Anti-breast cancer medicine and application

Abstract

The invention relates to the technical field of biology, relates to an anti-breast cancer medicine and application, and particularly discloses an anti-breast cancer medicine which comprises an active component, and the active component is a DECR1 inhibitor or a DECR1 binding agent; an application of a DECR1 inhibitor or a DECR1 binding agent in the preparation of a medicament for treating or preventing breast cancer; an application of a DECR1 inhibitor or a DECR1 binding agent in preparation of a drug for improving the sensitivity or treatment effect of an anti-breast cancer drug in treatment or prevention of breast cancer; wherein the DECR1 binding agent is Erigoster B, and the DECR1 binding agent is Erigoster B; according to the application disclosed by the invention, expression of DECR1 in breast cancer tissue cells is inhibited through Erigoster B, so that the proliferation, migration and invasion capabilities of breast cancer are reduced, the ferroptosis probability of breast cancer cells is increased, and a new treatment strategy and thought are provided for breast cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-breast cancer drug and application thereof. Background Art

[0002] Breast cancer is the most common tumor in women, and its morbidity and mortality rates remain high. Although breast cancer has been extensively studied, the treatment options for patients with advanced breast cancer are limited and the treatment effect is poor. Therefore, it is of great clinical significance and scientific research value to find new and effective targets for breast cancer treatment and develop potential anti-tumor drug candidates.

[0003] Metabolic reprogramming is considered to be one of the hallmarks of tumors, including glycolysis, oxidative phosphorylation, amino acid metabolism, and fatty acid metabolism. Generally, rapidly proliferating tumor cells require a large amount of fatty acids to synthesize phospholipids to promote cell membrane formation. In addition, fatty acids can also serve as raw materials for mitochondrial ATP production and play a role in regulating protein function. Therefore, fatty acids play an important role in the occurrence and development of tumors, and interfering with the fatty acid metabolism of tumor cells may become an effective strategy for tumor treatment.

[0004] Cell membranes and organelle membranes are rich in polyunsaturated fatty acids. However, excessive ROS generated by polyunsaturated fatty acid reactions can lead to cell damage and accumulation of lipid peroxides, resulting in ferroptosis of cells. Ferroptosis plays a vital role in various diseases, such as tumors, osteoporosis, and cardiovascular diseases. It is well known that lipid peroxidation induced by cellular oxidative stress is the basis for ferroptosis. Although tumor cells are more susceptible to oxidative stress, tumor cells can usually produce NADPH, GSH and other reductants to reduce cellular ROS levels and maintain cellular redox homeostasis, thereby avoiding ferroptosis. Strategies targeting tumor ferroptosis have shown exciting results, but their clinical application is still subject to many limitations due to the lack of clarity and depth of related research. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide an anti-breast cancer drug and its application, so as to provide a new idea for the treatment of breast cancer.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] An anti-breast cancer drug comprises an active ingredient, wherein the active ingredient is a DECR1 inhibitor or a DECR1 binder.

[0008] Use of a DECR1 inhibitor or a DECR1 binder in preparing a drug for treating or preventing breast cancer.

[0009] Application of DECR1 inhibitors or DECR1 binders in the preparation of drugs for improving the sensitivity or therapeutic effect of anti-breast cancer drugs in the treatment or prevention of breast cancer.

[0010] As an implementable embodiment, the DECR1 is highly expressed in breast cancer tissue; and the high expression of DECR1 is associated with a poor prognosis of breast cancer.

[0011] As an embodiment, knocking down the expression level of DECR1 in breast cancer reduces the proliferation, migration and invasion capabilities of breast cancer cell lines.

[0012] As an embodiment, knocking down the expression level of DECR1 in breast cancer can reduce the expression of mitochondrial ROS and Fe in breast cancer cells. 2+ The level and MDA content increased.

[0013] As an implementable method, knocking down the expression level of DECR1 in breast cancer increases the probability of ferroptosis of breast cancer tissue cells.

[0014] As an implementable method, knocking down the expression level of DECR1 in breast cancer reduces the PC content in breast cancer cells; knocking down the expression level of DECR1 in breast cancer increases the AA content in breast cancer cells; knocking down the expression level of DECR1 in breast cancer increases the PLA2G12A expression level in breast cancer cells;

[0015] The expression level of PLA2G12A in breast cancer cells is negatively correlated with the PC content in breast cancer cells, and positively correlated with the AA content in breast cancer cells; the expression level of PLA2G12A in breast cancer cells is positively correlated with the probability of ferroptosis of breast cancer cells.

[0016] In one embodiment, the DECR1 binder is Erigoster B.

[0017] As an embodiment, the ErigosterB forms hydrogen bonds with THR69, ASN144, THR196, LYS214, ILE243 and SER250, and binds to the DECR1.

[0018] The beneficial effects of the present invention are as follows: the present invention evaluates the specific role of DECR1 in the progression of breast cancer, identifies it as a therapeutic target for breast cancer, reveals that the regulatory effect on ferroptosis is the key mechanism of action of DECR1 in promoting the progression of breast cancer, inhibits the expression of DECR1 in breast cancer tissue cells by Erigoster B, reduces the proliferation, migration and invasion ability of breast cancer, increases the probability of ferroptosis of breast cancer tissue cells, and provides new therapeutic strategies and ideas for the treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the expression of fatty acid metabolism related genes in breast cancer tissues and normal tissues in the TCGA-BRCA-1096 dataset in Example 2 of the present invention.

