Composition for preventing or treating colon cancer containing daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient

Abstract

The present invention relates to a composition for the prevention or treatment of colorectal cancer, comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient, and confirms that daunorubicin exhibits an anticancer effect against colorectal cancer by inhibiting the Hedgehog pathway.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention or treatment of colorectal cancer comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient, and a composition for improving the therapeutic responsiveness of an anticancer agent against colorectal cancer, etc. Background Technology

[0003] The Hedgehog signaling pathway is a signaling pathway that transmits information necessary for proper cell differentiation to embryonic cells. The concentration of Hedgehog signaling proteins varies across different embryonic regions, and this pathway continues to play a role in adults. Diseases associated with dysfunction of this pathway have been reported to include cancer.

[0004] Activation of the Hedgehog pathway is associated with the development of cancer in various organs, including the brain, lungs, mammary glands, prostate, and skin. Basal cell carcinoma, the most common form of cancerous malignancy, is most closely linked to Hedgehog signaling. While the association between the Hedgehog signaling pathway and cancer development is highly complex, it is becoming clear that abnormal activation of Hedgehog signaling leads to the growth, proliferation, and invasion of tumor cells.

[0005] Several studies using various cancer models have revealed that the GLI transcription factor can be regulated by Hh ligands or PTCH / Smo, as well as oncogenic signaling pathways such as Raf / MEK / ERK and PI3K / Akt signaling. Therefore, the Hedgehog pathway is an important cancer therapeutic target.

[0006] To find a new class of Hedgehog pathway inhibitors, the inventors performed screening using a GLI-luciferase reporter assay and identified a compound that inhibits GLI1-mediated transcription with a novel mechanism of action, and intend to provide this as an invention. The problem to be solved

[0008] The technical problem to be solved by the present invention is to provide a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] Another technical objective of the present invention is to provide a composition for improving the responsiveness to anticancer drug treatment for colorectal cancer, comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0012] To solve the above problem, the inventors have completed the present invention by being the first to confirm that daunorubicin inhibits GLI-dependent transcription in colorectal cancer cells and that anticancer activity is partially mediated by the inhibition of the Hedgehog pathway, and provide a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] According to one aspect, the pharmaceutical composition can inhibit the proliferation of cancer cells or induce apoptosis.

[0014] According to one side, the above-mentioned daunorubicin can be administered in combination with an anticancer drug to enhance the activity of the anticancer drug.

[0015] According to one side, the anticancer drug comprises nitrogen mustard, imatinib, oxaliplatin, rituximab, panitumumab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, cemasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, 5-fluorouracil (5-FU), bevacizumab, cisplatin, cetuximab, aflibercept, regorafenib, viscolumab, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab touxetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, Procarbazine, Alprostadil, Holmium Nitrate Chitosan, Gemcitabine, Doxyfluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Leucovorin, Carmoper, Raltitrexed, Interferon Alpha-2a, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vincristine, Vinblastine, Tenifoside, Doxorubicin, Idarubicin, Epirubicin, Mithoxantrone, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, It may be one or more selected from the group consisting of aclarubicin, pepromycin, temsirolimus, temozolomide, 5-fluorouracil, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, nabelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, and carmustine.

[0016] According to one side, the above-mentioned daunorubicin can inhibit the Hedgehog pathway.

[0017] According to one side, the colorectal cancer may be one that overexpresses GLI1.

[0018] According to another embodiment of the present invention, a composition for improving the responsiveness to anticancer drug treatment for colorectal cancer is provided, comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0019] According to one side, the anticancer drug comprises nitrogen mustard, imatinib, oxaliplatin, rituximab, panitumumab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, cemasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, 5-fluorouracil (5-FU), bevacizumab, cisplatin, cetuximab, aflibercept, regorafenib, viscolumab, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab touxetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, Procarbazine, Alprostadil, Holmium Nitrate Chitosan, Gemcitabine, Doxyfluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Leucovorin, Carmoper, Raltitrexed, Interferon Alpha-2a, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vincristine, Vinblastine, Tenifoside, Doxorubicin, Idarubicin, Epirubicin, Mithoxantrone, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, It may be one or more selected from the group consisting of aclarubicin, pepromycin, temsirolimus, temozolomide, 5-fluorouracil, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, nabelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, and carmustine. Effects of the invention

[0021] The present invention relates to a pharmaceutical composition for the prevention or treatment of colorectal cancer or a composition for improving the responsiveness to anticancer drug treatment for colorectal cancer, comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient. According to the present invention, daunorubicin inhibits the Hedgehog pathway and has an anticancer effect on GLI1-overexpressing colorectal cancer, and in particular, when administered in combination with a commercially available anticancer drug, it produces a synergistic effect and can be usefully used for anticancer purposes alone or in combination.

