An anti-ovarian cancer drug composition and its target

Abstract

The present invention relates to the field of medicine and discloses an anti-ovarian cancer drug composition and its target. The anti-tumor drug composition comprises a Curcuma zedoaria extract and an Astragalus membranaceus extract. The combination of calycosin isoflavones and bisdemethoxycurcumin produces a synergistic effect, reducing drug dosage, side effects, and costs. The MCM2 gene, a target for the anti-ovarian cancer drug, has been discovered. This gene significantly influences the proliferation, migration, and invasion of ovarian cancer, providing guidance for the development and screening of drugs for the prevention or treatment of ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anti-ovarian cancer drug composition and its target. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Among gynecological tumors, ovarian cancer is relatively silent in its early stages, making it difficult to detect in time with most clinical screening methods. Approximately 70% of patients are diagnosed at an advanced stage, resulting in the highest mortality rate among gynecological malignancies. Currently, the standard treatment for ovarian cancer is surgical resection and platinum-based chemotherapy. Tumor resistance and the severe side effects of chemotherapy compromise patient quality of life, resulting in low overall survival rates.

[0004] Tumorigenesis begins with genetic alterations. Abnormal expression and activation of MCM family proteins directly impair DNA replication, leading to genomic instability and associated with tumorigenesis and malignancy. Overexpression of MCM family proteins has been detected in tumors such as breast cancer, hepatocellular carcinoma, and lung adenocarcinoma, suggesting that they can influence tumor progression and have diagnostic potential for related diseases. Minichromosome maintenance protein 2 (MCM2) is a key member of the MCM protein family. Absence of MCM2 expression indicates that cells have exited the cell cycle, meaning they have differentiated and matured or entered the G0 phase. Therefore, abnormalities in MCM2 not only provide a warning of carcinogenesis but also hold promise as a potential target for cancer therapy. MCM2 is also a marker of tumor proliferation, migration, invasion, and metastasis, positively correlating with the TNM stage and lymph node metastasis of many cancers. Studies in cholangiocarcinoma have shown that MCM2 overexpression promotes cholangiocarcinoma (CCA) progression by inhibiting the p53 signaling pathway. Increased MCM2 levels accelerate tumor cell proliferation, migration, and invasion and inhibit apoptosis. Research by KM Lau et al. has shown that upregulation of MCM2 promotes the proliferation, migration, and invasion of medulloblastoma (MB) cells. The prior art, "Inhibition of Minichromosome Support Protein 2 (MCM2) Expression Enhances Ovarian Cancer Sensitivity to Carboplatin," discloses that ovarian cancer cell sensitivity to carboplatin may be related to a P53-mediated apoptosis mechanism. Downregulating MCM2 expression can enhance ovarian cancer cell sensitivity to chemotherapy, providing a new approach for the treatment of ovarian cancer.

[0005] In recent years, with the in-depth study of traditional Chinese medicine, Chinese medicine has played an important role in the treatment of tumors, showing good therapeutic effects on tumor diseases. At the same time, it has also shown significant improvement in the adverse reactions caused by chemotherapy, improving the quality of life of patients. Because Chinese medicine contains a wide variety of active ingredients with different effects, exploring the extraction and compatibility of active ingredients in Chinese medicine for tumor treatment can provide a theoretical basis for the development of low-side effect and high-efficiency tumor treatment drugs, and is also of great significance to the development and utilization of anti-tumor Chinese medicine. Summary of the Invention

[0006] The purpose of the present invention is to provide an anti-ovarian cancer drug composition and its target for the defects of current anti-tumor drugs and methods, to provide an effective traditional Chinese medicine component drug for combating ovarian cancer and to provide guidance for the development of anti-ovarian cancer drugs.

[0007] The technical solutions of the present invention are as follows:

[0008] An anti-ovarian cancer pharmaceutical composition comprises a zedoary turmeric extract.

[0009] According to a preferred embodiment, it also includes an Astragalus extract.

[0010] According to a preferred embodiment, the Astragalus extract includes calycosin isoflavones, and the Curcuma zedoaria extract includes bisdemethoxycurcumin.

[0011] According to a preferred embodiment, the ratio of calycosin to bisdemethoxycurcumin is 16:1.

[0012] According to a preferred embodiment, the anti-ovarian cancer pharmaceutical composition targets MCM2.

[0013] Another aspect of the present invention provides the use of the MCM2 gene as a target of an anti-ovarian cancer drug composition in the development and screening of drugs for preventing or treating ovarian cancer. The drug targets the MCM2 gene to affect cell cycle regulation and the EMT pathway to inhibit ovarian cancer cells.

[0014] According to a preferred embodiment, the drug affects the levels of CDK4, Cyclin D1 and P21, causing the formation of CyclinD1-CDK4 / 6 complex, and arresting the cell cycle.

[0015] According to a preferred embodiment, the drug affects the levels of E-cadherin, N-cadherin and Vimentin to regulate the EMT pathway and reduce the proliferation, migration and invasion capabilities of ovarian cancer cells.

[0016] Compared with the existing technology, the beneficial effects of the present invention are:

[0017] 1. An anti-ovarian cancer pharmaceutical composition. The prior art provides the use of long-acting curcumin derivatives in the preparation of anti-tumor drugs. It is disclosed that curcumin, demethoxycurcumin, or bisdemethoxycurcumin, when used alone, can inhibit tumors such as ovarian cancer and cervical cancer. In the use of drugs, the synergistic effect of different drugs can significantly enhance the drug's efficacy while reducing the drug dosage and saving costs. However, no such results have been found for calycosin isoflavone. The inventors of this application have discovered that calycosin isoflavone and bisdemethoxycurcumin have a synergistic effect, and can work together to combat ovarian cancer, producing a better inhibitory effect than using bisdemethoxycurcumin alone. The combined use of the two to combat ovarian cancer can reduce the drug dosage, reduce drug side effects, and save costs.

[0018] 2. Application of the MCM2 gene as a target for anti-ovarian cancer drug compositions in the development and screening of drugs for the prevention or treatment of ovarian cancer. Research and analysis revealed that the MCM2 gene inhibits ovarian cancer cell proliferation, migration, cloning, and invasion by affecting CDK4, Cyclin D1, and P21 levels, leading to the formation of the Cyclin D1-CDK4 / 6 complex and arresting the cell cycle, and by regulating the EMT pathway through E-cadherin, N-cadherin, and Vimentin levels. This provides a novel mechanism of action for the MCM2 gene compared to existing technologies and confirms that the MCM2 gene also serves as a drug target for ovarian cancer drugs such as calycosin and bisdemethoxycurcumin, providing guidance for the screening of anti-ovarian cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of different concentrations of CA and BDMC on the proliferation of SKOV3 and Hey ovarian cancer cells in Example 1;

[0020] Figure 2 is the inhibition rate and combination coefficient of the combined administration of CA and BDMC in Example 1;

[0021] Figure 3 This is a box plot of the normalized data of each sample in each data chip in Example 2;

[0022] Figure 4 This is a Venn diagram of differentially expressed genes in the three data chips in Example 2;

[0023] Figure 5 This is the bubble chart of the KEGG gene pathway enrichment results in Example 2;

[0024] Figure 6 It is the key functional module of the protein interaction network in Example 2;

[0025] Figure 7This is the survival analysis of the FAM83D and MCM2 genes in Example 2;

[0026] Figure 8 The expression of MCM2 protein and mRNA in SKOV3 and Hey cells after MCM2 knockdown in Example 3 (ⅹ±s, n=3) Note: **P<0.01;

[0027] Figure 9 The expression of MCM2 protein and mRNA in SKOV3 and Hey cells after MCM2 overexpression in Example 3 (ⅹ±s, n=3) Note: **P<0.01;

[0028] Figure 10 The changes in proliferation ability of SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 3;

[0029] Figure 11 The changes in the clone formation of SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 3 (ⅹ±s, n=3);

[0030] Figure 12 The migration changes of SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 3 (ⅹ±s, n=3);

