Ellagic acid and terbinafine combination and its application in the preparation of drugs for treating lung cancer

Abstract

This invention relates to a composition of ellagic acid and terbinafine and its application in the preparation of drugs for treating lung cancer, belonging to the field of biomedical technology. To address the problem of increased SQLE feedback during terbinafine monotherapy for lung cancer in existing technologies, this invention provides a composition of ellagic acid and terbinafine, consisting of ellagic acid and terbinafine in a mass ratio of 4:7. The pharmaceutical composition provided by this invention is used to prepare drugs for treating lung cancer. This composition can target and inhibit SQLE through a dual mechanism, blocking cholesterol biosynthesis pathways and inhibiting tumor growth; the synergistic effect of the two significantly enhances the anti-tumor effect, while also reducing adverse reactions such as metabolic disorders caused by terbinafine, mitigating the drug toxicity of ellagic acid itself, and effectively preventing the development of drug resistance. This invention is based on the development of already marketed drugs, possessing the dual advantages of enhanced efficacy and improved safety, providing a more optimized treatment plan for lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a composition of ellagic acid and terbinafine and its application in the preparation of drugs for treating lung cancer. Background Technology

[0002] Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer accounting for 85% of all deaths. Current treatments, such as EGFR-TKI targeted therapy and chemotherapy, face challenges including secondary drug resistance and metabolic remodeling of the tumor microenvironment. Squalene epoxidase (SQLE) plays a crucial role in the cholesterol synthesis pathway, and recent studies have shown that SQLE is closely related to the occurrence, development, and metastasis of lung cancer. High SQLE expression is associated with poor prognosis in lung cancer patients and can promote tumor cell proliferation, survival, and migration.

[0003] Terbinafine, a traditional antifungal drug, has been shown to effectively inhibit the activity of SQLE. It has also demonstrated potential in lung cancer treatment. However, terbinafine alone leads to compensatory upregulation of SQLE expression levels. This feedback regulation mechanism results in decreased efficacy and side effects such as hepatotoxicity, limiting its further application in lung cancer treatment.

[0004] Ellagic acid, a natural polyphenol compound, possesses antioxidant, anti-inflammatory, and lipid metabolism-regulating effects. It can inhibit tumor cell growth, induce tumor cell apoptosis, and inhibit tumor angiogenesis through multiple pathways. However, its low bioavailability and dose-dependent toxicity limit its clinical application. Summary of the Invention

[0005] To address the issue of increased SQLE feedback during terbinafine monotherapy for lung cancer in existing technologies, this invention provides a combination of ellagic acid and terbinafine, and its application in the preparation of a lung cancer treatment drug.

[0006] The technical solution of this invention:

[0007] A composition of ellagic acid and terbinafine, wherein the mass ratio of ellagic acid to terbinafine is 4:7.

[0008] The application of an ellagic acid and terbinafine composition provided by the present invention in the preparation of a medicament for treating lung cancer.

[0009] Furthermore, the drug for treating lung cancer is administered orally.

[0010] Furthermore, the drug for treating lung cancer also contains a pharmaceutically acceptable carrier or excipient.

[0011] Furthermore, the drug for treating lung cancer is in the form of granules, tablets, capsules, pills, or oral liquid preparations.

[0012] Furthermore, the lung cancer in question is non-small cell lung cancer.

[0013] Furthermore, the non-small cell lung cancer mentioned is adenocarcinoma or large cell carcinoma of non-small cell lung cancer.

[0014] Furthermore, the drug for treating lung cancer can simultaneously inhibit SQLE enzyme activity and compensatory upregulate SQLE expression.

[0015] Furthermore, the drug for treating lung cancer can disrupt the lipid metabolism of lung cancer tumor cells and block cholesterol synthesis.