[0022] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by a person skilled in the art from the description below. Brief explanation of the drawing

[0024] Figure 1 shows the results of the GLI luciferase assay for various drugs. Figure 2 shows that daunorubicin and pralatrexate reduce GLI1 in HCT116. Figure 3 shows that daunorubicin inhibits Hedgehog signaling in HCT116 cells. Figure 4 shows that daunorubicin downregulates GL1 protein levels in other CRC cells, including HT29 and SNU283 cells. Figure 5 shows the caspase-dependent apoptosis effect induced by daunorubicin in HCT116 cells. Figure 6 shows that daunorubicin induces p53-mediated apoptosis and GLI1 downregulation in HCT-116 cells. Figure 7 shows that daunorubicin promotes GLI1 ubiquitination and proteasome degradation. Figure 8 shows that daunorubicin inhibits the Hedgehog pathway and induces apoptosis in an in vivo mouse model of HCT116 xenograft. Figure 9 shows the effect of co-administration of daunorubicin and a commercially available anticancer drug. Figure 10 shows a schematic diagram of the operation of the target path of the present invention. Specific details for implementing the invention

[0025] The inventors discovered that daunorubicin inhibits GLI1 by promoting β-TrCP-mediated GLI1 ubiquitination and subsequent proteasome degradation through the upregulation of P53, and confirmed that it has apoptotic and anticancer effects against colon cancer cells, thereby completing the present invention.

[0026] To solve the above problem, the present invention provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0027] In the present invention, the term “pharmaceuticalally acceptable salt” refers to a formulation of a compound that does not cause severe irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salt may be obtained by reacting the compound of the present invention with an inorganic acid such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, or an organic carboxylic acid such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc. In addition, the compound of the present invention may be obtained by reacting it with a base to form salts such as ammonium salts, alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucarmine, tris(hydroxymethyl)methylamine, and amino acid salts such as arginine and lysine.

[0028] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, or recurrence of cancer by administering the composition of the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of the said disease by administering the composition of the present invention.

[0029] In the present invention, the term "pharmaceutical composition" means that it is prepared for the purpose of preventing or treating the above disease, and can be formulated into various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated into external preparations, suppositories, and sterile injectable solutions.

[0030] In the present invention, "included as an active ingredient" means that the corresponding ingredient is included in an amount necessary or sufficient to realize the desired biological effect. In actual application, the amount included as an active ingredient is determined as an amount for treating the target disease, taking into account factors that do not cause other toxicities, and may vary depending on various factors such as, for example, the disease or condition being treated, the form of the composition administered, the size of the subject, or the severity of the disease or condition. A person skilled in the art to which the present invention pertains can empirically determine the effective amount of an individual composition without involving excessive experimentation.

[0031] In addition, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above, depending on each formulation.

[0032] The above-mentioned pharmaceutically acceptable carrier may be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and may further include other conventional additives such as antioxidants, buffers, and bacteriostatic agents as needed. Additionally, by additionally adding diluents, dispersants, surfactants, binders, and lubricants, it may be formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions, or into pills, capsules, granules, or tablets. Furthermore, it may be preferably formulated according to each disease or component by appropriate methods in the art or by using methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton, PA).

[0033] The composition of the present invention may be administered orally or parenterally in a pharmaceutically effective amount according to the intended method, and the term “pharmaceutically effective amount” of the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects, and the effective dose level may be determined based on factors including the patient’s health status, severity, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0034] In addition, the present invention may provide a method for preventing or treating cancer comprising the step of administering the daunorubicin or a pharmaceutically acceptable salt thereof to an individual.

