Use of urolithin a in the preparation of a medicament for treating ovarian cancer and medicament

Abstract

The application relates to application of urolithin A in preparation of a medicine for treating ovarian cancer and the medicine, and the medicine is used for inhibiting any one or more of proliferation, migration and invasion of ovarian cancer SKOV3 cells and A2780 cells, and / or inducing apoptosis of the ovarian cancer SKOV3 cells and A2780 cells. The application discloses a new use of urolithin A in treatment of ovarian cancer, and provides a new drug selection for treatment of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of urolithin A in the preparation of drugs for treating ovarian cancer and the drug itself. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the female reproductive system, ranking third in incidence among gynecological malignancies and consistently leading in mortality. Due to the deep anatomical location of the ovaries and the lack of typical early clinical manifestations, most patients are diagnosed at an advanced stage, missing the optimal window for radical surgical intervention. Although current clinical treatment has evolved into a comprehensive approach based on cytoreductive surgery combined with platinum-based chemotherapy, and targeted maintenance therapy strategies such as PARP inhibitors (polyadenosine diphosphate ribose polymerase inhibitors) are gradually being introduced, ovarian cancer patients still generally face high recurrence rates, strong drug resistance, and poor long-term prognosis, with a 5-year survival rate consistently below 50%. Therefore, developing novel anti-ovarian cancer drugs that combine efficacy and safety remains an important research direction in the field of gynecological oncology prevention and treatment.

[0003] Currently, screening for candidate molecules with antitumor activity from natural active ingredients has become an important approach to expanding the sources of antitumor drugs. Urolithin A (UA) is an important metabolite produced by ellagitannins and ellagic acid under the action of intestinal flora, and is widely present in the metabolic processes of foods rich in ellagitannins, such as pomegranates, strawberries, and walnuts. However, there is currently no research on the effects of UA on ovarian cancer. Therefore, developing anti-ovarian cancer drugs with UA as the active ingredient has significant clinical application value and market prospects. Summary of the Invention

[0004] To address the shortcomings of existing methods, this invention provides the application of urolithin A in the preparation of drugs for treating ovarian cancer and the drug itself.

[0005] The technical solution adopted by the present invention to solve its technical problem is: the application of urolithin A in the preparation of a drug for treating ovarian cancer, wherein the drug is used to inhibit any one or more of the proliferation, migration and invasion of ovarian cancer SKOV3 cells and A2780 cells, and / or induce apoptosis of ovarian cancer SKOV3 cells and A2780 cells.

[0006] Preferably, the drug can also be used to inhibit the growth of ovarian cancer xenografts.

[0007] Preferably, the drug is an oral or injectable formulation.

[0008] Preferably, the drug includes urolithiasis A as an active ingredient and pharmaceutically acceptable excipients.

[0009] A drug for treating ovarian cancer includes urolithiasis A as the active ingredient and pharmaceutically acceptable excipients.

[0010] Preferably, the drug is an oral or injectable formulation.

[0011] The beneficial effects of this invention are as follows: This invention is the first to apply UA to the field of anti-ovarian cancer, providing a new candidate drug for the treatment of ovarian cancer; in vitro cell experiments have confirmed that UA can significantly inhibit the proliferation, migration and invasion of ovarian cancer SKOV3 cells and A2780 cells, and induce apoptosis; Western blotting experiments have confirmed that UA can downregulate the protein expression of Bcl-2 and Cyclin D1, and upregulate the protein expression of Bax and Cleaved caspase-3; nude mouse xenograft models have confirmed that UA can significantly inhibit tumor growth and has no obvious toxic damage to major organs, demonstrating good in vivo safety. Attached Figure Description

