Use of caMKII-beta inhibitors in the preparation of medicaments for the treatment of ovarian cancer

Abstract

The application relates to application of a CaMKII-beta inhibitor in preparation of a drug for treating ovarian cancer. The application discloses that siRNA can obviously down-regulate the expression level of CaMKII-beta in ovarian cancer SK-OV-3 and OVCAR-8 cells, determines that CaMKII-beta is a potential treatment target of ovarian cancer, and identifies tannic acid as an effective CaMKII-beta inhibitor through virtual screening and in-vitro experiment. The experiment shows that the tannic acid can obviously inhibit the growth of ovarian cancer SK-OV-3 cells and OVCAR-8 cells, and the effect is similar to that of siRNA treatment. The in-vivo experiment of SK-OV-3 transplanted tumor proves that the tannic acid realizes the in-vivo tumor inhibition effect by inhibiting CaMKII-beta, and reveals the good tumor inhibition effect of the tannic acid on ovarian cancer.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy drug technology, and relates to a drug for treating ovarian cancer, particularly a drug that treats ovarian cancer by inhibiting the expression of CaMKII-β in ovarian cancer cells. The drug of this invention inhibits the proliferation of ovarian cancer cells by suppressing the kinase activity of CaMKII-β in ovarian cancer cells. Background Technology

[0002] Ovarian cancer, a malignant tumor originating from ovarian epithelial cells, is highly malignant and difficult to diagnose early, with approximately 75% of patients diagnosed at an advanced stage (stage III / IV). This tumor exhibits high heterogeneity and readily metastasizes extensively through peritoneal implantation, forming a characteristic "omental cake" lesion. Although cytoreductive surgery combined with platinum-based chemotherapy (such as carboplatin / paclitaxel) as the standard treatment regimen achieves a high initial response rate, approximately 70% of patients eventually face platinum-resistant relapse. Combined with the hematologic and neurotoxic effects of traditional chemotherapy and acquired resistance to targeted therapies, this constitutes a major bottleneck in current clinical treatment. Therefore, exploring new therapeutic targets and developing highly effective and low-toxicity treatment pathways has become an urgent need to address this challenge.

[0003] Calmodulin-dependent protein kinase II (CaMKII) families are key intracellular signaling hubs whose functions are closely related to tumorigenesis and development. The CaMKII family includes four isoforms: α, β, γ, and δ, and is a class of kinases dependent on calcium ions (Ca). 2+ The CaMKII family of protein kinases, including calcium ions and calmodulin (calmodulin), is widely distributed in various organisms. Members of this family have distinct functions. The α and β isoforms are primarily found in the nervous system and are crucial for regulating various neural functions, particularly learning and memory. The γ and δ isoforms exhibit broader tissue distribution; the δ isoform dominates cardiac function and is essential for maintaining vascular tone and regulating cardiac function. The γ isoform, most closely associated with cancer, is widely involved in regulating gene expression and cell proliferation. The entire CaMKII family can be activated by calcium ions / calmodulin and undergo autophosphorylation, thereby converting transient calcium signals into persistent physiological or pathological effects, making it a highly promising molecular target for exploring disease mechanisms.

[0004] To achieve precise intervention on specific targets such as CaMKII-γ, siRNA (small interfering RNA) technology provides a powerful tool.

[0005] siRNA is a short double-stranded RNA molecule, typically 20-25 nucleotides in length. siRNA plays a crucial role in gene silencing and is part of the RNA interference (RNAi) mechanism, which specifically inhibits the expression of target genes. siRNA binds to the RNA-induced silencing complex (RISC) within the cell, directing the complex to recognize and cleave mRNA sequences complementary to the siRNA, thereby preventing these mRNAs from being translated into proteins and downregulating the expression of specific target genes.

[0006] The discovery and development of siRNA technology has greatly advanced research in functional genomics and therapeutics. Scientists can design and synthesize specific siRNAs to target almost any gene of interest, for studying gene function, validating drug targets, and developing new treatments. For example, in cancer research, siRNAs are used to explore the function of oncogenes and assess their potential as therapeutic targets. Furthermore, siRNA therapy is emerging as a new treatment approach for targeting disease targets that are difficult to reach with traditional drugs.

[0007] Tannic acid is a natural polyphenolic compound derived from plants, composed of a glucose core and multiple gallate groups, with a molecular weight of approximately 1700 Da. Rich in phenolic hydroxyl groups, tannic acid is widely distributed in plants such as grapes, hawthorn, persimmon, raspberry, gallnut, and oak, and is used as a key component in leather tanning, iron ink production, and the fermentation and aging of red wine. Studies have confirmed that tannic acid possesses significant anti-inflammatory, antibacterial, and antiviral biological activities. At appropriate concentrations, it can be used to treat hemorrhoids, diarrhea, and other gastrointestinal diseases, as well as to assist in the treatment of severe burns, and shows potential for the prevention of neurodegenerative diseases.

[0008] In addition, thanks to the abundance of reactive functional groups in its molecules, tannic acid is also widely used as a highly efficient natural crosslinking agent in the field of biomaterials. It can significantly enhance the mechanical properties of natural or synthetic hydrogels and polymers, while simultaneously endowing the materials with inherent anti-inflammatory and antibacterial functions, thereby expanding its application prospects in biomedicine. Summary of the Invention

[0009] The purpose of this invention is to provide a CaMKII-β inhibitor and its application in the preparation of a drug for treating ovarian cancer.

[0010] To achieve the above-mentioned objectives, this invention first provides the application of CaMKII-β as a drug target in the screening and preparation of drugs for treating ovarian cancer.