[0035] In the present invention, the term “individual” is not limited to mammals such as livestock or humans that require prevention, treatment, and / or diagnosis of the disease, but preferably may be a human.

[0036] The term "administration" in the present invention means providing a specific substance to a patient by any appropriate method. The pharmaceutical composition of the present invention may be formulated into various forms for administration to an individual, and representative formulations for parenteral administration are injectable formulations, preferably isotonic aqueous solutions or suspensions. Injectable formulations may be prepared according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. For example, each component may be dissolved in saline solution or a buffer solution to be formulated for injection. Additionally, formulations for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers, and these formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and a lubricant (e.g., silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycol). The above tablet may include a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally may further include a disintegrant such as starch, agar, alginic acid or its sodium salt, an absorbent, a coloring agent, a flavoring agent and / or a sweetener. The above formulation may be prepared by conventional mixing, granulation, or coating methods.

[0037] In addition, the pharmaceutical composition of the present invention may further include adjuvants such as preservatives, hydrating agents, emulsification promoters, salts or buffers for osmotic pressure regulation, and other therapeutically useful substances, and may be formulated according to conventional methods.

[0038] The pharmaceutical composition according to the present invention may be administered via various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular, and the dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, the patient's age, gender, body weight, and severity of the patient. In addition, the composition of the present invention may be administered in combination with known compounds that can enhance the desired effect.

[0039] The pharmaceutical composition according to the present invention may be administered to humans and animals by a route of administration such as orally or parenterally, intravenously, subcutaneously, intranasally, or intraperitoneally, but preferably by intravenous administration.

[0040] In the pharmaceutical composition of the present invention, the total effective amount of daunorubicin or its pharmaceutically acceptable salt according to the present invention may be administered to a patient as a single dose, or administered by a fractionated treatment protocol in which multiple doses are administered over a long period. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease, but typically, based on adults, it may be administered repeatedly several times a day at an effective dose of 100 μg to 3,000 mg per single dose, but is not limited to such amounts. The concentration of daunorubicin or its pharmaceutically acceptable salt may be determined by considering various factors such as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, as well as the route of administration and frequency of treatment.

[0041] Furthermore, the pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention, and the pharmaceutical composition of the present invention may additionally include known agents as active ingredients in addition to daunorubicin and its pharmaceutically acceptable salts, and may be used in combination with other treatments known for the treatment of these diseases.

[0042] In particular, the inventors confirmed that daunorubicin shows a significant synergistic effect on colorectal cancer cell lines when administered in combination with an anticancer drug. Non-limiting examples of anticancer drugs include nitrogen mustard, imatinib, oxaliplatin, rituximab, panitumumab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, 5-fluorouracil (5-FU), bevacizumab, cisplatin, cetuximab, aflibercept, regorafenib, viscolumab, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab touxetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, Procarbazine, Alprostadil, Holmium Nitrate Chitosan, Gemcitabine, Doxyfluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Leucovorin, Carmoper, Raltitrexed, Interferon Alpha-2a, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vincristine, Vinblastine, Tenifoside, Doxorubicin, Idarubicin, Epirubicin, Mithoxantrone, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, It may be aclarubicin, pepromycin, temsirolimus, temozolomide, 5-fluorouracil, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, nabelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, and carmustine, etc., and preferably may be irinotecan, but is not limited thereto.

[0043] According to one aspect, the pharmaceutical composition may inhibit the proliferation of cancer cells or induce apoptosis. The colorectal cancer may be any one of adenocarcinoma, lymphoma, sarcoma, squamous cell carcinoma, rectal cancer, or metastatic lesions that have spread to the colon.

[0044] According to one side, the above-mentioned daunorubicin can inhibit the Hedgehog pathway.

[0045] According to one side, the colorectal cancer may be one that overexpresses GLI1.

[0046] As disclosed in Example 2 below, the inventors have confirmed that daunorubicin effectively inhibits GLI1 and exhibits a death effect on colorectal cancer cell lines, which can be used more effectively in colorectal cancer subtypes that overexpress GLI1.