[0016] The beneficial effects of this invention are:

[0017] This invention provides an anti-lung cancer drug composition based on the synergistic effect of ellagic acid and terbinafine. Experiments have demonstrated that this composition targets and inhibits SQLE through a dual mechanism: terbinafine directly inhibits SQLE enzyme activity, blocking cholesterol biosynthesis pathways and significantly inhibiting tumor growth; simultaneously, ellagic acid effectively inhibits the compensatory upregulation of SQLE expression induced by terbinafine monotherapy. The synergistic effect of the two significantly enhances the anti-tumor efficacy. This composition not only overcomes the limitations of monotherapy but also reduces adverse reactions such as metabolic disorders caused by terbinafine, mitigates the drug toxicity of ellagic acid itself, and avoids excessive stimulation by a single drug by regulating the body's metabolic balance. More importantly, this combination therapy strategy effectively prevents the development of drug resistance. Based on existing drugs, this invention combines the advantages of enhanced efficacy and improved safety, providing a more optimized treatment option for lung cancer. Attached Figure Description

[0018] Figure 1 This is a statistical graph showing the mRNA expression levels of SQLE in tumors and adjacent normal tissues from the TCGA+GTEx, GSE7670, GSE151102, and GSE31547 cohorts in Example 1. A represents the TCGA+GTEx cohort, B represents the GSE7670 cohort, C represents the GSE151102 cohort, and D represents the GSE31547 cohort.

[0019] Figure 2 The graph shows the relationship between SQLE mRNA expression and overall survival in the TCGA cohort and the GSE11969 cohort in Example 1. A represents the TCGA cohort, and B represents the GSE11969 cohort.

[0020] Figure 3Immunoblot images of SQLE protein expression levels in 8 pairs of tumor tissues (T) and paired adjacent normal tissues (N) from an independent cohort in Example 1;

[0021] Figure 4 The images show the SQLE protein expression levels of the SQLE knockdown lung cancer cell models and their control cell models in Example 2. A represents HCC827 cells, B represents H460 cells, and C represents A549 cells.

[0022] Figure 5 The image shows a comparison of cell growth curves for each group of SQLE-knocked lung cancer cell models and their control cell models in Example 2. A represents HCC827 cells, B represents H460 cells, and C represents A549 cells.

[0023] Figure 6 The images show a comparison of colony formation in each group of SQLE-knocked lung cancer cell models and their control cell models in Example 2. A is a photograph of colony formation, and B is a comparison of the number of colony-forming clones.

[0024] Figure 7 These are comparative images of wound healing in each group of SQLE-knockdown lung cancer cell models and their control cell models in Example 2. A represents HCC827 cells, B represents H460 cells, and C represents A549 cells.

[0025] Figure 8 This is a comparison of wound healing rates between the SQLE knockdown lung cancer cell models and their control cell models in Example 2. A represents HCC827 cells, B represents H460 cells, and C represents A549 cells.

[0026] Figure 9 The images show colonies formed after migration of the SQLE-knocked lung cancer cell models and control cell models in Example 2, along with a comparison of the number of migrated cells. A represents HCC827 cells, B represents H460 cells, and C represents A549 cells.

[0027] Figure 10 The images show the SQLE protein expression levels of the lung cancer cell models overexpressing SQLE and their control cell models in Example 2. A represents A549 cells, B represents LLC cells, and C represents PC9 cells.

[0028] Figure 11 This is a comparison of cell growth curves of lung cancer cell models overexpressing SQLE and their control cell models in Example 2. A represents A549 cells, B represents LLC cells, and C represents PC9 cells.

[0029] Figure 12The images show a comparison of colony formation in each group of lung cancer cell models overexpressing SQLE and their control cell models in Example 2. A is a photograph of colony formation, and B is a comparison of the number of colony-forming clones.

[0030] Figure 13 These are comparative photos of wound healing in each group of lung cancer cell models overexpressing SQLE and their control cell models in Example 2. A represents A549 cells, B represents LLC cells, and C represents PC9 cells.

[0031] Figure 14 This is a comparison of wound healing rates between the lung cancer cell models overexpressing SQLE and their control cell models in Example 2. A represents A549 cells, B represents LLC cells, and C represents PC9 cells.

[0032] Figure 15 The images show the colonies formed after migration of lung cancer cell models overexpressing SQLE and control cell models in Example 2, and the comparison of the number of migrated cells. A represents A549 cells, B represents LLC cells, and C represents PC9 cells.

[0033] Figure 16 The image shows a comparison of the growth of subcutaneous xenografts in mice in each group in Example 3. A represents the appearance of the tumor, B represents the volume of the tumor, and C represents the weight of the tumor.

[0034] Figure 17 This is a comparison of PCNA expression levels in subcutaneous xenografts of mice in Example 3. A is the empty vector control group, and B is the SQLE overexpression group.

[0035] Figure 18 The images show the immunoblot data of SQLE protein expression levels in each group of cells after treatment with terbinafine (TB) in Example 4. A represents H460 cells, and B represents A549 cells.