[0020] Figure 2 A is the Kaplan-Meier curve diagram of patients with DECR1 expression above and below the critical value (calculated by the "bestssurv" algorithm) in Example 2 of the present invention; B is the relationship between DECR1 expression and survival probability estimated by data in the TCGA-BRCA-1096 data set (n=788); C is the relationship between DECR1 expression and survival probability estimated by data in the GEO96058 data set.

[0021] Figure 3 Representative images of IHC staining of DECR1 in breast cancer tissues (n=140) and normal breast tissues (n=77) in Example 2 of the present invention.

[0022] Figure 4 This is a diagram showing the protein expression of DECR1 in breast cancer tissue and normal breast tissue according to the IHC staining results in Example 2 of the present invention.

[0023] Figure 5 This is a Kaplan-Meier curve diagram of OS of patients whose DECR1 expression is higher and lower than the critical value according to the IHC staining results in Example 2 of the present invention.

[0024] Figure 6 This is a probability diagram of the Nomogram predicting the OS of cancer patients in Example 2 of the present invention; related factors include age, site, pathological T stage (T), pathological N stage (N), American Joint Committee on Cancer (AJCC) stage and DECR1 expression listed on the dot axis. The 5-year and 10-year OS probabilities are plotted to calculate the OS incidence *P<0.05, **P<0.01.

[0025] Figure 7 This is a graph showing the proliferation of MDA-MB-231 cells after knocking down DECR1 in Example 2 of the present invention.

[0026] Figure 8 This is a proliferation diagram of BT474 cells after knocking down DECR1 in Example 2 of the present invention.

[0027] Fig. 9 This is a diagram of cell migration after DECR1 knockdown in breast cancer cells (MDA-MB-231 and BT474) observed by scratch experiment in Example 2 of the present invention.

[0028] Fig.10This is a diagram of an invasion assay using a Transwell chamber with a Matrigel-coated membrane in DECR1 knockdown MDA-MB-231 and BT474 cells in Example 2 of the present invention.

[0029] Fig.11 This is a graph showing the number of invaded cells in DECR1 knockdown MDA-MB-231 and BT474 cells using a Transwell chamber with a Matrigel-coated membrane in Example 2 of the present invention.

[0030] Fig.12 This is a diagram showing the effect of knocking down DECR1 on the tumorigenicity of breast cancer cells in a subcutaneous xenograft model established in NOD-SCID mice in Example 2 of the present invention; tumors derived from MDA-MB-231 and BT474 (NC, shDECR1-1, shDECR1-2) cells were isolated (n=5 in each group).

[0031] Fig.13 The tumor volume of the tumor derived from MDA-MB-231 cells in Example 2 of the present invention is about 50 mm 3 Growth graphs were drawn every two days thereafter; *P<0.05, **P<0.01.

[0032] Fig.14 The tumor volume of the tumor derived from BT474 cells in Example 2 of the present invention is about 50 mm 3 Growth graphs were drawn every two days thereafter; *P<0.05, **P<0.01.

[0033] Fig.15 This is a diagram of the mitochondrial morphology observed using TEM after DECR1 knockout in Example 2 of the present invention.

[0034] Fig.16 This is a diagram of quantifying mitochondrial ROS levels using MitoSOX staining in MDA-MB-231 cells with stable knockdown of DECR1 in Example 2 of the present invention; the fluorescence intensity was quantified using flow cytometry, and the mitochondrial ROS level was expressed as the mean fluorescence intensity value.

[0035] Fig.17 In Example 2 of the present invention, MitoSOX staining was used to quantify mitochondrial ROS levels in BT474 cells with stable knockdown of DECR1; flow cytometry was used to quantify fluorescence intensity, and mitochondrial ROS levels were expressed as mean fluorescence intensity values.

[0036] Fig.18 The intracellular Fe in MDA-MB-231 cells after knocking down DECR1 using the FerroOrange assay kit in Example 2 of the present invention is 2+Horizontal flow cytometry analysis.

[0037] Fig.19 The intracellular Fe in BT474 cells after knocking down DECR1 using the FerroOrange assay kit in Example 2 of the present invention is 2+ Horizontal flow cytometry analysis.

[0038] Fig. 20 This is a graph showing the measurement of MDA content in MDA-MB-231 cells with stable knockdown of DECR1 in Example 2 of the present invention, *P<0.05, **P<0.01.

[0039] Fig.21 This is a graph showing the measurement of MDA content in BT474 cells with stable knockdown of DECR1 in Example 2 of the present invention, *P<0.05, **P<0.01.

[0040] Fig. 22 This is a volcano plot of non-targeted metabolomics to identify dysregulated metabolites in DECR1 knockdown compared with negative control MDA-MB-231 and BT474 cells in Example 2 of the present invention.

[0041] Fig.23 This is an expression diagram of the metabolism-related gene PLA2G12A after knocking down DECR1 in Example 2 of the present invention.

[0042] Fig.24 This is a graph showing the PLA2G12A content after knocking down DECR1 in breast cancer cells in Example 2 of the present invention.