[0047] According to another embodiment of the present invention, a composition for improving the responsiveness to anticancer drug treatment for colorectal cancer is provided, comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0048] In the present invention, "therapeutic responsiveness" refers to whether a patient responds preferentially or non-preferentially to a therapeutic agent, such as an anticancer drug, or the risk of resistance to the anticancer drug, the patient's prognosis after treatment, etc. The composition for improving therapeutic responsiveness according to the present invention can achieve a synergistic effect by co-administering daunorubicin compared to the effect of known anticancer drugs.

[0049] In the present invention, the term "improvement" refers to any action that beneficially changes parameters related to the condition being treated, such as at least reducing the severity of symptoms or improving the disease.

[0050] As used herein, the term "resistance to anticancer drugs" refers to a situation where, when treating cancer patients with anticancer drugs, there is no therapeutic effect from the beginning of treatment, or although there is an initial therapeutic effect, the therapeutic effect is lost during the course of continuous treatment. For example, in anticancer drug therapy, the general assessment of therapeutic efficacy can be determined based on response evaluation criteria for solid tumor groups. According to the above criteria, the efficacy of cancer treatment can be classified into Complete Response (CR), Partial Response (PR), Progressive Disease (PD), or Stable Disease (SD) groups based on factors such as changes in tumor size.

[0051] According to one side, the anticancer drug comprises nitrogen mustard, imatinib, oxaliplatin, rituximab, panitumumab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, cemasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, 5-fluorouracil (5-FU), bevacizumab, cisplatin, cetuximab, aflibercept, regorafenib, viscolumab, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab touxetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, Procarbazine, Alprostadil, Holmium Nitrate Chitosan, Gemcitabine, Doxyfluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Leucovorin, Carmoper, Raltitrexed, Interferon Alpha-2a, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vincristine, Vinblastine, Tenifoside, Doxorubicin, Idarubicin, Epirubicin, Mithoxantrone, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, It may be one or more selected from the group consisting of aclarubicin, pepromycin, temsirolimus, temozolomide, 5-fluorouracil, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, nabelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, and carmustine.

[0053] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0054] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0055] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0056] Example 1. Experimental Method

[0057] Example 1-1. Cell Culture

[0058] NIH3T3 cell lines stably expressing the GLI-luciferase reporter construct were purchased from BPS Bioscience Inc. and maintained in DMEM, 10% fetal calf serum, and 1% penicillin / streptomycin. Human colorectal cancer cell lines HCT116, HT29, SNU283, DLD-1, and HCT8 were purchased from the Korean Cell Line Bank and cultured in RPMI 1640 medium. All cells were grown at 37°C in a 5% CO2 incubator.

[0060] Example 1-2. GLI-Luciferase Reporter Gene Analysis

[0061] NIH3T3 cell lines stably expressing the GLI-luciferase reporter construct were seeded in 96-well plates at a rate of 25,000 cells / 100 μL per well. After overnight incubation, 500 nL of the pre-plated compound dissolved in DMSO was pin-delivered to the cells using a Janus automated liquid handler workstation (PerkinElmer, Waltham, MA, USA) and incubated for 24 hours. Bright-Glo luciferase assay reagent was added to the cells, and luminescence emission was read using an Envision plate reader (PerkinElmer, Waltham, MA, USA).

[0063] Examples 1-3. Analysis of MTT cell viability

[0064] Cells (5,000 cells / 100 μL per well) were seeded into 96-well tissue culture treatment plates. After 1 day, the drug was treated with a 1 / 5 dilution from a peak concentration of 20 μM. After 24 hours, MTT reagent (Promega Corp., Fitchburg, WI, USA) was added according to the manufacturer's instructions, and the uptake signal was read using an Envision plate reader (PerkinElmer, Waltham, MA, USA). GI50 was calculated using Prism8 software (GraphPad Software Inc., San Diego, CA, USA).

[0066] Examples 1-4. Western blot

[0067] Cells were lysed in RIPA buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate, along with a cocktail of protease and phosphatase inhibitors. Equal amounts of protein were loaded onto SDS-PAGE and then transferred to a membrane. After transfer, the cells were blocked in 5% skim milk for 2 hours. Subsequently, the primary antibody was incubated overnight at 4°C and incubated with the HRP-labeled secondary antibody for 2 hours. Protein expression levels were detected using Enhanced Chemiluminescence (ECL).