[0011] Based on this, the present invention also provides the application of CaMKII-β inhibitors in the preparation of drugs for treating ovarian cancer.

[0012] Furthermore, the present invention screened and obtained a natural product, tannic acid, which can effectively inhibit the kinase activity of CaMKII-β, and therefore can be used to prepare CaMKII-β inhibitors.

[0013] Furthermore, the present invention also provides the application of this natural product tannic acid in the preparation of drugs for treating ovarian cancer.

[0014] Specifically, the medicinal mechanism of the natural product tannic acid is to inhibit the proliferation of ovarian cancer cells by inhibiting the kinase activity of CaMKII-β in ovarian cancer cells.

[0015] Based on the above technical solution, the present invention further provides a drug for treating ovarian cancer, wherein the drug contains a CaMKII-β inhibitor as the active pharmaceutical ingredient.

[0016] Furthermore, the drug for treating ovarian cancer specifically contains tannic acid, a natural product, as its active pharmaceutical ingredient.

[0017] This invention, through virtual screening combined with in vitro and in vivo efficacy verification, obtained a natural product, tannic acid, that can effectively inhibit the activity of CaMKII-β kinase. It has a significant inhibitory effect on the growth of ovarian cancer SK-OV-3 cells and OVCAR-8 cells, and also has a significant inhibitory effect on the growth of SK-OV-3 xenografts. This demonstrates that high expression of CaMKII-β can promote the growth and proliferation of ovarian cancer, and that tannic acid achieves its in vivo tumor-suppressing effect by inhibiting the kinase activity of CaMKII-β.

[0018] This invention reveals the role of CaMKII-β in ovarian cancer and provides a theoretical basis for developing novel anticancer drugs targeting CaMKII-β. Tannic acid, as a potential CaMKII-β inhibitor, has shown good tumor-suppressing effects. Attached Figure Description

[0019] Figure 1 The results are from a pan-cancer analysis of genes in the CAMKII family, showing that CAMK2B is significantly overexpressed in ovarian cancer.

[0020] Figure 2 This reflects the differences in expression of genes in the CAMKII family between healthy tissues and ovarian cancer patient tissues.

[0021] Figure 3 The results of the correlation analysis between CAMK2B expression levels and survival in ovarian cancer patients showed that CAMK2B expression levels were significantly negatively correlated with survival in ovarian cancer patients.

[0022] Figure 4 The difference in CaMKII-β expression levels between normal ovarian IOSE-80 cell lines and ovarian cancer SK-OV-3 and OVCAR-8 cell lines is shown.

[0023] Figure 5 This refers to the difference in CaMKII-β expression levels between normal ovarian tissue and ovarian cancer tissue.

[0024] Figure 6 This refers to the change in the expression level of CaMKII-β protein in the transfected ovarian cancer SK-OV-3 and OVCAR-8 cell lines compared to the control group.

[0025] Figure 7 This refers to the proliferative activity of ovarian cancer SK-OV-3 and OVCAR-8 cell lines in each transfection group compared to the control group.

[0026] Figure 8 The differences in body weight changes, final tumor size, tumor histopathological changes (HE staining), and Ki67 protein expression levels between transfected and control mice were compared.

[0027] Figure 9 Compared with healthy tissue, ovarian cancer patients showed an enrichment of related signaling pathways in their tissues.

[0028] Figure 10 This indicates an enrichment of related signaling pathways in ovarian cancer patients with high CAMK2B expression compared to those with low CAMK2B expression.

[0029] Figure 11 Compared with the control group, this study describes the changes in the expression levels of proteins related to the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in ovarian cancer SK-OV-3 and OVCAR-8 cell lines in each transfection group.

[0030] Figure 12 The structural formula of tannic acid and its IC50 inhibitory activity against CaMKII-β kinase are shown. 50 value.

[0031] Figure 13 The IC50 of tannic acid against ovarian cancer cell lines SK-OV-3 and OVCAR-8 is... 50 value.

[0032] Figure 14 Compared with the control group, the changes in the expression levels of proteins related to the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in ovarian cancer SK-OV-3 and OVCAR-8 cell lines after tannic acid treatment.

[0033] Figure 15 The differences in body weight changes, final tumor size, tumor histopathological changes (HE staining), and Ki67 protein expression levels between the treatment group and the control group were compared.

[0034] Figure 16 Compared with the control group, this refers to the changes in the expression levels of proteins related to the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in the tumors of mice in the treatment group. Implementation

[0035] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can better understand and utilize the present invention, and are not intended to limit the scope of protection of the present invention.

[0036] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0037] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0038] The applicant's previous research found that, compared with normal tissue, the expression of the gene encoding CaMKII-β, CAMK2B, was significantly increased in pathological tissues of ovarian cancer. Further survival curve analysis also revealed a significant negative correlation between CAMK2B expression levels and the survival of ovarian cancer patients. However, to date, no definitive research has confirmed the relationship between CAMK2B and ovarian cancer.

[0039] Based on the above findings, this invention utilizes siRNA as a tool to reduce the expression level of CaMKII-β in order to investigate its role in ovarian cancer. By selectively inhibiting CaMKII-β, this invention aims to observe the effects of this inhibition on ovarian cancer cell proliferation and signaling pathways, thereby revealing the role of CaMKII-β in the pathogenesis of ovarian cancer and evaluating its potential as a therapeutic target.

[0040] Therefore, the present invention first provides the application of CaMKII-β as a drug target in the screening and preparation of drugs for treating ovarian cancer.

[0041] This invention identifies CaMKII-β (calcium / calmodulin-dependent protein kinase II-β) as a potential therapeutic target for ovarian cancer through bioinformatics analysis.

