Composition of ellagic acid and terbinafine and application of composition in preparation of medicine for treating bladder cancer

Abstract

The invention relates to an ellagic acid and terbinafine composition and application thereof to preparation of a medicine for treating bladder cancer, and belongs to the technical field of biological medicine. The invention provides an ellagic acid and terbinafine composition which is used for solving the problem that the compensation of SQLE expression level is easily increased when terbinafine is used for treating bladder cancer, and the ellagic acid and terbinafine composition is prepared from ellagic acid and terbinafine in a mass ratio of 5: 8. The application is used for preparing the bladder cancer treatment medicine, terbinafine can directly inhibit SQLE enzyme activity, block cholesterol synthesis and inhibit tumor growth, ellagic acid can reduce SQLE expression compensatory up-regulation caused by independent treatment of terbinafine, metabolic side effects caused by terbinafine and medicine toxicity of ellagic acid can be reduced through synergism of terbinafine and ellagic acid, and the bladder cancer treatment medicine can be used for treating bladder cancer. The bladder cancer resisting effect is obviously enhanced, and the single drug resistance is overcome. Based on the development of medicines on the market, the traditional Chinese medicine composition has the double advantages of treatment synergy and safety improvement, and a more optimized treatment scheme is provided for bladder cancer treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a composition of ellagic acid and terbinafine and an application thereof in preparing a drug for treating bladder cancer. Background Art

[0002] Bladder cancer (BCa) is a common urinary tract malignancy worldwide. Current treatments include surgical resection, chemotherapy, and immunotherapy, but these are associated with high recurrence rates and drug resistance. In recent years, targeted therapy has emerged as a new research focus for bladder cancer. Squalene epoxidase (SQLE), a key enzyme in the cholesterol biosynthesis pathway, has been shown to be closely associated with the development and progression of various tumors.

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

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

[0005] In order to solve the problem that terbinafine treatment of bladder cancer easily causes compensatory increase in SQLE expression level, the present invention provides an ellagic acid and terbinafine composition and its use in preparing a drug for treating bladder cancer.

[0006] The technical solution of the present invention:

[0007] A composition of ellagic acid and terbinafine, wherein the mass ratio of the ellagic acid to terbinafine is 5:8.

[0008] The invention discloses an application of the ellagic acid and terbinafine composition in preparing a medicine 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 bladder urothelial carcinoma.

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

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

[0015] Beneficial effects of the present invention:

[0016] The present invention discovered that ellagic acid can effectively inhibit the increased SQLE expression levels caused by terbinafine treatment, thereby providing a synergistic pharmaceutical composition of ellagic acid and terbinafine. When used to treat bladder cancer, the pharmaceutical composition synergistically inhibits SQLE through a dual mechanism, blocking the cholesterol biosynthesis pathway, inhibiting tumor growth, and significantly enhancing the anti-bladder cancer effect.

[0017] The pharmaceutical composition of the present invention directly inhibits SQLE enzyme activity, blocking cholesterol synthesis, while ellagic acid reduces the compensatory upregulation of SQLE expression caused by terbinafine alone. The two ingredients synergistically reduce the metabolic side effects of terbinafine and the drug toxicity of ellagic acid, while also overcoming single-drug resistance. This invention, developed based on an already marketed drug, offers the dual advantages of enhanced therapeutic efficacy and safety, providing a more optimized treatment option for bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Statistical graphs of SQLE mRNA expression levels in tumors and adjacent adjacent tissues from different cohorts in Example 1. A represents the TCGA cohort, B represents the GSE3167 cohort, and C represents the GSE188715 cohort.

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

[0020] Figure 3 These are the immunoblot images and statistical graphs 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 4Representative immunofluorescence staining photos and relative fluorescence intensity statistical graphs of SQLE protein expression levels in tumor and adjacent adjacent tissues in Example 1. A is an immunofluorescence staining photo in which SQLE is orange and DAPI is blue; B is a relative fluorescence intensity statistical graph.

