Application of urolithin A in the preparation of drugs for treating ovarian cancer

Abstract

This invention discloses a nanodelivery system loaded with urolithin A and its application in the preparation of drugs for treating peritoneal metastases of ovarian cancer. The therapeutic effects of UA on ovarian cancer cells were evaluated in vitro and in vivo, and its potential mechanisms were explored. Furthermore, PLGA nanoparticles loaded with UA were constructed and used in combination with ABT263, which has anti-aging effects, to treat ovarian cancer in a mouse model with peritoneal metastases, demonstrating its important role in the treatment of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical applications, specifically to the use of urolithin A in the preparation of drugs for treating ovarian cancer. Background Technology

[0002] Ovarian cancer (OC) is one of the most threatening malignant tumors of the female reproductive system. Due to its often subtle early symptoms, most patients are diagnosed only when the disease has progressed to an advanced stage. Standard treatment for ovarian cancer typically includes tumor resection followed by platinum-based chemotherapy. However, 70% of patients experience recurrence within two years of treatment, developing platinum-resistant recurrent ovarian cancer (PROC), which significantly reduces treatment effectiveness, resulting in a five-year survival rate of less than 30%. Therefore, the development of new drugs and therapies for the treatment of ovarian cancer is of paramount importance.

[0003] Cellular senescence refers to a cell cycle arrest state entered by tumor cells under specific stress. This state was initially considered an important tumor suppressor mechanism, designed to prevent damaged cells from proliferating indefinitely and evolving into cancer cells. However, these senescent tumor cells are not harmless. Instead of being promptly eliminated, they continuously secrete large amounts of inflammatory factors, forming a senescence-associated secretory phenotype (SASP), and playing a complex role in the occurrence and development of cancer. The p53-p21 signaling pathway is one of the core mechanisms regulating cellular senescence. However, in cancer cells, the function of this pathway is often suppressed due to p53 mutations or deletions. Therefore, restoring or mimicking the function of this pathway has become an important strategy for anti-tumor therapy.

[0004] Urolithin A (UA) is a natural product produced by the metabolism of ellagitannins by gut microbes. Its main biological functions include activating mitophagy and exerting anti-inflammatory and antioxidant effects. UA also shows potential in anti-tumor therapy. In colorectal cancer cells, UA can inhibit cell growth, affect cell migration, and regulate the expression of matrix metalloproteinases in a dose- and time-dependent manner. Studies have found that UA can promote the proliferation of T-type memory stem cells and enhance anti-tumor immunity. Furthermore, UA can inhibit breast cancer development by activating TFEB-mediated mitophagy and reduce the harmful inflammatory response of tumor macrophages. Research has also found that UA can induce the interaction between apoptosis and autophagy in oral squamous cell carcinoma through the mTOR / AKT / ERK1 / 2 pathway. However, research on the role of UA in ovarian cancer, especially its role in regulating ovarian cancer cell senescence, remains an emerging and unresolved area.

[0005] Furthermore, after being absorbed by the body, UA undergoes a second stage of metabolism, forming inactive metabolites bound to glucuronic acid, resulting in low bioavailability. Therefore, optimizing UA delivery systems to improve the bioavailability of its active form is a current research focus. However, research on UA ​​delivery using biomaterials is limited. Zuoqin Yan's team constructed a functionalized hydrogel containing cerium oxide (CeO2) and UA for cartilage repair. Han Lu's team fabricated a uric acid nanomotor targeting microglia mitochondrial autophagy for the treatment of neuroinflammatory diseases. Changyou Gao's team created a therapeutic microgel for inflammatory bowel disease. In tumor treatment, Masoud Homayouni Tabrizi's team created a solid lipid nanocarrier containing methylurodollin A by coating folic acid-bound chitosan onto fatty acids, achieving cytotoxic effects in breast cancer cells. However, they have not verified its therapeutic effect on tumors in animal models, nor has it been used to treat ovarian cancer.

