Application of daphnetin as inhibitor in preparation of medicine for treating triple negative breast cancer

Abstract

The invention relates to the technical field of biological medicines, in particular to application of daphnetin as an inhibitor in preparation of a medicine for treating triple negative breast cancer. According to the application disclosed by the invention, technologies such as network pharmacology and high-throughput sequencing are comprehensively applied, the tumor suppression effect of the daphnetin on the triple-negative breast cancer and the possible molecular mechanism of the daphnetin are verified through in-vitro and in-vivo experiments from the multi-way, multi-channel and multi-site angles, and a theoretical basis is laid for clinical application of the daphnetin in the triple-negative breast cancer. Meanwhile, compared with a traditional chemotherapeutic drug, daphnetin possibly has unique multi-target and multi-path effect advantages and is worthy of further research and development of potential treatment objects, and the implementation of the project lays a foundation for development of novel efficient and low-toxicity antitumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of daphnetin as an inhibitor in the preparation of a drug for treating triple-negative breast cancer. Background Art

[0002] Currently, triple-negative breast cancer is the main cause of survival problems for patients due to its high malignancy, limited treatment options, and high recurrence and metastasis. Therefore, actively exploring new drugs for triple-negative breast cancer and exploring their functions and mechanisms has always been a hot topic in the field of breast cancer research.

[0003] Although chemotherapy is one of the most widely studied anticancer treatments, its effectiveness and safety remain major concerns due to its toxicity and other serious side effects. Furthermore, multidrug resistance is an even greater challenge. Medicinal herbs are a major source of new drugs, with over half of new chemicals derived directly or indirectly from natural products. In recent years, several natural products isolated from traditional Chinese medicines have been found to inhibit proliferation, induce apoptosis, inhibit angiogenesis, delay metastasis, and enhance chemotherapy, demonstrating anticancer potential both in vitro and in vivo. Daphnetin, also known as 7,8-dihydroxycoumarin, is a natural coumarin widely distributed in the genus Daphne. It exhibits multiple biological activities, including antitumor, analgesic, anti-inflammatory, antioxidant, anti-pathogenic and anti-parasitic, and immunomodulatory properties. It is also highly safe, has low toxicity, and is non-irritating and non-sensitizing. It is a first-of-its-kind natural drug monomer discovered in my country. In recent years, it has demonstrated excellent cytotoxic potential against various malignancies, including ovarian, liver, pancreatic, and breast cancer. Current research shows that it may exert its anti-cancer effects in various ways, such as inhibiting tumor growth and development by inducing cell apoptosis, reducing inflammation and oxidative stress, and inhibiting cancer cell growth. However, the specific mechanism of action of daphnetin on triple-negative breast cancer is still not fully understood. Therefore, exploring the role and mechanism of action of daphnetin in triple-negative breast cancer may become a new therapy for triple-negative breast cancer and provide a scientific basis and guidance for the clinical use of daphnetin.

[0004] With the rise of network pharmacology, the occurrence and development of diseases is explained from the perspective of systems biology and biological network balance, and the interaction between drugs and the body is understood from the perspective of improving or restoring network balance. This improves the therapeutic effect of drugs and reduces toxic side effects. This is an important way to develop innovative drugs. Due to its holistic and systematic characteristics, which are consistent with the holistic view and syndrome differentiation and treatment principles of traditional Chinese medicine, it has been widely used in the research of traditional Chinese medicine. As a new research method, network pharmacology can explain the mechanism of action of drugs in many aspects through bioinformatics, computer technology and other means. Summary of the Invention

[0005] The present invention provides the use of daphnetin as an inhibitor in the preparation of a drug for treating triple-negative breast cancer. Using network pharmacology methods, the potential molecular mechanism of daphnetin in triple-negative breast cancer is explored, which is expected to provide a theoretical basis for the treatment of triple-negative breast cancer with daphnetin.

[0006] The technical solutions of the present invention are as follows: Use of daphnetin as an inhibitor in the preparation of drugs for treating triple-negative breast cancer.

[0007] The daphnetin acts on MAPK8, a key target of triple-negative breast cancer, to regulate the MAPK signaling pathway.

[0008] The beneficial effects of the present invention are as follows: This study utilizes network pharmacology, high-throughput sequencing, and other technologies to validate the tumor-suppressing effects of daphnetin on triple-negative breast cancer and its potential molecular mechanisms through in vitro and in vivo experiments from a multi-pathway, multi-channel, and multi-site perspective. This study lays a theoretical foundation for the clinical application of daphnetin in triple-negative breast cancer. Furthermore, compared to traditional chemotherapy drugs, daphnetin may possess unique multi-target and multi-pathway advantages, making it worthy of further research and development as a potential therapeutic target. The implementation of this project will lay the foundation for the development of new, highly effective, and low-toxic anti-tumor drugs.

[0009] (1) Explore the targets of daphnetin on triple-negative breast cancer and its possible molecular mechanisms through network pharmacology, and understand the mechanism of action of daphnetin from a holistic perspective of multiple pathways, multiple channels, and multiple sites.

