Use of a natural source naphthoquinone compound in the preparation of a drug for preventing and treating bladder cancer

Abstract

The application discloses 10 natural naphthoquinone compounds in the preparation of drugs for preventing and treating bladder cancer, especially the application of juglone in the preparation of drugs for preventing and treating bladder cancer, wherein the bladder cancer is caused by T24 and UMUC3 cells. The application finds that the inhibitory effect of juglone on bladder cancer T24 and UMUC3 cells is the most significant through systematic activity screening. After 72 hours of treatment, the IC 50 of juglone on T24 cells is 0.51 muM, and the IC 50 of juglone on UMUC3 cells is 0.37 muM, which are better than those of other nine tested compounds. Mechanism research shows that juglone can inhibit the expression of key negative regulation proteins SLC7A11 and GPX4 of ferroptosis by targeting combination and down-regulation of BRD9 protein expression, and induce ferroptosis of bladder cancer cells. Therefore, juglone can be used for preparing drugs for preventing and treating bladder cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of 10 naturally derived naphthoquinone compounds in the preparation of drugs for the prevention and treatment of bladder cancer, particularly the application of juglone in the preparation of drugs for the prevention and treatment of bladder cancer caused by T24 and UMUC3 cells. Background Technology

[0002] Bladder cancer (BLCA) is one of the most common malignant tumors of the urinary system, characterized by high recurrence and mortality rates. Non-muscle-invasive bladder cancer (NMIBC) has a high recurrence rate, and some patients may progress to the more aggressive muscle-invasive bladder cancer (MIBC). Even after radical cystectomy, MIBC patients still face the risk of postoperative recurrence and metastasis. While chemotherapy and radiotherapy, as adjuvant therapies, can improve the survival rate of MIBC patients to some extent, they have many limitations, including bone marrow suppression, liver and kidney damage, radiation cystitis, and enteritis. Therefore, the search for novel, low-toxicity, and highly effective antitumor drugs is of significant clinical importance.

[0003] Natural active ingredients have attracted much attention in anti-tumor drug development due to their advantages such as multi-target effects, low toxicity, wide availability, and low cost. Naphthoquinones are a class of quinone derivatives widely found in nature, mainly distributed in higher plants such as those in the Juglandaceae, Boraginaceae, Plumbaginaceae, and Bignoniaceae families, as well as some fungi and marine organisms. Naturally derived naphthoquinones possess various biological activities, including anti-tumor, anti-inflammatory, antibacterial, antiviral, and cardiovascular protective activities, and have become an important source for anti-tumor drug development.

[0004] Juglone (5-hydroxy-1,4-naphthalenedione, molecular formula C) 10 H6O3 (molecular weight 174.15) is a typical naturally occurring naphthoquinone compound, widely found in the roots, leaves, shells, and bark of plants in the genus *Juglans mandshurica* (family Juglandaceae). Previous studies have shown that juglone has inhibitory activity against various tumor cell lines, including breast cancer, liver cancer, gastric cancer, glioblastoma, and esophageal cancer. Other naturally occurring naphthoquinone compounds, such as plumbagin, shikonin, lapachol, and β-lapachone, have also been reported to have varying degrees of antitumor activity, but current research lacks a systematic comparison of the anti-bladder cancer activity of these compounds.

[0005] Currently, research on the anti-bladder cancer effects of naturally derived naphthoquinones remains inadequate. On the one hand, the relative activity of different naphthoquinones in bladder cancer is unclear, lacking direct comparative data based on the same experimental platform. On the other hand, existing research reports limited data on the anti-bladder cancer activity of juglone, and its target and molecular mechanism have not been fully elucidated. To address these shortcomings, this invention systematically screened and compared the anti-bladder cancer activities of 10 representative naturally derived naphthoquinones, clarifying for the first time the relative activity of each compound. Juglone was found to have the most superior activity. Furthermore, through a combination of methods including mouse UMUC3 cell subcutaneous xenograft experiments, proteomics, thermal shift experiments, and BRD9 overexpression recovery experiments, it was elucidated that juglone exerts its anti-bladder cancer effect by targeting and inhibiting BRD9 expression, downregulating SLC7A11 and GPX4, and inducing ferroptosis in bladder cancer cells. Summary of the Invention

