WO2021194276A1 - Pharmaceutical composition, for prevention or treatment of cancer, comprising 13-hydroxyoctadecadienoic acid as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, for the prevention or treatment of cancer, comprising 13-hydroxyoctadecadienoic acid (13-HODE) as an active ingredient. The composition of the present invention exhibits efficacy of inhibiting the growth of cancer cells and killing cancer cells, and thus can be used as an anticancer agent.

Description

Pharmaceutical composition for preventing or treating cancer comprising 13-hydroxyoctadecadenoic acid as an active ingredient

The present invention was made by the project number 2015061639 under the support of the Ministry of Science, ICT and Future Planning. Development of source technology and establishment of hepatocyte-based toxicity and drug efficacy evaluation system”, hosted by the Korea Advanced Institute of Science and Technology, and the research period is from October 1, 2015 to September 30, 2016.

This patent application claims priority to Korean Patent Application No. 10-2020-0036500 filed with the Korean Intellectual Property Office on March 25, 2020, the disclosure of which is incorporated herein by reference.

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising 13-HODE (13-Hydroxyoctadecadienoic acid) as an active ingredient.

Cancer is the most common and serious disease that threatens human health, and research and development of anticancer drugs is important for prolonging the lifespan of cancer patients. In recent years, cancer treatment methods have made a lot of progress due to the rapid development of oncogenomics and molecular pharmacology and the development of new anticancer drugs, but there is still a need for the development of new therapeutic agents.

Endometrial cancer is one of the most common cancers in women. Although the incidence rate of endometrial cancer varies by region, it ranks fourth in the United States after breast cancer, lung cancer, and skin cancer. In 2015, US statistics showed 54,870 new cases and 10,170 deaths. In Korea, 20,859 patients occurred in 2015, and endometrial cancer is the 7th most common cancer among all female cancer patients, and the incidence rate is increasing.

The mortality rate of endometrial cancer has doubled compared to 20 years ago, and it has increased by about 8% compared to 10 years ago. Endometrial cancer mainly occurs during menopause in women and is known to occur more in developed countries than in developing countries. In particular, endometrial cancer is highly correlated with obesity, and 81% of patients with a BMI of 30 or higher are diagnosed with endometrial cancer. In addition, the incidence rate among younger women has been increasing over the past 10 years.

The treatment method for endometrial cancer may vary depending on the disease stage, tumor size, age and general condition of the patient, and whether or not a child is desired, but surgery is mostly performed as the primary treatment. Additional treatment methods may include radiation therapy or chemotherapy. Endometrial cancer is treated with hormone therapy with progesterone or chemotherapy without surgery. Chemotherapy uses doxorubicin and cisplatin, which leads to hematologic toxicity and side effects such as hair loss, dizziness, and vomiting. For recurrent or advanced endometrial cancer, taxanes and platinum-containing drugs (TC therapy) are administered adjuvantly.

mTOR (mechanistic target of rapamycin) protein is a serine/threonine kinase and is a key signaling factor that regulates cell growth, cell cycle, protein synthesis and glucose metabolism. In particular, as a key protein for regulating cell growth signaling, its activity is abnormally increased in 30% of solid cancer cells, and it is known that these emtors and higher-level factors (PI3K, Akt) are the most changed in cancer cells. Activation of emtor signals in cancer is caused by mutations in the emtor gene itself, high-level oncogenes (PI3K, Akt) that enhance the activity of emtor, and mutations in tumor suppressor factors (eg PTEN, TSC1/2). . Therefore, inhibition of emtor-associated signaling can lead to cell death by inhibiting protein synthesis, inhibiting lipid synthesis, and inhibiting cell growth. Therefore, there is a need for the development of an emtor-related signaling inhibitor for the inhibition of cancer cell growth and cancer cell death.

The present inventors made intensive research efforts to develop compounds for inhibiting cancer cell growth and killing cancer cells. As a result, the present invention was completed by identifying that the composition containing 13-HODE (13-Hydroxyoctadecadienoic acid) as an active ingredient is effective in treating cancer.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising 13-HODE (13-Hydroxyoctadecadienoic acid) as an active ingredient.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising 13-HODE (13-Hydroxyoctadecadienoic acid) as an active ingredient.

The 13-HODE corresponds to an mTOR signaling inhibitor.

The emtor signal transduction inhibitor refers to a substance that inhibits the activity of emtor itself or inhibits the activity of an upper-level factor that increases the activity of emtor. Targets whose activity is inhibited by the emtor signaling inhibitor may include, but are not limited to, emtor, PI3K, Akt, S6K, and S6.

