Anticancer agent containing 1,1-diethoxyethane as the active ingredient

Abstract

To provide novel anticancer drugs. [Solution] An anticancer agent containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient is provided.

Description

[Technical Field]

[0001] [References to related applications] This application claims priority to Korean Patent Application No. 10-2025-0010954, filed on 24 January 2025, and Korean Patent Application No. 10-2025-0150102, filed on 16 October 2025. The entire contents of each application are incorporated herein by reference.

[0002] The present invention relates to an anticancer agent containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient. [Background technology]

[0003] Leukemia is one of the most common cancers in children and adolescents, accounting for about one-third of all cancer cases diagnosed.[1] Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy and is classified into B-cell precursor ALL (B-ALL) and T-cell precursor ALL (T-ALL).[2,3] T-cell acute lymphoblastic leukemia (T-ALL) is a rare form in children, accounting for about 12% to 15% of newly diagnosed cases and is distinguished by distinct clinical and molecular characteristics. In the past, it had a worse prognosis than B-ALL, but advances in treatment have led to improved results, including an event-free survival (EFS) of about 85%.[4-6]

[0004] Leukemia can metastasize to various tissues and is complex to manage [7]. Therefore, a clearer understanding of the complexity and mechanisms of antitumor immunity can contribute to the development of tumor immunotherapy and cancer biology [8]. In the development of cancer therapeutics, it is important to evaluate the specificity for inducing cancer apoptosis [9]. While approaches to kill lymphoma cells are a unique therapeutic strategy, existing cell death inducers can cause serious side effects due to their heavy metal content. Therefore, it is essential to discover new cell death inducers with higher potency and lower toxicity [10, 11]. Oxidative stress is a major mediator of lymphoid cell death, and reactive oxygen species (ROS) are essential for various intracellular functions and selectively target polyunsaturated fatty acids, which are abundant in actively proliferating lymphocytes. It has been proposed that excessive ROS production promotes cell death via mitochondrial damage, which activates signaling networks that induce cell cycle arrest, DNA repair, and apoptosis. Alterations in cyclin expression play a crucial role in regulating cell proliferation and cancerous deformation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Thimoteo, RRC, et al., Microarray data analysis of antileukemic action of Cinnamoylated benzaldehyde LQB-461 in Jurkat cell line.Mol Biol Rep, 2024.51(1):p.187. [Non-Patent Document 2] Mirsanei, J.S., et al., Does Gold-Silver Core-Shell Nanostructure with Alginate Coating Induce Apoptosis in Human Lymphoblastic Tumoral (Jurkat) Cell Line Rep Biochem Mol Biol, 2023.12(2): p. 233-240.

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Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

[0006] A characteristic feature of cancer is abnormal cell proliferation, and the regulation of the cell cycle is carried out by cyclins, particularly cyclin-dependent kinases (CDKs) that regulate G1-S conversion and G2 progression. These cell cycle regulatory mechanisms play a crucial role in the proliferation and survival of cancer cells, and therapeutic strategies targeting them are needed.

[0007] The inventors continued their research to develop compounds that have anticancer effects by inhibiting the proliferation of cancer cells and inducing apoptosis. As a result, they confirmed that 1,1-diethoxyethane (1,1-DEE) exhibits a potent apoptosis effect in the T-lymphoblastic leukemia cell line (Jurkat E6.1) (Experimental Example 1). Specifically, the cell morphology changed to a rounder, more contracted form upon 1,1-DEE treatment, and increased Bax protein expression and decreased Bcl-2, caspase-3, and caspase-9 expression were observed by Annexin V / PI staining and Western blot analysis. On the other hand, no such effect was observed in the group treated with 1,2-DEE, an isomer of 1,1-DEE.

[0008] In addition, 1,1-DEE was demonstrated to induce the production of intracellular reactive oxygen species (ROS), thereby reducing the levels of CDK3, CDK4, and cyclin D1, D3, and E proteins, and inducing cell cycle arrest at the G0 / G1 phase (Experimental Example 2). It was confirmed that apoptosis and cell cycle arrest during pretreatment with ROS scavengers (NAC, Trolox, Ebselen) were suppressed, suggesting that the anti-cancer effect of 1,1-DEE is related to the ROS-mediated mechanism.

[0009] Furthermore, 1,1-DEE was confirmed to activate AMPK signaling, regulate cancer cell metabolic reprogramming, suppress the Warburg effect, thereby reducing the expression of glycolytic enzymes (LDHA, HK2) and glucose transporter (GLUT1), and maintaining mitochondrial oxidative phosphorylation (OXPHOS) activity (Experimental Examples 3 and 4). Such metabolic regulation shows an effect of suppressing cancer cell growth and survival in combination with apoptosis and cell cycle arrest.

[0010] Furthermore, in the NSG mouse model, it was confirmed that 1,1-DEE shows the effects of suppressing tumor growth, increasing survival rate, minimizing histological damage in major organs, and reducing leukemia-related inflammatory biomarker (IL-8) (Experimental Example 5).

[0011] Thus, the present invention presents that 1,1-DEE can effectively suppress cancer cell growth and survival through ROS generation, cell cycle regulation, AMPK-mediated metabolic reprogramming, and apoptosis induction, and provides a basis for the development of anti-cancer agent compositions and cancer prevention and treatment methods utilizing the same.

[0012] However, the problems to be solved by the present application are not limited to the problems described above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Means for Solving the Problems

[0013] The present invention discloses an anticancer agent containing 1,1 - diethoxyethane (1,1 - DEE) as an active ingredient.

[0014] In the present invention, the 1,1 - diethoxyethane (1,1 - DEE) has a molecular formula of C6H 14 O2 and is represented by the following Structural Formula 1, and is also called Acetaldehyde diethyl acetal or Ethyllidene diethyl ether.

[0015] [Chemical Formula]

[0016] In the present invention, the 1,1 - DEE can induce the generation of reactive oxygen species (ROS) in cancer cells.

[0017] In the present invention, the 1,1 - DEE can induce apoptosis in cancer cells.

[0018] In the present invention, the 1,1 - DEE can induce cell cycle arrest through the depletion of G2 / M - phase cells and the accumulation of G1 - phase cells in cancer cells, and can reduce the expression of CDK3, CDK (cyclin - dependent kinase) 4, and cyclin D1, D3, and E proteins.

[0019] In the present invention, the anticancer agent can be used for hematological malignancies or solid tumors.

[0020] In the present invention, the anticancer agent is used to treat squamous cell carcinoma, basal cell carcinoma, melanoma, tumors of the epithelial lining of glands or ducts, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma of the liver and bile ducts, hepatocellular carcinoma of the gastrointestinal tract, esophageal squamous cell carcinoma, esophageal adenocarcinoma, colorectal cancer, gastric cancer, airway tumors, bronchial carcinoma, small cell carcinoma, transitional cell carcinoma of the urinary tract, transitional cell carcinoma of the bladder, squamous cell carcinoma of the bladder, prostate cancer, cervical cancer, leukemia of blood cells and associated cells, acute and chronic lymphocytic leukemia, polycythemia vera, lymphoid tissue carcinoma, malignant lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, follicular lymphoma, diffuse lymphoma, It can be used for cancers selected from the group consisting of small lymphocytic lymphoma, large cell lymphoma, lymphoblastic lymphoma, multiple myeloma, connective tissue tumors, osteosarcoma, nervous system tumors, neuroblastoma, retinoblastoma, glioblastoma, tumor-forming virus-associated glioma, Burkitt lymphoma, B-cell lymphoma in immunocompetent individuals, nasopharyngeal cancer, esophageal and gastroesophageal cancer, squamous cell carcinoma, islet tumors, breast cancer, lung cancer, colorectal cancer, retinoblastoma, liver cancer, pancreatic cancer, brain cancer, malignant mesothelioma, hepatitis B virus hepatocellular carcinoma, endometrial cancer, ovarian cancer, head and neck cancer, thyroid cancer, and soft tissue-related cancers.

[0021] In the present invention, when the anticancer agent is used for leukemia, the leukemia may be a form selected from the group consisting of acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonuclear leukemia, acute minimally differentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, T-cell precursor lymphocytic leukemia, T-cell prolymphoblastic leukemia, Philadelphia chromosome-positive leukemia, FLT3 mutation-positive acute myeloblastic leukemia, and acute erythroleukemia.