[0006] The purpose of this invention is to provide the application of a naturally derived naphthoquinone compound in the preparation of a drug for the prevention and treatment of bladder cancer, specifically for the prevention and treatment of bladder cancer caused by T24 and UMUC3 cells.

[0007] To achieve the above objectives, the present invention provides the following technical method: The 10 naturally derived naphthoquinone compounds described in this invention include: shikonin, deoxyshikonin, β-lapachone, α-lapachone, lawsone, naphthazarin, lapachol, juglone, plumbagin, and vitamin K1 (Phylloquinone). The chemical structural formulas of these compounds are shown in Formula 1. Chemical Formula 1

[0008] This invention screened the anti-bladder cancer activity of the aforementioned 10 naturally derived naphthoquinone compounds and found that juglone had the most significant inhibitory effect on bladder cancer T24 and UMUC3 cells. After 72 hours of treatment, juglone showed a significant inhibitory effect on the IC50 of T24 cells. 50 The IC50 concentration of 0.51 μM was observed in UMUC3 cells. 50 The effective concentration was 0.37 μM, which was superior to the other nine tested compounds.

[0009] Mechanistic studies have shown that juglone exerts its anti-bladder cancer effect through the following pathways: (1) inhibiting the DNA synthesis and proliferation of bladder cancer cells and reducing the EdU positivity rate of T24 and UMUC3 cells; (2) inhibiting the migration ability of bladder cancer cells and reducing the number of cells crossing the Transwell membrane; (3) inhibiting tumor growth and reducing tumor volume and weight in a mouse UMUC3 cell subcutaneous xenograft model; (4) proteomics detection and KEGG enrichment analysis showed that differentially expressed proteins after juglone treatment were mainly enriched in pathways related to ferroptosis, oxidative stress and glutathione metabolism; (5) transmission electron microscopy and ferroptosis detection kit results showed that juglone can cause abnormal mitochondrial morphology in bladder cancer cells and enhance ferroptosis-related indicators; (6) Western blot results showed that juglone can reduce the expression of ferroptosis inhibition-related proteins SLC7A11 and GPX4; (7) molecular docking, molecular dynamics simulation and thermal displacement experiments showed that juglone can interact with BRD9, and overexpression of BRD9 can partially restore the ferroptosis-related protein expression changes induced by juglone. The above results confirm that juglone can inhibit the proliferation and migration of bladder cancer in vitro and in vivo, and induce ferroptosis in bladder cancer cells through the BRD9 / SLC7A11 / GPX4 axis, thereby exerting an anti-bladder cancer effect.

[0010] Therefore, this invention clarifies for the first time the relative strength of anti-bladder cancer activity among 10 naturally derived naphthoquinone compounds, with juglone being the most active compound, which can be used to prepare drugs for the prevention and treatment of bladder cancer caused by T24 and UMUC3 cells. Attached Figure Description

[0011] Figure 1 In vitro inhibitory activity of 10 naturally derived naphthoquinone compounds against human bladder cancer cells T24 (CCK-8 assay, 72 h) Figure 2 In vitro proliferative inhibitory activity of 10 naturally derived naphthoquinone compounds against human bladder cancer cells UMUC3 (CCK-8 assay, 72 h). Figure 3 EdU assay to detect the effect of juglone on the proliferation of human bladder cancer cells Figure 4 Transwell assay to detect the effect of juglone on the migration ability of human bladder cancer cells Figure 5 Tumorigenicity assay in nude mice to detect the endosome-mediated killing effect of juglone on tumors. Figure 6 KEGG enrichment analysis following proteomics assays revealed the impact of juglone on the mechanism of action in bladder cancer cells. Figure 7 Transmission electron microscopy to investigate the effect of juglone on ferroptosis in bladder cancer cells Figure 8Ferrocyte assay kit detects the effect of juglone on ferroptosis in bladder cancer cells. Figure 9 Western blot analysis of the effect of juglone on the expression of ferroptosis-related proteins in bladder cancer cells. Figure 10 Molecular docking to investigate the target proteins of juglone Figure 11 Western blot analysis revealed the reversal effect of BRD9 overexpression on juglone-induced ferroptosis. Detailed Implementation

