Anticancer agent comprising 1,1-diethoxyethene as active ingredient

Abstract

The present invention relates to an anticancer agent comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

Description

Anticancer drugs containing 1,1-diethoxyethane as an active ingredient

[0001] The present invention relates to an anticancer agent comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0002] Leukemia is one of the most common cancers in children and adolescents, accounting for about one-third of all diagnosed cancer cases [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, the prognosis was poor compared to B-ALL, but with advancements in treatment, it is showing improved results, with the event-free survival (EFS) reaching about 85% [4-6].

[0003] Leukemia is complex to manage because it can metastasize to various tissues [7]. Therefore, a better understanding of the complexity and mechanisms of anti-tumor immunity can contribute to the development of tumor immunotherapy and cancer biology [8]. In the development of cancer therapeutics, it is important to evaluate specificity for inducing cancer cell apoptosis [9]. While the approach of killing lymphoma cells is a unique therapeutic strategy, existing apoptosis inducers can cause serious side effects due to their heavy metal content. Therefore, it is essential to discover new apoptosis inducers with higher efficacy and lower toxicity [10, 11]. Oxidative stress is a major mediator of lymphocyte apoptosis, and reactive oxygen species (ROS) are essential for various intracellular functions, specifically targeting polyunsaturated fatty acids abundant in actively proliferating lymphocytes. Excessive production of ROS is suggested to promote apoptosis through mitochondrial damage, which activates signaling networks that induce cell cycle arrest, DNA repair, and apoptosis. Changes in cyclin expression play a crucial role in regulating cell growth and cancerous transformation.

[0004]

[0005] Prior art literature

[0006] [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.

[0007] [비특허문헌 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.

[0008] [비특허문헌 3] Caracciolo, D., et al., The emerging scenario of immunotherapy for T-cell Acute Lymphoblastic Leukemia: advances, challenges and future perspectives. Experimental Hematology & Oncology, 2023. 12(1): p. 5.

[0009] [비특허문헌 4] Raetz, E.A. and D.T. Teachey, T-cell acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program, 2016. 2016(1): p. 580-588.

[0010] [비특허문헌 5] Zhong, F., et al., Hirsutanol A inhibits T-acute lymphocytic leukemia Jurkat cell viability through cell cycle arrest and p53-dependent induction of apoptosis. Exp Ther Med, 2021. 22(1): p. 741.

[0011] [비특허문헌 6] Sheykhhasan, M., H. Manoochehri, and P. Dama, Use of CAR T-cell for acute lymphoblastic leukemia (ALL) treatment: a review study. Cancer Gene Therapy, 2022. 29(8): p. 1080-1096.

[0012] [비특허문헌 7] Basaiyye, S.S., et al., Molecular mechanism of apoptosis induction in Jurkat E6-1 cells by Tribulus terrestris alkaloids extract. J Tradit Complement Med, 2018. 8(3): p. 410-419.

[0013] [비특허문헌 8] Rostami, F., et al., PDL1 targeting by miR-138-5p amplifies anti-tumor immunity and Jurkat cells survival in non-small cell lung cancer. Scientific Reports, 2024. 14(1): p. 13542.

[0014] [비특허문헌 9] Kumar, N., et al., Comparison of cell-based assays to quantify treatment effects of anticancer drugs identifies a new application for Bodipy-L-cystine to measure apoptosis. Scientific Reports, 2018. 8(1): p. 16363.

[0015] [Non-patent Document 10] Zhu, XL, et al., Inhibitory effect of Embelin on human acute T cell lymphoma Jurkat cells through activation of the apoptotic pathway. Oncol Lett, 2015. 10(2): p. 921-926.

[0016] [Non-patent Document 11] Sun, YL, et al., A novel Bcl-2 inhibitor, BM-1197, induces apoptosis in malignant lymphoma cells through the endogenous apoptotic pathway. BMC Cancer, 2019. 20(1): p. 1.

[0017] Cancer is characterized by abnormal cell proliferation, and cell cycle regulation is mediated by cyclins, particularly cyclin-dependent kinases (CDKs) that regulate G1-S transition and G2 progression. These cell cycle regulatory mechanisms play a crucial role in the proliferation and survival of cancer cells, requiring therapeutic strategies that target them.

[0018]

[0019] As a result of continuous research to develop compounds with anticancer effects through the inhibition of cancer cell growth and the induction of apoptosis, the inventors confirmed that 1,1-diethoxyethane (1,1-DEE) exhibits a potent apoptotic effect in T-lymphoblastic leukemia cell lines (Jurkat E6.1) (Experimental Example 1). Specifically, upon treatment with 1,1-DEE, changes in cell morphology to become round and contracted were observed, and Annexin V / PI staining and Western blot analysis revealed an increase in Bax protein expression and a decrease in the expression of Bcl-2, caspase-3, and caspase-9. On the other hand, these effects were not observed in the group treated with 1,2-DEE, an isomer of 1,1-DEE.

[0020] In addition, it was demonstrated that 1,1-DEE induces the generation 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 in the G0 / G1 phase (Experimental Example 2). It was confirmed that apoptosis and cell cycle arrest were inhibited upon pretreatment with ROS scavengers (NAC, Trolox, Ebselen), suggesting that the anticancer action of 1,1-DEE is related to a ROS-mediated mechanism.

[0021] Furthermore, it was confirmed that 1,1-DEE activates AMPK signaling to regulate metabolic reprogramming in cancer cells, reduces the expression of glycolytic enzymes (LDHA, HK2) and glucose transporter (GLUT1) by inhibiting the Warburg effect, and maintains mitochondrial oxidative phosphorylation (OXPHOS) activity (Experimental Examples 3 and 4). This metabolic regulation, combined with apoptosis and cell cycle arrest, exerts an effect that inhibits the growth and survival of cancer cells.

[0022] In addition, it was confirmed that 1,1-DEE inhibited tumor growth and increased survival rates in an NSG mouse model, minimized histological damage in major organs, and reduced leukemia-related inflammatory biomarkers (IL-8) (Experimental Example 5).

[0023] Thus, the present invention suggests that 1,1-DEE can effectively inhibit the growth and survival of cancer cells through ROS generation, cell cycle regulation, AMPK-mediated metabolic reprogramming, and apoptosis induction, and provides a basis for the development of anticancer drug compositions and cancer prevention and treatment methods utilizing this.

