Ellagic acid and terbinafine composition and its application in the treatment of bladder cancer

Abstract

This invention relates to a composition of ellagic acid and terbinafine and its application in the preparation of drugs for treating bladder cancer, belonging to the field of biomedical technology. To address the problem that terbinafine treatment of bladder cancer easily causes compensatory increases in SQLE expression levels, this invention provides a composition of ellagic acid and terbinafine, consisting of ellagic acid and terbinafine in a mass ratio of 5:8. This invention uses this composition in the preparation of drugs for treating bladder cancer. Terbinafine can directly inhibit SQLE enzyme activity, blocking cholesterol synthesis and inhibiting tumor growth, while ellagic acid can reduce the compensatory upregulation of SQLE expression caused by terbinafine alone. The synergistic effect of the two can reduce the metabolic side effects caused by terbinafine and the drug toxicity of ellagic acid, significantly enhancing the anti-bladder cancer effect and overcoming monotherapy resistance. This invention is based on the development of already marketed drugs, possessing the dual advantages of therapeutic enhancement and improved safety, providing a more optimized treatment plan for bladder cancer.

Description

Technical Field

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

[0002] Bladder cancer (BCa) is a common malignant tumor of the urinary system worldwide. Current treatments include surgical resection, chemotherapy, and immunotherapy, but these methods suffer from high recurrence rates and strong drug resistance. In recent years, targeted therapy has become a new direction in bladder cancer research. Among these treatments, squalene epoxidase (SQLE), a key enzyme in the cholesterol synthesis pathway, has been shown to be closely related to the development and progression of various tumors.

[0003] Terbinafine, a traditional antifungal drug, has been found to effectively inhibit SQLE activity, showing potential application value in the treatment of bladder cancer. However, the use of terbinafine leads to a compensatory increase in SQLE expression levels. This feedback regulation mechanism not only reduces the efficacy of the drug but may also cause other side effects such as metabolic disorders.

[0004] Ellagic acid, a natural polyphenol compound, has been shown to possess significant antitumor activity in numerous studies. However, its low bioavailability, insufficient systemic exposure, and time-dependent increase in toxicity limit its therapeutic efficacy as an anticancer drug. Summary of the Invention

[0005] To address the issue that terbinafine treatment for bladder cancer can easily lead to a compensatory increase in SQLE expression levels, this invention provides a composition of ellagic acid and terbinafine, and its application in the preparation of drugs for treating bladder cancer.

[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 5:8.

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

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

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

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

[0012] Furthermore, the bladder cancer is urothelial carcinoma of the bladder.

[0013] Furthermore, the drug for treating bladder cancer can simultaneously inhibit SQLE enzyme activity and compensatory upregulate SQLE expression.

[0014] Furthermore, the drug for treating bladder cancer can disrupt the lipid metabolism of bladder cancer tumor cells and block cholesterol synthesis.

[0015] The beneficial effects of this invention are:

[0016] This invention discovers that ellagic acid can effectively inhibit the increase in SQLE expression levels induced by terbinafine treatment, thus providing a synergistic drug composition of ellagic acid and terbinafine. When this drug composition is used to treat bladder cancer, it synergistically inhibits SQLE through a dual mechanism, blocks cholesterol biosynthesis pathways, inhibits tumor growth, and significantly enhances the anti-bladder cancer effect.

[0017] In this invention, terbinafine directly inhibits SQLE enzyme activity, blocking cholesterol synthesis, while ellagic acid reduces the compensatory upregulation of SQLE expression caused by terbinafine alone. The two work synergistically to reduce the metabolic side effects of terbinafine and the drug toxicity of ellagic acid, while also overcoming monotherapy resistance. This invention, developed based on already marketed drugs, offers the dual advantages of synergistic therapeutic effects and improved safety, providing a more optimized treatment option for bladder cancer. Attached Figure Description

[0018] Figure 1 The graph shows the mRNA expression levels of SQLE in tumors and adjacent normal tissues from different cohorts in Example 1. A is the TCGA cohort, B is the GSE3167 cohort, and C is the GSE188715 cohort.

[0019] Figure 2 This is a graph showing the relationship between SQLE mRNA expression and overall survival in the TCGA cohort of Example 1;

[0020] Figure 3 The images show the immunoblot and statistical graph of SQLE protein expression levels in 8 pairs of tumors (T) and paired adjacent normal tissues (N) in an independent cohort in Example 1. A is the immunoblot image of SQLE protein expression levels, and B is the statistical graph of expression levels.

[0021] Figure 4The images show representative immunofluorescence staining photographs and relative fluorescence intensity statistics of SQLE protein expression levels in tumor and adjacent normal tissues in Example 1. A is an immunofluorescence staining photograph, in which SQLE is orange and DAPI is blue. B is a relative fluorescence intensity statistics.

[0022] Figure 5 The image shows representative SQLE immunohistochemical staining of bladder cancer tissue and adjacent normal tissue in tissue microarray analysis in Example 1. A represents adjacent normal tissue, and B represents tumor.