[0012] To better understand the present invention, the following detailed description of specific embodiments further illustrates the above-mentioned content of the present invention. However, this should not be construed as a limitation of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0013] 1. Experimental Materials 1.1 Test reagents: Ten natural naphthoquinone compounds, including Shikonin, Deoxyshikonin, β-Lapachone, α-Lapachone, Lawsone, Naphthazarin, Lapachol, Juglone, Plumbagin, and Phylloquinone, were purchased from Shanghai TargetMol Biotechnology Co., Ltd. (with a purity ≥98%). Each compound was dissolved in DMSO and then diluted to the required concentration with the corresponding culture medium, with a final DMSO concentration <0.1%.

[0014] 1.2 Cell lines: Human bladder cancer T24 cells and human bladder cancer UMUC3 cells were purchased from the American Type Culture Collection (ATCC).

[0015] 1.3 Culture medium: T24 cells were cultured in RPMI-1640 medium and UMUC3 cells were cultured in DMEM medium, both with 10% fetal bovine serum added, and cultured in an incubator with 37°C and 5% CO2 saturated humidity.

[0016] 2. Testing Methods The inhibitory effect of the compound on the proliferation of bladder cancer cells was determined using the CCK-8 assay. T24 and UMUC3 cells in logarithmic growth phase were used at a concentration of 8 × 10⁻⁶ cells / cell. 3Cells were seeded at a density of 100 μL per well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, 100 μL of culture medium containing different concentrations of the test compound (50, 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, 0.000128 μM, with an equal volume of DMSO as a solvent control) was added to each well, with 3 replicates, and the cells were incubated for another 72 hours. 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 hours. The optical density (OD) at 450 nm was then measured using an automated microplate reader. Cell viability was calculated using the following formula: (The text abruptly ends here, so the translation stops as well.) Cell viability (%) = (OD-treated group - OD blank group) / (OD control group - OD blank group) × 100% The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression fitting with the logarithm of compound concentration as the x-axis and cell viability as the y-axis. 50 The experiment was independently repeated 3 times.

[0017] 3. Experimental Results The results showed that among the 10 naturally derived naphthoquinone compounds tested, juglone exhibited the most significant inhibitory effect on bladder cancer T24 and UMUC3 cells. After 72 hours of treatment, the IC50 values ​​of each compound on T24 and UMUC3 cells were [not specified in the original text]. 50 The values ​​are shown in Table 1.

[0018] The above results indicate that among the 10 naturally derived naphthoquinone compounds, juglone exhibits the best anti-bladder cancer activity.

[0019] Table 1. Half-maximal inhibitory concentrations (IC50) of 10 naturally derived naphthoquinone compounds against human bladder cancer T24 and UMUC3 cells. 50 (μM, 72 h) Example 2: EDU assay to detect the effect of juglone on the proliferative capacity of bladder cancer

[0020] 1. Experimental Materials 1.1 Test reagents: Juglone was purchased from Shanghai Taoshu Biotechnology Co., Ltd. (TargetMol), with a purity ≥98%. Juglone was dissolved in DMSO to prepare a stock solution, which was then diluted with the corresponding cell culture medium to the required concentration before use, with a final DMSO concentration <0.1%.