[0036] Figure 19 This is a comparison of cell cholesterol concentrations in each group of cells after treatment with terbinafine (TB) in Example 4.

[0037] Figure 20 This is a comparison of cell growth curves after terbinafine (TB) treatment in each group of cells in Example 4. A represents H460 cells, and B represents A549 cells.

[0038] Figure 21 The images show a comparison of cell colony formation in each group of cells after terbinafine (TB) treatment in Example 4. A is a photograph of colony formation, and B is a comparison of the number of colony-forming clones.

[0039] Figure 22 The image shows a comparison of cell wound healing in each group of cells after treatment with terbinafine (TB) in Example 4. A is a photograph of wound healing, and B is the wound healing rate.

[0040] Figure 23 The images show the immunoblot data of SQLE protein expression levels in each group of cells after treatment with different drugs in Example 5. A represents H460 cells, and B represents A549 cells.

[0041] Figure 24 This is a comparison of cell growth curves after different drug treatments in each group of cells in Example 5. A represents H460 cells, and B represents A549 cells.

[0042] Figure 25 The images show a comparison of cell colony formation in each group of cells after treatment with different drugs in Example 5. A is a photograph of colony formation, and B is a comparison of the number of colony-forming clones.

[0043] Figure 26 These are wound healing images of cells in each group of cells after treatment with different drugs in Example 5. A represents H460 cells, and B represents A549 cells.

[0044] Figure 27 This is a comparison of the wound healing rates of cells in different groups after treatment with different drugs in Example 5. A represents H460 cells, and B represents A549 cells.

[0045] Figure 28 The image shows a comparison of the growth of subcutaneous xenografts in mice after different drug treatments in Example 5. A represents the appearance of the tumor, B represents the volume of the tumor, and C represents the weight of the tumor.

[0046] Figure 29 The image shows a comparison of SQLE expression levels in subcutaneous xenograft tumors of mice in different groups after treatment with different drugs in Example 5. A is the control solvent group, B is the terbinafine monotherapy group, C is the ellagic acid monotherapy group, and D is the ellagic acid combined with terbinafine synergistic treatment group. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0048] The statistical analysis method for the experimental data of Examples 1-5 of this invention is as follows:

[0049] All statistical analyses were performed using GraphPad Prism (version 8.0.1) or SPSS (version 21.0). Data are expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare multiple groups. Two-tailed Student's t-tests were used to compare two variables. Kaplan-Meier survival curves and OG-rank tests were used to assess the association between overall survival and expression levels. A p-value <0.05 was considered statistically significant.

[0050] Example 1

[0051] This embodiment confirms that SQLE is highly expressed in lung cancer and is associated with poor prognosis.

[0052] First, this embodiment uses a paired non-samples t-test to analyze the SQLE mRNA levels in tumor and normal tissues in the Cancer Genome Atlas (TCGA) and in normal tissues in the Genotype-Tissue Expression Project (GTEx) database. It also examines data from lung cancer-related datasets GSE7670, GSE151102, and GSE31547 from the GEO (Gene Expression Omnibus) database (official website: ncbi.nlm.nih.gov / geo / ).

[0053] Figure 1 This is a statistical plot of SQLE mRNA expression levels in tumors and adjacent normal tissues from the TCGA+GTEx, GSE7670, GSE151102, and GSE31547 cohorts. Figure 1 It can be seen that in the TCGA+GTEx cohort, as well as the GSE7670, GSE151102, and GSE31547 cohorts, the SQLE mRNA expression level in tumor tissues was higher than that in normal adjacent normal tissues.

[0054] This embodiment focuses on investigating the mRNA and protein expression levels of SQLE in tumor and adjacent normal tissues, and analyzing the association between SQLE expression levels and overall survival. In the survival analysis, 498 patients from TCGA and 82 patients from the GSE11969 dataset from the GEO database were divided into a high SQLE expression group and a low SQLE expression group, respectively. Kaplan-Meier curves were used to assess overall survival (OS), and the log-rank test was used to determine differences between groups.

[0055] Figure 2 This is a graph showing the relationship between SQLE mRNA expression and overall survival in the TCGA and GSE11969 cohorts; Figure 2It can be seen that in both the TCGA and GSE11969 cohorts, the overall survival rate of SQLE high-expression samples was significantly lower than that of low-expression samples.

