Application of cedarwood biflavone in preparation of EGFR (epidermal growth factor receptor) inhibitor

Abstract

The invention provides application of cedar biflavone in preparation of an EGFR (epidermal growth factor receptor) inhibitor, and belongs to the technical field of biomedicine. The cedarwood biflavone (CAS number: 3952-18-9) can obviously inhibit proliferation, migration and invasion of lung cancer cells and induce ferroptosis and epithelial-mesenchymal transition (EMT) reversion by regulating and controlling an EGFR (epidermal growth factor receptor) signal channel. In-vivo researches further prove that the cedarwood biflavone can effectively inhibit the growth of xenograft tumors in nude mice H1299, and has no obvious visceral toxicity. Mechanism research shows that the cedarwood biflavone plays an anti-tumor role by inhibiting EGFR phosphorylation, regulating and controlling a downstream signal channel and mediating lipid peroxidation, ferroptosis and EMT inhibition. In addition, the combined use of the cedarwood biflavone and the erlotinib can enhance the anti-tumor effect. Therefore, the invention provides a potential candidate compound for developing novel lung cancer treatment drugs and EGFR phosphorylation targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of cupressuflavone in the preparation of an EGFR inhibitor. Background Art

[0002] Cupressuflavone is a naturally occurring biflavonoid compound, which is mainly extracted from plants of the Cupressaceae family, such as plants of the genera Cupressus and Platycladus orientalis. In addition, it also exists in plants of the genus Cycas of the Cycadaceae family. As a special subclass of flavonoid compounds, the remarkable feature of biflavonoids is that two flavone molecules are linked together by covalent bonds to form a unique chemical structure. This structure endows cupressuflavone with high chemical stability and diverse biological activities, making it highly concerned in traditional medicine and modern drug research.

[0003] Cupressuflavone is considered to have important potential application values due to its wide range of biological activities. Research shows that it has significant antioxidant effects, can scavenge free radicals, and reduce the damage of oxidative stress to cells; at the same time, it also exhibits anti-inflammatory properties and can relieve inflammatory responses by inhibiting inflammation-related signaling pathways (such as the NF-κB and MAPK pathways). In addition, cupressuflavone also shows antibacterial and antiviral potentials, and can interfere with the metabolism of pathogens or destroy their cell membranes, thereby inhibiting the growth and reproduction of microorganisms.

[0004] However, the current research on the role of cupressuflavone in lung cancer is still in its infancy, and its specific mechanism and efficacy are not yet clear. In particular, there is no relevant report on the targeted regulatory effect of the key protein EGFR in the occurrence and development of lung cancer. Summary of the Invention

[0005] The purpose of the present invention is to provide a new application of cupressuflavone in the preparation of an EGFR inhibitor.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: In the first aspect, the present invention provides the application of cupressuflavone in the preparation of an inhibitor for targeted inhibition of EGFR phosphorylation, and the CAS number of the cupressuflavone is 3952-18-9.

[0007] In the second aspect, the present invention provides an inhibitor for targeted inhibition of EGFR phosphorylation, and the inhibitor is composed of cupressuflavone and a pharmaceutically acceptable carrier, and the CAS number of the cupressuflavone is 3952-18-9.

[0008] Preferably, in the inhibitor, the concentration of the cupressuflavone is 20-80 μM.

[0009] In a third aspect, the present invention provides the use of cupressuflavone in the preparation of a drug for inhibiting lung cancer metastasis, and the CAS number of the cupressuflavone is 3952-18-9.

[0010] Preferably, the cupressuflavone inhibits lung cancer metastasis by targeting and inhibiting EGFR phosphorylation.

[0011] In a fourth aspect, the present invention provides a drug for inhibiting lung cancer metastasis, which is composed of cupressuflavone and a pharmaceutically acceptable carrier, and the CAS number of the cupressuflavone is 3952-18-9; The concentration of the cupressuflavone is 20-80 μM.

