Use of convallatoxin in the preparation of drugs against prostate cancer
Convatoxin activates specific signaling pathways by binding to the UPP1-encoded protein, enhancing apoptosis in prostate cancer cells. By combining with UPP1 overexpression plasmids or reagents, it solves the problem of drug resistance in prostate cancer, achieving significant tumor growth inhibition and good in vivo tolerance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-11
- Publication Date
- 2026-07-14
AI Technical Summary
In the existing technology, the drug resistance problem in prostate cancer is mainly caused by androgen receptor splicing mutations and lineage plasticity, and drugs targeting the AR signaling pathway are difficult to overcome effectively. The mechanism of action of lilytoxin in prostate cancer has not been fully studied.
Convatins activate the p38 MAPK signaling pathway by interacting with the UPP1-encoded protein and maintain caspase-3-mediated apoptosis via the JNK signaling pathway. They also enhance the anti-prostate cancer effect by binding to UPP1 overexpression plasmids or agents that promote UPP1 expression.
The binding of lilytoxin to UPP1 enhances the apoptosis-inducing ability of prostate cancer cells. Overexpression of UPP1 significantly improves the efficacy of lilytoxin, with significant tumor growth inhibition effect and good tolerability in mouse models.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of lilytoxin in the preparation of anti-prostate cancer drugs. Background Technology
[0002] Prostate cancer (PCa) is one of the most common malignant tumors in men. Despite significant progress in novel endocrine therapies (such as abiraterone and enzalutamide) and PSMA-targeted therapies, drug resistance remains a core challenge. This resistance is primarily caused by androgen receptor (AR) splicing mutations (such as AR-V7) and lineage plasticity. Therefore, developing small molecule drugs that target novel pathways, rather than relying on the AR signaling pathway, has become crucial for overcoming prostate cancer drug resistance.
[0003] Urate phosphorylase 1 (UPP1) encodes a protein that serves as a key enzyme in pyrimidine nucleoside metabolism, providing an alternative energy source for tumor cells under glucose deprivation conditions. Simultaneously, UPP1 drives the formation of an immunosuppressive microenvironment by regulating the PD-L1 / TGF-β1 pathway. This mechanism also promotes the establishment of pre-metastatic niches and mediates chemotherapeutic resistance. Pan-cancer analysis results indicate that UPP1 is aberrantly expressed in various malignancies and is associated with poor patient prognosis. Existing research suggests that drugs targeting the UPP1-encoded protein have the potential to inhibit tumor growth and metastasis in various preclinical models. Furthermore, these drugs exhibit synergistic benefits when used in combination with immunotherapy or chemotherapy.
[0004] Convallatoxin is a natural cardiac glycoside compound that, at nanomolar concentrations, can inhibit the proliferation of various tumor cells, including human breast cancer (MCF-7, MDA-MB-231, MDA-MB-468), non-small cell lung cancer (A549), leukemia (K562), liver cancer, and gastric cancer. However, current research on the role and related mechanisms of convallatoxin in prostate cancer cells remains limited, especially regarding whether convallatoxin can exert its anti-prostate cancer effect by targeting the UPP1-encoded protein, which lacks systematic investigation. Summary of the Invention
[0005] To address the aforementioned shortcomings in the prior art, this invention provides the application of lilytoxin in the preparation of anti-prostate cancer drugs. Studies have shown that lilytoxin can interact with the UPP1-encoded protein, then activate the p38 MAPK signaling pathway and maintain caspase-3-mediated apoptosis via the JNK signaling pathway. This combination can enhance the apoptosis-inducing ability of lilytoxin on prostate cancer cells.
[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide the application of lilytoxin in the preparation of a medicament for treating prostate cancer. The structural formula of lilytoxin is as follows: .
[0007] Furthermore, the effective in vitro concentration of lilytoxin is 1.56~200 nM. In a mouse subcutaneous xenograft model, the dosage of lilytoxin is 150 μg / kg, administered via intraperitoneal injection.
[0008] Furthermore, lilytoxin can upregulate the expression of cleaved-caspase-3 and induce PARP cleavage.
[0009] Another object of the present invention is to provide the use of reagents that promote UPP1 expression in the preparation of formulations that enhance the sensitivity of prostate cancer to lilytoxin, in which high expression of UPP1 is not a therapeutic purpose itself, but a sensitivity condition that improves the binding / action efficiency of lilytoxin.