[0022] In the present invention, the anticancer agent may be administered in combination with one or more of the following: immunotherapy agents, monoclonal antibodies, chemotherapeutic agents, radioprotective agents, radiotherapy agents, and gene therapy agents. In this case, the immunotherapy agent may include immune barrier inhibitors including PD-1, PD-L1, and CTLA-4 inhibitors; cytokine therapy including interleukins and interferons; CAR-T cell therapy, oncolytic viruses, vaccines, or targeted therapies; the chemotherapeutic agent may include antimetabolites, platinum-based preparations, alkylating agents, or topoisomerase inhibitors; and the radiotherapy agent may include external beam radiation therapy, proximal radiation therapy, proton therapy, radiofrequency thermotherapy, stereotactic radiotherapy, or neutron therapy.

[0023] In the present invention, the anticancer agent can be used to prevent the onset of cancer. In the present invention, the anticancer agent can exhibit an energy-restoring effect during the course of cancer treatment. In the present invention, the anticancer agent may be used as a pharmaceutical composition for the prevention or treatment of cancer. The pharmaceutical composition may be administered by one or more routes of administration selected from oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernosal injection, intrathecal injection, epidural injection, and rectal administration. In the present invention, the anticancer agent may be used as a cosmetic composition for the prevention or improvement of cancer. The cosmetic composition may be prepared in one or more dosage forms selected from solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, packs, softening lotions, emulsions, makeup bases, essences, soaps, liquid cleansers, bath additives, sunscreens, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays. In the present invention, the anticancer agent may be used as a food composition for the prevention or improvement of cancer. The food may include meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen, other noodles, chewing gum, ice cream and dairy products, various soups, beverages, teas, coffee beverages, energy drinks, alcoholic beverages, or vitamin complexes. In the present invention, the anticancer agent may be used as a feed composition for the prevention or improvement of cancer. The feed may include powdered feed, solid feed, moist pellet feed, dry pellet feed, extruded pellet (EP) feed, or live feed. [Effects of the Invention]

[0024] The 1,1-diethoxyethane (1,1-DEE) according to the present invention was shown to decrease cell viability in a concentration-dependent manner in the T lymphoblastic leukemia cell line (Jurkat E6.1), induce cell morphological changes, and promote apoptosis by increasing Bax protein expression and decreasing Bcl-2, caspase-3, and caspase-9 expression (Experimental Example 1). Furthermore, it was confirmed that 1,1-DEE induces ROS generation, and that this effect can be suppressed by a ROS scavenging agent (Experimental Example 2). Cell cycle analysis results demonstrated that 1,1-DEE induces cell cycle arrest through the depletion of G2 / M phase cells and accumulation of G1 phase cells, and contributes to the suppression of cancer cell proliferation by decreasing CDK3, CDK4, and cyclin D1, D3, and E protein levels (Experimental Example 2).

[0025] Furthermore, 1,1-DEE was shown to suppress the Warburg effect and regulate metabolic reprogramming in cancer cells by activating AMPK signaling, suppressing the expression of glycolytic enzymes (LDHA, HK2) and glucose transporter (GLUT1) in cancer cells, and maintaining or enhancing mitochondrial oxidative phosphorylation (OXPHOS) activity (Experimental Examples 3 and 5).

[0026] In in vivo (NSG mouse) experiments, 1,1-DEE significantly suppressed tumor growth, increased survival rates, minimized histological damage to major organs, and reduced the expression of leukemia-associated inflammatory biomarker (IL-8), demonstrating its potential as a cancer treatment agent (Experimental Example 4).

[0027] Therefore, the present invention provides a novel anticancer treatment method using 1,1-DEE, and is expected to contribute to the development of a new type of anticancer agent that not only has lower toxicity and higher efficacy compared to existing therapeutic agents, but can also promote cancer prevention, treatment, and recovery through AMPK-mediated energy metabolism regulation.

[0028] On the other hand, the scope of the present invention is not limited by the effects described above. [Brief explanation of the drawing]

[0029] [Figure 1] The results of cell viability evaluation using MTT analysis are shown. Cells were treated with 1,1-DEE (A and B) and its structural isomer compound 1,2-DEE (C and D) at specified concentrations for 24 hours, followed by reaction in 10 μL of MTT solution for 2 hours. Each experiment was repeated three times, resulting in a total of three independent experiments. Data are expressed as mean ± standard error (SEM), where *p<0.05;****p<0.0001 indicates significance relative to the control group. (E) shows the morphological changes of Jurkat cells observed under a 20x inverted microscope after treatment with 1,1-DEE or 1,2-DEE at concentrations of 1, 5, and 10 mM, respectively, for 6, 12, and 24 hours. In particular, morphological changes such as cell shrinkage and cell rounding were clearly observed in cells treated with 1,1-DEE at concentrations of 5 mM and 10 mM for 24 hours. [Figure 2]The results of apoptosis analysis of Jurkat cells using flow cytometry are shown. Treatment with 1,1-DEE at different concentrations for 24 hours resulted in a significant increase in the number of apoptotic cells compared to the control group. (A) and (B) show the Annexin V and PI staining results and the ratio of apoptosis, and (C) and (D) show the results of Western blot analysis of pro-apoptotic and anti-apoptotic protein expression. The data are expressed as mean ± standard deviation (SD) from three independent experiments, and statistical significance was confirmed as p<0.0001. Statistical significance is indicated as ***p<0.0001 and ****p<0.00001, which are results compared to the control group. [Figure 3] The results of cell cycle analysis of Jurkat cells 24 hours after treatment with 1,1-DEE at concentrations of 1–10 mM are shown. (A) shows representative flow cytometry results for cell cycle arrest, and (B) shows the percentage of early apoptotic cells observed at the sub-G1 peak, along with the ratio of S phase and G2 / M phase cells. (C) and (D) show the results of Western blot analysis, with data from three independent experiments shown as mean ± standard deviation (SD). Statistical significance is indicated by *p<0.05, showing significance compared to the control group. [Figure 4]The results of ROS generation by 1,1-DEE treatment are shown. (A) shows the results of flow cytometry analysis of ROS generation in Jurkat cells that were pretreated with NAC (2 mM), Trolox (250 μM), and Ebselen (10 μM), and then treated with 1,1-DEE (5 mM) or H2O2 (2 mM). (B) shows the results of measuring ROS fluorescence intensity during 1,1-DEE and 1,2-DEE treatment and ROS scavenger treatment. (C) shows the results of evaluating the change in cell viability by ROS scavenger by MTT analysis after pretreating cells with 1-5 mM NAC, 125-500 μM Trolox, and 1-10 μM Ebselen for 1 hour, and then treating them with 5 mM 1,1-DEE for 24 hours. Statistical significance is indicated as follows: *p<0.05 indicates comparison with the control group, and #p<0.05 indicates comparison with the 1,1-DEE treatment group. Data are expressed as mean ± SD from three repeated experiments. [Figure 5] The results of AMPK activation in Jurkat cells induced by 1,1-DEE treatment are shown. Jurkat cells were treated with 1,1-DEE for 0–120 minutes, after which total AMPK and phosphorylated AMPK (p-AMPK) were analyzed by Western blotting. (A) shows time-dependent activation, and (C) shows concentration-dependent activation. (B) and (D) show p-AMPK / AMPK Western blot quantification data. Statistical significance is indicated by *p<0.05, meaning significance compared to the control group. Data are expressed as mean ± SD from three repeated experiments. [Figure 6]This paper shows mitochondrial protein expression and glycolytic metabolic changes induced by 1,1-DEE treatment. Mitochondrial protein expression was evaluated using an antibody cocktail targeting five mitochondrial oxidative phosphorylation (OXPHOS) complex proteins. Jurkat cells were treated with 1–5 mM 1,1-DEE and 5 mM 1,2-DEE (a negative control) for 2 hours, after which (A) glucose absorption and (B) changes in lactate production were measured. Jurkat cells were also treated with 1,1-DEE for 2–24 hours. (C) shows representative Western blot results, and (DH) shows the quantification results for OXPHOS complex proteins. (IJ) includes glycolysis analysis, and the expression of glycolytic enzymes HK2 and LDHA was also evaluated after 1,1-DEE treatment. (K) shows the results of RT-PCR analysis of time-dependent HK2 and GLUT1 expression, and (L) and (M) show the quantification of the RT-PCR results. Data are shown as mean ± SEM for three independent experimental results. Statistical significance is indicated as follows: *p<0.05 means comparison with the control group, and #p<0.05 means comparison with 1,1-DEE. [Figure 7] The results of mitochondrial membrane potential changes in Jurkat cells after 1,1-DEE treatment are shown. Jurkat cells were treated with 1-5 mM 1,1-DEE, and 1,2-DEE was used as a negative control. (A) is a representative confocal microscopy image showing the polarization phenomenon of mitochondrial membrane potential evaluated by JC-1 staining (30 minutes) after 1 hour of treatment, at a magnification of 200×. [Figure 8]The results of Jurkat cell proliferation in a xenograft mouse model are shown. (A) 2 × 10⁷ Jurkat cells were inoculated distally into the left leg of mice and subcutaneously injected into the bald thigh, after which tumor growth was monitored for 4 weeks. In the treatment group, 1,1-DEE was administered intratumorally at a dose of 110 mg / kg on days 2, 4, 6, 8, 10, 12, 14, and 16, while the control group was administered PBS. The collected data are as follows: (B) Tumor volume in the treatment and control groups, (C) Rate of increase in tumor volume, (D) Mouse body weight, (E) Kaplan-Meier survival curves of tumor-inoculated mice, and (F) H&E staining results of the heart, liver, spleen, lung, and kidney. A significant difference was observed in the survival curves between the PBS control group and the 1,1-DEE treatment group, and it was statistically significant compared to PBS at p<0.05. [Figure 9] The ROS levels measured at different time intervals using DCFDA staining are shown. The representative flow cytometry histogram shows the time-dependent accumulation of ROS, and is the result of treating Jurkat cells with 5 mM 1,1-DEE at different time intervals. [Figure 10] This study demonstrates the effects of 1,1-DEE on tumor growth and inflammatory cytokine levels in mice. (A) Comparison of tumor size between the control group (PBS) and the 1,1-DEE-treated group; (B) inflammatory cytokine IL-8 levels measured in normal mice, untreated tumor mice, and 1,1-DEE-treated mice; (C) Representative tumor images of PBS-treated and 1,1-DEE-treated mice. Data are expressed as mean ± SEM results from three independent experiments. Statistical significance is indicated by ***p<0.0001, meaning comparison with the normal group (N). [Modes for carrying out the invention]

