Application of EZH2 in diagnostic reagent and therapeutic drug for ovarian cancer

Abstract

The invention relates to application of EZH2 as a biomarker in preparation of an ovarian cancer diagnostic kit. The invention further provides an application of the inhibitor of the EZH2 in preparation of a medicine for treating the ovarian cancer, and the inhibitor of the EZH2 is GSK126 or Tazemetostat. The invention also provides an application of the Napabucasin in preparation of a medicine for treating ovarian cancer. The invention has the advantages that EZH2 is abnormally highly expressed in ovarian cancer and promotes the proliferation of ovarian cancer cells; the EZH2 inhibitor inhibits the proliferation of ovarian cancer cells; a new drug Napabucasin is found through high-throughput drug screening, the sensitivity of the new drug Napabucasin is obviously superior to that of a traditional inhibitor GSK126 or Tazemetostat of EZH2, and the new drug Napabucasin is verified in ovarian cancer cells. Meanwhile, on an in-vivo mouse model, the Napabucasin is found to be capable of remarkably inhibiting metastasis of the ovarian cancer with abnormally high expression. Important theoretical basis and experimental support are provided for developing a novel treatment strategy aiming at EZH2.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically, to the application of EZH2 in diagnostic reagents and therapeutic drugs for ovarian cancer. Background Art

[0002] Ovarian cancer is one of the common tumors of female reproductive organs. Its incidence rate ranks third after cervical cancer and endometrial cancer, accounting for about 4% of all female malignancies. However, the death toll caused by ovarian cancer ranks first among various gynecological tumors, posing a serious threat to women's lives. The cause of ovarian cancer is still unclear, and its occurrence may be related to age, fertility, blood type, mental factors, and environment, etc.

[0003] Ovarian cancer can occur at any age, but most cases occur during the period when ovarian function changes from exuberant to declining. It is generally more common in menopausal and postmenopausal women. The incidence of ovarian cancer is relatively low in women under 20 years old. The age distribution of different types of ovarian cancer is also different. The incidence of ovarian epithelial cancer increases rapidly after the age of 40, with the peak age being 50 - 60 years old, and gradually decreasing after the age of 70; sex cord-stromal tumors are similar to ovarian epithelial cancer and increase with age; germ cell tumors are more common in young women under 20 years old, and the incidence of ovarian cancer is high in single or childless women.

[0004] The main treatment methods for ovarian cancer are mainly comprehensive treatments including surgery and chemotherapy, and some patients can choose to add radiotherapy. Immunotherapy and traditional Chinese medicine treatment can be used to consolidate the curative effect.

[0005] EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2), responsible for catalyzing the monomethylation, dimethylation, and trimethylation of lysine 27 of histone H3 (H3K27me3), and this histone mark is related to compact chromatin and transcriptional repression.

[0006] High-throughput drug screening: It is a modern scientific technology based on experimental methods at the molecular level and cell level, with the main goal of drug research and development. As an important strategy for discovering new targets and active molecules, high-throughput screening is a very important link in the drug research and development process. It is based on the theories of multiple disciplines such as chemistry, biology, and computer science, and uses an automated system to control the execution of experiments, with the characteristics of microscale, rapidity, sensitivity, and accuracy. Through high-throughput and automated screening, researchers can obtain a large amount of experimental information, efficiently achieve the characterization and identification of active compounds, antibodies, genes, etc., thereby better promoting the development of drug research.

[0007] According to different experimental models, high-throughput screening methods can be roughly divided into two categories: screening based on the molecular level and screening based on the cellular level. Screening at the molecular level can detect changes in enzyme or receptor function, the binding of probes to proteins, and the kinetic characteristics of protein-ligand binding, etc. This type of screening method has the characteristics of a simple system, direct action, rapid detection, and convenient operation. Screening at the cellular level uses intact cells to find potential drug targets and discover promising active molecules through techniques such as cell viability detection, gene editing, reporter genes, and high-content imaging. The screening method at the cellular level has outstanding advantages such as visualization, dynamics, and comprehensive multi-parameter evaluation. In our project, screening at the cellular level is used.