[0010] Furthermore, the active ingredients of the reagents that promote UPP1 expression are small molecule compounds, UPP1 overexpression induction systems, UPP1 overexpression vectors, or CRISPRa / dCas9 fusion chemical induction systems.
[0011] Furthermore, convalescent glycosides enhance their anti-prostate cancer efficacy after binding to the UPP1-encoded protein.
[0012] Another object of the present invention is to provide the use of reagents that promote UPP1 expression and lilytoxin in the preparation of combination drugs for the treatment of prostate cancer.
[0013] Another object of the present invention is to provide a formulation that enhances the efficacy of lilytoxin against prostate cancer, comprising a UPP1 overexpression plasmid or an agent that promotes UPP1 expression, and pharmaceutically acceptable excipients thereof.
[0014] Another object of the present invention is to provide a combination drug for treating prostate cancer, comprising lilytoxin as an active ingredient and a UPP1 overexpression plasmid or agent for promoting UPP1 expression, and pharmaceutically acceptable adjuvants thereof.
[0015] Furthermore, the excipient is any one or a combination of several of the following: adhesive, disintegrant, lubricant, and flow aid.
[0016] Furthermore, the effective in vitro concentration of lilytoxin is 1.56~200 nM. In a mouse subcutaneous xenograft model, the dosage of lilytoxin is 150 μg / kg, administered via intraperitoneal injection.
[0017] Furthermore, the dosage form of the combination drug is tablets, pills, capsules, drops, granules, ointments, powders, or liquid preparations.
[0018] The beneficial effects of this invention are: This invention reveals that lilytoxin can interact with the UPP1-encoded protein, and this interaction generates a time-differential regulatory signal: the p38 MAPK signal is activated early, while the JNK signal is persistently activated. The binding of both enhances caspase-3 activity, thereby overcoming apoptosis resistance caused by a single signaling pathway.
[0019] Convalescence glycoside is derived from traditional Chinese medicine. Under the administration conditions described in this embodiment, no significant decrease in mouse body weight was observed, suggesting that this dose has a certain degree of in vivo tolerance. This invention also found that increasing UPP1 expression can enhance the toxicity of convalescence glycoside to prostate cancer cells. Compared with the control group treated with convalescence glycoside alone, the apoptosis rate induced by UPP1-overexpressing 22Rv1 cells treated with convalescence glycoside increased by approximately fourfold, indicating that increased UPP1 expression can enhance the apoptosis-inducing effect of convalescence glycoside on prostate cancer cells. Attached Figure Description
[0020] Figure 1 The results of transcriptomics analysis based on the GEO database are shown in the figure; where A is a volcano plot; B is a bubble plot of KEGG pathway enrichment analysis; C is a plot of differential gene expression analysis of UPP1 in primary and metastatic prostate cancer; and D is a Kaplan-Meier survival curve of UPP1 gene expression and recurrence-free survival. Figure 2 Figure showing the docking results of lilytoxin and UPP1 molecules; Figure 3 This is a graph showing the detection of cell thermal displacement. Figure 4 The image shows the results of detecting the enhancement of UPP1 by lilytoxin against the proliferation of prostate cancer cells 22Rv1. Figure 5 Figure showing the results of flow cytometry evaluation of apoptosis induced by lilytoxin in prostate cancer cells 22Rv1; Figure 6 The figure shows the results of Western blot analysis to evaluate the apoptosis induced by convalescent glycosides in prostate cancer cells 22Rv1. Figure 7 A diagram illustrating the mechanism by which convalescent glycosides promote apoptosis in prostate cancer cells, as detected by Western blot. Figure 8The figures show the results of the study on the effect of lilytoxin on the growth of prostate cancer cell xenografts in vivo; where A is a representative image of the appearance of subcutaneous xenografts in mice at the end of treatment; B is a graph of the weight change of mice during treatment; and C is a graph of the growth of xenografts during treatment. Detailed Implementation
[0021] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0022] Example 1 Through the GEO database official website ( https: / / www.ncbi.nlm.nih.gov / geo / Transcriptome data for hormone-sensitive and castration-resistant prostate cancer were retrieved and downloaded. The downloaded data were preprocessed using R software. Preprocessing steps included: removing duplicate values, eliminating genes with a deletion rate exceeding 50%, gene annotation and standardization of the cleaned data, and merging the data from different groups. Differentially expressed genes were screened using a threshold of |log2FC| ≥ 2 and a corrected P-value (adjP) < 0.01. Bayesian statistical analysis was performed on the fitting results to improve estimation accuracy. Genes obtained from the analysis were extracted and corrected using the False Discovery Rate (FDR) to screen for genes with significant differences. GO enrichment and KEGG enrichment analyses were performed on the differentially expressed genes to identify significantly different signaling pathways.