[0024]

[0025] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0026] The present invention discloses an anticancer agent comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0027] In the present invention, the 1,1-diethoxyethane (1,1-DEE) has the molecular formula C6H 14 It is represented by the following structural formula 1 as O2, and is also called acetaldehyde diethyl acetal or ethyllidene diethyl ether.

[0028] [Structural Formula 1]

[0029]

[0030] In the present invention, the 1,1-DEE can induce the generation of reactive oxygen species (ROS) in cancer cells.

[0031] In the present invention, the 1,1-DEE can induce apoptosis in cancer cells.

[0032] In the present invention, the 1,1-DEE can induce cell cycle arrest in cancer cells through depletion of the cell cycle G2 / M phase and accumulation of the G1 phase, and reduce the expression of CDK3, CDK4 and cyclin D1, D3, E proteins.

[0033] In the present invention, the anticancer agent may be used for hematological malignancy or solid tumors.

[0034] In the present invention, the anticancer agent is 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, large cell carcinoma of the urinary tract, transitional cell carcinoma of the bladder, bladder squamous cell carcinoma, prostate cancer, cervical cancer, leukemia of blood cells and related cells, acute and chronic lymphocytic leukemia, polycythemia vera, lymphoid tissue carcinoma, malignant lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, follicular lymphoma, diffuse lymphoma, small lymphocytic lymphoma, large cell lymphoma, lymphoblastic lymphoma, multiple myeloma, connective tissue tumors, osteosarcoma, nervous system tumors, neuroblastoma, retinoblastoma, glioblastoma, oncogenetic virus-related It can be used for cancers selected from the group consisting of oligodendroglioma, Burkitt lymphoma, immune-containing B-cell lymphoma, nasopharyngeal cancer, esophageal and gastroesophageal cancer, squamous cell carcinoma, pancreatic islet tumor, 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.

[0035] 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 myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythrocytic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell prolymphoid leukemia, T-cell prolymphoid leukemia, Philadelphia chromosome-positive leukemia, FLT3 mutation-positive acute myeloid leukemia, and acute erythrocytic leukemia.

[0036] In the present invention, the anticancer agent may be administered in combination with one or more of an immunotherapy agent, a monoclonal antibody, a chemotherapy agent, a radioprotective agent, a radiotherapy agent, and a gene therapy agent. In this case, the immunotherapy agent may include an immune checkpoint inhibitor including PD-1, PD-L1, and CTLA-4 inhibitors; a cytokine therapy including interleukin and interferon; CAR-T cell therapy, oncolytic virus, vaccine therapeutic agent, or targeted therapeutic agent; the chemotherapy agent may include an anmettotal agent, a platinum-based agent, an alkylating agent, or a topoisomerase inhibitor; and the radiotherapy agent may include external radiation therapy, brachytherapy, proton therapy, radiofrequency thermal therapy, stereotactic radiosurgery, or neutron therapy.

[0037] In the present invention, the anticancer agent can be used to prevent the onset of cancer.

[0038] In the present invention, the anticancer agent can exhibit a vitality recovery effect during the cancer treatment process.

[0039] 1,1-diethoxyethane (1,1-DEE) according to the present invention was shown to reduce cell viability in a concentration-dependent manner in T-lymphoblastic leukemia cell lines (Jurkat E6.1), induce changes in cell morphology, and promote apoptosis through increased Bax protein expression and decreased expression of Bcl-2, caspase-3, and caspase-9 (Experimental Example 1). In addition, it was confirmed that 1,1-DEE induces ROS generation and that this effect can be inhibited by a ROS scavenger (Experimental Example 2). Cell cycle analysis results demonstrated that 1,1-DEE induces cell cycle arrest through the depletion of the G2 / M phase and accumulation of the G1 phase, and contributes to the inhibition of cancer cell proliferation by reducing the levels of CDK3, CDK4, and cyclin D1, D3, and E proteins (Experimental Example 2).

[0040] Furthermore, 1,1-DEE was shown to activate AMPK signaling to inhibit the expression of glycolytic enzymes (LDHA, HK2) and glucose transporter (GLUT1) in cancer cells, and to inhibit the Warburg effect by maintaining or enhancing mitochondrial oxidative phosphorylation (OXPHOS) activity, thereby regulating metabolic reprogramming in cancer cells (Experimental Examples 3 and 5).

[0041] In in vivo (NSG mouse) experiments, 1,1-DEE significantly inhibited tumor growth and increased survival rates, minimized histological damage to major organs, and reduced the expression of the leukemia-related inflammatory biomarker (IL-8), thereby demonstrating its potential as a cancer treatment (Experimental Example 4).

[0042] Therefore, the present invention provides a new 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 low toxicity and high efficacy compared to existing treatments, but can also promote the prevention, treatment, and recovery of cancer through AMPK-mediated energy metabolism regulation.

[0043]

[0044] Meanwhile, the scope of the present invention is not limited by the effects described above.

[0045] Figure 1 shows the results of cell viability evaluation using the MTT assay. Cells were treated with 1,1-DEE (A and B) and its structural isomer, 1,2-DEE (B and D), at specified concentrations for 24 hours, followed by incubation with 10 μL of MTT solution for 2 hours. Each experiment was repeated three times, and a total of three independent experiments were conducted. 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 in Jurkat cells observed under an inverted microscope at 20x magnification 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, distinct morphological changes such as cell shrinkage and cell rounding were observed in cells treated with 1,1-DEE at concentrations of 5 mM and 10 mM for 24 hours.

[0046] Figure 2 shows the results of analyzing apoptosis in Jurkat cells using flow cytometry. As a result of treating with 1,1-DEE at different concentrations for 24 hours, the number of apoptotic cells significantly increased compared to the control group. (A) and (B) show the Annexin V and PI staining results and the apoptosis rate, while (C) and (D) show the results of Western blot analysis of the expression of pro-apoptotic and anti-apoptotic proteins. The data are presented 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.

[0047] Figure 3 shows the results of cell cycle analysis of Jurkat cells 24 hours after treatment with 1,1-DEE at concentrations of 1–10 mM. (A) shows representative flow cytometry results for cell cycle arrest, and (B) shows the proportion of S-phase and G2 / M-phase cells, as well as the proportion of early apoptotic cells observed at the sub-G1 peak. (C) and (D) show the results of Western blot analysis, and the data are presented as mean ± standard deviation (SD) of three independent experiments. Statistical significance is indicated as *p < 0.05, indicating significance compared to the control group.