[0012] Figure 1 This invention describes the effect of UA on the proliferation of different cell types. Figure A shows the effect of UA on SKOV3 cell viability; Figure B shows the effect of UA on A2780 cell viability; Figure C shows the effect of UA on L929 cell viability; Figure D shows the effect of UA on the colony-forming ability of SKOV3 and A2780 cells; Figure E shows the effect of UA on the proliferation ability of SKOV3 cells; Figure F shows the effect of UA on the proliferation ability of A2780 cells. Compared with Control... * P <0.05, ** P <0.01, *** P <0.001; Figure 2 This invention relates to the effect of UA on the migration and invasion of ovarian cancer cells. Figure A shows the effect of UA on the scratch healing ability of SKOV3 cells; Figure B shows the effect of UA on the scratch healing ability of A2780 cells; Figure C shows the effect of UA on the migration ability of both SKOV3 and A2780 cells; and Figure D shows the effect of UA on the invasion ability of both SKOV3 and A2780 cells. Compared to the Control group, ** P <0.01, *** P <0.001; Figure 3 This invention relates to the effect of UA on apoptosis in ovarian cancer cells. Figure A shows the ability of UA to induce apoptosis in SKOV3 cells; Figure B shows the ability of UA to induce apoptosis in A2780 cells. Compared to the Control group, ** P <0.01,*** P <0.001; Figure 4 This invention relates to the effect of UA on the expression of related proteins in ovarian cancer cells. Figure A shows the effect of UA on the expression of related proteins in SKOV3 cells; Figure B shows the effect of UA on the expression of related proteins in A2780 cells. Compared with the Control group, * P <0.05, ** P <0.01, *** P <0.001; Figure 5 This invention describes the effect of UA on SKOV3 cell xenografts in nude mice. Figure A shows the tumor volume change curve in nude mice; Figure B shows the body weight change curve in nude mice; Figure C shows representative images of the tumors; Figure D shows tumor quality statistics; and Figure E shows HE staining of important organs in nude mice. Compared to the Control group, ** P <0.01, *** P <0.001; Figure 6 This invention relates to the effect of UA on the positive expression of related proteins in nude mouse tumors. Detailed Implementation

[0013] To more clearly illustrate the objectives, technical solutions, and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It is clear and complete that the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0014] The application of urolithin A in the preparation of a drug for treating ovarian cancer, wherein the drug is used to inhibit any one or more of the proliferation, migration, and invasion of ovarian cancer SKOV3 and A2780 cells, and / or induce apoptosis of ovarian cancer SKOV3 and A2780 cells, the drug comprising urolithin A as the active ingredient and pharmaceutically acceptable excipients, the chemical structural formula of urolithin A being as follows: .

[0015] Example 1: Human ovarian cancer cells SKOV3 and A2780 in logarithmic growth phase and mouse fibroblasts L929 were harvested at 8 × 10⁻⁶ cells / year. 3Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C with 5% CO2. SKOV3 and A2780 cells were cultured in RPMI-1640 complete medium, while L929 cells were cultured in high-glucose DMEM complete medium. After cell adhesion, the control group (0 μmol / L) was treated with an equal volume of basal medium containing 0.1% DMSO, while the experimental groups were treated with different concentrations of UA (1, 2, 4, 8, 16, 32, and 64 μmol / L) prepared from the basal medium. The concentration of UA in L929 cells was set within 20 μM. Cell viability was assessed using the CCK-8 assay after 24 or 48 h of treatment.

[0016] Simultaneously, SKOV3 and A2780 cells were fed at a rate of 1.2 × 10⁻⁶. 3 Cells were seeded at a rate of [number] cells / well in 6-well plates and cultured in RPMI-1640 complete medium at 37°C with 5% CO2. When the number of cells per colony was greater than 20 under a microscope, the control group was treated with an equal volume of basal medium containing 0.1% DMSO. The experimental groups were treated with 50 nM paclitaxel (PTX), a low concentration (10 μM) of UA, and a high concentration (20 μM) of UA, respectively, forming the PTX group, UA-L group, and UA-H group. After 48 h of treatment, the medium was replaced with fresh complete medium, and the cells were cultured until visible colonies appeared. Colony formation was observed after fixation and crystal violet staining.

[0017] In addition, the EdU incorporation method was used to detect cell proliferation. SKOV3 and A2780 cells were cultured at 3 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in RPMI-1640 complete medium at 37°C with 5% CO2. After overnight culture, the control group was treated with an equal volume of basal medium containing 0.1% DMSO, while the experimental groups were treated with 50 nM PTX, 10 μM UA, and 20 μM UA prepared from basal medium, respectively, forming the PTX group, UA-L group, and UA-H group. After 12 h of treatment, EdU was added and incubated for 2 h. After fixation and staining, the cells were photographed under a fluorescence microscope for observation.

[0018] The results are as follows Figure 1 As shown: UA can inhibit the viability of SKOV3 cells and A2780 cells in a concentration-dependent manner. Figure 1 (A, 1B), where UA-L indicates a low concentration of UA and UA-H indicates a high concentration of UA. Based on the IC50 value, 10 μM and 20 μM were selected as the low and high concentrations of UA for subsequent experiments. Within the set concentration range, UA had little effect on the viability of L929 cells. Figure 1 C). Colony formation assay results showed that UA treatment significantly reduced the number and size of SKOV3 and A2780 cell colonies, effectively inhibiting their colony-forming ability. Figure 1 D). EdU experiment results showed that the proportion of EdU-positive cells in both cell lines was significantly reduced after UA treatment ( Figure 1 E, 1F). The above results indicate that UA can effectively inhibit the proliferation of ovarian cancer cells, while having little effect on normal cells L929, demonstrating a certain degree of selectivity.