[0023] Figure 6 This is a statistical graph of SQLE immunohistochemical staining in bladder cancer tissue and adjacent normal tissue in tissue microarray analysis in Example 1;

[0024] Figure 7 The image shows immunohistochemical staining representations of high, medium, and low expression of SQLE protein in bladder cancer samples from the tissue microarray analysis in Example 1. A represents low expression, B represents medium expression, and C represents high expression.

[0025] Figure 8 This is a graph showing the relationship between high, moderate, and low expression of SQLE protein and survival rate in bladder cancer samples in Example 1.

[0026] Figure 9 This is a comparison of SQLE protein expression levels in the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2. A represents UMUC3 cells, and B represents J82 cells.

[0027] Figure 10 The growth curves of the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2 are shown in Figure 2. A represents UMUC3 cells and B represents J82 cells.

[0028] Figure 11 The images show colony formation staining and statistical diagrams of the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2. A is the colony formation staining image, and B is the colony formation statistical diagram.

[0029] Figure 12 The images show a comparison of wound healing in the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2, along with a statistical chart of wound healing rate. A is a comparison of wound healing in UMUC3 cells, B is a comparison of wound healing in J82 cells, and C is a statistical chart of wound healing rate.

[0030] Figure 13 The images show the staining patterns of migrating cells and the relative number of migrating cells in the Transwell experimental control group and UMUC3 and J82 cells overexpressing SQLE in Example 2. A is the staining pattern of migrating cells, and B is the relative number of migrating cells.

[0031] Figure 14 The flow cytometry cell cycle diagrams for the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2 are shown. A represents UMUC3 cells and B represents J82 cells.

[0032] Figure 15 The flow cytometry analysis of cell cycle statistics of the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2 is shown in Figure 2. A represents UMUC3 cells and B represents J82 cells.

[0033] Figure 16 This is a comparison of SQLE protein expression levels in the control group and SQLE-knocked 647V and UMUC3 cells in Example 2. A represents 647V cells, and B represents UMUC3 cells.

[0034] Figure 17 The growth curves of the control group and SQLE-knocked 647V and UMUC3 cells in Example 2 are shown in Figure 2. A represents 647V cells and B represents UMUC3 cells.

[0035] Figure 18 The images show the colony formation staining and statistical diagrams of the control group and SQLE-knocked 647V and UMUC3 cells in Example 2. A is the colony formation staining image, and B is the colony formation statistical diagram.

[0036] Figure 19 The images show a comparison of wound healing in the control group and SQLE-knocked 647V and UMUC3 cells in Example 2, along with a statistical chart of wound healing rate. A is a comparison of wound healing in 647V cells, B is a comparison of wound healing in UMUC3 cells, and C is a statistical chart of wound healing rate.

[0037] Figure 20 The images show the staining patterns of migrating cells and the relative number of migrating cells in the control group and SQLE-knocked 647V and UMUC3 cells in the Transwell experiment of Example 2. A is the staining pattern of migrating cells, and B is the relative number of migrating cells.

[0038] Figure 21 The flow cytometry analysis of cell cycle diagrams in Example 2, which were obtained by analyzing cell number in the control group and SQLE-knocked 647V and UMUC3 cells, shows that A represents 647V cells and B represents UMUC3 cells.

[0039] Figure 22 The flow cytometry analysis of cell cycle statistics in Example 2 shows the control group and SQLE-knocked 647V and UMUC3 cells, with A representing 647V cells and B representing UMUC3 cells.

[0040] Figure 23 The images show actual tumor photographs and tumor volume and weight statistics of the empty vector control group and the subcutaneous xenografts derived from J82 cells overexpressing SQLE in Example 3. A is an actual tumor photograph, B is a tumor volume statistics, and C is a tumor weight statistics.

[0041] Figure 24 This is a comparison of the expression levels of PCNA and cyclin D1 proteins in the control group and the subcutaneous xenografts of J82 cells overexpressing SQLE in Example 3. A is the control group and B is the SQLE overexpression group.

[0042] Figure 25 The images show actual tumor photographs and tumor volume and weight statistics of the control group and the UMUC3 cell-derived subcutaneous xenografts with SQLE knockdown in Example 3. A is an actual tumor photograph, B is a tumor volume statistics, and C is a tumor weight statistics.

[0043] Figure 26 This is a comparison of the expression levels of PCNA and cyclin D1 proteins in the control group and the SQLE-knockdown UMUC3 cell-derived subcutaneous xenografts in Example 3. A is the control group and B is the SQLE-knockdown group.

[0044] Figure 27 From left to right, the images show representative MRI images, gross histological images, HE staining images, and magnified HE staining images of the bladders of Rosa26-Sqle mice and Sqle bladTg mice in Example 3. A represents Rosa26-Sqle mice, and B represents Sqle bladTg mice.

[0045] Figure 28 The graph shows the incidence and severity of bladder tumors in Rosa26-Sqle mice and Sqle bladTg mice in Example 3. A represents the incidence of tumors, and B represents the severity of disease.