[0008] Chinese patent document CN114796223A discloses the use of a selective EZH2 inhibitor in the preparation of a drug for treating Sjogren's syndrome, and the selective EZH2 inhibitor is GSK343 and pharmaceutically acceptable salts of GSK343. The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the compound and one or more pharmaceutically acceptable carriers. The present invention discovers for the first time that a selective EZH2 inhibitor can be applied to the preparation of a drug for treating Sjogren's syndrome.

[0009] Chinese patent document CN111905102A discloses the use of an EZH2 inhibitor in the treatment of glioma, especially in the radiosensitization of glioma radiotherapy. The inventors have studied and found that during the treatment of glioma, the EZH2 inhibitor has a synergistic effect with radiotherapy, and can effectively assist in improving the curative effect, or reducing the radiotherapy dose and reducing radiation damage without reducing the curative effect.

[0010] However, there is currently no report on the application of EZH2 in ovarian cancer diagnostic reagents and therapeutic drugs. Summary of the Invention

[0011] The object of the present invention is to provide, in view of the deficiencies in the prior art, EZH2 in ovarian cancer diagnostic reagents, inhibitors of EZH2 for treating ovarian cancer, and pharmaceutical compositions.

[0012] In the first aspect, the present invention provides the use of EZH2 as a biomarker in the preparation of an ovarian cancer diagnostic kit.

[0013] In the second aspect, the present invention provides the use of a reagent for detecting the content of EZH2 protein in the preparation of an ovarian cancer diagnostic kit.

[0014] In the third aspect, the present invention provides the use of an inhibitor of EZH2 in the preparation of a drug for treating ovarian cancer.

[0015] As a preferred example, the inhibitor of EZH2 is GSK126 or Tazemetostat.

[0016] In a fourth aspect, the present invention provides a pharmaceutical composition for treating ovarian cancer, which is composed of Napabucas in or composed of Napabucas in and Tazemetostat.

[0017] In a fifth aspect, the present invention provides the use of Napabucasin in the preparation of a medicament for treating ovarian cancer.

[0018] In a sixth aspect, the present invention provides the use of Napabucasin in the preparation of a medicament for treating ovarian cancer metastasis.

[0019] The advantages of the present invention are as follows: EZH2 is abnormally highly expressed in ovarian cancer and promotes the proliferation of ovarian cancer cells; EZH2 inhibitors inhibit the proliferation of ovarian cancer cells; a new drug Napabucasin is discovered through high-throughput drug screening, and its sensitivity is significantly better than that of the traditional EZH2 inhibitor GSK126 or Tazemetostat, and it has been verified in ovarian cancer cells. At the same time, it is found in an in vivo mouse model that Napabucasin can significantly inhibit the metastasis of ovarian cancer with abnormally high expression. We found that the combination of Napabucasin and the EZH2 inhibitor GSK126 or Tazemetostat has a significant synergistic effect, and it has been verified in ovarian cancer cells. It provides important theoretical basis and experimental support for the development of new treatment strategies targeting EZH2. Description of the Drawings

[0020] Figure 1 : EZH2 promotes the proliferation of ovarian cancer cells.

[0021] Figure 2 : EZH2 promotes the proliferation of ovarian cancer cells by activating the RAP1 signaling pathway.

[0022] Figure 3 : EZH2 inhibitors inhibit the proliferation of ovarian cancer cells.

[0023] Figure 4 : A new drug Napabucasin is discovered through high-throughput drug screening.