[0022] Figure 5 Representative images of SQLE immunohistochemical staining in bladder cancer tissue and adjacent paracancerous tissue in the tissue microarray analysis in Example 1, A is adjacent paracancerous tissue, B is tumor;

[0023] Figure 6 Statistical graph of SQLE immunohistochemical staining in bladder cancer tissue and adjacent paracancerous tissue in tissue microarray analysis in Example 1;

[0024] Figure 7 Representative immunohistochemical staining images of high, medium, and low expression of SQLE protein in bladder cancer samples analyzed by tissue microarray in Example 1, A represents low expression, B represents medium expression, and C represents high expression;

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

[0026] Figure 9 Comparison of SQLE protein expression levels in the control group and SQLE-overexpressing UMUC3 and J82 cells in Example 2, A for UMUC3 cells, B for J82 cells;

[0027] Figure 10 Figure 2 is a growth curve of the control group and UMUC3 and J82 cells overexpressing SQLE in Example 2, A is UMUC3 cells, B is J82 cells;

[0028] Figure 11 These are the colony formation staining images and colony formation statistics of the control group and SQLE-overexpressing UMUC3 and J82 cells in Example 2, A is the colony formation staining image, and B is the colony formation statistics image;

[0029] Figure 12 These are wound healing comparison graphs and wound healing rate statistics for the control group and SQLE-overexpressing UMUC3 and J82 cells in Example 2. A is a wound healing comparison graph for UMUC3 cells, B is a wound healing comparison graph for J82 cells, and C is a wound healing rate statistics graph.

[0030] Figure 13 Figures 2A and 2B are the migration cell staining images and relative migration cell number statistics for the Transwell experimental control group and SQLE-overexpressing UMUC3 and J82 cells in Example 2. A is the migration cell staining image, and B is the relative migration cell number statistics.

[0031] Figure 14 Flow cytometric cell cycle diagrams of the control group and SQLE-overexpressing UMUC3 and J82 cells in Example 2, A for UMUC3 cells, B for J82 cells;

[0032] Figure 15 Flow cytometric cell cycle statistics of the control group and SQLE-overexpressing UMUC3 and J82 cells in Example 2, A for UMUC3 cells, B for J82 cells;

[0033] Figure 16 Comparison of SQLE protein expression levels in the control group and SQLE-knockdown 647V and UMUC3 cells in Example 2, A for 647V cells, B for UMUC3 cells;

[0034] Figure 17 Figure 2 is a growth curve of the control group and SQLE-knockdown 647V and UMUC3 cells in Example 2, A is 647V cells, B is UMUC3 cells;

[0035] Figure 18 These are the colony formation staining images and colony formation statistics of the control group and SQLE-knockdown 647V and UMUC3 cells in Example 2, A is the colony formation staining image, and B is the colony formation statistics image;

[0036] Figure 19 Comparative graphs of wound healing and statistical graphs of wound healing rates of the control group and SQLE-knockdown 647V and UMUC3 cells in Example 2. A is a comparative graph of wound healing of 647V cells, B is a comparative graph of wound healing of UMUC3 cells, and C is a statistical graph of wound healing rates.

[0037] Figure 20 Figures 2A and 2B are the migration cell staining images and relative migration cell number statistics for the control group and SQLE-knockdown 647V and UMUC3 cells in the Transwell experiment in Example 2. A is the migration cell staining image, and B is the relative migration cell number statistics.

[0038] Figure 21 Flow cytometric analysis of cell cycle diagrams of the control group and SQLE-knockdown 647V and UMUC3 cells in Example 2, A is 647V cells, B is UMUC3 cells;

[0039] Figure 22 Flow cytometric analysis of cell cycle statistics of the control group and SQLE-knockdown 647V and UMUC3 cells in Example 2, A is 647V cells, B is UMUC3 cells;

[0040] Figure 23 Figures 1 and 2 show actual tumor photos and tumor volume and weight statistics for the empty vector control group and the subcutaneous transplanted tumor derived from J82 cells overexpressing SQLE in Example 3. (A) shows an actual tumor photo, (B) shows a tumor volume statistics chart, and (C) shows a tumor weight statistics chart.