[0046] Figure 29 The images show representative Ki-67 staining patterns and Ki-67 statistical graphs of the bladders of Rosa26-Sqle mice and Sqle bladTg mice in Example 3. A is a Ki-67 staining pattern, and B is a Ki-67 statistical graph.

[0047] Figure 30 This is a comparison of the expression levels of PCNA and cyclin D1 proteins in the bladder tissue of Rosa26-Sqle mice and Sqle bladTg mice in Example 3. A represents Rosa26-Sqle mice, and B represents Sqle bladTg mice.

[0048] Figure 31From left to right, the images show representative MRI images, gross histological images, HE staining images, and magnified HE staining images of the bladders of Sqlefl / fl mice and Sqle bladKO mice in Example 3. A represents Sqlefl / fl mice, and B represents Sqle bladKO mice.

[0049] Figure 32 The graph shows the incidence and severity of bladder tumors in Sqlefl / fl mice and Sqle bladKO mice in Example 3. A represents the incidence of tumors, and B represents the severity of disease.

[0050] Figure 33 The images show representative Ki-67 staining patterns and Ki-67 statistical graphs of the bladders of Sqlefl / fl mice and Sqle bladKO mice in Example 3. A is a Ki-67 staining pattern, and B is a Ki-67 statistical graph.

[0051] Figure 34 This is a comparison of the expression levels of PCNA and cyclin D1 proteins in the bladder tissue of Sqlefl / fl mice and Sqle bladKO mice in Example 3. A represents Sqlefl / fl mice, and B represents Sqle bladKO mice.

[0052] Figure 35 This is a statistical graph showing the cholesterol levels in each group after treatment with the control group and terbinafine in Example 4.

[0053] Figure 36 The image shows a comparison of the growth curves of cells in each group after treatment with the control solvent and terbinafine in Example 4. A represents J82 cells, B represents UMUC3 cells, C represents RT112 cells, and D represents TCCSUP cells.

[0054] Figure 37 The images show staining diagrams and statistical graphs of cell colony formation in each group after treatment with the control solvent and terbinafine in Example 4. A is a staining diagram of cell colony formation, and B is a statistical graph of cell colony formation.

[0055] Figure 38 The image shows a comparison of wound healing in each group of cells after treatment with the control solvent and terbinafine in Example 4. A represents J82 cells, B represents UMUC3 cells, C represents RT112 cells, and D represents TCCSUP cells.

[0056] Figure 39 This is a statistical graph showing the wound healing rate of cells in each group after treatment with the control solvent and terbinafine in Example 4.

[0057] Figure 40In Example 4, the actual tumor photographs and tumor volume and weight statistics of subcutaneous tumors after oral administration of the control solvent and terbinafine are shown in Figure 4. A is a photograph of the actual tumor, B is a statistical chart of tumor volume, and C is a statistical chart of tumor weight.

[0058] Figure 41 From left to right, the images show representative gross histological images, HE staining images, and magnified partial images of the bladders of mice with orthotopic bladder cancer treated with oral control solvent and terbinafine in Example 4. A represents the control solvent group, and B represents the terbinafine treatment group.

[0059] Figure 42 This is a statistical chart showing the incidence and severity of bladder tumors in mice with orthotopic bladder cancer treated with the oral control solvent and terbinafine in Example 4. A represents the incidence of tumors, and B represents the severity of disease.

[0060] Figure 43 The images show representative Ki-67 staining and Ki-67 statistical diagrams of the bladders of mice with orthotopic bladder cancer treated with the oral control solvent and terbinafine in Example 4. A is the Ki-67 staining image, and B is the Ki-67 statistical diagram.

[0061] Figure 44 This is a comparison of SQLE protein expression levels in cells of different groups after treatment with the control solvent and terbinafine in Example 4. A represents UMUC3 cells, B represents RT112 cells, and C represents TCCSUP cells.

[0062] Figure 45 The growth curves of cells in the control group and ellagic acid-treated groups in Example 5 are shown in the figure. A is UMUC3 cells, B is J82 cells, C is 647V cells, D is T24 cells, and E is RT112 cells.

[0063] Figure 46 The images show the colony formation staining diagrams and colony formation statistics of cells in the control group and after ellagic acid treatment in Example 5. A represents UMUC3 cells, B represents J82 cells, C represents 647V cells, D represents T24 cells, and E represents RT112 cells.

[0064] Figure 47 This is a statistical graph showing the colony formation of cells in the control group and ellagic acid-treated groups in Example 5.

[0065] Figure 48 This is a comparison of SQLE protein expression levels in cells of the control group and ellagic acid-treated groups in Example 5. A represents UMUC3 cells, B represents 647V cells, and C represents J82 cells.

[0066] Figure 49This is a comparison of SQLE protein expression levels in J82 and RT112 bladder cancer cells from different treatment groups in Example 6. A represents J82 cells, and B represents RT112 cells.