[0031] Figure 13 The changes in invasion of SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 3 (ⅹ±s, n=3)

[0032] Figure 14 The changes in tumor volume and the final tumor inhibition rate of tumor-bearing nude mice after intervention with DDP, CA, BDMC and their combination in Example 1 (x±s, n=8);

[0033] Figure 15 This is the molecular docking diagram of CA and BDMC with MCM2 protein in Example 4;

[0034] Figure 16 The expression of MCM2 protein in SKOV3 and Hey cells treated with CA and BDMC in Example 4 (ⅹ±s, n=3);

[0035] Figure 17 The clone formation of SKOV3 and Hey cells after CA, BDMC and combined drug intervention in MCM2 knockdown in Example 4 (ⅹ±s, n=3);

[0036] Figure 18 The results of the clone formation of SKOV3 and Hey cells with MCM2 overexpression treated with CA, BDMC and their combined administration in Example 4 (ⅹ±s, n=3);

[0037] Figure 19 The changes in cell migration and invasion after CA, BDMC and their combined administration intervened in MCM2-knockdown SKOV3 and Hey cells in Example 4 (ⅹ±s, n=3);

[0038] Figure 20 The changes in cell migration and invasion after CA, BDMC and their combined administration intervened in MCM2-overexpressing SKOV3 and Hey cells in Example 4 (ⅹ±s, n=3);

[0039] Figure 21 The changes in the cell cycle of SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 5 (ⅹ±s, n=3);

[0040] Figure 22 The expression of cell cycle-related proteins in SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 5 (ⅹ±s, n=3);

[0041] Figure 23 The expression of EMT-related proteins in SKOV3 and Hey cells after MCM2 knockdown and overexpression in Example 5 (ⅹ±s, n=3);

[0042] Figure 24 The percentage of cells in the G0 / G1 phase after CA, BDMC and combined drug intervention in MCM2 knockdown SKOV3 and Hey cells in Example 5 (ⅹ±s, n=3);

[0043] Figure 25 The percentage of cells in the G0 / G1 phase after CA, BDMC and their combined administration intervened in MCM2-overexpressing SKOV3 and Hey cells in Example 5 (ⅹ±s, n=3);

[0044] Figure 26 The changes in cell cycle and EMT-related proteins after CA, BDMC and combined drug intervention in MCM2-knockdown SKOV3 and Hey cells in Example 5 (ⅹ±s, n=3);

[0045] Figure 27 The changes in cell cycle and EMT-related proteins after CA, BDMC and their combined administration intervened in MCM2-overexpressing SKOV3 and Hey cells in Example 5 (ⅹ±s, n=3);

[0046] Figure 28 The immunohistochemistry results of cell cycle proteins in nude mouse tumors in Example 5 (400×);

[0047] Figure 29 The expression of cell cycle proteins in tumors of nude mice bearing tumors in Example 5;

[0048] Figure 30 This is the expression of EMT proteins in the tumors of tumor-bearing nude mice in Example 5. DETAILED DESCRIPTION

[0049] The features and properties of the present invention are further described in detail below with reference to the examples. Calycosin is abbreviated as CA; bisdemethoxycurcumin is abbreviated as BDMC. The human SKOV3 ovarian cancer cells, human Hey ovarian cancer cells, and human 293T cells used in the experiments were purchased from the Cell Bank of the Chinese Academy of Sciences. Unless otherwise specified, the experimental materials and instruments used in this application are commercially available or conventional reagents in the art. Unless otherwise specified, the experimental methods used in this application are conventional methods in the art.

[0050] Example 1 Synergistic effect of CA and BDMC on ovarian cancer cells

[0051] 1. CCK-8 detection of ovarian cancer cell proliferation

[0052] SKOV3 and Hey ovarian cancer cells in the logarithmic growth phase were digested with 0.1% trypsin to prepare a cell suspension, and the cell concentration was quantified at 10 4 Cells were plated at 200 μL per well in a 96-well plate. 200 μL of PBS was added to each well around the periphery to eliminate edge effects. The plates were incubated in a 37°C incubator with 5% CO₂ for 24 hours. After cell attachment, the culture medium was discarded, and the cells were rinsed three times with PBS buffer. Fresh culture medium containing various concentrations of CA (0, 50, 100, 200, 400, and 800 μmol / L) and BDMC (0, 3.13, 6.25, 12.5, 25, and 500 μmol / L) was added. Six replicate wells were set up for each group. After drug addition, the cells were cultured for 24 and 48 hours. The 96-well plates were removed and the original culture medium was discarded. The cells were rinsed three times with PBS and 200 μL of serum-free culture medium containing 10% CCK-8 was added. The cells were incubated in a 37°C incubator for 1 hour. The wells with a drug concentration of 0 μmol / L served as control wells, and the wells with only CCK-8 solution added were blank wells. The absorbance was measured at a wavelength of 450 nm using a microplate reader, and the IC50 value was calculated using GraphPad Prism 8 software. Cell viability (%) = [(control well - experimental well) / (control well - blank well)] × 100%.

[0053] Test results such as Figure 1 As shown, Figure 1 A and Figure 1 B shows the effects of CA and BDMC on the proliferation of SKOV3 ovarian cancer cells; Figure 1 C and Figure 1 D shows the effects of CA and BDMC on the proliferation of Hey ovarian cancer cells.* P<0.05, ** P < 0.01. Figure 1 The growth of SKOV3 and Hey ovarian cancer cells treated with 800, 400, 200, 100, and 50 μmol / L of CA and 50, 25, 12.5, 6.25, and 3.125 μmol / L of BDMC, respectively, was inhibited compared to the control group. The results showed that CA and BDMC inhibited the proliferation of both SKOV3 and Hey ovarian cancer cells, and their inhibitory effects increased with increasing drug concentration over the same time period, demonstrating a concentration-dependent pattern. At the same concentration, the proliferation inhibition rate after 48 hours of treatment was significantly higher than that after 24 hours of treatment. The inhibitory effects of CA and BDMC on SKOV3 and Hey ovarian cancer cells were time-dependent.

[0054] 2. Evaluation of the synergistic effect of CA and BDMC on ovarian cancer cells

[0055] Based on the IC50 values ​​of CA and BDMC on the proliferation inhibition rates of SKOV3 and Hey ovarian cancer cells, drug synergistic effect experiments were performed.

[0056] SKOV3 and Hey ovarian cancer cells in the logarithmic growth phase were digested with 0.1% trypsin to prepare a cell suspension, and the cell concentration was quantified at 10 4 Cells were plated at 400 μL / well, 200 μL per well, inoculated into 96-well plates. Six parallel wells were plated per group and incubated at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, the culture medium was discarded and the cells were rinsed three times with PBS buffer. 200 μL of a mixed solution containing CA (0, 50, 100, 200, 400, and 800 μmol / L) and BDMC (0, 3.13, 6.25, 12.5, 25, and 50 μmol / L) was added. Zero wells and blank wells were set up, with six parallel wells per group. The plates were then incubated in an incubator for another 24 hours. After incubation, the original culture medium was discarded, the cells were rinsed three times with PBS, and 200 μL of a serum-free medium containing 10% CCK-8 was added. The plates were incubated in a 37°C incubator for 1 hour. The wells with a drug concentration of 0 μmol / L served as controls, and the wells with only CCK-8 solution added served as blanks. The absorbance was measured at 450 nm using a microplate reader, and the inhibition rate was calculated. Compusyn software and the Chou-Talalay data processing model were used to calculate the combination index (CI) of the two drugs. When CI = 1, the two drugs have an additive effect; when CI > 1, the two drugs have an antagonistic effect; when CI < 1, the two drugs have a synergistic effect.

[0057] Test results such as Figure 2 As shown in Table 1 and Table 2. Figure 2A is the cell inhibition rate and combination coefficient of CA and BDMC combined with SKOV3 ovarian cancer cells; Figure 2 B is the cell inhibition rate and combination coefficient of CA and BDMC combined to Hey ovarian cancer cells.

