100 mm culture dish of 1 × 10 ³ to 1 × 10 ⁴ / well, respectively, Gosipol was treated at a concentration of 0 or 10 μM and then incubated for an additional 24 hours. Gosipol treated cells were obtained, fixed with 70% ethanol, washed with cold 1X PBS, and stained with PI + RNase solution (DPBS, pH7.4) in the dark for 30 minutes. The cells were subjected to flow cytometry (FACS) to analyze the ratio of cell cycles by DNA content. The results are shown in FIG.

Experimental results show that the combined ratio of sub G1 and GO / G1, which can be interpreted as apoptosis, decreased after goofypole treatment in mesodifferentiated cells (MKN28), whereas that in low-differentiated cells increased than before gosypole treatment (MKN45, AGS and HS746T) or maintained (SNU668 and Kato3).

Example 2 Confirmation of the Effect of Gosipol on Gastric Cancer Stem Cells

Example 2-1. Confirmation of Gosipol's Inhibitory Effect on Gastric Cancer Stem Cells

It can be seen from Example 1 that Gosipol has a significant effect on the inhibition of growth of low-differentiated cancer, and considering that low-differentiation cancer contains a large number of cancer stem cells, Gosipoul also inhibits the growth of cancer stem cells. The following experiment was conducted in anticipation of a significant effect.

First, two kinds of general gastric cancer cells (AGS, MKN-74) and two types of gastric cancer stem cells (MKN-1, SNU-484) were prepared to confirm the inhibitory effect of Gosipol on gastric cancer stem cells. It was. Of the individual cells in a 96-well plate at 1 X 10 ³ - with 1 X 10 ⁴ / well and incubated with for 24 hours, treated a notice of each pole in a concentration of 0, 10, 20, 40 or 60μM, more Incubate for 48 hours. The cells were then fixed with 50 wt% trichloroacetic acid (TCA) for 1 hour at 4 ° C., washed with distilled water, and dried at room temperature. Each well containing immobilized cells was stained with 100 μl of 0.4 wt% Sulforhodamine B dissolved in 1 vol% acetic acid solution for 10 min and washed 4 times with 1 vol% acetic acid solution. The stained plates were dried at room temperature and 100 μl of 10 mM Tris buffer (pH 7.5) was added to each well, and the eluted pigment was absorbed at 515 nm. 6 and 7 show the results of calculating the concentration of Gosipol from which the viability of each cell becomes IC50.

The experimental results showed that the IC50 Gosipol concentrations of MKN-1 and SNU-484, which are gastric cancer stem cells, were 2.6 mM and 2.7 mM, respectively, average 2.65mM. Were 5.6 mM and 4.5 mM, respectively, with an average of 5.05 mM. From the above results, gastric cancer stem cells were more sensitive to Gosipol than general gastric cancer cells, and it was found that the effect of inhibiting cancer cell growth by gosipol was more pronounced in gastric cancer stem cells than general cancer cells.

Example  2-2. For stem cells selected and isolated from cancer cells Gossip  Confirmation of growth inhibitory effect

Cancer cells that survived for 4 weeks of continuous metabolic stress from normal gastric cancer cell AGS (PAGS, parental AGS) were expressed as cancer stem cell markers, tumor spheroid forming ability, standard anticancer drug resistance, and tumor forming ability in immunosuppressive mice. The growth inhibition effect by gosipol was confirmed for AGS cells (SAGS, selected AGS, Cell Death Dis. 2015 Jul 2; 6: e1805) selected by phenotypic analysis. Cell culture and viability measurement method was performed in the same manner as in Example 2-1. The experimental results showed that the IC50 gopopol concentration of PAGS was 4.2 mM, whereas the IC50 gopopol concentration of SAGS, which has the characteristics of cancer stem cells, was 2.8 mM, depending on whether the cells of the same origin have the characteristics of stem cells. Therefore, it was found that drug sensitivity to Gosipol was significantly different. The results are shown in FIG. 8.

