KR20220046233A - Anticancer composition containing morusin - Google Patents

Abstract

The present invention relates to an anticancer composition containing morusin, and a method for preventing or treating cancer using the anticancer composition. Morusin of the present invention is a natural product-derived component, biocompatible, has no side effects, and thus can inhibit cancer growth by inducing autography.

Description

Anticancer composition comprising morusin {ANTICANCER COMPOSITION CONTAINING MORUSIN}

The present invention relates to an anti-cancer composition and a health functional food containing morusin.

Lung cancer is one of the leading causes of cancer-related death worldwide, with a 5-year survival rate of less than 15%. About 70% of lung carcinoma patients have metastasized at the time of diagnosis, which is a cause of high mortality from lung cancer. Patients with advanced stage lung tumors have been reported to die within 18 months of diagnosis. Lung cancer is divided into two histological classes, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), accounting for 80-85% of all lung cancer cases. Although significant progress has been made in the treatment of NSCLC over the past decades, such as epithelial growth factor receptor (EGFR) targeted therapy, angiogenesis inhibitors and immune checkpoint inhibitors, lung cancer patients still suffer from poor prognosis and aggressive disease progression. Therefore, it is necessary to discover new drugs for the treatment of NSCLC and establish more effective treatment strategies.

Autophagy is a cellular process that removes and recycles nonfunctional cytoplasmic components. During the autophagy process, damaged organelles or abnormally folded proteins in the cytoplasm are sequestered by autophagosomes, which are double-membrane endoplasmic reticulum. Autophagosomes finally fuse with lysosomes to form autolysosomes, and the isolated cytoplasmic components are degraded by lysosomal enzymes. Autophagy occurs in most tissues as a cytoplasmic quality-control mechanism for maintaining intracellular homeostasis. However, autophagy can be caused by various intracellular stresses such as nutrient deprivation, reactive oxygen species (ROS) production, hypoxia, and DNA damage. In addition, autophagy has been implicated in various human diseases, such as neurodegenerative diseases, liver diseases, muscle diseases, and pathogenic infections, as well as cancer. The role of autophagy in cancer progression depends on the tumor type, pathological stage, and clinical stage. In general, autophagy inhibits tumorigenesis by eliminating genomic instability and tissue damage at an early stage. However, in advanced stages of cancer, it promotes tumor growth and chemo-resistance. The role of autophagy according to stages in cancer may be a clinical solution by inducing or inhibiting autophagy in cancer treatment.

AMP-activated protein kinase (AMPK) is a well-known serine/threonine protein kinase that regulates metabolic pathways in response to cellular stress and energy deprivation. Activation of the AMPK pathway is known to play a central role in the autophagy process. When intracellular ATP is depleted and AMP levels increase, AMPK changes its conformation so that the upstream kinase LKB1 can readily access and activate AMPK. Activated AMPK then phosphorylates various substrates and transcription factors to regulate metabolism. Among its substrates, UNC-51-like kinase1 (ULK1) is known to promote autophagy under glucose starvation. Like other downstream proteins, regulatory proteins of mTOR (Raptor) and tuberous sclerosis complex 2 (TSC2) block the interaction between AMPK and ULK1, inactivating the mammalian target of rapamycin complex 1 (mTORC1), which inhibits ULK1. . Therefore, AMPK not only directly activates ULK1, but also triggers autophagy by interfering with the mTORC1 inhibitory effect on ULK1. Numerous studies have demonstrated that the AMPK/mTOR signaling association plays an essential role in the autophagy process.

Morusin is a marker component of the mulberry tree ( Morus alba L. ) and has been traditionally used in Korea for lung diseases such as cough, asthma and pulmonary congestion. Morusin exhibits various biological activities such as antioxidant, anticonvulsant, antibacterial, and anti-inflammatory.

Korean Patent Publication No. 10-2015-0083604

The inventors of the present invention have completed the present invention by discovering that morusin triggers autophagy in NSCLC cells and confirming its mechanism.

Accordingly, an object of the present invention is to provide an anti-cancer composition and health functional food containing morusin, and a method for preventing or treating cancer using the anti-cancer composition.