[0023] like Figure 1 As shown, transcriptomic analysis revealed that UPP1 was positively correlated with the malignant progression of prostate cancer and associated with poor prognosis.
[0024] Example 2 Molecular docking experiments between lilytoxin and the UPP1-encoded protein were performed using the AutoDock 4.2.6 software package. The specific steps included: 1. Molecular structure pretreatment The three-dimensional structure of the UPP1 receptor protein was obtained from the RCSB PDB database. Preprocessing was performed using AutoDock Tools (ADT) 1.5.6, including removing water of crystallization molecules and non-critical ligands, adding polar hydrogen atoms, calculating the Gasteiger-Marsili charge, and saving the receptor in PDBQT format. The lilytoxin structure was downloaded from the PubChem database, converted to a different format and minimized using OpenBabel 3.1.1, and then rotatable bonds were defined using ADT to generate a PDBQT file.
[0025] 2. Dating parameter settings The force field uses the default AD4 atom type, the van der Waals potential is described as the Lennard-Jones 12-6 potential, and the electrostatic potential is calculated using the Coulomb potential. The docking algorithm uses the Lamarckian Genetic Algorithm (LGA), with a population size of 150 and 100 independent docking attempts per ligand. Other genetic algorithm search parameters use the recommended or default settings from AutoDock 4 to ensure sufficient sampling of the conformation space.
[0026] 3. Results Analysis and Visualization The docking results were clustered (with a tolerance of 2.0 Å RMSD) to classify conformations, and the binding free energy (ΔG) and estimated dissociation constant (Ki) were calculated using the AutoDock semi-empirical scoring function. The top 5% of low-energy conformations were visualized using PyMOL 2.5.2. The hydrogen bond network, hydrophobic interactions, and spatial complementarity were analyzed to clarify the binding mode of lilytoxin and UPP1; the results are shown in […]. Figure 2 .
[0027] like Figure 2 As shown, the binding energy between lilytoxin and the protein encoded by UPP1 is -9.0 kcal / mol, indicating that it has a strong binding affinity.
[0028] Example 3 1. Cellular thermal displacement analysis to detect the effect of convalescent glycosides on the thermal stability of UPP1 encoded protein 22Rv1 cells were lysed with cell lysis buffer containing 0.1% protease inhibitor. The protein concentration was adjusted to 5 mg / mL using the BCA method. 50 μL of sample was taken and either 1 μM convalescentine or an equal volume of DMSO (as a control) was added, and the mixture was incubated at 4°C for 1 hour. The samples were heated using a PCR thermal cycler, with temperatures ranging from 37°C to 85°C, incremented every 3 minutes. The expression level of the UPP1-encoded protein was detected using Western blot; the Western blot experimental method is detailed in Example 4. The band intensity was quantified using ImageJ software, and melting curves were generated accordingly. The results are shown in [Figure 4]. Figure 3 .
[0029] like Figure 3 As shown, the melting curve of UPP1 in the lilytoxin-treated group shifted to the right compared to the DMSO group (ΔTm = 8.12℃), indicating that lilytoxin can improve the thermal stability of the UPP1 encoded protein, suggesting a direct interaction between the two.
[0030] 2. The sensitizing effect of UPP1 on lilytoxin was detected by the CCK-8 assay. The cell lines used were as follows: 22Rv1 cells transfected with the empty vector and 22Rv1 cells transfected with the UPP1 overexpression plasmid.
[0031] (1) Paving When the cells reached 80% confluence, they were digested with 0.25% Trypsin-EDTA solution. After digestion, the cells were collected by centrifugation at 900 rpm for 4 minutes. The cells were resuspended in RPMI 1640 medium and diluted to a density of 8.0 × 10⁻⁶. 3 Cells / mL. The diluted cell suspension was seeded into 96-well plates, 100 μL per well. The 96-well plates were placed in a cell culture incubator at 37°C and 5% CO2 and cultured for 12 hours.
