Method for analyzing new anti-breast cancer action mechanism of lalotinib induced ferroptosis based on network pharmacology and biological methods

Abstract

The invention discloses a method for analyzing a new anti-breast cancer action mechanism of lalotinib induced ferroptosis based on network pharmacology and biological methods, and belongs to the technical field of biomedicine. In combination with multiple disciplines of network pharmacology, molecular docking, bioinformatics, molecular biology and the like, a new anti-breast cancer mechanism of lalotinib is discussed, and the method comprises the following processes: obtaining lalotinib, breast cancer and ferroptosis related genes in a database; further screening out five core targets by utilizing network pharmacology, molecular docking and bioinformatics technologies; a new mechanism of lalotinib for resisting breast cancer, namely induction of ferroptosis of breast cancer cells, is disclosed by a molecular biological technology. Compared with the prior art, by combining a multidisciplinary analysis technology, the invention aims to widen the clinical application range of lalotinib and provide theoretical support for treating breast cancer by lalotinib.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and relates to a method for analyzing a new mechanism of action of larotrectinib in inducing ferroptosis against breast cancer based on network pharmacology and biological methods. Background Art

[0002] Breast cancer remains the leading cause of cancer death among women worldwide. Despite significant progress in early detection and multiple treatments (including surgery, chemotherapy, radiotherapy, and hormone therapy), the prognosis of advanced breast cancer remains poor, mainly due to cancer metastasis and drug resistance. Given the complex heterogeneity and genetic diversity of breast cancer cells, traditional therapies often fail to meet treatment needs. Therefore, there is an urgent need to develop more targeted and efficient treatment strategies.

[0003] Recent developments in molecular targeted therapy have opened up new avenues for cancer treatment and emphasized the importance of precision medicine. As a highly effective and selective inhibitor of TRK (tropomyosin receptor kinase) protein, larotrectinib has demonstrated excellent efficacy in treating tumors carrying NTRK gene fusion mutations. Although these fusions are relatively rare, they may occur in a variety of cancer types, including breast cancer. Studies have confirmed the effectiveness of larotrectinib in reducing the size of cancer tumors carrying TRK fusions and inhibiting their progression. However, the specific mechanism of action of larotrectinib in breast cancer, especially for those cancers that are not driven solely by NTRK fusions, still needs further in-depth study.

[0004] Ferroptosis is a regulated cell death mechanism driven by iron-dependent lipid peroxidation and has attracted much attention as a new strategy for cancer treatment. It is different from other forms of cell death, such as apoptosis and necrosis, and involves unique metabolic pathways. Recent studies have revealed that inducing ferroptosis can overcome the drug resistance mechanisms of various cancers, including those that are resistant to traditional therapies. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for analyzing the new mechanism of action of larotrectinib in inducing ferroptosis against breast cancer based on network pharmacology and biological methods, predicting the molecular targets and related pathways of larotrectinib through network pharmacology and molecular docking, inferring its potential mechanism for treating breast cancer, and jointly applying bioinformatics, molecular biology and other technologies to reveal the new mechanism of action of larotrectinib in inhibiting the progression of breast cancer by inducing ferroptosis, broadening the clinical application scope of larotrectinib, and providing theoretical support for future research on the treatment of breast cancer with larotrectinib.

[0006] In order to achieve the above object, the present invention provides a method for analyzing the new mechanism of action of larotrectinib inducing ferroptosis against breast cancer based on network pharmacology and biological methods, comprising the following steps:

[0007] 1. Obtain the targets of larotrectinib, breast cancer-related genes, and ferroptosis-related genes

[0008] The targets of larotrectinib were searched in the SwissTargetPrediction, BATMAN, Similarity ensemble approach (SEA) and PharmMapper databases respectively. Among them, in the SwissTargetPrediction database, Homo sapiens and probability*>0 were used as screening conditions. In the BATMAN database, Score cutoff≧5 and p<0.05 were used as screening conditions. The SEA database used "human" as the screening condition. The UniProt database (http: / / www.uniprot.org) was used to convert the UniProt ID obtained by PharmMapper into a standard gene name. Finally, the genes screened from the four databases were deduplicated to obtain the potential targets of larotrectinib.