[0012] In a fifth aspect, the present invention provides the use of cupressuflavone in the preparation of a drug for promoting ferroptosis of lung cancer cells, the CAS number of the cupressuflavone is 3952-18-9, and the cupressuflavone promotes ferroptosis of lung cancer cells by targeting and inhibiting EGFR phosphorylation.

[0013] In a sixth aspect, the present invention provides a drug for promoting ferroptosis of lung cancer cells, which is composed of cupressuflavone and a pharmaceutically acceptable carrier, and the CAS number of the cupressuflavone is 3952-18-9; The concentration of the cupressuflavone is 20-80 μM.

[0014] In a seventh aspect, the present invention provides the use of a composition in the preparation of a drug for inhibiting lung cancer metastasis and ferroptosis of lung cancer cells. The composition is composed of cupressuflavone and erlotinib. The CAS number of the cupressuflavone is 3952-18-9, and the CAS number of the erlotinib is 183321-74-6.

[0015] Preferably, in the composition, the concentration of the cupressuflavone is 80 μM, and the concentration of the erlotinib is 10 μM.

[0016] The beneficial effects of the present invention are as follows: 2. Cupressuflavone can induce ferroptosis of lung cancer cells by regulating the expression of ferroptosis-related proteins (such as GPX4, SLC7A11, TF, FTH1, and ALOX15). Transmission electron microscopy observation shows that after treatment with cupressuflavone, the mitochondrial cristae of lung cancer cells decrease and the membrane density increases, which are typical morphological changes of ferroptosis. In addition, after being combined with the ferroptosis inhibitor DFO, the anti-tumor effect of cupressuflavone is reversed, further verifying its mechanism of exerting anti-cancer effects through the ferroptosis pathway.

[0017] 3. Through screening with the SwissTarget Prediction database and RNA-seq sequencing analysis, combined with molecular docking verification, it was clarified that the core target of cupressuflavone against lung cancer is EGFR. At the same time, it was detected that cupressuflavone can directly target and inhibit the phosphorylation of EGFR, so it can be used as an EGFR phosphorylation-targeted inhibitor.

[0018] 4. By using it in combination with the EGFR tyrosine kinase inhibitor erlotinib, the anti-tumor effect of cupressuflavone is enhanced, so cupressuflavone and erlotinib can be combined to synergistically inhibit lung cancer metastasis and promote ferroptosis of lung cancer cells. Description of the Drawings