In order to achieve the above object, the present invention provides an anticancer composition comprising morusin as an active ingredient.

In one embodiment of the present invention, the anticancer composition may further include an autophagy inhibitor, an AMPK inhibitor, and a combination thereof.

In one embodiment of the present invention, the autophagy inhibitor may include Dorsomorphin (compound C), STO-609, GSK-690693, and combinations thereof.

In one embodiment of the present invention, the AMPK inhibitor may include Dorsomorphin (compound C), STO-609, GSK-690693, and combinations thereof.

In one embodiment of the present invention, the composition may have a preventive and therapeutic effect on a cancer selected from the group consisting of lung cancer, colorectal cancer, liver cancer, prostate cancer, rectal cancer, skin cancer, breast cancer, ovarian cancer and uterine cancer.

In addition, the present invention provides a health functional food for anticancer comprising morusin as an active ingredient.

The present invention also provides a method for preventing or treating cancer, comprising administering the anticancer composition according to the present invention to a patient.

Morusin of the present invention is a natural product-derived component, biocompatible and has no side effects, and can inhibit cancer growth by inducing autophagy. In addition, the present invention can provide a clue to understand the controversial role of autophagy in cancer progression by confirming the role of Morusin-induced autophagy in cancer cell growth, so that it is an effective anticancer agent or anticancer-related health functional food information for the development of

1 shows the induction of autophagy by anorusin in NSCLC cells: (A) Cell morphology taken under a microscope (×200 magnification). (B) LC3 puncta in cells immunostained with anti-LC3 antibody and fluorescent-dye conjugated secondary antibody. DAPI staining was performed to visualize the nuclei. LC3 puncta (green) and nuclei (blue) were photographed under a fluorescence microscope (×200 magnification) and combined. (C and D) Protein expression detected by Westen blot. Actin is the loading control. ImageJ software was used for densitometry analysis of the immunoblot. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. untreated control.
Figure 2 shows an increase in anorusin-induced apoptosis in NSCLC cells anmorusin-induced apoptosis by autophagy inhibition. H460 cells were treated with or without the autophagy inhibitor bafilomycin A1 (1 nM) for 12 h (A) or 72 h (BE) with anorucin. (A) Expression of LC3II detected by Western blot. Actin was used as an internal control. (B) Cell viability measured by MTT assay. (C) Proportion of viable cells assessed by trypan blue exclusion assay. (D) Proportion of sub-G1 phase cells (apoptotic cells) determined by performing cell cycle analysis by flow cytometry. (E) Apoptosis detection by Annexin V/PI double staining analysis. Annexin V(+) cells were determined to be apoptotic cells. *** P < 0.001 vs. each contrast. Mo, morucine; Baf, bafilomycin A1.
3 shows the role of the AMPK/mTOR pathway in Morusin-induced autophagy in NSCLC cells. (A) Protein expression detected by Western blot. Actin is the loading control. (B) Ratio between phosphorylated protein and total protein calculated after densitometry analysis of immunoblots using Image J software. *** P < 0.001 vs. untreated controls.
Figure 4 shows that morusin-induced apoptosis is increased in NSCLC cells by AMPK inhibition. H460 cells were treated with or without the AMPK inhibitor compound C (1 μM) for 24 h (A) or 72 h (BD) with morusin (5 μM). (A) Expression of proteins detected by Western blot analysis. Actin was used as an internal control. (B) Cell viability measured by MTT assay. (C) Proportion of viable cells assessed by trypan blue exclusion assay. (D) Proportion of sub-G1 phase cells (apoptotic cells) as determined by flow cytometry. *** P < 0.001 vs. each contrast.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to an anticancer composition and a health functional food comprising morusin as an active ingredient.

The morusin may further include an autophagy inhibitor, an AMPK inhibitor, and a combination thereof.

The autophagy inhibitor may be used without limitation as long as it is an autophagy inhibitor used in the art. For example, chloroquinine (CQ), hydroxychloroquinine (HCQ), etc., which are currently clinically approved autophagy inhibitors, may be used, or bafilomycin A1, SBI currently under development in preclinical or clinical stages. It may include, but is not limited to, various autophagy inhibitors such as -0206965 and 3-methyladenine.