[0056] In this embodiment, eight pairs of lung cancer tumors and their paired adjacent normal tissues from independent cohorts were collected, and the expression level of SQLE protein in the tumor tissues and adjacent normal tissues was examined using conventional immunoblotting analysis methods in the art.

[0057] All human lung cancer tumor tissues and adjacent normal tissues collected in this embodiment were sourced from the Cancer Hospital of Harbin Medical University. This research project has been approved by the Ethics Committee of the Cancer Hospital of Harbin Medical University, and informed consent was obtained from each participating patient.

[0058] Figure 3 Immunoblot images showing SQLE protein expression levels in 8 pairs of tumor tissues (T) and paired adjacent normal tissues (N) from independent cohorts; by Figure 3 It can be seen that in the eight pairs of tumors and their paired adjacent normal tissues in the independent cohort, the expression level of SQLE protein in the tumor tissue was significantly higher than that in the adjacent normal tissue.

[0059] Based on the above findings, it can be concluded that SQLE is overexpressed in human lung cancer and is associated with poor prognosis.

[0060] Example 2

[0061] This embodiment demonstrates through in vitro cell experiments that SQLE promotes the growth and migration of lung cancer cells.

[0062] The lung cancer cell lines used in this embodiment are:

[0063] The LLC cell line of lung cancer was purchased from ATCC (Manassas, Virginia, USA). A549, HCC827 and H460 cells were provided by Professor Sun Xilin of the Fourth Affiliated Hospital of Harbin Medical University, China, and PC9 cells were provided by Professor Zhao Liuyang of Chongqing Medical University.

[0064] The cell culture method in this embodiment is as follows:

[0065] (1) HCC827, H460 and PC9 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin;

[0066] (2) A549 cells were cultured in F-12K medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin;

[0067] (3) LLC cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin;

[0068] All culture conditions were maintained at 37°C in a humid environment containing 5% carbon dioxide. Furthermore, all cell cultures were confirmed to be free of mycoplasma contamination through appropriate testing.

[0069] This embodiment constructed SQLE knockdown lung cancer cell models and control cell models in HCC827, H460, and A549 cells, as well as SQLE overexpression lung cancer cell models and control cell models in A549, LLC, and PC9 cells. The SQLE overexpression lung cancer cell models and control cell models were constructed by transfecting the pCMV-SQLE expression plasmid and the pCMV empty vector plasmid, while the SQLE knockdown lung cancer cell models and control cell models were constructed by transfecting the lentiCRISPRv2-SQLE sgRNA vector and the lentiCRISPRv2 empty vector. All plasmid vectors in this embodiment were commercially available from OriGene.

[0070] The steps for overexpressing SQLE and empty vectors, SQLE knockdown, and transfection with empty vectors are as follows:

[0071] (1) Cell preparation: Select cells in the logarithmic growth phase and seed them in 6-well plates at an appropriate density 24 hours in advance. The cell confluence should be 60%-80% at the time of transfection.

[0072] (2) Preparation of transfection complex:

[0073] Nucleic acid dilution: Dilute 2 μg / well of the corresponding plasmid DNA in Opti-MEM for later use;

[0074] Transfection reagent dilution: Dilute 4 μl / well of lipo2000 in Opti-MEM for later use;

[0075] Reagent mixing: Add the transfection reagent to the diluted nucleic acid, mix gently, and let stand at room temperature for 10 minutes to form a DNA lipid complex.

[0076] (3) Transfection procedure: Remove the original culture medium. Add the obtained DNA lipid complex dropwise to the cell culture wells, gently shake the culture plate to distribute it evenly, and add serum-free culture medium to 2 ml / well. Perform routine transfection incubation for 6 hours, and replace with complete culture medium after 6 hours.

[0077] This embodiment investigated the SQLE expression levels in SQLE-knockdown lung cancer cell models (HCC827, H460, and A549 cells) and their control cell models using Western blotting analysis. The results are as follows:

[0078] (1) Electrophoretic separation: Take 25 μg of protein sample and separate it by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to distribute the protein according to its molecular weight.

[0079] (2) Transfer: Electrotransfer the proteins in the gel to a nitrocellulose membrane (NC membrane) to ensure that the protein bands are transferred intact.

[0080] (3) Blocking: Block with TBST solution containing 3% BSA at room temperature for 2 hours to reduce nonspecific binding.

