Application of D724-0491 as ovarian cancer cell inhibitor

Abstract

The invention discloses application of D724-0491 as an ovarian cancer cell inhibitor, and belongs to the technical field of biological medicines. The application of the D724-0491 as the ovarian cancer cell inhibitor is provided for the first time, and the application range of the D724-0491 is expanded. The D724-0491 is proposed for the first time to inhibit combination of HBXIP and KEAP1 of ovarian cancer cells and reduce the nuclear entry level of NRF2, so that oxidative stress of the ovarian cancer cells is induced, and death of the ovarian cancer cells is promoted. The invention provides D724-0491 as an active ingredient of the composition for inhibiting ovarian cancer cells for the first time, and D724-0491 has no cytotoxicity to human adrenal gland cells 293T.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of D724-0491 as an inhibitor of ovarian cancer cells. Background Technology

[0002] Ovarian cancer is the deadliest malignant tumor of the female reproductive system. It can occur at any age and is known as the "king of gynecological cancers." Because its early symptoms are often subtle and easily overlooked, it is also called the "silent killer" of women. In 2013, the World Ovarian Cancer Federation designated May 8th as "World Ovarian Cancer Day" to raise public awareness and understanding of ovarian cancer and to call on society to provide more care and support to patients. Early-stage ovarian cancer usually has no obvious symptoms, while later stages may present with symptoms such as abdominal distension, abdominal pain, and indigestion. The prognosis for ovarian cancer is poor because it is usually diagnosed at an advanced stage. Treatment methods include surgery and chemotherapy. Unfortunately, current clinical diagnostic and treatment levels for ovarian cancer cannot fully meet the treatment needs of patients; furthermore, patients' quality of life is significantly reduced due to traditional treatments such as surgery or chemotherapy. Therefore, in-depth research into the pathogenesis of ovarian cancer and the exploration and development of targeted therapies for ovarian cancer are of paramount importance.

[0003] Hepatitis B virus (HBV) not only infects liver cells, making it a major cause of liver cancer, but it can also infect extrahepatic cells, causing chronic inflammation and being closely related to the development of various extrahepatic tumors. The female reproductive organs are connected to the external environment through the vagina; viral nucleic acid can be detected in the vaginal secretions and ovarian tissue of HBV carriers. Some epidemiological studies show that HBV infection may increase the risk of gynecological malignancies and is associated with poor patient prognosis. Common gynecological malignancies include cervical cancer, endometrial cancer, and ovarian cancer.

[0004] Hepatitis B virus X protein-binding protein (HBXIP) is a membrane protein located on the surface of lysosomes, encoded by the Lamtor gene. It is highly expressed in various types of malignant tumors, including ovarian cancer, esophageal squamous cell carcinoma, and liver cancer, and is often a predictor of poor tumor stage and poor patient prognosis. Over the past decade, research on the oncogenic mechanisms of HBXIP in various tumor types has deepened. Studies have found that the tumor protein HBXIP competitively binds to KEAP1 to activate NRF2 and promote the growth and metastasis of ovarian cancer cells. Tumor cells have significantly higher levels of reactive oxygen species (ROS) than normal cells, which can promote malignant tumor progression by regulating signaling pathways and proteins. To adapt to high ROS levels, tumor cells enhance their antioxidant capacity through various mechanisms. Researchers have found that interfering with the protein-protein interaction between Kelch-like ECH-associated protein (KEAP1) and the nuclear transcription factor erythroid 2-associated factor 2 (NRF2) is one of the main mechanisms by which tumor cells enhance their antioxidant capacity. Normally, NRF2 is mainly located in the cytoplasm at physiological levels, and its primary fate is ubiquitination and degradation after binding to KEAP1. Only a small amount of NRF2 enters the nucleus and activates related antioxidant elements (AREs) to maintain ROS at normal physiological levels. However, under the stimulation of pathological conditions such as tumors, NRF2 no longer binds to KEAP1, but instead enters the nucleus in large quantities and acts on downstream AREs, thereby enhancing the antioxidant capacity of tumor cells. Therefore, in previous tumor-related research, it is logical to search for small molecule compounds that target the KEAP1-NRF2 protein and its protein-protein interactions.

