KR20190106315A - Composition for enhancing EGFR-TKI sensititivity for non small cell lung carcinoma including extract of Coptis chinesis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating non-small cell lung cancer that exhibits resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, which contains an extract of Coptis chinensis as an active component.

Description

Composition for enhancing EGFR-TKI sensititivity for non small cell lung carcinoma including extract of Coptis chinesis}

The present invention relates to a composition for enhancing sensitivity to epidermal growth factor receptor target anticancer agent of non-small cell lung cancer. Specifically, the present invention is directed to a composition for preventing or treating non-small cell lung cancer, which exhibits resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), which comprises a rhubarb extract as an active ingredient. It is about.

Lung cancer is the leading cause of cancer-related deaths worldwide. The most common form of lung cancer is non small cell lung carcinoma (NSCLC), which accounts for 80% of all lung cancers. In Korea, the incidence and mortality rate is increasing every year. According to the National Cancer Information Center, the incidence rate is fourth, but mortality is first.

Although lung cancer has doubled in the last 30 years through the development and application of various angle treatments such as surgery, chemotherapy and radiation therapy, lung cancer has a 15-year survival rate of around 15% due to the difficulty of early detection. Is not very good. Accordingly, there is a need for development of drugs and technologies that can be actively used for prevention and treatment along with biological basic knowledge of lung cancer.

Currently, various epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as Gefitinib, Erlotinib, Afatinib, and Osimertinib have been developed in lung cancer. Lung cancer patients with mutations in the genes have shown dramatic effects on treatment.

However, despite the initial response of cancer, there is a need to overcome new problems in that cancer cells acquire resistance to treatment within 6 months to 1 year. Such mechanisms of resistance acquisition have been identified, such as the acquisition of secondary mutations in EGFR, activation of various signaling pathways in various cells, avoidance of cell death, changes in microenvironment in cancer tissues.

As a treatment for overcoming such a resistance mechanism, a combination of drugs that may exhibit synergistic effects may be used. However, the combination of conventional chemical drugs are highly toxic to normal cells as well as cancer cells may affect not only cancer cells but also normal cells.

Natural products have minimal toxicity to normal cells, but may have anticancer effects specific to cancer cells. And since natural products are not monolithic and their toxicity is relatively weak, they can be a very effective approach when used as a combination therapy or adjuvant.

Coptis chinesis , a natural product, belongs to the family of potted yams . It brightens the eyes and stops tears from coming out, soothing the liver and removing heat poisoning. Your eyes become red and hard to see, but put them in your eyes when you are sick. It is written in the main text that it is surprising to treat heart pounding, anxiety, to help the phlegm, to treat the mouth and to get rid of children's sickness (drying disease). It is known to contain alkaloids (berberine), phenulic acid, and chlorogenic acid. These rhubarb extracts are known to be effective in treating allergies or inflammation, improving skin aging, and preventing respiratory diseases.

Accordingly, the present inventors have completed the present invention by continuing to study the drug sensitivity of lung cancer in which resistance to EGFR target anticancer drugs has occurred.

Korean Patent Application No. 10-2013-0070782 Korean Patent Application No. 10-2008-0055537

An object of the present invention is to provide a composition for the prevention or treatment of non-small cell lung cancer that exhibits resistance to EGFR tyrosine kinase inhibitors.

Another object of the present invention is to provide a composition for inhibiting resistance to EGFR tyrosine kinase inhibitors of non-small cell lung cancer.

The present invention provides a composition for the prevention or treatment of non-small cell lung cancer that exhibits resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, comprising a rye extract as an active ingredient.

As used herein, the term "non-small cell lung cancer" is a malignant cell that develops from epithelial cells of the lung and is a tumor cell observed by an observer, for example, a histopathologist under a microscope. Are classified according to their size and shape.

In the present invention, the term “epidermal growth factor receptor (EGFR)” is overexpressed in about 50-80% of non small cell lung carcinoma (NSCLC), and when overexpressed, the prognosis is poor.