[0009] Breast cancer targets were screened in GeneCards, Comparative Toxicogenomics Database (CTD), PharmGKB, OMIM, and Therapeutic Target Database (TTD) databases. In the GeneCards database, Relevance score ≧20 was used as the screening condition. In the CTD database, Inference Score ≧100 was used as the screening condition. After removing duplicate values, the disease targets of breast cancer were finally obtained.

[0010] FerrDb database was used to search for genes related to ferroptosis. Human was used as the screening principle, and after removing duplicate values, ferroptosis-related genes were obtained.

[0011] 2. Acquisition of target genes at the intersection of larotrectinib, ferroptosis and breast cancer

[0012] The genes of larotrectinib, ferroptosis and breast cancer were introduced into jvenn to draw the Venn diagram. The intersection genes obtained from the Venn diagram are the target genes of larotrectinib-induced ferroptosis for the treatment of breast cancer.

[0013] 3. Construct a protein interaction network (PPI) of the intersection target genes and perform GO and KEGG enrichment analysis on the PPI network

[0014] The intersection target genes obtained in 2 above were imported into Search Tool for the Retrieval of Interacting Genes (STRING) to construct a PPI network diagram. Topological analysis was performed using Cytoscape 3.9.1 software to screen the core targets in the PPI network. The core targets were visualized using R language. GO and KEGG enrichment analysis was performed on the core target proteins of the PPI network using the Metscape website, and the target proteins were associated with biological processes (BP), cellular components (CC), and molecular functions (MF) above the p<0.01 threshold to screen out pathways. Visual analysis of the top 20 KEGG pathways was performed.

[0015] 4. Molecular docking analysis of the affinity of larotrectinib to the core target protein

[0016] The first five target proteins in the above 3 were selected for molecular docking. The 2D structure of larotrectinib was converted into a 3D structure by Chem 3D software. In the PDB database, the structure of the docked protein was obtained according to the screening principles of Homo sapiens, Refinement Resolution ≦2.5 and 7.3≦pH≦7.4. The receptor protein was pre-processed by dehydration, residue removal, hydrogenation and charge calculation using PyMOL software, and the target protein structure was molecularly docked with larotrectinib using AutoDock software, and the results were visualized and analyzed using PyMOL software.

[0017] 5. Bioinformatics analysis of the correlation between core target proteins and clinical factors

[0018] The mRNA expression level and pathological stage of the core target protein in molecular docking were determined by the GEPIA database. The immunohistochemical staining data of normal tissues and breast cancer tissues were obtained using the Human Protein Atlas (HPA) database to detect protein expression levels. Clinical data and transcriptomics (RNA-seq) data of breast cancer patients and normal subjects were obtained in the Cancer Genome Atlas Genomic Data Commons Data Portal (TCGA GDC) database. The differential expression of specific genes in cancer patients and normal subjects was analyzed using the R software packages "limma" and "ggpubr", and box plots of clinical relevance were drawn.

[0019] 6. Cell biology technology reveals new mechanism of larotrectinib against breast cancer

[0020] 6.1 Cell culture

[0021] The present invention selects human breast cancer cell lines MCF7 and MDA-MB-231 cells as in vitro models. MCF7 and MDA-MB-231 cells are cultured in 10% FBS+RPMI 1640 and 10% FBS+DMEM culture media, respectively.

[0022] 6.2 CCK8 analysis

[0023] MCF7 or MDA-MB-231 cells were seeded in 96-well plates and cultured for 24 hours, treated with different reagents, and then incubated with CCK8 reagent for 1 hour. The absorbance at a wavelength of 450 nm was recorded using an ELISA reader.