[0067] Figure 50 This is a comparison of the growth curves of bladder cancer J82 and RT112 cells in each treatment group in Example 6. A represents J82 cells, and B represents RT112 cells.

[0068] Figure 51 This is a staining image showing colony formation of bladder cancer J82 and RT112 cells in each treatment group in Example 6;

[0069] Figure 52 This is a statistical diagram of colony formation of bladder cancer J82 and RT112 cells in each treatment group in Example 6;

[0070] Figure 53 These are photographs of the actual tumors of the subcutaneous xenografts in each treatment group in Example 6;

[0071] Figure 54 This is a statistical chart of tumor volume of subcutaneous xenografts in each treatment group in Example 6;

[0072] Figure 55 This is a statistical chart of tumor weight of subcutaneous xenografts in each treatment group in Example 6;

[0073] Figure 56 This is a comparison of SQLE protein levels in subcutaneous xenograft tumors of mice in each treatment group in Example 6. 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

[0074] 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.

[0075] The statistical analysis method for the experimental data of Examples 1-6 of this invention is as follows:

[0076] 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.

[0077] Example 1

[0078] This embodiment confirms that SQLE is highly expressed in bladder cancer and is associated with poor prognosis.

[0079] First, this embodiment uses paired-samples t-test to analyze the SQLE mRNA levels between tumor tissues and normal tissues in the Cancer Genome Atlas (TCGA), and uses unpaired-samples t-test to analyze data from datasets GSE3167 and GSE188715 from the GEO (GeneExpression Omnibus) database (official website: ncbi.nlm.nih.gov / geo / ).

[0080] Depend on Figure 1 It can be seen that the mRNA expression level of SQLE in tumor tissues of the TCGA cohort, GSE3167 cohort, and GSE188715 cohort is higher than that in normal adjacent normal tissues.

[0081] This embodiment investigated the expression levels of SQLE mRNA and protein in tumor tissues and adjacent normal tissues, and the relationship between different SQLE expression levels and overall survival. In the survival analysis, 407 patients from TCGA were divided into a high SQLE expression group and a low SQLE expression group. Kaplan-Meier curves were plotted to assess overall survival (OS), and the differences between groups were assessed using the log-rank test.

[0082] Depend on Figure 2 It can be seen that in the TCGA cohort, the overall survival rate of samples with high SQLE expression is significantly lower than that of samples with low SQLE expression.

[0083] In this embodiment, eight pairs of bladder 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.

[0084] All human bladder cancer tumor tissues and adjacent normal tissues collected in this study were obtained 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 has been obtained from each participating patient. The human bladder cancer tissue microarray (HBlaU079Su01) used in this study was purchased from OUTDO Biotechnology Co., Ltd. (Shanghai, China).

[0085] Depend on Figure 3 It can be seen that in the 8 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.

[0086] In this embodiment, immunohistochemical and immunofluorescence analyses were performed on human bladder cancer tumor tissue and adjacent normal tissue. The specific methods are as follows:

[0087] Immunohistochemistry:

[0088] Fresh tumors were fixed overnight in 4% paraformaldehyde and then transferred to 70% ethanol for 12 hours. Specimens were embedded in paraffin and sectioned into 4µm sections. Sections were dewaxed by baking at 60°C for 30 minutes. Paraffin-embedded tissue sections were dewaxed using xylene, treated sequentially with 100%, 90%, and 70% ethanol, and then rinsed with water. Antigen retrieval was performed on the sections. For immunohistochemical staining, endogenous peroxidase activity was inhibited for 15 minutes at room temperature with 3% hydrogen peroxide. Tissues were blocked with 5% goat serum for 1 hour and incubated with primary antibody overnight at 4°C. After washing with PBS-T, secondary antibody was applied for 1 hour at room temperature.

[0089] Immunofluorescence:

[0090] For tissue analysis, sections were dewaxed by baking at 60°C for 30 minutes. After dewaxing and rehydration, antigen retrieval was performed. Sections were incubated overnight at 4°C, then washed three times with PBST and incubated with fluorescently labeled secondary antibody for one hour. After washing three times with PBST, cell nuclei were stained with DAPI, and the tissues were imaged using a laser scanning confocal microscope (Zeiss LSM 800).

[0091] Depend on Figure 4 It can be seen that the relative fluorescence intensity of SQLE protein in tumor tissue is significantly higher than that in adjacent normal tissue. Figure 5 and Figure 6 It can be seen that, in tissue microarray analysis, the immunohistochemical score of SQLE protein expression in tumor tissue was higher than that in adjacent normal tissue. Figure 7 and Figure 8 It can be seen that the overall survival rate of tumor samples with high levels of SQLE protein expression is lower than that of samples with medium and low levels.

[0092] The above findings indicate that SQLE is overexpressed in human bladder cancer (BCa) and is associated with poor prognosis.

[0093] Example 2

[0094] This embodiment demonstrates through in vitro cell experiments that SQLE promotes the growth and migration of bladder cancer cells.