As used herein, the term “EGFR tyrosine kinase inhibitor (EGFR-TKI)” is an EGFR target anticancer agent, which selectively binds to tyrosine kinase in the EGFR intracellular domain to inhibit ATP binding. Block the signal path.

All of these terms are well known to those skilled in the art.

In the present invention, the "non-small cell lung cancer that exhibits resistance to EGFR-TKI" means that non-small cell lung cancer cells exhibit resistance or resistance to EGFR-TKI (EGFR-TKI-R) and EGFR-TKI is no longer treated. Non-small cell lung cancer that does not show an effect.

In the present specification, the "non-small cell lung cancer exhibiting resistance to EGFR-TKI" may be referred to as EGFR-TKI-R non-small cell lung cancer or EGFR-TKI-R lung cancer.

The EGFR-TKI can include any known one in the art. Specifically, Gefitinib, Erlotinib Afatinib, Daconitib, Ossimertinib, Ceritinib, CI-1033 (Canertinib), HKI-272 (Neratinib) or pharmaceutically acceptable salts thereof may be included, but is not necessarily limited thereto.

The non-small cell lung cancer may be squamous cell cancer, large cell cancer or adenocarcinoma.

The sulfur extract may include berberine or a pharmaceutically acceptable salt thereof. The berberine is a compound known as a component isolated from the extract of the yellow lotus.

In another aspect, the present invention provides a pharmaceutical composition comprising: i) yellow lotus extract; And ii) an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) or a pharmaceutically acceptable salt thereof as an active ingredient, an EGFR tyrosine kinase inhibitor of non-small cell lung cancer ( It provides a composition for inhibiting resistance to EGFR-TKI).

The meaning and type of the non-small cell lung cancer, EGFR-TKI are as described above.

The i) sulfur extract is, ii) a substance that increases the sensitivity of non-small cell lung cancer to EGFR-TKI.

In the present invention, the term “increased sensitivity of EGFR-TKI” means sensitizing non-small cell lung cancer cells to EGFR-TKI, thereby reducing the resistance of cancer cells to anticancer drugs (EGFR-TKI) and increasing the effects of anticancer drugs. do. This can increase the treatment efficiency of EGFR-TKI therapeutics, and when combined with cancer treatment, EGFR-TKI sensitivity of cancer cells can be enhanced to reduce the killing and proliferation of cancer cells while reducing the amount of conventional EGFR-TKI. Will be.

The non-small cell lung cancer may be resistant to EGFR-TKI.

The sulfur extract may include berberine or a pharmaceutically acceptable salt thereof.

Mutations in non-small cell lung cancer show resistance to EGFR-TKI (resistant), and EGFR-TKI is no longer effective for treating lung cancer. In the present invention, for the treatment of non-small cell lung cancer exhibiting such resistance, it was used together with the rhubarb extract that can synergistically improve the effect of EGFR-TKI treatment.

According to one embodiment of the invention, the composition of the invention may comprise i) sulfur extract and ii) EGFR-TKI in the same container, or each in a separate container.

The sulfur extract of the present invention inhibits EGFR phosphorylation activity (pEGFR) in lung cancer cells resistant to EGFR-TKI and inhibits the activity of AKT / pAKT, which is a downstream signaling system of EGFR, as well as inhibiting cell viability. Cell mobility and invasiveness are also effectively inhibited.

In addition, the extract of the present invention induces apoptosis, and inhibits the expression of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-xl through cleavage of caspase 3 and PARP. It was confirmed to induce apoptosis effectively.

These results can be seen that the extract of L. oleracea shows a particularly effective anti-cancer effect of non-small cell lung cancer and non-small cell lung cancer (EGFR-TKI-R non-small cell lung cancer) cell line. In addition, the extract of L. hull shows that it is applicable to the treatment of lung cancer and / or EGFR-TKI-R lung cancer cells or as an adjuvant treatment of EGFR-TKI.

