KR101803000B1 - Pharmaceutical compositions for preventing or treating lung cancer comprising apigenin, curcumin, and honokiol as active ingredients - Google Patents

Pharmaceutical compositions for preventing or treating lung cancer comprising apigenin, curcumin, and honokiol as active ingredients Download PDF

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KR101803000B1
KR101803000B1 KR1020160111383A KR20160111383A KR101803000B1 KR 101803000 B1 KR101803000 B1 KR 101803000B1 KR 1020160111383 A KR1020160111383 A KR 1020160111383A KR 20160111383 A KR20160111383 A KR 20160111383A KR 101803000 B1 KR101803000 B1 KR 101803000B1
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apigenin
curcumin
lung cancer
honokiol
present invention
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KR1020160111383A
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Korean (ko)
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김철우
장지영
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(주) 바이오인프라생명과학
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. cannabinols, methantheline
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/045Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05Phenols
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/12Ketones
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES
    • A23V2200/00Function of food ingredients
    • A23V2200/30Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention

Abstract

The present invention relates to pharmaceutical compositions for preventing or treating lung cancer comprising apigenin, curcumin, and honokiol as active ingredients. The compositions according to the present invention induces the death of cancer cells synergistically when treated with apigenin, curcumin or honokiol in mixture or combination than when treated with apigenin, curcumin or honokiol alone, and thus the pharmaceutical compositions for preventing or treating lung cancer comprising apigenin, curcumin, and honokiol as active ingredients increase an anticancer effect and induce cancer cell death, thereby exhibiting an excellent cancer therapeutic effect. Therefore, the compositions according to the present invention can be usefully used for preventing, ameliorating or treating lung cancer.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating lung cancer, which comprises apigenin, curcumin and honokiol as active ingredients,

The present invention relates to a pharmaceutical composition comprising apigenin or curcumin; And the effect of treating the lung cancer with the honeycomb mixture.

Cancer is a group of abnormal cells caused by continuous division and proliferation by destroying the balance between cell division and death due to various causes, and it is also called malignant tumor. In general, cancer develops in more than 100 parts of the body, including organs, leukocytes, bones, lymph nodes, etc., and develops into serious symptoms through the invasion of surrounding tissues and metastasis to other organs, eventually leading to death do. Among them, lung cancer, which is a malignant tumor originating from the lung, is divided into small cell lung cancer and non-small cell lung cancer according to its tissue type. Although the small cell lung cancer is classified as a part of lung cancer by the location of the onset tissue, it is distinguished from the other lung cancer in terms of clinical course, treatment method and prognosis. In addition, non-small cell lung cancer is classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma according to histology.

In particular, small-cell lung cancer is a large mass, grayish white, and is known to proliferate along the bronchial wall. In many cases, surgical resection is difficult at the time of diagnosis, and malignancy is rapidly growing. It is known that chemotherapy or radiotherapy has a remarkable therapeutic effect while it is easily transferred to whole body through blood circulation. The major organs in which the small cell lung cancer has metastasized are brain, liver, bone, lung, adrenal, kidney, etc. It is known that it first develops mainly in the airway (bronchus or bronchus)

On the other hand, as described above, non-small cell lung cancer is classified into adenocarcinoma, squamous cell acinoma and large-cell carcinoma. First, adenocarcinomas occur mainly in the lungs, and they occur frequently in women or non-smokers. In many cases, the adenocarcinoma is metastasized even if the size is small, and the incidence of the adenocarcinoma is increasing recently. Next, squamous cell carcinoma is found mainly in the central lung, and it is known to grow up to the lumen of the bronchial tree, blocking the airway, is common to men, and is closely related to smoking. Finally, large cell carcinomas arise mainly in the vicinity of the lung surface (the distal end of the lung), about half in large bronchi, accounting for 4 to 10% of all lung cancers, and generally large cell size, Proliferation and metastasis tend to be worse than other non-small cell lung cancer.