[0021] 1.2 Cell lines: human bladder cancer T24 cells and human bladder cancer UMUC3 cells, with cell source and culture conditions the same as in Example 1.

[0022] 1.3 Main reagents and consumables: EdU cell proliferation assay kit, Hoechst 33342 staining solution, 4% paraformaldehyde, 0.5% Triton X-100, PBS buffer, and cell culture plates suitable for fluorescence observation.

[0023] 2. Testing Methods T24 and UMUC3 cells in logarithmic growth phase were digested and a single-cell suspension was prepared. These suspensions were then seeded into 24-well plates or fluorescence detection plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, the cells were treated with culture medium containing different concentrations of juglone, with an equal volume of DMSO as a solvent control. Culture was continued for another 24 hours.

[0024] Before the end of the treatment, EdU working solution was added to each well according to the EdU cell proliferation assay kit instructions for incubation, allowing cells in the DNA synthesis phase to incorporate EdU. The culture medium was then discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then incubated in the dark with click reaction solution. Finally, the cell nuclei were counterstained using Hoechst 33342.

[0025] Observe and photograph under a fluorescence microscope, randomly select multiple fields of view to count the number of EdU-positive cells and the total number of cells, and calculate the EdU positivity rate according to the following formula. The experiment was independently repeated 3 times.

[0026] EdU positivity rate (%) = (Number of EdU-positive cells / Total number of cells) × 100% 3. Experimental Results See results Figure 3 Compared with the DMSO control group, the proportion of EdU-positive cells in T24 and UMUC3 cells was significantly reduced after treatment with juglone, indicating that juglone can inhibit DNA synthesis and cell proliferation in bladder cancer cells.

[0027] The above results indicate that juglone has a clear inhibitory effect on the in vitro proliferation of bladder cancer cells.

[0028] Example 3: Transwell assay to detect the effect of juglone on the migration ability of bladder cancer 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2.

[0029] 1.2 Cell lines: Human bladder cancer T24 cells and human bladder cancer UMUC3 cells, cultured under the same conditions as in Example 1.

[0030] 1.3 Main reagents and consumables: Transwell chambers, serum-free culture medium, complete culture medium containing 10% fetal bovine serum, 4% paraformaldehyde, crystal violet staining solution, PBS buffer, cotton swabs and microscope.

[0031] 2. Testing Methods T24 and UMUC3 cells in logarithmic growth phase were harvested, digested, resuspended in serum-free medium, and cell density was adjusted. The cell suspension was added to the upper chamber of a Transwell chamber, while the lower chamber was treated with complete medium containing 10% fetal bovine serum as chemotactic conditions. A DMSO control group and a juglone treatment group were included in the experiment.

[0032] After culturing for a certain period, remove the Transwell chamber, discard the culture medium in the upper chamber, and gently wipe away any unmigrated cells from the upper chamber with a cotton swab. Fix the cells that have migrated to the submembrane surface with 4% paraformaldehyde and then stain with crystal violet. After staining, wash with PBS, observe and photograph under a microscope.

[0033] Multiple fields of view were randomly selected to count the number of cells that had migrated through the membrane. The number of cells that had migrated through the membrane was used to evaluate cell migration ability. The experiment was independently repeated 3 times.

[0034] 3. Experimental Results See results Figure 4 Compared with the DMSO control group, the number of T24 and UMUC3 cells that crossed the Transwell membrane was significantly reduced in the juglone treatment group, suggesting that juglone can inhibit the migration ability of bladder cancer cells.

[0035] The above results further demonstrate that juglone not only has an inhibitory effect on proliferation, but also reduces the migration potential of bladder cancer cells.

[0036] Example 4: Experimental study on the therapeutic effect of juglone on subcutaneous xenografts of UMUC3 cells in mice. 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2. When used in animal experiments, juglone is dissolved in a suitable solubilizer and then diluted to the required concentration with the animal experimental drug medium. The model control group is given an equal volume of the solvent.