The AMPK inhibitor may include, but is not limited to, Dorsomorphin (compound C), STO-609, GSK-690693, and the like.

The anticancer composition may exhibit an anticancer effect against cancer such as lung cancer, colorectal cancer, liver cancer, prostate cancer, rectal cancer, skin cancer, breast cancer, ovarian cancer, uterine cancer, etc. For example, it may exhibit an anticancer effect on lung cancer, It is not limited thereto.

The anticancer composition comprising anmorusin according to the present invention as an active ingredient may include one or more pharmaceutically acceptable carriers, excipients or diluents in addition to a pharmaceutically effective amount of anorusin.

As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to exhibit the desired physiological or pharmacological activity when the physiologically active ingredient is administered to an animal or human. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of symptoms, the age, weight, health status, sex, administration route, and treatment period of the patient.

In addition, the term "pharmaceutically acceptable" as used herein means that when it is physiologically acceptable and administered to humans, it does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives and the like may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, and may be formulated for a variety of oral or parenteral administration. It can be formulated in the form Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The composition according to the present invention may be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on several factors such as the route of administration, the patient's age, sex, body weight, and the severity of the patient. may be appropriately selected according to In addition, the composition of the present invention may be administered in parallel with a known compound capable of enhancing the desired effect.

Furthermore, the composition according to the present invention can be used not only as a pharmaceutical composition as described above, but also as a health functional food. For example, it can be easily utilized as a main raw material of food, an auxiliary raw material, a food additive, a functional food or a beverage.

The “food” means a natural product or processed product containing one or more nutrients, and preferably means a state that can be eaten directly through a certain amount of processing, and in a conventional sense, food , which includes all food additives, functional foods and beverages.

Foods to which the composition for food can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, and the like. In addition, special nutritional foods (eg, formula milk, young, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasoned foods (eg, soy sauce) , soybean paste, red pepper paste, mixed paste, etc.), sauces, sweets (eg snacks), candy, chocolate, gum, ice cream, dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi) , pickles, etc.), beverages (eg, fruit beverages, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.), but are not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

In addition, the “functional food” or “health functional food” refers to a food group or food composition in which added values are added so that the food functions and expresses the function of the food for a specific purpose using physical, biochemical, or bioengineering methods, etc. It refers to food that is designed and processed to sufficiently express the body control functions related to defense rhythm control, disease prevention and recovery, etc. to the living body. Specifically, it may be a health functional food. The functional food may include a food supplementary additive that is pharmaceutically acceptable, and may further include an appropriate carrier, excipient and diluent commonly used in the manufacture of functional food.

The type of health supplement is not limited thereto, but may be in the form of powder, granule, tablet, capsule or beverage.

In addition, the present invention provides a method for preventing or treating cancer, comprising administering the anticancer composition according to the present invention to a patient.

The treatment method of the present invention comprises administering the pharmaceutical composition to a subject in a therapeutically effective amount. A specific therapeutically effective amount for a particular subject will depend on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the subject's age, weight, general health, sex and diet, time of administration; It is preferable to apply differently depending on various factors including the route of administration and secretion rate of the composition, the duration of treatment, the drug used together with or concurrently with the specific composition, and similar factors well known in the pharmaceutical field. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention in consideration of the foregoing.

The patient is applicable to any mammal, and the mammal includes not only humans and primates, but also domestic animals such as cattle, pigs, sheep, horses, dogs and cats.

Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the embodiments described below in detail. Hereinafter, the present invention will be described in detail by way of Examples. However, these examples are for describing the present invention in detail, and the scope of the present invention is not limited to these examples.