[0043] Fig.25 This is a graph showing the contents of seven PCs in MDA-MB-231 cells with stably knocked-down DECR1 determined by mass spectrometry in Example 2 of the present invention.

[0044] Fig.26 This is a graph showing the contents of seven PCs in BT474 cells with stably knocked-down DECR1 determined by mass spectrometry in Example 2 of the present invention.

[0045] Fig. 27 This is a graph showing the intracellular AA content measured after knocking down DECR1 using ELISA in Example 2 of the present invention.

[0046] Fig.28 This is a graph of AA content measured after further knocking out PLA2G12A using stable DECR1 knockout in breast cancer cells in Example 2 of the present invention.

[0047] Fig.29This is a graph showing the quantification of mitochondrial ROS levels using MitoSOX staining in MDA-MB-231 cells with stable DECR1 knockout and further PLA2G12A knockout in Example 2 of the present invention; the fluorescence intensity was quantified using flow cytometry, and the mitochondrial ROS level was expressed as the mean fluorescence intensity value.

[0048] Fig.30 This is a graph showing the quantification of mitochondrial ROS levels using MitoSOX staining in BT474 cells with stable DECR1 knockout and further PLA2G12A knockout in Example 2 of the present invention; the fluorescence intensity was quantified using flow cytometry, and the mitochondrial ROS level was expressed as the mean fluorescence intensity value.

[0049] Fig.31 The intracellular Fe2 + Horizontal flow cytometry analysis; *P<0.05, **P<0.01.

[0050] Fig.32 The intracellular Fe2 + Horizontal flow cytometry analysis; *P<0.05, **P<0.01.

[0051] Fig.33 This is a diagram of the binding pattern of DECR1 and erigoster B in the 2D model in Example 2 of the present invention.

[0052] Fig.34 This is a diagram of the binding pattern of DECR1 and erigoster B in the 3D model in Example 2 of the present invention.

[0053] Fig.35 It is a comparison diagram of the binding modes between DECR1 and NADP and between DECR1 and ErigosterB in Example 2 of the present invention.

[0054] Fig.36 This is the SPR analysis diagram of the binding affinity of DECR1-Erigoster-B in Example 2 of the present invention.

[0055] Fig.37 This is a diagram showing the proliferation of MDA-MB-231 and BT474 cells damaged by Erigoster B treatment in Example 2 of the present invention.

[0056] Fig.38The migration diagram of MDA-MB-231 and BT474 cells with and without Erigoster B treatment in wound healing assay in Example 2 of the present invention.

[0057] Fig.39 The figure is a diagram of the invasion assay in MDA-MB-231 and BT474 cells with and without Erigoster B treatment in Example 2 of the present invention (conducted in a transwell chamber with a Matrigel-coated membrane).

[0058] Fig.40 This is a graph showing the number of invaded cells measured in MDA-MB-231 and BT474 cells treated with and without Erigoster B in Example 2 of the present invention.

[0059] Fig.41 This is a diagram of establishing a subcutaneous xenograft model in NOD-SCID mice to evaluate the effect of Erigoster B on breast cancer progression in Example 2 of the present invention; the tumor was derived from 4T1 cells (n=5 in each group).

[0060] Fig.42 The tumor volume in Example 2 of the present invention is about 50 mm 3 After that, tumor growth was plotted every two days; *P<0.05, **P<0.01.

[0061] Fig.43 This is a schematic diagram of the evaluation of MDA-MB-231 cell death induced by 25 μM Erigoster B treatment in Example 2 of the present invention using ferroptosis inhibitor (ferrostatin-1, 1 μM), autophagy inhibitor (3-MA, 1 μM) and apoptosis inhibitor (Z-VAD-FMK, 5 μM).

[0062] Fig.44 This is a schematic diagram of the evaluation of BT474 cell death induced by 25 μM Erigoster B treatment in Example 2 of the present invention using a ferroptosis inhibitor (ferrostatin-1, 1 μM), an autophagy inhibitor (3-MA, 1 μM) and an apoptosis inhibitor (Z-VAD-FMK, 5 μM).

[0063] Fig.45 This is a quantitative graph of mitochondrial ROS levels in MDA-MB-231 cells treated with or without Erigoster B in Example 2 of the present invention using MitoSOX staining; the fluorescence intensity was quantified using flow cytometry, and the mitochondrial ROS level was represented by the mean fluorescence intensity value.

[0064] Fig.46This is a quantitative graph of mitochondrial ROS levels in BT474 cells treated with or without Erigoster B in Example 2 of the present invention using MitoSOX staining; the fluorescence intensity was quantified using flow cytometry, and the mitochondrial ROS level was expressed as the average fluorescence intensity value.

[0065] Fig.47 The intracellular Fe in MDA-MB-231 cells treated with or without Erigoster B was measured using the FerroOrange assay kit in Example 2 of the present invention. 2+ Horizontal flow cytometry analysis graph.

[0066] Fig.48 The intracellular Fe in BT474 cells treated with or without Erigoster B was measured using the FerroOrange assay kit in Example 2 of the present invention. 2+ Horizontal flow cytometry analysis graph.

[0067] Fig.49 This is a graph showing the AA content in breast cancer cells treated with or without Erigoster B using ELISA in Example 2 of the present invention.