[0042] To further explore the role of CaMKII-β in the specific pathogenesis of ovarian cancer, this invention conducted a series of experimental studies. First, using siRNA technology, the expression level of CaMKII-β was successfully and significantly downregulated in two ovarian cancer cell lines, SK-OV-3 and OVCAR-8. Further cell proliferation assays showed that this downregulation significantly inhibited the in vitro proliferation ability of these two cell lines, indicating that CaMKII-β may promote the growth of ovarian cancer cells. In vitro xenografts also showed the same experimental results. Subsequently, through bioinformatics prediction and Western blot analysis, it was further discovered that the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway may be a key pathway by which CaMKII-β regulates ovarian cancer progression. Specifically, after downregulating CaMKII-β expression, the expression levels of related proteins in this pathway changed significantly, suggesting that CaMKII-β may regulate ovarian cancer development by affecting the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway.

[0043] Secondly, the above experimental results suggest that CaMKII-β inhibitors can be used as drugs for the treatment of ovarian cancer. Based on this, this invention not only reveals for the first time the crucial role of CaMKII-β in the pathogenesis of ovarian cancer, but also provides a clear theoretical basis and experimental foundation for the development of novel anti-ovarian cancer drugs targeting CaMKII-β.

[0044] More importantly, this invention, through virtual screening combined with experimental verification, screened out a natural product from the natural product library that can effectively inhibit the activity of CaMKII-β kinase—tannic acid.

[0045] Therefore, the present invention further provides the application of the natural product tannic acid in the preparation of CaMKII-β inhibitors.

[0046] More specifically, this invention provides the use of the natural product tannic acid in the preparation of a medicament for treating ovarian cancer.

[0047] Clearly, the new research findings of this invention regarding the use of tannic acid in the treatment of ovarian cancer are significantly different from the traditional uses of tannic acid.

[0048] This invention demonstrates that tannic acid significantly inhibits the growth of ovarian cancer SK-OV-3 and OVCAR-8 cells, and also significantly inhibits the growth of SK-OV-3 xenografts. Tumor tissue treated with tannic acid was significantly smaller than that in the control group, and the changes in proteins related to the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in the tumor tissue were consistent with in vitro experiments. This indicates that tannic acid inhibits the proliferation of ovarian cancer cells by downregulating the expression of CaMKII-β in ovarian cancer cells, thereby achieving its in vivo tumor-suppressing effect.

[0049] This new discovery suggests that tannic acid may have a therapeutic effect on ovarian cancer by inhibiting CaMKII-β, which is completely different from the traditional application of tannic acid. It expands the application of tannic acid from a traditional material reagent to a potential therapeutic approach, especially for specific types of cancer such as ovarian cancer. This research not only provides new insights into the biological functions of tannic acid but also paves the way for the development of new anticancer drugs.

[0050] Furthermore, based on the above experimental studies, the present invention provides a drug for treating ovarian cancer, wherein the drug contains the above-mentioned CaMKII-β inhibitor, which inhibits the proliferation of ovarian cancer cells by downregulating the expression of CaMKII-β in ovarian cancer cells, as the active pharmaceutical ingredient.

[0051] Furthermore, the active pharmaceutical ingredient contained in the medicament for treating ovarian cancer provided by the present invention is preferably tannic acid, a natural product.

[0052] This invention demonstrates through experiments that the expression of CaMKII-β can promote the growth and proliferation of ovarian tumors, while tannic acid exerts its in vivo tumor-suppressing effect by inhibiting CaMKII-β. Similar to the results of siRNA treatment, both methods inhibit the proliferation of ovarian cancer cells by regulating protein expression in the DNA transcriptional regulatory signaling pathway mediated by the RAS-PI3K-AKT-NF-κB axis in cells. Example

[0053] Example 1

[0054] This embodiment uses bioinformatics to analyze the expression of the CAMKII family in healthy tissue samples and ovarian cancer patient tissue samples, and uses si-RNA to target and downregulate the expression of CaMKII-β.

[0055] I. Experimental Materials

[0056] The normal human ovarian epithelial IOSE80 cell line, the human ovarian cancer SK-OV-3 cell line, and the OVCAR-8 cell line were all purchased from Yimo Biotechnology Co., Ltd.

[0057] A si-RNA specifically targeting CAMK2B, synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., is used to knock down the expression level of CaMKII-β. Its specific gene sequence is as follows: Chain of Justice: 5'-CCAGCUCUACGAGGAUAUUTT-3'; Antonym chain: 3'-AAUAUCCUCGUAGAGCUGGTT-5'.

[0058] SPF-grade V-NSG mice, 4 weeks old and female, were purchased from Beijing Beiyou Biotechnology Co., Ltd. All mice were housed in an environment with a temperature of (22±2)℃, humidity of (60±5)℃, and alternating light and dark conditions for 12 hours.

[0059] II. Experimental Methods and Results

[0060] 1. Bioinformatics analysis of differences in expression levels of the CAMKII family in healthy tissue samples and tissue samples from different cancer patients.

[0061] First, a standardized pan-cancer dataset was downloaded from the UCSC database, and expression levels of CAMKII family genes in all samples were extracted. To further standardize the data, sample sources were screened, and the expression value of each gene was calculated using log2(...). x +0.001) conversion. Finally, cancer types with fewer than 3 samples within a single cancer type were excluded to ensure statistical stability of the analysis, ultimately yielding CAMKII family gene expression data covering 34 cancer types.

[0062] Finally passed Figure 1 The pan-cancer analysis results showed that CaMK2B was significantly overexpressed in ovarian cancer.