Example 3 Confirmation of Combination Effect of Gosipol and Existing Drugs on Gastric Cancer Cells

Example 3-1. Analysis of Cell Growth of Gastric Cancer Cells Treated with Gosipol and Existing Drugs

AGS, SNU484, SNU668, MKN28, Hs746T, MKN45, and SNU719 gastric cancer stem cells were inoculated in 96-well plates at 5 × 10 ³ to 4 × 10 ⁴ / well, respectively, and incubated for 24 hours. Each well was then administered with Gosipol, Phenformin, and Irinotecan drugs as shown in Table 3 and incubated for an additional 24 hours. Thereafter, the cells were fixed with 50 vol% TCA (trichloroacetic acid, final concentration of 10% TCA) for 1 hour at 4 ° C., washed with distilled water, and then dried at room temperature. Each well containing immobilized cells was stained with 100 μl of 0.4 wt% Sulforhodamine B dissolved in 1 vol% acetic acid solution for 10 min and washed 4 times with 1 vol% acetic acid solution. The stained plates were dried at room temperature and 100 μl of 10 mM Tris buffer (pH 7.5) was added to each well, and the eluted pigment was absorbed at 515 nm. The growth inhibitory effect of the gastric cancer cell line derived as a result is described in FIG. 9.

Experimental Example	Dosing medication
Control	No medication
Experimental Example 1	Gosipol 5μM administration
Experimental Example 2	Phenformin 100μM administration
Experimental Example 3	Gosipol 5μM + Penformin 100μM
Experimental Example 4	Irinotecan 1μM Administration
Experimental Example 5	Irinotecan 1μM + Gosipol 5μM
Experimental Example 6	Irinotecan 1μM + Penformin 100μM
Experimental Example 7	Irinotecan 1 μM + Gosipol 5 μM + Penformin 100 μM

As a result, it was shown that when Gosipol, phenformin, or irinotecan alone were administered alone, it showed various anticancer effects depending on the cell line. In the case of selectively using two of the three drugs, Experimental Example 3 (Gosypol + phenformin) had a relatively stable effect of inhibiting the growth of gastric cancer cells, but in other combinations the efficacy according to the cell line It appeared to be various. However, when all three drugs were used in combination (Experimental Example 7), it was found that all cell lines used in the experiment had a cell growth inhibition effect of 80% or more uniformly.

Example  3-2. Gossipol  And mitochondrial activity of gastric cancer cells treated with conventional drugs

Mitochondrial cell membrane activity analysis was performed on the control and Experimental Examples 1 to 7 cells of Example 3-1. Mitochondrial cell membrane activity is used as an indicator of intracellular energy production. First, 100 nM TMRE (tetramethylrodamine ester, ab113852, Abcam) was added to the culture medium 20 minutes before the end of drug treatment of each cell sample, the cells were washed three times with cold PBS, and then 585 nm (FL) with a flow cytometer. -2) the fluorescence intensity of the cells was measured using the channel. The results are shown in FIG.

As a result, the combination of gosypol, phenformin, and irinotecan showed a significant decrease in mitochondrial activity than that of gosypol and phenformin alone. This can be judged to lower the energy production of cancer cells.

Example 3-3. Analysis of Cell Death of Gastric Cancer Cells Treated with Gosipol and Conventional Drugs

For the control and Experimental Examples 1 to 7 cells of Example 3-1, samples were taken at 12 hours, 24 hours, 48 hours, and 72 hours after each drug administration, and cell death analysis was performed. First, the cells were washed with cold PBS, centrifuged at 1400 rpm for 3 minutes and then resuspended at a concentration of 1 x 10 ⁶ cells / ml. 100 μl of the cell suspension was transferred to a 5 ml culture tube, 5 μl of Annexin V-FITC and PI stain were added, respectively, and reacted for 15 minutes at dark room temperature. 400 μl of 1 × binding buffer was added to the FACS flow cytometer (BD Falcon, Bedford, Mass., USA). Graphs converted into FACS results and numerical values are shown in FIGS. 11 and 12. The results of the study showed that when Gossipol, Penformin, and Irinotecan were co-administered at all times, Gossipol, Penformin, or Itinotecan were administered alone, and Gossipol and Penformin were co-administered. More significantly, cancer cell death was shown to increase.