[0069] Examples 1-5. Immunoprecipitation test method

[0070] Cells were lysed with 300 μL of lysis buffer (1 mM PMSF, protease inhibitor, phosphatase inhibitor), and bicinchoninic acid content was analyzed. The primary antibody was incubated overnight at 4°C, after which Protein G PLUS-Agarose beads were added and incubated in a rotator for 1 hour. The immunoprecipitate was washed several times, 2X sample buffer was added at 15,000 rpm, and heated at 100°C for 5 minutes; finally, protein analysis was performed using Western blot.

[0072] Examples 1-6. Colony Formation Analysis

[0073] 5 x 10⁶ HCT-116 cells in a 6-well plate 2 After seeding cells / wells, they were cultured in a 5% CO2 incubator at 37°C. The medium was changed 2 to 3 times a week. After 2 weeks, the cells were washed with DPBS (Dulbecco phosphate-buffered saline), fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then stained with 0.5% crystal violet for 30 minutes.

[0075] Examples 1-7. Measurement of Cell Apoptosis

[0076] HCT116 cell lines were treated with daunorubicin for 24 hours. After 24 hours, the cells were converted into single cells using trypsin-EDTA. Then, after treatment with Annexin-FITC and propidium iodide, the cells were stained in the dark at 4°C for 30 minutes. Immediately after 30 minutes, cell apoptosis was measured using a flow cytometer.

[0078] Examples 1-8. PCR test method

[0079] RNA was extracted using the TRIzol reagent. Transcript amplification was performed using a reverse transcriptase PCR kit. qRT-PCR was performed on an Applied Biosystems® QuantStudio™ 6 Flex real-time PCR system using gene-specific Taqman™ probes (Applied Biosystems). mRNA expression was calculated using CT values.

[0081] Examples 1-9. Immunofluorescence staining

[0082] HCT-116 cells were seeded from glass coverslips in 12-well plates and treated with daunorubicin for 24 hours. After 24 hours, the cells were washed three times with PBS and fixed with 3.7% formaldehyde for 30 minutes at room temperature. Cells were treated with 0.5% Triton X-100 for 30 minutes at room temperature and then blocked with 1% BSA (bovine serum albumin) for 2 hours. Subsequently, HCT-116 cells were incubated overnight with the primary antibody at 4°C. The next day, the cells were treated with the Alexa Fluor® 594 conjugated secondary antibody for 15 minutes. Then, the nuclei (DAPI) were stained using 4',6-diamidino-2-phenylindole. After mounting with the mounting solution, the cells were observed under a fluorescence microscope.

[0084] Examples 1-10. Measurement of Caspase 3 / 7 Activity

[0085] 8 x 10 HCT116 cells per well in a 96-well plate 3 Canine cells were seeded. After treating with daunorubicin at concentrations of 0, 0.5, and 1 μM for 24 hours, 100 μL of caspase-Glo 3 / 7 reagent was added to each well. After incubation in the dark for 2 hours, caspase-3 / 7 activity was measured using a Varioskan Lux reader (Thermo Fisher).

[0087] Examples 1-11. Tumor xenograft

[0088] Four-week-old female BALB / c nude mice were purchased and reared in a specific pathogen-free environment. Prior to the experiment, the animals were reared for one week for acclimatization and were allowed free access to food and water. HCT116 cells (2×10⁶ 6 ) was mixed with Matrigel and subcutaneously implanted into 5-week-old BALB / c nude female mice. Tumor size 100 mm 3 After reaching [date], nude mice were intraperitoneally administered vehicle (DMSO) or daunorubicin (2 mg / kg) every other day for 15 days.

[0090] Example 2. Confirmation of inhibition of GLI1 activity by daunorubicin in CRC cells

