KR102333235B1 - Pharmaceutical Composition for Cancer Therapy Comprising ER Stress Inhibitors - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer comprising an ER stress inhibitor as an active ingredient. By using the pharmaceutical composition for cancer treatment comprising the ER stress inhibitor of the present invention as an active ingredient in combination with an anticancer agent, it is possible to improve the therapeutic effect according to the administration of the anticancer agent by removing the senescent cancer cells.

Description

Pharmaceutical composition for cancer treatment comprising ER stress inhibitor {Pharmaceutical Composition for Cancer Therapy Comprising ER Stress Inhibitors}

The present invention relates to a pharmaceutical composition for treating cancer comprising an ER stress inhibitor as an active ingredient.

The regulatory function of cells can be problematic for a variety of reasons. The problem with cell regulation is that abnormal cells that should normally die cause excessive proliferation, and in some cases, they invade surrounding tissues and organs, forming a mass and destroying or modifying the existing normal structure. Modern medicine defines this condition as cancer or malignancy. Cancer is a representative modern incurable disease, and its incidence is increasing due to various causes such as environmental pollution, excessive stress, and Western diet. Cancer is the number one cause of death in Korea as of 2016 and is predicted to become the number one cause of death worldwide by the end of the 21st century.

Surgical surgery, radiation therapy, and chemical therapy are currently used as methods of treating cancer. Surgical surgery is effective in removing cancer in its initial state, but in some cases, it prevents side effects such as the need to remove an organ and metastasis. There is a problem that radiation therapy cannot do it, and in the case of radiation therapy, the treatment of cancer in a specific area has the advantage of high therapeutic effect, while the new risk of cancer caused by radiation irradiation and the fact that metastasis cannot be prevented, and the patient's pain during treatment There is a problem with doing

In addition, chemotherapy is a method of using an anticancer agent, which is a chemotherapeutic agent, for the treatment of malignant tumors. The anticancer agent intervenes in the metabolic pathway of cancer cells to block DNA replication, transcription, and translation processes, or to interfere with the synthesis of nucleic acid precursors, By inhibiting cell division, it induces toxicity in cancer cells, which divide faster than normal cells. However, when anticancer drugs are used, various side effects may occur due to acquisition of resistance to cancer cells, generation of cancer stem cells, increase in cancer malignancy, and high concentration and continuous use of anticancer drugs, so the need for a new approach for cancer treatment is emerging.

A novel approach for cancer treatment relates to the concept of synthetic lethality. A cell can survive if there is a mutation or inhibition of function in only one of two genes (or both gene products), but synthetic lethality results in cell death if both genes are mutated or inhibited It is said In other words, "synthetic lethality" describes a situation in which mutations or drugs work together to cause the death of cancer cells. Targeting a synthetic lethal gene (or gene product) to a cancer-related mutation kills only cancer cells and allows normal cells to survive. Thus, synthetic lethality provides a framework for the development of anticancer-specific agents, and thus synthetic lethality may present a new direction for the elimination of cancer cells.

Braakman, I., and Bulleid, N. J. (2011). Protein folding and modification in the mammalian endoplasmic reticulum. Annu Rev Biochem 80, 71-99.

The present inventors have studied diligently to suggest a new direction to eliminate cancer cells and to improve the problems of conventional anticancer drugs, such as acquisition of resistance of cancer cells, generation of cancer stem cells, increase in cancer malignancy, and various side effects due to high concentration and continuous use of anticancer drugs. tried As a result, by removing cancer cells by using a pharmaceutical composition for the treatment of cancer containing an ER stress inhibitor as an active ingredient in combination with an anticancer agent, it was found that the side effects caused by the administration of the anticancer agent could be improved, and the present invention was completed. .

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating cancer comprising an ER stress inhibitor as an active ingredient.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for treating cancer comprising an ER stress inhibitor as an active ingredient.

As used herein, the term "ER stress" refers to an immature protein exceeding the ability of the ER to process by a physiological or pathological environment is introduced into the ER, or calcium in the ER is depleted, which means that a disorder occurs in the ER function. , mainly means a state in which the endoplasmic reticulum protein folding load is exceeded.

When ER stress occurs, cells have a defense mechanism for survival, which is called ER stress response. The ER stress response involves three signaling pathways present in the ER membrane, PERK (pancreatic ER kinase), IRE-1α/XBP-1 (inositol-requiring 1α/X-box binding protein 1), and ATF6 (activating transcription factor). , and its underlying substrate, eIF2-α pathway, is also involved.