In one embodiment of the present invention, the 13-HODE is 13-HODE, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

In one embodiment of the present invention, the cancer is a solid cancer.

As used herein, the term "solid cancer" has characteristics distinguishing it from blood cancer, and includes bladder, breast, intestine, kidney, lung, liver, brain, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate and skin. It is a cancer consisting of a mass caused by abnormal cell growth in various solid organs such as

In one embodiment of the present invention, the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer , nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, Pancreatic cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, exogenous female It is selected from the group consisting of gastric cancer, female urethral cancer, and skin cancer, but is not limited thereto. More specifically, the solid cancer is endometrial cancer, colorectal cancer, glioblastoma, breast cancer, liver cancer, and pancreatic cancer.

The brain tumor is selected from the group consisting of meningioma, pituitary tumor, hemangioblastoma, epidermoid tumor, glioma cyst, glioma, oligodendrocyte glioma, glioblastoma, and metastatic brain tumor.

In one embodiment of the present invention, the cancer is a hematologic cancer.

As used herein, the term “hematologic cancer” refers to cancer occurring in components constituting blood, and refers to malignant tumors occurring in blood, hematopoietic organs, lymph nodes, lymphatic organs, and the like.

In one embodiment of the present invention, the hematologic cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. is selected from, but not limited thereto.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the composition as an active ingredient.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration , intracerebral administration, intracranial administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe an effective dosage (a pharmaceutically effective amount) for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-mentioned diseases.

As used herein, the term “prevention” refers to the prevention or protective treatment of a disease or disease state. As used herein, the term “treatment” refers to reduction, suppression, sedation or eradication of a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. At this time, the dosage form may be prepared in various ways such as oral medicine, injection, etc., in the form of a solution, suspension or emulsion in oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, dispersant or stable Additional topics may be included.

According to an embodiment of the present invention, the pharmaceutical composition comprising 13-HODE of the present invention has an effect of inhibiting the emtor signaling process.

In addition, according to an embodiment of the present invention, the pharmaceutical composition of the present invention has the effect of inhibiting the growth of cancer cells and the effect of inhibiting the growth of tumors in vivo. Therefore, the pharmaceutical composition of the present invention has an anticancer effect on cancer, more specifically endometrial cancer.

According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising administering to a subject a pharmaceutical composition comprising the above-described 13-HODE of the present invention as an active ingredient. .

As used herein, the term "administration" or "administer" refers to directly administering a therapeutically or prophylactically effective amount of a composition of the present invention to a subject (individual) suffering from, or likely to suffer from, the subject disease. It means that the same amount is formed in the body of

The "therapeutically effective amount" of the composition means an amount of the composition sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is administered, and includes a "prophylactically effective amount".

In addition, as used herein, the term "subject (subject)" is a mammal, including humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons and rhesus monkeys. . Most specifically, the subject of the present invention is a human.

Since the method for preventing or treating cancer of the present invention includes administering the pharmaceutical composition according to an aspect of the present invention, the description thereof is omitted to avoid excessive redundancy of the present specification for overlapping content. .

The features and advantages of the present invention are summarized as follows:

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising 13-HODE (13-Hydroxyoctadecadienoic acid) as an active ingredient.

The composition of the present invention can be used as an anticancer agent by inhibiting the growth of cancer cells and exhibiting the efficacy of killing cancer cells.

1 shows the chemical formula of 13-HODE.

2 shows the results of analyzing the binding between the emtor protein and 13-HODE through a pull-down assay.

Figure 3a shows the results of analyzing the molecular position of the emtor protein to which 13-HODE binds through hydrogen-deuterium substitution mass spectrometry.

Figure 3b shows the catalytic cleft of the emtor protein, which is the molecular site at which 13-HODE binds.

Figure 4a shows the results of analyzing proteins related to emtor signaling in cells after 13-HODE was treated with respect to JHUEM7 cells through immunoprecipitation.

Figure 4b shows the results of analyzing proteins related to emtor signaling in cells after 13-HODE was treated with respect to JHUEM7 cells through immunoprecipitation.

Figure 4c shows the results of analysis of proteins related to emtor signaling in cells after 13-HODE was treated with respect to JHUEM7 cells through immunoprecipitation.

Figure 5a shows the results of analyzing proteins related to emtor signaling in cells after 13-HODE was treated with respect to JHUEM7 cells through western blotting.

Figure 5b shows the results of analyzing proteins related to emtor signaling in HCT116, SW480, HT29, MDA-MB-468, and U-373 MG cells after treatment with 13-HODE through Western blotting.