The composition of the present invention may be provided in various forms selected from pharmaceutical compositions or functional food compositions.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the sulfur extract. Such pharmaceutically acceptable carriers are lactose, textose, sucrose, acacia rubber, calcium phosphate, alginate, sorbitol, mannitol, starch, gelatin, polyvinylpyrrolidone, cellulose, silicic acid commonly used in pharmaceutical formulations. Calcium, microcrystalline cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, calcium silicate, microcrystalline cellulose and the like. However, it is not limited thereto. In addition, the pharmaceutical composition may further include a sweetener, lubricant, wetting agent, flavoring agent, sweetening agent, flavoring agent, emulsifier, suspending agent, preservative, and the like as an additive.

The method of administration of the pharmaceutical composition is determined according to the degree of symptoms, and in general, a topical administration method is recommended. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on conditions of disease, age, sex, weight, route of administration, etc., and may be administered from one time to several times daily.

The pharmaceutical composition may be administered to various mammals such as mice, rats, domestic animals, humans, and the like. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intrauterine dural, rectal or cerebrovascular injections.

The pharmaceutical composition may be prepared in unit dose form by formulation with a pharmaceutically acceptable carrier and excipient or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion, or may be in the form of an exercicide, a granule, a tablet, a warning agent, an excipient, a powder, a lotion, an ointment.

Oral dosage forms containing the composition of the present invention as an active ingredient may be formulated as tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Formulated into tablets and capsules, such as lactose, sorbitol, saccharose, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate It may include a lubricating oil, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient may be used in the form of injections such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method or aerosol for inhalation through the respiratory system. It can be formulated as. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into the suppository, it may be formulated into a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

In addition, when the composition of this invention is a functional food composition, there is no restriction | limiting in particular in the kind of said food.

When the sulfur extract of the present invention is administered in combination with EGFR-TKI, the therapeutically effective amount of the sulfur extract may be administered simultaneously or sequentially with EGFR-TKI.

The therapeutically effective amount means an amount that effectively enhances the sensitivity of the tumor in cancer cells to EGFR-TKI. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on various factors including the route of administration and the rate of release of the composition, the duration of treatment, and the radiation dose to be irradiated and similar factors well known in the medical arts. Therefore, the effective amount of the composition for enhancing radiation sensitivity suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters. The daily dosage of the composition of the present invention may be, for example, 0.0001-100 mg / kg.

The composition of the present invention provides a prophylactic or therapeutic effect of non-small cell lung cancer that is resistant to EGFR TKI. In addition, the composition of the present invention provides a combination formulation and combination use of the barberry extract and EGFR TKI for the prevention or treatment of non-small cell lung cancer resistant to EGFR TKI.

1 is a result of MTT analysis of (EGFR-TKI-R) non-small cell lung cancer cells resistant to (EGFR-TKI) EGFR-TKI according to an embodiment of the present invention.
Figure 2 shows the result of reduced mobility (A) and invasiveness (B) of EGFR-TKI-R non-small cell lung cancer cells according to an embodiment of the present invention.
3 to 7 show the apoptosis of the EGFR-TKI-R non-small cell lung cancer cells according to an embodiment of the present invention.
8 and 9 are the results confirming the synergistic effect of the combination treatment of the rhubarb extract and EGFR-TKI according to an embodiment of the present invention.
Figure 10 shows the results of the MTT analysis when treated with berberine in accordance with an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in more detail. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth. It should be noted that the embodiments of the present invention described below are intended to sufficiently convey the spirit of the present invention to those skilled in the art.