Example 1

Reagents and Antibodies

Morusin was purchased from ChemFaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) at 100 mM as a stock solution. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Duchefa (Haarlem, The Netherlands) and trypan blue was purchased from WelGENE (Daegu, Republic of Korea). 4,6-diamidino-2-phenylindole (DAPI), DMSO, propidium iodide (PI), RNase A, bafilomycin A1, and compound C were purchased from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies to autophagy-related 5 (Atg5), Beclin-1, LC3, phospho-AMPK (Thr172), AMPK, phospho-acetyl-coA carboxylase (ACC; Ser79), ACC, phospho-mTOR (Ser2448), and mTOR was purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against Atg12, phospho-p70S6 kinase (p70S6K, Ser434), p70S6K, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-mouse (Bethyl Laboratories, Montgomery, TX, USA) and goat anti-rabbit antibodies (Enzo Life Sciences, Farmingdale, NY, USA) were used as secondary antibodies.

Cell Lines and Cell Cultures

H1299 and H460 cells are human lung carcinoma cell lines (American Type Culture Collection; ATCC; Manassas, VA, USA). RPMI 1640 medium (WelGENE) was used for culturing cells and was added together with 10% FBS (WelGENE) and 1% penicillin-streptomycin solution (WelGENE). Cells were maintained in 5% CO ₂ and wet incubation at 37°C.

MTT analysis

H460 cells were seeded in 96-well plates at a density of 2.5×10 ³ cells/well and stabilized overnight. After cells were treated with malignant for 72 h, 10 μL of MTT stock solution (4 mg/mL) was added to culture medium (100 μL) to give a final concentration of 0.4 mg/mL. After incubation at 37° C. for 2 hours, the medium was completely aspirated. Then, 100 μL of DMSO was injected into each well. The 96-well plate was gently shaken for 30 minutes to completely dissolve the formazan. Then, the absorbance at 540 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Trypan blue exclusion analysis

H460 cells (2×10 ⁴ cells/well) were seeded in 12-well plates and stabilized overnight. Adhered cells were treated with drugs. After incubation for 72 hours, cells were harvested. 25 μL of cell suspension was mixed with 25 μL of 0.4% trypan blue solution. Then, unstained cells (viable cells) were counted under a microscope (Leica, Wetzlar, Germany) using a hemocytometer.

Immunofluorescence

H460 or H1299 cells were seeded in 6-well plates and allowed to settle overnight. After treatment with morucin (20 μM) for 6 or 48 hours, the cells attached to the coverslip were washed repeatedly with cold PBS (phosphate-buffered saline) and fixed with 100% methanol at -20°C for 10 minutes. It was then blocked with a 3% bovine serum albumin (BSA; GenDEPOT, Katy, TX, USA) solution at 4°C for 50 min. After washing with cold PBS, cells were incubated overnight at 4°C with primary antibody against LC3, followed by incubation with Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA, USA) for 50 min at room temperature. The cells were then counterstained with DAPI. Then, LC3 puncta and nuclei were observed under a fluorescence microscope (Leica, Wetzlar, Germany) at ×200 magnification.

flow cytometry

H460 cells (1x10 ⁵ cells/well) were inoculated into a 6-well plate and treated with drugs for 72 hours. To determine the sub-G1 phase, cell cycle analysis was performed. Cells were pelleted and fixed with cold 80% ethanol overnight at -20°C. Then, the cells were washed with PBS and stained with a PI staining solution (50 μg/mL PI in PBS containing 30 μg/mL RNase A) for 30 minutes in the dark. Thereafter, the stained cells were pelleted by centrifugation and resuspended in 500 μL of PBS. The DNA content of the cells was checked by flow cytometry (FACSCalibeur, BD Biosciences), and the ratio of each phase of the cell cycle (sub-G1, G1, S, G2/M) was calculated with CellQuest Pro software (version 5.1). For Annexin V-PI double staining analysis, cells were collected, stained with Annexin V-FITC apoptosis detection kit I (BD Biosciences; PharMingen), and analyzed by flow cytometry. Annexin V(+) cells were determined to be apoptotic cells.