[0041] Figure 24 This is a comparison of the expression levels of PCNA and cyclin D1 proteins in the control group and the subcutaneous transplanted tumors derived from J82 cells overexpressing SQLE in Example 3, A is the control group, and B is the SQLE overexpression group;

[0042] Figure 25 Figures 1 and 2 show the actual tumor photos and tumor volume and weight statistical graphs of the subcutaneous transplanted tumors derived from the control group and SQLE-knockdown UMUC3 cells in Example 3. A shows the actual tumor photo, B shows the tumor volume statistical graph, and C shows the tumor weight statistical graph.

[0043] Figure 26 Comparison of PCNA and cyclin D1 protein expression levels in the control group and SQLE-knockdown UMUC3 cell-derived subcutaneous transplanted tumors in Example 3, A is the control group, B is the SQLE-knockdown group;

[0044] Figure 27 From left to right are representative MRI images, gross histological images, HE staining images, and partial magnifications of HE staining images of the bladders of Rosa26-Sqle mice and Sqle bladTg mice in Example 3. A is a Rosa26-Sqle mouse, and B is a Sqle bladTg mouse.

[0045] Figure 28 Figure 3 shows the statistical graphs of bladder tumor incidence and disease severity in Rosa26-Sqle mice and Sqle bladTg mice, with A representing tumor incidence and B representing disease severity.

[0046] Figure 29 Representative Ki-67 staining images and Ki-67 statistical graphs of the bladders of Rosa26-Sqle mice and Sqle bladTg mice in Example 3, A is the Ki-67 staining image, and B is the Ki-67 statistical graph;

[0047] Figure 30 Comparison of PCNA and cyclin D1 protein expression levels in bladder tissues of Rosa26-Sqle mice and Sqle bladTg mice in Example 3. A is a Rosa26-Sqle mouse, and B is a Sqle bladTg mouse.

[0048] Figure 31From left to right are representative MRI images, gross histological images, HE staining images, and partial magnifications of HE staining images of the bladders of Sqlefl / fl mice and SqlebladKO mice in Example 3. A is a Sqlefl / fl mouse, and B is a SqlebladKO mouse.

[0049] Figure 32 Figure 3 shows the incidence and severity of bladder tumors in Sqlefl / fl mice and SqlebladKO mice. Figure A shows the incidence of tumors and Figure B shows the severity of the disease.

[0050] Figure 33 Representative Ki-67 staining images and Ki-67 statistical graphs of the bladders of Sqlefl / fl mice and SqlebladKO mice in Example 3, A is the Ki-67 staining image, and B is the Ki-67 statistical graph;

[0051] Figure 34 Comparison of PCNA and cyclin D1 protein expression levels in bladder tissues of Sqlefl / fl mice and SqlebladKO mice in Example 3. A: Sqlefl / fl mice, B: SqlebladKO mice.

[0052] Figure 35 Statistical graph of cholesterol levels in the control group and in each group after terbinafine treatment in Example 4;

[0053] Figure 36 Comparison of the growth curves of cells in each group after treatment with the control solvent and terbinafine in Example 4, A is J82 cells, B is UMUC3 cells, C is RT112 cells, and D is TCCSUP;

[0054] Figure 37 Figures 4A and 4B are colony formation staining images and colony formation statistics of each group of cells after treatment with a control solvent and terbinafine, A is a colony formation staining image, and B is a colony formation statistics image;

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

[0056] Figure 39 Statistical graph of wound healing rates of cells in each group after treatment with control solvent and terbinafine in Example 4;

[0057] Figure 40Photos of subcutaneous tumors and statistical graphs of tumor volume and weight after oral administration of a control solvent and terbinafine in Example 4, A is a photo of the tumor, B is a statistical graph of tumor volume, and C is a statistical graph of tumor weight;

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

[0059] Figure 42 Figure 4 shows the statistical graphs of bladder tumor incidence and disease severity in the orthotopic bladder cancer model mice treated with oral control solvent and terbinafine in Example 4, with A representing tumor incidence and B representing disease severity.