[0024] Figure 5 : Napabucasin (napabucardin) can significantly inhibit the metastasis of ovarian cancer with abnormally high expression of EZH2. Detailed Embodiments

[0025] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0026] Example 1

[0027] II. Experimental materials

[0028] Ovarian cancer cell lines were purchased from the American Type Culture Collection (ATCC); RPMI-1640 medium, DMEM medium, fetal bovine serum, penicillin-streptomycin, and dimethyl sulfoxide (DMSO) were purchased from Gibco; TRYPSIN 0.25% EDTA was purchased from Thermo Fisher Scientific; RNAi Plus universal RNA extraction and lysis reagent TRIzo1 was purchased from TaKaRa; reverse transcription kit and SYBR fluorescence quantitative reagent were purchased from ABclonal; transfection reagent was purchased from Life Technologies; EZH2-control, EZH2-0E, EZH2-scramble, and EZH2-shRNA were purchased from Addgene; other commonly used reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd.; real-time quantitative PCR instrument was purchased from ABI; flow cytometer was purchased from BD; microplate reader was purchased from Bio-rad; laminar flow hood was purchased from Suzhou Sutai Purification Equipment Engineering Co., Ltd.; Cell Counting Kit-8 reagent and compound library were purchased from MCE.

[0029] III. Experimental methods

[0030] 2.1 Cell culture:

[0031] HEY, A2780, and OV90 were placed in DMEM medium, and SKOV3 and OVCAR8 were cultured in RPMI-1640 medium. The medium contained 10% fetal bovine serum and 1% penicillin + streptomycin. They were cultured in an incubator at 37°C and 5% CO2. The medium was changed every other day, and subculture was performed every 2 days.

[0032] 2.2 Lentivirus preparation: 293T cells were transduced with 8 μg of EZH2-control, EZH2-0E, EZH2-scramble, EZH2-shRNA1, EZH2-shRNA2, EZH2-shRNA3 and packaging plasmids (5.34 μg of psPAX2 and 2.67 μg of pMD2.G). The supernatant was collected after 48 hours, concentrated by filtration, and finally used to infect cells such as HEY, OVCAR8, SKOV3, and A2780 for further experiments.

[0033] 2.3 Real-time PCR detection:

[0034] Total mRNA of the target cells was extracted using TRIzo1 reagent, and cDNA was reverse transcribed according to the instructions of the reverse transcription kit. Real-time PCR was performed on the target gene. The reaction conditions were set according to the kit instructions: 2 min at 37°C, 3 min at 95°C, 5 s at 95°C, and 30 s at 60°C, for a total of 40 cycles. GAPDH was used as a reference. The data collection was mainly calculated by the fluorescence quantitative PCR reaction instrument software to obtain the starting cycle number (Ct) of all standards and samples, and a standard curve was drawn based on the Ct values of the standards. Quantitative analysis was then performed according to the 2-ΔΔCt method.

[0035] 2.4 Western blot detection:

[0036] Whole cells were lysed with RIPA lysis buffer, and the supernatant was collected after sonication and centrifugation. Protein loading buffer was added to the supernatant, and the samples were separated by 10% SDS-PAGE protein electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1.5 h, then incubated with the primary antibody overnight at 4°C, washed with TBST, incubated with the corresponding secondary antibody in the dark at room temperature for 1 h, washed with TBST, and developed with ECL.

[0037] 2.5 CCK8 detection of cell proliferation rate:

[0038] The target cells were digested and counted, and 1500 cells / well were seeded in a 96-well plate. Then, 10 μL of CCK-8 solution was added to each well at 24 h, 48 h, 72 h, and 96 h of culture. After culturing in an incubator for 2 h, the absorbance (A) value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. The cell proliferation rate (%) = (A experimental group / A solvent control group - 1) × 100%. Each group was set with 5 replicate wells, and the experiment was repeated 3 times.

[0039] 2.5 Colony formation assay

[0040] 1000 target cells were seeded in a 6-well plate at 500 cells per well. After culturing for 14 days, the cells were fixed with 4% paraformaldehyde for 30 minutes, stained with 0.5% crystal violet solution for 30 minutes, and colonies larger than 1 mm were counted using IMAGE J.

[0041] 2.6 High-throughput drug screening:

[0042] The above-mentioned cells to be tested (EZH2-control and EZH2-0E cells) were placed on a 96-well plate (10,000 cells / well) containing kinase inhibitors and FDA library compounds at a final concentration of 1 μM. After incubation in a 37 °C, 5% CO₂ incubator for 24 hours, cell viability was evaluated using Cell Counting Reagent SF (07553-44, Nacalaitesque) on an Infinite F200Pro microplate reader (Tecan). The sensitivity index of EZH2-0E cells was determined by calculating the ratio of the A value to the B value, where the A value is the viability of EZH2-0E and the B value is the viability of EZH2-control cells.