[0095] The bladder cancer cell lines used in this embodiment are:

[0096] Bladder cancer cell line J82 was purchased from ATCC (Manassas, Virginia, USA), while 647V and UMUC3 cells were provided by Professor Xu Wanhai of the Cancer Hospital of Harbin Medical University, China. All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin under humidified conditions of 37 °C and 5% carbon dioxide. Furthermore, all cell cultures were confirmed to be free of mycoplasma contamination through appropriate assays.

[0097] This embodiment constructed bladder cancer cell models overexpressing SQLE in UMUC3 and J82 cells and their control cell models, as well as bladder cancer cell models with SQLE knockdown in 647V and UMUC3 cells and their control cell models. The SQLE-overexpressing bladder cancer cell models and their control cell models were constructed by transfecting the pRL-cyto-megalovirus (pCMV)-SQLE plasmid and the pCMV-empty vector plasmid. The SQLE knockdown bladder cancer cell models and their control cell models were constructed by transfecting SQLE sgRNA (lentiCRISPRv2-SQLE) and the empty vector plasmid (lentlCRISPPR v2). All plasmids were purchased from OriGene.

[0098] The steps for overexpressing SQLE and empty vectors, SQLE knockdown, and transfection with empty vectors are as follows:

[0099] (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.

[0100] (2) Preparation of transfection complex:

[0101] Nucleic acid dilution: Dilute 2 μg / well of the corresponding plasmid DNA in Opti-MEM for later use;

[0102] Transfection reagent dilution: Dilute 4 μl / well of lipo2000 in Opti-MEM for later use;

[0103] Reagent mixing: Add the transfection reagent to the diluted nucleic acid, mix gently, and let stand at room temperature for 20 minutes to form a DNA lipid complex.

[0104] (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.

[0105] This embodiment investigated the SQLE expression levels in a bladder cancer cell model overexpressing SQLE in UMUC3 and J82 cells, as well as a control cell model, using Western blotting analysis. The results are as follows:

[0106] 25 µg of protein was loaded and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a nitrocellulose membrane. After blocking with TBST solution containing 3% bovine serum albumin (BSA) for 2 hours at room temperature, the membrane was incubated overnight at 4°C with primary antibody in 1.5% BSA, followed by incubation with secondary antibody for 2 hours at room temperature. The target protein was visualized using an enhanced chemiluminescence reagent (Omin-ECL, Yaxin, China). All experiments were performed in triplicate.

[0107] like Figure 9 As shown, compared with the empty vector control group, the SQLE protein expression level of each group of bladder cancer cell models overexpressing SQLE was significantly increased, indicating that the bladder cancer cell model overexpressing SQLE in this embodiment was successfully constructed.

[0108] This embodiment evaluated the cell proliferation activity of UMUC3 cells and J82 cells overexpressing SQLE in a bladder cancer cell model and its control cell model using the MTT assay. The specific method is as follows:

[0109] Cells were seeded into 96-well plates. At specific time points, 10 μl of MTT solution (5 mg / ml) was added to each well. Subsequently, the optical density (OD490) was measured at 490 nm using a spectrophotometer to construct cell growth curves. All experiments were performed in triplicate.

[0110] like Figure 10 As shown, compared with the empty vector control group, the bladder cancer cell model overexpressing SQLE exhibited enhanced cell proliferation activity.

[0111] This embodiment investigated the colony formation assay of bladder cancer cell models overexpressing SQLE in UMUC3 and J82 cells and their control cell models. The specific methods are as follows:

[0112] In colony formation assays, cells were seeded at a density of 1000 cells per well in 6-well plates. After an incubation period of 8 to 14 days, cells were fixed with 70% ethanol and then stained with 0.5% crystal violet solution for 15 minutes. Colonies containing more than 50 cells were counted. All experiments were performed in triplicate.

[0113] like Figure 11 As shown, compared with the empty vector control group, the number of cells proliferating in the bladder cancer cell model overexpressing SQLE was significantly increased.

[0114] This embodiment investigated wound healing in bladder cancer cell models overexpressing SQLE, specifically UMUC3 and J82 cells, and their control cell models. The specific methods are as follows:

[0115] Cells were seeded in 6-well plates and incubated overnight at 37°C. Wounds were created in the cell monolayer using pipette tips. Images were taken immediately at different time intervals during cell migration to assess wound closure. Migration distances were then compared. All experiments were performed in triplicate.

[0116] like Figure 12 As shown, compared with the empty vector control group, the bladder cancer cell model overexpressing SQLE exhibited enhanced cell migration ability.

[0117] This example investigated the Transwell migration assay of bladder cancer cell models overexpressing SQLE in UMUC3 and J82 cells, as well as their control cell models. The specific methods are as follows:

[0118] Transwell migration assays were performed using Transwell chambers according to established protocol. Cells were resuspended in 200 µl of serum-free starved medium and placed in the upper chamber, while 600 µl of complete medium was added to the lower chamber. Cells were incubated at 37°C for 24 to 48 hours, followed by fixation with 4% formaldehyde and staining with 0.5% crystal violet. All experiments were performed in triplicate.