[0060] Figure 43 Representative Ki-67 staining images and Ki-67 statistical graphs of the bladders of mice with orthotopic bladder cancer treated with a control solvent and terbinafine in Example 4, A is the Ki-67 staining image, and B is the Ki-67 statistical graph;

[0061] Figure 44 Comparison of SQLE protein expression levels in cells 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 Figure 5 is a comparison of the growth curves of the control group and the cells in each group after ellagic acid treatment in Example 5. A is UMUC3 cells, B is J82 cells, C is 647V cells, D is T24 cells, and E is RT112 cells.

[0063] Figure 46 Figures 2 and 3 show colony formation staining and colony formation statistics of the control group and each group of cells after ellagic acid treatment in Example 5. A shows UMUC3 cells, B shows J82 cells, C shows 647V cells, D shows T24 cells, and E shows RT112 cells.

[0064] Figure 47 Statistical graph of colony formation of cells in the control group and each group after ellagic acid treatment in Example 5;

[0065] Figure 48 Comparison of SQLE protein expression levels in the control group and the cells in each group after ellagic acid treatment in Example 5. A is UMUC3 cells, B is 647V cells, and C is J82 cells.

[0066] Figure 49Comparison of SQLE protein expression levels in bladder cancer J82 and RT112 cells in each treatment group in Example 6, A for J82 cells, B for RT112 cells;

[0067] Figure 50 Comparison of the growth curves of bladder cancer J82 and RT112 cells in each treatment group in Example 6, A for J82 cells, B for RT112 cells;

[0068] Figure 51 The colony formation staining images of bladder cancer J82 and RT112 cells in each treatment group in Example 6;

[0069] Figure 52 Statistical graphs of colony formation of bladder cancer J82 and RT112 cells in each treatment group in Example 6;

[0070] Figure 53 These are photos of subcutaneous transplanted tumors in each treatment group in Example 6;

[0071] Figure 54 Figure 6 is a statistical graph of the tumor volume of subcutaneous transplanted tumors in each treatment group;

[0072] Figure 55 Statistical graph of tumor weight of subcutaneous transplanted tumors in each treatment group in Example 6;

[0073] Figure 56 6 is a comparative diagram of the SQLE protein levels of subcutaneous transplanted tumors in mice in each treatment group in Example 6, A is the control solvent group, B is the terbinafine single-drug group, C is the ellagic acid single-drug group, and D is the ellagic acid combined with terbinafine co-treatment group. DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0075] The statistical analysis method of the experimental data of Examples 1 to 6 of the present invention is as follows:

[0076] All statistical analyses were performed using GraphPad Prism (version 8.0.1) or SPSS (version 21.0) software. Data are presented as mean ± standard deviation (SD). Multiple groups were compared using one-way analysis of variance (ANOVA). Two-way comparisons of two variables were performed using the two-sided Student's t-test. Kaplan-Meier survival curves and the oG-rank test were used to assess the association between overall survival and expression levels. A P value < 0.05 was considered significant.

[0077] Example 1

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

[0079] First, this example used a paired sample t-test to analyze the SQLE mRNA levels between tumor tissues and normal tissues in The Cancer Genome Atlas (TCGA). An unpaired sample t-test was used to analyze data from datasets GSE3167 and GSE188715 from the GEO (Gene Expression 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 TCGA cohort, GSE3167 cohort and GSE188715 cohort is higher than that in normal adjacent cancer tissues.

[0081] This example examined the expression levels of SQLE mRNA and protein in tumor tissues and adjacent adjacent tissues, and the relationship between different SQLE expression levels and overall survival. For survival analysis, 407 patients from the TCGA were divided into high and low SQLE expression groups. Kaplan-Meier curves were plotted to estimate overall survival (OS), and inter-group differences 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 was significantly lower than that of samples with low SQLE expression.