[0068] Fig.50 This is a graph showing the contents of seven PCs measured by mass spectrometry in MDA-MB-231 cells treated with or without Erigoster B in Example 2 of the present invention; *P<0.05, **P<0.01.

[0069] Fig.51 This is a graph showing the contents of seven PCs measured by mass spectrometry in BT474 cells treated with or without Erigoster B in Example 2 of the present invention; *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0070] The present invention is further described in detail below in conjunction with specific embodiments.

[0071] It should be noted that these embodiments are only used to illustrate the present invention rather than to limit the present invention. Simple improvements to the method based on the concept of the present invention all fall within the scope of protection claimed by the present invention.

[0072] Example 1 Methods and Materials

[0073] 1.1 Bioinformatics analysis

[0074] Fatty acid metabolism (FAM)-related genes were derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG; https: / / www.genome.jp / kegg / ). Data from an independent cohort (TCGA-BRCA) containing 1096 cases were downloaded from The Cancer Genome Atlas (TCGA) and imported into R (version 4.1) (https: / / bioconductor.org / bioclite.r). In this cohort, 1045 cases were retained for further analysis because they had complete clinical follow-up data. A data set containing 3409 cases (GSE96058) was obtained from the GEO database, and the data of this data set were used to verify the gene expression and prognostic value of DECR1 in breast cancer patients. Survival analysis refers to normalizing the gene expression data and centering them with the mean, and drawing Kaplan-Meier curves to show the value of FAM-related genes in predicting the overall survival (OS) of patients. The outcome event was defined as “death”. The cutoff expression level of each gene was estimated using the best separation algorithm according to the R package “surfminer”. The log-rank test was used to analyze the data of the Kaplan-Meier plots and the results of the univariate Cox regression analysis. The correlation between DECR1 expression and OS rate was further evaluated by multivariate Cox regression analysis, and a nomogram was generated with the “rms” package based on the results of the multivariate Cox regression analysis.

[0075] Protein-protein interaction network: FAM-related genes were imported into the STRING database (https: / / cn.string-db.org / ) to obtain the protein-protein interaction network diagram. The degree, topological coefficient, and neighborhood connectivity of the genes were then scored using Cytoscape (version 3.7.0), and genes with all three scores higher than the median score were selected.

[0076] 1.2 Tissue microarray and immunohistochemical staining

[0077] A total of 140 breast cancer tissues and 77 paracancerous tissues were collected based on the completeness of clinical and pathological information and the availability of follow-up information. The diagnosis of these specimens was further verified by a pathologist. To construct the tissue microarray, a representative area was selected from the paraffin tissue of each sample. In addition, microarray sections were obtained by tissue microarray paraffin blocks. The Medical Ethics Committee of Shanghai Aoduo Biotechnology Co., Ltd. granted ethical approval for this project (approval number: SHYJC-CP-1901002; SHYJC-CP-1607006). The tissue microarray was incubated with rabbit monoclonal anti-DECR1 antibody (Abcam, ab198848) at a dilution of 1:100 at 4°C. The EnVision system was used for immunohistochemical staining. Diaminobenzidine (Dako, Glostrup, Denmark) was used for visualization of antibody binding. The staining intensity of DECR1 was calculated by Image ProPlus (version 6.0.0.260, USA).

[0078] 1.3 Cell culture

[0079] Cell culture: Human breast cancer cell lines MDA-MB-231 and BT474 were purchased from the American Type Culture Collection (ATCC). All cells were maintained in Dulbecco's modified Eagle medium (DMEM) or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, 10437028) and 1% penicillin / streptomycin (Gibco, 10378016) at 37°C in a humidified atmosphere of 5% CO2.

[0080] shDECR1 cell line construction.

[0081] 1.4 Cell function detection (cell viability, cell migration, cell invasion)

[0082] Cell viability: The cell counting kit-8 (CCK-8) cell proliferation assay kit (Solarbio, CA1210) was used to assess breast cancer cell viability. 5 The density of individual cells / mL is inoculated in 96-well plates and cultivated.After incubation for 48 hours with DMSO, 10 μL CCK-8 solution is added in culture medium, and cells are incubated for another 2 hours.OD is then measured at 450nm using a multi-well spectrophotometer (microplate reader).Cell viability is represented by the percentage of control (untreated) cell viability.

[0083] Cell migration: When MDA-MB-231 and BT474 cells were 100% confluent, a wound was made in the central area of ​​each well using a 200 μL pipette tip. The wounded area was photographed at 0 and 24 h. Each experiment was repeated three times.

[0084] Cell invasion: Breast cancer cells were cultured in serum-free medium for 12 h. The cell concentration was adjusted to 4-5×10 5 Cells / mL. Transwell chamber (Corning, NY, USA) with Matrigel coating membrane is placed in 24-well plates. RPMI-1640 medium (500 μL) is placed in the bottom compartment, and 200 μL of cell suspension is placed in the upper compartment. Then, the cells are incubated for 24 hours. The cells on the lower surface of the membrane are fixed with glacial acetic acid for 15-30min, stained with crystal violet for 30 minutes, and counted. The experiment was repeated three times independently.