[0006] Poly(lactic-glycolic acid) (PLGA) is a copolymer formed by the polymerization of lactic acid (LA) and glycolic acid (GA). Due to its excellent biocompatibility, biodegradability, and drug release properties, it has become one of the most widely studied carriers in the field of nanomedicine. It received clinical use approval from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) on April 9, 2010. PLGA nanoparticles can deliver anticancer drugs to tumor sites, thereby increasing drug concentrations in tumor tissue and reducing toxic side effects on healthy tissues. For example, researchers have encapsulated doxorubicin (DOX) in PLGA nanoparticles to achieve targeted therapy for breast cancer. Furthermore, PLGA nanoparticles have also been used to simultaneously deliver carboplatin and sensitizers to restore the sensitivity of ovarian cancer to platinum-based chemotherapy. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide the use of urolithin A in the preparation of a drug for treating ovarian cancer.

[0008] To achieve the above objectives, the present invention provides the following technical solution: Application of urolithin A in the preparation of drugs for treating ovarian cancer.

[0009] Application of urolithin A in the preparation of drugs that inhibit the proliferation and migration of ovarian cancer cells.

[0010] Application of urolithin A in the preparation of drugs that promote the senescence of ovarian cancer cells.

[0011] Preferably, the drug is based on a nanodelivery system of urolithin A.

[0012] Preferably, in this invention, the nanodelivery system uses a mixture of N,N-dimethylformamide and acetonitrile as the organic solvent, and PLGA38000-PEG10000, PLGA, and urolithin A as solutes. After mixing, an organic solvent phase is formed. The organic solvent phase is then added dropwise to a dispersed phase solution (ultrapure water) in an ice bath using a syringe. The dropwise addition process must be carried out on a magnetic stirrer. The resulting liquid is centrifuged, and the supernatant is collected as a PLGA-UA nanoparticle suspension. The obtained nanoparticles have a particle size of 197.68 ± 41.61 nm.

[0013] Preferably, the mass-to-volume ratio of the solute to the organic solvent is 11:1 (mg:ml).

[0014] Preferably, the volume ratio of N,N-dimethylformamide to acetonitrile is 1:1.

[0015] Preferably, the mass ratio of PLGA38000-PEG10000, PLGA and urolithin A is 3.33:16.67:2.

[0016] Preferably, the drug is a dual nanodelivery system of urolithin A and aging scavenger ABT263.

[0017] The beneficial effects of this invention are as follows: This invention provides the application of urolithin A in the preparation of drugs for treating ovarian cancer. We evaluated the therapeutic effect of UA on ovarian cancer cells in vitro and in vivo, and explored its potential mechanism. Furthermore, we constructed PLGA nanoparticles loaded with UA and used them in combination with ABT263, which has anti-aging effects, to treat ovarian cancer in a mouse model with peritoneal metastasis. This invention is the first to apply UA to the treatment of ovarian cancer; it also proposes for the first time the molecular mechanism by which UA induces senescence in ovarian cancer cells through the GSK3β-MDM2-p53-p21 pathway; and it utilizes a nanodelivery system that delivers both UA and the senescence scavenger ABT263 to synergistically treat peritoneal metastasis of ovarian cancer in a mouse model. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Schematic diagram of nanoparticle preparation; Figure 2Characterization of the nanoparticles (A: Transmission electron microscopy images showing the morphology and particle size of PLGA and PLGA-UA nanoparticles in the dry state; scale bar 300 nm; B: Particle size of the two nanoparticles when dissolved in water by dynamic light scattering; C: Zeta potential of PLGA and PLGA-UA nanoparticles; D: UA content in nanoparticles determined by high performance liquid chromatography; E, F: Cytotoxicity of different nanoparticles in A2780 and ID8 cell lines, respectively).