[0019] Figure 1 Effects of cupressuflavone on the proliferation viability of lung cancer cells (n = 4) (A) Changes in the proliferation viability of A549 after treatment with cupressuflavone for 24 and 48 h; (B) Changes in the proliferation viability of H1299 after treatment with cupressuflavone for 24 and 48 h. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001; Figure 2 Effects of cupressuflavone on the proliferation viability of BEAS-2B cells (n = 4); compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 3 shows the effects of cupressuflavone on the colony formation ability of A549 and H1299 cells (n = 3); (A) Representative pictures of colony formation after 14 days of cells; (B) Quantitative analysis of cell clone formation rate; compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 4 Effects of cupressuflavone on the scratch healing ability of A549 and H1299 cells (n = 3); (A) Representative pictures of cell scratch healing; (B) Quantitative analysis of cell scratch healing rate; compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 5 shows the effects of cupressuflavone on the migration ability of A549 and H1299 cells (n = 5); (A) Representative pictures of cell migration; (B) Quantitative analysis of relative cell migration rate; compared with the control group, * p < 0.05, ** p < 0.01, *** p< 0.001; Figure 6 shows the effect of cupressuflavone on the invasion ability of A549 and H1299 cells (n = 5); (A) Representative pictures of cell invasion; (B) Quantitative analysis of relative cell invasion rate; compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 7 shows the effect of cupressuflavone on the mitochondrial membrane potential of A549 and H1299 cells (n = 3); (A) Representative pictures of the change in mitochondrial membrane potential detected by flow cytometry; (B) Quantitative analysis of the ratio of JC-1 red and green fluorescence intensities; compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 8 shows the effect of cupressuflavone on apoptosis of A549 and H1299 cells (n = 5); (A) Representative pictures of cell apoptosis detected by flow cytometry; (B) Quantitative analysis of cell apoptosis rate; compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 9 shows the analysis of differentially expressed genes and pathway enrichment after treatment of H1299 cells with cupressuflavone; (A) Volcano plot of differentially expressed genes; (B) KEGG enrichment analysis plot of differentially expressed genes; (C) GO enrichment analysis plot of differentially expressed genes; Figure 10 shows the effect of cupressuflavone on the mitochondrial structure of A549 and H1299 cells observed by transmission electron microscopy; blue arrows indicate mitochondria, scale bar = 5 μm; Figure 11 shows the effect of cupressuflavone combined with ferroptosis inhibitor on the expression of ferroptosis-related proteins GPX4, SLC7A11, TF, FTH1 and ALOX15 in A549 and H1299 cells; Representative bands of the effect of different doses of cupressuflavone on ferroptosis-related proteins in A549 and H1299 cells; Figure 12 shows the effect of cupressuflavone combined with ferroptosis inhibitor on cell viability and intracellular Fe 2+ levels; (A) Changes in cell viability after combination with DFO; (B) Quantitative analysis of intracellular Fe 2+ concentration; Model group (Model, 0 μM), Cupressuflavone group (CPF, 80 μM), Deferoxamine group (DFO, 20 μM), Cupressuflavone combined with Deferoxamine group (CPF + DFO); compared with the control group, * p< 0.05, ** p < 0.01, *** p < 0.001; Compared with the cupressuflavone group, # p < 0.05, ## p < 0.01, ### p < 0.001; Figure 13 This is the effect of cupressuflavone on the expression of EMT-related proteins ZO-1, N-cadherin, E-cadherin, Vimentin and Snail in A549 cells; (A-F) Representative bands and corresponding quantitative statistical results of EMT-related proteins in A549 cells; (G) Representative fluorescence images of EMT-related proteins in A549 cells; Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 14 shows the effect of cupressuflavone on the expression of EMT-related proteins ZO-1, N-cadherin, E-cadherin, Vimentin and Snail in H1299 cells; (A-F) Representative bands and corresponding quantitative statistical results of EMT-related proteins in H1299 cells, (G) Representative fluorescence images of EMT-related proteins in H1299 cells; Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 15 shows the effect of cupressuflavone on the growth of subcutaneous xenografts of lung cancer H1299 in nude mice; (A) Pictures of nude mice in each group; (B) Pictures of intact tumors; (C) Tumor volume growth within 16 days after administration; (D) Quantitative analysis of tumor weight; (E) Representative pathological section images of tumors in nude mice in each group; Model group (Model, 0.5% normal saline), cupressuflavone group (CPF, 160 mg / kg), deferoxamine group (DFO, 100 mg / kg), cupressuflavone combined with deferoxamine group (CPF+DFO); Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001; Compared with the cupressuflavone group, # p < 0.05, ## p < 0.01, ### p < 0.001; Figure 16 shows the effect of cupressuflavone on the proliferation of subcutaneous transplanted tumor cells of lung cancer H1299 nude mice; (A) Representative fluorescence images of EdU staining of transplanted tumor tissues of nude mice in each group; (B) Quantitative analysis of the number of EdU-positive cells in transplanted tumor tissues; Model group (Model, 0.5% normal saline), Cupressuflavone group (CPF, 160 mg / kg), Deferoxamine group (DFO, 100 mg / kg), Cupressuflavone combined with Deferoxamine group (CPF+DFO); Compared with the control group, * p <0.05, ** p <0.01, *** p <0.001; Compared with the cupressuflavone