When such lung cancer is diagnosed early, it can be recovered through medication or radiation therapy. However, if the disease progresses to a certain level, the lesion is removed through surgery, and treatment with medication or radiation therapy . However, the number of drugs approved for the treatment of lung cancer has been limited to a few, so it is difficult to perform appropriate treatment for the patients, and the side effects are also becoming a major problem. Most primary tumors are observed At the time of diagnosis, cancer metastasis has already been found and the success rate of treatment after surgery is low.

Accordingly, as a new approach for cancer treatment, efforts have been made to develop cancer-suppressing agents and cancer treatment agents that have a low side effect from natural substances having relatively low toxicity and are excellent in efficacy of inhibiting or reducing cancer metastasis. However, The fundamental disadvantage that the therapeutic effect is not excellent is not solved (Korean Patent Publication No. 10-2012-0109139).

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a formulation that can treat lung cancer more safely and effectively, and as a result, apigenin or curcumin; The present inventors have found that the compound of the present invention can be useful as a composition for treating lung cancer by confirming that a mixture of a compound of the present invention and a hornokiol can cause the death of lung cancer cells and inhibit cancer cell proliferation.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems. The present inventors have found that the reduction of the action rate of lung cancer cell by the administration of apigenin, curcumin or honokiol alone or in combination, inhibition of ANT2 expression, inhibition of HK2 expression, L1 expression and ATP production, and thus the present invention has been completed on the basis thereof.

Accordingly, an object of the present invention is to provide a pharmaceutical composition comprising apigenin or curcumin; And a pharmaceutical composition for the prophylaxis or treatment of lung cancer which comprises as an active ingredient honkiol.

Another object of the present invention is to provide a pharmaceutical composition comprising apigenin or curcumin; And a health functional food composition for preventing or ameliorating lung cancer comprising honkiol as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition comprising apigenin or curcumin; And a pharmaceutical composition for the prophylaxis or treatment of lung cancer which comprises as an active ingredient honkiol.

Another object of the present invention is to provide a pharmaceutical composition comprising apigenin or curcumin; And a health functional food composition for preventing or ameliorating lung cancer comprising honkiol as an active ingredient.

In one embodiment of the present invention, the composition may comprise an apigenin and a honeycomb mixture as an active ingredient.

In another embodiment of the present invention, the composition may comprise a curcumin and a honeycomb mixture as an active ingredient.

In another embodiment of the present invention, the composition may reduce the expression of the ANT2 (Adenine nucleotide translocator 2) gene.

In another embodiment of the present invention, the composition may reduce the expression of HK2 (hexokinase 2) or PD-L1 (Programmed death-ligand 1) protein.

Furthermore, the present invention provides a method of preventing or treating lung cancer comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating lung cancer.

The present invention relates to a pharmaceutical composition comprising apigenin or curcumin; And a pharmaceutical composition for preventing or treating lung cancer comprising as an active ingredient. The composition according to the present invention synergistically induces apoptosis of cancer cells when treated with apigenin, curcumin or honokiol alone or in combination with each other, as compared with apigenin or curcumin of the present invention. And a pharmaceutical composition for the prevention or treatment of lung cancer, which comprises a mixture of a hornokiol as an active ingredient, exhibits an excellent cancer therapeutic effect by inducing the killing of cancer cells by raising the anticancer effect. Therefore, the composition according to the present invention can be usefully used for preventing, ameliorating or treating lung cancer.

FIG. 1 shows the results of confirming lung cancer cell killing effect in the case of treatment with honokiol, apigenin and curcumin alone or in combination.
FIG. 2 shows the results of confirming the inhibitory effect of ANC2 on the expression of lung cancer cells in the treatment of monocytoin, apigenin and curcumin alone or in combination.
FIG. 3 shows the results of confirming the effect of reducing the ATP production of lung cancer cells by treating alone or in combination with honokiol, apigenin and curcumin.
FIG. 4 shows the results of confirming the inhibitory effect of HK2 on the expression of lung cancer cells in the treatment with honokiol, apigenin and curcumin alone or in combination.
FIG. 5 is a graph showing the effect of reducing the glycolysis rate of lung cancer cells in the treatment with honokiol, apigenin and curcumin alone or in combination.
FIG. 6A shows the results of confirming the inhibitory effect of PD-L1 on lung cancer cell line A549 in the treatment with honokiol, apigenin and curcumin alone or in combination.
FIG. 6B shows the results of confirming the effect of suppressing the expression of PD-L1 in lung cancer cell line H2228 upon treatment with honokiol, apigenin, curcumin alone or in combination.