6a shows the results of analyzing the viability of JHUEM7 cells after treatment with 13-HODE.

Figure 6b shows the results of analyzing the viability of MDA-MB-468 cells after treatment with 13-HODE.

7a shows the results of analyzing the degree of cancer cell colony proliferation after treatment with 13-HODE for JHUEM7 cells.

Figure 7b shows the results of analyzing the degree of cancer cell colony proliferation after 13-HODE treatment with respect to MDA-MB-468 cells.

Figure 8a shows the results of analyzing the size change of the tumor after treatment with 13-HODE JHUEM7 cells xenografted into mice.

Figure 8b shows the results of observing the size change of the tumor after treatment with 13-HODE JHUEM7 cells xenografted into mice.

9 shows the results of analysis of tumor samples extracted from mice through Western blotting.

Figure 10a shows the results of analyzing the size change of the tumor after treatment with 13-HODE of MDA-MB-468 cells xenografted into mice.

Figure 10b shows the results of observing the size change of the tumor after treatment with 13-HODE of MDA-MB-468 cells xenografted into mice.

11a shows the results of analyzing significant genes using RNA-seq analysis after treating JHUEM7 cells with DMSO control drug and 150 μM 13-HODE for 48 hours, respectively.

11b shows the results of performing pathway enrichment analysis using RNA-seq analysis after treating JHUEM7 cells with DMSO control drug and 150 μM 13-HODE for 48 hours, respectively.

11c shows the results of performing pathway enrichment analysis using RNA-seq analysis after treating JHUEM7 cells with DMSO control drug and 150 μM 13-HODE for 48 hours, respectively.

12 shows the results of quantitative analysis of the viability of 13-HODE-treated JHUEM7 cells and the amount of 13-HODE inside the cells.

13 shows the results of analyzing proteins related to emtor signaling in JHUEM7 cells overexpressing ALOX15 through western blotting.

14 shows the results of analyzing the change in tumor size after transplanting JHUEM7 cells overexpressing ALOX15 into mice.

15A shows the results of analyzing proteins related to emtor signaling in MCF7 cells overexpressing ALOX15 through Western blotting.

15B shows the results of analyzing the degree of colony proliferation of MCF7 cells overexpressing ALOX15.

Figure 16a shows the results of analyzing proteins related to emtor signaling in cells after 13-HODE was treated with respect to HEC59 cells through western blotting.

Figure 16b shows the results of analyzing the viability of HEC59 cells after treatment with 13-HODE.

Figure 16c shows the results of analyzing the degree of cancer cell colony proliferation after 13-HODE treatment on HEC59 cells.

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

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1: Cell culture method

HEK293, HEK293T, MDA-MB-468, MCF7, HEC59 and U373MG cells were cultured in high-glucose DMEM medium supplemented with 2 mM glutamine, 100 µg/ml penicillin-streptomycin and 10% fetal bovine serum (FBS, Atlas Biological). cultured. JHUEM7 cells were cultured in DMEM/F12 (Thermo Fisher Scientific) supplemented with 1X non-essential amino acids (Sigma Aldrich, Missouri, USA) and 10% FBS. SW480 and HT29 cells were cultured in RPMI1640 medium supplemented with 2 mM glutamine, 100 μg/ml penicillin-streptomycin and 10% fetal bovine serum (FBS, Atlas Biological). All cell lines were cultured at 37 °C, 5% CO ₂ conditions.

HEK293 and HEK293T cells were purchased from ATCC (American Type Culture Collection, Virginia, USA), and JHUEM7 cells were purchased from RIKEN (Saitama, Japan).

Example 2: Analysis of the relationship between Emtor and 13-HODE through pull-down assay

JHUEM7 cells were lysed with 1x CHAPS lysis buffer. The composition of the CHAPS buffer is 50 mM HEPES, 150 mM NaCl, 0.4% CHAPS, 50 mM NaF, 10 mM Na-pyrophosphate, 100 mM β-glycerophosphate at pH 7.4, and a protease inhibitor.

2 mg of total cell lysate were prepared with 1) 10 μM biotin, 2) 10 μM biotin-labeled 13-HODE (US, Michigan, Cayman Chemical), 3) 10 μM biotin and 10 μM biotin-labeled 13- HODE, or 4) 10 μM biotin labeled 13-HODE and 20 μM unlabeled 13-HODE. Streptavidin beads (Streptavidin bead) were added to the reaction mixture and mixed for an additional hour. Then, 10 μL sample buffer was added to stop the reaction, and after heating for 5 minutes, analysis was performed by Western blotting.