[0085] 1.5 Transmission electron microscopy

[0086] The morphological characteristics of breast cancer cells with ferroptosis were observed by transmission electron microscopy. The main steps are fixation: the sample was pre-fixed with 3% glutaraldehyde and then fixed with 1% osmium tetroxide; dehydration: acetone dehydration step by step, the concentration gradient of the dehydrating agent was 30%→50%→70%→80%→90%→95%→100% (100% concentration was changed 3 times); infiltration and embedding: the ratio of dehydrating agent and Epon812 embedding agent was 3:1, 1:1, 1:3, and finally Ep812 embedding. Ultrathin sections: Use an ultrathin slicer to prepare ultrathin sections of about 60 to 90 nm, spread the sections, and then scoop them onto a copper grid; Staining: First stain with uranyl acetate for 10 to 15 minutes, then stain with lead citrate for 1 to 2 minutes, and stain at room temperature; Electron microscopy observation: Use a JEM-1400FLASH transmission electron microscope produced by JEOL to capture images of the copper grid. Each copper grid is first observed at 6000 times, and the area to be observed is selected to capture images to observe the specific morphology of cells and organelles.

[0087] 1.6 Transcriptomic analysis

[0088] Transcriptome sequencing was performed using the previously constructed MDA-MB-231 and BT474 cell lines with stable knockdown of DECR1. First, 1×107 cells were collected for each group, with 3 replicates per group, and then total RNA was extracted using the Trizol method. Then, the transcriptome sequencing library was constructed using the Illumina RNA library preparation kit (NEB, USA). Next, the library was sequenced on the Illumina NovaSeq platform. The raw reads in fastq format were processed. Then, the read counts of each gene were calculated using the featureCounts (version: 1.5.0) software package.

[0089] 1.7 Non-targeted metabolomics analysis

[0090] For non-targeted metabolomics, 1 × 10 7The cells were placed in EP tubes and the samples were resuspended in pre-cooled 80% methanol by vortexing. Then, the samples were thawed on ice and centrifuged for 30 seconds. After 6 minutes of ultrasonic treatment, the samples were centrifuged at 5000rpm, 4°C for 1 minute. The supernatant was freeze-dried and dissolved in 10% methanol. Finally, the solution was injected into the LC-MS / MS system for analysis. UHPLC-MS / MS analysis was performed using a Vanquish UHPLC system (ThermoFisher, Germany) coupled to an Orbitrap Q ExactiveTM HF mass spectrometer (ThermoFisher, Germany) of Novogene Co., Ltd. (Beijing, China). The raw data files generated by UHPLC-MS / MS were processed using Compound Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking and quantification of each metabolite. The KEGG database, the Human Metabolome Database (HMDB; https: / / hmdb.ca / metabolites ), and the LIPIDMaps database ( http: / / www.lipidmaps.org / ) were used.

[0091] 1.8 Mass spectrometry analysis

[0092] The control or DECR1 knockdown MDA-MB-231 or BT474 cells were collected, and the cell number was about 1×10 7 Each group was repeated 5 times, and after collection, 70% methanol / water solution was added to grind and crush the cells. Then, the metabolites were extracted with chloroform solution. The metabolites containing chloroform were further concentrated in a concentration device. The concentrate was further dissolved in 300 μL of isopropanol: methanol (1:1) solution (containing 100 ng / mL internal standard PC (15:0 / 18:1-7)), centrifuged for 15 minutes, and the supernatant was retained for analysis. In addition, the PC (16:0-18:1) standard solution (500 ng / ml) was analyzed, and the internal standard concentration was 100 ng / ml. Chromatographic analysis was performed on Waters Acquity UPLC using an Acquity UPLC BEH amide column (1.7 μm, 2.1 mm×100 mm). The chromatographic separation conditions were: column temperature 45°C; flow rate, 0.300 mL / min. Metabolomic analysis was performed with mobile phase A (H2O: acetonitrile 5 mM; NH4AC, 95 / 5, v / v) and mobile phase B (H2O: methanol 5 mM; NH4AC 50 / 50, v / v, pH adjusted to 8.0 with aqueous ammonia) with the following elution gradient: 0–1 min, 0% B; 1–2 min, 50% B; 2–4 min, 100% B; 4–6 min, 0% B.

[0093] In addition, mass spectrometry analysis was performed on an AB SCIEX 5500 triple quadrupole (QQQ)-mass spectrometer using an electrospray ionization (ESI) source. Mass spectrometry was performed under the following conditions: curtain gas, 35 arb; collision gas, 9 arb; ion spray voltage, 4500 volts; temperature 450°C; ion source gas 1, 50 arb; and ion source gas 2, 50 arb. The final results were integrated using MultiQuant software, and the metabolite content was calculated using the internal standard method.

[0094] 1.9 Arachidonic acid content detection

[0095] MDA-MB-231, BT474 cells after DECR1 knockdown or treatment with Erigoster B (5 μM, 48 h) were collected. Then 150-200 μL PBS was added to each 106 breast cancer cells for resuspension. The cells were lysed by ultrasound, and the supernatant was tested with an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. Arachidonic acid (AA) assay kit (E-EL-0051c) was purchased from Elabscence.

[0096] 1.10qRT-PCR

[0097] Total RNA was extracted and purified using TRIzol reagent (Magen, R4801-01). Next, reverse transcription was performed using a transcription kit (Vazyme, R122-01). Quantitative RT-PCR was performed using an ABI 7900 real-time PCR system. Finally, the data were analyzed using the 2-ΔΔCt method. The mRNA levels of the target genes were normalized to the levels of β-actin or GAPDH.