Example 1 Experimental Methods and Materials

1-1: Preparation of Rhubarb Extract

Purchased from Dongguk University Ilsan Hospital (Kyunggi-do, South Korea) and selected under the sentiment of Professor Eun-Jung Kim (Department of Oriental Medicine) was selected and used. 10 g of rhubarb is placed in a 3L round flask equipped with a reflux condenser, and extracted by adding 10-fold water (w / v) solvent (water, ethanol) for 24 hours at room temperature, and then boiling for 4 hours at a temperature below boiling point (90 ℃, ethanol extraction was extracted at 70 ℃). The extract was filtered using a 0.2 μm pore size Wattpan paper, and the filtrate was concentrated under reduced pressure using a rotary vacuum concentrator (EYELA, Japan). After lyophilization using a lyophilizer (EYELA, Japan) was used as a sample. The weight and yield of the obtained extract were 1.26 g / 12.6% of ethanol solvent.

1-2: Reagent

EGFR-TKI, Gefitinib, was purchased from Selleck chemical. Antibodies to EGFR, AKT, p-AKT, Caspase 3, PARP were purchased from Cell signaling technology. Antibodies against p-EGFR, MET, BCL2, MCL-1, BCL-xl and β-actin were purchased from Santa Cruz Biotechnology. Secondary antibodies, goth anti-mouse IgG and goth anti-rabbit IgG, were purchased from Santa Cruz biotechnology.

1-3: Cell Culture

Human normal lung epithelial cell lines BEAS-2B and human non-small cell lung cancer cell lines PC9, PC9GR, A549, and A549GR were each provided by Dr. Jin-Kyung Roh of Asan Hospital, University of Ulsan. Wherein PC9GR and A549GR cells are in turn PC9G resistant cell lines and A549 resistant non-small cell lung cancer cell lines, respectively. Berberine was purchased from Sigma. 10% FBS (fetal bovin serum, Welgene) and 1% penicillin / streptomycin (penicillin / Streptomycin) added to the cells in 37% at 37 ℃ in a 5% CO 2 incubator in RPMI medium (Welgene).

1-4: Cell Viability and Drug Toxicity Analysis

Cell viability was performed using MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) assay. Cells (1 × 10 ³ ) are seeded in 96-well cell culture plates and cultured overnight, and then treated or not treated with the corresponding reagent EGFR-TKI (zeftinib) or rhubarb extract or berberine. After 72 hours of cell culture, 50 uL of MTT solution (0.5 mg / ml) (Sigma-aldrich) is added. After 4 hours of incubation, the MTT solution was removed and dimethyl Sulfoxide (DMSO) was added. Absorbance at 570 nm was specified using a microplate reader (SpectraMax Plus 384). Drug combination effects were measured by calculating the Fa (fraction affected) and CI (combinatin index) values using CompuSyn Software (www.combosvn.com). A CI value of <1 means a complementary (synergy) effect, a value of 1 means addicitve, and a value of 1 means antithesis.

1-5: Cell migration assay

Cell migration was performed using wound healing analysis. Cells ( ⁵ × 10 ⁵ ) are seeded in 6-well plates and incubated for 24 hours, followed by scraping the worms with sterile micropipette tips. The cells are washed with PBS and then a fixed amount of drug (Rhubarb extract of Example 1-1) is added. From this point of view, the degree of healing of the wound for a predetermined time was observed under a microscope, and then the degree of healing of the wound was compared.

1-6: Cell Infiltration Assay

Infiltration analysis was performed using transwell chambers with a polycarbonate nucleopore membrane (Costar). Precoated filters [6.5 mm in diameter, 8-μm pore size, Matrigel (100 μg / cm 2)] were rehydrated with 100 μl medium. Next, 1 × 10 ⁵ cells were seeded at the top of each chamber, while the bottom was filled with 600 μl of the medium. After incubation at 37 ° C. for 24 hours, non-invasive cells on the filter top surface were removed with a cotton swab, the invaded cells on the filter bottom surface were fixed and hematoxylin / eosin stained. Invasiveness was measured by counting cells in five random microscope regions per well, and the chipping range was expressed as the average number of cells per microscope region.