Antibodies used in the Western blotting were anti-mTOR antibody (Cell Signaling Technology #2972, Massachusetts, USA) and anti-raptor antibody (Bethyl Laboratories A300-553A, Alabama, USA).

Biotin-labeled 13-HODE was bound to emtor and raptor (regulatory protein that forms a protein complex with emtor). However, biotin-labeled 13-HODE showed a low binding affinity to emtor and raptor in the presence of unlabeled 13-HODE ( FIG. 2 ).

From the above results, it can be seen that 13-HODE and emtor interact.

Example 3: Analysis of the Relationship Between Emtor and 13-HODE by Hydrogen-Deuterium Substitution Mass Spectrometry

In order to more specifically confirm the interaction between 13-HODE and emtor confirmed in Example 2, hydrogen-deuterium substitution mass spectrometry was performed.

Through hydrogen-deuterium substitution mass spectrometry, structural and conformational changes at the C-terminus of Emtor to which 13-HODE binds were observed, and the mechanism of action of 13-HODE and the molecular position of binding to Emtor were confirmed.

Protein sequence coverage maps were obtained using undeuterated samples as follows: 3.5 μL of ~50 μM Emtor C-terminal fragment, 25 mM HEPES, pH 7.4, 50 mM KCl, 10 mM MgCl ₂ , ice-cold 10% glycerol diluted with 96.5 µL quench solution (100 mM glycine, 25 mM TCEP, pH 2.5).

The sample was subjected to Waters HDX nanoAcquity UPLC (Waters, Milford, Mass.) and subjected to in-line pepsin treatment (Waters Enzymate BEH pepsin 40 column). Peptide fragments were separated through an Acquitty UPLC BEH C18 peptide trap and column. After separation, the peptides were eluted directly with a Waters Synapt G2 mass spectrometer (Waters, Milford, MA) using a gradient of 5% to 35% acetonitrile (0.1% formic acid). MS ^E data were obtained via 20 to 30 V lamp trap CE. Peptides were analyzed using Waters' ProteinLynx Global Server 2.5.1 (PLGS).

The following samples were used to analyze the Emtor C-terminus to which 13-HODE binds: 3.5 µL of -50 µM Emtor C-terminal fragment, 25 mM HEPES, pH 7.4, 50 mM KCl, 10 mM MgCl ₂ , 10% glycerol, 1% DMSO, 130 μM 13-HODE. As a control, a sample not containing 13-HODE was used.

_{D 2} 0 was added to the sample to initiate a deuterium substitution reaction. The deuterium substitution reaction was performed under various conditions (10 seconds, 1 minute, 10 minutes and 2 hours).

Back exchange correction was performed by adding 6 M deuterated guanidine DCI (DCI) before quenching. Quenching was performed using a 65 μL solution containing chilled 100 mM glycine buffer, 5 mM TCEP. Finally, deuterium uptake (% D) in hydrogen-deuterium substitution was analyzed by reaction time through Waters DynamX 3.0 software.

The normalized percentage of deuterium uptake (% D) at incubation time t for the peptide was calculated as follows: % D = (100 (mt-m0)) / (mf-m0). (mt: central mass at incubation time t, m0: central mass of non-deuterated control, mf: central mass of fully deuterated control) The % D was used to calculate the percent deuterated plot (Δ% D) . Confidence intervals for the Δ% D plot were determined and adjusted for percent deuterated using a fully deuterated control.

In the presence of 13-HODE, some regions exhibited altered deuterium uptake kinetics, mainly the kinase domain (Fig. 3a).

Therefore, by combining the above results with the previously known crystal structure of Emtor, it was confirmed that the binding site of 13-HODE spanned across the catalytic cleft of the kinase protein ( FIG. 3b ).

Example 4: Treatment and analysis of 13-HODE on Emtor kinase isolated through immunoprecipitation

To perform the Emtor kinase assay, HEK293T cells were washed once with ice-cold PBS and lysed with ice-cold lysis buffer. The composition of the lysis buffer is HEPES 40 mM, 120 mM NaCl, 2 mM EDTA, 10 mM pyrophosphate, 10 mM sodium fluoride, 0.3% CHAPS, IX protease inhibitor cocktail at pH 7.4.

The cell lysate was incubated at 4° C. for 10 minutes and centrifuged at 13,000 rpm for 10 minutes to collect the supernatant. 2 μg of anti-mTOR antibody (Cell Signaling Technology #2972, Massachusetts, USA) was added to 2 mg of the supernatant and spin-incubated at 4° C. for 1.5 hours. 20 μL of agarose beads (Pierce) was added and incubated for an additional hour.