[0081] (4) Primary antibody incubation: Place the membrane in a primary antibody diluted with 1.5% BSA and incubate overnight at 4°C to allow the antibody to specifically bind to the target protein.

[0082] (5) Secondary antibody incubation: Incubate with HRP-labeled secondary antibody at room temperature for 2 hours to enhance signal detection sensitivity.

[0083] (6) Development: The target protein signal was captured by a chemiluminescence imaging system using an ultrasensitive chemiluminescence reagent (Omin-ECL, YARIC, China). All experiments were performed in triplicate.

[0084] Figure 4 Immunoblot images of SQLE protein expression levels in SQLE-knockdown lung cancer cell models and their control cell models; by Figure 4 As can be seen, compared with the control cell model group, the SQLE protein expression level of each group of SQLE knockdown lung cancer cell models showed a significant decrease, indicating that the SQLE knockdown lung cancer cell model in this embodiment was successfully constructed.

[0085] This embodiment evaluated the cell proliferation activity of SQLE knockdown lung cancer cell models (HCC827, H460, and A549 cells) and control cell models using the MTT assay. The specific method is as follows:

[0086] Cell suspensions were quantitatively seeded into 96-well plates, and 10 μL of MTT reagent (5 mg / mL) was added to each well at the pre-defined observation points. After a 6-hour reaction, the absorbance (OD490) was measured at 490 nm using a spectrophotometer, and the cell proliferation kinetics curve was finally generated. All experiments were performed in triplicate.

[0087] Figure 5 Comparison of cell growth curves for SQLE-knockdown lung cancer cell models and their control cell models; by Figure 5 It can be seen that, compared with the control cell model group, the SQLE knockdown lung cancer cell model showed reduced cell proliferation activity.

[0088] This embodiment investigated the colony formation assay of SQLE knockdown lung cancer cell models (HCC827, H460, and A549 cells) and control cell models. The specific methods are as follows:

[0089] Single-cell suspensions were seeded at a density of 1000 cells / well in 6-well plates, with 3 parallel wells per group. The plates were incubated at 37°C with 5% CO2 for 8–14 days, with the medium replaced every 3 days. After incubation, the medium was discarded, and the cells were fixed with 70% ethanol at room temperature for 15 minutes, followed by staining with 0.5% crystal violet solution for 15 minutes. Colonies containing ≥50 cells were counted, and the colony formation rate was calculated. All experiments were performed in triplicate.

[0090] Figure 6 Comparison of colony formation in SQLE-knockdown lung cancer cell models and their control cell models; by Figure 6 It can be seen that, compared with the control cell model group, the number of cells proliferating in the SQLE knockdown lung cancer cell model was significantly reduced.

[0091] This embodiment investigated wound healing experiments using SQLE knockdown lung cancer cell models (HCC827, H460, and A549 cells) and control cell models. The specific methods are as follows:

[0092] Cells were seeded at an appropriate density in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to form a monolayer. Straight wounds were made on the cell monolayer using a 200 μL sterile pipette tip (ensuring consistent wound width). Images of the wound areas were taken using an inverted microscope at different time points. The wound width was measured at different time points, and cell migration rate was calculated. All experiments were performed in triplicate.

[0093] Figure 7 and Figure 8 The images show comparative photos and wound healing rates of SQLE-knocked lung cancer cell models and their control cell models, respectively. Figure 7 , Figure 8 It can be seen that, compared with the control cell model group, the SQLE knockdown lung cancer cell model has weakened cell migration ability.

[0094] This example investigated the Transwell migration assay of SQLE-knockdown lung cancer cell models (HCC827, H460, and A549 cells) and control cell models. The specific methods are as follows:

[0095] The Transwell migration assay was performed using Transwell chambers according to the established protocol. The cells were resuspended in 200 µl of serum-free starvation medium and placed in the upper chamber, while 600 µl of complete medium was added to the lower chamber. The cells were incubated at 37 °C for 48 hours, then fixed with 4% formaldehyde and stained with 0.5% crystal violet. All experiments were repeated three times.

[0096] Figure 9 It is a comparison graph of cell migration of lung cancer cell models with SQLE knockdown and their control cell models for each group; Figure 9 It can be seen that compared with the control cell model group, the cell invasion ability of the lung cancer cell model with SQLE knockdown is weakened.