[0030] The following describes in detail an anticancer agent containing 1,1-diethoxyethane as an active ingredient, based on a specific embodiment of the invention. However, this is presented as an example of the invention and does not limit the scope of the invention's rights. It will be obvious to those skilled in the art that various modifications are possible to the embodiment within the scope of the invention's rights. Unless otherwise specified throughout this specification, "contains" or "includes" means to include a certain component (or constituent) without any limitation and should not be interpreted as excluding the addition of other components (or constituents).

[0031] As used herein, the term “treatment” means any form of therapy or prevention that provides an effect to an individual who is afflicted with a disease or at risk of developing a disease, including an improvement in the individual’s condition, a delay in disease progression, a delay in the onset of symptoms, or a slowing of symptom progression. Therefore, the term “treatment” includes preventive treatment of an individual that prevents the onset of symptoms. Furthermore, “treatment” and “prevention” are not intended to mean a cure or complete elimination of symptoms.

[0032] As used herein, the term “improvement” may mean any action that alleviates or treats a symptom, such as any action that at least reduces the severity of the symptom.

[0033] As used herein, the term “subject” means an animal, including animals such as cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, the subject may be a mammal, in particular a human.

[0034] 1. Anticancer drugs The present invention aims to provide an anticancer agent containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0035] The aforementioned 1,1-DEE can induce the generation of reactive oxygen species (ROS) in cancer cells.

[0036] The aforementioned 1,1-DEE can induce apoptosis in cancer cells.

[0037] The aforementioned 1,1-DEE can induce cell cycle arrest in cancer cells through the depletion of G2 / M phase cells and the accumulation of G1 phase cells, and can reduce the expression of CDK3, CDK4, and cyclin D1, D3, and E proteins.

[0038] The aforementioned 1,1-DEE can activate AMPK signaling and suppress the expression of glycolytic enzymes (LDHA, HK2) and glucose transporters (GLUT1) in cancer cells.

[0039] The aforementioned 1,1-DEE can maintain or promote mitochondrial membrane potential (ΔΨm) and oxidative phosphorylation (OXPHOS) activity.

[0040] The aforementioned 1,1-DEE can reduce the expression of leukemia-related inflammatory biomarker (IL-8).

[0041] The aforementioned anticancer agent can be used for hematological malignancies or solid tumors.

[0042] The aforementioned anticancer agent can be used for leukemia, multiple myeloma, or lymphoma. For example, the anticancer agent can be used for squamous cell carcinoma, basal cell carcinoma, melanoma, tumors of the epithelial lining of glands or ducts, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma of the liver and bile ducts, hepatocellular carcinoma of the gastrointestinal tract, esophageal squamous cell carcinoma, esophageal adenocarcinoma, colorectal cancer, gastric cancer, airway tumors, bronchial carcinoma, small cell carcinoma, transitional cell carcinoma of the urinary tract, transitional cell carcinoma of the bladder, squamous cell carcinoma of the bladder, prostate cancer, cervical cancer, leukemia of blood cells and associated cells, acute and chronic lymphocytic leukemia, polycythemia vera, lymphoid tissue carcinoma, malignant lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, follicular lymphoma, diffuse lymphoma, small cell carcinoma It can be used for cancers selected from the group consisting of mphancytic lymphoma, large cell lymphoma, lymphoblastic lymphoma, multiple myeloma, connective tissue tumors, osteosarcoma, nervous system tumors, neuroblastoma, retinoblastoma, glioblastoma, tumor-forming virus-associated glioma, Burkitt lymphoma, B-cell lymphoma in immunocompetent individuals, nasopharyngeal cancer, esophageal and gastroesophageal cancer, squamous cell carcinoma, islet tumors, breast cancer, lung cancer, colorectal cancer, retinoblastoma, liver cancer, pancreatic cancer, brain cancer, malignant mesothelioma, hepatitis B virus hepatocellular carcinoma, endometrial cancer, ovarian cancer, head and neck cancer, thyroid cancer, and soft tissue-related cancers.

[0043] When the aforementioned anticancer drug is used for leukemia, the leukemia may be a form selected from the group consisting of acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonuclear leukemia, acute minimally differentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, T-cell precursor lymphocytic leukemia, T-cell prolymphoblastic leukemia, Philadelphia chromosome-positive leukemia, FLT3 mutation-positive acute myeloblastic leukemia, and acute erythroleukemia.

[0044] The anticancer agent may be administered alone or in combination with one or more of the following: immunotherapy agents, monoclonal antibodies, chemotherapeutic agents, radioprotective agents, radiotherapeutic agents, and gene therapy agents such as microRNAs. In this case, the immunotherapy agent may include immune barrier inhibitors including PD-1, PD-L1, and CTLA-4 inhibitors; cytokine therapy including interleukins and interferons; CAR-T cell therapy, oncolytic viruses, vaccine therapies, or targeted therapies; the chemotherapeutic agent may include antimetabolists, platinum-based preparations, alkylating agents, or topoisomerase inhibitors; and the radiotherapeutic agent may include external beam radiation therapy, proximal therapy, proton therapy, radiofrequency thermotherapy, stereotactic radiotherapy, or neutron therapy. In particular, the transmission of the anticancer agent may occur before, during, or after administration of one or more known antitumor agents, including, but not limited to, hypomethylating agents, PD-1 inhibitors, PD-L1 inhibitors, mustard compounds, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, floxuridine, methotrexate, vincristine, vinblastine, taxol, etoposide, temiposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitomycin, cisplatin, carboplatin, estramustine, phosphate hydroxyurea, BCNU, procabazine, VM-26, interferon, and all-trans retinoic acid (ATRA) or other retinoids. Suitable hypomethylating agents may include decitabine, guancitabine, and azacitidine. Suitable examples of PD-1 inhibitors include pembrolizumab and nivolumab, and suitable PD-L1 inhibitors include atezolizumab, avelumab, and davalumab. In the anticancer agent according to the present invention, the anticancer agent may include a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition.