[0063] 2. Bioinformatics analysis of the expression levels of the CAMKII family in healthy tissue samples and ovarian cancer patient tissue samples.

[0064] Statistical analysis was performed using R (version 4.3.2), and microarray data from the Gene Expression Comprehensive Database (GEO) for ovarian cancer were analyzed, including data from the control (CON) and tumor (Tumor) groups. Ovarian cancer transcriptome sequencing data were obtained from the Genotype-Tissue Expression Project (GTEx) and The Cancer Genome Atlas (TCGA), including 88 normal samples (all from the GTEx dataset) and 379 tumor samples (all from the TCGA dataset). The analyzed data were in RNA-seq format.

[0065] Initially, the "normalized intermediate array" function in the "limma" package was used to standardize the sequencing depth across samples, reducing technical biases that could affect subsequent analyses. Subsequently, the four members of the CAMKII family—CAMK2A, CAMK2B, CAMK2D, and CAMK2G—were examined, and the "characteristic state map" function in the "scprep" package was used to assess the differential expression levels of these genes in different samples.

[0066] Ultimately, through Figure 2 Differential gene expression analysis showed that CAMK2B expression was significantly upregulated in ovarian cancer patient tissue samples compared with healthy tissue samples.

[0067] 3. Bioinformatics analysis of the correlation between CAMK2B expression levels and survival in ovarian cancer patients

[0068] The ovarian cancer clinical dataset from the TCGA database was used for analysis. The optimal cutoff value of the RiskScore was calculated using the R package maxstat. Based on this, patients were divided into high and low groups. The prognostic differences between the two groups were further analyzed using the survfit function of the "survival" package. The logrank test method was used to evaluate the significance of the prognostic differences between the different groups.

[0069] Survival analysis showed that ovarian cancer patients with high CaMK2B expression had significantly shorter overall survival compared to those with low CaMK2B expression. Figure 3 As shown in the figure, the expression level of CAMK2B is significantly negatively correlated with the survival of ovarian cancer patients, and CaMKII-β protein is a potential target for the treatment of ovarian cancer.

[0070] 4. Cell culture and Western blot analysis of differences in CaMKII-β expression levels

[0071] Normal ovarian epithelial cell line 0IOSE80 and ovarian cancer cell line SK-OV-3 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody, while ovarian cancer cell line OVCAR-8 was cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody.

[0072] Normal ovarian cells and ovarian cancer cells in the logarithmic growth phase were harvested, washed three times with PBS buffer, and lysed on ice with RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein content of the cell samples was detected using a BCA kit.

[0073] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; proteins were transferred to PVDF membranes by wet transfer method and blocked with protein-free rapid blocking solution.

[0074] The PVDF membrane was reacted overnight at 4°C with CaMKII-β primary antibody dilution buffer and GAPDH primary antibody dilution buffer, followed by three TBST washes for 10 min each. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0075] Figure 4 The results showed that, compared with normal ovarian cells IOSE-80, ovarian cancer cells SK-OV-3 and OVCAR-8 showed high expression of CaMKII-β protein.

[0076] 5. Immunohistochemical staining of normal ovarian tissue and ovarian cancer tissue

[0077] Paraffin-embedded samples of pathologically confirmed normal ovarian tissue and ovarian cancer tissue were collected and sectioned (4-5 μm) onto processed glass slides. After dewaxing with xylene and hydration with graded ethanol, the sections underwent heat-induced antigen retrieval in citrate buffer. Endogenous peroxidase (3% H2O2) was blocked and serum was applied. Rabbit anti-human CAMK2B primary antibody working solution (incubated overnight at 4°C) and PBS negative control were added to the sections respectively. The next day, the sections were washed with PBS and incubated with HRP-labeled polymeric secondary antibody (30-60 min at room temperature). After washing, DAB chromogenic reaction was performed, with the degree of chromogenic reaction controlled under a microscope and distilled water used to stop the reaction. Finally, the cell nuclei were counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The expression localization (cytoplasm / nucleus) and intensity of CAMK2B protein in normal and cancerous tissues were observed and compared under an optical microscope.

[0078] Figure 5 In ovarian cancer tissue, the CaMKII-β protein is also highly expressed compared to normal ovarian tissue.

[0079] 6. Cell transfection

[0080] SK-OV-3 cells and OVCAR-8 cells were divided into siRNA transfection group and blank control group, respectively. Transfection was performed in six-well cell culture plates when the cell binding rate was about 60-70%.

[0081] SK-OV-3 cells were first plated using OPTI-reduced serum medium. After 0.5-3 hours, 200 nM si-RNA and Lipofectamine 3000 transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the Lipofectamine 3000 instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0082] First, OVCAR-8 cells were plated using standard culture medium. Then, 100 nM si-RNA and Lipofectamine 3000 transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the Lipofectamine 3000 instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0083] 7. Western blot analysis of CaMKII-β expression levels

[0084] Cells were harvested 72 h after transfection, washed three times with PBS buffer, lysed on ice with RIPA lysis buffer, and the supernatant was collected by centrifugation. The protein content of the cell samples was detected using a BCA kit.

[0085] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; proteins were transferred to PVDF membranes by wet transfer method and blocked with protein-free rapid blocking solution.

[0086] The PVDF membrane was reacted overnight at 4°C with CaMKII-β primary antibody dilution buffer and GAPDH primary antibody dilution buffer, followed by three TBST washes for 10 min each. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0087] The expression levels of CaMKII-β protein in each transfection group were compared. Figure 6 In the study, compared with the blank control group, the expression level of CaMKII-β protein was significantly downregulated in the si-RNA transfection groups of SK-OV-3 cells and OVCAR-8 cells transfected with si-RNA.