[0024] 6.3 Real-time fluorescence quantitative PCR

[0025] Total RNA was extracted from cells using a total RNA extraction kit (R1200, Solarbio, China) according to the manufacturer's instructions. cDNA was synthesized using the III RT SuperMix for qPCR (+gDNA wiper) kit (R223-01, Vazyme, China). RT-qPCR was performed using the PCR 2×Taq Plus Master Mix II kit (Dye Plus, P213-01, Vazyme) on an ABI 7500 real-time fluorescence quantitative PCR system (Applied Biosystem, USA). The primers used in the RT-qPCR reaction are as follows:

[0026] GAPDH: Forward-AAATCCCATCACCATCTTCCAG, Reverse-AGGGGCCATCCACAGTCTTCT;

[0027] PTGS2: Forward-TAAGTGCGATTGTACCCGGAC, Reverse-TTTGTAGCCATAGTCAGCATTGT;

[0028] EGFR: Forward-ATGCTTCACAACCCACCA, Reverse-GCCCTTCGCACTTCTTACAC;

[0029] HRAS: Forward-AGCAGGTGGTCATTGATGG, Reverse-GTTTGATCTGCTCCCTGTACT;

[0030] HMOX1: Forward-AAGACTGCGTTCCTGCTCAAC, Reverse-AAAGCCCTACAGCAACTGTCG.

[0031] 6.4Western blot

[0032] Total protein was extracted with RIPA lysis buffer (C1053, Applygen, China) containing protease cocktail inhibitor (4693132001, Roche, USA). Protein concentration was determined with BCA protein assay reagent (23228, Thermo Fisher Scientific, USA). Total protein at a concentration of 10 μg / μL was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane (1620177, Millipore, USA). After incubation with specific primary and secondary antibodies, the blot was detected using a chemiluminescent imager (Fusion SoloS.EDGE, VILBER, France) using Immobilon Wester chemilum HRP substrate (WBKLS0500, Millipore). The antibodies used in western blotting were as follows: anti-α-tubulin antibody (B1052, Biodragon, China), anti-HRAS antibody (AF7131, Beyotime, China), anti-EGFR antibody (AG1810, Beyotime), anti-PTGS2 antibody (AF1924, Beyotime), anti-HMOX1 antibody (AF1333, Beyotime), goat anti-rabbit IgG H&L (HRP) (ab97051, Abcam, USA).

[0033] 6.5 Analysis of intracellular lipid reactive oxygen species (Lipid ROS) levels

[0034] MCF7 cells were seeded in 12-well plates and cultured for 24 hours. After treatment with different reagents, 10 μM Lipid Peroxidation Senser (D3861, Invitrogen, USA), incubate for 30 min in the dark, wash the cells with PBS and analyze by flow cytometry.

[0035] 6.6 Analysis of cellular iron levels

[0036] MCF7 cells were inoculated in 6-well plates and cultured for 24 hours. After treatment with different reagents, the intracellular ferrous and total iron contents were determined using the Iron Assay kit (ab83366, Abcam). After incubation at 37°C for 60 minutes, the absorbance was measured at 593 nm using a microplate reader.

[0037] 7. Statistical Analysis

[0038] The in vitro experimental data were presented in three independent replicates, and all statistical analyses were performed using GraphPad Prism 8.0 and Image J software. One-way ANOVA analysis was performed followed by Tukey's multiple comparisons tests for comparisons among multiple groups. P values ​​less than 0.05 were considered statistically significant.

[0039] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0041] Figure 1 The present invention is a method for predicting the core genes of larotrectinib-induced ferroptosis against breast cancer. (A) Veen diagram of target genes of larotrectinib, (B) Veen diagram of targets of breast cancer, (C) Veen diagram of larotrectinib-ferroptosis-breast cancer intersection genes, (D) PPI network diagram of larotrectinib-induced ferroptosis against breast cancer was drawn using the STRING database, (E) Topological analysis network of target genes of larotrectinib-induced ferroptosis against breast cancer was drawn using Cytoscape 3.9.1 software, (F) Visual analysis of core targets from low to high levels was performed using R software.