Emtor immunoprecipitates were washed twice with the lysis buffer and twice with kinase wash buffer. The composition of the kinase wash buffer is 25 mM HEPES, 20 mM potassium chloride, pH 7.4.

Then, the kinase assay was performed at 37° C. for 15 minutes using 15 μL of mTOR Complex 1 kinase buffer and 150 ng of S6K1 substrate. The reaction was stopped by adding 10 μL sample buffer, heated for 5 minutes, and then analyzed by Western blotting.

Antibodies used in the Western blotting were anti-T389 p-S6K antibody (Cell Signaling Technology #9205, Massachusetts, USA), anti-S473 p-Akt antibody (Cell Signaling Technology #9271), and anti-S6K antibody (Cell Signaling Technology). Technology #9202), Anti-Akt Antibody (Cell Signaling Technology #9272), Anti-mTOR Antibody (Cell Signaling Technology #2972,), Anti-mLST Antibody (Cell Signaling Technology #3274,), Anti-raptor Antibody (Bethyl Laboratories) A300-553A, Alabama, USA), an anti-rictor antibody (Bethyl Laboratories A400-458A).

In the presence of 13-HODE, the Emtor Cl substrate, S6K1, was dose-dependently inhibited (Fig. 4a). In the presence of 13-HODE, phosphorylation of Akt, a substrate of mTOR C2 (mTOR Complex 2) at S473, was further blocked ( FIG. 4b ). The above results show the inhibitory activity of metabolites for Emtor C1 and Emtor C2, which 13-HODE exhibits.

In addition, with respect to the concentration of 13-HODE required for Emtor C1 inhibition, a smaller amount was required to achieve the same level of inhibition as Emtor C2, indicating that 13-HODE has a stronger activity on Emtor C1. Means that.

For further confirmation that 13-HODE directly targets the emtor kinase domain, an in-vitro kinase assay using a recombinant emtor C-terminal fragment comprising amino acids 1362 to 2549 forming the kinase domain of emtor was performed. As a result, 13-HODE inhibited phosphorylation of S6K1 ( FIG. 4c ).

These results indicate that 13-HODE directly inhibits the kinase activity of emtor and directly targets the emtor kinase domain portion.

Example 5: Emtor kinase analysis of 13-HODE-treated cells by Western blotting

Example 5-1. Emtor kinase analysis of 13-HODE-treated JHUEM7 cells by Western blotting

In order to analyze the activity of emtor signaling in JHUEM7 cells, emtor kinase was analyzed.

35 mm cell culture dishes were seeded with ^{JHUEM7 3×10 5 cells.} The next day, 68 or 135 uM of 13-HODE or 10 nM Torin1 was mixed with the cell culture medium and treated, and then 48 hours later, emtor signaling was analyzed by western blotting. The 13-HODE or Torin1 mixed culture medium was freshly replaced every 24 hours.

The cells were lysed with NP-40 lysis buffer. The composition of NP-40 is 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% NP-40 substitute, 1 mM EDTA at pH 8.0, 50 mM NaF, 10 mM Na-Pi lophosphate (Na-pyrophosphate), 15 mM Na ₃ VO ₄ , 100 mM β-glycerophosphate, and a protease inhibitor.

The whole cell lysate was mixed with 5X SDS sample buffer to adjust the final concentration to 1-2 μg/μL and then heated for 5 minutes. Then, Western blotting was performed with the heated cell lysate.

Antibodies used in the Western blotting were anti-T389 p-S6K antibody (Cell Signaling Technology #9205, Massachusetts, USA), anti-S240/244 p-S6 antibody (Cell Signaling Technology #5364), anti-T37/46 p-4EBP1 antibody (Cell Signaling Technology #9459), anti-S473 p-Akt antibody (Cell Signaling Technology #9271), anti-T308 p-Akt antibody (Cell Signaling Technology #4056), anti-T638/641 p-PKCa Antibody (Cell Signaling Technology #9375), Anti-S6K Antibody (Cell Signaling Technology #9202), Anti-S6 Antibody (Cell Signaling Technology #2217), Anti-4EBP1 Antibody (Cell Signaling Technology #9452), Anti-Akt Antibody ( Cell Signaling Technology #9272), anti-PKCa antibody (Cell Signaling Technology #2056).

Through the above experiment, it was confirmed that when 13-HODE was treated in JHUEM7 cells, phosphorylation of T389 S6K1 was reduced, that is, the emtor signaling process was inhibited ( FIG. 5a ).