[0010] (2) Based on preliminary screening of network pharmacology and combined with the results of RNA transcriptome sequencing, daphnetin may have an inhibitory effect on triple-negative breast cancer by regulating the MAPK signaling pathway through the key target MAPK8. This indicates that daphnetin can interfere with the key biological processes of triple-negative breast cancer, thereby inhibiting its growth and spread, and is expected to become an effective means of treating triple-negative breast cancer.

[0011] (3) Daphneline may affect the progression of triple-negative breast cancer through multiple targets and pathways, and has some unique advantages, suggesting that Daphneline may have a different mechanism of action or better drug properties than traditional chemotherapy drugs. The implementation of this project will lay the foundation for the development of new and highly effective anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of the in vitro functional study of the present invention's daphnetin on the inhibition of triple-negative breast cancer.

[0013] Figure 2 Schematic diagram of the in vivo functional study of the present invention's daphnetin on the inhibition of triple-negative breast cancer.

[0014] Figure 3 Schematic diagram of obtaining the potential target of daphnetin in triple-negative breast cancer of the present invention: Figure 3 A Schematic diagram of the 3D structure of daphnetin; Figure 3 B. Schematic diagram of potential targets of daphnetin in triple-negative breast cancer; Figure 3 C “Drug active ingredient-target” network diagram.

[0015] Figure 4 A is the PPI diagram of protein-protein interaction between daphnetin and triple-negative breast cancer; Figure 4 B is the GO enrichment analysis diagram.

[0016] Figure 5 Schematic diagram of the transcriptomic analysis of the effect of daphnetin on triple-negative breast cancer cells 4T1. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0018] Use of daphnetin as an inhibitor in the preparation of drugs for treating triple-negative breast cancer.

[0019] The daphnetin acts on MAPK8, a key target of triple-negative breast cancer, to regulate the MAPK signaling pathway.

[0020] The present invention reveals the mechanism of action of daphnetin in inhibiting triple-negative breast cancer: In recent years, it has been found that daphnetin has excellent cytotoxic potential against various tumors, such as ovarian cancer, liver cancer, and breast cancer. In theory, it also has the same effect on triple-negative breast cancer. Subsequently, in order to verify the hypothesis of the present invention, human triple-negative breast cancer cells MDA-MB-231 and mouse triple-negative breast cancer cells 4T1 were selected. The present invention used the CCK-8 method to detect the effects of different concentrations of daphnetin on the two cell lines and found that both could induce apoptosis of triple-negative breast cancer cells. Figure 1 BC. Through Edu staining analysis, the present invention found that after treatment with daphnetin, the DNA replication ability of both triple-negative breast cancer cells decreased significantly. Figure 1DE, provides strong evidence for exploring the key role of daphnetin in the growth of triple-negative breast cancer. In order to comprehensively study the potential effects of daphnetin on the migration and invasion ability of triple-negative breast cancer cells, the present invention used the Transwell method to conduct extensive detection. The experimental data showed that after the cells were treated with daphnetin, the migration and invasion abilities of MDA-MB-231 and 4T1 cells were significantly reduced. Figure 1 FG. In addition, the present invention evaluated the effect of daphnetin on the cloning potential of triple-negative breast cancer cells through a plate colony formation experiment. The results showed that daphnetin significantly inhibited the cloning ability of triple-negative breast cancer cells. Figure 1 HI, further emphasizes the core role of daphnetin in the proliferation and colony formation of triple-negative breast cancer cells. In vitro experiments confirmed the inhibitory effect of daphnetin on triple-negative breast cancer.

[0021] In order to transform the research results of the present invention from the laboratory into practical applications, the present invention adopted a more physiological animal model and successfully established a subcutaneous triple-negative breast cancer model in C57BL / 6 mice using 4T1 cells. Subsequently, the present invention divided the experimental mice into a Con group and a Daphnetin group. The Daphnetin group mice were treated with intratumoral injection of daphnetin every day after the 4T1 cell injection on the 6th day, while the Con group was injected with an equal volume of normal saline at the same time point. At specific time points (8th, 10th, 12th, and 14th days), the changes in subcutaneous tumors in the two groups were carefully observed and measured ( Figure 1 A). On the 14th day, 6 mice were randomly selected from each group, and the subcutaneous tumors were removed and photographed ( Figure 1 A). In addition, according to the results of HE and Ki67 immunohistochemistry experiments, the present invention found that the proliferation ability of mice in the Daphnetin group was significantly reduced compared with that in the Con group ( Figure 2 B). During the period of continuing feeding until the mice died naturally, the present invention conducted a detailed statistical analysis of the survival rates of the two groups of mice. The results showed that the survival ability of the mice in the Daphnetin protein group was significantly better than that of the mice in the Con group ( Figure 2 C). In addition, statistical analysis of the weight and volume of the subcutaneous tumors of the two groups of mice revealed that the growth of the subcutaneous tumors in the Daphnetin group was significantly smaller than that in the Con group.