[0058] Table 1 Inhibitory rate and combination coefficient of SKOV3 cells after combined administration of CA and BDMC

[0059]

[0060]

[0061] Table 2 Hey cell inhibition rate and combination coefficient of combined administration of CA and BDMC

[0062]

[0063] When CA:BDMC was used at a ratio of 16:1 to intervene in SKOV3 and Hey ovarian cancer cells, the combination index CI value of the two drugs was lower than 1 at different dosages, indicating that the two drugs had a synergistic effect.

[0064] 3. Animal Experiments to Detect the Synergistic Effects of CA and BDMC on Ovarian Cancer Cells

[0065] (1) Construction of tumor-bearing nude mouse model

[0066] Newly caged BALB / c nude mice were placed in a laminar flow rack in an SPF-grade breeding room for adaptive breeding for 1 week to adjust the experimental animal status. Hey ovarian cancer cells were cultured to the logarithmic growth phase, the original culture medium was discarded, and 0.1% trypsin was added to wash with PBS buffer for digestion. The corresponding culture medium was added to terminate the digestion, and after centrifugation at 1000 rpm for 3 minutes, PBS buffer was added to make a cell suspension. The cells were counted using a hemocytometer, and the quantitative cell concentration was 5×107 / mL. After grasping and fixing the nude mouse with the left hand, the skin at the injection site under the armpit of the right forelimb of the nude mouse was disinfected with a 75% alcohol cotton ball. A disposable 1mL syringe was used to obliquely pierce the needle and subcutaneously inject about 100μL of cell suspension behind the armpit of the right forelimb, so that each nude mouse was injected with about 5×10 6 After inoculation, gently press the needle with a cotton ball to prevent the cell suspension from leaking from the needle eye.

[0067] (II) Treatment of tumor-bearing nude mice

[0068] The growth of the tumor at the inoculation site of the nude mice was observed daily. Approximately one week later, the tumor at the inoculation site was firm and light red in color, indicating successful tumor growth. The nude mice were then randomly divided into five groups: the control group, which received intraperitoneal injections of normal saline once daily; the DDP group, which received intraperitoneal injections of cisplatin (3 mg / kg) once daily; the CA group, which received intraperitoneal injections of CA (80 mg / kg) once daily; the BDMC group, which received intraperitoneal injections of BDMC (5 mg / kg) once daily; and the CA+BDMC group, which received intraperitoneal injections of CA (80 mg / kg) and BDMC (5 mg / kg) once daily, with 8 mice in each group.

[0069] Tumor volume was calculated by measuring the long diameter (L) and short diameter (W) of the tumors with a vernier caliper every three days. After 15 days of dosing, nude mice were sacrificed by cervical dislocation, and tumor tissue was dissected and weighed to calculate the tumor inhibition rate. Two batches of samples were stored at -80°C and fixed with 4% paraformaldehyde.

[0070]

[0071]

[0072] The changes in tumor volume and the final tumor inhibition rate of tumor-bearing nude mice after intervention with DDP, CA, BDMC and their combined administration are shown in Figure 2. Figure 14 As shown, Figure 14 A: Images of nude mice in each group; B: Images of tumors in each group; C: Tumor volume at each measurement in each group; D: Final tumor inhibition rate in each group; E: Weight of nude mice at each measurement in each group. **P < 0.01 compared with the control group; &&P < 0.01 compared with the CA+BDMC group.

[0073] like Figure 14 As shown, the tumor volume ( Figure 14 -C) The tumor inhibition rate in the DDP group, CA group, BDMC group and CA+BDMC group was significantly lower than that in the Control group (P<0.01); the tumor inhibition rate in the CA+BDMC group was significantly lower than that in the CA group and BDMC group (P<0.01). Figure 14 -D) The body weights of the DDP, CA, BDMC, and CA+BDMC groups were significantly larger than those of the control group (P<0.01); the body weights of the CA+BDMC group were significantly larger than those of the CA and BDMC groups (P<0.01). This experiment shows that the combined administration of CA and BDMC can inhibit tumor proliferation in tumor-bearing nude mice, and the combined administration is more effective than either drug alone. There were no significant differences in body weight among the groups.

[0074] Example 2 Screening of the MCM2 gene, a key gene that plays a role in the proliferation, migration, invasion and cell cycle of ovarian cancer cells

[0075] 1. GEO database download and data preprocessing

[0076] Access the GEO database from https: / / www.ncbi.nlm.nih.gov / geo / and filter according to the following criteria: (1) samples must be derived from ovarian cancer patient tissue; (2) samples must include both normal ovarian tissue and ovarian cancer samples; and (3) the number of samples must be as large as possible. Based on the information contained in the chips, select the required chips for download. After screening and downloading, three data chips were obtained. For each sample, background processing was performed using signal values, missing data was filled, and data normalization was performed before subsequent differential gene comparison.

[0077] like Figure 3 The medians of the samples are basically on the same level, indicating that the normalization degree between the samples is good and subsequent differential gene comparison can be performed.

[0078] 2. Differential gene enrichment analysis

[0079] The obtained differential genes were imported into the KEGG database to screen out the biological characteristics and signal pathways of the differential genes between ovarian cancer and normal ovarian tissues.

[0080] like Figure 4 As shown, there are 229 genes at the intersection of the three data chip sets, which means that there are 229 differentially expressed genes in the three data chips.

[0081] To further explore the functions of the 229 differentially expressed genes, the differentially expressed genes from the three data chips were imported into the KEGG database for signal pathway enrichment analysis. Figure 5 and as shown in Table 3. Figure 5 The size of the bubble indicates the number of genes involved in the pathway, and the colors from blue to red represent the P-values ​​from highest to lowest. The differentially expressed genes primarily exert biological effects through the following four signaling pathways: cell cycle, tyrosine metabolism, retinol metabolism, and drug metabolism-cytochrome P450. The cell cycle pathway showed the highest correlation.

[0082] Table 3 KEGG pathway analysis results

[0083]

[0084] Using the STRING database, 229 differentially expressed genes were imported, and "Homo sapiens" was selected in the species category to perform interaction network analysis of the differentially expressed genes. By analyzing the protein interaction network, key node proteins in the differential network can be screened. The Cytoscape software was used for visualization and the MCODE plug-in in the software was used to screen key modules in the protein interaction network with a "Degree Cutoff" of 2 and a "Node Score Cutoff" of 0.2. Protein interaction network data was obtained. The protein interaction data constructed from the STRING database was visualized using Cytoscape software, and the MCODE plug-in was used to perform key functional module analysis, resulting in a key module containing 25 node proteins and 286 protein pairs. Figure 6 As shown, Figure 6 The larger and darker the bubbles, the more proteins they interact with. The proteins expressed by these differentially expressed genes may be key sites that are affected or activated or inhibited during the carcinogenesis and progression of normal ovarian cancer tissue.

[0085] 3. Survival analysis of key genes

[0086] Enter the genes in the obtained key module into the KM Plotter database, select "ovarian cancer" in the disease, enter the gene name in the key module in "Use multiple genes", select the survival option to be studied as "OS" in "survival", perform survival analysis, and select "Draw Kaplan-Meierplot" to output the survival curve.

[0087] The genes of the key functional modules screened by the MCODE plug-in in Cytoscape software were used for survival analysis using the Kaplan-Meier Plotter database to predict the association between these genes and the prognosis of ovarian cancer. The KM curve is shown in Figure 2. Figure 7 The results showed that the key genes FAM83D and MCM2 in the module were significantly correlated with survival (P<0.05).