western blot

Cells were washed with cold RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitors (100 mM NaF and 1 mM Na ₃ VO ₄ ). After dissolving on ice for 50 minutes, the supernatant was collected by centrifugation. Protein concentrations were measured with the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). 20 μg of protein was separated on an 8-13% acrylamide gel by SDS-PAGE, and transferred to a polyvinyl difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) at 60 V for 2 hours. After blocking the membrane with 3% BSA at room temperature for 30 min, a 1:500-1:1000 diluted primary antibody was added to the membrane and incubated at 4°C overnight. Membrane was washed repeatedly with TBST for 50 min and probed with secondary antibody diluted 1:10000 for 50 min at room temperature. Protein expression was detected using the D-Plus ECL Femto System (Donginbio, Seoul, Korea). ImageJ software (ImageJ 1.38; National Institutes of Health) was used for densitometry analysis of immunoblots.

statistical analysis

Results are presented as mean ± standard deviation (SD) of results obtained from three replicates. Statistical analysis was performed by paired Student's t-test. In all cases, differences with P -value < 0.05 were considered statistically significant.

Example 2

Morusin-induced autophagy induction in NSCLC cells

It was found that many vacuoles were formed in the cytoplasm of H460 and H1299 human lung carcinoma cells after morusin treatment. Vacuoles were detected from 6 hours after anorucine treatment and were maintained until 48 hours ( FIG. 1A ). In a previous study, it was reported that autophagy vacuoles similar to the vacuoles observed in the present invention appeared during autophagy, so it was checked whether these vacuoles were related to autophagy induction. It is well known that LC3II formed by conjugation of lipid phosphatidylethanolamine (PE) with cytosolic LC3I is recruited to the autophagosome membrane during autophagy and is expressed as LC3 puncta. As can be seen in Figure 1B, morucine treatment significantly increased the formation of LC3 puncta in H460 and H1299 cells. LC3 puncta remained constant from early (6 h) to late (48 h) after morusin treatment, which is consistent with the results of Fig. 1A, suggesting that the observed cytoplasmic vacuoles are associated with the formation of autophagosomes ( 1B).

In addition, it was confirmed whether the expression of autophagy-related proteins was changed by morusin. Beclin-1 plays an important role in the initiation of autophagosomes, and the Atg5-Atg12 complex expands the autophagosome membrane. Experimental results of the present invention showed that morusin increased the expression of Atg5, Atg12, beclin-1, and LC3II in a time-dependent manner in two cell lines (FIG. 1C). In addition, the accumulation of LC3II was maintained up to 48 hours after morusin treatment (Fig. 1D). Taken together, we demonstrated that morucin maintained autophagy in autophagy NSCLC cells.

Inhibition of autophagy increases Morusin-induced apoptosis in NSCLC cells.

In order to determine what role Morusin-induced autophagy plays in the survival of NSCLC cells, bafilomycin A1, which inhibits autophagy by blocking autophagosome-lysosomal fusion as well as lysosomal acidification, was used. As can be seen in FIG. 2A , the expression of LC3II was increased by treatment with morusin for 12 hours and was further increased by co-treatment with bafilomycin A1, indicating that bafilomycin works well ( FIG. 2A ). The results of MTT analysis showed that the cell viability was significantly reduced in H460 cells co-treated with anorusin and bafilomycin A1 compared to when anorusin alone was treated ( FIG. 2B ). A trypan blue exclusion assay, which measures the percentage of viable cells, also showed similar results (Fig. 2C). In addition, co-treatment with anorusin and bafilomycin A1 synergistically increased the proportion of sub-G1 phase cells in H460 cells, indicating that inhibition of autophagy clearly stimulated anmorusin-induced apoptosis (Fig. 2D). The proportion of annexin V-positive cells increased significantly to 72.28% in H460 cells co-treated with anvil and bafilomycin A1, whereas only 45.7% of annexin V-positive cells were shown in cells treated with anvil, which was consistent with the experimental results. (Fig. 2E). In summary, these findings demonstrate that morusin-induced autophagy protects NSCLC cells from apoptosis.