1-7: Flow Cytometry

To test cell cycle distribution, cells dissociated into single cells were fixed with 75% ethanol, reacted at −20 ° C. for at least 1 hour, and washed three times with PBS. Then, resuspended in PBS containing 0.1 mg / mL RNase-A, 50 mg / mL propidium iodide and 0.05% Triton X-100 for 40 minutes at room temperature, washed with PBS And FACS Canto 2 (Becton Dickinson).

1-8: Apoptosis Assay

Cell death was performed by terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis. Cells were inoculated onto the cover slide containing the complete medium, and the addition of the rhubarb extract was added or not. After 24 and 48 hours of incubation, the cells were fixed with 4% paraformaldehyde at 4 ° C. for 10 minutes and washed twice with PBS at room temperature. Cells were then permeated for 5 minutes at room temperature with 0.2% Triton X-100 dissolved in PBS and washed twice with PBS. Stained cells were identified under fluorescence microscopy according to the manufacturer's protocol (DeadEnd ™ Fluorometric TUNEL System; Promega).

1-8: Reverse Transcriptase-Polymerase Chain Reaction

Total RNA (1 μg / μl) was isolated from cells using TRIzol (Molecular Research Center) according to the manufacturer's protocol. Complimentary DNA (cDNA) was synthesized using cDNA synthesis kit (Applied biosystmes). Oligonucleotide primer sequences are as follows:

BCL2 sense: 5'-AAGGGGGAAACACCAGAATC-3 '(SEQ ID NO: 1),

BCL2 antisense: 5'-ATCCTTCCCAGAGGAAAAGC-3 '(SEQ ID NO: 2);

MCL-1 sense: 5′-TGCTGGAGTAGGAGCTGGTT-3 ′ (SEQ ID NO: 3),

MCL-1antisense: 5′-CCTCTTGCCACTTGCTTTTC-3 ′ (SEQ ID NO: 4),

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense: 5′-GCCATCGTCACCAACTGGGAC-3 ′ (SEQ ID NO: 5),

GAPDH antisense: 5′-CGATTTCCCGCTCGGCCGTGG-3 ′ (SEQ ID NO: 6).

1-8: Western blot analysis

Cells were dissolved in 1 × RIPA buffer [50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA (ethylenediaminetetraacetic acid), 0.5% Nonidet P-40 and protease inhibitor cocktail tablet (Roche), and Bio-Rad protein assay reagent Protein concentration was measured by (Bio-Rad). Total protein aliquots (30 mg protein / lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrocellulose at 100 V for 2 hours. Electrophoresis was carried out to the membrane (Millipore). The membrane was blocked at room temperature for 1 hour with 5% BSA dissolved in TBST [20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.01% Tween 20], and then reacted at 4 ° C. overnight with primary antibody. After washing the membrane intensively with TBST, the membrane was probed for 1 hour at room temperature with a secondary antibody conjugated with horseradish peroxidase. After three washes for 15 minutes with TBST, the membrane was visualized by chemiluminescence (Amersham) according to the manufacturer's protocol.

1-9: Statistical Analysis

Statistical analysis was performed using an independent sample t-test. The deviation was determined to be statistically significant at P <0.05.

Example 2: Results

2-1: Viability and Mobility Inhibition of EGFR-TKI-R Lung Cancer Cells by Huang Lian Extract

The present inventors first tested the effect of rhubarb extract on the viability, mobility and invasiveness of lung cancer, EGFR-TKI-R lung cancer cells and normal bronchial epithelial cells. As a method for specifying the viability of the cells, the value of the half maximal inhibitory concentration (IC50) was measured using a well-known MTT assay. In normal bronchial epithelial cells, about 80% of cells survive at 100 mg / ml (IC50: 178u / ml or more), whereas the IC50 of each lung cancer cell line was 70ug / ml and 30ug / ml. IC50 of the EGFR-TKI-R lung cancer cells constructed by 33ug / ml, 20ug / ml, respectively, it was confirmed that the most effective inhibition of survival of EGFR-TKI-R lung cancer cells (Fig. 1).