group, # p <0.05, ## p <0.01, ### p <0.001; Figure 17 shows the effect of cupressuflavone on various organs of nude mice with subcutaneous transplanted tumors of lung cancer H1299; Representative images of pathological changes in the heart, liver, spleen, lung, and kidney of nude mice in each group detected by H&E staining; Model group (Model, 0.5% normal saline), Cupressuflavone group (CPF, 160 mg / kg), Deferoxamine group (DFO, 100 mg / kg), Cupressuflavone combined with Deferoxamine group (CPF+DFO); Figure 18 shows the effect of cupressuflavone on the expression of ferroptosis-related proteins GPX4, SLC7A11, TF, FTH1, and ALOX15 in the tissues of subcutaneous transplanted tumors of lung cancer H1299 nude mice; Representative images of the expression of ferroptosis-related proteins detected by immunohistochemical staining in the transplanted tumor tissues of nude mice in each group; Model group (Model, 0.5% normal saline), Cupressuflavone group (CPF, 160 mg / kg), Deferoxamine group (DFO, 100 mg / kg), Cupressuflavone combined with Deferoxamine group (CPF+DFO); Figure 19 shows the effect of cupressuflavone on the expression of EMT-related proteins E-cadherin and Vimentin in the tissues of subcutaneous transplanted tumors of lung cancer H1299 nude mice; (A) (B) Representative fluorescence images of E-cadherin in the transplanted tumor tissues of nude mice in each group and corresponding quantitative analysis of fluorescence intensity; (C) (D) Representative fluorescence images of Vimentin in the transplanted tumor tissues of nude mice in each group and corresponding quantitative analysis of fluorescence intensity; Model group (Model, 0.5% normal saline), Cupressuflavone group (CPF, 160 mg / kg), Deferoxamine group (DFO, 100 mg / kg), Cupressuflavone combined with Deferoxamine group (CPF+DFO); Compared with the control group, * p <0.05, **p < 0.01, *** p < 0.001; Compared with the cupressuflavone group, # p < 0.05, ## p < 0.01, ### p < 0.001; Figure 20 For the acquisition of potential targets of cupressuflavone against lung cancer and pathway enrichment analysis; (A) Potential targets of cupressuflavone against lung cancer; (B) Venn diagram of the intersection of potential targets of cupressuflavone against lung cancer and differential targets screened by RNA-seq sequencing; (C) KEGG enrichment analysis diagram of 105 potential targets of cupressuflavone against lung cancer; Figure 21 For the screening of core targets of cupressuflavone against lung cancer; (A) Differential expression analysis of TNKS2, TNKS, PTPRS, IGF1R, EGFR and AXL; (B-G) Prognostic survival analysis of TNKS2, TNKS, PTPRS, IGF1R, EGFR and AXL; (H) Molecular docking of EGFR and cupressuflavone; (I) Binding stability of EGFR and cupressuflavone by molecular dynamics simulation; (J) Detection of the binding stability of EGFR and cupressuflavone by surface plasmon resonance experiment (SPR); (K) Evaluation of the binding affinity of cupressuflavone and EGFR by microscale thermophoresis experiment (MST); Figure 22 For the effects of cupressuflavone combined with Erlotinib and EGF on the protein expressions of EGFR and p-EGFR in A549 and H1299 cells; Figure 23 For the effects of EGFR signaling on the cytotoxicity of cupressuflavone against lung cancer cells; Model group (Model, 0 μM), Cupressuflavone group (CPF, 80 μM), Erlotinib group (Erlotinib, 10 μM), Cupressuflavone combined with Erlotinib group (CPF+Erlotinib), Epidermal growth factor group (EGF, 50 ng / mL), Cupressuflavone combined with epidermal growth factor group (CPF+EGF); * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 24Effect of EGFR signaling on the ability of cupressuflavone to promote lipid peroxidation; (A)(B) Representative fluorescence images of intracellular ROS levels in A549 and H1299 cells detected by flow cytometry; (C)(D) Quantitative analysis of the fluorescence intensity of the BODIPY 581 / 591 C11 probe in cells; Model group (Model, 0 μM), Cupressuflavone group (CPF, 80 μM), Erlotinib group (Erlotinib, 10 μM), Cupressuflavone combined with Erlotinib group (CPF+Erlotinib), Epidermal growth factor group (EGF, 50 ng / mL), Cupressuflavone combined with Epidermal growth factor group (CPF+EGF); * p <0.05, ** p <0.01, *** p <0.001; Figure 25 Effect of EGFR signaling on the expression of ferroptosis-related proteins regulated by cupressuflavone in lung cancer cells; (A-B) Representative fluorescence images of GPX4 protein expression in A549 cells and corresponding quantitative analysis of fluorescence intensity; (C-D) Representative fluorescence images of FTH1 protein expression in A549 cells and corresponding quantitative analysis of fluorescence intensity; (E-F) Representative fluorescence images of GPX4 protein expression in H1299 cells and corresponding quantitative analysis of fluorescence intensity; (G-H) Representative fluorescence images of FTH1 protein expression in H1299 cells and corresponding quantitative analysis of fluorescence intensity; *p<0.05, **p<0.01, ***p<0.001; Figure 26 Effect of EGFR signaling on the expression of EMT-related proteins regulated by cupressuflavone in lung cancer cells; (A)(B) Representative fluorescence images of E-cadherin in A549 cells and corresponding quantitative analysis of fluorescence intensity; (C)(D) Representative fluorescence images of Vimentin in A549 cells and corresponding quantitative analysis of fluorescence intensity; (E)(F) Representative fluorescence images of E-cadherin in H1299 cells and corresponding quantitative analysis of fluorescence intensity; (G)(H) Representative fluorescence images of Vimentin in H1299 cells and corresponding quantitative analysis of fluorescence intensity; * p <0.05, ** p <0.01, *** p <0.001. Specific implementation manners