[0005] A primary approach is to identify compounds that target and interfere with the protein-protein interactions between Keap1 and NRF2. Recent research reports that HBXIP can actively bind to the Keap1 protein via the GLNLG motif, thereby forcing NRF2 into the nucleus and activating the expression of related antioxidant proteins.

[0006] Currently, there is no evidence to suggest that D724-0491 can be used as an inhibitor of ovarian cancer cells. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide the application of D724-0491 as an inhibitor of ovarian cancer cells, thus expanding the application scope of D724-0491. D724-0491 inhibits the binding of HBXIP and KEAP1 in ovarian cancer cells, reduces the level of NRF2 nuclear translocation, and thereby induces oxidative stress in ovarian cancer cells, promoting ovarian cancer cell death.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the application of D724-0491 as an inhibitor of ovarian cancer cells, wherein the chemical name of D724-0491 is 2-(2-{[3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]amino}ethoxy)ethan-1-ol. The structural formula of D724-0491 is as follows.

[0009] .

[0010] Based on the above technical solution, the D724-0491 further suppresses the binding of HBXIP and KEAP1, reducing the level of NRF2 entry into the core.

[0011] Based on the above technical solution, D724-0491 further promotes the increase of ROS level in the ovarian cancer cells.

[0012] Based on the above technical solution, the inhibitor is further described as an oral or injectable formulation.

[0013] In a first aspect, the present invention provides a composition for inhibiting ovarian cancer cells, wherein the active ingredient of the composition includes D724-0491.

[0014] Based on the above technical solution, further, the only active ingredient in the composition is D724-0491.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to propose the application of D724-0491 as an inhibitor of ovarian cancer cells, thus expanding the application scope of D724-0491.

[0016] 2. This invention is the first to propose that D724-0491 inhibits the binding of HBXIP and KEAP1 in ovarian cancer cells, reduces the level of NRF2 entering the nucleus, and thereby induces oxidative stress in ovarian cancer cells and promotes the death of ovarian cancer cells.

[0017] 3. This invention is the first to propose D724-0491 as an active ingredient in a composition for inhibiting ovarian cancer cells. D724-0491 has no cytotoxicity to human adrenal cortex cells 293T. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0019] Figure 1 This is a diagram showing the results of D724-0491 inhibiting HBXIP protein expression in ovarian cancer cells in Example 1 of the present invention; Figure 2 The following graphs show the results of D724-0491 inhibiting the proliferation rate of ovarian cancer cells in Example 1 of the present invention: A is a line graph of the proliferation rate of ovarian cancer cells, and B is a bar graph of the proliferation rate of ovarian cancer cells. Figure 3 The following are the results of D724-0491 inhibiting the size and number of ovarian cancer cell clone clusters in Example 1 of the present invention: A is a staining result of ovarian cancer cells, and B is a bar chart of staining result of ovarian cancer cells; Figure 4 This is a diagram showing the results of ovarian cancer cells after treatment with KEAP1 co-precipitation of D724-0491 in Example 2 of the present invention. Figure 5 The following diagrams show the localization results of NRF2 in the cell nucleus after treatment with D724-0491 in Example 2 of the present invention: A is a fluorescence result diagram of NRF2 in the cell nucleus, and B is a bar chart of the localization results of NRF2 in the cell nucleus. Figure 6 The following figures illustrate the effects of D724-0491 on the Keap-NRF2-related signaling pathway and HBXIP expression in SK-OV-3 ovarian cancer cells, as verified in Example 3 of this invention: A is a graph of protein expression results, and B is a bar chart of protein expression results. Figure 7 The following is a graph showing the results of verifying the effect of D724-0491 on the Keap-NRF2-related signaling pathway and HBXIP expression in ovarian cancer cells CAOV-3 in Example 3 of the present invention; A is a graph of protein expression results, and B is a bar graph of protein expression results. Figure 8 The following graphs show the results of verifying the effect of D724-0491 on promoting the accumulation of ROS levels in ovarian cancer cells in Example 4 of the present invention: A is a fluorescence result graph of ROS level accumulation, and B is a bar graph of immunofluorescence result graph of ROS level accumulation. Figure 9 The following graphs show the results of WB verification of the silencing effect of siRNA on HBXIP in Example 5 of this invention: A is a graph of protein expression results, and B is a bar chart of protein expression results. Figure 10 This is a graph showing the proliferation rate of ovarian cancer cells after silencing HBXIP in Example 5 of the present invention. Figure 11 The following are the results of clonal proliferation of ovarian cancer cells after silencing HBXIP in Example 5 of the present invention: A is a staining result of ovarian cancer cells, and B is a bar chart of staining result of ovarian cancer cells. Figure 12The following are the results of verifying that silencing HBXIP can affect KEAP1-NRF2 binding and NRF2 core localization in Embodiment 6 of the present invention: A is the result of silencing HBXIP affecting KEAP1-NRF2 binding, B is the result of silencing HBXIP affecting NRF2 core localization, and C is a bar chart of the result of silencing HBXIP affecting NRF2 core localization. Figure 13 The following are the results of verifying the effect of silencing HBXIP on the KEAP1-NRF2 related signaling pathway in Example 7 of the present invention: A is a graph of protein expression results, and B is a bar chart of protein expression results; Figure 14 The following graphs show the results of verifying that silencing HBXIP promotes the accumulation of ROS levels in ovarian cancer cells in Example 8 of this invention: A is a fluorescence result graph of ROS level accumulation, and B is a bar graph of the fluorescence result graph of ROS level accumulation. Figure 15 The following diagrams illustrate the results of the inhibition of ovarian cancer cells and the toxicity experiment of D724-0491 on human adrenal epithelial cells in Example 9 of this invention: A is a schematic diagram of the molecular docking between D724-0491 and the site of action of ovarian cancer cells; B is the chemical structural formula of D724-0491; C is the result of the CCK8 cytotoxicity experiment; and D is the result of the toxicity experiment of D724-0491 on human adrenal epithelial cells 293T. Note: ns: no statistical difference; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Detailed Implementation

