WO2018043989A1 - Pharmaceutical composition comprising apigenin, curcumin, and honokiol as active ingredients for preventing or treating lung cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising, as active ingredients, apigenin or curcumin, and honokiol for preventing or treating lung cancer. In the composition according to the present invention, when compared with the treatment with apigenin, curcumin, or honokiol alone, the treatment with a mixture or combination thereof synergistically induces the apoptosis of cancer cells, and thus the pharmaceutical composition comprising, as active ingredients, apigenin or curcumin, and honokiol for preventing or treating lung cancer of the present invention increases an anticancer effect to induce the apoptosis of cancer cells, thereby exhibiting an excellent cancer treatment increasing effect. Therefore, the composition according to the present invention can be advantageously used in the prevention, alleviation, or treatment of lung cancer.

Description

Pharmaceutical composition for the prevention or treatment of lung cancer containing apigenin, curcumin and honokiol as active ingredients

The present invention is apigenin or curcumin; And the therapeutic effect of lung cancer of the honokiol mixture.

Cancer refers to a group of abnormal cells caused by continuous division and proliferation due to the disruption of the balance between cell division and death by various causes, also called malignant tumor. In general, cancer develops in more than 100 different parts of the body, including organs, white blood cells, bones, lymph nodes, etc., and develops severe symptoms through infiltration into surrounding tissues and metastases to other organs, leading to death. do. Among them, lung cancer, a malignant tumor originating in the lung, is largely classified into small cell lung cancer and non-small cell lung cancer according to its tissue type. The small cell lung cancer is classified as a part of the lung cancer by the location of the diseased tissue, but is distinguished as described above because it is distinctly distinguished from other lung cancer in terms of clinical course, treatment and prognosis. In addition, non-small cell lung cancer is classified into adenocarcinoma, squamous cell carcinoma and large cell carcinoma according to histologic type.

Specifically, small cell lung cancer is generally known to have a large mass, gray-white, and proliferate along the bronchial wall, and in most cases, surgical resection is difficult to progress at the time of diagnosis. In addition, it is easily transmitted to the whole body through blood circulation, while it is known to have a remarkable therapeutic effect by chemotherapy or radiotherapy. Brain, liver, bone, lung, adrenal gland, kidney, etc. have been reported as the main organs for the metastasis of small cell lung cancer, and it is known that it occurs first in the airways (bronchi or bronchioles).

In contrast, non-small cell lung cancer is classified into adenocarcinoma, squamous cell acrcinoma, and large-cell carcinoma as described above. First, adenocarcinoma mainly occurs in the peripheral part of the lungs, and also occurs well in women or non-smokers, and even if the size is small, the metastasis is often, the frequency of its occurrence is increasing recently. Next, squamous cell carcinoma is mainly found in the center of the lungs, grows mainly into the lumen of the bronchus, shows symptoms of blocking the bronchus, is common in men, and is known to be closely related to smoking. Finally, large cell carcinoma occurs mainly near the lung surface (peripheral lung), about half occurs in large bronchus, accounting for 4-10% of all lung cancers, and large cell sizes, some of which It tends to proliferate and metastasize rapidly, resulting in a poor prognosis compared to other non-small cell lung cancers.

When such lung cancer is diagnosed early, it can be recovered through drug treatment and radiation therapy, but when the condition is worsened to a certain level, the lesion is removed through surgery and treatment through drug treatment and radiation therapy. However, there are a few limited drugs that can be used for the treatment of lung cancer, so it is difficult to carry out appropriate treatment for patients, and the side effects are also a big problem, and most primary tumors are observed. Cancer metastasis has already been found at this point, and the success rate after surgery is low.

Accordingly, efforts have been made to develop cancer suppressors and cancer therapeutic agents which have a low side effect from relatively low toxicity products and are excellent in inhibiting or reducing metastasis of cancer species as new approaches for cancer treatment. The fundamental disadvantage that the therapeutic agent is not excellent in the therapeutic effect is not solved (Korean Patent Publication No. 10-2012-0109139).

Against this background, the present inventors have made intensive research efforts to develop an agent that can safely and effectively treat lung cancer, resulting in apigenin or curcumin; And it was confirmed that the mixture of the honokiol can cause the death of lung cancer cell line, inhibit the proliferation of cancer cells, found that it can be usefully used as a composition for treating lung cancer, and completed the present invention.