The endoplasmic reticulum stress response consists of four steps: (a) inhibiting the translation from mRNA to protein in the ribosome (translational attenuation) to reduce the influx of new proteins into the endoplasmic reticulum; (b) inducing the expression of ER chaperones, such as BiP (also known as HSPA5), which is required to fold proteins, thereby enhancing the folding ability of the ER; (c) removing the unfolded or misfolded protein from the ER by ER stress-asoociated degradation (ERAD) through the intracytoplasmic ubiquitin-proteasome system; (d) When the ER stress becomes so severe that the ER stress cannot be overcome by the above three steps and the ER cannot restore its function, the apoptosis pathway is activated to remove damaged cells.

As used herein, the term ER stress inhibitor refers to a substance that inhibits some or all steps of the ER stress response.

In one embodiment of the present invention, the ER stress inhibitor can be applied to any material as long as it inhibits the ER stress response, specifically, salubrinal, GSK2656157 ( 1-[5-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-4-fluoro-2,3-dihydro-1H-indol-1-yl]- 2-(6-methyl-2-pyridinyl)-ethanone), STF-083010 (N-[(2-Hydroxy-1-naphthyl)methylene]-2-thiophenesulfonamid), 4-PBA (4-phenylbutyrate), Tudca ( Tauroursodeoxycholic acid), Ceapin-A7 (N-(1-{[2,4-bis(Trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-1,2 -oxazole-3-carboxamide), and PF-429242 (4-[(Diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-(3R)-3-pyrrolidinyl-benzamide dihydrochloride), preferably For example, but not limited to salubrinal.

More specifically, salubrinal suppresses the ER stress response by inhibiting the eIF2-α phosphatase enzyme. GSK2656157 inhibits the ER stress response by acting as an inhibitor of PERK. STF-083010 suppresses the endoplasmic reticulum stress response by inhibiting the endonuclease activity of Ire1 and blocking XBP1 mRNA splicing. 4-PBA and Tudca act as chemical chaperones to inhibit the ER stress response. Ceapin-A7 is a potential inhibitor of ATF6-alpha and inhibits the endoplasmic reticulum stress response. PF-429242 is an S1P inhibitor and inhibits the endoplasmic reticulum stress response.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to achieve cancer therapeutic efficacy or cancer preventive efficacy of the aforementioned endoplasmic reticulum stress inhibitor. The upper limit of the quantity of the endoplasmic reticulum stress inhibitor contained in the composition of the present invention may be selected by those skilled in the art within an appropriate range.

As demonstrated in the examples below, salubrinal, an endoplasmic reticulum stress inhibitor of the present invention, exhibited an effect of inhibiting cellular senescence and increasing apoptosis of tumor cells in combination with an anticancer agent. In particular, it showed a synergistic effect when used in combination with doxorubicin, cisplatin, and paclitaxel. Breast cancer cell lines (MCF-7, MDA-MB-231), prostate cancer cell lines (PC-3), lung cancer cell lines (A549) ), gastric cancer cell line (MKN28) and liver cancer cell line (SK-HEP1).

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, 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. 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, and in the case of parenteral administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intranasal administration, intramucosal administration, intrathecal administration, intraperitoneal administration It can be administered by administration, intraocular administration, etc., and specifically, it can be administered orally.