The composition according to the present invention may comprise apigenin or curcumin; And Hunochiol as an active ingredient and confirmed the prevention, amelioration or therapeutic effect of lung cancer by reducing the action rate of lung cancer cell line, inhibiting ANT2 expression, inhibiting HK2 expression, inhibiting PD-L1 expression and ATP production , Thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition comprising apigenin or curcumin; And a composition for the prophylaxis or treatment of lung cancer, which comprises as an active ingredient honkiol. Such compositions include pharmaceutical and health functional food compositions.

As used herein, the term "prophylactic " means any action that inhibits or delays the onset of a lung cancer-related disease by administration of the pharmaceutical composition according to the present invention.

The term "treatment" as used in the present invention means all the actions for improving or alleviating symptoms of lung cancer by administration of the pharmaceutical composition according to the present invention.

"Cancer", a disease to be prevented and treated by the composition of the present invention, is a disease in which a cell grows indefinitely in a living tissue to cause a malignant tumor. More specifically, the cancer is not limited thereto.

As used herein, "honokiol" is a polyphenolic compound, which can be isolated, purified or extracted from Magnolia species and, more specifically, can be isolated, purified or extracted from barberry But are not limited to, chemically synthesized compounds or both commercially available ones. It may also be, but is not limited to, a monochiol derivative or analog.

As used herein, the term "apigenin (5,7,4'-trihydroxyflavone)" is a flavonoid-based compound that is found not only in the acacia tree extract but also in fruits such as oranges, apples, cherries, grapes, , Celery, barley, tomato, etc., vegetables, tea, wine, etc., and may be isolated, purified or extracted therefrom, but the present invention is not limited thereto, and chemically synthesized compounds or commercially available products You can use all of them. Also, it may be an apigenin derivative or an analogue, but is not limited thereto.

As used herein, "curcumin" is an alkaloid compound, which may be isolated, purified or extracted from turmeric or corn, but not limited thereto, and may be any chemically synthesized compound or commercially available . It may also be a curcumin derivative or analog, but is not limited thereto.

In addition, the composition according to the present invention may be a composition for preventing, improving or treating lung cancer comprising an apigenin and a honeycomb mixture as an active ingredient, but is not limited thereto.

In addition, the composition according to the present invention may be a composition for prevention, improvement or treatment of lung cancer comprising curcumin and a honeycomb mixture as an active ingredient, but is not limited thereto.

In addition, the composition according to the present invention is characterized by decreasing the expression of the ANT2 (Adenine nucleotide translocator 2) gene.

The "ANT2 (Adenine nucleotide translocator 2)" of the present invention is a gene having important functions in survival, proliferation and metastasis of cancer cells. The adenine nucleotide transreactase / transurocatheter (ANT) 2 protein is the most abundant protein in the mitochondrial inner membrane and is formed by six membrane perforations. ANT plays an important role in cellular energy metabolism by catalyzing changes from mitochondrial ATP to cytoplasmic ADP, thereby affecting mitochondrial oxidative phosphorylation. All mammals have an isoform of ANT1 to ANT3 and the isoform family ANT2 appears to increase the uptake of cellular ATP produced by the process in the conversion to intramitochondrial ADP, It can maintain the Condylar membrane potential, and knockdown of ANT2 reduces membrane potential difference in cancer cells.

In addition, the composition according to the present invention is characterized by decreasing the expression of HK2 (hexokinase 2) or PD-L1 (Programmed death-ligand 1) protein.