The present invention has been made to solve the above problems, the inventors of the present invention is to reduce the rate of action of lung cancer cell line by a single or mixed administration of apigenin, curcumin, honokiol, inhibition of ANT2 expression, inhibition of HK2 expression, PD- The effect of inhibiting L1 expression and reducing ATP production was confirmed, and thus the present invention was completed.

Accordingly, an object of the present invention is apigenin or curcumin; And it provides a pharmaceutical composition for preventing or treating lung cancer comprising honokiol as an active ingredient.

Further, another object of the present invention is apigenin or curcumin; And it provides a health functional food composition for preventing or improving lung cancer comprising honokiol 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.

In order to achieve the object of the present invention as described above, the present invention is apigenin or curcumin; And it provides a pharmaceutical composition for preventing or treating lung cancer comprising honokiol as an active ingredient.

Another object of the present invention is apigenin or curcumin; And it provides a health functional food composition for preventing or improving lung cancer comprising honokiol as an active ingredient.

In one embodiment of the invention, the composition may comprise apigenin and honokiol as an active ingredient.

In another embodiment of the present invention, the composition may include curcumin and honokiol as an active ingredient.

In another embodiment of the present invention, the composition may reduce the expression of the Adenine nucleotide translocator 2 (ANT2) 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 for preventing or treating lung cancer, comprising administering the pharmaceutical composition to a subject.

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

The present invention is apigenin or curcumin; And it relates to a pharmaceutical composition for the prevention or treatment of lung cancer comprising a honokiol as an active ingredient. The composition according to the present invention induces the death of cancer cells synergistically when combined or combined treatment than when treated with apigenin, curcumin or honokiol alone, apigenin or curcumin of the present invention; And lung cancer preventive or therapeutic pharmaceutical composition comprising a mixture of Honokiol as an active ingredient shows an excellent cancer treatment enhancement effect by increasing the anticancer effect to induce the death of cancer cells. Therefore, the composition according to the present invention can be usefully used for preventing, improving or treating lung cancer.

1 is a result confirming the effect of killing lung cancer cell line when treated alone or mixed with Honokiol, apigenin, curcumin.

Figure 2 is a result confirming the inhibitory effect of ANT2 expression in lung cancer cell lines when treated alone or in combination of Honokiol, apigenin, curcumin.

Figure 3 is a result confirming the effect of reducing the ATP production of lung cancer cell line when treated alone or mixed with Honokiol, apigenin, curcumin.

Figure 4 is a result confirming the inhibitory effect of HK2 expression in lung cancer cell line when treated alone or in combination of Honokiol, apigenin, curcumin.

5 is a result confirming the effect of reducing the glycolysis rate (glycolysis rate) of lung cancer cell lines when treated alone or in combination of Honokiol, apigenin, curcumin.

Figure 6a is a result confirming the inhibitory effect of PD-L1 expression in lung cancer cell line A549 when treated alone or in combination with Honokiol, apigenin, curcumin.

Figure 6b is a result confirming the inhibitory effect of PD-L1 expression in lung cancer cell line H2228 when treated with Honokiol, apigenin, curcumin alone or in combination.

Compositions according to the invention include apigenin or curcumin; And a mixture of Honokiol as an active ingredient, reducing the corresponding action rate of the lung cancer cell line, inhibition of ANT2 expression, inhibition of HK2 expression, inhibition of PD-L1 expression and ATP production, thereby confirming the effects of preventing, improving or treating lung cancer. Based on this, the present invention has been completed.

Hereinafter, the present invention will be described in detail.

The present invention is apigenin or curcumin; And it provides a composition for the prevention or treatment of lung cancer comprising honokiol as an active ingredient. The composition includes a pharmaceutical and nutraceutical composition.

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

As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms for lung cancer by administration of the pharmaceutical composition according to the present invention.

"Cancer", which is a disease to be prevented and treated by the composition of the present invention, is a disease that causes malignant tumors by unlimited proliferation of cells in living tissues, and more specifically, lung cancer, but is not limited thereto.

As used herein, "honokiol" is a polyphenol-based compound, which may be isolated, purified or extracted from Magnolia species, and more specifically, may be isolated, purified or extracted from hummus. Without limitation, chemically synthesized compounds or both commercially available may be used. It may also be a honokiol derivative or analog, but is not limited thereto.

"Apigenin (5,7,4'-trihydroxyflavone)" used in the present invention is a flavonoid-based compound, which is not only found in locust extract, but also fruits such as oranges, apples, cherries, grapes, and onions, parsley , Abundantly present in vegetables such as celery, barley, tomatoes, beverages such as tea, wine, etc., can be separated, purified or extracted therefrom, but is not limited thereto, chemically synthesized compounds or commercially available You can use both. In addition, it may be an apigenin derivative or an analog, but is not limited thereto.