[0019] Example 2: SKOV3 and A2780 cells in logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶. 5 Cells were seeded at a rate of 10 cells / well in 6-well plates and cultured in RPMI-1640 complete medium at 37°C with 5% CO2. Once cell confluence reached 90% or higher, scratches were made using a 200 μL sterile pipette tip. After washing with PBS, the control group (Control group) received an equal volume of basal medium containing 0.1% DMSO. The experimental groups received 50 nM PTX, 10 μM UA, and 20 μM UA prepared from basal medium, corresponding to the PTX group, UA-L group, and UA-H group, respectively. Scratch healing was recorded by photographing at 0 h, 24 h, and 48 h.

[0020] Simultaneously, the Transwell assay was used to detect cell migration and invasion abilities. SKOV3 and A2780 cells in logarithmic growth phase were used at a concentration of 3 × 10⁻⁶ cells / mL. 5 Cells were seeded at a rate of [number] cells / well in 6-well plates and cultured in RPMI-1640 complete medium at 37°C with 5% CO2. After overnight culture, the control group was treated with an equal volume of basal medium containing 0.1% DMSO, while the experimental groups were treated with 50 nM PTX, 10 μM UA, and 20 μM UA prepared from basal medium to form the PTX, UA-L, and UA-H groups, respectively. After 24 h of treatment, cells from each group were digested and collected, resuspended in basal medium, counted, and the cell density was adjusted to 5 × 10⁶ cells / well. 4 1 well / well (migration experiment) or 1×10 5 Cells per well (invasion assay) were seeded in the upper chamber of a Transwell chamber, and 600 μL of complete culture medium (containing 10% FBS) was added to the lower chamber. After culturing for 36 h (migration) or 48 h (invasion), the cells were fixed, stained, and the number of cells that had penetrated the membrane was counted. The chambers used for the invasion assay were pre-coated with Matrigel (1:8 dilution).

[0021] The results are as follows Figure 2As shown: the scratch results indicate that the scratch healing rate of SKOV3 cells and A2780 cells was significantly slower after UA treatment, and a wider scratch area remained compared with the Control group within the same time period. Figure 2 A, 2B); Transwell assay results showed that UA treatment significantly reduced the number of transmembrane cells in both cell lines, and significantly decreased cell migration and invasion activity. Figure 2 (C, 2D); The above results indicate that UA can effectively inhibit the migration and invasion of ovarian cancer cells.

[0022] Example 3: SKOV3 and A2780 cells in logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a rate of [number] cells / well in 6-well plates and cultured in RPMI-1640 complete medium at 37°C with 5% CO2. After overnight culture, the control group (Control group) was treated with an equal volume of basal medium containing 0.1% DMSO, while the experimental groups were treated with 50 nM PTX, 10 μM UA, and 20 μM UA prepared from basal medium to form the PTX group, UA-L group, and UA-H group, respectively. After 48 h of treatment, cells (including floating and adherent cells) were collected, and the apoptosis rate was detected using Annexin V-FITC / PI double staining.

[0023] The results are as follows Figure 3 As shown: Compared with the Control group, the proportion of apoptotic cells in both SKOV3 and A2780 cells was significantly increased after UA treatment, mainly due to an increase in late-stage apoptotic cells. Figure 3 (A, 3B); the results showed that UA can effectively induce apoptosis in ovarian cancer cells.

[0024] Example 4: SKOV3 and A2780 cells in logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶. 5 Cells were seeded at a rate of [number] cells / well in 6-well plates and cultured in RPMI-1640 complete medium at 37°C with 5% CO2. When cell confluence reached 70%-80%, the control group (Control group) was treated with an equal volume of basal medium containing 0.1% DMSO. The experimental groups were treated with 50 nM PTX, 10 μM UA, and 20 μM UA prepared from basal medium, corresponding to the PTX group, UA-L group, and UA-H group, respectively. After 48 h of treatment, total protein was extracted, and the protein expression levels of Bcl-2, Bax, Cleaved caspase-3, and Cyclin D1 were detected by Western blotting.