[0088] 8. Cell proliferation activity detection

[0089] CellTiter-Glo ® The proliferation activity of transfected cells was detected by luminescence assay, and the assays were performed within 1-6 days after transfection.

[0090] Cells were seeded in 96-well plates, with 5000 cells and 100 μL of culture medium added to each well. After adding 100 μL of CellTiter, the plates were shaken for 10 min to allow for full reaction. The luminescence value of each well was measured using a microplate reader. Each group was repeated 3 times.

[0091] Relative proliferation activity (%) = luminescence value of transfected group / luminescence value of blank control group × 100%.

[0092] Figure 7 The results showed that, compared with the blank control group, the proliferation of SK-OV-3 cells and OVCAR-8 cells transfected with si-RNA was significantly slower.

[0093] 9. Animal Model Establishment and Processing

[0094] Transfected ovarian cancer SK-OV-3 cells and normal ovarian cancer SK-OV-3 cells were collected and made into single-cell suspensions with PBS. The viable cell count was determined by trypan blue staining, and the cell density was adjusted to 5 × 10⁶ cells / year. 7 / mL.

[0095] 0.2 mL of cell suspension was inoculated into the right abdomen of V-NSG mice, and their body weight was measured before the operation. The mice were observed daily after inoculation. Once a visible xenograft appeared, the maximum longitudinal diameter (a) and maximum transverse diameter (b) of the xenograft were measured using calipers. The tumor volume was calculated as 0.5 × a × b. 2 .

[0096] Thirty days later, mice were euthanized by cervical dislocation, photographed, and the morphology and size of the tumor mass were observed and recorded. The mass was dissected, weighed, and stored at -80℃ for later use. Subsequently, the largest cross-sectional tissue blocks from the transfected group and the normal group were taken and immediately fixed in 4% paraformaldehyde (PFA) for 24 hours. After graded ethanol dehydration, xylene clearing, and paraffin embedding, 5μm thick serial sections were prepared. Hematoxylin-eosin (HE) staining and Ki67 immunohistochemical staining were performed on the sections. Two pathologists evaluated the sections without knowing the grouping: HE staining was used to observe pathological features such as tissue necrosis, inflammation, and vacuolar degeneration; the Ki67 positivity rate was obtained by randomly selecting five 400× fields and calculating the percentage of brown-nucleated positive cells.

[0097] After a period of experimental observation, compared with the control group, the tumor volume of mice in the transfection treatment group was significantly smaller, but the body weight of mice in both groups did not change significantly. Further immunohistochemical staining results showed that HE staining revealed increased tumor structural destruction in the transfection group, while the proportion of Ki67-positive cells was significantly reduced, indicating that transfection treatment can effectively inhibit tumor proliferation and promote cancer cell death. Figure 8 As shown.

[0098] Cancer is a highly heterogeneous disease; even within the same type of cancer, tumor cells can exhibit significant differences between patients. Using two or more cell lines can help validate the consistency of the effects of drugs, gene editing, or other interventions. If the same results are observed in multiple cell lines, the findings are considered more reliable.

[0099] To verify the reliability of the experimental results, the ovarian cancer cell lines SK-OV-3 and OVCAR-8 were selected for this study. The results showed that siRNA significantly downregulated the expression level of CaMKII-β in both ovarian cancer cell lines SK-OV-3 and OVCAR-8. Further cell proliferation assays indicated that downregulating CaMKII-β expression with siRNA significantly inhibited the in vitro proliferation of both SK-OV-3 and OVCAR-8 cell lines. The same conclusion was also obtained using an in vivo transplantation model. These results suggest that CaMKII-β may be an important molecular target regulating the proliferation of ovarian cancer cells.

[0100] Example 2

[0101] This embodiment utilizes bioinformatics analysis to enrich signaling pathways in tissue samples from ovarian cancer patients. By using siRNA to target and downregulate the expression of CaMKII-β in ovarian cancer cell lines, changes in signaling pathway-related proteins were detected.

[0102] 1. Bioinformatics analysis of signaling pathways significantly enriched in ovarian cancer patient tissues

[0103] Statistical analysis was performed using R4.3.2, and the ovarian cancer dataset from the Gene Expression Comprehensive Database (GEO) was examined. An adjusted p-value less than 0.05 was used to control for false positives in multiple comparisons. A log-fold change (logFC) greater than 2 indicated that the gene expression level in the tumor sample was at least upregulated by 2-fold (or downregulated by 0.5-fold) compared to the normal sample.

[0104] Based on the above stringent criteria, a total of 632 genes were identified as significantly upregulated in tumor samples. To further explore the functions of these differentially expressed genes, KEGG pathway enrichment analysis was performed using the R package "clusterProfiler". The differentially expressed genes were mapped to the KEGG pathway database, and the enrichment significance of each pathway was calculated. KEGG entries with p-values ​​< 0.05 were selected as significantly enriched pathways.

[0105] The identified significantly enriched pathways were exported as Excel files for subsequent analysis and visualization. A dotplot (or bubble plot) was created using the dotplot function to visually represent the results of the KEGG enrichment analysis. The plot uses the size and color of the dots to represent the number of genes in the pathway and the enrichment significance (-log10(p-value)), respectively, thus helping to identify signaling pathways closely related to ovarian cancer.

[0106] Through the above bioinformatics analysis, it was found that the DNA transcriptional regulatory signaling pathway mediated by the RAS-PI3K-AKT-NF-κB axis was significantly enriched in healthy tissues and ovarian cancer tissues, as well as in ovarian cancer tissues with high and low CAMK2B expression. Specific results are as follows: Figure 9 , 10 As shown.