[0083] In this example, 8 pairs of bladder cancer tumors and paired adjacent normal tissues were collected from independent cohorts, and the SQLE protein expression levels in the tumor tissues and adjacent normal tissues were examined using conventional immunoblotting analysis methods in the art.

[0084] All human bladder cancer tumor tissues and adjacent adjacent tissues collected in this example were obtained from the Affiliated Cancer Hospital of Harbin Medical University. This research project was approved by the Ethics Committee of the Affiliated Cancer Hospital of Harbin Medical University, and informed consent was obtained from each participating patient. The human bladder cancer tissue microarray (HBlaU079Su01) used in this study was purchased from OUTDO Biotechnology (Shanghai, China).

[0085] Depend on Figure 3 It can be seen that in 8 pairs of tumors and paired adjacent cancer tissues in an independent cohort, the SQLE protein expression level in tumor tissues was significantly higher than that in adjacent cancer tissues.

[0086] In this example, immunohistochemical analysis and immunofluorescence analysis were performed on human bladder cancer tumor tissue and adjacent adjacent tissues. 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 cut into 4-µm sections. Sections were deparaffinized by baking at 60°C for 30 minutes. Paraffin-embedded tissue sections were deparaffinized in xylene, sequentially treated with 100%, 90%, and 70% ethanol, and then rinsed in water. Sections underwent antigen retrieval. For immunohistochemical staining, endogenous peroxidase activity was inhibited with 3% hydrogen peroxide for 15 minutes at room temperature. Tissues were blocked with 5% goat serum for 1 hour and incubated with primary antibodies overnight at 4°C. After washing with PBS-T, secondary antibodies were applied for 1 hour at room temperature.

[0089] Immunofluorescence:

[0090] For tissue analysis, sections were deparaffinized by baking at 60°C for 30 minutes. After deparaffinization and rehydration, antigen retrieval was performed. Sections were incubated overnight at 4°C, then washed three times with PBST and incubated with fluorochrome-conjugated secondary antibodies for one hour. After three washes with PBST, cell nuclei were stained with DAPI, and 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 tissue. Figure 5 and Figure 6 It can be seen that the immunohistochemical score of SQLE protein expression in tumor tissues was higher than that in adjacent tissues in tissue microarray analysis. 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] These results indicate that SQLE is overexpressed in human bladder cancer (BCa) and is associated with poor prognosis.

[0093] Example 2

[0094] This example 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 example are:

[0096] Bladder cancer cell line J82 cells were purchased from ATCC (Manassas, VA, USA). 647V and UMUC3 cells were provided by Professor Xu Wanhai of the Affiliated Cancer Hospital of Harbin Medical University, China. All cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% carbon dioxide. All cell cultures were confirmed to be free of mycoplasma contamination through appropriate testing.

[0097] In this example, SQLE-overexpressing bladder cancer cell models and control cell models were constructed for UMUC3 cells and J82 cells, as well as SQLE-knockdown bladder cancer cell models and control cell models for 647V cells and UMUC3 cells. The SQLE-overexpressing bladder cancer cell model and control cell model were constructed by transfection with pRL-cyto-megalovirus (pCMV)-SQLE plasmids and pCMV-empty vector plasmids, while the SQLE-knockdown bladder cancer cell model and control cell model were constructed by transfection with SQLE sgRNA (lentiCRISPRv2-SQLE) and an empty vector plasmid (lentlCRISPPR v2). All of the above plasmids were purchased from OriGene.

[0098] The transfection steps for overexpressing SQLE and empty vector, and for knocking down SQLE and empty vector are as follows:

[0099] (1) Cell preparation: Select cells in the logarithmic growth phase and plate them in a 6-well plate at an appropriate density 24 hours in advance. The cell confluence should be 60%-80% during 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 lipo2000 in Opti-MEM;

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

[0104] (3) Transfection procedure: Remove the original culture medium. Add the resulting DNA-lipid complex dropwise to the cell culture wells, gently shake the culture plate to evenly distribute it, and add serum-free culture medium to a volume of 2 ml / well. Incubate for 6 hours as per conventional transfection. After 6 hours, replace with complete culture medium.