From the results of Examples 1 to 3 above, it was found that Gosipol inhibits the proliferation of cancer cells including cancer stem cells and promotes cell death. In addition, when using a combination of phenformin and irinotecan in gosipol, it was confirmed that the cancer cell killing effect is significantly increased.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

The present invention relates to a pharmaceutical composition for treating cancer comprising a polyphenol compound as an active ingredient, wherein the pharmaceutical composition according to the present study is for treating cancer tissue including cancer stem cells or cancer stem cells in large amounts such as low-differentiated cancer. And very effective in improving prognosis. In addition, polyphenol compounds, biguanide-based compounds, and anticancer agents are used in combination with cancer cells, and thus, the effects of inhibiting cancer cell growth are significantly increased compared to those administered alone, and are expected to be greatly utilized in the field of cancer treatment.

Claims (19)

1. Cancer preventive or therapeutic pharmaceutical composition comprising a polyphenol compound as an active ingredient.
2. The method of claim 1,

   The polyphenolic compound is any one or more selected from the group consisting of safrole, Gossypol, and coumarins, pharmaceutical composition.
3. The method of claim 1,

   The polyphenol compound is contained in an amount of 0.5 to 500 mM, pharmaceutical composition.
4. The method of claim 1,

   The pharmaceutical composition is a pharmaceutical composition further comprises a biguanide compound.
5. The method of claim 4, wherein

   The biguanide-based compound is any one or more selected from the group consisting of metformin (metformin), buformin (buformin), and phenformin (phenformin), pharmaceutical composition.
6. The method of claim 4, wherein

   The pharmaceutical composition further comprises an anticancer agent, pharmaceutical composition.
7. The method of claim 6,

   The anticancer agent is Irinotecan (Irinotecan), a pharmaceutical composition.
8. The method of claim 1,

   Wherein the cancer comprises cancer stem cells, pharmaceutical compositions.
9. The method of claim 1,

   The cancer is gastric cancer, pharmaceutical composition.
10. The method of claim 1,

    The treatment inhibits the increase in the number of cancer cells, including cancer stem cells, inhibits quantitative proliferation, kills the cells, reduces or maintains the size of cancer tissue, including cancer stem cells, or cancer A pharmaceutical composition comprising inhibiting neovascular development in cancerous tissue including stem cells.
11. A pharmaceutical composition for inhibiting cancer metastasis comprising a polyphenol compound as an active ingredient.
12. The method of claim 11,

    The polyphenolic compound is any one or more selected from the group consisting of safrole, Gossypol, and coumarins, pharmaceutical composition.
13. The method of claim 11,

    The polyphenol compound is contained in an amount of 0.5 to 500 mM, pharmaceutical composition.
14. The method of claim 11,

    The pharmaceutical composition is a pharmaceutical composition further comprises a biguanide compound.
15. The method of claim 14,

    The biguanide-based compound is any one or more selected from the group consisting of metformin (metformin), buformin (buformin), and phenformin (phenformin), pharmaceutical composition.
16. The method of claim 14,

    The pharmaceutical composition further comprises an anticancer agent, pharmaceutical composition.
17. The method of claim 16,

    The anticancer agent is Irinotecan (Irinotecan), a pharmaceutical composition.
18. The method of claim 11,

    Wherein the cancer comprises cancer stem cells, pharmaceutical compositions.
19. The method of claim 11,

    The cancer is gastric cancer, pharmaceutical composition.

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