[0097] In this example, the same immunoblot analysis method was used to investigate the SQLE expression levels of lung cancer cell models with overexpressed SQLE and their control cell models in A549 cells, LCC cells, and PC9 cells. [[ID=X]] Figure 10 It is an immunoblot of the SQLE protein expression levels of lung cancer cell models with overexpressed SQLE and their control cell models for each group; Figure 10 It can be seen that compared with the control cell model group, the SQLE protein expression levels of lung cancer cell models with overexpressed SQLE in each group showed a significant increase, indicating that the lung cancer cell models with overexpressed SQLE in this example were successfully constructed.

[0098] In this example, the cell proliferation activities of lung cancer cell models with overexpressed SQLE and their control cell models in A549 cells, LCC cells, and PC9 cells were evaluated by the MTT assay. Figure 11 It is a comparison of the cell growth curves of lung cancer cell models with overexpressed SQLE and their control cell models for each group; Figure 11 It can be seen that compared with the control cell model group, the cell proliferation activity of the lung cancer cell model with overexpressed SQLE was enhanced.

[0099] In this example, the colony formation assays of lung cancer cell models with overexpressed SQLE and their control cell models in A549 cells, LCC cells, and PC9 cells were investigated. Figure 12 It is a comparison graph of colony formation of lung cancer cell models with overexpressed SQLE and their control cell models for each group; Figure 12 It can be seen that compared with the control cell model group, the number of proliferating cells in the lung cancer cell model with overexpressed SQLE was significantly increased.

[0100] In this example, the wound healing experiments of lung cancer cell models with overexpressed SQLE and their control cell models in A549 cells, LCC cells, and PC9 cells were investigated. Figure 13 and Figure 14The images show comparative photos and wound healing rates of lung cancer cell models overexpressing SQLE and their control cell models, respectively. Figure 13 , Figure 14 It can be seen that, compared with the control cell model group, the lung cancer cell model overexpressing SQLE has enhanced cell migration ability.

[0101] This embodiment investigated the Transwell migration assays of lung cancer cell models overexpressing SQLE, including A549, LCC, and PC9 cells, and their control cell models. Figure 15 Comparison of cell migration in lung cancer cell models overexpressing SQLE and their control cell models; by Figure 15 It can be seen that, compared with the control cell model group, the lung cancer cell model overexpressing SQLE has enhanced cell invasion ability.

[0102] The experimental data in this embodiment show that the expression level of SQLE is positively correlated with the malignant phenotype of lung cancer cells.

[0103] Example 3

[0104] This embodiment demonstrates through animal model experiments that SQLE promotes the development and progression of lung cancer in vivo.

[0105] In this embodiment, a xenograft human lung cancer mouse model was established using an A549 cell line with stable SQLE overexpression. The specific method for constructing the model is as follows:

[0106] Six-week-old male Balb / c nude mice were selected for subcutaneous xenograft experiments. Cells from the A549-empty vector control group and the A549-SQLE overexpression group (cells suspended in 0.05 ml PBS and 0.05 ml Matrigel Matrix, each group containing 3 × 10⁻⁶ cells) were used. 6 (1 cell) was subcutaneously injected into the right dorsal side of nude mice. The experimental animals were then treated at designated time points, and the mice were sacrificed and examined on day 18 after transplantation.

[0107] Figure 16 This is a comparison chart of the growth of subcutaneous xenografts in each group of mice. Figure 16 It can be seen that, compared with the control group, the tumors induced by cells in the A549-SQLE overexpression group were significantly larger in size and weight than those in the control group.

[0108] Figure 17 This is a comparison of PCNA expression levels in subcutaneous xenografts of mice in different groups. Figure 17 It can be seen that the expression level of PCNA (proliferating cell nuclear antigen) in subcutaneous xenografts induced by A549-SQLE overexpression group was significantly higher than that in the control group.

[0109] The above findings strongly suggest that SQLE plays a carcinogenic role in the development of lung tumors in vivo.

[0110] Example 4

[0111] This embodiment demonstrates through in vitro cell experiments that terbinafine inhibits the growth of lung cancer cells and increases the expression level of SQLE protein.

[0112] This embodiment used H460 cells and A549 cells as experimental cells to investigate the synthesis of SQLE protein and cholesterol in cells treated with terbinafine. The specific experimental method is as follows:

[0113] Cells were seeded at 50% confluence in 6-well plates and treated the following day with either DMSO (solvent control) or 60 µM terbinafine. After 48 hours, cells were collected for cholesterol content determination and protein extraction analysis.