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 a dosage effective for the desired treatment or prophylaxis. According to a specific embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-1000 mg/kg. The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.1-1000 mg/kg, 0.1-900 mg/kg, 0.1-800 mg/kg, 0.1-700 mg/kg, 0.1-600 mg/kg, 0.1 -500 mg/kg, 0.1-400 mg/kg, 0.1-300 mg/kg, 0.1-200 mg/kg, 0.1-100 mg/kg, 0.1-50 mg/kg, 0.1-30 mg/kg, 0.1- 20 mg/kg, 0.1-10 mg/kg, 0.1-7 mg/kg, 0.1-5 mg/kg, 1-1000 mg/kg, 1-900 mg/kg, 1-800 mg/kg, 1-700 mg/kg, 1-600 mg/kg, 1-500 mg/kg, 1-400 mg/kg, 1-300 mg/kg, 1-200 mg/kg, 1-100 mg/kg, 1 -50 mg/kg, 1-30 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-7 mg/kg, 1-5 mg/kg, more specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. In yet another embodiment, the daily dose of the pharmaceutical composition of the present invention is, for example, 100-900 mg/kg, 100-800 mg/kg, 100-700 mg/kg, 100-600 mg/kg, 100-500 mg/kg, 100-400 mg/kg, 100-300 mg/kg, 100-200 mg/kg, 200-900 mg/kg, 200-800 mg/kg, 200-700 mg/kg kg, 200-600 mg/kg, 200-500 mg/kg, 200-400 mg/kg, 200-300 mg/kg, 300-900 mg/kg, 300-800 mg/kg, 300-700 mg/kg, 300-600 mg/kg, 300-500 mg/kg, 300-400 mg/kg, 400-900 mg/kg, 400-800 mg/kg, 400-700 mg/kg, 400 -600 mg/kg, 400-500 mg/kg, more specifically 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, It may be 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg, but is not limited thereto.

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. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

In one embodiment of the present invention, the pharmaceutical composition may be administered in combination with a known anticancer agent having an effect of preventing and treating cancer, and the anticancer agent includes a compound or a pharmaceutical composition.

In one embodiment of the present invention, the anticancer agent is doxorubicin, cisplatin, paclitaxel, docetaxel, cabazitaxel, carboplatin, etoposide ), irinotecan, gemcitabine, 5-fluorouracil, capecitabine, gefitinib, sorafenib, and imatinib It may be selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the anticancer agent is ACTH (adrenocorticotropic hormone) producing tumor, acute lymphocytic or lymphoblastic leukemia, acute or chronic lymphocytic leukemia, acute non-lymphocytic leukemia, bladder cancer, brain tumor, breast cancer, uterus Cervical cancer, chronic myelogenous leukemia, lymphoma, endometriosis, esophageal cancer, Ewing's sarcoma, tongue cancer, Hodgkin's lymphoma, Kaposi's sarcoma, renal cancer, liver cancer, lung cancer, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, It is a therapeutic agent for osteosarcoma, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, penile cancer, retinoblastoma, skin cancer, gastric cancer, thyroid pressure, uterine cancer, testicular cancer, Wilms' tumor and trophoblastoma.

In one embodiment of the present invention, the pharmaceutical composition of the present invention is a cell senescence-associated beta-galactosidase assay (SA-β-gal assay) SA-β-gal positive cancer cells increased by the use of an anticancer agent reduce the number

As used herein, the term “cellular senescence” refers to a phenomenon in which normal somatic cells can no longer divide after dividing a certain number of times. Senescence-associated β-galactosidase (SA-β-gal) activity increases with the morphological features of aging cells becoming larger and flatter, heterochromatin increases in the nucleus, and vacuoles in the cytoplasm. The amount of proteins that inhibit cell growth such as p53, p16INK4, and p21CIP1/WAF1 increases, and insulin-like growth factor binding proteins (IGFBPs), interleukin-6 (interleukin-6), transforming growth It secretes various inflammatory proteins such as transforming growth factorβ (TGF-β) and interferon.

As used herein, the term "apoptosis" refers to cell destruction or suicide in eukaryotic cells. Apoptosis occurs in response to various external and internal stimuli, along with the progress of apoptosis, cell membrane and cytoplasm destruction, or cell membrane blebbing, cytoskeletal change, cell contraction, chromosome condensation, and DNA Characteristic changes such as segmentation occur.

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

(a) The present invention provides a pharmaceutical composition for treating cancer comprising an ER stress inhibitor as an active ingredient.

(B) The pharmaceutical composition for treating cancer comprising the ER stress inhibitor of the present invention as an active ingredient may be used in combination with an anticancer agent.