[0020] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0021] Example 1 This embodiment verifies the inhibitory effect of D724-0491 on the proliferation and colony formation ability of ovarian cancer cells SK-OV-3 and CAOV-3.

[0022] Ovarian cancer cells treated with 0 μmol / L, 5 μmol / L, and 10 μmol / L D724-0491 were subjected to Western blot experiments. Ovarian cancer cells treated with 0 μmol / L D724-0491 served as the control group, while those treated with 5 μmol / L and 10 μmol / L D724-0491 served as the treatment groups. The specific steps are as follows.

[0023] 1. Cell treatment: Cells from the treatment group and control group were digested with trypsin and transferred to spare EP tubes. After centrifugation, the supernatant was discarded, PBS was added, and centrifugation was repeated. After the supernatant was discarded, RIPA protein lysis buffer (Solepro) was added, and the cells were lysed by rotation at 4°C for 30 minutes. Subsequently, the cells were centrifuged in a low-temperature centrifuge, and the supernatant was placed in spare EP tubes.

[0024] 2. Protein quantification: The concentration of the extracted protein was detected using the BCA protein quantification kit. After calculation, 5× loading buffer was added and the protein was boiled in boiling water for 5 minutes to denature it. The protein was then stored in a -20℃ refrigerator for later use.

[0025] 3. Electrophoresis: Prepare PAGE gels of the required concentration in advance, load protein samples of equal mass according to the set grouping, and perform electrophoresis at an appropriate voltage for the corresponding time according to the molecular weight of the target protein.

[0026] 4. Transfer: After electrophoresis, cut the target protein into the molecular weight range specified by the Maker, then place it in the transfer tank and transfer it at an appropriate voltage for a certain period of time according to the molecular weight of the target protein.

[0027] 5. Sealing: After the transfer is complete, place the PVDF membrane in a 5% milk sealing solution and seal for 1.5 hours.