The expression "HK2 (hexokinase 2)" of the present invention is an enzyme which converts 6-carbon sugar having 6 carbon atoms to 6-phosphoric acid by phosphorylation. HK2 is a small amount of expression in normal cells but is highly expressed in most cancer cells In addition to phosphorylation of glucose, it binds with VDAC protein of mitotonidia and inhibits apoptosis, thereby enhancing the viability of cancer cells. Therefore, it can be effectively used for cancer treatment targeting HK2.

Also, the "PD-L1 (Programmed death-ligand 1)" protein of the present invention is a protein present on the surface of cancer cells, and the T cell having the killing effect of cancer cells is a protein When combined with -L1, T cells do not attack cancer cells and lose the cancer cell killing effect. Immune anticancer drugs block the binding of the PD-L1 protein by the antibody, causing T cells to attack cancer cells themselves. The anticancer agent enhances the human immune system and has no side effects or resistance to conventional anticancer agents. Therefore, it is possible to suppress the function of tumor-specific T lymphocyte cells by changing the immune checkpoint function through modulation of PD-L1 expression as one of immunity avoidance strategies of cancer cells.

In one embodiment of the present invention, the killing effect of cancer cells was confirmed by treating monoclonal or monocyte-treated cells with lung cancer cells alone (see Example 1), and monokinin, apigenin, curcumin alone or in combination (See Example 2), ATP production reduction effect (see Example 3), HK2 expression inhibition effect (see Example 4), corresponding action rate reduction effect (see Example 5), and PD-L1 expression inhibitory effect (see Example 6).

Thus, apigenin or curcumin of the present invention; And a prodrug of the present invention are useful as a preventive, ameliorating, or therapeutic agent for lung cancer, which is effective for reducing the action rate of lung cancer cells, inhibiting ANT2 expression, inhibiting HK2 expression, inhibiting PD- And may also be used as an active ingredient of the composition.

The pharmaceutical composition according to the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. Herein, pharmaceutically acceptable carriers are those conventionally used at the time of formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate and excretion rate of the active ingredient in the body, type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In addition, the composition of the present invention may be added to a health functional food for the purpose of preventing or ameliorating lung cancer-related diseases. When the compound having the lung cancer prevention or ameliorating effect of the present invention is used as a food additive, the compound can be added as it is, or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the compound of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In another aspect of the present invention, the present invention provides a method for treating lung cancer comprising administering the pharmaceutical composition to a subject. The term " individual "as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse and cattle .

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example 1. Effect of Honkiol, Apigenin, Curcumin alone or Mixed on Lung Cancer Cells

In order to confirm the effect of Hunokiol, apigenin and curcumin on the lung cancer cell death, human lung cancer cell line A549 (adenocarcinomic human alveolar basal epithelial cells) was administered alone or mixed with honokiol, apigenin, and curcumin (Sigma-Aldrich) Lt; / RTI > Concentrations of hornokiol, apigenin and curcumin in human lung cancer cells were determined by the dose-concentration curve using the IC 20 (inhibitory concentration 20) mu M, alone or in combination. After 24 hours of treatment, lung cancer cell killing effect was analyzed using CCK8 assay (Cell Counting Kit-8, Dojindo Molecular Technologies, Inc).

As a result, as shown in Fig. 1, the cell killing effect was remarkably increased in the mixed treatment, compared with the monoclonal treatment with honokiol, apigenin or curcumin. Specifically, compared to the results of cell death by 19% of Honokeol alone, 22% of apigenin-only treatment, and 20% of apoptosis by curcumin alone treatment, Cell death was 79% and cell death by hornokiol + curcumin mixture was 83%. It can be confirmed that the cell killing effect by the mixture is not a simple additive effect but a synergistic effect. From the above, it is expected that the combined administration of honokiol + apigenin or honokiol + curcumin in the treatment of patients with lung cancer, compared with monotherapy of honokiol, apigenin and curcumin, may enhance lung cancer therapeutic efficacy.