[0048] Figure 4 shows the results of ROS generation following 1,1-DEE treatment. (A) shows the results of analyzing ROS generation using flow cytometry in Jurkat cells pretreated with NAC (2 mM), Trolox (250 μM), and Ebselen (10 μM) followed by treatment with 1,1-DEE (5 mM) or H₂O₂ (2 mM). (B) shows the results of measuring ROS fluorescence intensity upon treatment with 1,1-DEE and 1,2-DEE and treatment with ROS scavengers. (C) shows the results of evaluating changes in cell viability induced by ROS scavengers via MTT analysis after pretreating cells with 1-5 mM NAC, 125-500 μM Trolox, and 1-10 μM Ebselen for 1 hour, followed by treatment with 5 mM 1,1-DEE for 24 hours. Statistical significance is indicated as *p < 0.05 compared to the control group and #p < 0.05 compared to the 1,1-DEE treatment group, and data are presented as the mean ± SD of the results from three repeated experiments.

[0049] Figure 5 shows the results of AMPK activation in Jurkat cells following 1,1-DEE treatment. Total AMPK and phosphorylated AMPK (p-AMPK) in Jurkat cells were analyzed by Western blot after 0-120 minutes of 1,1-DEE treatment. (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 as *p < 0.05, signifying significance compared to the control group, and the data are presented as mean ± SD of the results from three replicate experiments.

[0050] Figure 6 shows changes in mitochondrial protein expression and glycometabolic changes following 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 as a negative control for 2 hours, after which (A) glucose uptake and (B) lactate production changes were measured. Additionally, Jurkat cells were treated with 1,1-DEE for 2-24 hours. (C) shows representative Western blot results, and (DH) shows the quantification results of OXPHOS complex proteins. (IJ) includes glycolysis, and the expression of the corresponding enzymes, HK2 and LDHA, was also evaluated after 1,1-DEE treatment. (K) represents the results of RT-PCR analysis of time-dependent HK2 and GLUT1 expression, and (L) and (M) represent the quantification of the RT-PCR results. Data are presented as the mean ± SEM of three independent experiments. Statistical significance is indicated as *p < 0.05 compared to the control group, and #p < 0.05 compared to 1,1-DEE.

[0051] Figure 7 shows the results of changes in mitochondrial membrane potential in Jurkat cells following 1,1-DEE treatment. 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 of mitochondrial membrane potential evaluated by JC-1 staining (30 min) after 1 hour of treatment, with a magnification of 200×.

[0052] Figure 8 shows the results of Jurkat cell growth in a xenograft mouse model. (A) 2 × 10⁶ Jurkat cells were unilaterally inoculated into the left leg of mice and subcutaneously injected into the shaved thigh, after which tumor growth was monitored for 4 weeks. In the treatment group, 1,1-DEE was administered at 110 mg / kg via intratumoral injection 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 of the treatment and control groups, (C) Tumor volume growth rate, (D) Mouse body weight, (E) Kaplan-Meier survival curves of tumor-inoculated mice. (F) H&E staining results of the heart, liver, spleen, lungs, and kidneys. A significant difference was observed in the survival curves between the PBS control group and the 1,1-DEE treatment group, and was statistically significant compared to PBS with a p < 0.05.

[0053] Figure 9 shows ROS levels measured at different time intervals using DCFDA staining. Representative flow cytometry histograms show time-dependent accumulation of ROS and are the results of treating Jurkat cells with 5 mM 1,1-DEE at different time intervals.

[0054] Figure 10 shows 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, and (C) Representative tumor images of PBS-treated and 1,1-DEE-treated mice. Data are presented as mean ± SEM of the results from three independent experiments. Statistical significance is indicated as ***p < 0.0001, meaning relative to the normal group (N).

[0055] Hereinafter, an anticancer agent comprising 1,1-diethoxyethane as an active ingredient according to a specific embodiment of the invention will be described in detail. However, this is presented as one example of the invention and does not limit the scope of the invention, and it is obvious to those skilled in the art that various modifications to the embodiment are possible within the scope of the invention. Throughout this specification, unless otherwise specifically stated, "includes" or "contains" refers to the inclusion of any component (or constituent) without any particular limitation and should not be interpreted as excluding the addition of other components (or constituents).

[0056] As used herein, the term "treatment" means any form of treatment or prevention that provides effects, including improvement of the individual's condition, delay of disease progression, delay of symptom onset, or slowing of symptom progression, to an individual who suffers from a disease or is at risk of developing a disease. Accordingly, the term "treatment" includes preventive treatment of the individual that prevents the onset of symptoms. Furthermore, the terms "treatment" and "prevention" are not intended to mean the cure or complete elimination of symptoms.

[0057] As used in this specification, the term "improvement" may mean any action that at least reduces parameters related to the alleviation or treatment of a condition, such as the degree of symptoms.

[0058] As used herein, the term “object” means an animal including animals such as cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, the object may be a mammal, particularly a human.

[0059]

[0060] 1. Anticancer drugs

[0061] The present invention

[0062] We intend to provide an anticancer drug containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0063] The above 1,1-DEE can induce the generation of reactive oxygen species (ROS) in cancer cells.

[0064] The above 1,1-DEE can induce apoptosis in cancer cells.

[0065] The above 1,1-DEE can induce cell cycle arrest in cancer cells through depletion of the cell cycle G2 / M phase and accumulation of the G1 phase, and reduce the expression of CDK3, CDK4 and cyclin D1, D3, E proteins.

[0066] The above 1,1-DEE can inhibit the expression of glycolytic enzymes (LDHA, HK2) and glucose transporters (GLUT1) in cancer cells by activating AMPK signaling.

[0067] The above 1,1-DEE can maintain or promote mitochondrial membrane potential (ΔΨm) and oxidative phosphorylation (OXPHOS) activity.

[0068] The above 1,1-DEE can reduce the expression of leukemia-related inflammatory biomarkers (IL-8).

[0069] The above anticancer agent can be used for hematological malignancy or solid tumors.

[0070] The above anticancer agent can be used for leukemia, multiple myeloma, or lymphoma. For example, the above anticancer agent is 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, large cell carcinoma of the urinary tract, transitional cell carcinoma of the bladder, bladder squamous cell carcinoma, prostate cancer, cervical cancer, leukemia of blood cells and related cells, acute and chronic lymphocytic leukemia, polycythemia vera, lymphoid tissue carcinoma, malignant lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, follicular lymphoma, diffuse lymphoma, small lymphocytic lymphoma, large cell lymphoma, lymphoblastic lymphoma, multiple myeloma, connective tissue tumors, osteosarcoma, nervous system tumors, neuroblastoma, retinoblastoma, glioblastoma, oncogeneic virus-related It can be used for cancers selected from the group consisting of oligodendroglioma, Burkitt lymphoma, immune-containing B-cell lymphoma, nasopharyngeal cancer, esophageal and gastroesophageal cancer, squamous cell carcinoma, pancreatic islet tumor, 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.