[0042] Figure 2 The method for analyzing the GO function and KEGG pathway of the core target genes of larotrectinib-induced ferroptosis against breast cancer in the present invention. (A) Bubble chart of GO function enrichment analysis of core target genes, (B) Bar chart of KEGG pathway analysis of core target genes.

[0043] Figure 3 The method for analyzing the mechanism of action of larotrectinib in inducing ferroptosis against breast cancer in the present invention. (A) Heat map of molecular docking results, (B) docking sites of larotrectinib with HRAS, EGFR, ALB, PTGS2, and HMOX1. (C) Violin plot showing the relationship between mRNA expression level and pathological stage of breast cancer.

[0044] Figure 4 This is a method for analyzing the clinical relevance of core target genes in the present invention. (AE) Box plots show the correlation between ALB, PTGS2, EGFR, HRAS and HMOX1 levels and age, gender, stage, M stage, N stage and T stage, respectively.

[0045] Figure 5 This is a method for analyzing the expression level of core target genes in the present invention. (A) Box plots show the mRNA expression levels of ALB, PTGS2, EGFR, HRAS and HMOX1, and (B) immunohistochemistry results show the protein expression levels of ALB, PTGS2, EGFR, HRAS and HMOX1.

[0046] Figure 6 The present invention experimentally verifies the method of larotrectinib inducing ferroptosis of breast cancer cells. (A) The CCK8 experiment was used to detect the effects of different doses of larotrectinib on the viability of MCF7 cells and MDA-MB-231 cells, (B) The CCK8 experiment was used to detect the effects of different doses of Fer-1 on the viability of MCF7 cells and MDA-MB-231 cells, (C) The expression level of mRNA in MCF7 cells was detected by RT-qPCR, (D) The expression level of protein in MCF7 cells was detected by Western blot, (E) The level of Lipid ROS in MCF7 cells was detected by BODIPY581 / 591C11 fluorescent probe, and (F) The iron ion content in MCF7 cells was detected by Iron Assay kit. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below with reference to the accompanying drawings. This embodiment is implemented based on the technical solution of the present invention, and provides detailed implementation methods and specific operating procedures. The chemical reagents used in the embodiments are all conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0048] Please also read Figure 1-Figure 6 This example is used to illustrate a method for analyzing the new mechanism of action of larotrectinib in inducing ferroptosis against breast cancer based on network pharmacology and biological methods, including the following steps:

[0049] Using network pharmacology to explore the new mechanism of larotrectinib against breast cancer

[0050] The present invention first obtains the PubChem CID, Canonical SMILES, 2D structure and other related information of larotrectinib from the PubChem database. In order to obtain the target gene of larotrectinib, the present invention collected 107, 38, 8 and 299 targets of larotrectinib in the SwissTargetPrediction database, BATMAN database, SEA database and PharmMapper database, respectively. Among them, when analyzing the results obtained in the PharmMapper database, the researchers converted the UniProt ID into a standard gene name through the UniProt database, and obtained a total of 289 genes. Finally, the genes obtained by screening the four databases were deduplicated, and a total of 403 targets of larotrectinib were obtained ( Figure 1 A). A total of 812, 870, 189, 247 and 120 breast cancer-related genes were screened from the GeneCards database, CTD database, PharmGKB database, OMIM database and TTD database ( Figure 1 B). After removing duplicate values, a total of 1723 breast cancer genes were obtained. The present invention obtained 214 target genes related to ferroptosis in the FerrDb database. By drawing a Veen diagram of the genes related to larotrectinib, breast cancer and ferroptosis, 15 common genes among the three were found ( Figure 1 C).