[0019] Figure 3 To demonstrate the inhibitory effect of urolithin A on the proliferation and migration of ovarian cancer cells in vitro (A: molecular structure of urolithin A; B: cell proliferation curve representing the half-inhibitory concentration (IC50) of UA in different cell types; C and D: colony formation assay (C) and quantitative analysis (D) were used to determine the number of colonies formed after treatment with different concentrations of UA; E and F: spheroid formation assay (E) and quantitative analysis (F) were used to determine the number of OC stem cell spheroids formed after treatment with different concentrations of UA; scale bar is 100 μm. G and H: Transwell staining and histogram quantitative detection of the invasive ability (G) and quantitative results (H) of different OC cell groups were performed. Scale bar is 100 μm. I and J: Real-time qPCR and Western blotting were used to detect the gene and protein expression of E-cadherin in different OC cell groups, respectively.

[0020] Figure 4 To demonstrate the effect of UA on the inhibition of ovarian cancer growth in mice (A shows the experimental procedure in a subcutaneous mouse model; B is a statistical graph of the body weight of different groups of mice (n = 5); C: stereoscopic photographs of different tumors from the subcutaneous mouse model; D: statistical graph of the body weight of two groups of tumors (n = 5); E: SA-βGal staining images of different tumors (with or without UA treatment); scale bar is 100 micrometers; F: quantitative statistics of the proportion of senescent cells calculated based on (E) (n = 3)).

[0021] Figure 5To illustrate how UA induces senescence in ovarian cancer cells by upregulating the p53 signaling pathway (A: SA-βGal staining images of different OC cell groups treated with different concentrations of UA; unit: 100 μm; B: Quantitative statistics based on the data in Figure A, showing the proportion of senescent cells (n = 3); C, D: Gene heatmaps selected based on RAN-seq analysis, showing that the expression of genes related to cell senescence, SASP, and genes related to the p53 signaling pathway were upregulated in the UA group; color bars indicate the scale of gene expression; E: Gene set enrichment analysis (GSEA) showed that the p53 signaling pathway was enriched after UA treatment; F: KEGG enrichment analysis after uric acid treatment; G: Detection of p21 gene expression by real-time qPCR in different OC cell groups; H: Detection of p21 and p53 protein expression by Western blotting in different OC cell groups).

[0022] Figure 6 To illustrate how UA promotes cellular senescence by inhibiting the activity of the GSK3β-MDM2 axis (A: Venn diagram showing overlapping targets between the chemical and disease target sets; B: Molecular docking diagram showing the direct interaction between UA and GSK3β; C: Drug affinity response target stability (DARTS) assay showing that UA protects GSK3β from protease degradation; D, E: Co-precipitation immunoblot images showing MDM2 phosphorylation and p53 ubiquitination in A2780 cells after UA treatment; F: Mechanism diagram summarizing the molecular mechanism by which UA promotes senescence in ovarian cancer cells).

[0023] Figure 7 To demonstrate that UA-loaded nanoparticles significantly inhibited ovarian cancer progression in vivo (A: Schematic diagram of the experimental procedure in a mouse model of peritoneal metastasis; B: Luciferase signal detection results in ovarian cancer tissues of these 5 groups; C: Statistical results of fluorescence intensity obtained from B (n = 5); D: Statistical chart of mouse body weight in different groups (n = 5)). Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] Example 1: Preparation of Nanoparticles Urolithin A (UA) and PLGA38000-PEG10000 were purchased from MedChemExpress, USA, with catalog numbers HY-100599 and HY-176499, respectively. PLGA (Mw=38000) was purchased from Yuanye Pharmaceutical Co., Ltd., China, with catalog number S28133. The preparation method of PLGA-UA nanoparticles was described in reference [J Nanobiotechnology. 2016;14:32], using a phase separation precipitation method (…). Figure 1 The specific steps are as follows: Take a mixture of 500 μL N,N-dimethylformamide and 500 μL acetonitrile as the organic solvent. The organic solvent solutes are 3.33 mg PLGA38000-PEG10000, 16.67 mg PLGA, and 2 mg UA. Stir the above components overnight at 4°C on a magnetic stirrer until fully dissolved and homogeneous to obtain the organic solvent phase. Then, use a 1 mL syringe to dropwise add the organic solvent phase to a 20 mL ice-bathed dispersion solution (ultrapure water). The dropwise addition process must be carried out on a magnetic stirrer with high-speed stirring for 3 hours. Centrifuge the resulting liquid at 15000 g for 15 minutes. Transfer the supernatant to a new centrifuge tube to obtain a PLGA-UA nanoparticle suspension, which is stored at 4°C for later use.