[0071] When the above 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 myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythrocytic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell prolymphoid leukemia, T-cell prolymphoid leukemia, Philadelphia chromosome-positive leukemia, FLT3 mutation-positive acute myeloid leukemia, and acute erythrocytic leukemia.

[0072] The above anticancer agent may be administered alone or in combination with one or more of immunotherapies, monoclonal antibodies, chemotherapy agents, radioprotective agents, radiotherapeutic agents, and gene therapies such as microRNA. In this case, the immunotherapies may include immune checkpoint inhibitors including PD-1, PD-L1, and CTLA-4 inhibitors; cytokine therapies including interleukin and interferon; CAR-T cell therapy, oncolytic viruses, vaccine therapies, or targeted therapies, the chemotherapy agents may include antimetabolites, platinum-based agents, alkylating agents, or topoisomerase inhibitors, and the radiotherapeutic agents may include external radiation therapy, brachytherapy, proton therapy, radiofrequency thermal therapy, stereotactic radiosurgery, or neutron therapy. In particular, the delivery of the above anticancer agent is, before, during, or after administration of one or more known antitumor agents comprising, 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, methoctrexate, vincristine, vinblastine, taxol, etoposide, temifoside, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitomycin, cisplatin, carboplatin, estramustine phosphate hydroxyurea, BCNU, procarbazine, VM-26, interferol, and all-trans retinoic acid (ATRA), or other retinoids. It may occur. Suitable hypomethylation agents may include decitabine, guancitabine, azacitidine, etc. Suitable examples of PD-1 inhibitors include pembrolizumab and nivolumab, and suitable PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab.

[0073] The above anticancer agent may be administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, epithelially, locally, vaginally, pulmonaryly, rectally, sublingually, buccally, transdermally, ocularly, inhaled, intracavernously, intrathecally, epidurally, and rectally. When administered orally, for example, the above anticancer agent may be formulated as a tablet, or the active agent may be coated or protected from degradation in the stomach. Additionally, the active substance of the above anticancer agent may be administered by any device capable of delivering it to target cells. The route of administration may vary depending on the general condition and age of the subject being treated, the nature of the treatment conditions, and the selected active ingredient.

[0074] The above anticancer agent may be carried on a carrier, and the carrier may include one or more selected from virus particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, and exosomes, but is not limited thereto.

[0075] The appropriate dosage of the above anticancer agent varies depending on factors such as the formulation method, method of administration, patient's age, weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response responsiveness, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the above anticancer agent may be administered as a single or multiple doses, or divided into 1 to 4 doses per day. For example, the above anticancer agent may include 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.

[0076] The above anticancer agent may be manufactured in a unit dose form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation 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, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer. Furthermore, the above anticancer agent may be manufactured for administration to mammals, more preferably for administration to humans.

[0077] The above pharmaceutically acceptable carrier may be solid or liquid and may be one or more selected from excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavorings, lubricants, release regulators, wetting agents, stabilizers, suspending agents, and lubricants. Additionally, the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

[0078] In one embodiment, 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).

[0079] In one embodiment, suitable binders may include, but are not limited to, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gums, sucrose, starch, or mixtures thereof.

[0080] In one embodiment, 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.

[0081] In one embodiment, suitable disintegrant may be sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or a mixture thereof, but is not limited thereto.

[0082] In one embodiment, 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.

[0083] In one embodiment, suitable glidant may be silica, colloidal silicon dioxide, talc, etc., but is not limited thereto.

[0084] In one embodiment, 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 wax, or mixtures thereof.

[0085]

[0086] 2. Methods for preventing or treating cancer

[0087] The present invention

[0088] The present invention aims to provide a method for preventing or treating cancer comprising the step of administering an anticancer agent containing 1,1-diethoxyethane (1,1-DEE) as an active ingredient to an individual.

[0089] The above-mentioned individuals may include, but are not limited to, humans, cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs.

[0090] The route of administration, dosage, and frequency of administration of the above anticancer agent may be administered to the subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal method of administration, dosage, and frequency of administration can be selected within an appropriate range by a person skilled in the art. In the present invention, the preferred dosage of the above anticancer agent may be in the range of 0.001 mg / kg to 100 mg / kg per day for adults, depending on the patient's condition, body weight, gender, age, severity of the patient, and route of administration. Administration may be carried out once a day or divided into several doses. Such dosage shall not be interpreted as limiting the scope of the present invention in any aspect.

[0091] Various embodiments are presented below to aid in understanding the invention. The following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of protection of the invention to the following embodiments.

[0092]

[0093] Materials and Methods

[0094] 1. Cell culture

[0095] 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 RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator maintained at 37°C and 5% CO₂.

[0096]

[0097] 2. Measurement of cell viability

[0098] Cells were plated at a density of 2 x 10⁴ cells (100 μl) per well in RPMI medium supplemented with 10% FBS and 1% penicillin & streptomycin. The next day, 1,1-DEE from Sigma-Aldrich (St. Louis, USA) and 1,2-DEE from TCI (Tokyo Chemical Industry Co. Ltd, Japan) were sequentially diluted and added to each well over a period of 24 hours (total 120 μl). Subsequently, 10 μl of MTT (DoGenBio Co., Ltd.) was added to each well of the plate and incubated at 37°C for 2 hours. Cell viability was analyzed by measuring absorbance at 450 nm using a microplate spectrophotometer (Epoch, Biotek, USA).

[0099]

[0100] 3. Western Blot

[0101] To extract proteins from Jurkat cells (5 × 10⁶ / mL) cultured in 60 × 15 mm cell culture dishes (SPL Life Sciences, Gyeonggi-do, Republic of Korea), 150 μL of Pro-PREP™ protein extraction solution from iNtRON Biotechnology (Gyeonggi-do, South Korea) was added to each plate. Materials such as polyvinylidene fluoride (PVDF) membranes and Western chemiluminescent HRP substrates were purchased from Millipore Corporation (Billerica, MA, USA). After isolating 30 μg of total protein, it was separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to PVDF membranes for further analysis. The membrane was blocked for 1 to 2 hours by adding 0.1% Tween-20 to TBST containing 5% skim milk, and then incubated overnight at 4°C with the addition of a primary antibody (1:1000) to TBST. After washing three times with TBST for 10 minutes each, a secondary antibody (1:2000) conjugated with horseradish peroxidase was used to detect immunoreactive proteins via chemiluminescence.For Western blot experiments, the following antibodies provided by Cell Signaling Technology (Danvers, MA, USA) were used: 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), Total OXPHOS Cocktail (Abcam, 110411), and anti-β (5125S). Monoclonal antibodies. To measure total protein concentration, the blotted membrane was washed with Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific, Meridian Rd., Rockford, IL, USA) at 56°C for 30 minutes.