Example 2 Inhibitory Effect of Honkiol, Apigenin and Curcumin on the ANT2 Expression of Lung Cancer Cells

In order to confirm the effect of inhibiting the ANT2 expression of honokiol, apigenin and curcumin, human lung cancer cell line A549 was treated with honokiol, apigenin and curcumin alone or in combination. After that, qRT-PCR analysis was performed to confirm that the expression of ANT2 mRNA in lung cancer cells was inhibited. More specifically, RNAs were extracted (TRIzol® RNA Isolation Reagents, Thermo Sientific), the reverse transcription reaction was performed 24 hours after the human lung cancer cell line A549 was treated with 10 μM of Honkiol, Apigenin and Curcumin alone or mixed Real-time polymerase chain reaction (qPCR) was performed using SYBR (SYBR Premix Ex Taq, Takara) after cDNA synthesis (PrimeScript 1st strand cDNA Synthesis Kit, Takara) Respectively. ANT2 (adenine nucleotide translocase 2) is a well known gene that plays an important role in the survival, proliferation and metastasis of cancer cells.

Analysis of the results of qRT-PCR experiments was performed using the following formula.

(fold change = 2 - ΔΔCt, where ΔΔCt = (Ct of gene of interest, treated - Ct of HK gene, treated) number and HK was the house-keeping gene.]

The base sequences of the primers used in qPCR are shown in Table 1 below.

primer sequence ANT2 Forward 5'-ACGTGTCTGTGCAGGGTATT-3 ' Reverse 5'-GTGTCAAATGGATAGGAAG-3 ' GAPDH Forward 5'-AAGGTGAAGGTCGGAGTCAA-3 ' Reverse 5'-AATGAAGGGGTCATTGATGG-3 '

As a result, as shown in Fig. 2, ANT2 expression was decreased by the treatment of monocytoin, apigenin, and curcumin alone or mixed, and compared with monokine treatment with honokiol, apigenin and curcumin, Or the effect of inhibiting the expression of ANT2 was relatively high in the honokiol + curcumin treatment.

From the above, a therapeutic effect similar to that of cancer treatment by shRNA (short hairpin RNA) or siRNA (short interfering RNA), which is an ANT2 small interference RNA, can be expected by the combination of honokiol + apigenin or honokiol + curcumin . In addition, antinociceptive effects of honokiol, apigenin, curcumin, or curcumin alone or with mixed treatment reduce the ANT2 using small interfering RNAs, resulting in the delivery of siRNA or shRNA into the body delivery system) that can be used to solve these problems.

Example 3 Reduction of ATP Production in Lung Cancer Cells by Honokeol, Apigenin, Curcumin alone or Mixed Treatment

As a result of the above Example 2, it was confirmed that ANT2 expression was effectively inhibited when human lung cancer cells were treated with monocytoin, apigenin, curcumin alone or in combination. Therefore, antineoplastic activity of ANT2 is thought to play a crucial role in the ATP production of cancer cells, suggesting that treatment of monoclonal, apigenin, curcumin alone or in combination would reduce ATP production in cancer cells.

In order to confirm the effect of inhibiting ANT2 expression, ATP production of lung cancer cells was evaluated by using CellTiter-Glo ™ Luminescent Cell Viability assay kits (Promega ).

As a result, as shown in FIG. 3, it was confirmed that ATP production of human lung cancer cell line was effectively reduced when monoclonal, apigenin and curcumin were treated alone or in combination. In particular, , The effect of reducing ATP production relative to single administration was relatively increased.

From the above, it can be concluded that the reduction of ATP, which is an essential energy of survival, inhibits the growth of cancer cells and ultimately induces the death of cancer cells, so that the administration of monoclonal, apigenin, curcumin alone or in combination causes ATP production , And thus, it is expected that an improved cancer treatment effect can be expected by inhibiting ATP production in cancer cells.

Example 4 Inhibitory Effect of Honkiol, Apigenin and Curcumin on the Expression of HK2 Expression in Lung Cancer Cells

To determine whether the inhibition of ANT2 expression by either alone or in combination with honokiol, apigenin, and curcumin modulates the expression of hexokinase 2 (HK2), the enzyme involved in the action, human lung cancer cell line A549 Jenine, and curcumin, either singly or in combination, and then analyzed using qRT-PCR.

Analysis of the results of qRT-PCR experiments was performed using the following formula.