[0107] 2. Cell culture and transfection; Western blotting was used to detect changes in the expression levels of related proteins in the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway.

[0108] Ovarian cancer SK-OV-3 cell line was cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody according to standard methods, and ovarian cancer OVCAR-8 cell line was cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody according to standard methods.

[0109] SK-OV-3 cells and OVCAR-8 cells were divided into siRNA transfection group and blank control group, respectively. Transfection was performed in six-well cell culture plates when the cell binding rate was about 60-70%.

[0110] SK-OV-3 cells were first plated using OPTI-reduced serum medium. After 0.5-3 hours, 200 nM si-RNA and Lipofectamine 3000 transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the Lipofectamine 3000 instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0111] First, OVCAR-8 cells were plated using standard culture medium. Then, 200 nM si-RNA and Lipofectamine 3000 transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the Lipofectamine 3000 instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0112] Cells were harvested 72 h after transfection, washed three times with PBS buffer, and the four groups of cells were lysed on ice with RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein content of the cell samples was measured using a BCA kit.

[0113] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; the protein was transferred to a PVDF membrane by wet transfer method and blocked with a protein-free rapid blocking solution.

[0114] The PVDF membrane was reacted overnight at 4°C with dilution buffers for primary antibodies RAS, PI3K, AKT, NF-κB, p-PI3K, p-AKT, p-NF-κB, and GAPDH, followed by three TBST washes for 10 min each. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0115] Figure 11 In the study, compared with the blank control group, the expression level of CaMKII-β protein in si-RNA transfected cells of SK-OV-3 cells and OVCAR-8 cells was significantly downregulated. The expression levels of PI3K, AKT and NF-κB proteins in the DNA transcriptional regulatory signaling pathway mediated by the RAS-PI3K-AKT-NF-κB axis did not change significantly, while the expression levels of RAS, p-PI3K (phosphorylated PI3K), p-AKT (phosphorylated AKT) and p-NF-κB (phosphorylated NF-κB) proteins were downregulated.

[0116] The above results indicate that both the CAMK2B and RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathways are significantly overexpressed in ovarian cancer patient tissues. Using siRNA technology, the expression level of CaMKII-β in ovarian cancer cell lines (including SK-OV-3 cells and OVCAR-8 cells) can be effectively downregulated, while significantly reducing the phosphorylation level of related proteins in the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway.

[0117] This finding suggests that CAMK2B may play an important role in regulating the activity of DNA transcriptional regulatory signaling pathways mediated by the RAS-PI3K-AKT-NF-κB axis. Its mechanism may involve directly or indirectly affecting the phosphorylation status of these proteins, thereby participating in the occurrence and development of ovarian cancer.

[0118] Example 3

[0119] This embodiment utilizes CaMKII-β as a drug target to screen natural products that have the effect of targeting and inhibiting CaMKII-β.

[0120] I. Experimental Materials

[0121] The ADP-Glo™ kinase assay kit was purchased from Promega, Inc., USA.

[0122] Natural products library, purchased from MCE.

[0123] Tannic acid (CAS: 1401-55-4), purchased from MCE.

[0124] II. Experimental Methods and Results

[0125] 1. Screening of CaMKII-β inhibitors

[0126] First, a virtual screening system was constructed, and the structure of the CaMKII-β (PDB ID: 3BHH) protein was flexibly molecularly docked with 3704 natural products in a natural product library using virtual screening software. Then, the ADP-Glo™ kinase assay kit was used to verify the impact on CaMKII-β kinase activity of the top ten screened natural products, in order to screen for potential inhibitors that can effectively inhibit CaMKII-β.

[0127] This experiment used autocamtide-3 as the peptide substrate for CaMKII-β. When CaMKII-β is activated, it catalyzes the phosphorylation of the substrate and converts ATP into ADP. The ADP-Glo™ kinase assay kit indirectly reflects kinase activity by detecting the level of ADP generated in the reaction system. Its working principle is as follows: first, chemical reagents are used to convert ADP in the system into a stable chemical form, thereby preventing further metabolism or degradation of ADP; then, a specific enzyme mixture is added to quench unconsumed ATP and convert the remaining ADP into a product that generates a luminescent signal; finally, the luminescence intensity is detected to achieve precise quantification of kinase activity.

[0128] The total volume of the reaction system was 10 μL. The reaction buffer consisted of: 25 mmol / L Tris-HCl (pH 7.5), 0.2 mmol / L calcium chloride, 10 mmol / L magnesium chloride, 0.035 mmol / L ATP, 200 μmol / L autocamtide-3, 30 nmol / L calmodulin (CaM), and 0.1 mg / mL bovine serum albumin (BSA). First, add 1 μL of the drug to be tested to each well, followed by 2 μL (1.5 ng / μL) of human CaMKII-β recombinant protein, and incubate for 10 min to ensure sufficient interaction between the drug and protein. Then, add 7 μL of reaction buffer and allow the kinase reaction to proceed at room temperature (25-27℃) for 30 min. Finally, process the reaction system according to the ADP-Glo™ kinase assay kit instructions to stop the kinase reaction and remove unreacted ATP. ADP is converted to ATP through an enzymatic reaction, and the luminescence reaction between ATP and luciferin is catalyzed by luciferase to generate a light signal proportional to the amount of ADP, thereby reflecting the phosphotransferase activity of CaMKII-β.