[0020] To clearly illustrate the technical features of this solution, the following is an elaboration of this solution through specific implementation manners.

[0021] Example 1 Evaluation of the anti-lung cancer effect of cupressuflavone in vitro Cupressuflavone (CPF) used in the present invention: molecular weight 538.46, CAS No. 3952-18-9, purchased from Chengdu Lemeitian Medical Technology Co., Ltd., batch number: DSTDB031901, purity (HPLC≥98%).

[0022] The experimental cells used in the present invention are as follows: lung cancer cell A549 (resource number: B26030), lung cancer cell H1299 (resource number: B26058), purchased from Sichuan Bio-One Biotechnology Co., Ltd. Normal lung epithelial cell BEAS-2B (product number: CL0044), purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0023] (1) Detect the effect of cupressuflavone on the proliferation of lung cancer cells and normal BEAS-2B cells To determine the effect of cupressuflavone on the proliferation activity of lung cancer cells, first, the CCK-8 method was used to detect the survival rates of A549 and H1299 cells after treatment with different doses of cupressuflavone for 24 and 48 h. The results obtained are as Figure 1 shown.

[0024] Meanwhile, to determine the safe dose range of cupressuflavone, we used human normal lung epithelial cell BEAS-2B as the control cell and detected the effect of different doses of cupressuflavone on the cell survival rate of BEAS-2B after 24 h of treatment using the CCK-8 method. The results obtained are as Figure 2 shown.

[0025] From Figure 1 the results, it can be seen that cupressuflavone at a concentration of 40 μM and above can significantly inhibit the cell viability of A549 and H1299 cells after 24 h or 48 h of treatment, and as the administration concentration increases, the cell viability gradually decreases. However, compared with 48 h, the cells are more sensitive to the treatment of cupressuflavone at 24 h of treatment time. Therefore, we selected 24 h as the subsequent administration time.

[0026] From Figure 2 the results, it can be seen that within the dose range of 0-80 μM, the cell damage of cupressuflavone to BEAS-2B is less than 10%. When it exceeds 80 μM, cupressuflavone shows a certain cytotoxic effect on BEAS-2B. Therefore, we used 80 μM as the highest concentration for the subsequent experiments. The above results indicate that cupressuflavone can selectively inhibit the proliferation of lung cancer cells A549 and H1299 in a concentration-dependent manner.

[0027] (2) Detect the inhibitory effect of cupressuflavone on the colony formation of lung cancer cells To determine the effect of cupressuflavone on the clonogenic ability of lung cancer cells, the present invention used a plate colony formation assay to detect the formation of cell colonies after culturing single-dispersed lung cancer cells for 14 days following treatment with cupressuflavone. Using a dosing concentration of 0 μM as the control group, the results obtained were as Figure 3 shown.