1 shows MCF-7 cells corresponding to human breast cancer cell lines exposed to doxorubicin (100 nM), cisplatin (5 μM), paclitaxel (5 nM) for 5 days and then SA-β-gal The results of measuring cellular senescence by the assay are shown.
Figure 2 is a human breast cancer cell line MDA-MB-231 cells that were exposed to doxorubicin (30 nM), cisplatin (5 μM), paclitaxel (2 nM) for 5 days and SA-β Shows the result of measuring cellular senescence by -gal assay.
3 shows MCF-7 cells corresponding to human breast cancer cell lines doxorubicin (100 nM), cisplatin (5 μM), and anticancer agents such as paclitaxel (5 nM) and ER stress The inhibitor, salubrinal (10 μM) was used in combination and exposed for 5 days, and cellular senescence was measured by SA-β-gal assay.
Figure 4 shows MDA-MB-231 cells corresponding to human breast cancer cell lines with anticancer agents such as doxorubicin (30 nM), cisplatin (5 μM), and paclitaxel (2 nM) and endoplasmic reticulum stress (ER) stress) inhibitor salubrinal (10 μM) was used in combination and exposed for 5 days, and cellular senescence was measured by SA-β-gal assay.
Figure 5 is a human breast cancer cell line MCF-7 cells doxorubicin (doxorubicin) (100 nM), cisplatin (5 μM), paclitaxel (paclitaxel) (5 nM), such as anticancer agents and ER stress inhibitors After 3 days of exposure to salubrinal (10 μM) in combination, the results of colony forming assay for measuring cancer cell proliferation on the 7th day are shown.
Figure 6 is a human breast cancer cell line MDA-MB-231 cells doxorubicin (doxorubicin) (5 nM), cisplatin (cisplatin) (500 nM), paclitaxel (paclitaxel) (1 nM), such as anticancer agents and ER stress (ER stress) Shows the results of colony forming assay for measuring cancer cell proliferation on the 7th day after exposure to 3 days using the inhibitor salubrinal (2 μM) in combination.
Figure 7 is a human prostate cancer cell line PC-3 cells doxorubicin (doxorubicin) (5 nM), cisplatin (cisplatin) (10 μM), paclitaxel (paclitaxel) (2 nM), such as anticancer agents and ER stress inhibitors After 3 days of exposure to salubrinal (10 μM) in combination, the results of colony forming assay for measuring cancer cell proliferation on the 7th day are shown.
8 is a human lung cancer cell line A549 cells doxorubicin (doxorubicin) (30 nM), cisplatin (cisplatin) (1 μM), paclitaxel (paclitaxel) (2 nM), such as anti-cancer agents and ER stress inhibitor salubri The results of colony forming assay for measuring cancer cell proliferation on the 7th day after exposure for 3 days using salubrinal (10 μM) are shown.
Figure 9 is a human gastric cancer cell line MKN28 cells doxorubicin (doxorubicin) (10 nM), an anticancer agent such as paclitaxel (2 nM) and ER stress inhibitor salubrinal (10 μM) The results of colony forming assay for measuring cancer cell proliferation on the 7th day after exposure for 3 days in combination are shown.
Figure 10 is a human liver cancer cell line SK-HEP1 cells doxorubicin (doxorubicin) (2 nM), cisplatin (cisplatin) (1 μM), paclitaxel (paclitaxel) (0.5 nM), such as anticancer agents and ER stress inhibitors Shows the results of colony forming assay for measuring cancer cell proliferation on the 7th day after daily exposure with salubrinal (2 μM).
11 is an anticancer agent such as doxorubicin (100 nM), cisplatin (5 μM), and ER stress inhibitor salubrinal (10) MCF-7 cells corresponding to the breast cancer cell line μM) in combination and PI staining results are shown.
12 shows MCF-7 cells corresponding to breast cancer cell lines doxorubicin (100 nM), cisplatin (5 μM), and anticancer agents such as paclitaxel (5 nM) and ER stress inhibitor The results of confirming the expression level of cleaved caspase 7, an apoptosis-related protein, through western blotting after exposure to 1 day in combination with phosphorus salubrinal (10 μM) are shown.

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

Example 1: Cell culture method

MCF-7 (human breast cancer cell line) and SK-HEP-1 (human liver cancer cell line) cells were cultured in DMEM medium containing 10% fetal bovine serum (Hyclone) and 1% antibiotic-fungal agent (Gibco).

MDA-MB-231 (human breast cancer cell line), PC3 (human prostate cancer cell line), MKN28 (human gastric cancer cell line), A549 (human lung cancer cell line) cells were treated with 10% fetal bovine serum (Hyclone) and 1% antibiotic-fungicide ( Gibco) in RPMI-1640 medium.