[0028] 6. Antibody Incubation: After blocking, place the PVDF membrane in the appropriate primary antibody dilution buffer and incubate overnight at 4°C on a shaker. The next day, wash the membrane four times with PBST for 10 minutes each time. Then add the appropriate secondary antibody and incubate for 1 hour, followed by washing with PBST four times for 10 minutes each time. (Antibodies used in this experiment include: GAPDH, LaminA / C, HBXIP, KEAP1, NQO-1, and NRF2.) 7. Development: Add an appropriate amount of ECL luminescent solution (New Semiconductor) to the PVDF film and develop the color using a gel imaging system (BIO-RAD).

[0029] like Figure 1 As shown, it was found that the expression level of HBXIP protein decreased with increasing concentration of the inhibitor (D724-0491).

[0030] Ovarian cancer cells treated with D724-0491 at concentrations of 0 μmol / L, 5 μmol / L, and 10 μmol / L were counted continuously for four days using the CCK-8 assay. The specific procedure is as follows.

[0031] 1. Plating: Ovarian cancer cell lines SK-OV-3 and CAOV-3 were seeded into 96-well plates, with 4000 cells per well. The plates were then incubated in an incubator until the cell density reached approximately 70%-80%.

[0032] 2. Drug administration: Calculate the required dosage according to the preset drug concentration gradient, and after drug administration, place the cells in an incubator to continue culturing for one day.

[0033] 3. Treatment: The following day, discard the culture medium and add 10 μl of 10% CCK-8 (Ape Bio, K1018) solution and 90 μl of culture medium to each well, and continue incubation in an incubator for 2 hours. After incubation, the absorbance of the cells in each well was measured at 450 nm using a microplate reader, and the data were analyzed.

[0034] like Figure 2 As shown, the results revealed that, compared to the control group (without inhibitors), the proliferation of ovarian cancer cells was significantly inhibited after treatment, and this inhibition was dose-dependent.

[0035] Cell cloning experiments were performed on ovarian cancer cells treated with D724-0491 at concentrations of 0 μmol / L, 5 μmol / L, and 10 μmol / L. The specific procedures are as follows.

[0036] 1. Plate seeding: Ovarian cancer cells SK-OV-3 and CAOV-3 were seeded into six-well plates at a density of 800 cells per well and incubated in an incubator for 24-48 hours.

[0037] 2. Treatment: After the culture period, the cells in the treatment group were transfected with siRNA or administered drugs according to the different treatment objectives. After treatment, the cells were placed in an incubator and cultured for another 10-15 days, during which the number and size of ovarian cancer cell clones were observed daily.

[0038] 3. Photography: Once the number and size of the ovarian cancer cell clones have reached a certain level, discard the original culture medium. Rinse twice with PBS, then fix the cells with 1 mL of methanol for 30 minutes. After fixation, discard the methanol, stain with pre-cooled 0.1% crystal violet for 30 minutes, then rinse thoroughly with water, air dry, and photograph.

[0039] like Figure 3 As shown, the results indicate that the size and number of ovarian cancer cell clone clusters also decreased with increasing D724-0491 concentration.

[0040] In summary, it was found that D724-0491 inhibits the proliferation and colony formation of ovarian cancer cells by suppressing the expression of HBXIP protein.

[0041] Example 2 This embodiment verifies that D724-0491 can interfere with the HBXIP-Keap1-Nrf2 signaling pathway in ovarian cancer cells SK-OV-3 and CAOV-3 and affect the nuclear localization of NRF2.

[0042] The Keap1-NRF2 signaling pathway is a major mechanism for regulating intracellular ROS. Studies have shown that HBXIP can bind to Keap1 and activate NRF2 to enter the cell nucleus, thereby enhancing the antioxidant capacity of cancer cells.

[0043] Therefore, Keap1 was used for immunoprecipitation of NRF2 and HBXIP, and the specific procedure is as follows.

[0044] 1. The reagents used for immunoprecipitation were purchased from MCE.

[0045] 2. Bead Pretreatment: After obliquely cutting the pipette tip, pipette approximately 100 μL of beads into an EP tube, add 400 μL of ice-cold PBS, and pipette several times. Centrifuge at 1000g for 3 minutes. Discard the supernatant PBS, add 400 μL of PBS again, and repeat the above operation 4 times. After the final wash, add 400 μL of PBS and store at 4°C for later use. Mix well before each use, and add 80 μL of the bead and PBS mixture to every 1 ml of protein sample.