(fold change = 2 - ΔΔCt, where ΔΔCt = (Ct of gene of interest, treated - Ct of HK gene, treated) number and HK was the house-keeping gene.]

The base sequences of the primers used in qPCR are shown in Table 2 below.

primer sequence HK2 Forward 5'-CAAAGTGACAGTGGGTGTGG-3 ' Reverse 5'-GCCAGGTCCTTCACTGTCTC-3 ' GAPDH Forward 5'-AAGGTGAAGGTCGGAGTCAA-3 ' Reverse 5'-AATGAAGGGGTCATTGATGG-3 '

As a result, as shown in FIG. 4, it was confirmed that the expression of HK2 was effectively reduced when monoclonal or monocyte treated with honokiol, apigenin, or curcumin was effectively treated, and compared with monoclonal, apigenin, The effect of inhibiting the expression of HK2 was relatively high when mixed with apigenin or honokiol + curcumin.

Although the expression of HK2 enzyme is low in normal cells, the expression level of HK2 is very high in most cancer cells. In addition, the HK2 enzyme not only phosphorylates glucose but also binds to VDAC protein of mitoton dria to inhibit apoptosis, thereby enhancing the viability of cancer cells. Therefore, it is expected that the treatment effect of HK2-targeted cancer will be enhanced by remarkably inhibiting HK2 expression due to the treatment of a mixture of Honkiol + Apigenin or Honkiol + Curcumin.

Example 5 Reduction of the glycolysis rate of lung cancer cells by treatment with honokiol, apigenin, curcumin alone or in combination

In the results of Example 4, the effect of inhibiting HK2 expression by the treatment of monoclonal, apigenin and curcumin alone or mixed was confirmed. In order to confirm whether the treatment of Hunochiol, apigenin and curcumine alone or in combination reduces the rate of action of lung cancer cells, human lung cancer cell line A549 was treated with either alone or in combination with honokiol, apigenin and curcumin And then analyzed using the Lactate Assay Kit (Biovision).

As a result, as shown in Fig. 5, the effect of reducing the action of human lung cancer cell line was confirmed by the treatment of monoclonal, apigenin and curcumin alone or in combination. In particular, as compared with the administration of monocytoin, apigenin, It was confirmed that the effect of reducing the action was increased when the combination of honokiol + apigenin or honokiol + curcumin was administered.

On the other hand, in the glucose metabolism of cancer cells, the ATP productivity through the process is more ineffective than the oxidative phosphorylation, assuming that the oxidative phosphorylation pathway of mitochondria is defective and that ATP will be produced mainly by the process alone, Should perform the process at a very high level to produce sufficient ATP. Therefore, metabolic inhibition through inhibition of cancer-related enzymes is highly valued as a target of cancer therapy, and the effect of reducing the action of the combination of honokiol + apigenin or honokiol + It is expected to be linked to treatment.

Example 6 Inhibitory Effect of Honkiol, Apigenin, Curcumin alone or Mixed Treatment on PD-L1 Expression in Lung Cancer Cells

In order to examine the effect of inhibiting the expression of PD-L1 in human lung cancer cell lines due to the treatment of monoclonal, apoptotic, and curcumin alone or in combination, the human lung cancer cell lines A549 and H2228 were treated with monoclonal, apigenin, (PD-L1 antibody; cell-signaling, Actin antibody: santa cruze). The expression level of PD-L1 at the protein level was analyzed by Western blot.

As shown in FIG. 6A, PD-L1 expression was very low (PD-L1 low ) in lung cancer cell line A549 used in Examples 1 to 5, L1 expression in the lung was difficult to confirm, and the lung cancer cell line H2228 (PD-L1 high ), in which the expression of PD-L1 was relatively high , was used.

As a result, as shown in Fig. 6 (b), it was observed that the expression of PD-L1 was effectively inhibited when honokiol, apigenin, curcumin alone or in combination. In particular, the decrease in the expression of PD-L1 was observed very weakly when monocytes treated with monocytoin, apigenin or curcumin, while the expression of PD-L1 was very strongly inhibited in the mixed treatment of monocytoin + apigenin or honokiol + curcumin .