[0105] In this example, the SQLE expression levels in UMUC3 and J82 SQLE-overexpressing bladder cancer cell models and their control cell models were investigated by Western blotting analysis. The Western blotting analysis was as follows:

[0106] 25 µg of protein was loaded and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin (BSA) in TBST for 2 hours at room temperature, the membrane was incubated with primary antibodies in 1.5% BSA overnight at 4°C, followed by incubation with secondary antibodies for 2 hours at room temperature. Target proteins were visualized using enhanced chemiluminescence (Omin-ECL, Yazyme, 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, which shows that the bladder cancer cell model overexpressing SQLE in this example was successfully constructed.

[0108] In this example, the cell proliferation activity of SQLE-overexpressing bladder cancer cell models of UMUC3 cells and J82 cells and their control cell models was evaluated by MTT assay. The specific method is as follows:

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

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

[0111] This example investigates colony formation assays of SQLE-overexpressing bladder cancer cell models of UMUC3 and J82 cells and their control cell models. The specific methods are as follows:

[0112] For colony formation assays, cells were seeded at a density of 1000 cells per well in 6-well plates. After a culture period of 8 to 14 days, cells were fixed with 70% ethanol and subsequently stained with 0.5% crystal violet solution for 15 minutes. Colonies containing 50 or more 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 cell proliferation in the bladder cancer cell model overexpressing SQLE was significantly increased.

[0114] This example investigates wound healing experiments using UMUC3 and J82 cells, SQLE-overexpressing bladder cancer cell models, 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 a pipette tip. Images were taken immediately at various 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, the cell migration ability of the bladder cancer cell model overexpressing SQLE was enhanced compared with the empty vector control group.

[0117] This example investigates the Transwell migration assay of SQLE-overexpressing bladder cancer cell models of UMUC3 and J82 cells and their control cell models. The specific method is as follows:

[0118] Transwell migration assays were performed using Transwell inserts according to established protocols. 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. Cells were incubated at 37°C for 24 to 48 hours, then fixed with 4% formaldehyde and stained with 0.5% crystal violet. All experiments were performed in triplicate.

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

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

[0121] Cells were serum-starved overnight and then stimulated with complete 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 in the S phase increased and the number of cells in the G0 / G1 phase decreased.

[0123] In this example, the same immunoblotting analysis method was used to examine the SQLE expression levels in SQLE knockdown bladder cancer cell models of 647V cells and UMUC3 cells and control cell models. Figure 16 As shown, the SQLE protein expression levels of each group of SQLE knockdown bladder cancer cell models showed a significantly reduced trend, which indicates that the SQLE knockdown bladder cancer cell model of this example was successfully constructed.

[0124] In this example, the cell proliferation activity of SQLE knockdown bladder cancer cell models of 647V cells and UMUC3 cells and control cell models was evaluated by MTT assay. Figure 17 As shown in Figure 3, the cell proliferation activity of the SQLE knockdown bladder cancer cell model was weakened compared with the empty vector control group.

[0125] This example investigates the colony formation assay of SQLE knockdown bladder cancer cell models of 647V cells and UMUC3 cells and a control cell model. Figure 18 As shown, the number of cell proliferation in the SQLE knockdown bladder cancer cell model was significantly reduced compared with the empty vector control group.

[0126] This example investigates wound healing experiments in SQLE knockdown bladder cancer cell models of 647V and UMUC3 cells and control cell models. Figure 19 As shown in Figure 3, the cell migration ability of the SQLE knockdown bladder cancer cell model was weakened compared with the empty vector control group.

[0127] This example investigates the Transwell migration assay of SQLE knockdown bladder cancer cell models of 647V and UMUC3 cells and control cell models. Figure 20 As shown in Figure 3, the cell migration ability of the SQLE knockdown bladder cancer cell model was weakened compared with the empty vector control group.