[0026] In the preparation of the control group empty carrier nanoparticles, no UA component was added to the solute in the organic dissolved phase, while all other parameters remained the same. A PLGA nanoparticle suspension was obtained and stored at 4°C for later use.

[0027] Example 2: Characterization of Nanoparticles Nanoparticle concentration: 10.00 mL of each of the two nanoparticle suspensions prepared above was taken, freeze-dried, and weighed to obtain 3.67 mg of PLGA-UA nanoparticles and 2.00 mg of PLGA empty nanoparticles, respectively. The calculated concentrations of the two nanoparticle suspensions were 0.36 mg / mL and 0.20 mg / mL, respectively. The concentration of both nanoparticles was finally adjusted to 0.20 mg / mL for later use.

[0028] Nanoparticle size and Zeta potential: First, the morphology and dry particle size of two groups of nanoparticles were examined using transmission electron microscopy (Talos F200C, Czech Republic). Figure 2 According to ImageJ software calculations, the particle sizes of PLGA and PLGA-UA nanoparticles are 186.78 ± 38.60 nm and 197.68 ± 41.61 nm, respectively.

[0029] In addition, 1 mL of each of the two nanoparticle suspensions was placed in a dedicated cuvette and analyzed using a dynamic light scattering and Zeta potential analyzer (Zetatronix 939SZ, Opptronix, China). The results showed that the particle sizes of PLGA and PLGA-UA nanoparticles in suspension were 131.93±3.47 nm and 111.90±3.56 nm, respectively. Figure 2 B); the Zeta potentials were -25.90±0.44 mV and -23.20±3.06 mV, respectively. Figure 2 C).

[0030] Determination of UA carrying capacity in nanoparticles: An appropriate amount of UA standard was accurately weighed, dissolved in a suitable solvent, and diluted to 0.5 mg / mL as the reference solution. Detection was performed using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: methanol-water was used as the mobile phase, and separation was performed according to a gradient elution program: within 0 to 70 minutes, the methanol proportion linearly increased from 25% to 100%, and the water proportion decreased from 75% to 0%; the flow rate was 1.5 mL / min; the detection wavelength was 210 nm; and the injection volume was 50 μL. Under these conditions, the retention time of the UA standard was approximately 27.7 minutes. The results showed that 1 mg of PLGA-UA nanoparticles contained 7.89 μg of UA (…). Figure 2 D).

[0031] Cytotoxicity of Nanoparticles: To detect the cytotoxicity of two types of nanoparticles, human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8 were selected for cell viability assays. Cells were counted and seeded in 96-well plates at 3000 cells per well. The nanoparticle concentration gradients were 0 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL. The nanoparticle treatment times were 24, 48, 72, and 96 h. Cell viability was assessed using a CCK-8 assay kit (HY-K0301, MedChemExpress, USA) according to the provided instructions. After incubation at 37°C for 2 hours, absorbance at 450 nm and 630 nm was measured using a microplate reader (SpectraMax M5, Molecular Devices). Relative cell proliferation rate = (OD450 - OD630 of sample) / (OD450 - OD630 of control).

[0032] The results showed that no cytotoxicity of PLGA nanoparticles was detected in either A2780 or ID8 cells. Figure 2The presence of PLGA nanoparticles (E) indicates that the compound PLGA possesses good biocompatibility. Furthermore, PLGA-UA nanoparticles significantly inhibited the proliferation of ovarian cancer cells, demonstrating the effective cytotoxicity of the PLGA-UA nanodelivery system. Figure 2 F).