[0046] 3. Plating: Add 8 ml of DMEM complete culture medium and 500 μL of cell suspension to each dish, mix gently several times, and then incubate in an incubator until the cell density is about 70%-80% before administering the drug.

[0047] 4. Drug administration: Calculate the required drug volume according to the predetermined drug concentration, add it to the treatment group, mix well, and place in an incubator to continue incubation for 24 hours.

[0048] 5. Cell harvesting: Follow the same steps as cell passage. After centrifugation, resuspend the cells in PBS, then centrifuge at 2000 rpm for 3 minutes. Discard the supernatant, add Western blotting and IP cell lysis buffer, and lyse at 4°C for 40 minutes. Then centrifuge at 12000 rpm for 20 minutes. Transfer the supernatant to labeled control and treatment tubes.

[0049] 6. Incubation: Take 40 μL of supernatant from both the control and treatment groups, add 10 μL of 5× loading buffer, mix thoroughly, and boil for 5 minutes to denature. Store at -20°C. Add the calculated volume of HBXIP antibody to the remaining supernatant and incubate at 4°C for at least 4 hours. After incubation, add 80 μL of the treated beads and PBS mixture and incubate overnight for at least 16 hours.

[0050] 7. Processing: After incubation, centrifuge at 5000 rpm for 10 minutes in a low-temperature centrifuge. Discard the supernatant, add 1 ml of PBS, and wash by rotation at 4°C for 10 minutes. Then centrifuge at 5000 rpm for 10 minutes in a low-temperature centrifuge, discard the supernatant, and repeat the above operation twice. After completion, add 8 μL of 5× loading buffer and boil to denature.

[0051] 8. Electrophoresis: Add appropriate amounts of sample according to the grouping. After electrophoresis, transfer the membrane, block it, and add the corresponding primary and secondary antibodies before developing.

[0052] like Figure 4 As shown, compared with the control group, the binding of NRF2 to Keap1 was significantly increased after treatment with D724-0491, while the binding of HBXIP to Keap1 was significantly decreased.

[0053] In addition, previous studies have shown that NRF2 dissociated from Keap1 can translocate from the cytoplasm to the nucleus.

[0054] Therefore, the nuclear translocation of NRF2 after the action of the inhibitor was investigated by immunofluorescence technology. The specific process is as follows.

[0055] 1. Slide preparation: Place the slides in a six-well plate one day in advance, add an appropriate amount of alcohol, and sterilize overnight in a laminar flow hood. The next day, discard the alcohol, add PBS to rinse three times, and then use as is.

[0056] 2. Plate seeding: Ovarian cancer cells SK-OV-3 and CAOV-3 were seeded into six-well plates at a cell density of 1×10⁶ cells per well and incubated in an incubator for 24 hours.

[0057] 3. Administration: The next day, calculate the required drug volume according to the target concentration, gently mix after administration and place in an incubator for 24 hours.

[0058] 4. Treatment: The next day, aspirate the original culture medium and wash with PBS three times for 5 minutes each. Fix the cells with 4% polymethyl methacrylate for 15 minutes, then wash with PBS three times for 5 minutes each. Add 0.5% permeabilization buffer (PBS + Triton-100 mixture) to permeabilize the cells for 10 minutes, and wash with PBS three times for 5 minutes each after permeabilization.

[0059] 5. Blocking: Block with normal goat serum for 30 minutes at room temperature. After completion, wash with PBS 3 times * 5 minutes each time.

[0060] 6. Incubation: Add NRF2 antibody dilution buffer and incubate overnight on a shaker at 4°C. The next day, discard the antibody dilution buffer and wash three times with PBS for 5 minutes each time. Add secondary antibody and incubate in the dark for 1 hour. After incubation, wash three times with PBS for 5 minutes each time.

[0061] 7. Staining: Add DAPI dilution buffer and stain for 5 minutes in the dark, then rinse with PBS 4 times for 5 minutes each time.

[0062] 8. Taking photos: Remove the slide, add an appropriate amount of anti-fluorescence quencher before sealing, and take photos in a dark room using a fluorescence microscope.