[0128] This example investigates the cell cycle analysis of SQLE knockdown bladder cancer cell models of 647V cells and UMUC3 cells and control cell models. Figure 21 、 Figure 22 As shown, compared with the empty vector control group, SQLE knockdown arrested the cell cycle of the bladder cancer cell model, 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] These findings indicate that SQLE promotes malignant behavior in bladder cancer cell lines.

[0130] Example 3

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

[0132] In this example, a xenograft human bladder cancer mouse model was established using a J82 SQLE overexpression stable cell line and a UMUC3 SQLE knockdown stable cell line. The specific method for constructing the model was 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 (3×10 6 100 cells) were subcutaneously injected into the right dorsal flank of 6-week-old male Balb / c nude mice. Treatments were performed at the indicated time points. SQLE-overexpressing mice were sacrificed 22 days after transplantation, while SQLE-knockdown mice were sacrificed 33 days after transplantation and examined.

[0134] like Figure 23 、 Figure 24 As shown, the tumor volume and weight induced by SQLE-overexpressing J82 cells 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 transplanted tumors were higher than those in the control group.

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

[0136] This example also constructs a bladder cancer-specific SQLE overexpression mouse model for bladder carcinoma in situ. The specific construction method is as follows:

[0137] To activate Sqle overexpression, SqlebladTg mice (n = 5) were first injected with tamoxifen (100 mg / kg, intraperitoneal injection) once daily 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, mice were given water containing 0.05% BBN for 12 weeks and then carcinogen-free water for 8 weeks to establish bladder cancer. Mice were sacrificed at 20 weeks from the start of carcinogen (BBN) treatment. Histological scoring was performed by a pathologist blinded to the nature of the samples after hematoxylin and eosin staining. Similarly, Sqlefl / fl mice (n = 6) and SqlebladKO mice (n = 6) were established and bladder cancer was induced using similar methods.

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

[0139] like Figures 31-34 As shown in the results, compared with Sqlefl / fl mice, SqlebladKO knockdown mice had a lower tumor incidence rate, relatively milder cancerous changes, slower tumor cell proliferation and lower PCNA and cyclin D1 expression levels.

[0140] These findings suggest that SQLE plays an oncogenic role in bladder tumorigenesis in vivo.

[0141] Example 4

[0142] This example 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] In this example, J82 cells, UMUC3 cells, RT112 cells, and tccsup cells were used as experimental cells to investigate the cholesterol synthesis in cells after treatment with terbinafine. The specific experimental method was as follows:

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

[0145] The cholesterol content was determined as follows: 10 6Cholesterol concentration was quantified using a cholesterol quantification kit (ab65359, Abcam) per cell. All experiments were performed in triplicate.

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

[0147] In this example, J82 cells, UMUC3 cells, RT112 cells, and tccsup cells were used as experimental cells to investigate the cell proliferation activity and clone formation of cells after treatment 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. MTT assay or colony formation ability assessment were then performed, respectively.

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

[0150] In this example, J82 cells, UMUC3 cells, RT112 cells, and tccsup cells were used as experimental cells to investigate the migration ability of cells after treatment with terbinafine. The specific experimental method was as follows:

[0151] Cells were seeded at 80% confluence. The next day, wounds were created in the cell monolayer using a pipette tip. Cells were treated with DMSO (solvent control) or 60 µM terbinafine (TB), and migration was observed and recorded at different time points.

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

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

[0154] like Figures 40-43 As shown in the results, oral administration of terbinafine inhibited the formation of subcutaneous tumors, reducing the tumor volume and weight to lower levels than the control group; it also reduced the incidence and severity of bladder carcinoma in situ and slowed down the proliferation of tumor cells. Figure 44 As shown in Figure 3, terbinafine treatment increased the expression level of SQLE protein in bladder cancer cells.