[0037] 1.2 Cell line: Human bladder cancer UMUC3 cells, with cell source and culture conditions the same as in Example 1. Cells in logarithmic growth phase and in good condition were used to establish a subcutaneous xenograft model.

[0038] 1.3 Experimental Animals: BALB / c nude mice were adapted for use in establishing a subcutaneous xenograft model of human bladder cancer UMUC3 cells. Animal experiments were conducted in accordance with ethical requirements for laboratory animals.

[0039] 1.4 Main reagents and consumables: PBS buffer, Matrigel or serum-free culture medium, sterile syringe, vernier caliper, electronic balance, tumor tissue fixative and routine animal experimental consumables.

[0040] 2. Testing Method: UMUC3 cells in logarithmic growth phase were collected, digested, washed with PBS, and a cell suspension was prepared. The cell suspension was then subcutaneously inoculated into immunodeficient mice to establish a UMUC3 cell subcutaneous xenograft model. Once the subcutaneous tumors formed and reached the experimentally predetermined volume, the mice were randomly divided into a model control group and a juglone treatment group.

[0041] The juglone treatment group was administered juglone according to the experimental protocol, while the model control group received an equal volume of solvent. During the administration period, the general condition and weight changes of the mice were observed regularly, and the long and short diameters of the tumors were measured using calipers. The tumor volume was calculated using the following formula: Tumor volume = Long axis × Short axis 2 / 2 After the experiment, mice were euthanized, subcutaneous tumor tissue was dissected, and the size and weight of the tumor were observed and photographed. Further tumor tissue could be collected for subsequent protein expression or histological analysis. The tumor inhibition rate was calculated using the following formula: Tumor inhibition rate (%) = (mean tumor weight of the model control group - mean tumor weight of the juglone treatment group) / mean tumor weight of the model control group × 100% 3. Experimental Results See results Figure 5 Compared with the model control group, the growth rate of subcutaneous xenografts in mice treated with juglone was slowed, the rate of tumor volume increase was reduced, and the tumor weight at the experimental endpoint was decreased, suggesting that juglone can inhibit the growth of UMUC3 cell subcutaneous xenografts in vivo. No significant abnormalities were observed in the general condition of the mice during the administration period, indicating that juglone has a good in vivo anti-bladder cancer effect under the experimental conditions.

[0042] The above results indicate that juglone can not only inhibit the proliferation and migration of bladder cancer cells in vitro, but also inhibit the growth of subcutaneous xenografts of bladder cancer in vivo.

[0043] Example 5: Effect of KEGG enrichment analysis after proteomics detection on the mechanism of juglone in bladder cancer cells 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2.

[0044] 1.2 Cell samples: T24 and UMUC3 cells in logarithmic growth phase were collected and set up as DMSO control group and juglone treatment group, respectively.

[0045] 1.3 Main reagents and materials: protein lysis buffer, protease inhibitor, BCA protein quantification kit, trypsin, liquid chromatography-tandem mass spectrometry detection system and proteomics data analysis software.

[0046] 2. Testing Methods T24 and UMUC3 cells in logarithmic growth phase were treated with juglone, with a DMSO control group included. After treatment, cells were collected, washed with pre-cooled PBS, and lysing buffer containing protease inhibitors was added to extract total protein. Protein concentration was determined using the BCA method, and equal volumes of protein were then subjected to reduction, alkylation, and trypsin digestion to obtain peptide samples.

[0047] Peptide samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the obtained mass spectrometry data were used for protein identification and quantification. Differentially expressed proteins were screened based on the difference in protein expression levels between the juglone-treated group and the control group, and KEGG pathway enrichment analysis was performed on the differentially expressed proteins.

[0048] The potential molecular mechanism by which juglone affects bladder cancer cells was evaluated by the significance of enrichment pathways and the distribution of differentially expressed proteins.