[0045] (1) Pharmaceutical composition According to the present invention, the anticancer agent may be used as a pharmaceutical composition for the prevention or treatment of cancer. In the pharmaceutical composition according to the present invention, the pharmaceutical composition can be administered by oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernosal injection, intrathecal injection, epidural injection, and rectal administration. When administered orally, for example, the pharmaceutical composition can be formulated as an uncoated tablet or by coating the active agent or protecting it from degradation in the stomach. The composition may also be administered by any device that allows the active substance to move to target cells. The route of administration can vary depending on the general conditions and age of the patient being treated, the nature of the treatment condition, and the selected active ingredient. In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be supported on a carrier, and the carrier may include, but is not limited to, one or more selected from viral particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, and exosomes. In the pharmaceutical composition according to the present invention, the appropriate dosage of the pharmaceutical composition varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and response sensitivity. A skilled physician can usually easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the pharmaceutical composition can be administered in a single or multiple dose, divided into one to four doses per day. For instance, the pharmaceutical composition may contain 0.01 mg / kg to 100 mg / kg per adult, preferably 0.02 mg / kg to 90 mg / kg, and more preferably 0.03 mg / kg to 80 mg / kg. In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be manufactured in unit volume form or by being contained in a multi-volume container by formulating with pharmaceutically acceptable carriers and / or excipients in a manner readily available to a person with ordinary skill in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may contain additional dispersants or stabilizers. The pharmaceutical composition may also be administered in the form of a suppository, spray, ointment, cream, gel, inhalant or skin patch. Furthermore, the pharmaceutical composition may be manufactured for mammalian administration, more preferably for human administration. In the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier may be a solid or a liquid, and may be one or more selected from excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release regulators, wetting agents, stabilizers, suspending agents, and lubricants. The pharmaceutically acceptable carrier may also be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

[0046] In one specific example, suitable fillers may include, but are not limited to, sugars (e.g., dextrose, sucrose, maltose, and lactose), starch (e.g., corn starch), sugar alcohols (e.g., mannitol, sorbitol, maltitol, erythritol, and xylitol), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose), or mixtures thereof.

[0047] In one specific example, suitable binders may include, but are not limited to, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gums, sucrose, starch, or mixtures thereof.

[0048] In one specific example, suitable preservatives may include, but are not limited to, benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorbutol, gallate, hydroxybenzoate, EDTA, or mixtures thereof.

[0049] In one specific example, suitable disintegrants may include, but are not limited to, sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or mixtures thereof.

[0050] In one specific example, suitable sweeteners may include, but are not limited to, sucralose, saccharin, sodium or potassium or calcium saccharin, acesulfame potassium or sodium cyclamate, mannitol, fructose, sucrose, maltose, or mixtures thereof.

[0051] In one specific example, suitable glidants include, but are not limited to, silica, colloidal silicon dioxide, and talc. In one specific example, suitable lubricants may include, but are not limited to, long-chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyceride waxes, or mixtures thereof.

[0052] (2) Cosmetic composition According to the present invention, the anticancer agent may be used as a cosmetic composition for the prevention or improvement of cancer. In the cosmetic composition according to the present invention, the cosmetic composition may further contain a dermatologically acceptable carrier. The dermatologically acceptable carrier may include, but is not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, hygroscopic agent, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, preservative, lower alcohol, etc., and its type and concentration can vary and can be modified within the scope of the present invention by those skilled in the art. In the cosmetic composition according to the present invention, in addition to the active ingredient of the present invention, the cosmetic composition may optionally contain functional substances such as whitening agents, moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, ultraviolet blocking agents, antibiotics, acne inhibitors, perfumes, and dyes, which can be included in the cosmetic composition according to the present invention in amounts commonly used in the cosmetics field. In order to enhance its functional effect, the cosmetic composition of the present invention may further contain one or more moisturizing active ingredients having the same or equivalent function as the composition of the present invention. In the cosmetic composition according to the present invention, the cosmetic composition can be manufactured in the form of a general emulsifier and a solubilizer. Examples of cosmetics in the emulsifier form include nourishing lotions, creams, and essences, and examples of cosmetics in the solubilizer form include softening lotions. In addition to the active ingredient of the present invention, the cosmetic composition may be manufactured in the form of an auxiliary agent that can be applied topically or systemically and is commonly used in the industry, by containing a dermatologically acceptable medium or base. Suitable cosmetic dosage forms may include, for example, solutions, gels, solids or paste anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, fine granules, or ionic (liposomes) or nonionic camphor dispersants, creams, lotions, powders, ointments, sprays, or conceal sticks. It can also be manufactured in the form of a foam or an aerosol composition further containing a compressed propellant. In the cosmetic composition according to the present invention, the cosmetic composition can be formulated into one or more of the following: a solution, an external ointment, a cream, a foam, a nourishing lotion, a softening lotion, a perfume, a pack, a softening water, an emulsion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.

[0053] (3) Food composition As used herein, the term "food" means a natural product or processed product containing one or more nutrients, preferably one that has undergone some processing steps and is ready to be eaten directly, and can generally include all foods, food additives, functional foods and beverages. As used herein, the terms "functional food" or "health functional food" refer to a group of foods to which added value has been added using physical, biochemical, or biotechnological methods to act on and express the functions of the food in question for a specific purpose, or foods that have been designed and processed to fully express in the body the internal regulatory functions related to the regulation of biological defense rhythms, disease prevention and recovery, etc. Specifically, these may be health functional foods. The above functional foods may contain food-grade acceptable food additives and may further contain appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods. The types of the above health supplements are not limited to these, but may be in the form of powder, granules, tablets, capsules, or beverages. According to the present invention, the anticancer agent may be used as a food composition for the prevention or improvement of cancer. The food composition according to the present invention is characterized in that the food is meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, coffee beverages, energy drinks, alcoholic beverages, or vitamin complexes. In the food composition according to the present invention, the food composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These components can be used independently or in combination. In the food composition according to the present invention, the term "functional food" or "health functional food" means a group of foods to which added value has been added using physical, biochemical, or biotechnological methods to act and express the functions of the food in question for a specific purpose, or a food composition that has been designed and processed to fully express in the body the internal regulatory functions related to the regulation of biological defense rhythms, disease prevention and recovery, etc. Specifically, it can be a health functional food. The functional food may contain food-grade acceptable food additives and may further contain appropriate carriers, excipients, and diluents that are commonly used in the manufacture of functional foods.

[0054] (4) Feed composition According to the present invention, the anticancer agent may be used as a feed composition for the prevention or improvement of cancer. In the feed composition according to the present invention, the feed contains nutrients necessary for animals, such as energy, protein, lipids, vitamins, and minerals, and may be, but is not limited to, plant-based feed such as grains, root fruits, food processing by-products, algae, fiber, oils and fats, starches, gourds, and grain by-products, or animal-based feed such as proteins, inorganic substances, mineral substances, oils and fats, and single-cell proteins. In the feed composition according to the present invention, the feeds include, but are not limited to, powdered feeds, solid feeds, moist pellet feeds, dry pellet feeds, EP (Extruder Pellet) feeds, and live feeds. In the feed composition according to the present invention, the feed composition may include binders, emulsifiers, and preservatives added to prevent deterioration of quality, and the feed composition may also include feed additives. Examples of additives to the feed to increase efficacy include amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, silicates, buffers, extractants, and oligosaccharides. Other materials such as feed mixtures may also be included, but are not limited to these.

[0055] 2. Methods of cancer prevention or treatment The present invention aims to provide a method for preventing or treating cancer, which includes the step of administering an anticancer agent containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient to an individual.

[0056] The aforementioned individuals may include, but are not limited to, humans, cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs.

[0057] The administration route, dosage, and frequency of the aforementioned anticancer agent can be administered to the target in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency can be selected within an appropriate range by a skilled technician. The preferred dosage of the aforementioned anticancer agent in this invention may be in the range of 0.001 mg / kg to 100 mg / kg per day for adults, depending on the patient's condition, weight, sex, age, severity of the patient's condition, and administration route. Administration can be carried out once a day or in several divided doses. Such dosages should not be construed in any way as limiting the scope of this invention.