[0155] The above research results show 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 example 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] In this example, UMUC3 cells, J82 cells, 647V cells, T24 cells, and RT112 cells were used as experimental cells to investigate the cell proliferation activity and colony formation of cells after treatment with ellagic acid. The specific experimental method was 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. MTT assay or colony formation ability assessment were then performed, respectively.

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

[0161] In this example, UMUC3 cells, J82 cells, 647V cells, T24 cells, and RT112 cells were used as experimental cells to investigate the SQLE expression level in cells after ellagic acid treatment. The specific experimental method was as follows:

[0162] Cells were seeded in 6-well plates at 50% confluence and treated with DMSO (solvent control) or 50 μM ellagic acid (EA) the next day. 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 research results show 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 example 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 treatment alone, and further significantly improve the growth inhibitory effect on bladder cancer.

[0167] In this example, J82 cells and RT112 cells were used as experimental cells to investigate the SQLE expression levels of cells after treatment with different drugs. The specific experimental methods were as follows:

[0168] Cells were seeded in 6-well plates at 50% confluence and treated the next 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). Cells were collected 48 hours later for protein extraction and analysis.

[0169] like Figure 49 As shown in the results, 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 treatment effectively reduced the compensatory increase in SQLE protein expression level caused by terbinafine monotherapy.

[0170] In this example, J82 cells and RT112 cells were used as experimental cells to investigate the cell proliferation activity and clone formation of cells after treatment with different drugs. The specific experimental methods were as follows:

[0171] Cells were seeded in 96-well plates the day before and 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) the next day. MTT assay or colony formation ability assessment were then performed.

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

[0173] In this example, a human bladder cancer cell line (J82) was used to establish a xenograft human BCa mouse model. Six-week-old male Balb / c nude mice were randomly divided into four groups at designated time points. Mice received daily oral gavage with phosphate-buffered saline (PBS) (control), terbinafine (TB) (80 mg / kg / day), ellagic acid (EA) (50 mg / kg / day), and ellagic acid (EA) (50 mg / kg / day) combined with terbinafine (TB) (80 mg / kg / day). At designated time points, mice were sacrificed and examined.

[0174] like Figure 53-55 As shown in the figures, 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 tumor weight.

[0175] like Figure 56 As shown in the results, compared with the control group, terbinafine monotherapy increased the expression level of SQLE, and ellagic acid combined with terbinafine in the 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 inhibitory effect on bladder cancer.

Claims

1. A composition of ellagic acid and terbinafine, characterized in that: The mass ratio of the ellagic acid to terbinafine is 5:

8.

2. Use of the ellagic acid and terbinafine composition according to claim 1 in the preparation of a medicament for treating bladder cancer.

3. Use of the ellagic acid and terbinafine composition according to claim 2 in preparing a medicament for treating bladder cancer, characterized in that: The drug for treating bladder cancer is administered orally.

4. Use of the ellagic acid and terbinafine composition according to claim 3 in preparing a medicament for treating bladder cancer, characterized in that: The medicine for treating bladder cancer also contains a pharmaceutically acceptable carrier or excipient.

5. Use of the ellagic acid and terbinafine composition according to claim 4 in preparing a medicament for treating bladder cancer, characterized in that: The medicine for treating bladder cancer is in the form of granules, tablets, capsules, pills or oral liquid preparations.

6. Use of the ellagic acid and terbinafine composition according to claim 5 in preparing a medicament for treating bladder cancer, characterized in that: The bladder cancer is bladder urothelial carcinoma.

7. Use of the ellagic acid and terbinafine composition according to claim 6 in preparing a medicament for treating bladder cancer, characterized in that: The drug for treating bladder cancer can simultaneously inhibit SQLE enzyme activity and compensatory upregulation of SQLE expression.

8. Use of the ellagic acid and terbinafine composition according to claim 7 in preparing a medicament for treating bladder cancer, characterized in that: The medicine for treating bladder cancer can destroy the lipid metabolism of bladder cancer tumor cells and block cholesterol synthesis.

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