[0088] Example 3 Effects of the MCM2 gene on proliferation, migration, invasion, and cell cycle of ovarian cancer cells

[0089] 1. Effect of MCM2 gene on the proliferation of ovarian cancer cells

[0090] (I) MCM2 knockdown and overexpression lentiviral infection of SKOV3 and Hey ovarian cancer cells

[0091] SKOV3 and Hey ovarian cancer cells were subcultured in 6cm culture dishes and infected with lentivirus when they reached 50%-60% growth and were in good growth condition. SKOV3 and Hey ovarian cancer cells were divided into five groups: MCM2 knockdown group (sh-MCM2), MCM2 knockdown virus negative control group (sh-NC), MCM2 overexpression group (OE-MCM2), MCM2 overexpression virus negative control group (OE-NC), and blank control group (Control). The MCM2 knockdown virus and its corresponding negative virus, as well as the MCM2 overexpression virus and its corresponding negative virus, were removed from a -80°C freezer and placed on ice to thaw naturally. Each SKOV3 and Hey ovarian cancer cell culture dish was removed from each group, and an appropriate volume of the corresponding lentiviral solution was added to each dish. The dish was then placed in an incubator and incubated for 24 hours to allow the lentivirus to infect the cells. After the infection period, the culture medium was aspirated and 3 mL of PBS buffer was added. The culture flask was gently shaken to completely cover the cells and washed 3-4 times. The PBS buffer was then removed and the cells were washed with the corresponding culture medium containing 10% FBS and incubated for another 24 hours. After the incubation period, the culture medium was aspirated and replaced with the corresponding culture medium containing 10% FBS and 2 μg / mL puromycin. After the cells in the control group had completely died, the culture medium from the other groups was discarded. The cells were washed twice with PBS and incubated with culture medium containing 10% FBS for further incubation. Fluorescence expression of the cells was observed using an inverted microscope. MCM2 protein and mRNA expression was detected by Western blotting and RT-qPCR. The infected cells were then passaged, expanded, and cryopreserved for future experiments.

[0092] MCM2 knockdown results Figure 8 As shown in Figure 2, after SKOV3 and Hey ovarian cancer cells were infected with lentivirus and MCM2 was knocked down, the MCM2 protein and mRNA levels in the sh-MCM2 group were significantly decreased compared with those in the Control group and sh-NC group (P<0.01); there was no significant change in the MCM2 protein and mRNA levels in the Control group and sh-NC group. Figure 9 As shown in the results, after SKOV3 and Hey ovarian cancer cells were infected with lentivirus to overexpress MCM2, the MCM2 protein and mRNA levels of the OE-MCM2 group cells were significantly increased compared with those of the Control group and OE-NC group (P<0.01); there was no significant change in the MCM2 protein and mRNA levels of the Control group and OE-NC group cells.

[0093] (II) CCK-8 assay to detect cell proliferation

[0094] SKOV3 and Hey ovarian cancer cells in logarithmic growth phase with MCM2 knockdown (Control, sh-NC, and sh-MCM2) or MCM2 overexpression (Control, OE-NC, and OE-MCM2) were cultured, the original culture medium discarded, washed with PBS, and digested with 0.1% trypsin. Cells were then added to the corresponding culture medium to prepare a cell suspension. Cells were counted using a hemocytometer and quantified at a cell concentration of 104 cells / mL. 200 μL of the suspension was plated per well in a 96-well plate. Six replicate wells per group were plated and incubated in a 37°C incubator with 5% CO2 for 0, 24, 48, and 72 hours. After the corresponding incubation period, serum-free culture medium containing 10% CCK-8 was prepared in advance. The corresponding 96-well plate was removed, the original culture medium carefully aspirated, and the cells were gently washed three times with PBS. 200 μL of the corresponding 10% CCK-8 mixture was added to each well and incubated in a 37°C constant temperature incubator for 1 hour. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader to detect cell proliferation under the above time.

[0095] The results are as follows Figure 10 As shown, knockdown of MCM2 reduced the proliferation capacity of SKOV3 and Hey cells ( Figure 10 -A, B), the cell proliferation ability of the sh-MCM2 group was significantly decreased compared with the control group and sh-NC group (P<0.01); the cell proliferation ability of the control group and sh-NC group did not change significantly. The experimental results of MCM2 overexpression cells were consistent with those of MCM2 knockdown cells. MCM2 overexpression promoted the proliferation ability of SKOV3 and Hey cells ( Figure 10 (C, D) The OE-MCM2 group showed a significant increase in cell proliferation compared with the control and OE-NC groups (P < 0.01). There was no significant change in cell proliferation between the control and OE-NC groups. This experiment demonstrated that MCM2 knockdown inhibited ovarian cancer cell proliferation, while MCM2 overexpression promoted ovarian cancer cell proliferation. Negative virus had no significant effect on cell proliferation. Compared with the control group, **P < 0.01; compared with the sh-NC group or OE-NC group, &&P < 0.01.

[0096] Effects of the MCM2 gene on the proliferation and colony formation of ovarian cancer cells

[0097] The original culture medium of SKOV3 and Hey ovarian cancer cells with MCM2 knockdown (Control group, sh-NC group, sh-MCM2 group) and MCM2 overexpression (Control group, OE-NC group, OE-MCM2 group) in the logarithmic growth phase was discarded, and the cells were washed with PBS buffer and digested with 0.1% trypsin. The corresponding culture medium was added to prepare cell suspension, and the cells were counted using a hemocytometer, and the cell concentration was quantified at 10 3 Cells were plated at 1500 μL per well in a 6-well plate. Three parallel wells were set up for each group and cultured in a 37°C incubator with 5% CO₂ for 12 days, with the medium changed every three days. After the incubation period, the 6-well plate was removed, the original culture medium was discarded, and each well was fixed with an appropriate amount of 4% paraformaldehyde for 30 minutes. The paraformaldehyde was discarded, and the wells were washed twice with PBS buffer. The wells were then stained with an appropriate amount of 1% crystal violet solution for 10 minutes. The crystal violet solution was discarded, and the wells were washed twice with PBS buffer. The number of cell colonies formed was photographed and counted.

[0098] The results are as follows Figure 11 As shown in Figure 3, knockdown of MCM2 reduced the colony-forming and proliferation abilities of SKOV3 and Hey cells ( Figure 11 -A, B), the number of clone-forming plaques in the sh-MCM2 group was significantly decreased compared with the control group and sh-NC group (P<0.01); there was no significant change in the colony-forming ability and proliferation ability of cells in the control group and sh-NC group. MCM2 overexpression promoted the colony-forming ability and proliferation ability of SKOV3 and Hey cells ( Figure 11 -C, D). The number of colony-forming plaques in the OE-MCM2 group was significantly increased compared with the Control and OE-NC groups (P < 0.01). There was no significant change in the colony-forming and proliferation abilities of cells in the Control and OE-NC groups. This experiment demonstrated that MCM2 knockdown inhibited ovarian cancer cell proliferation and colony formation, while MCM2 overexpression promoted ovarian cancer cell proliferation and colony formation. Negative virus had no significant effect on cell proliferation and colony formation.

[0099] Effects of MCM2 gene on migration and invasion of ovarian cancer cells

[0100] The night before cell collection, chambers were placed in 24-well plates. Matrigel was removed from a -20°C freezer and allowed to thaw naturally on ice. Thawed Matrigel was then mixed with serum-free medium at a ratio of 1:5 and 100 μL was added to each chamber. After paving, the 24-well plates were placed in an incubator overnight. The next day, the culture medium of logarithmically growing SKOV3 and Hey ovarian cancer cells with MCM2 knockdown (Control, sh-NC, and sh-MCM2 groups) and MCM2 overexpression (Control, OE-NC, and OE-MCM2 groups) was discarded, the cells were washed with PBS, and then digested with 0.1% trypsin. Cell suspensions were then added to the corresponding serum-free medium. Cells were counted using a hemocytometer and quantified at a cell concentration of 5 × 104 cells / mL. Remove the 24-well plate, place a chamber in the well for the migration experiment and a 24-well plate with matrix gel for the invasion experiment, add 200 μL of the corresponding cell suspension to the upper chamber, add 700 μL of the corresponding culture medium containing 10% FBS to the lower chamber, and continue to culture the 24-well plate in the incubator. The 24-well plate for the migration experiment was removed after 24 hours of culture, and the 24-well plate for the invasion experiment was removed after 48 hours of culture. Pour out the culture medium within 24 hours, wipe the inside of the chamber clean with a cotton swab, wash twice with PBS buffer, place the chamber in 4% paraformaldehyde for 30 minutes, and after fixation, place it in 1% crystal violet solution for staining for 10 minutes. Remove the chamber, wash twice with PBS buffer, wipe dry with absorbent paper, and observe and count under a microscope.