[0025] The results are as follows Figure 4As shown: In SKOV3 and A2780 cells, UA intervention downregulated Cyclin D1 protein expression and upregulated the Bax / Bcl-2 ratio and Cleaved caspase-3 protein expression. Figure 4 (A, 4B); The above results indicate that UA can inhibit the expression of Bcl-2 and Cyclin D1 in ovarian cancer cells, and promote the expression of Bax and Cleaved caspase-3, thereby inhibiting cell proliferation and inducing apoptosis.

[0026] Example 5: SKOV3 cells in the logarithmic growth phase were mixed with Matrigel at a 1:1 ratio to prepare 2×10⁻⁶ cells / cells. 7 Cell suspension at 0.1 mL per animal (containing 2 × 10⁶ cells / mL) 6 (Number of cells) were inoculated subcutaneously on the back of the left hind limb of nude mice; when the tumor volume grew to approximately 100 mm³, the nude mice were randomly divided into 4 groups: control group, positive control group (paclitaxel, 10 mg / kg), low-dose urolithin A group (UA-L, 20 mg / kg), and high-dose urolithin A group (UA-H, 100 mg / kg), with 5 mice in each group; the control group was given an equal volume of 0.5% CMC-Na solution by gavage; the PTX group was injected with paclitaxel via tail vein once a week; the UA groups were given the corresponding dose of urolithin A by gavage five times a week; the administration was continued for 3 weeks, and the tumor volume was measured and the weight of the nude mice was recorded every 3 days during the administration period; after the administration was completed, the tumor tissue was dissected, weighed, and photographed, and the heart, liver, spleen, lung, and kidney were collected for HE staining. The results are as follows Figure 5 As shown: Compared with the control group, tumor growth in nude mice in each treatment group was slow, and tumor volume growth was significantly inhibited, while the body weight of nude mice did not decrease significantly. Figure 5 A, 5B); After the administration was completed, the tumors in each treatment group were significantly smaller and the tumor weight was significantly reduced (A, 5B). Figure 5 C, 5D). HE staining results showed no significant pathological differences in organ tissue structure among the drug-treated groups, and UA did not cause significant toxic damage to major organs such as the heart, liver, spleen, lungs, and kidneys in nude mice. Figure 5 E); the results showed that UA could significantly inhibit the growth of xenografts in nude mice and had good in vivo safety.

[0027] Example 6: Tumor tissues from nude mice in each group of Example 5 were taken and subjected to immunohistochemical staining to detect the positive expression of Ki67, Bcl-2, Cleaved caspase-3 and Cyclin D1.

[0028] The results are as follows Figure 6As shown, compared with the control group, the positive expression of Ki67, Bcl-2 and Cyclin D1 in the tumor tissues of nude mice in each drug administration group was significantly reduced, while the positive expression of Cleaved caspase-3 was significantly increased; the results indicate that UA can also inhibit tumor cell proliferation and induce apoptosis in vivo, thereby exerting a tumor-suppressive effect.

[0029] This invention discloses the application of UA in the preparation of drugs for treating ovarian cancer. In vitro cell experiments confirmed that UA significantly inhibits the proliferation, migration, and invasion of SKOV3 and A2780 ovarian cancer cells and induces apoptosis. Western blotting experiments confirmed that UA downregulates the protein expression of Bcl-2 and Cyclin D1, and upregulates the protein expression of Bax and Cleaved caspase-3. Nude mouse xenograft models confirmed that UA significantly inhibits tumor growth and has no significant toxic damage to major organs, demonstrating good in vivo safety. Furthermore, paclitaxel, as a positive control, showed the expected changes in all functional experiments, further supporting the reliability and effectiveness of the in vitro and in vivo evaluation system established in this application.

[0030] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of urolithin A in the preparation of drugs for treating ovarian cancer, characterized in that, The drug is used to inhibit any one or more of the proliferation, migration and invasion of ovarian cancer SKOV3 cells and A2780 cells, and / or induce apoptosis in ovarian cancer SKOV3 cells and A2780 cells.

2. The use of urolithin A according to claim 1 in the preparation of drugs for treating ovarian cancer, characterized in that, The drug can also be used to inhibit the growth of ovarian cancer xenografts.

3. The use of urolithin A according to claim 1 in the preparation of drugs for treating ovarian cancer, characterized in that, The drug is an oral or injectable formulation.

4. The use of urolithin A according to claim 1 in the preparation of drugs for treating ovarian cancer, characterized in that, The drug includes urolithiasis A as the active ingredient and pharmaceutically acceptable excipients.

5. A drug for treating ovarian cancer, characterized in that, This includes urolithiasis A as the active ingredient and pharmaceutically acceptable excipients.

6. The medicament for treating ovarian cancer according to claim 5, characterized in that, The drug is an oral or injectable formulation.

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