[0058] The following examples are provided to aid in understanding the present invention. These examples are provided to make the invention easier to understand, and the scope of protection of the invention is not limited to these examples. [Examples]

[0059] <Materials and Methods> 1.Cell culture Jurkat E6-1 (KCLB No: 40152) cells were purchased from the Korean Cell Line Bank (Daehak-ro, Jongno-gu, Seoul, South Korea) and cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator maintained at 37°C and 5% CO2.

[0060] 2. Measurement of cell viability The cells were placed in a 96-well plate containing RPMI medium supplemented with 10% FBS and 1% penicillin & streptomycin, with 2 × 10 cells per well. 4 Cells were smeared at a density of 100 μl. The following day, Sigma-Aldrich (St. Louis, USA) 1,1-DEE and TCI (Tokyo Chemical Industry Co. Ltd, Japan) 1,2-DEE were sequentially diluted and added to each well for 24 hours (total 120 μl). Subsequently, 10 μl of MTT (DoGenBio Co., Ltd.) was added to each well of the plate, and the cells were cultured at 37°C for 2 hours. Cell viability was analyzed by measuring absorbance at 450 nm using a microplate spectrophotometer (Epoch, Biotek, USA).

[0061] 3. Western Blot Jurkat cells (5×10) cultured in a 60×15mm cell culture dish (SPL Life Sciences, Gyeonggi-do, Republic of Korea) 5 To extract proteins from ( / mL), iNtRON Biotechnology (Gyeonggi-do, South Korea) uses Pro-PREP TMProtein extraction solution was added to each plate in 150 μL portions. Materials such as polyvinylidene fluoride (PVDF) membranes and Western chemiluminescent HRP substrates were purchased from Millipore Corporation (Billerica, MA, USA). After separating 30 μg of total protein, it was separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to PVDF membranes for further analysis. The membranes were blocked for 1-2 hours in TBST containing 5% skim milk with 0.1% Tween-20, and then incubated overnight at 4°C with primary antibody (1:1000) added to the TBST. After washing three times with TBST for 10 minutes each, horseradish peroxidase-conjugated secondary antibody (1:2000) was used to detect immunoreactive proteins via chemiluminescence. The following antibodies, provided by Cell Signaling Technology (Danvers, MA, USA), were used for the Western blot experiments: anti-phospho-AMPK (#2535S), anti-AMPK (#2532S), anti-Bax (2772S), anti-Cyclin D1 (#29226S), anti-Cyclin D3 (#2936S), anti-HXK II (B-8, SC374091), anti-LDHA (E-9, SC137243), anti-Bcl2 (SC509), anti-Cyclin E (SC377100), anti-Caspase3 (SC373730), anti-Caspase9 (SC56073), anti-CDK2 (SC-6248), anti-CDK4 (SC-23896), and Total OXPHOS. Cocktail (Abcam, 110411) and anti-β-actin (5125S) monoclonal antibody. To measure total protein concentration, the blotted membranes were washed with Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific, Meridian Rd., Rockford, IL, USA) at 56°C for 30 minutes.

[0062] 4. Measurement of Apoptosis (Cell Apoptosis) - Annexin V / FITC Assay To confirm the potential for apoptosis in Jurkat cells, the Annexin V fluorescence kit (BD Pharmingen, San Diego, USA) was used according to the manufacturer's protocol. Cells were placed in a 6-well plate containing RPMI medium with 10% FBS, at a rate of 2.0 × 10⁴ cells per well. 5 Cells were dispensed at the specified cell density for 24 hours. After dispensing, they were treated with the desired concentration of 1,1-DEE and cultured at 37°C for 24 hours. After treatment, the cells were harvested, pelletized, resuspended in 400 μl of binding buffer, and stained with 5 μl of FITC-Annexin-V and 10 μl of PI as provided in the kit. Subsequently, cells were analyzed for flow cytometry using a BD FACS Calibur (BD Biosciences, CA, USA). Data plotting and analysis were performed using FlowJo TM The procedure was performed using software (BD Biosciences, CA, USA).

[0063] 5. Cell cycle analysis Jurkat cells were placed in a 6-well plate at a rate of 5 × 10⁶ cells per 1 ml. 5 Cells were aliquoted and cultured at 37°C for 24 hours, then treated with 1,1-DEE at the desired concentration for 24 hours. Cells were separated using trypsin-EDTA, centrifuged, washed with 1× cold PBS, and fixed in 70% ethanol at 4°C for 30 minutes. To prepare cells for analysis, they were washed with 1× PBS and centrifuged at 1,200 rpm for 5 minutes. The cells were then placed in a 500 μl solution containing RNase A (Sigma-Aldrich) 10 μg / ml and propidium iodide (Sigma-Aldrich) 75 μM and cultured in a dark room at room temperature for 1 hour. After detecting DNA fluorescence intensity using flow cytometry with BD FACS Verse, FlowJo TM We used software to plot and analyze the data.

[0064] 6.ROS generation measurement The intracellular H2O2 level was measured using 5- and 6-chloromethyl 2’,7’-dichlorodichlorofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR, USA). Jurkat cells were cultured in RPMI supplemented with 10% FBS until they reached 80% confluence. To evaluate the ROS generation effect activated by 1,1-DEE, the cells were treated with 2 mM NAC, 250 μM Trolox, and 10 μM Ebsleen 1 hour before compound treatment. Then, the cells were cultured with 10 μM DCFDA for 15 minutes and then observed using a laser scanning confocal microscope (Carl Zeiss, Jena, Germany). DCFDA fluorescence was excited at 488 nm using an argon laser, and the emission was captured with a long-pass filter at 515 nm. After culturing with DCFDA, the cells were analyzed for ROS by flow cytometry using a BD FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA). The data were plotted and analyzed using FlowJo TM software version 10.10 (BD Biosciences, San Jose, CA, USA).

[0065] 7. Animal management and drug administration Four-week-old immunodeficient male mice (NOD.Cg-PrkdcscidIL2rgtm1Wjl / Szj, NSG) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed at the Experimental Animal Resource Center of Chonnam National University (CNU IACUC-H-2024-42) in Korea. The mice were housed at 23°C with 60% humidity on a 16-hour light / 8-hour dark cycle and provided with food and water until the experiment proceeded. Five mice were housed per cage, and their body weights were measured three times a week. All experiments were conducted in accordance with the institutional guidelines of Chonnam National University. Jurkat cells cultured in RPMI medium containing 10% FBS were resuspended in 100 μl of PBS at 2 × 10 7The drug was subcutaneously injected into the right flank of each mouse at the specified cell concentration. The mice were divided into a control group and a treatment group. Four weeks after tumor induction, the treatment group received intratumoral injections of 1,1-DEE (110 mg / kg) every other day from day 2 to day 16. The control group received injections of PBS. Eighteen days after drug administration, the animals were used for survival studies. Tumor tissue and major organs were also processed for histological analysis by H&E staining.

[0066] 8. Enzyme-Linked Immunosorbent Assay (ELISA) In in-vivo studies, plasma was collected from blood samples obtained via cardiac puncture of anesthetized NSG mice. Cytokine (IL-8) levels were measured using an ELISA kit (R&D Systems, USA) with a microplate spectrophotometer (Epoch) and a microplate strip washer (ELx50, BioTek Instruments, USA). The optical density of IL-8 concentration was calculated to create a standard curve, which was expressed in pg / ml.

[0067] 9. Measurement of lactic acid production (Lactate Production Assay) Jurkat cells were placed in a 96-well plate at a rate of 1 × 10⁶ cells per well. 4 Cells were dispensed at a concentration of 100 μl. Once the cells reached confluence, they were treated with 1,1-DEE 1,2,5 mM and 1,2-DEE 5 mM for 2 hours. Subsequently, the culture medium and cells were collected separately and subjected to Lactate-Glo according to the manufacturer's protocol. TM Lactic acid production was measured using an Assay (Promega, USA). Luminescence was measured using a GloMax plate reading photometer (Promega, USA).