[0101] The results are as follows Figure 12 , 13 (the scale bar in the figure is 10 μM), the migration of SKOV3 and Hey cells after MCM2 knockdown ( Figure 12 -A, B) and invasion ( Figure 13 -A, B) ability of SKOV3 and Hey cells was significantly decreased. The number of migration and invasion cells in the sh-MCM2 group was significantly decreased compared with the control group and sh-NC group (P<0.01); the number of migration and invasion cells in the control group and sh-NC group did not change significantly. Figure 12 -C, D) and invasion ( Figure 13 -C, D) significantly increased the migration and invasion capacity of cells in the OE-MCM2 group compared with the Control and OE-NC groups (P < 0.01). There was no significant change in the migration and invasion of cells in the Control and OE-NC groups. This experiment shows that MCM2 knockdown inhibits the migration and invasion of ovarian cancer cells, while MCM2 overexpression promotes the migration and invasion of ovarian cancer cells. Negative virus has no significant effect on cell migration and invasion.

[0102] Example 4 CA and BDMC use MCM2 as a target for inhibiting ovarian cancer

[0103] 1. Molecular docking experiment

[0104] The molecular docking visualization results were analyzed by Pymol software ( Figure 15 ). Figure 15 Figure A shows the docking diagram of CA and MCM2; Figure B shows the docking diagram of BDMC and MCM2. The gray structure represents the MCM2 protein, the red represents the small molecule ligand CA, the blue represents the small molecule ligand BDMC, and the green represents the amino acid residues connecting the protein and its components. The visualization results show that CA and BDMC can tightly bind to multiple amino acids on the MCM2 protein through hydrogen bonds.

[0105] 2. Effects of CA and BDMC on the expression of MCM2 protein in ovarian cancer cells

[0106] The CA group (100 μmol / L CA), the BDMC group (6.25 μmol / L BDMC), and the CA+BDMC group (100 μmol / L CA+6.25 μmol / L BDMC) used in Example 1 were treated with SKOV3 and Hey ovarian cancer cells for 24 hours. The expression of MCM2 protein in the two ovarian cancer cells was affected as shown in the figure. Figure 16 As shown, Figure 16 Figure A shows MCM2 protein expression in SKOV3 cells; Figure B shows MCM2 protein expression in Hey cells. Compared with the control group, #P<0.05, ##P<0.01; compared with the CA+BDMC group, &P<0.05, &&P<0.01. In both ovarian cancer cell lines, MCM2 protein expression was significantly decreased compared with the control group (P<0.05, P<0.01). Furthermore, MCM2 protein expression in the CA+BDMC group was significantly decreased compared with either the CA or BDMC groups (P<0.01). This study demonstrates that CA, BDMC, and combined intervention can reduce MCM2 protein expression in ovarian cancer cells, with the combined group exhibiting superior effects compared with either single-agent group.

[0107] 3. CA, BDMC, and their combined administration affect ovarian cancer cell proliferation and colony formation through MCM2

[0108] After MCM2 knockdown in SKOV3 and Hey ovarian cancer cells, CA, BDMC and combined drug intervention were used. Figure 17 shown. Figure 17 In the figure, A shows the colony formation of SKOV3 cells; B shows the colony formation of Hey cells. Compared with the sh-NC group, #P<0.05, ##P<0.01; compared with the sh-NC+CA+BDMC group, &P<0.05, &&P<0.01; **P<0.01.

[0109] from Figure 17 As shown, treatment with CA, BDMC, and combined drug delivery reduced the colony-forming and proliferation abilities of sh-NC cells, which are lentiviral vectors derived from SKOV3 and Hey ovarian cancer cells. The number of colony-forming plaques in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups for both cell lines was significantly decreased compared with the sh-NC group (P<0.05, P<0.01). The number of colony-forming plaques in the combined drug delivery group, sh-NC+CA+BDMC, was significantly decreased compared with the sh-NC+CA and sh-NC+BDMC groups (P<0.05, P<0.01). Following MCM2 knockdown, treatment with CA, BDMC, and combined drug delivery did not significantly alter the colony-forming and proliferation abilities of the cells. The number of colony-forming plaques in the sh-MCM2+CA, sh-MCM2+BDMC, and sh-MCM2+CA+BDMC groups for both cell lines was not significantly different compared with the sh-MCM2 group. The number of clone-forming plaques in the combined drug-treated group (sh-MCM2+CA+BDMC) showed no significant difference compared with the sh-MCM2+CA and sh-MCM2+BDMC groups. Meanwhile, under the same drug-treated conditions, the number of clone-forming plaques in the sh-MCM2 cell-treated group was significantly decreased compared with the sh-NC cell-treated group (P<0.01).

[0110] The results of drug intervention experiments on MCM2-overexpressing cells are as follows Figure 18 shown. Figure 18 In the figure, A shows the colony formation of SKOV3 cells; B shows the colony formation of Hey cells. Compared with the OE-NC group, #P<0.05, ##P<0.01; compared with the OE-NC+CA+BDMC group, &P<0.05, &&P<0.01; *P<0.05; compared with the OE-MCM2+CA+BDMC group, ΔP<0.05, ΔΔP<0.01; *P<0.05, **P<0.01.

[0111] from Figure 18As shown, treatment with CA, BDMC, and combined administration reduced the colony-forming and proliferation abilities of OE-NC cells, which are lentiviral vectors of SKOV3 and Hey ovarian cancer cells. The number of colony-forming plaques in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups for both cell lines was significantly decreased compared with the OE-NC group (P<0.05, P<0.01). The number of colony-forming plaques in the combined administration group (OE-NC+CA+BDMC) was significantly decreased compared with the OE-NC+CA and OE-NC+BDMC groups (P<0.05, P<0.01). After MCM2 overexpression, the colony-forming and proliferation abilities of cells did not change significantly after CA and BDMC monotherapy. The number of clone-forming plaques in the OE-MCM2+CA and OE-MCM2+BDMC groups did not change significantly compared with the OE-MCM2 group. However, the number of clone-forming plaques in the combined drug group OE-MCM2+CA+BDMC was significantly decreased compared with the OE-MCM2, OE-MCM2+CA, and OE-MCM2+BDMC groups (P<0.05, P<0.01). Furthermore, under the same drug treatment conditions, the number of clone-forming plaques in OE-MCM2 cells was significantly increased compared with that in OE-NC cells (P<0.05, P<0.01). This experiment showed that CA, BDMC and combined drug intervention can reduce the colony-forming ability and proliferation ability of ovarian cancer cells, and the effect of the combined drug group is better than that of each single drug group; the drug efficacy weakened after MCM2 knockdown and overexpression, and there was no significant change in the number of cell clone-formed plaques in each group after MCM2 knockdown and drug intervention. After MCM2 overexpression, only the combined drug group showed drug efficacy.

[0112] 4. CA, BDMC, and their combined administration affect ovarian cancer cell migration and invasion via MCM2

[0113] The results of the effects of CA, BDMC and combined administration on cell migration after MCM2 knockdown in SKOV3 and Hey ovarian cancer cells are shown in the figure. Figure 19 shown. Figure 19 The scale bar represents 10 μM. A shows the migration of SKOV3 cells; B shows the migration of Hey cells; C shows the invasion of SKOV3 cells; and D shows the invasion of Hey cells. Compared with the sh-NC group, ##P<0.01; compared with the sh-NC+CA+BDMC group, &&P<0.01; **P<0.01.