[0102]

[0103] 4. Measurement of Cell Apoptosis - Annexin V / FITC Assay

[0104] To confirm the apoptotic potential of Jurkat cells, the Annexin V fluorescence kit (BD Pharmingen, San Diego, USA) was used according to the manufacturer's protocol. Cells were seeded into 6-well plates containing RPMI medium with 10% FBS at a density of 2.0 × 10 cells per well for 24 hours. After seeding, the cells were treated with the desired concentration of 1,1-DEE and incubated 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 provided in the kit. Subsequently, the cells were analyzed for flow cytometry using a BD FACS Calibur (BD Biosciences, CA, USA). Data plotting and analysis were performed using FlowJo™Software (BD Biosciences, CA, USA).

[0105]

[0106] 5. Cell cycle analysis

[0107] Jurkat cells were seeded into 6-well plates at a density of 5 x 10⁶ cells per ml and incubated at 37°C for 24 hours, followed by treatment with the desired concentration of 1,1-DEE for 24 hours. Cells were detached using trypsin-EDTA and centrifuged; after washing with 1X cold PBS, they were fixed in 70% ethanol at 4°C for 30 minutes. To prepare cells for analysis, they were washed with 1X PBS and centrifuged at 1,200 rpm for 5 minutes. Subsequently, the cells were placed in 500 μl of a solution containing 10 μg / ml of RNase A (Sigma-Aldrich) and 75 μM of propidium iodide (Sigma-Aldrich) and incubated in a dark room at room temperature for 1 hour. DNA fluorescence intensity was detected using a flow cytometer equipped with BD FACS Verse, and the data were plotted and analyzed using FlowJo™ software.

[0108]

[0109] 6. ROS Generation Measurement

[0110] Intracellular H₂O₂ levels were measured using 5- and 6-amino 2',7'-dichlorodichlorofluoresceindiacetate (DCFDA; Molecular Probes, Eugene, OR, USA). Jurkat cells were cultured in RPMI supplemented with 10% FBS until 80% confluence was reached. To evaluate the ROS-generating effect activated by 1,1-DEE, cells were treated with 2 mM NAC, 250 μM Trolox, and 10 μM Ebsleen 1 hour prior to compound treatment. Subsequently, 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 emission was captured at 515 nm using a long-pass filter. After culturing with DCFDA, ROS was analyzed by flow cytometry using a BD FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA). Data were plotted and analyzed using FlowJo™ software version 10.10 (BD Biosciences, San Jose, CA, USA).

[0111]

[0112] 7. Animal Care and Drug Administration

[0113] Four-week-old male immunodeficient mice (NOD.Cg-PrkdcscidIL2rgtm1Wjl / Szj, NSG) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed at the Chonnam National University Laboratory Animal Resource Center (CNU IACUC-H-2024-42). Mice were housed at 23°C and 60% humidity on a 16-hour light / 8-hour dark cycle and provided with food and water until the experiment began. Five mice were housed per cage, and body weight was measured three times a week. All experiments adhered to the institutional guidelines of Chonnam National University. Jurkat cells cultured in RPMI medium containing 10% FBS were subcutaneously injected into the right flank of each mouse at a concentration of 2 x 10⁶ cells in 100 μl of PBS. Mice were divided into a control group and a treatment group. Four weeks after tumor induction, the treatment group was injected intratumorally with 1,1-DEE (110 mg / kg) every other day from day 2 to day 16. The control group was injected with PBS. Eighteen days after drug administration, animals were used for a survival study. Additionally, tumor tissues and major organs were processed for histological analysis using H&E staining.

[0114]

[0115] 8. Enzyme-Linked Immunosorbent Assay (ELISA)

[0116] In the in vivo study, plasma was collected from blood drawn via cardiac puncture of anesthetized NSG mice. Cytokine (IL-8) levels were measured using an ELISA kit (R&D Systems, USA) with an Epoch microplate spectrophotometer and an ELx50 microplate strip washer (BioTek Instruments, USA). A standard curve was constructed by calculating the optical density of IL-8 concentration and expressed in pg / ml.

[0117]

[0118] 9. Lactate Production Assay

[0119] Jurkat cells were seeded into 96-well plates at a concentration of 10⁴ cells (100 μl) per well. 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 lactate production was measured using the Lactate-Glo™Assay (Promega, USA) according to the manufacturer's protocol. Luminescence was measured using a GloMax plate-reading photometer (Promega, USA).

[0120]

[0121] 10. Glucose Uptake Assay

[0122] Jurkat cells were seeded into 96-well plates at a density of 10⁴ cells (100 μl) per well. Once the cells reached confluence, 1,1-DEE 1, 2, 5 mM and 1,2-DEE 5 mM were treated for 2 hours. Glucose uptake was evaluated using the Glucose Uptake-Glo™Assay (Promega, USA) according to the manufacturer's instructions. Luminescence was recorded using a GloMax® Luminometer (Promega, USA) with an integration time of 0.5 seconds according to the Glucose Uptake-Glo™ protocol.

[0123]

[0124] 11. Polymerase Chain Reaction (RT-PCR)

[0125] 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 a primer set for β-actin and IL-6 with PCR master mix solution (iNtRON, Korea). The primers used are as follows.

[0126] β-actin forward: 5′CAG GAG TAT GAC GAG TC-3′

[0127] β-actin reverse: 5′TTC ATA CAT CTC AAG TT-3′(561 bp)

[0128] LDHA forward: 5′CAT GAT TAA GGG TCA TTA C-3′

[0129] LDHA reverse: 5′TCA GAG ATT CCA TTC TG-3′(87 bp)

[0130] HK2 forward: 5′CCA CCA CTC ACC CTA CT-3′

[0131] HK2 reverse: 5′GGC ATT CGG CAA TGT G-3′(249 bp)

[0132] GLUT1 forward: 5′TCT ACA ACC AGA CAT GG-3′

[0133] GLUT1 reverse: 5′GTT CAT CAT CAG CAT TG-3′(179 bp)

[0134] PCR conditions were performed with denaturation at 94°C for 30 seconds, annealing at 54°C for 20 seconds, and extension at 72°C for 30 seconds.