Example 5-2. Emtor kinase analysis of 13-HODE-treated HCT003, SW480, HT29, MDA-MB-468 and U-373MG cells by Western blotting

In order to analyze the activity of emtor signaling in HCT003, SW480, HT29, MDA-MB-468, and U-373MG cells, emtor kinase was analyzed.

The experimental method was the same as the method described in Example 5-1, except that HCT003, SW480, HT29, MDA-MB-468, and U-373MG cells were used instead of JHUEM7 cells.

Through the experiment, it was confirmed that phosphorylation of T389 S6K1, S240/244, and T37/46 was reduced when 13-HODE was treated with HCT116 cells, SW480 cells, HT29 cells, MDA-MB-468 cells, and U-373 MG cells. , that is, it was confirmed that the emtor signaling process was inhibited (FIG. 5b).

Example 6: Viability analysis of cells treated with 13-HODE

To confirm the anticancer effect of 13-HODE, the viability of 13-HODE-treated JHUEM7 cells and MDA-MB-468 cells was analyzed.

JHUEM7 cells or MDA-MB-468 cells (1×10 ⁴ cells/mL) were seeded in 96 well plates. After overnight incubation, cells were treated with DMSO or 135 μM of 13-HODE for various times. Then, 20 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (2.5 mg/mL in PBS) was added to each well, Then, incubated at 37 °C for an additional 1 hour. The OD (optical density) value of each well was measured at 492 nm with a SpectraMax Paradigm Reader (Molecular Devices). For colony formation test, cells (4 x 10 ⁴ cells/well) were seeded in 24 well plates and cultured overnight, and cells were cultured with DMSO or 135 μM of 13-HODE for 4 days. The resulting colonies were then fixed with methanol at -20 °C for 30 min and stained with 1% crystal violet for 15 min.

Through the experiment, it was confirmed that the cell proliferation rate in the wells treated with 13-HODE was significantly lower than that in the wells not treated with 13-HODE ( FIGS. 6A and 6B ).

These results indicate that 13-HODE has a very good anticancer effect.

Example 7: Colony proliferation analysis of 13-HODE-treated cells

In order to confirm the association between 13-HODE and cancer cell colony growth rate in JHUEM7 cell line and MDA-MB-468 cell line, the cancer cell colony growth rate was examined when 13-HODE was added to wells culturing JHUEM7 cells or MDA-MB-468 cell line.

JHUEM7 cells or MDA-MB-468 cells (4 x 10 ⁴ cells/well) were seeded in a 24-well plate and cultured overnight, and the cells were cultured with DMSO or 135 μM of 13-HODE for 4 days. The resulting colonies were then fixed with methanol at -20 °C for 30 min and stained with 1% crystal violet for 15 min.

Through the experiment, it was confirmed that the cell proliferation rate in the wells treated with 13-HODE was significantly lower than that in the wells not treated with 13-HODE. These results indicate that 13-HODE has a very excellent anticancer effect ( FIGS. 7a and 7b ).

Example 8: Analysis of the anticancer effect of 13-HODE confirmed through animal experiments

Example 8-1. The anticancer effect of 13-HODE confirmed through the experiment of nude mice implanted with JHUEM7 cells

In order to confirm the anticancer effect of 13-HODE in the mouse xenograft model, the size of the tumor was examined after treatment with 13-HODE in the tumor-transplanted mice.

Animal experiments were performed according to guidelines approved by the Animal Experimentation Ethics Committee of the Korea Advanced Institute of Science and Technology. JHUEM7 cells (4 x 10 ⁶ ) were implanted by subcutaneous injection into 8-12 week old female BALB/c nude mice. After the mean tumor volume ^{reached 50 mm 3} , mice were randomly assigned to 3 different groups (6 mice/group). The mouse weight and tumor diameter were measured once every two days. Tumor volume using a caliper was evaluated according to the following formula ^{0.5x (width) 2 x (Length} ), a Student's T test was used to determine the P-values. For 13-HODE treatment, mice received intratumoral or intravenous injection of 13-HODE at 10 mg/kg every 2 days after initiation of the experiment.

Through the experiment, it was confirmed that the growth of the tumor in the mouse xenograft model treated with 13-HODE was inhibited when compared to the control group ( FIGS. 8a and 8b ).

In addition, it was confirmed that phosphorylation of T389 S6K1 and phosphorylation of S473 AKT were decreased in the 13-HODE-treated tumor through Western blotting ( FIG. 9 ).

Example 8-2. The anticancer effect of 13-HODE confirmed through an experiment in nude mice implanted with MDA-MB-468 cells

In order to confirm the anticancer effect of 13-HODE in the mouse xenograft model, the size of the tumor was examined after treatment with 13-HODE in the tumor-transplanted mice.