[0129] All procedures were strictly performed in accordance with the instructions for the ADP-Glo™ kinase assay kit. Finally, the luminescence value of each well was detected using a microplate reader, and the potential inhibitory ability of the compound was evaluated by quantitative analysis of its inhibitory effect on protein kinase activity.

[0130] Drug inhibition rate (%) = (Chemical emission value of drug group - Clinical emission value of negative control group) / (Chemical emission value of positive control group - Clinical emission value of negative control group) × 100%

[0131] Based on the aforementioned virtual screening system, a large-scale screening was conducted on 3704 natural products included in the MCE natural product library, initially identifying the top 10 compounds with good inhibitory effects on CaMKII-β. Through comprehensive analysis, tannic acid, the natural product with the best inhibitory effect, was ultimately determined as the inhibitor of CaMKII-β. Figure 12 The structure of tannic acid and its IC50 for CaMKII-β protein are given. 50 value.

[0132] 2. Cell proliferation activity test results

[0133] After confirming that the drug exhibits inhibitory and selective activity against CaMKII-β protein, the next step is to evaluate its effectiveness in inhibiting cell activity. For this purpose, a sample size of 5.0 × 10⁻⁶ was selected. 3Ovarian cancer cells (SK-OV-3 or OVCAR-8 cell lines) were seeded into 96-well plates and treated with a series of drug concentration gradients (1 nM to 100 μM). The total volume per well was set at 100 μL, and the cells were then cultured for 48 h. After culture, cell viability in each group was detected using the CellTiter-Glo® luminescence assay.

[0134] Add 100 μL of CellTiter-Glo® reagent to each well, then shake the 96-well plate on a shaker for 10 min to ensure sufficient reaction between the reagent and cells. Measure the luminescence value of cells at different drug concentrations using a microplate reader. Each experiment was repeated three times to improve data reliability. The IC50 value of the drug against ovarian cancer cells (SK-OV-3 and OVCAR-8 cell lines) was calculated based on dose-response curve fitting. 50 The value reflects the overall inhibitory effect of the drug at the cellular level.

[0135] Compared with the blank control group, cell proliferation was reduced in the tannic acid-treated group in a concentration-dependent manner. Based on the effects of different concentrations of tannic acid on cells, the IC50 of the cells was obtained. 50 Value, result as Figure 13 As shown.

[0136] The above results demonstrate that tannic acid significantly inhibits the activity of CaMKII-β protein at a low concentration of approximately 217 nM, exhibiting excellent inhibitory effect and high selectivity against the target protein. Simultaneously, at the cellular level, tannic acid significantly inhibited the proliferation of ovarian cancer cell lines (SK-OV-3 and OVCAR-8) at concentrations of 1.448 μM and 1.399 μM, showing a clear concentration-dependent effect, fully demonstrating its excellent inhibitory ability against tumor cells. Therefore, tannic acid not only exhibits highly efficient inhibitory effects on CaMKII-β protein but also demonstrates good anti-tumor activity in cell experiments, providing strong evidence for its potential role as a CaMKII-β inhibitor.

[0137] Example 4

[0138] This embodiment uses cell experiments to detect changes in the expression levels of related proteins in the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in cells after tannic acid administration, verifying the role of tannic acid as a natural product that targets and inhibits CaMKII-β in ovarian cancer.

[0139] I. Experimental Materials

[0140] p-CaMKII-β primary antibody was purchased from Cell Signaling Technology.

[0141] The HRP-labeled secondary antibody was purchased from Abcam.

[0142] Other experimental materials are the same as in Examples 1 and 2.

[0143] II. Experimental Methods and Results

[0144] After learning that tannic acid has an effect on the IC50 of ovarian cancer cells 50 After the value was obtained, the cells were cultured with the corresponding concentration of tannic acid for 48 h. Then the cells were harvested, washed three times with PBS buffer, and the cells in each group were lysed on ice with RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein content of the cell samples was measured using a BCA kit.

[0145] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; proteins were transferred to PVDF membranes by wet transfer method and blocked with protein-free rapid blocking solution.

[0146] The PVDF membrane was reacted overnight at 4°C with dilution buffers of primary antibodies CaMKII-β, p-CaMKII-β, RAS, PI3K, AKT, NF-κB, p-PI3K, p-AKT, p-NF-κB, and GAPDH primary antibody. The membrane was washed three times with TBST for 10 min each time. It was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each time. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0147] Figure 14 The changes in the expression levels of proteins related to the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in each group after drug treatment are presented.

[0148] Compared with the blank control group, the expression level of CaMKII-β protein in cells of the tannic acid-treated group did not change significantly, while the expression level of p-CaMKII-β (phosphorylated CaMKII-β) protein decreased. In the DNA transcriptional regulatory signaling pathway mediated by the RAS-PI3K-AKT-NF-κB axis, the expression levels of PI3K, AKT and NF-κB proteins did not change significantly, while the expression levels of RAS, p-PI3K, p-AKT and p-NF-κB proteins were downregulated.

[0149] The above results indicate that drug treatment may affect the DNA transcriptional regulatory signaling pathway mediated by the RAS-PI3K-AKT-NF-κB axis by inhibiting the kinase activity of CAMK2B. This result is consistent with the results observed in siRNA experiments, further verifying the key role of CAMK2B in this signaling pathway.

[0150] Example 5

[0151] This embodiment uses in vivo experiments in tumor-bearing mice to verify the in vivo tumor-suppressing effect of tannic acid as a natural product that targets and inhibits CaMKII-β on ovarian cancer.

[0152] I. Experimental Materials

[0153] SPF-grade V-NSG mice, 4-6 weeks old and female, were purchased from Beijing Beiyou Biotechnology Co., Ltd. All mice were housed in an environment with a temperature of (22±2)℃, humidity of (60±5)℃, and 12 hours of alternating light and dark conditions.