[0033] Example 3: Construction of a dual nanodelivery system of UA and ABT263 The aging scavenger ABT263 was purchased from MedChemExpress (HY-10087), and hyaluronic acid (HA) (Mw=1,000,000) was provided by Professor Xu Pengpeng of Shandong Aeronautical University. The final concentration of the HA hydrogel was 1% (w / v), and the concentration of ABT263 was 1 mM. 9.746 mg of ABT263 was weighed and dissolved in 0.1 mL of sterile DMSO to obtain a stock solution with a concentration of 100 mM. 24 μL of this stock solution was added to 2.4 mL of PLGA-UA nanoparticle suspension, and finally 2.4 mg of HA (Mw=1,000,000) was added. The mixture was thoroughly mixed using a pipette and incubated at 4°C on ice.

[0034] Example 4: UA inhibits the proliferation and migration of ovarian cancer cells in in vitro experiments. UA is a small molecule compound of diterpenoid derivative (Figure 3, A). We first evaluated the half-maximal inhibitory concentration (IC50) of UA in ovarian cancer cell lines. The results showed that the IC50 for A2780 and SKOV3 cells were 23.82 μM and 29.32 μM, respectively. Figure 3 (B). Clonogenesis experiments showed that UA inhibited the proliferation of A2780 and SKOV3 cells in a dose-dependent manner. Figure 3 (C~D). Furthermore, UA also inhibited the proliferative capacity of ovarian cancer stem cell spheres ( Figure 3 (E~F).

[0035] Cancer cell metastasis is also a significant factor contributing to poor prognosis in ovarian cancer. This study investigated the effect of UA on the migration ability of two ovarian cancer cell lines, A2780 and SKOV3. Transwell assays showed that UA significantly inhibited the migration ability of ovarian cancer cells. Figure 3 In addition, the expression of E-cadherin in ovarian cancer cells after UA treatment was detected by real-time quantitative PCR and Western blotting. The results showed that UA significantly upregulated its expression at both the mRNA and protein levels. Figure 3 ,I~J).

[0036] All of the above results indicate that UA has a dose-dependent inhibitory effect on the proliferation and migration of ovarian cancer cells.

[0037] Example 5: UA inhibits ovarian cancer growth and induces cancer cell senescence in a mouse subcutaneous tumor-bearing model. An ovarian cancer subcutaneous transplantation model was constructed in C57BL / 6 mice using ID8 cells, and the cells were treated under different conditions (with or without UA). Figure 4 Weight monitoring showed that the current dose of UA had low drug toxicity (A). Figure 4 (B) Stereoscopic imaging and tumor weight measurements showed that UA significantly inhibited the growth and progression of ovarian cancer in vivo. Figure 4 (C, 4, D). Furthermore, the results showed a significant increase in cellular senescence induced by UA in the tumor mass, which was confirmed by SA-βGal staining. Figure 4 These results suggest that UA may inhibit ovarian cancer progression by activating the aging process.

[0038] Example 6: UA induces senescence in ovarian cancer cells by upregulating the p53 signaling pathway. Focusing on cellular senescence, ovarian cancer cell lines A2780 and SKOV3 were treated with different concentrations of amino acids (UA), and SA-βGal staining was performed on the cells in vitro. The results showed that UA significantly induced senescence in ovarian cancer cells in a dose-dependent manner (Figure 5, A-B). To explore the underlying mechanism, A2780 cells were treated with 60 μM UA, and RNA sequencing was performed. Heatmap analysis showed that genes related to the senescence-associated secretory phenotype (SASP) were upregulated in the UA group, indicating that the cells exhibited a senescence phenotype after UA treatment. Figure 5 Furthermore, heatmap and KEGG enrichment analysis showed that genes involved in the p53 signaling pathway were significantly upregulated in the UA group (C). Figure 5 Gene set enrichment analysis (GSEA) further showed that the gene set of the p53 signaling pathway was significantly enriched in the urate group (D~F). Figure 5 Real-time quantitative qPCR and Western blotting results further demonstrated that UA can upregulate the expression of p53 and p21 in ovarian cancer cells. Figure 5 These results indicate that UA induces senescence in ovarian cancer cells by activating the p53-p21 pathway.