[0063] like Figure 5 As shown, after treatment with D724-0491, NRF2 located in the cell nucleus decreased significantly.

[0064] In summary, it was found that D724-0491 can increase the binding of NRF2 to KEAP1, decrease the binding of HBXIP to KEAP1, and decrease the nuclear localization of NRF2.

[0065] Example 3 This embodiment verifies that D724-0491 can affect the Keap-NRF2-related signaling pathway and HBXIP expression in ovarian cancer cells SK-OV-3 and CAOV-3.

[0066] Studies have shown (Zhou XL, Zhu CY, Wu ZG, Guo X, Zou W. The oncoprotein HBXIP competitively binds KEAP1 to activate NRF2 and enhance breast cancer cell growth and metastasis. Oncogene. 2019;38(21):4028-4046. doi:10.1038 / s41388-019-0698-5 and Li D, Hong X, Zhao F, Ci X, Zhang S. Targeting Nrf2 may reverse the drug resistance in ovarian cancer. Cancer Cell Int. 2021;21(1):116. doi:10.1186 / s12935-021-01822-1) that NRF2 entering the cell nucleus can activate the expression of downstream related antioxidant elements ARE, such as NQO1, thereby improving the antioxidant capacity of tumor cells. Furthermore, it is worth noting that NRF2 in the cell nucleus can also activate the promoter of HBXIP, thereby promoting HBXIP expression and thus forming a positive feedback loop.

[0067] Therefore, to investigate the changes in related proteins after treatment with D724-0491, ovarian cancer cells treated with the inhibitor were subjected to nucleo-cytoplasmic separation, and the changes in related proteins were detected by Western blot. The specific process of nucleo-cytoplasmic separation is as follows. The specific Western blot process is the same as in Example 1.

[0068] The reagent kit was purchased from Wanlleibo.

[0069] 1. Plate preparation: Ovarian cancer cells SK-OV-3 and CAOV-3 were seeded at an appropriate density in culture dishes and incubated in an incubator until ready for use.

[0070] 2. Drug administration: When the cell density is about 60%-70%, calculate the required dosage according to the target drug concentration, mix gently, and place in an incubator for continued culture.

[0071] 3. Cell harvesting: After trypsin digestion of cells, transfer them to new EP tubes, centrifuge and discard the supernatant. Resuspend in PBS and centrifuge again for use.

[0072] 4. Prepare reagents: Each culture dish requires 200 μL of plasma A, 10 μL of plasma B, and 50 μL of nucleus C. Calculate the required total amount, and then prepare plasma A cell membrane permeation buffer, plasma B washing buffer, and nucleus C nuclear lysis buffer according to the instructions of the nucleocytoplasmic separation kit. Place them on ice for later use.

[0073] 5. Processing: Add 200 μL of plasma A, vortex for 15 seconds, and incubate on ice for 15 minutes. After incubation, add 10 μL of pre-chilled plasma B, vortex for a few seconds, and incubate on ice for 1 minute. After completion, vortex again for a few seconds, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Collect the supernatant in a new EP tube and record it as cytoplasm. Add 50 μL of nucleus C to the precipitate, vortex for 15 seconds, and incubate on ice for 10 minutes. Repeat the above steps a total of 4 times. After completion, centrifuge at 12,000 rpm for 10 minutes, collect the supernatant in a new EP tube, and record it as nuclei.

[0074] 6. Protein quantification: The concentration of extracted cell nuclei and cytoplasm was measured using the BCA protein quantification kit. Calculated 5× loading buffer was added, and the mixture was boiled for 5 minutes and then placed in a -20℃ freezer for later use.

[0075] like Figure 3 As shown in Figure A, after treatment with D724-0491, the content of NRF2 in the cell nucleus decreased significantly; in addition, the expression of downstream NRF2-related proteins such as NQO1 and HBXIP also decreased. D724-0491 can inhibit the expression of NRF2, NQO-1, and HBXIP.

[0076] Example 4 This embodiment verifies that D724-0491 promotes changes in ROS levels within ovarian cancer cells SK-OV-3 and CAOV-3.

[0077] The changes in ROS levels in ovarian cancer cells after treatment with D724-0491 were analyzed using immunofluorescence technology.