In addition, wound healing analysis and invasiveness analysis were used as a method for determining whether the extracts of the yellow lotus have the effect of inhibiting the mobility and invasiveness of the cells, which are characteristic of cancer cells. The mobility of the cells was confirmed that the mobility of the cells was inhibited by slowing the healing rate of the epidermis in a concentration-dependent and treatment time-dependent manner of the L. extract (FIG. 2A). In addition, it was confirmed that the invasiveness of EGFR-TKI-R lung cells also decreased in a concentration-dependent manner (Figure 2B). These results indicate that rhubarb extract effectively inhibits the viability, mobility and invasiveness of lung cancer and EGFR-TKI-R lung cancer cells.

2-2: Rhizome Extract Arrests G2 / M Phase in the EGFR-TKI-R Lung Cancer Cell Cycle and Induces Apoptosis

We tested whether rhubarb extract induces death of EGFR-TKI-R lung cancer cells. As a result of cell cycle analysis, the fraction of subG1 increased in time-dependently in EGFR-TKI-R lung cancer cells. As a result of observing the treatment with the rhubarb extract under an optical microscope, it was confirmed that the cells treated with the rhubarb extract are dying compared to the cells without the rhubarb extract (FIGS. 4A and 4B). Confirmation of these results by immunocytochemical analysis showed that the extract of the barberry extract improved the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells (FIG. 5). ). These results indicate that rhubarb extract induces apoptosis in EGFR-TKI-R lung cancer cells, at least in part.

2-3: Lactobacillus extract inhibits EGFR-AKT signaling and antiapoptotic proteins Mcl-1, Bcl-2 and Bcl-xl in EGFR-TKI_R lung cancer cells to inhibit cell survival Judo

In order to identify the fundamental molecular mechanisms that induce survival inhibition and apoptosis in EGFR-TKI-R lung cancer cells by the extract of the rye, we investigated the expression patterns of important signal molecules related to cell survival and apoptosis. First, Western blot was used to investigate the factors whose expression was changed in EGFR-TKI_R lung cancer cells (PC9GR and A549GR) compared to the parent lung cancer cells (PC9 ALC A549) .The activation of AKT and AKT, the lower signaling factors of the EGFR signaling system, was investigated. It was observed that the expression of Mcl-1 or Bcl-2 was increased in the phosphorylated pAKT and bcl-2 family of anti-cell death molecules (FIG. 6). When the L. extract was treated to EGFR-TKI-R lung cancer cells, cell survival was inhibited by inhibiting the expression of EGFR and AKT signaling systems and their activators, pEGFR and pAKT, in a time and concentration-dependent manner, and EGFR-TKI-R Inhibiting the expression of bcl-xl as well as the increased Mcl-1 and Bcl-2 in lung cancer cells was confirmed to induce apoptosis while increasing the cleavage form of caspase 3 and PARP (Fig. 7).

2-4: Rhubarb Extract Synergically Increases Sensitivity of EGFR-TKI in EGFR-TKI-R Lung Cancer Cells

Since the extract of L. japonicum inhibits the survival of EGFR-TKI-R lung cancer cells and particularly induces apoptosis, it was tested whether lung cancer cells could increase the sensitivity to EGFR-TKI, a drug that is resistant. The present inventors have observed the viability of the cells by treating single or a combination of rhubarb extract and EGFR-TKI at selected concentrations by MTT assay, and confirmed that the viability of the cells was much reduced when the combination was treated. In addition, it was confirmed by Western blot that such a decrease in viability induced reduction of Mcl-1 and Bcl-2 of EGFR / AKT signaling factor and anti-cell death factor (FIG. 8). In order to verify that the combination of these drugs is complementary (synergeical), a median effect analysis was performed with Fa-CI Plot (Chu-Talalay Plot; www.combosyn.com). Combination index (combination index) through this analysis as a result of less than 1 was confirmed that the combination of the extract and EGFR-TKI synergistic effect (Fig. 9).