As used herein, "curcumin" is an alkaloid-based compound, which may be separated, purified, or extracted from turmeric or turmeric, but is not limited thereto. Any chemically synthesized compound or commercially available one can be used. Can be. 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 including apigenin and honokiol as an active ingredient, 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 including curcumin and honokiol as active ingredients, but is not limited thereto.

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

"ANT2 (Adenine nucleotide translocator 2)" of the present invention is a gene having an important function in the survival, proliferation and metastasis of cancer cells. Adenine nucleotide translocase / translocator (ANT) 2 protein is the most protein in the mitochondrial lining and is formed by six transmembrane helices. ANT plays an important role in cellular energy metabolism by catalyzing the change from mitochondrial ATP to cytoplasmic ADP, thereby affecting mitochondrial oxidative phosphorylation. All mammals have isoforms of ANT1 to ANT3, and the isoform family, ANT2, appears to increase the uptake of cellular ATP produced by glycolysis in the conversion to intramitochondrial ADP, resulting in mitochondria. Membrane potential can be maintained, and knockdown of ANT2 reduces membrane potential difference in cancer cells.

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

"HK2 (hexokinase 2)" of the present invention is an enzyme that phosphorylates hexasaccharides containing 6 carbons and converts them into hexasaccharides. HK2 is less expressed in normal cells but is highly expressed in most cancer cells. In addition to phosphorylating glucose, it has the function of binding to mitochondrial VDAC protein and inhibiting apoptosis to enhance cancer cell viability. Thus, it can be effectively used for cancer treatment targeting HK2.

In addition, "PD-L1 (Programmed death-ligand 1)" protein of the present invention is a protein present on the surface of cancer cells, T-cells having a killing effect of cancer cells, PD-L1 is a protein on the surface of cancer cells and When bound, T cells do not attack cancer cells and lose their cancer cell killing effect. Immune anticancer drugs block the binding of the PD-L1 protein, causing T cells to attack cancer cells on their own. The anticancer drug is to strengthen the human immune system, so there are no side effects or resistance to conventional anticancer drugs. Therefore, as one of the immune evasion strategies of cancer cells, it is possible to suppress the function of tumor-specific T lymphocyte cells through the change of immune checkpoint function through regulation of PD-L1 expression.

In one embodiment of the present invention confirmed the killing effect of the cancer cells by treating alone or mixed treatment with honokiol, apigenin, curcumin in the lung cancer cell line (see Example 1), single or mixed treatment of honokiol, apigenin, curcumin ANT2 expression inhibitory effect (see Example 2), ATP production inhibitory effect (see Example 3), HK2 expression inhibitory effect (see Example 4), glycolysis effect reduction effect (see Example 5) and PD -L1 expression inhibition effect (see Example 6) was confirmed.

Thus the apigenin or curcumin of the present invention; And a pharmaceutical composition comprising honokiol as an active ingredient reduces the corresponding action rate of the lung cancer cell line, inhibits ANT2 expression, inhibits HK2 expression, inhibits PD-L1 expression and reduces ATP production, and prevents, improves or treats lung cancer. It can also be used as an active ingredient of.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, it may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative and the like.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, 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 refers to the type of disease, the severity, the activity of the drug, It may be determined according to the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum 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 according to the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 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 divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

In addition, the composition of the present invention may be added to a dietary supplement for the purpose of preventing or improving lung cancer-related diseases. When the compound having a lung cancer prevention or improvement effect of the present invention is used as a food additive, the compound may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverages the compounds of the invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for health and hygiene or for health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of foods to which the substance may be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and includes all of the dietary supplements in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. Natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion 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 includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonating agents used in carbonated drinks. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually chosen 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 invention, the invention provides a method of treating lung cancer comprising administering the pharmaceutical composition to a subject. As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse and cow, which is human or non-human. .

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  One. Honokiol , Apigenin , Curcumin  Lung cancer cell line killing effect alone or in combination

To confirm the effects of adenocarcinoma cell death killing of Honokiol, Apigenin, and Curcumin, Honokiol, Apigenin, and Curcumin (Sigma-Aldrich) were used alone or in a mixture of human lung cancer cell lines A549 (adenocarcinomic human alveolar basal epithelial cells). Treatment. Concentrations treated with Honokiol, Apigenin, and Curcumin in human lung cancer cell lines were determined by the dose-concentration curve, which is a concentration of 20% (IC ₂₀ : inhibitory concentration 20). Treatments were made alone or in combination with μM. After 24 hours of treatment, the killing effect of lung cancer cell lines was analyzed using CCK8 assay (Cell Counting Kit-8, Dojindo Molecular Technologies, Inc).