[0135]

[0136] 12. Histological analysis (H&E staining, Histology)

[0137] After measuring tumor volume and weight, animals were sacrificed for histological studies. The tissues were dehydrated twice with 95% ethanol for 0.5 hours, immersed in xylene at 60–70° for 1 hour, and paraffin-embedding for 12 hours. 6 μm thick frozen sections were prepared and fixed with frozen section solution (FSC 22 Clear, Leica). The tissue sections were stained with Harris’ hematoxylin solution at 60–70° for 6 hours and rinsed with tap water until colorless. Then, a mixture of 10% acetic acid and 85% ethanol in water was applied twice for 2 and 10 hours, respectively, followed by rinsing the tissues with tap water to induce differentiation. For bluing, the sections were immersed in a saturated lithium carbonate solution for 12 hours and rinsed again with tap water. Finally, counterstaining was performed for 48 hours with an eosin Y ethanol solution.

[0138]

[0139] 13. Measurement of mitochondrial membrane potential (JC-1 staining, Flow Cytometry)

[0140] Mitochondrial membrane potential was measured using JC-1 dye (Beyotime, Seoul, Korea). Jurkat cells were cultured for 24 hours, treated with the indicated drug concentrations, and then incubated with JC-1 (20 nM) in the dark at 37°C for 30 minutes. After washing with PBS, cells were dispensed into confocal dishes (Carl Zeiss, Germany) for imaging or collected by trypsin treatment and analyzed using a BD FACS Calibur flow cytometer (BD Biosciences, USA). Data were processed using FlowJo™ software (BD Biosciences, USA).

[0141]

[0142] 14. Statistical Analysis

[0143] Each data value represents three separate experiments and is expressed as mean ± standard deviation (SD). Results were visualized using GraphPad Prism software (Version 8.0). Multivariable analyses were performed using ANOVA with Tukey's multiple comparison test, and values ​​of p < 0.05 (#, *, ^, +, @), p < 0.01 (##, **, ^^, ++, @@), p < 0.001 (###, ***, ^^^, +++, @@@), and p < 0.0001 (####, ****, ^^^^, ++++, @@@@) were considered statistically significant. Student t-tests were used for the analysis of tumor volume and weight, and Log-Rank tests were used for the comparison of survival rates. The significance level for all tests was p < 0.05.

[0144]

[0145] <Result>

[0146] Experimental Example 1. Evaluation of whether 1,1-DEE treatment induces cell growth inhibition and apoptosis in ALL cell lines

[0147] To confirm the antiproliferative and apoptosis-inducing effects of 1,1-DEE, evaluations were performed using the Jurkat E6.1 cell line. MTT analysis results showed that 1,1-DEE reduced the viability of Jurkat E6.1 cells in a concentration-dependent manner even at concentrations below 10 mM (Figs. 1A, 1B). In contrast, 1,2-DEE was used as a negative control, and no significant change in cell viability was observed (Figs. 1C, 1D).

[0148] To evaluate the low-dose toxicity of 1,1-DEE, Jurkat E6.2 cells were treated with various concentrations of 1,1-DEE for 6, 12, and 24 hours. As a result, the Jurkat E6.2 cells exhibited a round and contracted shape, and these morphological changes were observed more distinctly as the concentration and treatment time of 1,1-DEE increased (Fig. 1E). In contrast, no such morphological changes were observed in the 1,2-DEE treatment group (Fig. 1E). In particular, a change of approximately twofold was observed at 6 and 12 hours of treatment, and a strong inhibitory effect of approximately threefold was confirmed at 24 hours of treatment.

[0149] To evaluate whether apoptosis was induced, Jurkat E6.1 cells were cultured with 1,1-DEE for 24 hours and then stained with Annexin V and PI (Fig. 2A). Analysis results showed that apoptosis significantly increased with 1,1-DEE treatment, and the proportion of cells in the Q2 range increased in a concentration-dependent manner (Fig. 2B).

[0150] In addition, Western blot analysis confirmed that 1,1-DEE treatment increased the expression of Bax, a cell death-promoting protein, while decreasing the expression of Bcl-2, caspase-9, and caspase-3, which are cell death-inhibiting proteins. On the other hand, no significant changes were observed in the Compound C (10 μM, AMPK inhibitor) or 1,2-DEE treatment groups, which were used as positive controls, compared to the untreated control group (Fig. 2C, Fig. 2D).

[0151] Therefore, from the above results, it was confirmed that 1,1-DEE treatment induces apoptosis in Jurkat E6.1 cells and exhibits an inhibitory effect on cell growth.

[0152]

[0153] Experimental Example 2. Evaluation of whether 1,1-DEE treatment induces cell cycle arrest in ALL cell lines through ROS generation

[0154] It is known that an increase in intracellular concentrations of reactive oxygen species (ROS) induces oxidative stress, leading to cell cycle arrest and apoptosis in cancer cells. Furthermore, ROS have been reported to disrupt intracellular redox balance and increase the expression of CDK inhibitory proteins such as p21 and p27, thereby arresting the cell cycle at the G0 / G1 or G2 / M phases. Accordingly, this study evaluated whether 1,1-DEE treatment inhibits tumor growth by inducing ROS generation and causing cell cycle arrest.

[0155] First, changes in the cell cycle following treatment with 1,1-DEE were analyzed using Jurkat E6.1 cells. Jurkat E6.1 cells were treated with 1,1-DEE at concentrations of 1–10 mM for 24 hours, and the cell cycle was analyzed via flow cytometry. As a result, in the 1,1-DEE treated group, a decrease in the ratio of cells in the G2 / M phase and an accumulation of cells in the G1 phase were observed (Figs. 3A, 3B).

[0156] 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 (Figs. 3C, 3D). Notably, significant accumulation of G1 phase cells was observed even at the lowest concentration of 1 mM 1,1-DEE treatment, confirming that 1,1-DEE induces cell cycle arrest in a concentration-dependent manner.

[0157] ROS accumulation is known to play an important role in the regulation of cell proliferation and apoptosis. Accordingly, in this study, the degree of ROS accumulation after treatment with 1,1-DEE was evaluated using the fluorescent probe DCFH-DA. As a result, distinct ROS accumulation was induced within 30–60 minutes by treatment with 1,1-DEE (5 mM) (Fig. 9).