2-5: Anticancer Effect of Berberin, an Alkylating Substance, in Huang Lian Extract

As a result of analyzing the cell survival inhibitory effect of berberine, which is an alkylating substance, which is considered as an active ingredient in the extract of the rye, using MTT, it was confirmed that the survival of lung cancer cells was suppressed (FIG. 10). In particular, berberine can be seen that the cell survival inhibition in EGFR-TKI-R lung cancer cell line as well as general lung cancer cell line.

According to the present invention, as a result of confirming that rhubarb extract and berberine show cancer cell-specific anti-cancer effects in lung cancer cells and EGFR-TKI-R lung cancer cells, the effect of killing cells in normal bronchial epithelial cells is insignificant, but lung cancer and EGFR-TKI- It was confirmed that specifically inhibiting the activity of the EGFR / AKT signaling system in R lung cancer cells to inhibit cell survival and induce apoptosis pathway by inhibiting the expression of Bcl-2, Mcl-1, Bcl-xL. In addition, when EGFR-TKI and rhubarb extract at the same time treatment was confirmed that there is a complementary synergistic effect. Therefore, the extract can be used in parallel with EGFR-TKI to enhance the sensitivity of EGFR-TKI in lung cancer and EGFR-TKI-R lung cancer cells.

The present inventors completed the present invention by investigating whether rhubarb extract has an anticancer effect specifically for EGFR-TKI-R lung cancer cells and can enhance the effect of EGFR-TKI when used concurrently with EGFR-TKI. . The present inventors have anti-cancer effects by inhibiting cell survival and inducing apoptosis of lung cancer and EGFR-TKI-R lung cancer cells with little effect on the survival of normal bronchial epithelial cells, and simultaneously with EGFR-TKI When used, it was confirmed to enhance the efficacy of EGFR-TKI.

While the above has been shown and described with respect to preferred embodiments of the present invention, the present invention is not limited to the specific embodiments described above, it is usually in the technical field to which the invention belongs without departing from the spirit of the invention claimed in the claims. Various modifications can be made by those skilled in the art, and these modifications should not be individually understood from the technical spirit or prospect of the present invention.

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Claims (10)

A composition for preventing or treating non-small cell lung cancer, which exhibits resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, comprising a rye extract as an active ingredient.
The method of claim 1,
The EGFR tyrosine kinase inhibitor is Gefitinib, Erlotinib, Erfatinib, Afatinib, Daconitib, osimertinib, Seritib, Ceritinib, CI-1033. (Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof
The method of claim 1,
The non-small cell lung cancer is characterized in that the squamous cell cancer, large cell cancer or adenocarcinoma.
The method of claim 1,
The sulfur extract is a composition containing berberine or a pharmaceutically acceptable salt thereof.
i) rhubarb extract; And ii) an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, a composition for inhibiting resistance to an EGFR tyrosine kinase inhibitor of non-small cell lung cancer.
The method of claim 5,
Wherein i) the barberry extract increases the sensitivity of non-small cell lung cancer to the EGFR tyrosine kinase inhibitor.
The method of claim 5,
The non-small cell lung cancer is a non-small cell lung cancer showing resistance to EGFR tyrosine kinase inhibitor.
The method of claim 5,
The EGFR tyrosine kinase inhibitors are Gefitinib, Erlotinib Afatinib, Daconitib, ossimertinib, Ceritinib, CI-1033 ( Canertinib), HKI-272 (Neratinib) or a pharmaceutically acceptable salt thereof.
The method of claim 5,
The non-small cell lung cancer is characterized in that the squamous cell cancer, large cell cancer or adenocarcinoma.
The method of claim 5,
The sulfur extract is a composition containing berberine or a pharmaceutically acceptable salt thereof.

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