[0119] like Figure 13 As shown, compared with the empty vector control group, the bladder cancer cell model overexpressing SQLE exhibited enhanced cell migration ability.

[0120] This embodiment investigated the cell cycle analysis of bladder cancer cell models overexpressing SQLE in UMUC3 and J82 cells and their control cell models. The specific methods are as follows:

[0121] Cells were starved of serum overnight, then stimulated with complete culture medium for 4 to 8 hours. After stimulation, cells were fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. All experiments were performed in triplicate.

[0122] like Figure 14 , Figure 15 As shown, compared with the empty vector control group, the number of bladder cancer cells overexpressing SQLE increased in the S phase and decreased in the G0 / G1 phase.

[0123] This embodiment used the same immunoblotting analysis method to examine the SQLE expression level in a SQLE-knockdown bladder cancer cell model (647V and UMUC3 cells) and a control cell model. Figure 16 As shown, the expression level of SQLE protein in each group of SQLE knockdown bladder cancer cell models showed a significant decrease, indicating that the SQLE knockdown bladder cancer cell model in this embodiment was successfully constructed.

[0124] This embodiment evaluated the cell proliferation activity of SQLE knockdown bladder cancer cell models (647V and UMUC3 cells) and control cell models using the MTT assay. Figure 17 As shown, compared with the empty vector control group, the SQLE knockdown bladder cancer cell model exhibited reduced cell proliferation activity.

[0125] This embodiment investigated the colony formation assay of SQLE knockdown bladder cancer cell models (647V and UMUC3 cells) and control cell models, such as... Figure 18 As shown, compared with the empty vector control group, the number of cells proliferating in the SQLE knockdown bladder cancer cell model was significantly reduced.

[0126] This embodiment investigated wound healing experiments in SQLE knockdown bladder cancer cell models (647V and UMUC3 cells) and control cell models. Figure 19 As shown, compared with the empty vector control group, the SQLE knockdown bladder cancer cell model exhibited reduced cell migration ability.

[0127] This example investigated the Transwell migration assay of SQLE-knockdown bladder cancer cell models (647V and UMUC3 cells) and control cell models. Figure 20 As shown, compared with the empty vector control group, the SQLE knockdown bladder cancer cell model exhibited reduced cell migration ability.

[0128] This embodiment examined cell cycle analysis of SQLE knockdown bladder cancer cell models (647V and UMUC3 cells) and control cell models, such as... Figure 21 , Figure 22 As shown, compared with the empty vector control group, the SQLE knockdown bladder cancer cell model showed cell cycle arrest, resulting in a decrease in the number of cells in the S phase and an increase in the number of cells in the G0 / G1 phase.

[0129] The above findings suggest that SQLE promotes malignant behavior in bladder cancer cell lines.

[0130] Example 3

[0131] This embodiment demonstrates through in vivo animal model experiments that SQLE promotes the occurrence and development of bladder cancer in vivo.

[0132] In this embodiment, xenograft human bladder cancer mouse models were established using J82 cell lines with SQLE overexpression and UMUC3 cell lines with SQLE knockdown. The specific construction method of the models is as follows:

[0133] J82-empty vector control group and J82-SQLE overexpression group cells; and UMUC3-empty vector control group and UMUC3-SQLE knockdown group cells (containing 3×10⁻⁶ cells in 0.05 ml PBS and 0.05 ml Matrigel Matrix) were respectively prepared. 6 SQLE-overexpressing mice were subcutaneously injected into the right dorsal side of 6-week-old male Balb / c nude mice (1 cell). Treatments were administered at specified time points. SQLE-overexpressing mice were sacrificed on day 22 after transplantation, and SQLE-knockout mice were sacrificed on day 33 after transplantation and examined.

[0134] like Figure 23 , Figure 24 As shown, the tumor volume and weight induced by J82 cells overexpressing SQLE were significantly higher than those in the control group, and the expression levels of PCNA (proliferating cell nuclear antigen) and cyclin D1 (G1 / S-specific cyclin-D1) in subcutaneous xenografts were higher than those in the control group.

[0135] like Figures 25-26 As shown, the tumor volume and weight induced by UMUC3 cells with SQLE knockdown were lower than those in the control group, and the expression levels of PCNA and cyclin D1 in subcutaneous xenografts were lower than those in the control group.

[0136] This embodiment also constructed a bladder cancer-specific SQLE overexpression mouse model to establish bladder in situ carcinoma. The specific construction method is as follows:

[0137] To activate Sqle overexpression, tamoxifen (100 mg / kg, intraperitoneal injection) was first injected once daily into SqlebladTg (n = 5) mice for five days to activate CreERT2. Rosa26-Sqle control mice (purchased from Biocytogen, Beijing, China) (n = 6) were also treated with tamoxifen at 6 weeks of age. Seven days after the last tamoxifen injection, the mice were given water containing 0.05% BBN for 12 weeks, followed by carcinogen-free water for 8 weeks to establish bladder cancer. Mice were sacrificed at week 20 from the start of BBN treatment. Histological scoring was performed by a pathologist unaware of the sample characteristics after HE staining. Similarly, Sqlefl / fl mice (n = 6) and SqlebladKO mice (n = 6) were established, and bladder cancer was induced using a similar method.