Example 2: Confirmation of cellular senescence through SA-β-gal assay

SA-β-gal staining is a staining method for measuring cellular senescence, and 2x10 ⁵ cells were seeded in a 35 mm culture dish to perform SA-β-gal staining. Cells seeded in a 35 mm culture dish were washed with 1X PBS and fixed with 0.2% glutaraldehyde solution at room temperature for 20 minutes. After washing 3 times with 1X PBS solution, cells were stained with SA-β-gal staining solution [1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (5-bromo -4-chloro-3-indolyl-β-D-galactopyranoside), 5 mM K ₃ Fe(CN) ₆ , and PBS containing 2 mM MgCl ₂ , pH 6.0], and overnight cultured at 37 ° C. . Cells were analyzed with an inverted fluorescence microscope (Eclipse 80i; Nikon, Melville, NY). A total of 300 cells were counted in 15 random fields to determine the percentage of SA-β-gal staining positive cells.

salubrinal (Cat.No.2347), doxorubicin (Cat.No.2252), cisplatin (Cat.No.2251), paclitaxel (Cat.No.1097) It was purchased from Tocris and used.

Example 2-1. MCF-7 and MDA-MB-231 cell senescence when treated with anticancer drugs alone

The present inventors exposed MCF-7 cells, which are human breast cancer cell lines, to doxorubicin (100 nM), cisplatin (5 μM), and paclitaxel (5 nM) for 5 days, followed by SA-β-gal Cell senescence was measured by assay.

As a result, as shown in FIG. 1, SA-β-gal staining positive cells were treated with doxorubicin, cisplatin, and paclitaxel at the above concentrations compared to the culture cell line corresponding to the control culture condition. It was confirmed that there was a significant increase.

The present inventors exposed MDA-MB-231 cells, which are human breast cancer cell lines, to doxorubicin (30 nM), cisplatin (5 μM), and paclitaxel (2 nM) for 5 days, and SA-β Cell senescence was measured by -gal assay.

As a result, as shown in FIG. 2, SA-β-gal staining positive cells were treated with doxorubicin, cisplatin, and paclitaxel at the above concentrations compared to the culture cell line corresponding to the control culture condition. It was confirmed that there was a significant increase.

Example 2-2. Inhibition of MCF-7 and MDA-MB-231 cell senescence by combined treatment with anticancer drugs and ER stress inhibitors

The present inventors analyzed MCF-7 cells corresponding to human breast cancer cell lines with anticancer agents such as doxorubicin (100 nM), cisplatin (5 μM), and paclitaxel (5 nM) and ER stress The inhibitor, salubrinal (10 μM) was used in combination with exposure for 5 days, and cellular senescence was measured by SA-β-gal assay.

As a result, as shown in FIG. 3 , it was confirmed that SA-β-gal staining positive cells were decreased when the anticancer agent and the ER stress inhibitor were used in combination, rather than when the anticancer agent was treated alone.

The present inventors analyzed MDA-MB-231, which is a human breast cancer cell line, and an anticancer agent such as doxorubicin (30 nM), cisplatin (5 μM), paclitaxel (2 nM) and endoplasmic reticulum stress ( ER stress) inhibitor salubrinal (10 μM) was used in combination and exposed for 5 days, and cellular senescence was measured by SA-β-gal assay.

As a result, as shown in FIG. 4 , it was confirmed that SA-β-gal staining-positive cells were reduced when treated with an anticancer agent and an ER stress inhibitor than when an anticancer agent was treated alone. The above results confirmed that cell senescence caused by the anticancer agent is converted into apoptosis by the ER stress inhibitor by treatment with the anticancer agent and the ER stress inhibitor.

Example 3: Confirmation of inhibition of proliferation of tumor cells by treatment with anticancer drugs and ER stress inhibitors through colony forming assay

In order to perform the colony forming assay, MCF-7, MDA-MB-231, PC-3, A549, MKN28, SK-HEP1 cells were seeded in a 6 well plate and 24 hours under standard conditions. incubated during Next, each cell was treated with anticancer agents such as doxorubicin, cisplatin, and paclitaxel and ER stress inhibitor salubrinal for 3 days in combination. Thereafter, the cells were washed with 1X PBS and cultured in fresh medium for 1 week. Cells were washed twice with 1X PBS and fixed with 0.2% glutaraldehyde solution at room temperature for 20 minutes. After fixation, cells were washed twice with 1X PBS and 0.25% crystal violet (Sigma-Aldrich) The solution was stained for 30 minutes, and the number of colonies was counted using the Image J program.