[0091] To identify novel antagonists of the Hedgehog (Hh) pathway, an FDA-approved drug library consisting of 1,018 drugs was screened on commercially available NIH3T3 cell lines stably expressing a GLI-dependent firefly luciferase reporter. It was discovered that 10 drugs inhibited GLI-driven luciferase activity (Fig. 1). As shown in Fig. 2, among the 10 drugs, only daunorubicin and pralatrexate reduced GLI1 in the colorectal cancer cells HCT116. It was confirmed that daunorubicin inhibits the activity of GLI-driven luciferase (Figs. 3A and B). Next, HCT116 cells were treated with daunorubicin (0, 0.5, and 1 μM) for 24 hours, and the protein levels of components involved in the standard Hh pathway were investigated. The protein expression levels of GLI1 were significantly downregulated (Figs. 3C and E). The protein levels of SHH, GLI2, GLI3, Smo, and Ptch1 were not significantly altered by daunorubicin (Fig. 3C). Additionally, daunorubicin was shown to downregulate GL1 protein levels in other CRC cells, including HT29 and SNU283 cells (Fig. 4). Furthermore, it was confirmed that the GLI1 target gene was reduced (Fig. 3D). Western blot analysis of cytoplasmic and nuclear extracts from HCT116 cells showed that daunorubicin treatment downregulated cytoplasmic and nuclear expression of GLI1 compared to the control group (Fig. 3F). These results demonstrate that daunorubicin inhibits GLI1 expression.

[0093] Example 3. Induction of Caspase-Dependent Apoptosis by Daunorubicin in HCT116 Cells

[0094] First, to investigate the cytotoxicity of daunorubicin in CRC cells (Fig. 5A), various CRCs were cultured with various concentrations (0–20 μM) of daunorubicin for 24 hours. The viability of CRC cells was reduced by daunorubicin in a dose-dependent manner. Additionally, to confirm the reduced cell proliferation of CRC cells by daunorubicin, a colony formation assay was performed on HCT116 cells. As shown in Fig. 5B, colony formation in HCT116 cells was potently inhibited. Furthermore, to determine whether the cell death induced by treatment was apoptosis, Annexin V-propidium iodide (PI) staining was performed on HCT116 cells treated with 0, 0.5, and 1 μM of daunorubicin for 24 hours. In flow cytometry analysis, apoptosis was increased by daunorubicin (Fig. 5C), and caspase-3 / 7 activity increased in a dose-dependent manner (Fig. 5D). Daunorubicin increased the levels of cleaved PARP, caspase-8, caspase-9, and caspase-3, which were inhibited by combination therapy with the pan-caspase inhibitor z-VAD-fmk, indicating that daunorubicin induced caspase-dependent apoptosis in HCT116 cells (Fig. 5E). Therefore, these results demonstrate that daunorubicin induces caspase-dependent apoptosis in CRC.

[0096] Example 4. Induction of p53-mediated apoptosis and GLI1 downregulation by daunorubicin in HCT-116 cells

[0097] We investigated which pathways are regulated by daunorubicin (1 μM) using a human phosphokinase antibody array. Interestingly, among the phosphorylations of the protein array, p53 phosphorylation (S15, S46, S392) was increased by daunorubicin treatment (Fig. 6A). This indicates p53 activation induced by daunorubicin. In addition, daunorubicin enhanced the phosphorylation of Chk2 (T68) and p27 (T198), which are involved in cell cycle arrest (Fig. 6A).

[0098] The levels of apoptosis-related marker proteins were investigated. Consistent with Figure 6A, daunorubicin increased the levels of apoptosis-promoting proteins such as p53, Bim, Bak, Bax, and cleaved PARP, while decreasing the levels of anti-apoptotic Puma and Cyclin D1 (Figure 6B). Daunorubicin-induced PARP cleavage was reduced by p53 knockdown, suggesting that daunorubicin induced apoptosis through p53 activation (Figure 6C). Activation of p300 and PCAF is required for the p53-dependent response to genotoxic stress according to general mechanisms. As previously reported, it has been demonstrated that p53-induced upregulation of PCAF is necessary to inhibit GLI1 in response to DNA damage. We investigated whether the expression of PCAF and P300 was altered by daunorubicin. Daunorubicin increased PCAF expression in a dose-dependent manner (Figure 6D).

[0099] To determine whether PCAF-dependent inhibition of GLI1 occurred, PCAF was knocked down using PCAF siRNA. By demonstrating that GLI1 is still reduced by daunorubicin despite PCAF inhibition, these results show that inhibition of GLI1 by daunorubicin is not dependent on PCAF (Fig. 6E).