[0098] 1.11 Animal experiments

[0099] NOD-SCID mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. The animals were housed in cages at 23 ± 2 °C and fed with standard laboratory diet and tap water throughout the experiment.

[0100] Orthotopic xenograft model: Mice were anesthetized with 4% chloral hydrate and 100 μL of 1x10 7 The cancer breast cell suspension of cells was injected into the mammary fat pad to establish an orthotopic xenograft model. For tumorigenesis assays, negative control (NC) MDA-MB-231 and BT474 cells, DECR1 knockdown (shDECR1) MDA-MB-231 and BT 474 cells, and 4T1 cells were injected. Tumors were measured every two days, and tumor volume was calculated as follows: Tumor volume = (length × width 2 ) / 2.

[0101] 1.12 Detection of ferroptosis-related indicators

[0102] To evaluate the ferroptosis of each group, MDA, ROS and Fe 2+ According to the instructions of the kit, a lipid peroxidation (MDA) assay kit (Beyotime Biotechnology, S0131S) was used.

[0103] 1.13 Computer-Assisted Virtual Screening

[0104] Receptors for virtual screening: Using the Protein Preparation Wizard Panel ( 2021) optimized the protein structure of DECR1 by correcting the bond order, adding hydrogen atoms, distributing charges, and predicting the protonation state (pH 7.0). Constrained energy optimization was performed using the OPLS4 force field to eliminate atomic conflicts in the structure, and the root mean square deviation (RMSD) of heavy atoms converged to And optimize the side chain position to obtain a reasonable side chain structure.

[0105] Preparation of compound libraries: Using the LigPrep panel ( 2021) were used to treat compounds from L6020. First, the compounds were protonated and desalted at pH 7.0 ± 2.0 using the Epik program to generate tautomers and maintain the original atomic chirality. To ensure the conformational diversity of small molecules during virtual screening, a maximum of 32 conformations were generated for each small molecule.

[0106] The following screening method was used: Glide HTVS [High-Throughput Virtual Screening Mode] → GGlide SP [Standard Precision Mode] → GGlide XP [Extra Precision]. The protein structure was set to be rigid, the small molecule was set to be flexible, and other parameters were set to default values. The docked structures were subjected to energy optimization, and the structures with docking scores within the top 50% were retained for the next round of screening. Duplicates were removed and visual inspection was performed after filtering for drug-like properties. After the initial screening, approximately 15,000 small molecule compounds were obtained. After removing duplicate structures, 2,497 compounds were retained. Filtering was performed using QikProp based on predicted absorption, distribution, metabolism, and excretion (ADME) properties (specified criteria: 0 < H-bond donors < 6, 150 < Mol Weight < 725). Compounds with a molecular weight greater than 725 and more than 6 H-bond donors were removed, resulting in a final set of 1,258 compounds. Then, Canvas was used to generate binary fingerprints for these 1,258 compounds, and hierarchical clustering was performed based on the Tanimoto similarity metric. Compounds with the highest docking score (in kcal / mol, where a lower value indicates a higher affinity between the receptor and ligand) were selected from each cluster. This process yielded a final set of 100 small molecule compounds representing different chemical structures.

[0107] 1.14 Surface Plasmon Resonance (SPR) Assay

[0108] The interaction between the analyte and the ligand was evaluated at room temperature using an OpenSPR instrument (Nicoya, Canada). First, according to the standard procedure, a COOH sensor chip was installed in the OpenSPR instrument. The buffer was run at the maximum flow rate and degassing bubble parameters after reaching the signal baseline. Then, 10 mM glycine-HCl (pH 7.4) was injected onto the surface of the sensor chip. The chip was activated, and the prepared EDC and NHS solutions were injected to complete the functionalization of the chip surface. Then, the sample was injected at a flow rate of 20 μL / min.

[0109] Finally, different concentrations of the analyte were injected separately onto the surface of the ligand chip (specific concentrations are provided in the experimental results), and the analyte was sampled at a rate of 20 μL / min. The binding time of the analyte to the ligand was 240 s, and the natural dissociation time was 360 s. In each cycle, a 200 μL volume of the sample flowed through the chip at a constant flow rate of 20 μL / min for 240 s. The bound compounds were dissociated from the coated surface by treatment with acidic and basic solutions. After each measurement, treatment with 10 mM glycine-HCl (pH 1.5) was used to separate the small molecule and the target protein. Finally, the kinetic parameters of the binding reaction were calculated and analyzed using Trace Drawer software (Ridgeview Instruments AB, Sweden).

[0110] Example 2 Analysis Results

[0111] 2.1DECR1 is overexpressed in clinical breast cancer samples and is associated with poor prognosis in patients

[0112] See also Figure 1-6 , analyzed the expression of 84 fatty acid metabolism-related genes in breast cancer and adjacent tissues. First, 19 key genes were screened out by constructing a protein-protein interaction network based on Dgree and Neibo rankings. Then, the expression analysis of these 19 key genes showed that only DECR1 was expressed higher in breast cancer tissues than in adjacent tissues, and it was statistically significant. In addition, both TCGA and GEO96058 datasets showed that high expression of DECR1 was associated with poor prognosis of patients, so it was speculated that DECR1 has the potential to be a therapeutic target for breast cancer.