The experimental method is the same as that described in Example 8-1, except that MDA-MB-468 cells were used instead of JHUEM7 cells.

Through the experiment, it was confirmed that the growth of the tumor in the mouse xenograft model treated with 13-HODE was inhibited when compared to the control group ( FIGS. 10a and 10b ).

These results indicate that 13-HODE inhibits the emtor signaling process and has a very excellent anticancer effect.

Example 9: Analysis of significant gene expression and mechanism of 13-HODE-treated cells (RNA-seq assay)

Genes and related mechanisms that are significantly expressed through RNA-seq experiments, Differentially Expressed Genes analysis (DEG analysis) and Pathway enrichment analysis, to confirm the association between the drug mechanism of 13-HODE and genes analysis was performed.

DEG analysis is an analysis that selects a candidate group for a significant gene (Differentially Expressed Genes) with a difference in expression between the control group and the control group by measuring and statistically processing the expression value of the gene. The result of analyzing significant genes using RNA-seq analysis after treating JHUEM7 cells with DMSO control drug and 150 μM 13-HODE for 48 hours, respectively. In the 13-HODE treatment group, 3729 genes were decreased and 3842 genes were increased ( FIG. 11a ).

The above results indicate that there is a change in the gene group related to the emtor signal transduction pathway by 13-HODE treatment.

In addition, through the pathway enrichment analysis, cancer-related regulatory pathways such as liver cancer, pancreatic cancer, and breast cancer were detected ( FIG. 11b ), and cell cycle, aging, and fatty acid metabolism-related mechanistic pathways were also detected ( FIG. 11c ).

Example 10: Viability of cells treated with 13-HODE and 13-HODE measured inside cells

In order to check whether 13-HODE flows well into the cell, the amount of 13-HODE inside the 13-HODE-treated JHUEM7 cells was quantitatively analyzed.

As a result of the experiment, 13-HODE-treated cells compared to the DMSO-treated control group showed a higher amount of 13-HODE in the cells than the control group ( FIG. 12 ).

The amount of 13-HODE inside the cell was measured as follows.

13-HODE was extracted from ~1.4 mL cell supernatant using solid phase extraction (SPE). A 60 mg Oasis HLB (Waters) SPE cartridge was washed and pre-conditioned sequentially with ethyl acetate, methanol and (0.1 % acetic acid + 5 % methanol) aqueous solution. 10 μL of 0.2 mg/mL EDTA and BHT with MeOH:H ₂ O (50:50) as solvents were added to the adsorbent bed of the SPE column. An equal volume (0.1% acetic acid + 5% MeOH) aqueous solution was added to the cell supernatant. 50 μL of 500 nM 13-HODE-d4 was also added to the sample solution as an internal standard. The solution was added to the SPE column. After washing the column with 2 column volumes (0.1 % acetic acid + 5 % MeOH) aqueous solution, the column was dried using vacuum. Finally, 13-HODE was eluted with 0.5 mL methanol followed by 1.5 mL ethyl acetate. The sample solution was dried using a vacuum centrifuge and then stored at -20°C until analysis. The dried material was reduced with 40 μL of methanol before LC-MS/MS analysis. 13-HODE was determined using an LC-MS/MS system equipped with an Agilent 1290 HPLC (Agilent) and QTRAP 5500 mass spectrometry (AB Sciex). A reversed-phase column (Pursuit5 C18, 150×2.1 mm) was used with mobile phase A (0.1 % aqueous acetic acid solution) and mobile phase B (0.1 % acetic acid in acetonitrile/methanol (84/16) solvent). LC was run at 250 μL/min and 35°C. The separation gradient is as follows: 35% B at 0 min, 35% B hold for 0.25 min, 35% to 45% of B for 0.75 min, 45% hold of B for 2 min, 45% of B for 5.5 min. to 56% of B, from 56% to 65% of B for 5.5 min, hold 65% of B for 6 min, hold 65% to ~95% of B for 1.5 min, hold 95% of B for 5.5 min, 0.1 From 95% to 35% of B for min and hold for 2.9 min at 35% of B. Multiple reaction monitoring (MRM) mode was performed in anion mode of 13-HODE, and extracted ion chromatograms corresponding to specific transitions of 13-HODE were used for quantification (Q1/Q3 = 295/195, RT = 13- 18.09 min for HODE; Q1/Q3 = 299/198, RT = 17.93 min for 13-HODE-d4). The calibration range for 13-HODE is 1-10000 nM (r2 ≥ 0.99). Data analysis was performed using Analyst 1.5.2.