[0101] Example 5. Promotion of GLI1 ubiquitination of daunorubicin and proteasome degradation

[0102] To investigate whether the decrease in GLI1 was due to proteasome or lysosomal degradation, bortezomib and leupeptin were administered. GLI1 expression, which was reduced by daunorubicin, was increased by bortezomib and remained unchanged by leupeptin (Fig. 7A). This data demonstrates that the decrease in GLI1 caused by daunorubicin was due to proteosomal degradation. Additionally, HCT116 cells were treated with cyclohexamine (CHX), a protein synthesis inhibitor. GLI1 expression was significantly reduced when HCT116 cells were exposed to daunorubicin and CHX compared to CHX alone, implying that daunorubicin reduced GLI1 levels through ubiquitin-proteasome degradation (Fig. 7B). These results indicate that daunorubicin promotes the ubiquitination of GLI1 (Fig. 7C).

[0103] To investigate which E3 ligase is responsible for GLI1 ubiquitination, immunoprecipitation was performed to measure the interaction between GLI1 and E3 ligases. As indicated in Fig. 7D, daunorubicin increased the interaction between GLI1 and β-TrCP. To investigate whether β-TrCP-dependent GLI1 ubiquitination occurred, β-TrCP was knocked down using β-TrCP siRNA. Knockdown of β-TrCP restored the reduction of GLI1 caused by daunorubicin, and GLI1 ubiquitination also remained unchanged (Fig. 7E). These data suggest that daunorubicin induces GLI1 ubiquitination via β-TrCP.

[0105] Example 6. Inhibition of Hedgehog pathway and induction of apoptosis by daunorubicin in an HCT116 xenograft in vivo mouse model

[0106] HCT116 cells (2×10⁶ 6 ) was injected subcutaneously into BALB / c nude mice. The tumor size was 100 mm 3Nude mice were treated with intraperitoneal injection of daunorubicin (2 mg / kg) every 2 days for 15 days. Tumor growth and body weight were inhibited in the daunorubicin-treated group compared to the control group (Fig. 8 A, B, and C), while body weight did not differ significantly between the control and daunorubicin-treated groups (Fig. 8 D). These data indicate apoptosis induced by daunorubicin. Consistent with in vitro results, immunohistochemistry showed potent inhibition of GLI1 levels in the daunorubicin-treated group, while p53 levels were elevated (Fig. 8 E). TUNNEL analysis revealed apoptosis induced by daunorubicin in tumor tissue (Fig. 8 F). Taken together, daunorubicin exhibited an anticancer effect in CRC xenograft mice, partially through the downregulation of GLI1.

[0108] Example 7. Confirmation of combination effect between daunorubicin and a commercially available anticancer drug

[0109] We aimed to determine whether there was a synergistic effect when oxaliplatin or irinotecan, representative anticancer drugs used in the treatment of colorectal cancer, were combined with daunorubicin. In the case of oxaliplatin, no synergistic effect was observed when combined with daunorubicin, whereas a synergistic effect was confirmed with irinotecan (Fig. 9A). Since the combination of irinotecan 10 μM and daunorubicin 0.5 μM showed the greatest synergistic effect with a combination index of 0.27381, we examined the changes in cleaved PARP expression at this concentration. The results confirmed that the expression of cleaved PARP was significantly increased with combination treatment compared to monotherapy (Fig. 9B).

[0111] Although the embodiments have been described above with reference to the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or if the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from described, or replaced or substituted by other components or equivalents.

[0112] Therefore, other embodiments, other manufacturing examples, and equivalents to the claims also fall within the scope of the claims set forth below.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of colorectal cancer comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient and intended for co-administration with irinotecan. Claim 2 In claim 1, the pharmaceutical composition is a pharmaceutical composition that inhibits the proliferation of cancer cells or induces apoptosis. Claim 3 A pharmaceutical composition according to claim 1, wherein the daunorubicin is administered in combination with irinotecan to enhance the activity of irinotecan. Claim 4 delete Claim 5 A pharmaceutical composition according to claim 1, wherein the daunorubicin inhibits the Hedgehog pathway. Claim 6 A pharmaceutical composition according to claim 1, wherein the colorectal cancer overexpresses GLI1. Claim 7 A composition for improving the therapeutic responsiveness of irinotecan to colorectal cancer, comprising daunorubicin or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 8 delete

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