[0032] (2) Drug treatment Convallinoside dosage was set using a gradient concentration, starting at 200 nM and serially diluted to 1.56 nM, with a 0 concentration control included. After cells adhered and were cultured for 12 hours, the cells were treated with the drug according to the group settings. The cells were then placed in a cell culture incubator at 37°C and 5% CO2 and incubated for another 24 hours.
[0033] (3) Cell viability assay Discard the sample solution in the 96-well plate. Add basal medium containing 10% CCK-8 reagent, incubate for 1 to 2 hours, and measure the absorbance of each well at 450 nm.
[0034] (4) Data processing Export and save the absorbance results obtained from the test in Excel. Calculate cell viability using the following formula: Cell Viability = (OD) Drug -OD Blank ) / (OD Control -OD Blank )×100% Among them, OD Drug OD values for the drug-treated group and the model group; OD Blank OD values for cell-free blank wells (containing only culture medium and CCK-8, with the drug-treated groups containing additional drug solution); OD Control This represents the OD value of the normal group.
[0035] like Figure 4 As shown, the IC50 after 24 hours of lilytoxin exposure... 50 The values were 54.45 nM (22Rv1) and 45.57 nM (22Rv1-UPP1-OE), respectively. These results confirm that UPP1 overexpression can enhance the sensitivity of lily of the valley to prostate cancer cells.
[0036] 3. Flow cytometry analysis revealed that UPP1-sensitized lilytoxin effectively induced apoptosis in prostate cancer cells. The specific steps are as follows: After treating each group of cells with lilytoxin for 24 hours, cells were collected and washed once with pre-chilled phosphate-buffered saline (PBS). Cells were digested using trypsin without EDTA. After digestion, cells were collected by centrifugation, resuspended in PBS, and counted (density approximately 5 × 10⁻⁶). 6 (cells / mL). Afterwards, resuspend in 195 μL of 1× binding buffer (included in the Annexin V-APC / PI Apoptosis Detection Kit), add 5 μL of Annexin V-APC and 10 μL of PI staining solution, mix gently, and incubate at room temperature in the dark for 20 minutes. Subsequently, flow cytometry was used to detect and analyze apoptosis in the APC and PerCP channels. Results are shown in [Figure number missing]. Figure 5 .
[0037] like Figure 5 As shown, compared with the group treated with lilytoxin alone, treatment of 22Rv1 cells overexpressing UPP1 with lilytoxin induced an approximately 4-fold increase in the apoptosis rate of cancer cells, indicating that UPP1 enhanced the ability of lilytoxin to promote apoptosis in prostate cancer cells.
[0038] Example 4 The mechanism by which UPP1 enhances lilytoxin-induced apoptosis in prostate cancer cells was analyzed using Western blot. The specific steps are as follows: After treating 22Rv1 cells with lily toxin for 12 to 48 hours, the cells were lysed using RIPA lysis buffer containing protease inhibitors, and total protein was extracted. Protein samples were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1 hour. The following primary antibodies were added: p-p38, p38, p-JNK, JNK, caspase-3, cleaved-caspase-3, PARP, cleaved-PARP, and β-actin. The membranes were incubated overnight at 4°C. The membranes were washed three times with TBST buffer for 10 minutes each time. HRP-labeled secondary antibody was added, and the membranes were incubated at room temperature for 1 hour. Development was performed using ECL developing solution, and protein bands were recorded using an imaging system. The results are shown in [Figure number missing]. Figure 6 and Figure 7 .
[0039] like Figure 6 As shown, UPP1 enhanced the ability of lilytoxin to promote apoptosis in prostate cancer cells. Both promote apoptosis in prostate cancer cells by promoting the cleavage of caspase-3. From Figure 7 As can be seen, after convalescent glycosides bind to the UPP1-encoded protein, they first activate the p38 MAPK signaling pathway (0 to 12 hours), and then maintain the apoptosis of prostate cancer cells via the JNK pathway (12 to 24 hours).