Example 3-1. MCF-7 following anticancer drug and ER stress inhibitor treatment inhibition of cell proliferation

The present inventors used the human breast cancer cell line, MCF-7 cells, with anticancer agents such as doxorubicin (100 nM), cisplatin (5 μM), and paclitaxel (5 nM) and ER stress inhibitors. After 3 days of exposure to salubrinal (10 μM) in combination, colony forming assay for measuring cancer cell proliferation was performed on the 7th day.

As a result, as shown in FIG. 5, when treated with an anticancer agent alone or an anticancer agent and an ER stress inhibitor, the number of colonies is different from the control culture conditions or the case of treatment with the ER stress inhibitor alone It was confirmed that there was a significant decrease. In addition, it was confirmed that the number of colonies was further reduced when the anticancer agent and the ER stress inhibitor were used in combination compared to the case where the anticancer agent was treated alone.

Example 3-2. Inhibition of proliferation of MDA-MB-231 cells by treatment with anticancer drugs and ER stress inhibitors

The present inventors tested the human breast cancer cell line MDA-MB-231 cells with an anticancer agent such as doxorubicin (5 nM), cisplatin (500 nM), and paclitaxel (1 nM) and ER stress After 3 days of exposure to the inhibitor salubrinal (2 μM) in combination, colony forming assay for measuring cancer cell proliferation was performed on the 7th day.

As a result, as shown in FIG. 6, when treated with an anticancer agent alone or an anticancer agent and an ER stress inhibitor, the number of colonies was different from the control culture conditions or the case of treatment with the ER stress inhibitor alone. It was confirmed that there was a significant decrease. In addition, it was confirmed that the number of colonies was further reduced when the anticancer agent and the ER stress inhibitor were used in combination compared to the case where the anticancer agent was treated alone.

Example 3-3. Inhibition of proliferation of PC-3 cells by treatment with anticancer drugs and ER stress inhibitors

The present inventors used PC-3 cells, a human prostate cancer cell line, as an anticancer agent such as doxorubicin (5 nM), cisplatin (10 μM), and paclitaxel (2 nM) and an ER stress inhibitor. After 3 days of exposure to salubrinal (10 μM) in combination, colony forming assay for measuring cancer cell proliferation was performed on the 7th day.

As a result, as shown in FIG. 7 , when treated with an anticancer agent alone or an anticancer agent and an ER stress inhibitor, the number of colonies was different from the control culture conditions or the case of treatment with the ER stress inhibitor alone. It was confirmed that there was a significant decrease. In addition, it was confirmed that the number of colonies was further reduced when the anticancer agent and the ER stress inhibitor were used in combination compared to the case where the anticancer agent was treated alone.

Example 3-4. Inhibition of proliferation of A549 cells by treatment with anticancer drugs and ER stress inhibitors

The present inventors used human lung cancer cell line A549 cells with anticancer agents such as doxorubicin (30 nM), cisplatin (1 μM), and paclitaxel (2 nM) and ER stress inhibitor salubri Colony forming assay for measuring cancer cell proliferation was performed on the 7th day after exposure for 3 days using a combination of salubrinal (10 μM).

As a result, as shown in FIG. 8, when treated with an anticancer agent alone or an anticancer agent and an ER stress inhibitor, the number of colonies was different from the control culture conditions or the case of treatment with the ER stress inhibitor alone It was confirmed that there was a significant decrease. In addition, it was confirmed that the number of colonies was further reduced when the anticancer agent and the ER stress inhibitor were used in combination compared to the case where the anticancer agent was treated alone.

Example 3-5. Inhibition of proliferation of MKN28 cells by treatment with anticancer drugs and ER stress inhibitors

The present inventors treated MKN28 cells, a human gastric cancer cell line, an anticancer agent such as doxorubicin (10 nM) and paclitaxel (2 nM) and ER stress inhibitor salubrinal (10 μM). Colony forming assay for measuring cancer cell proliferation was performed on the 7th day after exposure for 3 days in combination.

As a result, as shown in FIG. 9, when treated with an anticancer agent alone or an anticancer agent and an ER stress inhibitor, the number of colonies was different from the control culture condition or the case of treatment with the ER stress inhibitor alone. It was confirmed that there was a significant decrease. In addition, it was confirmed that the number of colonies was further reduced when the anticancer agent and the ER stress inhibitor were used in combination compared to the case where the anticancer agent was treated alone.