As a result, as shown in Figure 1, apoptosis effect was significantly increased during the mixed treatment compared to the treatment with Honokiol, apigenin or curcumin alone. Specifically, compared to the result of 19% cell death by Honokiol alone, 22% cell death by Apigenin alone, and 20% cell death by Curcumin alone, Honokiol + apigenin mixture Cell death by 79% and cell death by the Honokiol + Curcumin mixture showed 83%. The cell killing effect by the mixture can be confirmed to be a synergistic effect, not a simple additive effect. From the above, it is expected that, in the treatment of lung cancer patients, a combination of honokiol + apigenin, or honokiol + curcumin may enhance the efficacy of lung cancer compared to the administration of honokiol, apigenin, curcumin alone.

Example  2. Honokiol , Apigenin , Curcumin  Lung cancer cell caution when used alone or in combination ANT2  Expression inhibitory effect

To confirm the inhibitory effect of hon2ol, apigenin, and curcumin on ANT2 expression, the human lung cancer cell line A549 was treated with honokiol, apigenin, curcumin alone or in combination. Then, qRT-PCR analysis was performed to confirm that ANT2 mRNA expression in lung cancer cell lines is suppressed. More specifically, after treatment or treatment with 10 μM of Honokiol, Apigenin, Curcumin alone or mixed in human lung cancer cell line A549, RNAs were extracted (TRIzol® RNA Isolation Reagents, Thermo Sientific), reverse transcription (RT) : Synthesis of cDNA by reverse transcription (PrimeScript 1st strand cDNA Synthesis Kit, Takara) and real-time polymerase chain reaction (qPCR) using SYBR (SYBR Premix Ex Taq, Takara) It was. 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 the qRT-PCR experiment using the following formula.

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

In addition, the base sequences of the primers used in qPCR are shown in Table 1 below.

primer		sequence	SEQ ID NO:
ANT2	Forward direction	5-ACGTGTCTGTGCAGGGTATT-3	One
	Reverse	5-GTGTCAAATGGATAGGAAG-3	2
GAPDH	Forward direction	5-AAGGTGAAGGTCGGAGTCAA-3	3
	Reverse	5-AATGAAGGGGTCATTGATGG-3	4

As a result, as shown in Figure 2, it was confirmed that the expression of ANT2 was reduced by the treatment or treatment of Honokiol, apigenin, curcumin alone, Honokiol + apigenin, compared to Honokiol, apigenin, curcumin alone treatment, Or Honokiol + curcumin treatment, it was confirmed that the expression inhibitory effect of ANT2 is relatively high.

From the above, by the Honokiol + apigenin, or Honokiol + curcumin mixed administration, the therapeutic effect similar to the cancer treatment effect by ANT2 short interfering RNA (shRNA) or short interfering RNA (siRNA) can be expected It seems to be. In addition, the ANT2 reduction effect by Honokiol, Apigenin, Curcumin alone or in combination treatment reduces ANT2 using small interfering RNA, resulting in the delivery of siRNA or shRNA into the body, which is a problem with cancer treatment. It is expected to solve the problems related to the delivery system.

Example  3. Honokiol , Apigenin , Curcumin  Reduction of ATP Production in Lung Cancer Cell Lines When Treated Alone or Mixed

As a result of Example 2, it was confirmed that ANT2 expression is effectively suppressed when the human lung cancer cell line is treated alone or in combination with honokiol, apigenin, curcumin. Therefore, it was expected that the treatment of honokiol, apigenin, curcumin alone or in combination would reduce the ATP production of cancer cells, since the function of ANT2 plays a critical role in ATP production of cancer cells.

Therefore, in order to confirm the effect of reducing ATP production of lung cancer cell lines due to inhibition of ANT2 expression, after treatment with Honokiol, apigenin, curcumin alone or in combination with human lung cancer cell line A549, CellTiter-Glo ™ Luminescent Cell Viability assay kits (Promega ).

As a result, as shown in FIG. 3, when the single or mixed treatment of honokiol, apigenin, curcumin alone or mixed treatment, it was confirmed that the result of effectively reducing the ATP production of human lung cancer cell line, especially Honokiol + apigenin, honokiol + curcumin In the mixed administration of, the effect of decreasing ATP production was relatively increased compared to the single administration.