[0113] To further clarify the role of DECR1 in breast cancer, immunohistochemical staining analysis was performed on tissue microarrays constructed from 140 breast cancer clinical samples and 77 adjacent normal breast tissues. The results showed that compared with adjacent tissues, DECR1 was highly expressed in breast cancer tissues, and high expression of DECR1 was positively correlated with poor prognosis of patients.

[0114] 2.2 Effect of DECR1 on the malignant phenotype of tumor cells

[0115] Bioinformatics analysis was used to identify whether DECR1 has the potential to be a therapeutic target for breast cancer. A breast cancer cell line with stable knockdown of DECR1 was constructed by lentiviral transfection to clarify the effect of inhibiting DECR1 expression on the malignant phenotype of breast cancer cells. Figure 7-14 Compared with the control group, the proliferation, migration and invasion abilities of the DECR1 knockdown cell lines were inhibited. In addition, an orthotopic xenograft model of breast cancer was constructed by injecting breast cancer cells of different treatment groups into the mammary fat pad of NOD-SCID mice to determine the role of DECR1 in vivo. The results showed that after DECR1 expression was inhibited, the growth rate of tumor transplants decreased, and the tumor volume and size were significantly lower than those of the control group. In summary, these results suggest that DECR1 promotes the progression of breast cancer both in vivo and in vitro.

[0116] 2.3 Targeting DECR1 to induce ferroptosis in breast cancer cells

[0117] The protein encoded by DECR1 is an auxiliary enzyme for fatty acid β-oxidation. The degradation of PUFA requires the help of multiple auxiliary enzymes, including δ2, δ3-enoyl-CoA isomerase and NADPH-dependent 2,4-dienoyl-CoA reductase (DECR1). DECR1 can act as a negative regulator of androgen receptor in prostate cancer. Inhibition of DECR1 can lead to the accumulation of PUFA in cells, triggering cellular oxidative stress and lipid peroxidation, thereby inducing cell ferroptosis. PUFA-related lipid peroxidation is a characteristic and driving factor of ferroptosis. To explore whether the expression level of DECR1 in breast cancer cells will interfere with cell ferroptosis. See Figure 15-21 First, transmission electron microscopy was used to observe whether the morphological changes of cells after DECR1 knockdown were consistent with the characteristics of ferroptosis. Electron microscopy results showed that after DECR1 knockdown, the mitochondrial membrane density was concentrated, the number of mitochondrial cristae was reduced or absent, the mitochondrial outer membrane was ruptured, and the cell nucleus size was normal. The above characteristics are consistent with the characteristics of breast cancer cells undergoing ferroptosis. Therefore, inhibition of DECR1 expression will induce ferroptosis in breast cancer cells. In addition, mitochondrial ROS and Fe in DECR1 knockdown MDA-MB-231 and BT474 cells were detected. 2+ The levels of mitochondrial ROS and Fe in DECR1 knockdown MDA-MB-231 and BT474 cells were measured to indicate the level of ferroptosis. The mean fluorescence intensity showed that compared with the negative control cells, the levels of mitochondrial ROS and Fe in DECR1 knockdown MDA-MB-231 and BT474 cells were significantly higher than those in the negative control cells. 2+ The levels of DECR1 and DECR1 were significantly increased. In addition, the content of MDA, a product of cellular lipid oxidation, was detected, and it was found that the MDA content increased in breast cancer cells with stable knockdown of DECR1. These findings all indicate that DECR1 knockdown may induce ferroptosis of breast cancer cells.

[0118] 2.4 Targeting DECR1 induces ferroptosis in breast cancer cells by promoting phosphatidylcholine metabolism

[0119] To further explore the potential mechanism by which DECR1 promotes cancer progression by interfering with cellular ferroptosis, transcriptome sequencing was performed on DECR1-knockdown MDA-MB-231 and BT474 cells to identify genes that were dysregulated after DECR1 knockdown. Considering the key role of DECR1 in fatty acid oxidation and its profound impact on cellular metabolism, metabolomics analysis was also performed to identify metabolites with significant changes. Figure 22-32Through multi-omics combined analysis, it was found that after DECR1 knockdown, PC metabolism was activated, PC content decreased, and conversely, AA content increased. At the same time, increased PLA2G12A expression levels were observed in breast cancer cells with DECR1 knockdown. In addition, mass spectrometry was used to measure the content of various phospholipids in breast cancer cells with DECR1 knockdown. The results showed that inhibiting the expression of DECR1 significantly reduced the content of various PCs and increased the content of AA.

[0120] AA is an omega-6 PUFA with the widest distribution and important biological activities in the body. Under physiological conditions, AA is mainly bound to PC or phosphatidylethanolamine glycerol. Under certain stimuli, phospholipase A2 (PLA2) is activated and catalyzes the hydrolysis of phospholipid diacyl groups, resulting in the release of AA into cells in a free form. The AA metabolic pathway in the KEGG database showed that PC hydrolysis produced a large amount of AA under the catalysis of PLA2G12A. DECR1 knockdown cells showed an increase in PLA2G12A levels. In cells with stable knockdown of DECR1, further knockdown of PLA2G12A was accompanied by a decrease in intracellular AA content, and breast cancer cells were protected from ferroptosis. These results indicate that targeting the DECR1-PLA2G12A signaling axis in breast cancer can promote AA production by regulating phospholipid metabolism, activate cellular ferroptosis, and thus inhibit tumor progression.