[0040] Example 5 The animal tumor model was constructed as follows: 1. Tumor cell culture Human prostate cancer cell lines (22Rv1 cells and 22Rv1-UPP1-OE cells) in the logarithmic growth phase (80% confluence) were placed in 15cm culture dishes, approximately 2 × 10⁶ cells per dish. 7 Add 3 mL of 0.25% Trypsin-EDTA solution to each cell, mix gently, and digest at 37°C for 2 minutes with 5% CO2. Then, add 3 mL of RPMI 1640 complete medium to terminate the digestion reaction. After that, add 1 volume of PBS, centrifuge at 1000 rpm for 5 minutes, and repeat the centrifugation and washing with PBS solution twice. Then, resuspend the cells in ice-chilled, phenol red-free RPMI 1640 medium and adjust the cell concentration to 5 × 10⁶ cells / mL. 7 per mL.
[0041] 2. Cells were inoculated into nude mice. Male BALB / c nude mice (7 weeks old, weighing 27±2 g) were acclimatized for 7 days under SPF-grade animal experimental conditions. The resuspended cells obtained in step (1) were then mixed with an equal volume of ice-chilled Matrigel cells. TMThe matrix gel was mixed. The mixture was administered subcutaneously to the groin region of the right hind limb of mice, with each mouse receiving 0.1 mL (containing 2.5 × 10⁻⁶ g / mL of the matrix gel). 6 Nude mice were randomly divided into 5 groups (including 3 groups injected with 22Rv1 cells: 22Rv1+PBS, 22Rv1+convastatin, 22Rv1+enzalutamide, and 22Rv1-UPP1-OE+PBS and 22Rv1-UPP1-OE+convastatin) with 5 mice in each group. They were fed standardly with free access to food and water. Injection was initiated when a tumor could be palpated at the inoculation site (tumor volume approximately 50-100 mm). 3 This indicates that the modeling was successful.
[0042] 3. Drug therapy Each group received intraperitoneal injections every two days of PBS (control group), lilytoxin (150 μg / kg), or enzalutamide (Enza, positive control, 25 mg / kg). Mice survival was observed and recorded over 15 days, and tumor growth curves and changes in mouse body weight were calculated. Tumor volume was defined as length × width × width × 0.5. Data were statistically analyzed using t-tests or ANOVA. P <0.05 indicates a significant difference between the two groups. The animal care and use procedures were approved by the Ethics Committee of Zhejiang University and complied with all applicable institutional and government regulations concerning the ethical use of animals (ZJU20250605).
[0043] The results are as follows Figure 8 As shown, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001. Convatinin showed a more significant tumor growth inhibition effect in the UPP1 overexpression group compared with the single drug group, and under the administration conditions of this example, the body weight of mice did not decrease significantly, suggesting that it has a certain degree of in vivo tolerance at this dose.
[0044] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.
Claims
1. Application of convalescent glycoside in the preparation of drugs for treating prostate cancer.
2. The application according to claim 1, characterized in that, The effective in vitro concentration of lilytoxin is 1.56~200 nM. In a mouse subcutaneous xenograft model, the dosage of lilytoxin is 150 μg / kg, administered via intraperitoneal injection.
3. Application of reagents that promote UPP1 expression in the preparation of formulations that enhance the sensitivity of prostate cancer to lilytoxin.
4. The application according to claim 3, characterized in that, The reagents that promote UPP1 expression include small molecule compounds, UPP1 overexpression induction systems, UPP1 overexpression vectors, or CRISPRa / dCas9 fusion chemical induction systems.
5. The application according to claim 3, characterized in that, Convatinin enhances its anti-prostate cancer efficacy after binding to the UPP1-encoded protein.
6. Application of reagents that promote UPP1 expression and lilytoxin in the preparation of combination drugs for the treatment of prostate cancer.
7. A formulation for enhancing the efficacy of lilytoxin against prostate cancer, characterized in that, This includes UPP1 overexpression plasmids or reagents that promote UPP1 expression as active ingredients, as well as pharmaceutically acceptable excipients, carriers, or excipients.
8. A combination drug for treating prostate cancer, characterized in that, This includes lilytoxin as an active ingredient and UPP1 overexpression plasmids or agents that promote UPP1 expression, as well as pharmaceutically acceptable excipients.
9. The combination drug according to claim 8, characterized in that, The effective in vitro concentration of lilytoxin is 1.56~200 nM. In a mouse subcutaneous xenograft model, the dosage of lilytoxin is 150 μg / kg, administered via intraperitoneal injection.
10. The combination drug according to claim 8, characterized in that, The dosage form of the combined drugs is tablets, pills, capsules, drops, granules, ointments, powders, or liquid preparations.