AMPK/mTOR Pathway Mediates Morusin-Induced Autophagy in NSCLC Cells

It is well known that AMPK/mTOR signaling cooperation plays an important role in the progression of autophagy. Activation of AMPK inhibits activation of mTOR, inducing autophagy and inhibiting cell growth. To determine whether the AMPK/mTOR pathway is involved in morusin-induced autophagy, the phosphorylation levels of AMPK, mTOR, and their downstream were measured. As can be seen in FIG. 3 , phosphorylation of AMPK was clearly increased by morusin in a time-dependent manner in H460 and H1299 cells. The expression of p-ACC, which is downstream of AMPK, also increased up to 24 hours after morusin treatment, and although it decreased at 48 hours, this seems to be due to a decrease in total protein. In contrast, the phosphorylation/total ratio of mTOR and its downstream p70S6K was significantly reduced in a time-dependent manner after morusin treatment in H460 and H1299 cells ( FIGS. 3A and 3B ). Taken together, these results demonstrate that morusin-induced autophagy is mediated by activation of AMPK and inhibition of the mTOR pathway.

Inhibition of AMPK Activity Increases Morusin-Induced Apoptosis in NSCLC Cells

In a previous study, it was reported that AMPK activation triggers not only autophagy but also apoptosis in cancer cells (Li W et al., Saud SM, Young MR, Chen G, Hua B. Targeting AMPK for cancer prevention and treatment. Oncotarget 2015 ;6:7365-78). [Park HJ et al., Induction of apoptosis by morusin in human non-small cell lung cancer cells by suppression of EGFR/STAT3 activation. Biochem Biophys Res Commun2018;505:194-200], it was confirmed whether morucine-induced AMPK activation was related to autophagy as a survival mechanism or apoptosis induction as a death mechanism in NSCLC cells. As can be seen in Fig. 4A, phosphorylation of both AMPK and ACC was significantly reduced by compound C, an inhibitor of AMPK, indicating that compound C effectively inhibits the kinase activity of AMPK (Fig. 4A). In addition, the results of MTT analysis ( FIG. 4B ) and trypan blue exclusion analysis ( FIG. 4C ) showed that treatment with compound C increased the cytotoxic effect of anorusin in H460 cells. Cell viability in H460 cells co-treated with anorusin and compound C was significantly reduced compared to cells treated with anorucine alone ( FIGS. 4B and 4C ). In addition, inhibition of AMPK by compound C increased anmorusin-induced apoptosis in H460 cells. The proportion of sub-G1 group was significantly increased to 23.78% by co-treatment with anorucine and compound C, whereas only 10.81% increased in cells treated with anmorucine alone ( FIG. 4D ). Taken together, these findings appear to induce autophagy in NSCLC cells, in which AMPK activation by anmorusin acts as a defense mechanism against anorusin-induced apoptosis.

conclusion

The present invention is the first study to confirm the role and underlying mechanism of anmorusin-induced autophagy in NSCLC cells. In addition, the present invention highlighted the role of AMPK in autophagy induction and survival of NSCLC cells under cytotoxic stress. Activation of AMPK and subsequent inactivation of mTOR mediates Morusin-induced autophagy, ultimately protecting NSCLC cells from apoptosis.

Although autophagy acts as a tumor suppressor by inducing autophagy cell death of type II programmed cell death (PCD) in cancer cells, the results of the present invention clearly demonstrate that morusin-induced autophagy acts as a survival mechanism in NSCLC cells. This has been demonstrated, suggesting that the efficacy of Morusin will be increased by combination with other autophagy inhibitors. Currently, chloroquine (CQ) and hydroxychloroquine (HCQ) are clinically acceptable autophagy inhibitors. They have been reported to deacidify lysosomes and block fusion between lysosomes and autophagosomes. There are numerous killing trials showing that the combination of CQ or HCQ with other chemotherapy improves clinical outcomes, such as prolonging overall survival and increasing cancer progression-free survival.