[0049] 3. Experimental Results See results Figure 6 Proteomics analysis revealed altered expression of multiple proteins in bladder cancer cells after juglone treatment. KEGG enrichment analysis showed that differentially expressed proteins were mainly enriched in ferroptosis, oxidative stress, glutathione metabolism, cysteine ​​metabolism, and tumor-related signaling pathways.

[0050] The above results suggest that the inhibitory effect of juglone on bladder cancer cells is closely related to the ferroptosis-related pathway, providing a basis for subsequent verification of juglone-induced ferroptosis by transmission electron microscopy, ferroptosis detection kits, and Western blot.

[0051] Example 6: Investigating the effect of juglone on ferroptosis in bladder cancer cells using transmission electron microscopy 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2.

[0052] 1.2 Cell lines: Human bladder cancer T24 cells and human bladder cancer UMUC3 cells, cultured under the same conditions as in Example 1.

[0053] 1.3 Main reagents and consumables: 2.5% glutaraldehyde fixative, osmium tetroxide, ethanol gradient dehydration reagent, epoxy resin embedding agent, ultramicrotome, copper mesh, uranium lead staining reagent and transmission electron microscope.

[0054] 2. Testing Methods T24 and UMUC3 cells in logarithmic growth phase were collected and divided into a DMSO control group and a juglone treatment group, respectively. After treatment, cells were collected, washed with PBS, and fixed with 2.5% glutaraldehyde. Subsequently, they were post-fixed with osmium tetroxide, dehydrated with graded ethanol, embedded in resin, ultra-thinly sectioned, and uranium-lead double-stained.

[0055] The prepared ultrathin sections were placed under a transmission electron microscope to observe the cell ultrastructure, focusing on ferroptosis-related morphological features such as mitochondrial volume, membrane density, mitochondrial cristae, and mitochondrial membrane integrity.

[0056] 3. Experimental Results See results Figure 7 Compared with the DMSO control group, T24 and UMUC3 cells treated with juglone showed obvious mitochondrial morphological abnormalities, including reduced mitochondrial volume, increased mitochondrial membrane density, and reduced or absent mitochondrial cristae, which are ferroptosis-related ultrastructural features.

[0057] The above results indicate that juglone can induce typical ferroptosis morphological changes in bladder cancer cells.

[0058] Example 7: Detection of the effect of juglone-induced ferroptosis in bladder cancer cells using a ferroptosis detection kit 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2.

[0059] 1.2 Cell lines: Human bladder cancer T24 cells and human bladder cancer UMUC3 cells, cultured under the same conditions as in Example 1.

[0060] 1.3 Main reagents and consumables: Ferroptosis-related detection kit, PBS buffer, cell culture plates, ELISA reader, fluorescence microscope or flow cytometer.

[0061] 2. Testing Methods T24 and UMUC3 cells in logarithmic growth phase were seeded into cell culture plates. After cell attachment, juglone was added for treatment, and a DMSO control group was set up. After treatment, ferroptosis-related assays were performed according to the instructions of the assay kit.

[0062] Depending on the type of test kit, intracellular ferrous ions, lipid peroxidation levels, malondialdehyde content, or glutathione levels and other ferroptosis-related indicators were measured, and the results were read using an enzyme-linked immunosorbent assay (ELISA) reader, fluorescence microscope, or flow cytometry.

[0063] The ability of juglone to induce ferroptosis in bladder cancer cells was evaluated by the changes in ferroptosis-related indicators between the juglone-treated group and the DMSO control group. The experiment was independently repeated 3 times.

[0064] 3. Experimental Results See results Figure 8 Compared with the DMSO control group, ferroptosis-related indicators were significantly increased in T24 and UMUC3 cells after juglone treatment, suggesting increased intracellular iron load and lipid peroxidation levels. These results indicate that juglone can promote ferroptosis in bladder cancer cells, consistent with the mitochondrial morphological changes observed by transmission electron microscopy in Example 6.