[0078] 1. Slice climbing: The steps are the same as in Example 2.

[0079] 2. Plate formation: Ovarian cancer cells SK-OV-3 and CAOV-3 were seeded at a density of (1.2-1.5) × 10⁶ per well in six-well plates covered with glass slides and incubated in an incubator.

[0080] 3. Treatment: Once the cell density reaches the required level, the treated group is transfected with siRNA or drug-induced. After treatment, the cells are placed in an incubator for further culture.

[0081] 4. Staining: Discard the original culture medium, wash twice with PBS, and add 1 mL of incomplete culture medium. Under light-protected conditions, add 1 μL of DCFH-DA to each well, incubate for 35 minutes, and wash three times with PBS for 3 minutes after the reaction.

[0082] 5. Photographing: Fix cells with 4% paraformaldehyde for 15 minutes, then rinse three times with PBS for 3 minutes each time. Stain with diluted DAPI for 5 minutes, then rinse four times with PBS for 5 minutes each time. Remove the slide, add an appropriate amount of anti-fluorescence quencher, and mount. Photograph using a fluorescence microscope in a dark room.

[0083] like Figure 8 As shown, the green fluorescence representing ROS significantly increased after treatment with D724-0491, indicating that D724-0491 treatment significantly promoted the increase of ROS levels in ovarian cancer cells.

[0084] Based on the data from Examples 1 to 4, it was found that D724-0491 can interfere with the Keap1-NRF2 signaling pathway by targeting and inhibiting the function of HBXIP protein, thereby increasing the ROS level of ovarian cancer cells and achieving a killing effect.

[0085] Example 5 This embodiment verifies whether D724-0491 plays a role in the action of HBXIP on ovarian cancer cells SK-OV-3 and CAOV-3, and explores whether the effect of silencing HBXIP on ovarian cancer cells is consistent with the effect of the inhibitor group.

[0086] siRNA-HBXIP was transfected into ovarian cancer cell lines SK-OV-3 and CAOV-3 using liposome transfection technology, and the silencing effect of HBXIP was verified by Western blot. The specific procedure for liposome transfection is as follows. The specific procedure for Western blot is the same as in Example 1. 1. Purchase siRNA required for the experiment from Ribobio, and purchase Lipofectamine 2000 transfection reagent from Thermo Fisher Scientific. 2. Plating: Seed an appropriate number of ovarian cancer cells SK-OV-3 and CAOV-3 into 6-well plates, then incubate in an incubator for 12-14 hours. Transfection is performed when the cell density is about 30%-40%.

[0087] 3. Transfection: Prepare the transfection mixture according to the instructions (Solution A per well: 250 μl serum-free medium + 5 μl siRNA; Solution B per well: 250 μl serum-free medium + 5 μl Lipofectamine 2000). Let solutions A and B stand for 5 minutes each, then mix them thoroughly and incubate at room temperature for 25 minutes. Aspirate the old culture medium from the six-well plate, add 1500 μL of antibiotic-free and serum-free medium to each well, add the incubated transfection mixture to the six-well plate, and incubate for 48 hours.

[0088] like Figure 9 As shown, the expression of HBXIP protein was significantly inhibited.

[0089] To verify the effect of HBXIP on the proliferation capacity of ovarian cells, the HBXIP-silenced ovarian cancer cell lines SK-OV-3 and CAOV-3 were continuously counted for 4 days using the CCK-8 assay. The specific procedure was the same as in Example 1.

[0090] like Figure 10 As shown, compared with the control group, the proliferation ability of ovarian cancer cells was significantly inhibited after silencing HBXIP, and silencing the expression of HBXIP protein can inhibit the proliferation ability of ovarian cancer cells.

[0091] like Figure 11 As shown, the results of the cell cloning experiment were the same as in Example 1. After silencing the expression of HBXIP, the size and number of cell clones of ovarian cancer cell lines SK-OV-3 and CAOV-3 were significantly smaller than those of the control group. The clonal proliferation ability of ovarian cancer cells was weakened after silencing HBXIP.

[0092] In conclusion, this study verifies that silencing HBXIP expression can inhibit the proliferation of ovarian cancer cells.