Example 3-6. Inhibition of proliferation of SK-HEP1 cells by treatment with anticancer drugs and ER stress inhibitors

The present inventors used human liver cancer cell line SK-HEP1 cells with anticancer agents such as doxorubicin (2 nM), cisplatin (1 μM), paclitaxel (0.5 nM) and ER stress inhibitors. After daily exposure to salubrinal (2 μM) in combination, colony forming assay for measuring cancer cell proliferation was performed on the 7th day.

As a result, as shown in FIG. 10, when treated with an anticancer agent alone or an anticancer agent and an ER stress inhibitor, the number of colonies was different from the control culture conditions or the case of treatment with the ER stress inhibitor alone It was confirmed that there was a significant decrease. In addition, it was confirmed that the number of colonies was further reduced when the anticancer agent and the ER stress inhibitor were used in combination compared to the case where the anticancer agent was treated alone. The above results show that when the anticancer agent and the ER stress inhibitor are treated in combination, the death of cancer cells is effectively induced than when the anticancer agent is treated alone.

Example 4: Confirmation of apoptosis through Propidium iodide (PI) staining

PI staining is a staining method using Propidium Iodide (PI), a fluorescent material that can be used to stain cells. Because PI stains DNA but does not cross the cell membrane, it can be used to identify metaphase and late apoptosis.

2x10 ⁵ cells were seeded in a 6-well plate, and each cell was cultured for 24 hours under standard conditions. After 24 hours, the cells were treated with an anticancer agent such as doxorubicin, cisplatin, and paclitaxel, and ER stress inhibitor, salubrinal, in combination for 3 days. Thereafter, the cells were washed twice with cold 1X PBS solution, and PI was added to the cell suspension at 2 μg/ml before microscopic observation. Cells were analyzed with an inverted fluorescence microscope (Eclipse 80i; Nikon, Melville, NY).

MCF-7 cells corresponding to the breast cancer cell line were treated with anticancer drugs such as doxorubicin (100 nM) and cisplatin (5 μM) and ER stress inhibitor salubrinal (10 μM). were exposed in combination.

As a result, as shown in FIG. 11 , by treatment with an anticancer agent and an ER stress inhibitor, the number of cancer cells that have advanced to mid- and late-stage apoptosis is increased than when the anticancer agent is treated alone.

Example 5: Identification of apoptosis-related proteins through Western blot

The present inventors analyzed MCF-7 cells corresponding to breast cancer cell lines with anticancer agents such as doxorubicin (100 nM), cisplatin (5 μM), and paclitaxel (5 nM) and ER stress inhibitors. After exposure to 1 day with phosphorus salubrinal (10 μM), the expression level of cleaved caspase 7, an apoptosis-related protein, was confirmed through western blotting. The α-tubulin antibody was purchased from calbiochem, and the caspase7 (cleaved form) (#9491) antibody was purchased from Cell Signaling Technology.

As a result, as shown in FIG. 12, unlike the case corresponding to the control culture conditions, when the anticancer agent and ER stress inhibitor salubrinal were treated together, the apoptosis-related protein cleaved caspase 7 Expression was significantly increased.

The above results show that when the anticancer agent and the ER stress inhibitor are treated together, the death of cancer cells is increased compared to the case where the anticancer agent is treated alone.

Claims (6)

In the pharmaceutical composition for cancer treatment comprising salubrinal and paclitaxel as active ingredients,
The cancer is an anticancer drug-resistant cancer selected from the group consisting of breast cancer, gastric cancer, prostate cancer, lung cancer, and liver cancer, wherein resistance is induced by cellular senescence by the paclitaxel treatment.
In the pharmaceutical composition for cancer treatment comprising salubrinal and cisplatin as active ingredients,
The cancer is an anticancer drug-resistant cancer selected from the group consisting of breast cancer, prostate cancer, lung cancer, and liver cancer, in which resistance is induced by cellular aging by the cisplatin treatment, a pharmaceutical composition.
delete delete delete 3. The method of claim 1 or 2,
The pharmaceutical composition is a cellular senescence-associated beta-galactosidase assay (SA-β-gal assay) to reduce the number of SA-β-gal positive cancer cells increased by the use of an anticancer agent in the pharmaceutical composition.

Images