From the above, the reduction of ATP, which is an essential energy source of survival, inhibits the growth of cancer cells and ultimately induces the death of cancer cells, so the administration of Honokiol, Apigenin, Curcumin alone or in combination of ATP production due to inhibition of ANT2 expression It is believed that the reduction was induced, thereby improving the therapeutic effect of cancer through the inhibition of ATP production in cancer cells.

Example  4. Honokiol , Apigenin , Curcumin  Inhibitory Effects of HK2 Expression in Lung Cancer Cell Lines When Treated Alone or Mixed

To determine whether inhibition of ANT2 expression by single or mixed treatment of honokiol, apigenin, and curcumin regulates the expression of hexokinase 2 (HK2), an enzyme involved in the action, Honokiol, api in human lung cancer cell line A549 Xenin and curcumin were treated alone or in combination and then analyzed using qRT-PCR.

Analysis of the results of the qRT-PCR experiment using the following formula.

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

In addition, the base sequences of the primers used for qPCR are shown in Table 2 below.

primer		sequence	SEQ ID NO:
GAPDH	Forward direction	5-AAGGTGAAGGTCGGAGTCAA-3	3
	Reverse	5-AATGAAGGGGTCATTGATGG-3	4
HK2	Forward direction	5-CAAAGTGACAGTGGGTGTGG-3	5
	Reverse	5-GCCAGGTCCTTCACTGTCTC-3	6

As a result, as shown in Figure 4, when treated alone or mixed with Honokiol, apigenin, curcumin, it was confirmed that the expression of HK2 is effectively reduced, compared to Honokiol, apigenin, curcumin alone treatment Honokiol + When apigenin or honokiol + curcumin were mixed, the effect of inhibiting the expression of HK2 was relatively high.

The HK2 enzyme is less expressed in normal cells, but the amount of HK2 is very high in most cancer cells. In addition, the HK2 enzyme not only phosphorylates glucose, but also binds to the VDAC protein of mitochondria, inhibits apoptosis, and as a result, functions to enhance the viability of cancer cells. Thus, by significantly inhibiting HK2 expression due to the treatment of the honokiol + apigenin mixture, or the honokiol + curcumin mixture, it is expected to increase its therapeutic effect in the treatment of cancers targeting HK2.

Example  5. Honokiol , Apigenin , Curcumin  Effect of Glycolysis Rate Reduction on Lung Cancer Cell Lines When Used alone or in Mixed Treatment

In the results of Example 4, it was confirmed that the inhibitory effect of HK2 expression due to single or mixed treatment of honokiol, apigenin, curcumin. Thus, to determine whether the treatment of honokiol, apigenin, curcumin alone or mixed treatment reduces the glycolysis rate of lung cancer cell lines, treatment with a single or mixed honokiol, apigenin, curcumin in human lung cancer cell line A549 After that, it was analyzed using Lactate Assay Kit (Biovision).

As a result, as shown in Figure 5, due to the treatment alone or mixed with Honokiol, apigenin, curcumin, the effect of reducing the glycolysis of human lung cancer cell line was confirmed, in particular than the administration of Honokiol, apigenin, curcumin alone, When the mixed administration of Honokiol + apigenin or Honokiol + curcumin, it was confirmed that the effect of reducing the action is increased.

On the other hand, in the glucose metabolism of cancer cells, because there is a defect in the oxidative phosphorylation pathway of the mitochondria, assuming that the production of ATP mainly by the process alone, ATP productivity through the process is inefficient than oxidative phosphorylation, cancer cells In order to produce sufficient ATP, the process must be performed at a very high level. Therefore, metabolism inhibition through the inhibition of enzymes related to cancer metabolism is considered to be very valuable as a target for cancer treatment. Therefore, the effect of reducing the action due to the mixed treatment of Honokiol + Apigenin or Honokiol + Curcumin is effective for lung cancer. It is expected to lead to treatment.

Example  6. Honokiol , Apigenin , Curcumin  Inhibitory Effect of Lung Cancer Cell Lines on PD-L1 Expression in Single or Mixed Treatments

In order to confirm the inhibitory effect of PD-L1 expression of human lung cancer cell lines due to the treatment of Honokiol, Apigenin, Curcumin alone or in combination, Honokiol, Apigenin, Curcumin alone or mixed in human lung cancer cell lines A549 and H2228 After treatment, the expression level at the protein level of PD-L1 was analyzed using Western-blot (PD-L1 antibody; cell-signaling, Actin antibody: santa cruze).