[0093] Example 6 This embodiment verifies that silencing HBXIP can affect the binding of KEAP1-NRF2 and the nuclear localization of NRF2.

[0094] Keap1 was used for immunoprecipitation of NRF2 and HBXIP. The specific procedure was the same as in Example 2.

[0095] like Figure 12 As shown in Figure A, compared to the control group, silencing HBXIP significantly increased the binding of NRF2 to Keap1, while conversely, the binding of HBXIP to Keap1 significantly decreased.

[0096] The nuclear translocation of NRF2 after inhibitor treatment was investigated using immunofluorescence technology. The specific procedure was the same as in Example 2.

[0097] like Figure 12 As shown in B and C, silencing HBXIP significantly reduced NRF2 localized in the cell nucleus.

[0098] Example 7 This embodiment verifies that silencing HBXIP affects the KEAP1-NRF2 related signal pathway.

[0099] like Figure 13 As shown, ovarian cancer cells after HBXIP silencing underwent nuclear-cytoplasmic separation, following the same procedure as in Example 3. Changes in related proteins were detected using Western blot, following the same procedure as in Example 1. The results showed that silencing HBXIP significantly reduced the content of NRF2 in the cell nucleus; furthermore, the expression of downstream NRF2 proteins such as NQO1 and HBXIP also decreased.

[0100] Example 8 This embodiment verifies that silencing HBXIP can promote the accumulation of ROS levels in ovarian cancer cells SK-OV-3 and CAOV-3.

[0101] To investigate whether the changes in the binding of HBXIP, KEAP1, and NRF2 in ovarian cancer cells after HBXIP silencing were as expected, the immunofluorescence procedure was the same as in Example 2. The changes in ROS levels in the ovarian cancer cell lines SK-OV-3 and CAOV-3 after HBXIP silencing were detected by immunofluorescence.

[0102] like Figure 14 As shown, after silencing HBXIP, the green fluorescence representing ROS increased significantly, and the ROS levels in the ovarian cancer cell lines SK-OV-3 and CAOV-3 were significantly higher than those in the control group.

[0103] Example 9 This embodiment verifies the inhibitory effect of D724-0491 on ovarian cancer cells SK-OV-3 and CAOV-3, and its toxicity to human adrenal epithelial cells.

[0104] Using molecular docking technology, D724-0491 mainly acts on the hydrophobic region of HBXIP, forming strong hydrogen bond interactions with Gly28 and Asn30. At the same time, the long-chain hydroxyl groups of D724-0491 also form hydrogen bond interactions with Asp39 and Gly43, respectively, indicating that hydrogen bonds play an important role in maintaining the binding between D724-0491 and HBXIP.

[0105] like Figure 15 As shown in Figures A and B, targeting this site, the inhibitor D724-0491 (C17H19CIN4O2) inhibits the binding of HBXIP to KEAP1, reduces the level of NRF2 nuclear translocation, and thus induces oxidative stress in tumor cells.

[0106] like Figure 15 As shown in C, the CCK8 cytotoxicity experiment was conducted using the same procedure as the CCK-8 method in Example 1. The results showed that D724-0491 could effectively kill ovarian cancer cell lines SK-OV-3 and CAOV-3.

[0107] like Figure 15 As shown in Figure D, D724-0491 is not toxic to human adrenal epithelial cells 293T.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of D724-0491 as an inhibitor of ovarian cancer cells, wherein the chemical name of D724-0491 is 2-(2-{[3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]amino}ethoxy)ethan-1-ol.

2. The application according to claim 1, characterized in that, The D724-0491 inhibits the binding of HBXIP to KEAP1, reducing the level of NRF2 nuclear entry.

3. The application according to claim 1, characterized in that, The D724-0491 promotes an increase in ROS levels within the ovarian cancer cells.

4. The application according to claim 1, characterized in that, The inhibitor is an oral or injectable formulation.

5. A composition for inhibiting ovarian cancer cells, characterized in that, The active ingredient of the composition includes D724-0491.

6. The composition for inhibiting ovarian cancer cells according to claim 5, characterized in that, The sole active ingredient of the composition is D724-0491.

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