[0114] like Figure 19As shown, CA, BDMC, and combined drug treatment affected the migration and invasion abilities of sh-NC cells, which were infected with empty lentiviral vectors from SKOV3 and Hey ovarian cancer cells. The number of migrating and invasive cells in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups of both cell lines was significantly decreased compared with the sh-NC group (P<0.01). The number of migrating and invasive cells in the combined drug treatment group, sh-NC+CA+BDMC, was significantly decreased compared with the sh-NC+CA and sh-NC+BDMC groups (P<0.01). After MCM2 knockdown, the number of migrating and invasive cells did not change significantly after CA, BDMC, or combined drug treatment. The number of migrating and invasive cells in the sh-MCM2+CA, sh-MCM2+BDMC, and sh-MCM2+CA+BDMC groups of both cell lines did not change significantly compared with the sh-MCM2 group. The numbers of migrating and invasive cells in the combined drug-treated group (sh-MCM2+CA+BDMC) showed no significant changes compared with the sh-MCM2+CA and sh-MCM2+BDMC groups. Furthermore, under the same drug-treated conditions, the numbers of migrating and invasive cells in the sh-MCM2 cell-treated group were significantly decreased compared with the sh-NC cell-treated group (P<0.01).

[0115] The results of the effects of CA, BDMC and combined administration on cell migration in SKOV3 and Hey ovarian cancer cells after MCM2 overexpression are shown in Figure 2. Figure 20 shown. Figure 20 The scale bar represents 10 μM. A shows the migration of SKOV3 cells; B shows the migration of Hey cells; C shows the invasion of SKOV3 cells; and D shows the invasion of Hey cells. Compared with the OE-NC group, ##P < 0.01; compared with the OE-NC + CA + BDMC group, &&P < 0.01; compared with the OE-MCM2 + CA + BDMC group, ΔP < 0.05, ΔΔP < 0.01; **P < 0.01.

[0116] from Figure 20As shown, CA, BDMC, and combined drug treatment affected the migration and invasion abilities of OE-NC cells, which are lentiviral vectors of SKOV3 and Hey ovarian cancer cells. The number of migrating and invasive cells in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups for both cell lines was significantly decreased compared with the OE-NC group (P<0.01). The number of migrating and invasive cells in the combined drug treatment group, OE-NC+CA+BDMC, was significantly decreased compared with the OE-NC+CA and OE-NC+BDMC groups (P<0.01). After MCM2 overexpression, the migration and invasion abilities of cells did not change significantly after CA and BDMC monotherapy. The number of migrating and invasive cells in the OE-MCM2+CA and OE-MCM2+BDMC groups did not change significantly compared with the OE-MCM2 group. However, the number of migrating and invasive cells in the combined OE-MCM2+CA+BDMC group was significantly decreased compared with the OE-MCM2, OE-MCM2+CA, and OE-MCM2+BDMC groups (P<0.01). Furthermore, under the same treatment conditions, the number of migrating and invasive cells in OE-MCM2 cells was significantly increased compared with that in OE-NC cells (P<0.01). This experiment showed that CA, BDMC and combined drug intervention can reduce the migration and invasion ability of ovarian cancer cells, and the effect of the combined drug group is better than that of each single drug group; the efficacy of the drug weakened after MCM2 knockdown and overexpression, and there was no significant change in the number of cell migration and invasion cells in each group after MCM2 knockdown and drug intervention. After MCM2 overexpression, only the combined drug group showed drug efficacy.

[0117] Example 5: MCM2 gene as a tumor drug target to inhibit tumor cells

[0118] 1. Effects of the MCM2 gene on the cell cycle

[0119] The original culture medium of SKOV3 and Hey ovarian cancer cells with MCM2 knockdown (Control group, sh-NC group, sh-MCM2 group) and MCM2 overexpression (Control group, OE-NC group, OE-MCM2 group) in the logarithmic growth phase was discarded, the cells were washed with PBS buffer, and then digested with 0.1% trypsin. The corresponding culture medium was added to make a cell suspension, and the cells were counted using a hemocytometer, and the cell concentration was quantified at 5×10 4Cells were plated at a concentration of 1500 μL per well in a 6-well plate. Three replicate wells were plated per group and incubated in a 37°C incubator with 5% CO₂ for 24 hours. After the incubation period, the 6-well plate was removed and the remaining culture medium was aspirated. The cells were gently washed three times with PBS buffer. 500 μL of 0.1% trypsin was added to each well. Once the cells could be pipetted free, the remaining culture medium was added to terminate the digestion. The cells were gently pipetted free from the wall of the 6-well plate. The cell suspension was aspirated and transferred to a centrifuge tube. Centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant discarded, and 1 mL of pre-chilled PBS buffer was added. The cells were gently resuspended and transferred to a sterile 1.5 mL centrifuge tube. Centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant discarded, and the bottom of the tube gently flicked to disperse the cells. 1 mL of pre-chilled 70% ethanol was added and mixed thoroughly by pipetting to form a cell suspension. The suspension was then fixed in a 4°C refrigerator for 24 hours. After fixation, open the water bath in advance, set the temperature to 37°C, centrifuge at 4°C, 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of pre-cooled PBS buffer and gently resuspend the washed cells. Centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and gently flick the bottom of the centrifuge tube to disperse the cells. Add 500 μL of PI staining working solution containing RNase to each cell sample to be tested and place it in a light-proof water bath at 37°C for 30 minutes. Use a flow cytometer to detect red fluorescence at an excitation wavelength of 488 nm and detect light scattering at the same time. Use modfit3.2 version to analyze the cell DNA cycle.

[0120] Cell cycle assays were performed after MCM2 knockdown and overexpression in SKOV3 and Hey ovarian cancer cells. Figure 21 As shown in Figure 2, the G0 / G1 phase ratios of SKOV3 and Hey cells increased significantly after MCM2 knockdown ( Figure 21 -A, B), the G0 / G1 phase ratio of cells in the sh-MCM2 group was significantly increased compared with the control group and sh-NC group (P<0.01); there was no significant change in the cell cycle of cells in the control group and sh-NC group. After MCM2 overexpression, the G0 / G1 phase ratio of SKOV3 and Hey cells was significantly increased ( Figure 21 -C, D). The G0 / G1 phase ratio in the OE-MCM2 group was significantly decreased compared with the Control and OE-NC groups (P < 0.01). There was no significant change in the cell cycle in the Control and OE-NC groups. This experiment showed that MCM2 knockdown increased the G0 / G1 phase ratio in ovarian cancer cells and arrested the cell cycle in the G0 / G1 phase; MCM2 overexpression decreased the G0 / G1 phase ratio in ovarian cancer cells and accelerated cell cycle progression; negative virus had no significant effect on the cell cycle.

[0121] Effects of MCM2 on the expression of cell cycle and EMT-related proteins in ovarian cancer cells

[0122] Effects of MCM2 knockdown and overexpression on cell cycle and EMT-related proteins in SKOV3 and Hey cells Figure 22 、 23 Expression of cell cycle-related proteins in SKOV3 and Hey cells after MCM2 knockdown ( Figure 22 -A, B): The expression of CDK4 and Cyclin D1 in the sh-MCM2 group was significantly decreased compared with the Control group and sh-NC group (P<0.01); the expression of P21 in the sh-MCM2 group was significantly increased compared with the Control group and sh-NC group (P<0.01). There was no significant change in the expression of CDK4, Cyclin D1 and P21 in the Control group and sh-NC group. Expression of EMT-related proteins in SKOV3 and Hey cells after MCM2 knockdown ( Figure 23 -A, B): The expression of E-cadherin in the sh-MCM2 group was significantly increased compared with the Control group and sh-NC group (P<0.01); the expression of N-cadherin and Vimentin in the sh-MCM2 group was significantly decreased compared with the Control group and sh-NC group (P<0.01). There was no significant change in the expression of E-cadherin, N-cadherin and Vimentin in the Control group and sh-NC group cells.