[0043] IV. Experimental results

[0044] 4.1 EZH2 is abnormally highly expressed in ovarian cancer and promotes the proliferation of ovarian cancer cells. First, RNA-Seq was used to detect the expression of EZH2 in ovarian cancer samples from clinical ovarian cancer patients. Second, stable cell lines with overexpression and knockdown of EZH2 were constructed. The overexpression and knockdown efficiencies were detected by Western blot and Real-time PCR. Subsequently, the effect on cell proliferation was detected by CCK-8. The results showed that EZH2 was abnormally highly expressed in ovarian cancer, and overexpression of EZH2 promoted the proliferation of ovarian cancer cells, while knockdown inhibited the proliferation of ovarian cancer cells (see Figure 1 ).

[0045] 4.2 EZH2 promotes the proliferation of ovarian cancer cells by activating the RAP1 signaling pathway. First, RAN-Seq was performed on HEY-EZH2-control and HEY-EZH2-0E cells, and subsequent enrichment analysis found that the RAP1 signaling pathway was significantly upregulated after overexpression of EZH2. Then, the expression of RAP1 was verified by Western blot and Real-time PCR. Finally, the effect of RAP1 on the proliferation of ovarian cancer cells was detected by CCK8 and rescue experiments. The results showed that the expression of RAP1 was upregulated after overexpression of EZH2, and knocking down RAP1 on the basis of EZH2-0E could reverse the promoting effect of EZH2 overexpression on the proliferation of ovarian cancer cells (see Figure 2 ).

[0046] 4.3 EZH2 inhibitors inhibit the proliferation of ovarian cancer cells

[0047] Ovarian cancer cells were treated with traditional and commonly used EZH2 inhibitors (GSK126 and Tazemetostat), and the effects on the proliferation of ovarian cancer cells were detected. First, the IC50 of GSK126 and Tazemetostat was detected by CCK-8, and then the effects on cell proliferation were detected by Real-time PCR. The results showed that GSK126 and Tazemetostat could partially inhibit the proliferation of ovarian cancer cells, but neither alone was sufficient to kill ovarian cancer cells (see Figure 3 ).

[0048] 4.4 Discovery of a new drug Napabucasin through high-throughput drug screening and verification in ovarian cancer cells

[0049] 4.5 Napabucasin can significantly inhibit the metastasis of ovarian cancer with abnormally high expression of EZH2. On the basis of the above research, in order to further find a way to inhibit the proliferation of ovarian cancer cells and kill ovarian cancer cells, we found through high-throughput compound screening experiments that the STAT3 inhibitor (Napabucasin) can kill ovarian cancer cells more sensitively and inhibit the proliferation of ovarian cancer cells. Then, the effects of Napabucasin on the proliferation of ovarian cancer cells were detected by CCK-8 assay and colony formation assay. The results showed that Napabucasin was more sensitive than the EZH2 inhibitors (GSK126 and Tazemetostat) and had a stronger effect on inhibiting the proliferation of ovarian cancer cells with high EZH2 expression (see Figure 4 ).

[0050] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Application of EZH2 as a biomarker in the preparation of ovarian cancer diagnostic kit.

2. Application of reagents for detecting EZH2 protein content in the preparation of ovarian cancer diagnostic kits.

3. Application of EZH2 inhibitors in the preparation of drugs for the treatment of ovarian cancer.

4. The use according to claim 3, characterized in that: The EZH2 inhibitor is GSK126 or Tazemetostat.

5. A pharmaceutical composition for treating ovarian cancer, characterized in that: The pharmaceutical composition consists of Napabucasin and or Napabucasin and Tazemetostat.

6. Application of Napabucasin in the preparation of drugs for treating ovarian cancer.

7. Application of Napabucasin in the preparation of drugs for treating ovarian cancer metastasis.

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