[0121] 2.5DECR1 inhibitor Erigoster B exhibits anti-tumor activity

[0122] It is shown that inhibition of DECR1 may be a promising strategy for treating breast cancer. Therefore, computer-assisted virtual screening and molecular docking methods were used to search for small molecules targeting DECR1 to preliminarily explore their potential as candidate drugs. Figure 33-42After screening and molecular docking scoring, the compound Erigoster B was found to be the most suitable for binding to DECR1 protein. The main amino acid residues for DECR1 to interact with Erigoster B are GLY66, THR69, GLY70, LEU71, GLY72, ASN144, ALA145, ALA146, ASN148, ILE150, ILE195, THR196, THR197, LYS214, PRO240, GLY241, PRO242, ILE243, THR245 and ARG251. In addition, Erigoster B forms hydrogen bonds with THR69, ASN144, THR196, LYS214, ILE243 and SER250, and these hydrogen bonds play a vital role in the binding of DECR1 and ErigosterB. In vitro SPR experiments verified the binding ability of Erigoster B to DECR1, and the results showed that DECR1 captured on the COOH chip could bind to Erigoster B with an affinity constant of 40.5 μM. In addition, the anti-tumor effect of Erigoster B was evaluated and it was found that it could inhibit the proliferation of breast cancer cells.

[0123] The effect of Erigoster B on the malignant phenotype of breast cancer cells was detected. The results showed that after 48 hours of Erigoster B treatment, the migration and invasion ability of breast cancer cells were significantly inhibited. After preliminary evaluation of drug treatment, it was found that the IC50 of Erigoster B in MDA-MB-231 cells was 17.12μM and in BT474 cells was 16.36μM. ErigosterB treatment can also effectively inhibit tumor growth in vivo, and its effect is comparable to that of paclitaxel. These findings suggest that Erigoster B has the potential to treat breast cancer and can be further studied as a candidate drug against breast cancer.

[0124] 2.6 Erigoster B induces ferroptosis in breast cancer cells

[0125] Erigoster B is a phenolic acid compound that was first identified in a systematic study of the phytochemistry and active components of Erigosterone. This compound has been shown to have antioxidant activity comparable to that of baicalin. Fig.43 In an attempt to rescue Erigoster B-induced cell death, different cell death inhibitors were used and it was found that ferroptosis was the main mechanism of Erigoster B-induced breast cancer cell death. As expected, Erigoster B treatment reduced mitochondrial ROS and Fe 2+levels, indicating that Erigoster B may play an anti-tumor role by inducing ferroptosis in breast cancer cells. In addition, we found that after ErigosterB treatment of breast cancer cells, intracellular PC content decreased and AA content increased. These findings suggest that Erigoster B has anti-tumor activity and induces ferroptosis by regulating PC metabolism and increasing AA content.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An anti-breast cancer drug, characterized in that: The invention comprises an active ingredient, wherein the active ingredient is a DECR1 inhibitor or a DECR1 binding agent.

2. Use of a DECR1 inhibitor or a DECR1 binder in the preparation of a drug for treating or preventing breast cancer.

3. Use of DECR1 inhibitors or DECR1 binders in the preparation of drugs for improving the sensitivity or therapeutic effect of anti-breast cancer drugs in the treatment or prevention of breast cancer.

4. The medicine according to claim 1 or the use according to claim 2 or 3, characterized in that: The DECR1 is highly expressed in breast cancer tissue; the high expression of DECR1 is associated with a poor prognosis of breast cancer.

5. The medicine according to claim 1 or the use according to claim 2 or 3, characterized in that: Knockdown of DECR1 expression level in breast cancer reduced the proliferation, migration and invasion abilities of breast cancer cell lines.

6. The medicine according to claim 1 or the use according to claim 2 or 3, characterized in that: Knockdown of DECR1 expression in breast cancer reduces mitochondrial ROS and Fe 2+ The level and MDA content increased.

7. The medicine according to claim 1 or the use according to claim 2 or 3, characterized in that: Knocking down the expression level of DECR1 in breast cancer increases the probability of ferroptosis of breast cancer cells.

8. The medicine according to claim 1 or the use according to claim 2 or 3, characterized in that: Knocking down the expression level of DECR1 in breast cancer reduces the PC content in breast cancer cells; knocking down the expression level of DECR1 in breast cancer increases the AA content in breast cancer cells; knocking down the expression level of DECR1 in breast cancer increases the expression level of PLA2G12A in breast cancer cells; The expression level of PLA2G12A in breast cancer cells is negatively correlated with the PC content in breast cancer cells, and positively correlated with the AA content in breast cancer cells; the expression level of PLA2G12A in breast cancer cells is positively correlated with the probability of ferroptosis of breast cancer cells.

9. The medicine according to claim 1 or the use according to claim 2 or 3, characterized in that: The DECR1 binder is Erigoster B.

10. The medicine according to claim 9 or the use according to claim 2 or 3, characterized in that: The ErigosterB forms hydrogen bonds with THR69, ASN144, THR196, LYS214, ILE243 and SER250, and binds to the DECR1.

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