[0051] Construction of a protein interaction network of larotrectinib-ferroptosis-breast cancer

[0052] The 15 common target genes of larotrectinib, ferroptosis and breast cancer were imported into the STRING website to obtain the PPI network diagram ( Figure 1 D) and downloaded the TSV format file. The PPI network has 15 nodes, 45 edges and an average node degree of 6. The CytoNCA plug-in of Cytoscape3.9.1 software was used for topological analysis ( Figure 1 E), and visualize the core targets using R language ( Figure 1 F). Five core genes were screened by the principles of Closeness≧0.00296784084670864, ​​Betweenness≧173.894736842104 and Degree≧28.5029239766081, namely HRAS, EGFR, ALB, PTGS2 and HMOX1.

[0053] GO and KEGG enrichment analysis of “Larotrectinib-ferroptosis-breast cancer”

[0054] The 15 common target genes were subjected to GO function and KEGG pathway enrichment analysis by using the Metascape tool. In the process of GO enrichment analysis, 234 biological processes, 14 cellular components and 21 molecular functions were obtained, and the top 5 functions were visualized and analyzed ( Figure 2 A). While using the Metascape tool to obtain 87 KEGG pathways, the top 20 KEGG pathways were also visualized and analyzed ( Figure 2 B).

[0055] Molecular docking and bioinformatics exploration of the mechanism of "larotrectinib-ferroptosis-breast cancer"

[0056] Molecular docking was performed on the five core genes in the PPI network. HRAS (PDB ID: 1CLU), EGFR (PDB ID: 5XDK), ALB (PDB ID: 4IW2), PTGS2 (PDB ID: 5F1A) and HMOX1 (PDB ID: 5BTQ) were identified as docking proteins. The molecular docking results showed that the binding energy scores of larotrectinib with the five target proteins were all less than -5.0 kcal / mol ( Figure 3 A). The docking sites of larotrectinib and five target proteins are also shown in the attached figure ( Figure 3 B). The results of molecular docking demonstrated that larotrectinib can exert its anti-breast cancer therapeutic effect by acting on the cell ferroptosis pathway.

[0057] The GEPIA database was used to analyze the relationship between the expression levels of the five core genes and the pathological stages of breast cancer. The results showed that the mRNA level of PTGS2 changed significantly with the pathological stage of breast cancer, and was significantly reduced in stages IV and Ⅹ ( Figure 3 C). Clinical correlation analysis showed that the expression levels of ALB, PTGS2, EGFR, HRAS and HMOX1 were significantly correlated with the clinical characteristics of breast cancer. The expression levels of ALB, PTGS2, EGFR, HRAS and HMOX1 were significantly different according to T stage ( Figure 4 AE). The expression levels of PTGS2 and EGFR differed significantly by age and gender ( Figure 4 B, C). In addition, the expression levels of PTGS2 in different stages and M stages were significantly different ( Figure 4 B). The mRNA expression levels of ALB, PTGS2, EGFR, HRAS, and HMOX1 were analyzed using the GEPIA database. Compared with normal tissues, the mRNA of ALB, PTGS2, and EGFR in breast tissues was significantly downregulated ( Figure 5A). The immunohistochemistry results of the HPA database showed that compared with normal breast tissue, the expression of ALB, PTGS2 and HRAS in breast cancer tissue was higher than that in normal tissue, while EGFR was more highly expressed in normal tissue ( Figure 5 B).

[0058] In vitro experiments exploring the mechanism of larotrectinib-induced ferroptosis in breast cancer cells

[0059] In order to explore the molecular mechanism of larotrectinib against breast cancer, the present invention cultured breast cancer cell lines MCF7 cells and MDA-MB-231 cells in vitro, and induced the death of MCF7 cells and MDA-MB-231 cells with larotrectinib. In order to find the effective concentration of larotrectinib, the present invention first treated MCF7 cells and MDA-MB-231 cells with different concentrations of larotrectinib, and analyzed the cell viability by CCK8 experiment after 24 hours. The results showed that larotrectinib induced MCF7 cell death in a dose-dependent manner, where 100 μM larotrectinib treatment could cause more than 50% of MCF7 cell death ( Figure 6 A). However, 100 μM larotrectinib caused less than 50% cell death in MDA-MB-231 cells ( Figure 6 A). In order to demonstrate that larotrectinib exerts its anti-breast cancer effect by inducing ferroptosis, the present invention co-treated MCF7 cells and MDA-MB-231 cells with 100 μM larotrectinib and different concentrations of ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1) for 24 hours, and detected cell viability by CCK8 assay. We found that compared with Vehicle, treatment with 100 μM larotrectinib significantly induced MCF7 cell death, and Fer-1 could rescue MCF7 cell death induced by larotrectinib in a dose-dependent manner ( Figure 6 B). Compared with MCF7 cells, MDA-MB-231 cells are less sensitive to larotrectinib and Fer-1 ( Figure 6 B). Therefore, MCF7 cells were selected as a cell model of breast cancer.