[0138] like Figures 27-30 As shown, compared with Rosa26-Sqle mice, Sqle bladTg overexpression mice had a higher tumor incidence, more severe carcinogenesis, more active tumor cell proliferation, and higher levels of PCNA and cyclin D1 expression.

[0139] like Figures 31-34 As shown, compared with Sqlefl / fl mice, Sqle bladKO knockdown mice had a lower tumor incidence, a relatively milder degree of carcinogenesis, slower tumor cell proliferation, and lower levels of PCNA and cyclin D1 expression.

[0140] The above findings indicate that SQLE plays a carcinogenic role in the development of bladder tumors in vivo.

[0141] Example 4

[0142] This embodiment demonstrates through in vitro cell experiments and in vivo animal model experiments that terbinafine inhibits the growth of BCa and increases the expression level of SQLE protein.

[0143] This embodiment used J82 cells, UMUC3 cells, RT112 cells, and tccsup cells as experimental cells to investigate cholesterol synthesis in cells treated with terbinafine. The specific experimental method is as follows:

[0144] Cells were seeded at 50% confluence in 6-well plates and treated the following day with either DMSO (solvent control) or 60 µM terbinafine (TB). Cells were collected 48 hours later for cholesterol content determination.

[0145] The method for determining cholesterol content is as follows: Use 10 6Cholesterol concentration was quantified using individual cells and measured using a cholesterol quantification kit (ab65359, Abcam). All experiments were performed in triplicate.

[0146] like Figure 35 As shown, compared with the control group, the cholesterol content of bladder cancer cells decreased after terbinafine treatment, indicating that terbinafine can reduce the cholesterol level in tumor cells.

[0147] This embodiment used J82 cells, UMUC3 cells, RT112 cells, and tccsup 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:

[0148] Cells were seeded in 96-well plates the day before, and treated with DMSO (solvent control) or 60 µM terbinafine (TB) the next day, followed by MTT assay or colony formation capacity assessment.

[0149] like Figure 36 , Figure 37 As shown, compared with the control group, the proliferation activity and colony formation of bladder cancer cells were significantly reduced after terbinafine treatment, indicating that terbinafine can significantly reduce the proliferation ability of bladder cancer tumor cells.

[0150] This embodiment used J82 cells, UMUC3 cells, RT112 cells, and tccsup cells as experimental cells to investigate the migration ability of cells after terbinafine treatment. The specific experimental method is as follows:

[0151] 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 (TB). Migration was observed and recorded at different time points.

[0152] like Figure 38 , Figure 39 As shown, compared with the control group, the wound healing rate of bladder cancer cells was significantly reduced after terbinafine treatment, indicating that terbinafine can significantly reduce the migration ability of bladder cancer tumor cells.

[0153] In this embodiment, a xenograft human BCa mouse model was established using the human bladder cancer cell line (RT112). Six-week-old male Balb / c nude mice were randomly divided into two groups at specified time points. Mice were treated with TB (terbinafine) (80 mg / kg / day) and phosphate-buffered saline (PBS) (control) via daily oral gavage. Mice were sacrificed and examined at specified time points. The therapeutic effect of terbinafine was also evaluated in the orthotopic mouse model. At six weeks of age, wild-type (C57BL / 6J background) mice were given water containing 0.05% BBN for 20 weeks to induce BCa. After 10 weeks of BBN induction, mice were randomly divided into two groups. Mice were treated with terbinafine (80 mg / kg / day) and PBS via daily oral gavage until the end of the experiment.

[0154] like Figures 40-43 As shown, oral terbinafine inhibited the formation of subcutaneous tumors, resulting in tumor volume and weight lower than the control group; it also reduced the incidence and severity of bladder carcinoma in situ, and slowed tumor cell proliferation. However, as... Figure 44 As shown, terbinafine treatment increased the expression level of SQLE protein in bladder cancer cells.

[0155] The above results indicate that terbinafine inhibits the growth of BCa in vitro and in vivo, but increases the expression level of SQLE protein.

[0156] Example 5

[0157] This embodiment demonstrates through in vitro cell experiments that ellagic acid inhibits the proliferation of bladder cancer cells in vitro and downregulates the expression level of SQLE protein.

[0158] This embodiment used UMUC3 cells, J82 cells, 647V cells, T24 cells, and RT112 cells as experimental cells to investigate the cell proliferation activity and colony formation of cells after ellagic acid treatment. The specific experimental methods are as follows:

[0159] Cells were seeded in 96-well plates the day before, and treated with DMSO (solvent control) or 50 µM ellagic acid (EA) the next day, followed by MTT assay or colony formation ability assessment.