[0154] II. Experimental Methods and Results

[0155] 1. Establishment, grouping, and administration of animal models

[0156] SK-OV-3 ovarian cancer cells in the logarithmic growth phase were collected, and a single-cell suspension was prepared with PBS. The viable cell count was determined using trypan blue staining, and the cell density was adjusted to 5 × 10⁶ cells / year. 7 / ml.

[0157] 0.2 ml of cell suspension was inoculated into the right abdomen of V-NSG mice, and their body weight was measured before the operation. The mice were observed daily after inoculation. Once a visible xenograft appeared, the xenograft was measured with calipers. The xenograft was considered complete when its volume reached 100 mm². 3 Around 1000 mice, tumor-bearing mice were randomly divided into a control group and a tannic acid group based on the principle of balanced tumor volume and body weight.

[0158] The tannic acid group received a dose of 50 mg / kg via intraperitoneal injection, three times a week. The blank control group received an equal volume of normal saline.

[0159] One day after the drug treatment ended, the mice were euthanized by cervical dislocation, photographed, and the shape and size of the mass were observed and recorded. The mass was dissected, weighed, and stored at -80℃ for later use.

[0160] 2. Tumor inhibition rate measurement

[0161] The maximum longitudinal diameter (a) and maximum transverse diameter (b) of the tumor were measured using vernier calipers. The tumor volume was calculated as 0.5 × a × b. 2 .

[0162] The in vivo tumor inhibition rate of the compound was calculated using the following formula:

[0163] Tumor inhibition rate (%) = (Tumor volume of control group - Tumor volume of treatment group) / Tumor volume of control group × 100%.

[0164] After a period of experimental observation, such as Figure 15As shown in the figure, compared with the control group, the tumor volume of mice in the tannic acid treatment group was significantly smaller, but the body weight of mice in both groups did not change significantly. This result indicates that tannic acid has a significant antitumor effect in vivo and does not produce obvious systemic toxicity in mice.

[0165] 3. Tumor histopathology and immunohistochemical analysis

[0166] At the end of the experiment, mice in each group were sacrificed, and the transplanted tumor tissue was completely dissected. The largest cross-sectional tissue block of the tumor was taken, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and then prepared into sections for hematoxylin-eosin (HE) staining and Ki67 immunohistochemical staining. All sections were evaluated by two pathologists in a blinded manner. HE staining was used to observe pathological features such as necrosis, inflammation, and vacuolar degeneration. The Ki67 positivity rate was obtained by counting the percentage of brown-nucleated positive cells in five randomly selected 400× fields.

[0167] Figure 15 The results showed that, compared with the blank control group, the tumor tissue in the drug-treated group showed intact tissue structure and no obvious pathological damage when stained with HE; at the same time, the proportion of Ki67 positive cells was significantly reduced, indicating that the drug effectively inhibited the proliferation of tumor cells without causing obvious toxic reactions.

[0168] 4. Western blot analysis of changes in the expression levels of proteins in the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway in mouse tumors.

[0169] Tumor tissue was obtained, ground, and total protein was extracted from the tumor tissue specimen. The tissue was lysed on ice for 30 min with RIPA lysis buffer, and the supernatant was collected by centrifugation. The protein content of the tumor tissue specimen was measured using a BCA kit.

[0170] Using GAPDH as an internal control, tissue samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; the protein was transferred to a PVDF membrane by wet transfer method and blocked with a protein-free rapid blocking solution.

[0171] The PVDF membrane was reacted overnight at 4°C with dilution buffers of primary antibodies CaMKII-β, p-CaMKII-β, RAS, PI3K, AKT, NF-κB, p-PI3K, p-AKT, p-NF-κB, and GAPDH primary antibody. The membrane was washed three times with TBST for 10 min each time. It was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each time. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0172] Compared with the control group, the expression level of CaMKII-β protein in tumors of mice treated with tannic acid did not change significantly, while the expression level of p-CaMKII-β protein decreased. In the RAS-PI3K-AKT-NF-κB axis-mediated DNA transcriptional regulatory signaling pathway, the expression levels of PI3K, AKT, and NF-κB proteins did not change significantly, while the expression levels of RAS, p-PI3K (phosphorylated PI3K), p-AKT (phosphorylated AKT), and p-NF-κB (phosphorylated NF-κB) proteins were downregulated. The results are as follows: Figure 16 As shown.

[0173] The above results indicate that tannic acid has a significant inhibitory effect on the growth of SK-OV-3 ovarian cancer xenografts. The tumor tissue treated with tannic acid was significantly smaller than that in the control group, indicating that CaMKII-β expression can promote the growth and proliferation of ovarian cancer, and that tannic acid achieves its tumor-suppressing effect in vivo by inhibiting CaMKII-β.

[0174] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of CaMKII-β as a drug target in screening and preparing drugs for the treatment of ovarian cancer.

2. Application of CaMKII-β inhibitors in the preparation of drugs for treating ovarian cancer.

3. Application of natural product tannic acid in the preparation of CaMKII-β inhibitors.

4. Application of natural product tannic acid in the preparation of drugs for treating ovarian cancer.

5. The application according to claim 4, characterized in that: The natural product tannic acid inhibits the proliferation of ovarian cancer cells by downregulating the expression of CaMKII-β.

6. A medicament for treating ovarian cancer, wherein the medicament contains the inhibitor of claim 2.

7. A medicament for treating ovarian cancer, wherein the medicament contains tannic acid, a natural product as described in claim 3.

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