As shown in Figure 6a, the expression of PD-L1 in the lung cancer cell line A549 used in Examples 1 to 5 is very low (PD-L1 ^low ), so that PD- by Honokiol, apigenin, curcumin It was determined that it was difficult to confirm whether or not the expression of L1 was reduced, and thus, lung cancer cell line H2228 (PD-L1 ^high ), which has a relatively high expression of PD-L1, was used.

As a result, as shown in FIG. 6B, it was observed that the expression of PD-L1 was effectively suppressed in the treatment of Honokiol, Apigenin, Curcumin alone or in a mixture. In particular, the decrease in the expression of PD-L1 is observed very weakly when treated with Honokiol, Apigenin or Curcumin alone, while the expression of PD-L1 is very strongly inhibited when Honokiol + Apigenin or Honokiol + Curcumin are mixed. Was observed.

When T cells having cancer cell death effects are combined with PD-L1, a protein on the surface of cancer cells, T cells cannot attack cancer cells and lose cancer cell death effects. Immune anticancer drugs block the binding of the PD-L1 protein, causing T cells to attack cancer cells on their own. These anticancer drugs enhance the human immune system, so there are no side effects or resistance to conventional anticancer drugs. Therefore, as one of the immune evasion strategies of cancer cells, it is possible to suppress the function of tumor-specific T lymphocyte cells through the change of immune checkpoint function through regulation of PD-L1 expression. That is, the tumor-specific T-lymphocyte cell attack is avoided by activating such inhibitory immunoassay in tumor cells. Recently, anti-tumor effects can be obtained by enhancing the activity and effect of tumor specific T-lymphocyte cells by inhibiting their function using monoclonal antibodies against PD-L1. It shows very effective clinical efficacy which has not been seen so far.

From the above, inhibition of ANT2 or HK2 expression by the mixed administration of Honokiol + Apigenin or Honokiol + Curcumin not only induces direct cell death, but also overcomes the immune avoidance mechanism of lung cancer cells through PD-L1 inhibition. Ultimately, it is expected to increase the efficacy of cancer treatment.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The composition according to the present invention induces the death of cancer cells synergistically when combined or combined treatment than when treated with apigenin, curcumin or honokiol alone, apigenin or curcumin of the present invention; And lung cancer preventive or therapeutic pharmaceutical composition comprising a mixture of Honokiol as an active ingredient shows an excellent cancer treatment enhancement effect by increasing the anticancer effect to induce the death of cancer cells. Therefore, the composition according to the present invention can be usefully used for preventing, improving or treating lung cancer.

Claims (12)

1. Apigenin or curcumin; And Honokiol as an active ingredient, lung cancer prevention or treatment pharmaceutical composition.
2. The method of claim 1,

   The composition comprises apigenin and honokiol as an active ingredient, lung cancer prevention or treatment pharmaceutical composition.
3. The method of claim 1,

   The composition comprises curcumin and Honokiol as an active ingredient, lung cancer prevention or treatment pharmaceutical composition.
4. The method of claim 1,

   The composition is characterized in that for reducing the expression of the Adenine nucleotide translocator 2 (ANT2) gene, pharmaceutical composition.
5. The method of claim 1,

   The composition is characterized in that for reducing the expression of HK2 (hexokinase 2) or PD-L1 (Programmed death-ligand 1) protein.
6. Apigenin or curcumin; And administering to the individual a pharmaceutical composition comprising honokiol as an active ingredient, lung cancer prevention or treatment method.
7. Apigenin or curcumin; And lung cancer prevention or treatment of the pharmaceutical composition comprising honokiol as an active ingredient.
8. Apigenin or curcumin; And comprising a honokiol as an active ingredient, lung cancer prevention or improvement health functional food composition.
9. The method of claim 8,

   The composition comprises apigenin and honokiol as an active ingredient, lung cancer prevention or improvement health functional food composition.
10. The method of claim 8,

    The composition comprises curcumin and Honokiol as an active ingredient, lung cancer prevention or improvement for health food composition.
11. The method of claim 8,

    The composition is characterized in that to reduce the expression of ANT2 (Adenine nucleotide translocator 2) gene, dietary supplement composition.
12. The method of claim 8,

    The composition is characterized in that to reduce the expression of HK2 (hexokinase 2) or PD-L1 (Programmed death-ligand 1) protein, dietary supplement composition.

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