[0068] 10. Measurement of glucose absorption (Glucose Uptake Assay) Jurkat cells were placed in a 96-well plate at a rate of 1 × 10⁶ cells per well. 4 Cells were dispensed in 100 μl portions. Once the cells reached confluence, they were treated with 1,1-DEE 1,2,5 mM and 1,2-DEE 5 mM for 2 hours. Glucose uptake was measured according to the manufacturer's guidelines. TMThe evaluation was performed using an Assay (Promega, USA). Luminescence was measured using Glucose Uptake-Glo TM GloMax with an integration time of 0.5 seconds according to the protocol. (登録商標) The data was recorded using a luminometer (Promega, USA).

[0069] 11. Polymerase Chain Reaction (RT-PCR) Total RNA extraction from HT-29 cells was performed using TRIzol reagent (Invitrogen, Carlsbad, USA). Primary complementary DNA (cDNA) was synthesized using 1 μg of total RNA with random primers and M-MLV transcriptase (Promega, USA). cDNA was amplified using PCR master mix solution (iNtRON, Korea) with primer sets for β-actin and IL-6. The primers used are as follows:

[0070] β-actin forward:5'-AAG CAG GAG TAT GAC GAG TC-3' β-actin reverse:5'-GCC TTC ATA CAT CTC AAG TT-3'(561 bp) LDHA forward:5'-CAC CAT GAT TAA GGG TCA TTA C-3' LDHA reverse:5'-AGG TCA GAG ATT CCA TTC TG-3'(87 bp) HK2 forward:5'-GAG CCA CCA CTC ACC CTA CT-3' HK2 reverse:5'-CCA GGC ATT CGG CAA TGT G-3'(249 bp) GLUT1 forward:5'-AGT TCT ACA ACC AGA CAT GG-3' GLUT1 reverse:5'-CAG GTT CAT CAT CAG CAT TG-3'(179 bp) The PCR procedure involved denaturation at 94°C for 30 seconds, annealing at 54°C for 20 seconds, and extension at 72°C for 30 seconds.

[0071] 12.Histological analysis (H&E staining, Histology) After measuring tumor volume and weight, animals were sacrificed for histological study. The tissue was dehydrated twice in 95% ethanol for 0.5 hours, immersed in xylene at 60-70°C for 1 hour, and then paraffin embedded for 12 hours. 6 μm thick frozen tissue sections were prepared, fixed with frozen section solution (FSC 22 Clear, Leica). The tissue sections were stained with Harris' hematoxylin solution at 60-70°C for 6 hours and rinsed with tap water until colorless. Next, a mixture of 10% acetic acid and 85% ethanol was applied twice each at 2 and 10 hours, respectively, followed by rinsing the tissue with tap water to induce differentiation. For blue staining, the sections were immersed in saturated lithium carbonate solution for 12 hours and then rinsed again with tap water. Finally, counterstaining was performed with eosin Y ethanol solution for 48 hours.

[0072] 13. Measurement of mitochondrial membrane potential (JC-1 staining, flow cytometry) Mitochondrial membrane potential was measured using JC-1 dye (Beyotime, Seoul, Korea). Jurkat cells were cultured for 24 hours, treated with the indicated drug concentration, and then cultured with JC-1 (20 nM) at 37°C in the dark for 30 minutes. After washing with PBS, cells were aliquoted into confocal dishes (Carl Zeiss, Germany), imaged or trypsinized, and collected for analysis using flow cytometry with BD FACS Calibur (BD Biosciences, USA). Data were processed using FlowJo™ software (BD Biosciences, USA).

[0073] 14.Statistical Analysis Each data point represents three separate experiments and is expressed as mean ± standard deviation (SD). Results were presented using GraphPad Prism software (Version 8.0). For multivariable analyses, ANOVA with Tukey's multiple comparison test was used, and p<0.05 (#,*,^,+,@), p<0.01 (##,**,^^,++,@@), p<0.001 (###,***,^^^,+++,@@@), and p<0.0001 (####,****,^^^^,++++,@@@@) were considered statistically significant. The Student t-test was used for the analysis of tumor volume and weight, and the Log-Rank test was used for the comparison of survival rates. The significance level for all tests was p<0.05.

[0074] <Result> Experimental Example 1. Evaluation of whether 1,1-DEE treatment inhibits cell growth and induces apoptosis in ALL cell lines. To confirm the antiproliferative and apoptosis-inducing effects of 1,1-DEE, the JurkatE6.1 cell line was used for evaluation. MTT analysis showed that 1,1-DEE reduced the viability of JurkatE6.1 cells in a concentration-dependent manner, even at concentrations below 10 mM (Figures 1A and 1B). On the other hand, 1,2-DEE was used as a negative control group, and no significant changes in cell viability were observed (Figures 1C and 1D).

[0075] To evaluate the low-dose toxicity of 1,1-DEE, JurkatE6.2 cells were treated with various concentrations of 1,1-DEE for 6, 12, and 24 hours. As a result, JurkatE6.2 cells exhibited a rounded and contracted morphology, and such morphological changes were observed more clearly as the concentration and treatment time of 1,1-DEE increased (Figure 1E). In contrast, no such morphological changes were observed in the 1,2-DEE-treated group (Figure 1E). In particular, changes of approximately twofold were observed after 6-hour and 12-hour treatments, and a strong inhibitory effect of approximately threefold was confirmed after 24-hour treatment.

[0076] To evaluate the presence or absence of apoptosis induction, JurkatE6.1 cells were cultured with 1,1-DEE for 24 hours and then stained with Annexin V and PI (Figure 2A). Analysis revealed that 1,1-DEE treatment significantly increased apoptosis, and the proportion of cells in the Q2 interval increased in a concentration-dependent manner (Figure 2B).

[0077] Furthermore, Western blot analysis revealed that 1,1-DEE treatment increased the expression of Bax, an apoptosis-promoting protein, while decreasing the expression of Bcl-2, caspase-9, and caspase-3, which are apoptosis-inhibiting proteins. On the other hand, no significant changes were observed in the groups treated with Compound C (10 μM, AMPK inhibitor) or 1,2-DEE, which were used as positive control groups, compared to the untreated control group (Figure 2C, Figure 2D).

[0078] Therefore, the above results confirm that 1,1-DEE treatment induces apoptosis in JurkatE6.1 cells and exhibits an inhibitory effect on cell proliferation.

[0079] Experimental Example 2.1: Evaluation of whether 1-DEE treatment induces cell cycle arrest via ROS generation in ALL cell lines. It is known that increased intracellular concentrations of reactive oxygen species (ROS) induce oxidative stress, leading to cell cycle arrest and apoptosis in cancer cells. Furthermore, ROS have been reported to disrupt the intracellular redox balance, increase the expression of CDK inhibitory proteins such as p21 and p27, and arrest the cell cycle at the G0 / G1 or G2 / M stage. Therefore, this study evaluated whether 1,1-DEE treatment induces ROS generation, induces cell cycle arrest, and suppresses tumor growth.

[0080] First, we analyzed cell cycle changes induced by 1,1-DEE treatment using JurkatE6.1 cells. JurkatE6.1 cells were treated with 1-10 mM 1,1-DEE for 24 hours, and the cell cycle was analyzed by flow cytometry. As a result, in the 1,1-DEE-treated group, a decrease in the proportion of G2 / M cells and an accumulation of G1 cells were observed (Figures 3A and 3B).

[0081] Western blot analysis confirmed that 1,1-DEE treatment reduced the expression of CDK2, CDK4, cyclin D1, cyclin D3, and cyclin E proteins. In contrast, no significant changes were observed in the positive control groups treated with cytochrome c (10 μM) or 1,2-DEE compared to the untreated control group (Figures 3C and 3D). In particular, significant accumulation of G1 stage cells was observed even with the lowest concentration of 1 mM 1,1-DEE, confirming that 1,1-DEE induces cell cycle arrest in a concentration-dependent manner.

[0082] ROS accumulation is known to play a crucial role in the regulation of cell proliferation and apoptosis. Therefore, in this study, the degree of ROS accumulation after 1,1-DEE treatment was evaluated using the DCFH-DA fluorescence probe. The results showed that clear ROS accumulation was induced within 30–60 minutes of 1,1-DEE (5 mM) treatment (Figure 9).