When a T cell that has a killing effect on a cancer cell binds to a protein PD-L1 on the surface of the cancer cell, the T cell can not attack the cancer cell, and the cancer cell killing effect is lost. Immune anticancer drugs block the binding of the PD-L1 protein by the antibody, causing T cells to attack cancer cells themselves. These anticancer drugs strengthen the body's immune system and have no side effects or resistance to conventional anticancer drugs. Therefore, it is possible to suppress the function of tumor-specific T lymphocyte cells by changing the immune checkpoint function through modulation of PD-L1 expression as one of immunity avoidance strategies of cancer cells. In other words, the inhibition of tumor-specific T-lymphocyte cells is avoided by activating such inhibitory immune detection in tumor cells. Recently, it has been known that an anti-tumor effect can be obtained by enhancing the tumor-specific T-lymphocyte cell activity and effect by inhibiting its function by using a monoclonal antibody against PD-L1, and the therapeutic method targeting PD- Has demonstrated a very effective clinical efficacy not previously available.

From the above, inhibition of ANT2 or HK2 expression by mixed administration of honokiol + apigenin or honokiol + curcumin not only induces direct cell death, but also overcomes the immunological evasion mechanism of lung cancer cells through PD-L1 inhibition , And is expected to ultimately increase cancer treatment efficacy.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (10)

  1. Apigenin or curcumin; And a pharmaceutical composition for preventing or treating lung cancer.
  2. The method according to claim 1,
    Wherein the composition comprises apigenin and honokiol as an active ingredient.
  3. The method according to claim 1,
    Wherein the composition comprises curcumin and honokiol as an active ingredient.
  4. The method according to claim 1,
    Wherein the composition reduces the expression of the ANT2 (Adenine nucleotide translocator 2) gene.
  5. The method according to claim 1,
    Wherein the composition reduces the expression of HK2 (hexokinase 2) or PD-L1 (Programmed death-ligand 1) protein.
  6. Apigenin or curcumin; And a health functional food composition for preventing or ameliorating lung cancer.
  7. The method according to claim 6,
    Wherein the composition comprises apigenin and honokiol as an active ingredient.
  8. The method according to claim 6,
    Wherein said composition comprises curcumin and honokiol as an active ingredient.
  9. The method according to claim 6,
    Wherein the composition reduces the expression of the ANT2 (Adenine nucleotide translocator 2) gene.
  10. The method according to claim 6,
    Wherein the composition reduces the expression of HK2 (hexokinase 2) or PD-L1 (Programmed death-ligand 1) protein.
KR1020160111383A 2016-08-31 2016-08-31 Pharmaceutical compositions for preventing or treating lung cancer comprising apigenin, curcumin, and honokiol as active ingredients KR101803000B1 (en)

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WO2012159085A2 (en) 2011-05-19 2012-11-22 Glax L.L.C. Compositions and methods for treating and preventing cancer by targeting and inhibiting cancer stem cells
US20160015709A1 (en) 2012-04-05 2016-01-21 The Regents Of The University Of California Compositions and methods for treating cancer and diseases and conditions responsive to cell growth inhibition

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US8916541B2 (en) * 2011-01-05 2014-12-23 Better Health Publishing, Inc. Synergistic combination of honokiol and modified citrus pectin in cancer therapy
US20160129127A1 (en) * 2013-05-20 2016-05-12 Sree Chitra Tirunal Institute For Medical Sciences And Technology Development of Soluble Albuminated Curcumin for Application in Cancer Therapy

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WO2012159085A2 (en) 2011-05-19 2012-11-22 Glax L.L.C. Compositions and methods for treating and preventing cancer by targeting and inhibiting cancer stem cells
US20160015709A1 (en) 2012-04-05 2016-01-21 The Regents Of The University Of California Compositions and methods for treating cancer and diseases and conditions responsive to cell growth inhibition

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Tumor Biol. Volume 37, Issue 10, pp 13871-13883 (2016.08.02.)

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