Example 11: Emtor kinase analysis of JHUEM7 cells overexpressing ALOX15 (human lipoxygenase15-1)

The effect of 13-HODE on emtor signaling was analyzed using JHUEM7 cells overexpressing ALOX15 (human lipoxygenase15-1), which is known to catalyze the synthesis of 13-HODE.

The experimental method is the same as the method described in Example 5, except that the cells used and 13-HODE were not treated. As a control group, cells not overexpressing ALOX15 were used.

As a result of the experiment, it was confirmed that the phosphorylation of T389 S6K1, S473, and T308 was decreased in the cells overexpressing ALOX15, that is, the emtor signaling process was inhibited (FIG. 13).

Example 12: Anticancer effect of 13-HODE confirmed through an experiment in nude mice implanted with JHUEM7 cells overexpressing ALOX15 (human lipoxygenase15-1)

In order to confirm the anticancer effect of 13-HODE in a mouse xenograft model, the tumor size change in mice transplanted with ALOX15 overexpressing cells was examined.

The experimental method was the same as that described in Example 8, except that ALOX15 overexpressing JHEUM7 cells were used instead of JHUEM7 cells and 13-HODE was not treated. As a control group, nude mice transplanted with cells not overexpressing ALOX15 were used.

Through the experiment, it was confirmed that the growth of the tumor in the mouse xenograft model transplanted with ALOX15 overexpressing JHEUM7 cells was inhibited when compared to the control group ( FIG. 14 ).

These results indicate that 13-HODE inhibits the emtor signaling process and has an anticancer effect.

Example 13: Emtor kinase of MCF7 cells overexpressing ALOX15 (human lipoxygenase15-1) and colony proliferation analysis of cells

Emtor kinase and cell colony proliferation assays were performed using MCF7 cells overexpressing ALOX15.

The experimental method is the same as that described in Examples 5 and 7, except that the cells used and 13-HODE were not treated. For the experimental group, MCF7 cells overexpressing wild-type ALOX15 were used, and as a control group and a comparison group, a control overexpressing cell incapable of enzyme expression and a mutant ALOX15 T560M cell in which enzyme activity was lost were used as the control group.

As a result of the experiment, it was confirmed that phosphorylation of S240/244, S473, and T308 was decreased in wild-type ALOX15 overexpressing JHEUM7 cells. That is, it was confirmed that the emtor signal transduction process was inhibited. This effect was not observed in the ALO15 T560M mutant cell line ( FIG. 15A ). Colony growth inhibition was observed in the wild-type ALOX15-expressing MCF7 cell line through colony growth analysis, but there was no colony growth inhibitory effect in the T560M mutant cell line ( FIG. 15b ).

These results indicate that the emtor signaling process and the proliferation of cancer cells are inhibited by 13-HODE.

Example 14: Cellular Emtor kinase, Cell Viability, Cell Colony Proliferation Analysis Using HEC59 Cells

Emtor kinase, cell viability, and cell colony proliferation assays performed in Examples 5, 6, and 7 were performed using HEC59 cells. HEC59 cells are cancer cells (endometrial cancer cells) and are cells with the emtor mutation (mTOR ^E1799K ).

The experimental method is the same as that described in Examples 5, 6, and 7 except for the cells used.

As a result of the experiment, it was confirmed that when 13-HODE was treated in HEC59 cells, phosphorylation of T389 S6K1, S240/244, and S473 was reduced, that is, the emtor signaling process was inhibited ( FIG. 16a ).

In addition, it was confirmed that the cell proliferation rate in the wells treated with 13-HODE was significantly lower than that in the wells not treated with 13-HODE ( FIGS. 16B and 16C ).

The above results indicate that 13-HODE exerts an anticancer effect on various cancers such as colorectal cancer, breast cancer, glioblastoma, endometrial cancer, liver cancer, and pancreatic cancer.

Claims (6)

A pharmaceutical composition for preventing or treating cancer comprising 13-HODE (13-Hydroxyoctadecadienoic acid) as an active ingredient.

The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a solid cancer.

According to claim 2, wherein the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer, nasopharyngeal cancer , salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer Cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, external genital cancer, A pharmaceutical composition for preventing or treating cancer, which will be selected from the group consisting of female urethral cancer and skin cancer.

The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a blood cancer.

5. The method of claim 4, wherein the hematologic cancer is selected from the group consisting of acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma. The pharmaceutical composition for the prevention or treatment of cancer.

A method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition comprising 13-HODE as an active ingredient.

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