[0114] The method for determining cholesterol content is as follows: Use 10 6 Cholesterol concentration was quantified using individual cells and measured using a cholesterol quantification kit (ab65359, Abcam). All experiments were performed in triplicate.

[0115] Figure 18 Immunoblot images of SQLE protein expression levels in each group of cells after terbinafine treatment; by Figure 18 It can be seen that, compared with the control group, the expression level of SQLE protein in lung cancer cells increased after treatment with terbinafine, thus proving that the use of terbinafine alone leads to compensatory upregulation of SQLE expression level.

[0116] Figure 19 This is a comparison of cholesterol concentrations in cells of different groups after terbinafine treatment; Figure 19 It can be seen that, compared with the control group, the cholesterol content of lung cancer cells decreased after terbinafine treatment, indicating that terbinafine can reduce the cholesterol level in tumor cells.

[0117] This embodiment used H460 cells and A549 cells as experimental cells to investigate the cell proliferation activity and colony formation of cells treated with terbinafine. The specific experimental method is as follows:

[0118] Cells were seeded in 96-well plates the day before, and treated with DMSO (solvent control) or 60 µM terbinafine the next day. MTT assay or colony formation ability assessment was then performed, respectively.

[0119] Figure 20 and Figure 21 The images show comparisons of cell growth curves and colony formation after terbinafine (TB) treatment in each group. Figure 20 , Figure 21 It can be seen that, compared with the control group, the proliferation activity and colony formation of lung cancer cells were significantly reduced after terbinafine treatment, indicating that terbinafine can significantly reduce the proliferation capacity of lung cancer tumor cells.

[0120] This embodiment used H460 cells and A549 cells as experimental cells to investigate the cell migration ability of cells treated with terbinafine. The specific experimental method is as follows:

[0121] Cells were seeded at 80% confluence. The next day, wounds were created in the cell monolayer using pipette tips, and the cells were treated with DMSO (solvent control) or 60 µM terbinafine. Migration was observed and recorded at different time points.

[0122] Figure 22 This is a comparison chart showing the wound healing status of cells in different groups after treatment with terbinafine (TB). Figure 22 It can be seen that, compared with the control group, the wound healing rate of lung cancer cells was significantly reduced after terbinafine treatment, indicating that terbinafine can significantly reduce the migration ability of lung cancer tumor cells.

[0123] The above results indicate that terbinafine inhibits the growth of lung cancer in vitro and in vivo, but increases the expression level of SQLE protein.

[0124] Example 5

[0125] This embodiment demonstrates the efficacy of combined treatment with ellagic acid and terbinafine through in vitro cell experiments and in vivo animal model experiments.

[0126] This embodiment used H460 cells and A549 cells as experimental cells to investigate the synthesis of SQLE protein in cells after combined treatment with terbinafine (TB) and ellagic acid (EA). The specific experimental method is as follows:

[0127] Cells were seeded at a density of 50% into 6-well plates. The following day, cells were treated with DMSO (as a solvent control), 60 µM terbinafine, 40 µM ellagic acid, or a combination of 60 µM terbinafine and 40 µM ellagic acid, respectively. Cells were collected and proteins were extracted for analysis after 48 hours.

[0128] Figure 23 Immunoblot images of SQLE protein expression levels in cells of different groups after treatment with different drugs; by Figure 23 It can be seen that, compared with the control group, terbinafine monotherapy increased the SQLE expression level in lung cancer cells, ellagic acid monotherapy decreased the SQLE expression level, and the synergistic treatment of ellagic acid and terbinafine effectively reduced the compensatory increase in SQLE protein expression caused by terbinafine monotherapy.

[0129] This embodiment used H460 cells and A549 cells as experimental cells to investigate the proliferation activity and colony formation of cells after combined treatment with terbinafine (TB) and ellagic acid (EA). The specific experimental method is as follows:

[0130] Cells were seeded in 96-well plates the day before, and treated the following day with DMSO (as a solvent control), 60 µM terbinafine, 40 µM ellagic acid, or a combination of 60 µM terbinafine and 40 µM ellagic acid, respectively. Cells were collected 48 hours later for MTT assay and colony formation capacity assessment.