[0160] like Figures 45-47 As shown, compared with the control group, ellagic acid treatment can inhibit the growth of UMUC3, J82, 647V, T24 and RT112 cells, and weaken the proliferation ability of tumor cells.

[0161] This embodiment used UMUC3 cells, J82 cells, 647V cells, T24 cells, and RT112 cells as experimental cells to investigate the SQLE expression level in cells treated with ellagic acid. The specific experimental method is as follows:

[0162] Cells were seeded at 50% confluence in 6-well plates and treated the following day with either DMSO (solvent control) or 50 µM ellagic acid (EA). Cells were collected 48 hours later for protein extraction and analysis.

[0163] like Figure 48 As shown, compared with the control group, ellagic acid treatment reduced the expression level of SQLE protein in UMUC3 cells, J82 cells, 647V cells, T24 cells and RT112 cells.

[0164] The above results indicate that ellagic acid inhibits the growth of bladder cancer cells in vitro and downregulates the expression level of SQLE protein.

[0165] Example 6

[0166] This embodiment demonstrates through in vitro cell experiments and in vivo animal model experiments that ellagic acid combined with terbinafine treatment can reduce the compensatory upregulation of SQLE expression caused by terbinafine alone, and further significantly improve the growth inhibition effect on bladder cancer.

[0167] This embodiment used J82 cells and RT112 cells as experimental cells to investigate the SQLE expression level in cells after different drug treatments. The specific experimental method is as follows:

[0168] Cells were seeded at 50% confluence in 6-well plates. The next day, the cells were treated with DMSO (solvent control), 60 µM terbinafine (TB), 50 µM ellagic acid (EA), or 60 µM terbinafine (TB) combined with 50 µM ellagic acid (EA). Cells were collected for protein extraction and analysis after 48 hours.

[0169] like Figure 49 As shown, compared with the control group, terbinafine monotherapy increased the expression level of SQLE, ellagic acid monotherapy decreased the expression level of SQLE, and ellagic acid combined with terbinafine synergistic treatment effectively reduced the compensatory increase in SQLE protein expression level caused by terbinafine monotherapy.

[0170] This embodiment used J82 cells and RT112 cells as experimental cells to investigate cell proliferation activity and colony formation after different drug treatments. The specific experimental methods are as follows:

[0171] Cells were seeded in 96-well plates the day before, and treated the following day with DMSO (solvent control), 60 µM terbinafine (TB), 50 µM ellagic acid (EA), or 60 µM terbinafine (TB) combined with 50 µM ellagic acid (EA). MTT assay or colony formation ability assessment was then performed.

[0172] like Figures 50-52 As shown, compared with the control group, synergistic treatment with ellagic acid and terbinafine further reduced the proliferation capacity of tumor cells.

[0173] In this embodiment, a xenograft human BCa mouse model was established using the human bladder cancer cell line (J82). At specified time points, 6-week-old male Balb / c nude mice were randomly divided into four groups. Mice were administered daily via oral gavage to either phosphate-buffered saline (PBS) (control), terbinafine (TB) (80 mg / kg / day), ellagic acid (EA) (50 mg / kg / day), or a combination of ellagic acid (EA) (50 mg / kg / day) and terbinafine (TB) (80 mg / kg / day), respectively. Mice were sacrificed and examined at specified time points.

[0174] like Figures 53-55 As shown, compared with the control group, terbinafine monotherapy group, and ellagic acid monotherapy group, ellagic acid combined with terbinafine synergistic treatment had the most significant inhibitory effect on tumors, and the lowest tumor volume and tumor weight.

[0175] like Figure 56 As shown, compared with the control group, terbinafine monotherapy increased the expression level of SQLE. Ellagic acid combined with terbinafine synergistic treatment of subcutaneous tumors effectively reduced the compensatory increase in SQLE protein expression level caused by terbinafine monotherapy.

[0176] The above research results indicate that ellagic acid combined with terbinafine treatment can reduce the compensatory upregulation of SQLE expression levels caused by terbinafine alone, and further significantly improve the growth inhibition effect on bladder cancer.

Claims

1. The use of a composition of ellagic acid and terbinafine in the preparation of a medicament for treating bladder cancer, characterized in that, The mass ratio of ellagic acid to terbinafine is 5:

8.

2. The use of the ellagic acid and terbinafine composition according to claim 1 in the preparation of a medicament for treating bladder cancer, characterized in that, The medication for treating bladder 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 bladder cancer, characterized in that, The medication for treating bladder 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 bladder cancer, characterized in that, The medication for treating bladder 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 bladder cancer, characterized in that, The bladder cancer mentioned is urothelial carcinoma of the bladder.

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

7. The use of the ellagic acid and terbinafine composition according to claim 6 in the preparation of a medicament for treating bladder cancer, characterized in that, The drug used to treat bladder cancer can disrupt the lipid metabolism of bladder cancer tumor cells and block cholesterol synthesis.

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