[0022] Triple-negative breast cancer is a highly heterogeneous disease, the occurrence and development of which involve abnormalities in multiple signaling pathways and molecular mechanisms. The applicant previously obtained the 3D structure of daphnetin through the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ) (Working basis, Figure 3A), the Swiss Target Prediction database (http: / / www. swisstargetprediction.ch / ) was imported to predict potential targets of daphnetin, and targets with a probability of "0" were eliminated. At the same time, the TCGA database (http: / / cancergenome.nih.gov / ), GeneCards database (http: / / www.genecards.org), and GEO database (http: / / www.ncbi.nlm.nih.gov / geo) were screened to obtain targets related to triple-negative breast cancer, and the intersection with the targets of daphnetin was taken. Finally, 79 potential targets were obtained (working basis, Figure 3 B), such as MAPK8, Bax, Bcl-2 and other targets. Cytoscape (version 3.9.1) software was used to establish a multi-level mechanism of action map of "drug active ingredient-disease-target" and to analyze its structure and function (work basis, Figure 3 C).

[0023] In order to understand the interaction relationship between each target, the intersection targets of daphnetin and triple-negative breast cancer were imported into the STRING database (https: / / string-db.org / ), and single target genes that could not interact were eliminated. At the same time, the protein-protein interaction map (PPI map) of daphnetin-triple-negative breast cancer was finally constructed using Cytoscape (version 3.9.1) software (Working basis, Figure 4 A) The selected core targets were imported into the DAVID database (https: / / david.ncifcrf.gov) and GO and KEGG enrichment analysis was performed on the anti-tumor targets of daphneline. Using P<0.01 as the screening condition, the signaling pathways with significant differences in the key biological processes and mechanisms of action related to daphneline anti-tumor were screened out. GO enrichment ( Figure 4Biological processes in B) include oxidative stress and metal ion reactions; KEGG pathway enrichment primarily targets the PI3K-Akt signaling pathway and the MAPK signaling pathway in triple-negative breast cancer. The PI3K / Akt signaling pathway, a key intracellular signaling pathway, promotes breast proliferation and apoptosis, invasion, and metastasis in breast cancer and is closely associated with chemotherapy resistance. The MAPK signaling pathway primarily consists of the JNK / SAPK and p38MAPK pathways. The former is primarily involved in cellular stress and apoptosis, while the latter primarily regulates cell proliferation. MAPK8, also known as c-Jun N-terminal kinase 1 (JNK1), is closely associated with the JNK signaling pathway. As a key enzyme in stress responses, it is activated by various intracellular and extracellular stimuli and participates in various cellular processes, playing a crucial role in apoptosis and inflammatory responses. The MAPK signaling pathway is closely associated with the development and progression of numerous diseases, particularly cancer, and also plays a crucial role in drug therapy. Inhibiting the JNK / SAPK signaling pathway is considered a promising anti-cancer approach. These results suggest that daphnetin may exert its effects through multiple genes and signaling pathways, completing the construction of a "daphnetin-key target-triple-negative breast cancer" network. Therefore, the present invention comprehensively understands the mechanism of action of daphnetin in inhibiting triple-negative breast cancer from a multi-pathway, multi-channel, and multi-site perspective.

[0024] Although network pharmacology has preliminarily analyzed the possible mechanism of action of daphnetin, we continued to explore the possible molecular mechanism of action of daphnetin. RNA transcriptome sequencing was performed on 4T1 cells treated with PBS and stimulated with daphnetin supernatant. First, the present invention found that 1627 genes were differentially expressed after daphnetin stimulation compared with the control group (DEGs, foldchange>1.5, p value<0.05), of which 835 genes were upregulated and 792 genes were downregulated ( Figure 5 A). GO functional enrichment analysis showed that after stimulation by daphneline, the main effects on cell protein binding, biological regulation and cell development process are closely related ( Figure 5 B) KEGG pathway enrichment analysis of differentially expressed genes showed that after stimulation with daphnetin, it mainly activated the cytokine-cytokine receptor interaction signaling pathway and the TNF signaling pathway in triple-negative breast cancer cells, and inhibited the MAPK signaling pathway, glycolysis / gluconeogenesis signaling pathway, cell ferroptosis-related signaling pathway, HIF-1 signaling pathway, etc. ( Figure 5C). Based on the results of network pharmacology and transcriptome sequencing, we suggest that the MAPK signaling pathway has an important inhibitory effect on triple-negative breast cancer. We selected key targets that play an important role in this pathway as candidates and preliminarily analyzed the molecular mechanism by which daphnetin may regulate the MAPK signaling pathway to inhibit triple-negative breast cancer through the key target MAPK8.

[0025] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the novel spirit and scope of the present invention. Such changes and improvements fall within the scope of the invention claimed. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. The application of daphnetin as an inhibitor in the preparation of drugs for the treatment of triple-negative breast cancer.

2. The use of daphnetin as an inhibitor in the preparation of a drug for treating triple-negative breast cancer according to claim 1, characterized in that The daphnetin acts on MAPK8, a key target of triple-negative breast cancer, to regulate the MAPK signaling pathway.

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