[0123] Expression of cell cycle-related proteins in SKOV3 and Hey cells after MCM2 overexpression ( Figure 22 -C, D): The expression of CDK4 and Cyclin D1 in the OE-MCM2 group was significantly increased compared with the Control group and OE-NC group (P<0.01); the expression of P21 in the OE-MCM2 group was significantly decreased compared with the Control group and OE-NC group (P<0.01). There was no significant change in the expression of CDK4, Cyclin D1 and P21 in the Control group and OE-NC group. Expression of EMT-related proteins in SKOV3 and Hey cells after MCM2 overexpression ( Figure 23-C, D): E-cadherin expression in the OE-MCM2 group was significantly decreased compared with the control and sh-NC groups (P<0.01). N-cadherin and vimentin expressions in the OE-MCM2 group were significantly increased compared with the control and OE-NC groups (P<0.01). There was no significant change in E-cadherin, N-cadherin, or vimentin expression in the control and OE-NC groups. This experiment shows that MCM2 knockdown and overexpression can affect the expression of cell cycle and EMT-related proteins in ovarian cancer cells; negative virus has no significant effect on the expression of cell cycle proteins and EMT-related proteins.

[0124] 3. CA, BDMC, and combined administration affect the cell cycle of ovarian cancer cells through MCM2

[0125] The results of cell cycle changes after MCM2 knockdown in SKOV3 and Hey ovarian cancer cells were shown in Figure 2. Figure 24 Figure 2. A shows the cell cycle of SKOV3 cells; B shows the cell cycle of Hey cells. Lentiviral empty vector-infected SKOV3 and Hey ovarian cancer cells, sh-NC cells, were arrested in the G0 / G1 phase after treatment with CA, BDMC, and combined drug therapy. The percentage of cells in the G0 / G1 phase in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups was significantly increased compared with the sh-NC group (P<0.01). The percentage of cells in the G0 / G1 phase in the combined drug therapy group, sh-NC+CA+BDMC, was significantly increased compared with the sh-NC+CA and sh-NC+BDMC groups (P<0.05, P<0.01). After MCM2 knockdown, the percentage of cells in the G0 / G1 phase did not change significantly after CA, BDMC, or combined drug intervention. The percentage of G0 / G1 cells in the sh-MCM2+CA, sh-MCM2+BDMC, and sh-MCM2+CA+BDMC groups did not change significantly compared with the sh-MCM2 group. In the combined drug group, the percentage of G0 / G1 cells in the sh-MCM2+CA+BDMC group did not change significantly compared with the sh-MCM2+CA and sh-MCM2+BDMC groups. Furthermore, under the same drug treatment conditions, the percentage of cells in the G0 / G1 phase in the sh-MCM2-treated group was significantly increased compared with the sh-NC-treated group (P < 0.01).

[0126] The results of cell cycle changes after CA, BDMC and combined drug intervention in SKOV3 and Hey ovarian cancer cells with MCM2 overexpression are shown in Figure 2. Figure 25Figures are shown. A shows the cell cycle of SKOV3 cells; B shows the cell cycle of Hey cells. SKOV3 and Hey ovarian cancer cell lines infected with empty lentiviral vectors, OE-NC cells, were arrested in the G0 / G1 phase after treatment with CA, BDMC, and combined drug therapy. The percentages of G0 / G1 cells in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups were significantly increased compared with the OE-NC group (P<0.05, P<0.01). The percentage of G0 / G1 cells in the combined drug therapy group, OE-NC+CA+BDMC, was significantly increased compared with the OE-NC+CA and OE-NC+BDMC groups (P<0.01). After MCM2 overexpression, the percentage of G0 / G1 cells in the cells treated with CA or BDMC alone did not change significantly. The percentage of G0 / G1 cells in the OE-MCM2+CA and OE-MCM2+BDMC groups did not change significantly compared with the OE-MCM2 group. However, the percentage of G0 / G1 cells in the combined OE-MCM2+CA+BDMC group was significantly increased compared with the OE-MCM2, OE-MCM2+CA, and OE-MCM2+BDMC groups (P<0.05, P<0.01). At the same time, under the same treatment conditions, the percentage of G0 / G1 cells in OE-MCM2 cells was significantly decreased compared with that in OE-NC cells (P<0.05, P<0.01). This experiment showed that CA, BDMC and combined drug intervention can increase the percentage of ovarian cancer cells in the G0 / G1 phase and block cells in the G0 / G1 phase. The effect of the combined drug group is better than that of each single drug group. The efficacy of the drug weakened after MCM2 knockdown and overexpression. After MCM2 knockdown and then drug intervention, there was no significant change in the percentage of cells in the G0 / G1 phase in each group. After MCM2 overexpression, only the combined drug group showed drug efficacy.

[0127] 4. CA, BDMC, and their combined administration affect the expression of cell cycle and EMT-related proteins in ovarian cancer cells through MCM2

[0128] The results of the intervention of CA, BDMC and combined administration on the cell cycle and EMT-related proteins in SKOV3 and Hey ovarian cancer cells after MCM2 knockdown are shown in the figure. Figure 26 As shown. Expression of cell cycle related proteins ( Figure 26-A, C): Compared with the sh-NC group, the expression of CDK4 and CyclinD1 in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups of SKOV3 and Hey ovarian cancer cells was significantly decreased (P<0.05, P<0.01). Compared with the sh-NC group and sh-NC+BDMC groups, the expression of CDK4 and Cyclin D1 in the combined drug group sh-NC+CA+BDMC was significantly decreased (P<0.05, P<0.01). Compared with the sh-NC group, the expression of P21 in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups was significantly increased (P<0.01). Compared with the sh-NC group, the expression of P21 in the combined drug group sh-NC+CA+BDMC was significantly increased (P<0.05, P<0.01). After MCM2 knockdown, the expression of CDK4, Cyclin D1, and P21 in cells treated with CA, BDMC, or a combination of these drugs remained unchanged. In the sh-MCM2+CA, sh-MCM2+BDMC, and sh-MCM2+CA+BDMC groups, CDK4, Cyclin D1, and P21 expressions were not significantly different from those in the sh-MCM2 group. In the combined drug group, sh-MCM2+CA+BDMC, CDK4, Cyclin D1, and P21 expressions were not significantly different from those in the sh-MCM2+CA and sh-MCM2+BDMC groups. Furthermore, under the same drug treatment conditions, CDK4 and Cyclin D1 expressions in the sh-MCM2 group were significantly decreased compared with those in the sh-NC group (P<0.05, P<0.01). However, P21 expression in the sh-MCM2 group was significantly increased compared with the sh-NC group (P<0.05, P<0.01).

[0129] Expression of EMT-related proteins ( Figure 26-B, D): E-cadherin expression in SKOV3 and Hey ovarian cancer cells in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups was significantly increased compared with the sh-NC group (P<0.01). E-cadherin expression in the combined drug-treated sh-NC+CA+BDMC group was significantly increased compared with the sh-NC+CA and sh-NC+BDMC groups (P<0.05, P<0.01). N-cadherin and vimentin expressions in the sh-NC+CA, sh-NC+BDMC, and sh-NC+CA+BDMC groups were significantly decreased compared with the sh-NC group (P<0.05, P<0.01). N-cadherin and vimentin expressions in the combined drug-treated sh-NC+CA+BDMC group were significantly decreased compared with the sh-NC+CA and sh-NC+BDMC groups (P<0.05, P<0.01). After MCM2 knockdown, the expression of E-cadherin, N-cadherin, and Vimentin in cells treated with CA, BDMC, or combined drug intervention did not change significantly. The expression of E-cadherin, N-cadherin, and Vimentin in the sh-MCM2+CA, sh-MCM2+BDMC, and sh-MCM2+CA+BDMC groups did not change significantly compared with the sh-MCM2 group. The expression of E-cadherin, N-cadherin, and Vimentin in the combined drug group, sh-MCM2+CA+BDMC, did not change significantly compared with the sh-MCM2+CA and sh-MCM2+BDMC groups. At the same time, under the same treatment conditions, the expression of E-cadherin in the sh-MCM2 cell treatment group was significantly increased compared with the sh-NC cell treatment group (P<0.05, P<0.01); the expression of N-cadherin and Vimentin in the sh-MCM2 cell treatment group was significantly decreased compared with the sh-NC cell treatment group (P<0.05, P<0.01).