[0083] Furthermore, to confirm whether ROS accumulation induced by 1,1-DEE treatment plays an important role in inhibiting cell proliferation, cell viability was evaluated after treating cells with 1,1-DEE together with ROS scavengers (NAC, Trolox, Ebselon) or ROS inducers (H2O2) (Figure 4A). In the absence of NAC, 1,1-DEE treatment induced ROS accumulation and inhibited cell proliferation. On the other hand, in the presence of NAC, 1,1-DEE-induced ROS accumulation decreased, and it was confirmed that this increased cell viability (Figures 4C, 4D).

[0084] Therefore, these results confirm that ROS generation plays a crucial role in the cell proliferation inhibitory effect of 1,1-DEE.

[0085] Experimental Example 3.1: Evaluation of whether 1-DEE treatment suppresses the Warburg effect in ALL cells by regulating AMPK signaling. AMPK (AMP-activated protein kinase) is a conserved serine / threonine phosphorylation enzyme that serves as a central factor in regulating intracellular energy homeostasis. AMPK activation is known to alter the bioenergetic properties of tumor cells by modulating major signaling pathways that induce metabolic rearrangement in tumor cells. Saito et al. reported that AMPK plays a crucial role in maintaining the viability of leukemia cells in the bone marrow by inhibiting glucose transporter (GLUT)-mediated glucose absorption and promoting ROS accumulation. On the other hand, genetic deficiencies in AMPK are known to result in leukemia cells becoming more sensitive to metabolic stress due to impaired glucose utilization.

[0086] Therefore, in this study, we evaluated whether 1,1-DEE activates AMPK using JurkatE6.1 cells. As shown in Figures 5A and 5B, 1,1-DEE treatment activated AMPK in a time-dependent manner, and a significant increase in AMPK activity was observed 60 minutes after treatment. Furthermore, 1,1-DEE induced AMPK phosphorylation in a concentration-dependent manner (Figures 5C and 5D), with the most pronounced activation observed at a concentration of 5 mM.

[0087] On the other hand, tumor cells are known to exhibit metabolic reprogramming, a phenomenon in which they rewire nutrient utilization pathways to secure the energy necessary for growth. In particular, cancer cells have been reported to exhibit the Warburg effect, converting glucose to lactate even in the presence of sufficient oxygen. Therefore, this study evaluated whether 1,1-DEE-mediated AMPK activation can suppress the Warburg effect.

[0088] As a result, when JurkatE6.1 cells were treated with 1,1-DEE at concentrations of 1 mM, 2 mM, and 5 mM, AMPK was activated while glucose absorption was significantly reduced (p<0.05) (Figure 6A). In particular, a clear inhibitory effect was observed even at the lowest concentration of 1 mM, confirming that 1,1-DEE exhibits a potent and concentration-dependent inhibitory effect on glycolytic activity. Furthermore, when JurkatE6.1 cells were treated with 1,1-DEE at concentrations of 2 mM and 5 mM, lactate production was significantly reduced, suggesting inhibition of the glycolytic metabolic flux (Figure 6B).

[0089] Therefore, these results confirm that 1,1-DEE treatment suppresses the Warburg effect by promoting AMPK phosphorylation, and that upward regulation of AMPK alone can induce a decrease in intracellular glycolytic activity.

[0090] Experimental Example 4.1: Evaluation of whether 1-DEE treatment modulates AMPK and alters membrane potential and glycolytic properties in ALL cells. Aerobic glycolysis in cancer cells is known to reduce oxidative phosphorylation (OXPHOS) activity by primarily utilizing a metabolic pathway that converts pyruvate to lactate without transferring it to the mitochondrial TCA cycle. Such metabolic reversals limit ROS production within mitochondria and maintain mitochondrial structural stability, while titration-level ROS play a role in tumor progression and survival. Therefore, targeting rate-limiting enzymes in glucose absorption and glycolysis, as well as mitochondrial oxidative phosphorylation, is considered a strategic approach to suppress the Warburg effect and block metabolic reprogramming in tumor cells.

[0091] Therefore, this study aimed to determine whether 1,1-DEE exerts an anti-Warburg effect by regulating the expression and activity of such glycolysis-related factors.

[0092] First, to evaluate the effect of 1,1-DEE on mitochondrial OXPHOS activity, JurkatE6.1 cells were treated with 1,1-DEE (2 mM), and Western blot analysis was performed using an OXPHOS antibody cocktail containing antibodies against cytochrome c oxidase subunit 2 (CO2) encoded in mitochondrial DNA (mtDNA) and four polypeptides encoded in nuclear genes (NDUFB8, SDHB, UQCRC2, ATP5A) (Figures 6C-6H). As a result, the expression of these complex-specific proteins increased in a time-dependent manner in the 1,1-DEE-treated group, suggesting improved mitochondrial respiratory activity compared to the untreated control group. These results indicate that 1,1-DEE promotes mitochondrial oxidative phosphorylation and enhances the bioenergetic function of cells.

[0093] Furthermore, to evaluate whether 1,1-DEE directly regulates the expression of the glucose transporter GLUT1, JurkatE6.1 cells were treated with 1,1-DEE (2 mM) for 4 hours, and GLUT1 protein levels were analyzed. As a result, GLUT1 expression was significantly reduced (Figure 7I-7K), indicating that 1,1-DEE inhibits GLUT1 expression at both the transcriptional and translational levels. Therefore, it was concluded that 1,1-DEE also effectively suppresses GLUT1-mediated glycolysis.

[0094] On the other hand, overexpression of LDHA (lactate dehydrogenase A) and HK2 (hexokinase 2) is known to be closely associated with high lactate production, increased tumor invasiveness, and resistance to anticancer drugs and radiotherapy. In this study, it was confirmed that LDHA and HK2 protein expression was significantly reduced when treated with 1,1-DEE (2 mM) for 4 hours compared to the untreated control. This suggests that 1,1-DEE effectively suppresses a major regulator of glycolysis and inhibits the aerobic glycolytic activity of cancer cells.

[0095] Mitochondrial membrane potential (ΔΨm) is known as an important indicator for evaluating cell viability and function. To assess this, the JC-1 staining method was used. JC-1 is a cationic cyanine dye, and its property of converting from a green monomer to a red fluorescent aggregate when the mitochondrial membrane potential is high allows for fluorescence-based analysis of the depolarization or hyperpolarization state of intracellular mitochondria.

[0096] The results showed that when JurkatE6.1 cells were treated with 1,1-DEE, the red fluorescence ratio increased in a concentration-dependent manner, indicating improved mitochondrial polarization. On the other hand, in the 1,2-DEE-treated group, the red / green fluorescence ratio was balanced, and no significant changes in membrane potential were observed. In particular, strong red fluorescence was predominantly observed in the 1,1-DEE-treated group (Figure 7), suggesting that 1,1-DEE supports mitochondrial function by maintaining or enhancing mitochondrial membrane potential.

[0097] Experimental Example 5.1: Evaluation of whether ALL cell proliferation is suppressed in vivo by treatment with 1-DEE. To further confirm whether 1,1-DEE inhibits tumor growth in vivo, a subcutaneous tumor model was constructed using 4-week-old NSG mice. Jurkat cells were stably subcutaneously injected, and tumor volume was measured 4 weeks after injection. Subsequently, 1,1-DEE at the desired concentration was injected directly into the tumor site at 2-day intervals for a total of 8 times, and changes in tumor volume were continuously observed.

[0098] As a result, we confirmed that 1,1-DEE significantly suppressed tumor growth in vivo (Figure 8A). Furthermore, when comparing the tumor growth curves of each group, tumor volume was significantly reduced in the 1,1-DEE-treated group 18 days after cell injection (Figures 8B, C). Simultaneously, the body weight of the mice was measured (Figure 8D), and tumor size and survival rate were compared in both the saline-treated group and the 1,1-DEE-treated group after 18 days. The survival curves for each group are shown in Figure 8E, and Kaplan-Meier survival analysis showed that the 1,1-DEE-treated group had a significantly increased survival period compared to the untreated group.

[0099] Subsequently, detailed histopathological examinations were performed 18 days after 1,1-DEE treatment to assess potential long-term toxicity. Pathological changes in major organs such as the heart, liver, kidneys, spleen, and lungs were evaluated, and representative microscopic images are shown in Figure 8F.