[0039] Example 7: UA promotes cellular senescence by inhibiting the activity of the GSK3β-MDM2 axis. For drug targets of UA, we screened 546 potential UA targets (chemical targets) using the TargetNet, SuperPred, SwissTargetPrediction, and BATMAN-TCM databases, and 110 ovarian cancer disease targets using the DisGeNET, GeneCards, OpenTargets, and CTDs databases. Screening the intersection of these two datasets revealed 37 common targets, including glycogen synthase kinase 3β (GSK3β). Figure 6 Molecular docking revealed a direct interaction between UA and GSK3β (Figure 6, B). Drug affinity response target stability (DARTS) assays further validated the binding of UA to GSK3β. Figure 6 (C) These results indicate that UA can bind directly to GSK3β.

[0040] It is well known that phosphorylated MDM2 can ubiquitinate and degrade p53 protein, thereby relieving the inhibitory effect of p53 on tumor growth. GSK3β is one of the phosphatases capable of phosphorylating MDM2 [Mol Cell Biol.25(16): 7170-80.]. Immunoprecipitation experiments showed that UA treatment significantly reduced the phosphorylation level of MDM2 (…). Figure 6 (D), indicating that GSK3β activity was inhibited in the UA group. Therefore, p53 ubiquitination was significantly reduced in the UA group ( Figure 6 In summary, we found that UA can bind to GSK3β and promote the hypophosphorylation of MDM2, thereby reducing p53 ubiquitination and degradation, and promoting cellular senescence in ovarian cancer cells. Figure 6 F).

[0041] Example 8: Nanoparticles loaded with UA significantly inhibited the progression of ovarian cancer in in vivo experiments. Cellular senescence may inhibit tumorigenesis and contribute to achieving ideal anticancer therapeutic effects. However, if not cleared in time, senescent cells may have a significantly higher tumor initiation potential under certain conditions [Nature, 2018. 553(7686): p. 96-100.]. Therefore, in a mouse model of peritoneal metastasis, we will use UA and the senescent cell scavenger ABT263 in combination to treat ovarian cancer ( Figure 7In vivo imaging and fluorescence intensity statistics showed that at week 3, administration of UA nanoparticles alone or in combination with ABT263 significantly inhibited peritoneal tumor metastasis, with better results than the PBS, PLGA / HA, and PLGA / HA-ABT263 groups (Figure 7, B-C). Body weight curve statistics showed no significant differences among the groups. Figure 7 HE staining results showed no significant differences in organs among the groups of mice (D). Figure 7 These results indicate that PLGA-UA nanoparticles and HA hydrogels have no significant toxic effects on mouse organs, and that this dual nanodelivery system is safe and effective for the treatment of peritoneal metastases from ovarian cancer in mice.

[0042] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Application of urolithin A in the preparation of drugs for treating ovarian cancer.

2. Application of urolithin A in the preparation of drugs that inhibit the proliferation and migration of ovarian cancer cells.

3. Application of urolithin A in the preparation of drugs that promote the senescence of ovarian cancer cells.

4. The application according to claim 1, characterized in that: The drug is based on a nanodelivery system for urolithin A.

5. The application according to claim 1, characterized in that: The nanodelivery system uses a mixture of N,N-dimethylformamide and acetonitrile as the organic solvent, and PLGA38000-PEG10000, PLGA, and urolithin A as solutes. After mixing, an organic solvent phase is formed. The organic solvent phase is then added dropwise to the dispersed phase solution (ultrapure water) in an ice bath using a syringe. The dropwise addition process must be carried out on a magnetic stirrer. The resulting liquid is centrifuged, and the supernatant is collected as a PLGA-UA nanoparticle suspension. The obtained nanoparticles have a particle size of 197.68 ± 41.61 nm.

6. The application according to claim 5, characterized in that: The mass-to-volume ratio of the solute to the organic solvent is 11:1 (mg:ml).

7. The application according to claim 5, characterized in that: The volume ratio of N,N-dimethylformamide to acetonitrile is 1:

1.

8. The application according to claim 5, characterized in that: The mass ratio of PLGA38000-PEG10000, PLGA and urolithin A is 3.33:16.67:

2.

9. The application according to claim 1, characterized in that: The drug is a dual nanodelivery system of urolithin A and aging scavenger ABT263.

Images