[0130] The results of the intervention of CA, BDMC and combined administration on the cell cycle and EMT-related proteins in SKOV3 and Hey ovarian cancer cells after MCM2 overexpression are shown in the figure. Figure 27 As shown. Expression of cell cycle related proteins ( Figure 27-A, C): CDK4 and Cyclin D1 expressions in SKOV3 and Hey ovarian cancer cells in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups were significantly decreased compared with the OE-NC group (P<0.05, P<0.01). In the combined drug administration group, CDK4 and Cyclin D1 expressions in the OE-NC+CA+BDMC group were significantly decreased compared with the OE-NC+CA and OE-NC+BDMC groups (P<0.05, P<0.01). P21 expressions in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups were significantly increased compared with the OE-NC group (P<0.05, P<0.01). The expression of P21 in the combined drug administration group OE-NC+CA+BDMC was significantly increased compared with the OE-NC+CA group and OE-NC+BDMC group (P<0.05, P<0.01). After MCM2 overexpression, the expression of CDK4, Cyclin D1 and P21 in cells was not significantly changed after CA and BDMC monotherapy. The expression of CDK4, Cyclin D1 and P21 in the OE-MCM2+CA and OE-MCM2+BDMC groups did not change significantly compared with the OE-MCM2 group. In the combined drug group, the expression of CDK4 and Cyclin D1 in the OE-MCM2+CA+BDMC group was significantly decreased compared with the OE-MCM2 group, OE-MCM2+CA group and OE-MCM2+BDMC group (P<0.05, P<0.01). The expression of P21 in the OE-MCM2+CA+BDMC group was significantly increased compared with the OE-MCM2 group, OE-MCM2+CA group and OE-MCM2+BDMC group (P<0.05, P<0.01). At the same time, under the same treatment conditions, the expressions of CDK4 and Cyclin D1 in the OE-MCM2 cell-treated group were significantly increased compared with those in the OE-NC cell-treated group (P<0.01); the expression of P21 in the OE-MCM2 cell-treated group was significantly decreased compared with that in the OE-NC cell-treated group (P<0.05, P<0.01).

[0131] Expression of EMT-related proteins ( Figure 27-B, D): E-cadherin expression in SKOV3 and Hey ovarian cancer cells in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups was significantly increased compared with the OE-NC group (P<0.01). In the combined drug-treated group, E-cadherin expression in the OE-NC+CA+BDMC group was significantly increased compared with the OE-NC+CA and OE-NC+BDMC groups (P<0.05, P<0.01). N-cadherin and vimentin expressions in the OE-NC+CA, OE-NC+BDMC, and OE-NC+CA+BDMC groups were significantly decreased compared with the OE-NC group (P<0.05, P<0.01). In the combined drug-treated group, N-cadherin and vimentin expressions in the OE-NC+CA+BDMC group were significantly decreased compared with the OE-NC+CA and OE-NC+BDMC groups (P<0.05, P<0.01). After MCM2 overexpression, the expression of CDK4, Cyclin D1 and P21 in cells did not change significantly after CA and BDMC monotherapy intervention. The expression of E-cadherin, N-cadherin and Vimentin in the OE-MCM2+CA and OE-MCM2+BDMC groups did not change significantly compared with the OE-MCM2 group. In the combined drug group, the expression of E-cadherin in the OE-MCM2+CA+BDMC group was significantly increased compared with the OE-MCM2 group, OE-MCM2+CA group and OE-MCM2+BDMC group (P<0.05, P<0.01); the expression of N-cadherin and Vimentin in the OE-MCM2+CA+BDMC group was significantly decreased compared with the OE-MCM2 group, OE-MCM2+CA group and OE-MCM2+BDMC group (P<0.05, P<0.01). At the same time, under the same treatment conditions, E-cadherin expression in the OE-MCM2 cell-treated group was significantly decreased compared with the OE-NC cell-treated group (P<0.01); N-cadherin and Vimentin expressions in the OE-MCM2 cell-treated group were significantly increased compared with the OE-NC cell-treated group (P<0.05, P<0.01). This experiment shows that CA, BDMC, and combined drug intervention can affect the expression of cell cycle and EMT-related proteins in ovarian cancer cells, and the combined drug group has a better effect than each single drug group. The drug efficacy is weakened after MCM2 knockdown and overexpression. After MCM2 knockdown, there is no significant change in the drug intervention group. After MCM2 overexpression, only the combined drug group shows drug efficacy.

[0132] 5. Immunohistochemical Detection of Cell Cycle-Related Proteins in Tumors of Tumor-Bearing Nude Mice

[0133] After the nude mice were treated with DDP, CA, BDMC and their combined administration, the immunohistochemical status of cell cycle proteins in the nude mice tumors was as follows: Figure 28 The figure shows images at 400X magnification, and the scale bar is 10 μM. Compared with the control group, the expression of MCM2, CDK4, and Cyclin D1 proteins decreased in the CA, BDMC, and CA+BDMC groups; the expression of MCM2, CDK4, and Cyclin D1 proteins decreased in the CA+BDMC group compared with the CA and BDMC groups. Compared with the control group, the expression of P21 protein increased in the CA, BDMC, and CA+BDMC groups; the expression of P21 protein increased in the CA+BDMC group compared with the CA and BDMC groups.

[0134] VI. Western Blot assay to detect cell cycle and EMT-related proteins in tumor-bearing nude mice

[0135] The expression of cell cycle proteins and EMT-related proteins in tumor-bearing nude mice after intervention with DDP, CA, BDMC and their combination Figure 29 In the figure, ① is the Control group; ② is the DDP group; ③ is the CA group; ④ is the BDMC group; and ⑤ is the CA+BDMC group.

[0136] Expression of cell cycle-related proteins: Compared with the control group, the expression of MCM2, CDK4, and Cyclin D1 proteins in the CA, BDMC, and CA+BDMC groups was significantly decreased (P<0.05, P<0.01). Compared with the control group, the expression of CDK4 protein in the DDP group was significantly decreased (P<0.01). Compared with the CA and BDMC groups, the expression of MCM2, CDK4, and Cyclin D1 proteins in the CA+BDMC group was significantly decreased (P<0.05, P<0.01). Compared with the control group, the expression of P21 protein in the CA, BDMC, and CA+BDMC groups was significantly increased (P<0.05, P<0.01). Compared with the CA and BDMC groups, the expression of P21 protein in the CA+BDMC group was significantly increased (P<0.05).

[0137] Expression of EMT-related proteins Figure 30 As shown in the figure: ① is the Control group; ② is the DDP group; ③ is the CA group; ④ is the BDMC group; ⑤ is the CA+BDMC group.

[0138] Compared with the control group, E-cadherin protein expression was significantly increased in the CA, BDMC, and CA+BDMC groups (P<0.05, P<0.01). E-cadherin protein expression was significantly increased in the CA+BDMC group compared with the CA and BDMC groups (P<0.05, P<0.01). N-cadherin and vimentin protein expression was significantly decreased in the CA, BDMC, and CA+BDMC groups compared with the control group (P<0.05, P<0.01). Compared with the CA and BDMC groups, N-cadherin and vimentin protein expression was significantly decreased in the CA+BDMC group (P<0.05, P<0.01). This study demonstrates that combined administration of CA, BDMC, and CA+BDMC can affect the expression of cell cycle and EMT-related proteins in tumor-bearing nude mice, with combined administration being more effective than single-agent therapy.

[0139] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. An anti-ovarian cancer pharmaceutical composition, characterized in that: The invention consists of the isoflavone and bisdemethoxycurcumin in a molar ratio of 16:1 per unit volume.

2. The anti-ovarian cancer pharmaceutical composition according to claim 1, characterized in that: Targeting the MCM2 gene.

3. The anti-ovarian cancer pharmaceutical composition according to claim 2, characterized in that: The drug targets the MCM2 gene to block cell cycle regulation and affect the EMT pathway to inhibit ovarian cancer cells.

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