[0065] Example 8: Western blot analysis of the effect of juglone on the expression of ferroptosis-related proteins in bladder cancer cells. 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2.

[0066] 1.2 Cell lines: Human bladder cancer T24 cells and human bladder cancer UMUC3 cells, cultured under the same conditions as in Example 1.

[0067] 1.3 Main reagents and consumables: protein lysis buffer, BCA protein quantification kit, SDS-PAGE gel preparation reagent, PVDF membrane, blocking buffer, SLC7A11 antibody, GPX4 antibody, β-actin or GAPDH internal control antibody, HRP-labeled secondary antibody and ECL chemiluminescence reagent.

[0068] 2. Testing Methods T24 and UMUC3 cells treated with juglone were washed with PBS after the culture medium was discarded, and total protein was extracted by adding protein lysis buffer. After determining the protein concentration using the BCA method, an equal volume of protein sample was taken for SDS-PAGE electrophoresis and transferred to a PVDF membrane.

[0069] After blocking, PVDF membranes were incubated overnight at 4°C with SLC7A11, GPX4, and internal control protein antibodies. The following day, secondary antibodies were added and the membranes were developed using ECL chemiluminescence immunoassay. The grayscale values ​​of the protein bands were analyzed using image analysis software.

[0070] Using β-actin or GAPDH as internal controls, the expression levels of SLC7A11 and GPX4 proteins were standardized and calculated. The experiment was independently repeated three times.

[0071] 3. Experimental Results See results Figure 9 Compared with the DMSO control group, the expression levels of SLC7A11 and GPX4 proteins in T24 and UMUC3 cells were significantly reduced after treatment with juglone. SLC7A11 and GPX4 are important proteins that inhibit ferroptosis, and their reduced expression suggests a decrease in the cells' ability to resist lipid peroxidation.

[0072] The above results indicate that juglone can promote ferroptosis in bladder cancer cells by downregulating SLC7A11 and GPX4.

[0073] Example 9: Molecular docking to investigate the target protein of juglone. 1. Experimental Materials 1.1 Small molecule structure: The two-dimensional or three-dimensional structure files of juglone are obtained from public compound databases and have been processed by energy minimization.

[0074] 1.2 Protein Structure: The three-dimensional structure file of the BRD9 protein was obtained from a public protein structure database. Before use, water molecules and irrelevant ligands were removed, and hydrogenation, charge addition, and structural optimization were performed.

[0075] 1.3 Main software and reagents: molecular docking software, molecular dynamics simulation software, binding free energy analysis tools, reagents related to cell thermal displacement experiments, BRD9 antibody and reagents related to Western blot.

[0076] 2. Testing Methods The potential binding ability of juglone to BRD9 protein was analyzed using molecular docking. After structural pretreatment of the BRD9 protein receptor and the juglone small molecule ligand, docking activity pockets and parameters were set. Juglone and BRD9 were then molecularly docked, and the binding energy, binding conformation, and key amino acid residue interactions were analyzed.

[0077] Molecular dynamics simulations were performed using the juglone-BRD9 complex obtained by molecular docking as the initial conformation. The root mean square deviation, root mean square fluctuation, number of hydrogen bonds, and binding free energy of the complex were analyzed to evaluate the stability of the complex.

[0078] Further cell thermal displacement assays were used to verify the interaction between juglone and BRD9. T24 and UMUC3 cell lysates or cell samples were treated with juglone or DMSO, respectively. Soluble proteins were collected after heating at different temperature gradients, and Western blot was used to detect changes in the stability of BRD9 protein under different temperature conditions.

[0079] 3. Experimental Results See results Figure 10 .

[0080] Molecular docking results showed that juglone can form a relatively stable binding conformation with the BRD9 protein and generate hydrogen bonds, hydrophobic interactions, or other non-covalent interactions with key amino acid residues of BRD9. Molecular dynamics simulation results showed that the juglone-BRD9 complex maintained a stable overall conformation during the simulation, suggesting that juglone has the potential to bind to BRD9.