[0060] PTGS2 and HMOX1 are important molecular markers of ferroptosis. The present invention used 100 μM larotrectinib and 10 μM Fer-1 to treat MCF7 cells for 24 hours, and detected the expression levels of PTGS2, EGFR, HRAS and HMOX1 by RT-qPCR and Western blot methods, respectively. The results showed that larotrectinib treatment increased the mRNA and protein levels of PTGS2, EGFR and HMOX1 in MCF7 cells, while Fer-1 could reduce their expression levels ( Figure 6C, D). The increase in intracellular Lipid ROS levels is a hallmark event of ferroptosis. The present invention uses BODIPY 581 / 591C11 fluorescent probe to detect the level of LipidROS in MCF7 cells. The flow cytometry test results show that larotrectinib can cause an increase in intracellular Lipid ROS levels, while co-treatment with Fer-1 and larotrectinib can significantly reduce the intracellular Lipid ROS level ( Figure 6 E). The accumulation of intracellular divalent iron ions is the main cause of ferroptosis. The present invention detected the intracellular iron ion concentration and found that the concentrations of Fe2+ and Fe3+ increased in MCF7 cells treated with larotrectinib, while the addition of Fer-1 successfully reduced the intracellular Fe2+ and Fe3+ concentrations ( Figure 6 F).

[0061] These results suggest that larotrectinib induces lipid peroxidation by causing iron overload in breast cancer cells, thereby inducing ferroptosis.

[0062] The embodiments of the present invention have been described above, and the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, equivalent replacements or changes can be made according to the technical solutions and inventive concepts of the present invention, and all such changes or replacements should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method based on network pharmacology and biological methods to analyze the new mechanism of action of larotrectinib in inducing ferroptosis against breast cancer.

2. According to claim 1, a method for analyzing the new mechanism of action of larotrectinib inducing ferroptosis against breast cancer based on network pharmacology and biological methods, characterized in that: The method combines a variety of technical means such as network pharmacology, molecular docking, bioinformatics analysis and molecular biology to analyze the new mechanism of the drug larotrectinib in fighting breast cancer.

3. The method for analyzing the new mechanism of action of larotrectinib inducing ferroptosis against breast cancer based on network pharmacology and biological methods according to claim 2, characterized in that: Network pharmacology technology was used to prove that the drug larotrectinib has common target genes with ferroptosis and breast cancer, and 5 core genes were screened out.

4. The method for analyzing the new mechanism of action of larotrectinib inducing ferroptosis against breast cancer based on network pharmacology and biological methods according to claim 2, characterized in that: Molecular docking technology was used to prove that the drug larotrectinib has good binding ability with the core gene.

5. The method for analyzing the new mechanism of action of larotrectinib inducing ferroptosis against breast cancer based on network pharmacology and biological methods according to claim 2, characterized in that: Bioinformatics technology was used to prove that core genes were differentially expressed in breast cancer.

6. The method for analyzing the new mechanism of action of larotrectinib in inducing ferroptosis against breast cancer based on network pharmacology and biological methods according to claim 2, characterized in that: Molecular biological techniques were used to demonstrate that the drug larotrectinib triggers lipid peroxidation by causing iron ion overload in breast cancer cells, thereby inducing ferroptosis.

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