[0158] In addition, to determine whether ROS accumulation induced by 1,1-DEE treatment plays a significant role in inhibiting cell growth, cell viability was evaluated after treatment with 1,1-DEE in combination with ROS scavengers (NAC, Trolox, Ebselon) or ROS inducers (H₂O₂) (Fig. 4A). Under conditions without NAC, treatment with 1,1-DEE induced ROS accumulation and inhibited cell growth. Conversely, in the presence of NAC, ROS accumulation induced by 1,1-DEE decreased, and it was confirmed that cell viability increased accordingly (Figs. 4C, 4D).

[0159] Therefore, from these results, it was confirmed that ROS generation plays an important role in the cell growth inhibitory effect of 1,1-DEE.

[0160]

[0161] Experimental Example 3. Evaluation of whether 1,1-DEE treatment inhibits the Warburg effect in ALL cells through the regulation of AMPK signaling

[0162] AMPK (AMP-activated protein kinase) is a conserved serine / threonine kinase that serves as a central factor regulating intracellular energy homeostasis. AMPK activation is known to alter the bioenergetic properties of tumor cells by regulating key signaling pathways that induce metabolic reorganization in tumor cells. Saito et al. reported that AMPK plays a crucial role in maintaining the viability of leukemia cells within the bone marrow by inhibiting glucose transporter (GLUT)-mediated glucose uptake and promoting ROS accumulation. Conversely, genetic defects in AMPK are known to result in leukemia cells becoming sensitive to metabolic stress due to impaired glucose utilization capacity.

[0163] Accordingly, in this study, AMPK activation by 1,1-DEE was evaluated using Jurkat E6.1 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 particularly after 60 minutes of treatment. In addition, 1,1-DEE induced phosphorylation of AMPK in a concentration-dependent manner (Figures 5C and 5D), with the most distinct activation confirmed at a concentration of 5 mM.

[0164] Meanwhile, tumor cells are known to exhibit metabolic reprogramming, a phenomenon in which they rewire nutrient utilization pathways to secure the energy required for growth. In particular, cancer cells have been reported to exhibit the Warburg effect, converting glucose into lactic acid even in the presence of sufficient oxygen. Therefore, this study evaluated whether AMPK activation by 1,1-DEE could inhibit the Warburg effect.

[0165] As a result, when 1,1-DEE was treated at concentrations of 1 mM, 2 mM, and 5 mM, AMPK was activated in Jurkat E6.1 cells, and glucose uptake was significantly reduced (p < 0.05) (Fig. 6A). In particular, a distinct 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 Jurkat E6.1 cells were treated with 1,1-DEE at concentrations of 2 mM and 5 mM, lactate production was significantly reduced, suggesting inhibition of glycolytic flux (Fig. 6B).

[0166] Therefore, from these results, it was confirmed that 1,1-DEE treatment inhibits the Warburg effect by promoting AMPK phosphorylation, and that upregulation of AMPK alone can induce a decrease in intracellular glycolytic activity.

[0167]

[0168] Experimental Example 4. Evaluation of whether 1,1-DEE treatment regulates AMPK in ALL cells and alters membrane potential and glycolytic properties

[0169] Aerobic glycolysis in cancer cells is known to reduce oxidative phosphorylation (OXPHOS) activity by primarily utilizing a metabolic pathway that converts pyruvate to lactate instead of transferring it to the mitochondrial TCA cycle. This metabolic shift maintains mitochondrial structural stability by limiting ROS production within mitochondria, while appropriate levels of ROS play a role in supporting tumor progression and survival. Therefore, targeting rate-limiting enzymes of glucose uptake and glycolysis, as well as mitochondrial oxidative phosphorylation, is considered a strategic approach to inhibit the Warburg effect and block metabolic reprogramming in tumor cells.

[0170] Accordingly, this study aimed to determine whether 1,1-DEE exhibits anti-Warburg effects by regulating the expression and activity of these relevant factors.

[0171] First, to evaluate the effect of 1,1-DEE on mitochondrial OXPHOS activity, Jurkat E6.1 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 (NDUFB8, SDHB, UQCRC2, ATP5A) encoded in nuclear genes (Figs. 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 that mitochondrial respiratory activity was enhanced compared to the untreated control group. These results indicate that 1,1-DEE promotes mitochondrial oxidative phosphorylation, thereby enhancing the bioenergetic functions of the cell.

[0172] In addition, to evaluate whether 1,1-DEE directly regulates the expression of the glucose transporter GLUT1, GLUT1 protein levels were analyzed after treating Jurkat E6.1 cells with 1,1-DEE (2 mM) for 4 hours. As a result, GLUT1 expression was significantly reduced (Figs. 7I–7K), indicating that 1,1-DEE inhibited GLUT1 expression at both transcriptional and translational levels. Therefore, it was determined that 1,1-DEE also effectively inhibits GLUT1-mediated glycolysis.

[0173] Meanwhile, 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 radiation therapy. In this study, we confirmed that when treated with 1,1-DEE (2 mM) for 4 hours, the expression of LDHA and HK2 proteins was significantly reduced compared to the untreated control group. This suggests that 1,1-DEE effectively inhibits key regulators of glycolysis, thereby inhibiting aerobic glycolysis in cancer cells.

[0174] Mitochondrial membrane potential (ΔΨm) is known as an important indicator for evaluating cell viability and function. To evaluate this, the JC-1 staining method was used. JC-1 is a cationic cyanine dye that utilizes the characteristic of switching from a green monolayer to a red fluorescent aggregate when the mitochondrial membrane potential is high, allowing for the fluorescence-based analysis of the depolarization or hyperpolarization state of mitochondria within the cell.

[0175] As a result, when Jurkat E6.1 cells were treated with 1,1-DEE, the ratio of red fluorescence increased in a concentration-dependent manner, indicating an improvement in mitochondrial polarization. In contrast, in the 1,2-DEE treatment group, the ratio of red to green fluorescence was balanced, and no significant changes in membrane potential were observed. In particular, strong red fluorescence was dominant in the 1,1-DEE treatment group (Fig. 7), suggesting that 1,1-DEE supports mitochondrial function by maintaining or enhancing the mitochondrial membrane potential.

[0176]

[0177] Experimental Example 5. Evaluation of whether ALL cell growth is inhibited in vivo by treatment with 1,1-DEE

[0178] To further confirm whether 1,1-DEE inhibits tumor growth in vivo, a subcutaneous implantation tumor model was established using 4-week-old NSG mice. Jurkat cells were stably injected subcutaneously, and the 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.