[0131] Figure 24 and Figure 25 The images show comparisons of cell growth curves and colony formation after different drug treatments in each group. Figure 24 , Figure 25 It can be seen that, compared with terbinafine and ellagic acid monotherapy, the synergistic treatment of ellagic acid combined with terbinafine significantly reduced the proliferation capacity of tumor cells.

[0132] This embodiment used H460 cells and A549 cells as experimental cells to investigate the cell migration ability of cells after combined treatment with terbinafine (TB) and ellagic acid (EA). The specific experimental method is as follows:

[0133] Cells were seeded at 80% confluence. The next day, wounds were created in the cell monolayer using pipette tips, and the cells were treated with DMSO (as a solvent control), 60 µM terbinafine, 40 µM ellagic acid, or a combination of 60 µM terbinafine and 40 µM ellagic acid. Migration was observed and recorded at different time points.

[0134] Figure 26 and Figure 27 The images show photographs and comparison charts of wound healing rates in cells treated with different drugs in each group. Figure 26 , Figure 27 It can be seen that, compared with terbinafine and ellagic acid monotherapy, the synergistic treatment of ellagic acid combined with terbinafine significantly reduced the migration ability of tumor cells.

[0135] This embodiment uses the human lung cancer cell line A549 to construct a xenograft human lung cancer mouse model. The specific construction method is as follows:

[0136] At designated time points, 6-week-old male Balb / c nude mice were randomly divided into four groups. Each group received either terbinafine (70 mg / kg / day), ellagic acid (40 mg / kg / day), a combination of ellagic acid (40 mg / kg / day) and terbinafine (70 mg / kg / day), or phosphate-buffered saline (PBS, as a control) via oral gavage. Mice were sacrificed and examined on day 21 after transplantation.

[0137] Figure 28 This is a comparison chart showing the growth of subcutaneous xenograft tumors in mice after different drug treatments. Figure 28 It can be seen that, compared with the control group, the terbinafine monotherapy group, and the ellagic acid monotherapy group, the synergistic treatment of ellagic acid combined with terbinafine had the most significant inhibitory effect on tumors, with the lowest tumor volume and weight.

[0138] Figure 29 This is a comparison of SQLE expression levels in subcutaneous xenografts of mice in different groups after treatment with different drugs. Figure 29 It can be seen that, compared with the control group, terbinafine monotherapy increased the expression level of SQLE, while the synergistic treatment of ellagic acid combined with terbinafine effectively reduced the compensatory increase in SQLE protein expression caused by terbinafine monotherapy.

[0139] In conclusion, ellagic acid combined with terbinafine treatment can reduce the compensatory upregulation of SQLE expression levels caused by terbinafine alone and significantly enhance the inhibitory effect on lung cancer growth.

Claims

1. The use of a composition of ellagic acid and terbinafine in the preparation of a medicament for treating lung cancer, characterized in that, The mass ratio of ellagic acid to terbinafine is 4:

7.

2. The use of the ellagic acid and terbinafine composition according to claim 1 in the preparation of a medicament for treating lung cancer, characterized in that, The medication for treating lung cancer is administered orally.

3. The use of the ellagic acid and terbinafine composition according to claim 2 in the preparation of a medicament for treating lung cancer, characterized in that, The drug for treating lung cancer also contains pharmaceutically acceptable carriers or excipients.

4. The use of the ellagic acid and terbinafine composition according to claim 3 in the preparation of a medicament for treating lung cancer, characterized in that, The medication for treating lung cancer is in the form of granules, tablets, capsules, pills, or oral liquid preparations.

5. The use of the ellagic acid and terbinafine composition according to claim 4 in the preparation of a medicament for treating lung cancer, characterized in that, The lung cancer in question is non-small cell lung cancer.

6. The use of the ellagic acid and terbinafine composition according to claim 5 in the preparation of a medicament for treating lung cancer, characterized in that, The non-small cell lung cancer mentioned refers to adenocarcinoma or large cell carcinoma of non-small cell lung cancer.

7. The use of the ellagic acid and terbinafine composition according to claim 5 in the preparation of a medicament for treating lung cancer, characterized in that, The drug for treating lung cancer can simultaneously inhibit SQLE enzyme activity and compensatory upregulate SQLE expression.

8. The use of the ellagic acid and terbinafine composition according to claim 5 in the preparation of a medicament for treating lung cancer, characterized in that, The drug used to treat lung cancer can disrupt the lipid metabolism of lung cancer tumor cells and block cholesterol synthesis.

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