[0081] The results of the thermal displacement experiment showed that the thermal stability of BRD9 protein changed after treatment with juglone compared with the DMSO control group, further indicating that juglone can interact with BRD9. These results suggest that BRD9 may be an important target protein for juglone to exert its anti-bladder cancer effect.

[0082] Example 10: Western blot analysis of the reversal effect of BRD9 overexpression on juglone-induced ferroptosis 1. Experimental Materials 1.1 Test reagent: Juglone, source, purity and preparation method are the same as in Example 2.

[0083] 1.2 Cell lines: Human bladder cancer T24 cells and human bladder cancer UMUC3 cells, cultured under the same conditions as in Example 1.

[0084] 1.3 Main reagents and consumables: BRD9 overexpression plasmid or lentiviral vector, empty vector control, transfection reagent, protein lysis buffer, BCA protein quantification kit, BRD9 antibody, SLC7A11 antibody, GPX4 antibody, β-actin or GAPDH internal control antibody, HRP-labeled secondary antibody and ECL chemiluminescence reagent.

[0085] 2. Testing Methods BRD9 overexpression plasmids or lentiviral vectors were transfected into T24 and UMUC3 cells, with an empty vector control group included. After successful BRD9 overexpression, cells were treated with juglone. Cells were then collected, and total protein was extracted.

[0086] Western blot was used to detect the expression levels of BRD9, SLC7A11, and GPX4 proteins. β-actin or GAPDH was used as an internal control. The gray values ​​of each protein band were standardized and analyzed, and the differences between the empty vector control group, the juglone treatment group, and the BRD9 overexpression combined with juglone treatment group were compared.

[0087] The role of BRD9 in juglone-induced ferroptosis in bladder cancer cells was evaluated by observing whether BRD9 overexpression could restore the decrease in SLC7A11 and GPX4 induced by juglone.

[0088] 3. Experimental Results See results Figure 11 Juglone treatment reduced the expression of BRD9, SLC7A11 and GPX4 proteins in bladder cancer cells. Compared with the juglone treatment group alone, BRD9 overexpression significantly increased BRD9 protein levels and partially restored the expression of SLC7A11 and GPX4.

[0089] The above results indicate that BRD9 overexpression can partially reverse the changes in ferroptosis-related protein expression induced by juglone, suggesting that juglone induces ferroptosis in bladder cancer cells at least partially by inhibiting BRD9 expression and downregulating SLC7A11 and GPX4.

Claims

1. This invention relates to the application of a naturally derived naphthoquinone compound in the preparation of drugs for the prevention and treatment of bladder cancer, characterized in that, The 10 naturally derived naphthoquinone compounds are selected from any one or more of the following compounds: shikonin, deoxyshikonin, β-lapachone, α-lapachone, lawsone, Naphthazarin, Lapachol, Juglone, Plumbagin, and Phylloquinone; the bladder cancer is bladder cancer caused by T24 and / or UMUC3 cells.

2. The application according to claim 1, characterized in that, The naturally sourced naphthoquinone compound is juglone, and its chemical structure is shown in formula (I) below: Formula (I); 3. The application according to claim 2, characterized in that, After 72 hours of treatment, the half-maximal inhibitory concentration (IC50) of the juglone against bladder cancer T24 cells and UMUC3 cells was determined. 50 The concentrations were 0.51 μM and 0.37 μM, respectively.

4. The application according to claim 2, characterized in that, Juglone inhibits the proliferation and migration of bladder cancer cells by inducing ferroptosis in bladder cancer cells.

5. The application according to claim 4, characterized in that, Juglone induces ferroptosis in bladder cancer cells by targeting and downregulating the expression of the BRD9 protein, thereby inhibiting the expression of key negative regulators of ferroptosis, SLC7A11 and GPX4.

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