AMPK plays an important role in the process of autophagy and is recognized as a novel target for cancer therapy. Many studies have reported a close relationship between AMPK and tumor progression. Basically, AMPK blocks the activity of mTORC1, thereby attenuating cell proliferation. As 30-50% of NSCLCs carry LKB1 mutations, it is hypothesized that the inability to activate AMPK results in uninhibited cell proliferation in NSCLCs. Indeed, it has been reported that AMPK activation in NSCLC is associated with a better prognosis and increased overall survival. In addition, various AMPK agonists such as metformin and AICAR also exhibit strong anti-tumor activity. Conversely, induction of autophagy by AMPK activation can cause active growth of tumors by maintaining energy production, suggesting a potential problem of AMPK activation in cancer therapy. Since the present inventors' previous papers and the experimental results of the present invention show that anmorusin triggers both apoptosis and cytoprotective autophagy in NSCLC cells, anmorusin-induced AMPK activation is not dependent on either cell survival or apoptotic cell death. It was confirmed that it is related. Experimental results show that inhibition of AMPK increases the cytotoxic effect of anorusin in NSCLC cells, and AMPK acts as a defense mechanism against anorusin-induced apoptosis, thereby inducing autophagy in NSCLC cells. is to propose Several candidates, such as EGFR, mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3), have been identified as mediators of morusin-triggered apoptosis in cancer cells. It has been proposed instead of AMPK activation.

Interestingly, in our previous study, we demonstrated that methylene chloride extract of Morus alba L. (MEMA) triggers autophagy in tNSCLC cells as a survival mechanism through ROS production, and that morusin also triggers methylene chloride of Morus tree. It was shown to be contained as a label compound in the extract. Therefore, we propose that morusin may be an active compound in methylene chloride extract of Morus tree to induce autophagy in NSCLC cells. Considering that the accumulation of ROS has a contradictory effect on the survival of cancer cells by activating AMPK, it is likely to be a mediator of morusin-induced autophagy in NSCLC cells. However, as our previous data showed that pretreatment of N-acetylcysteine (NAC) in NSCLC cells increased anmorucine-induced apoptosis (data not shown), this finding was consistent with MEMA-induced autophagy. It is suggested that the specific mechanism underlying the morusine-induced autophagy may be different.

An important question is whether Morusin is effective against chemoresistant NSCLC cells. In this regard, a previous study by the present inventors demonstrated that morusin induces apoptosis in EGFR tyrosine kinase inhibitor (TKI)-resistant NSCLC cells as well as untreated NSCLC cells [Park et al., Induction of apoptosis by morusin in human non-small cell lung cancer cells by suppression of EGFR/STAT3 activation. Biochem Biophys Res Commun 2018;505:194-200]. Although the present invention did not focus on the chemo-resistance-overcoming activity of anmorucine, autophagy is known as a possible mechanism involved in drug resistance in NSCLC. Therefore, co-treatment with morusin and autophagy inhibitors will be effective for chemoresistant NSCLC cells.

In conclusion, in the present invention, it was demonstrated that anmorusin induces autophagy in NSCLC cells through AMPK activation as a defense mechanism against anmorusin-induced apoptosis. In addition, the present invention provides insight into the controversial roles of autophagy and AMPK in cancer progression. In addition, the present invention suggests that the combined treatment of anmorusin and an autophagy inhibitor or an AMPK inhibitor can improve the clinical efficacy of anmorusin in NSCLC.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (7)

An anticancer composition comprising morusin as an active ingredient. The anticancer composition of claim 1, further comprising an autophagy inhibitor, an AMPK inhibitor, and a combination thereof. The method according to claim 2, wherein the autophagy inhibitor is selected from the group consisting of chloroquinine (CQ), hydroxychloroquinine (HCQ), bafilomycin A1, SBI-0206965, 3-methyladenine, and combinations thereof. an anticancer composition. The anticancer composition of claim 2, wherein the AMPK inhibitor is selected from the group consisting of Dorsomorphin (compound C), STO-609, GSK-690693, and combinations thereof. According to claim 1, wherein the composition is lung cancer, colon cancer, liver cancer, prostate cancer, rectal cancer, skin cancer, breast cancer, ovarian cancer and the anticancer composition to have a therapeutic effect on the cancer selected from the group consisting of cancer. A health functional food for anticancer that contains Morusin as an active ingredient. A method for preventing or treating cancer comprising administering the anticancer composition of any one of claims 1 to 5 to a patient other than a human.

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