[0100] Hepatocytes from mice with solid tumors showed clear morphological changes compared to those from normal mice. In the liver tissue of normal mice, the central vein and portal vein were regularly arranged, whereas in tumor-bearing mice, lymphocyte infiltration and irregular central vein and portal vein structures were observed. However, in tumor-bearing mice treated with 1,1-DEE, the slightly disrupted liver tissue structure was restored, with the central vein and portal vein being properly maintained and cell infiltration minimized.

[0101] In kidney tissue, acute vacuolation, epithelial wall swelling, nuclear degeneration, necrosis, and epithelial degeneration were observed. In cardiac tissue, chemodectoma, toxic myocarditis, reddish-brown atrophy, and yellowish-brown pigmentation were observed, which are thought to correspond to lipofuscin granules, presumably the residues of organelles and cytoplasmic material. In lung tissue, vacuolation, central vein degeneration, inflammation, hemorrhage, deformed cellular structures, hemosiderophages, and lesions were observed.

[0102] Therefore, the results of this study showed that 1,1-DEE treatment restores liver tissue structure similar to that of normal animals. Furthermore, H&E staining of solid tumor tissue revealed that the cardiac tissue of the 1,1-DEE-treated group maintained a normal tissue structure, while the cardiac tissue of tumor-carrying mice showed an irregular structure.

[0103] Recent studies have shown that IL-8 is a key biomarker associated with disease progression and poor prognosis in various leukemias. Meanwhile, AMPK activation has been reported to suppress IL-8 expression through NF-κB inhibition. Therefore, this study investigated whether 1,1-DEE can suppress IL-8 expression and mitigate leukemia-related inflammatory responses by activating AMPK (Figure 10). ELISA analysis revealed increased IL-8 expression in control mice, but significantly decreased IL-8 expression in the 1,1-DEE-treated group.

[0104] As a result, this study demonstrates that 1,1-DEE suppresses the Warburg effect by increasing ΔΨm (mitochondrial membrane potential) and promoting OXPHOS activity, while simultaneously reducing the expression of major glycolytic enzymes, including LDHA and HK2, thereby suppressing the glycolytic flux. Due to these time- and concentration-dependent metabolic regulatory effects, 1,1-DEE exhibits potent anticancer activity and can be presented as a promising therapeutic candidate targeting metabolic regulation.

[0105] Having described in detail the specific parts of the present invention above, it will be clear to those with ordinary skill in the art that these specific technologies are merely preferred examples and that the scope of the invention is not limited thereto. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

[0106] National research and development projects that supported this invention [Issue Number] 2018R1D1A1B06051438 [Department Name] Department of Science, Technology and ICT [Project Management (Specialized) Institution Name] Korea Research Foundation [Research Project Title] Regulation of the tumor suppressor protein PTEN by alcohol [Project Implementation Organization] Chonnam National University

Claims

1. An anticancer drug containing 1,1-diethoxyethane (1,1-DEE) as the active ingredient.

2. The anticancer agent according to claim 1, characterized in that the 1,1-DEE induces the generation of reactive oxygen species (ROS) in cancer cells.

3. The anticancer agent according to claim 1, characterized in that the 1,1-DEE induces apoptosis in cancer cells.

4. The anticancer agent according to claim 1, characterized in that 1,1-DEE induces cell cycle arrest in cancer cells through depletion of G2 / M phase cells and accumulation of G1 step cells, and reduces the expression of CDK3, CDK4 and cyclin D1, D3, and E proteins.

5. The anticancer agent according to claim 1, characterized in that it is used for hematological malignancies or solid tumors.

6. The aforementioned anticancer agents are used to treat squamous cell carcinoma, basal cell carcinoma, melanoma, tumors of the epithelial lining of glands or ducts, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma of the liver and bile ducts, hepatocellular carcinoma of the gastrointestinal tract, esophageal squamous cell carcinoma, esophageal adenocarcinoma, colorectal cancer, gastric cancer, airway tumors, bronchial carcinoma, small cell carcinoma, transitional cell carcinoma of the urinary tract, transitional cell carcinoma of the bladder, squamous cell carcinoma of the bladder, prostate cancer, cervical cancer, leukemia of blood cells and associated cells, acute and chronic lymphocytic leukemia, polycythemia vera, lymphoid tissue carcinoma, malignant lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, follicular lymphoma, diffuse lymphoma, small lymphocytic lymphoma, The anticancer agent according to claim 1, characterized in that it is used for cancers selected from the group consisting of large cell lymphoma, lymphoblastic lymphoma, multiple myeloma, connective tissue tumors, osteosarcoma, nervous system tumors, neuroblastoma, retinoblastoma, glioblastoma, tumor-forming virus-associated glioma, Burkitt lymphoma, B-cell lymphoma in immunocompetent individuals, nasopharyngeal cancer, esophageal and gastroesophageal cancer, squamous cell carcinoma, islet tumors, breast cancer, lung cancer, colorectal cancer, retinoblastoma, liver cancer, pancreatic cancer, brain cancer, malignant mesothelioma, hepatitis B virus hepatocellular carcinoma, endometrial cancer, ovarian cancer, head and neck cancer, thyroid cancer, and soft tissue-related cancers.

7. The anticancer agent according to claim 1, characterized in that, when the anticancer agent is used for leukemia, the leukemia is a form selected from the group consisting of acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonuclear leukemia, acute differentially differentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell precursor lymphocyte leukemia, T-cell prolymphoblastic leukemia, Philadelphia chromosome-positive leukemia, FLT3 mutation-positive acute myeloblastic leukemia, and acute erythroleukemia.

8. The aforementioned anticancer agent is administered in combination with one or more of the following: immunotherapy agents, monoclonal antibodies, chemotherapeutic agents, radioprotective agents, radiotherapy agents, and gene therapy agents. The immunotherapy agents include immune barrier inhibitors comprising PD-1, PD-L1, and CTLA-4 inhibitors; cytokine therapies comprising interleukins and interferons; CAR-T cell therapy, oncolytic viruses, vaccine therapies, or targeted therapies; The aforementioned chemotherapeutic agent comprises an antimetabolite, a platinum-based preparation, an alkylating agent, or a topoisomerase inhibitor. The anticancer agent according to claim 1, characterized in that the radiotherapy agent includes external beam radiation therapy, proximal radiation therapy, proton therapy, radiofrequency thermotherapy, stereotactic radiotherapy, or neutron therapy.

9. The anticancer agent according to claim 1, wherein the anticancer agent is for the purpose of preventing the onset of cancer.

10. The anticancer agent according to claim 1, wherein the anticancer agent exhibits an energy-restoring effect during the course of cancer treatment.

11. The anticancer agent according to claim 1, wherein the anticancer agent is used as a pharmaceutical composition for the prevention or treatment of cancer.

12. The anticancer agent according to claim 11, characterized in that the pharmaceutical composition is administered by one or more routes of administration from among oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal mucosa administration, transdermal administration, ocular administration, inhalation, intracavernosal injection, intrathecal injection, epidural injection, and rectal administration.

13. The anticancer agent according to claim 1, wherein the anticancer agent is used as a cosmetic composition for the prevention or improvement of cancer.

14. The anticancer agent according to claim 13, characterized in that the cosmetic composition is prepared in one or more dosage forms from among a solution, topical ointment, cream, foam, nourishing lotion, softening lotion, perfume, pack, softening lotion, emulsion, makeup base, essence, soap, liquid cleanser, bath additive, sunscreen cream, sunscreen oil, suspension, emulsion, paste, gel, lotion, powder, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, patch, and spray.

15. The anticancer agent according to claim 1, wherein the anticancer agent is used as a food composition for the prevention or improvement of cancer.

16. The anticancer agent according to claim 15, characterized in that the food includes meat, sausage, bread, chocolate, candy, snacks, cookies, pizza, ramen and other noodles, chewing gum, ice cream and dairy products, various soups, beverages, tea, coffee beverages, energy drinks, alcoholic beverages, or vitamin complexes.

17. The anticancer agent according to claim 1, wherein the anticancer agent is used as a feed composition for the prevention or improvement of cancer.

18. The anticancer agent according to claim 17, characterized in that the feed comprises powdered feed, solid feed, wet pellet feed, dry pellet feed, extruded pellet (EP) feed, or raw feed.

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