[0179] As a result, it was confirmed that 1,1-DEE significantly inhibited tumor growth in vivo (Fig. 8A). Furthermore, a comparison of the tumor growth curves of each group revealed a significant reduction in tumor volume in the 1,1-DEE-treated group 18 days after cell injection (Figs. 8B, C). Simultaneously, mouse body weight was measured (Fig. 8D), and after 18 days, tumor size and survival rates were compared between the physiological saline-treated group and the 1,1-DEE-treated group. The survival curves for each group are presented in Fig. 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.

[0180] Subsequently, 18 days after 1,1-DEE treatment, a detailed histopathological examination was performed to evaluate potential organ 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.

[0181] Distinct morphological changes were observed in hepatocytes of mice with solid tumors compared to 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 liver tissue structure, which had been slightly disrupted, appeared to be restored, with the central vein and portal vein properly maintained and cell infiltration minimized.

[0182] In kidney tissue, acute vacuolation, dilation of the epithelial wall, nuclear degeneration, necrosis, and epithelial degeneration were observed. In heart tissue, chemodectoma, toxic myocarditis, reddish-brown atrophy, and yellowish-brown pigmentation were observed, which appear to correspond to lipofuscin granules, presumed to be remnants of cellular organelles and cytoplasmic material. In lung tissue, vacuolation, central venous degeneration, inflammation, hemorrhage, altered cellular structures, hemosiderophages, and lesions were observed.

[0183] Therefore, the results of this study showed that 1,1-DEE treatment can restore liver tissue structure similar to that of normal animals. In addition, H&E staining of solid tumor tissues revealed that the heart tissue of the 1,1-DEE treated group maintained a normal tissue structure, whereas the heart tissue of tumor-bearing mice exhibited an irregular structure.

[0184]

[0185] According to several recent studies, IL-8 is known as a key biomarker associated with disease progression and poor prognosis in various types of leukemia. Meanwhile, it has been reported that the activation of AMPK inhibits IL-8 expression through the inhibition of NF-κ. Accordingly, this study investigated whether 1,1-DEE could inhibit IL-8 expression and alleviate leukemia-related inflammatory responses by activating AMPK (Fig. 10). ELISA analysis showed that IL-8 expression increased in control mice, whereas IL-8 expression was significantly decreased in the 1,1-DEE administration group.

[0186]

[0187] Consequently, this study demonstrates that 1,1-DEE inhibits the Warburg effect by increasing ΔΨm (mitochondrial membrane potential) and promoting OXPHOS activity, while simultaneously inhibiting glycolytic flux by reducing the expression of key glycolytic enzymes, including LDHA and HK2. Through these time- and concentration-dependent metabolic regulatory actions, 1,1-DEE exhibits potent anticancer activity and can be presented as a promising therapeutic candidate targeting metabolic regulation.

[0188]

[0189] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0190]

[0191] National R&D project that supported this invention

[0192] [Assignment No.] 2018R1D1A1B06051438

[0193] [Ministry Name] Ministry of Science and ICT

[0194] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0195] [Research Project Title] Regulation of the Cancer Suppressor Protein PTEN by Alcohol

[0196] [Name of Project Performing Organization] Chonnam National University

Claims

An anticancer agent containing 1,1,1-diethoxyethane (1,1-DEE) as an active ingredient.

2. In Paragraph 1, The above 1,1-DEE is an anticancer agent characterized by inducing the generation of reactive oxygen species (ROS) in cancer cells.

3. In Paragraph 1, The above 1,1-DEE is an anticancer agent characterized by inducing apoptosis in cancer cells.

4. In Paragraph 1, The above 1,1-DEE is an anticancer agent characterized by inducing cell cycle arrest in cancer cells through depletion of the cell cycle G2 / M phase and accumulation of the G1 phase, and reducing the expression of CDK3, CDK4, and cyclin D1, D3, and E proteins.

5. In Paragraph 1, The above anticancer agent is characterized by being used for hematological malignancy or solid tumors.

6. In Paragraph 1, The above anticancer drug is 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, large cell carcinoma of the urinary tract, transitional cell carcinoma of the bladder, bladder squamous cell carcinoma, prostate cancer, cervical cancer, leukemia of blood cells and related cells, acute and chronic lymphocytic leukemia, polycythemia vera, lymphoid tissue carcinoma, malignant lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, follicular lymphoma, diffuse lymphoma, small lymphocytic lymphoma, large cell lymphoma, lymphoblastic lymphoma, multiple myeloma, connective tissue tumors, osteosarcoma, nervous system tumors, neuroblastoma, retinoblastoma, glioblastoma, oncogeneic virus-associated oligodendroglioma, An anticancer agent characterized by being used for cancer selected from the group consisting of Burkitt lymphoma, immune-containing intracellular B-cell lymphoma, nasopharyngeal cancer, esophageal and gastroesophageal cancer, squamous cell carcinoma, pancreatic islet tumor, 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 cancer.

7. In Paragraph 1, When the above anticancer agent is used for leukemia, the leukemia is characterized in that it is a form selected from the group consisting of acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythrocytic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell prolymphocytic leukemia, T-cell prolymphocytic leukemia, Philadelphia chromosome-positive leukemia, FLT3 mutation-positive acute myeloid leukemia, and acute erythrocytic leukemia.

8. In Paragraph 1, The above anticancer agent is administered in combination with one or more of an immunotherapy agent, a monoclonal antibody, a chemotherapy agent, a radioprotective agent, a radiotherapy agent, and a gene therapy agent, and The above immunotherapeutic agents include immune checkpoint inhibitors including PD-1, PD-L1, and CTLA-4 inhibitors; cytokine therapies including interleukin and interferon; CAR-T cell therapy, oncolytic virus, vaccine therapeutics, or targeted therapeutics, and The above chemotherapeutic agent includes an anmetatalist, a platinum-based agent, an alkylating agent, or a topoisomerase inhibitor, and The above-mentioned radiotherapy agent is an anticancer agent characterized by including external radiation therapy, brachytherapy, proton therapy, radiofrequency thermal therapy, stereotactic radiosurgery, or neutron therapy.

9. In Paragraph 1, The above anticancer drug is an anticancer drug intended to prevent the onset of cancer.

10. In Paragraph 1, The above anticancer drug is an anticancer drug that exhibits a restorative effect during the cancer treatment process.

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