[0028] From Figure 3 the results, it can be seen that after treatment with cupressuflavone, the colony formation rates of A549 and H1299 cells decreased with increasing concentration. At a concentration of 80 μM, both the number and size of cell colonies were much lower than those of the control group. The results indicate that cupressuflavone can inhibit the colony formation ability of A549 and H1299 cells in a concentration-dependent manner.

[0029] (3)Detecting the inhibitory effect of cupressuflavone on the migration of lung cancer cells Cell migration is a basic feature of tumor metastasis. The present invention used a scratch assay and a Transwell assay to detect the effect of cupressuflavone on the migration ability of A549 and H1299 cells. Using a dosing concentration of 0 μM as the control group, the results obtained were as Figure 4 and Figure 5 shown.

[0030] From Figure 4 the results, it can be seen that 24 hours after administration of cupressuflavone, the scratch widths of A549 and H1299 cells decreased with increasing dosing concentration. At a concentration of 80 μM, the relative scratch healing rate of A549 was less than 15%, and that of H1299 was less than 10%.

[0031] From Figure 5 the results, it can be seen that cupressuflavone can significantly inhibit the migration of A549 and H1299 cells to the lower layer of the chamber. At a concentration of 80 μM, the relative migration rates of A549 and H1299 were approximately 50%. The above results indicate that cupressuflavone can inhibit the migration of A549 and H1299 cells in a concentration-dependent manner.

[0032] (4)Detecting the inhibitory effect of cupressuflavone on the invasion of lung cancer cells In addition to relying on migration, tumor metastasis is also closely related to cell invasion. Therefore, the present invention used a Transwell assay to detect the effect of cupressuflavone on the invasion ability of lung cancer cells. Using a dosing concentration of 0 μM as the control group, the results obtained were as Figure 6 shown.

[0033] From Figure 6As shown by the results, the higher the concentration of cupressuflavone, the fewer cells transfer to the lower chamber of Transwell after passing through Matrigel. At the concentration of 80 μM, the relative invasion rates of A549 and H1299 are both lower than 50%. The results indicate that cupressuflavone can inhibit the invasion of A549 and H1299 cells in a concentration-dependent manner.

[0034] (5)Detect the effect of cupressuflavone on the mitochondrial membrane potential of lung cancer cells As an important site for aerobic respiration in cells, mitochondria can generate a large amount of adenosine triphosphate (ATP) to provide energy for cells. During this process, a mitochondrial membrane potential (MMP) is formed, and the decrease of MMP will interfere with the normal physiological functions of cells. The effect of cupressuflavone on the mitochondrial membrane potential of A549 and H1299 cells was detected using JC-1 dye. With the administration concentration of 0 μM as the control group, the obtained results are as Figure 7 shown.

[0035] From Figure 7 the results, it can be seen that cupressuflavone can induce a decrease in the mitochondrial membrane potential of A549 and H1299 cells, and with the increase of concentration, the decrease of mitochondrial membrane potential is more significant. The results indicate that cupressuflavone can induce depolarization of the mitochondrial membrane potential of lung cancer cells in a concentration-dependent manner.

[0036] (6)Detect the effect of cupressuflavone on the apoptosis of lung cancer cells The decrease of mitochondrial membrane potential is closely related to apoptosis, autophagy, necrosis, etc. of cells. Based on the above experimental results, we first investigated whether cupressuflavone can induce apoptosis of lung cancer cells, and detected the apoptosis of cells after 24 h of treatment with cupressuflavone using Annexin V-FITC / PI double staining. With the administration concentration of 0 μM as the control group, the obtained results are as Figure 8 shown.

[0037] From Figure 8 it can be seen that the apoptosis rates of A549 and H1299 cells after treatment with different concentrations of cupressuflavone all increase. At the concentration of 80 μM, the apoptosis rate of cells is only about 10%. The above results indicate that cupressuflavone can induce apoptosis of lung cancer cells, but it is not the main way to induce the death of lung cancer cells.

[0038] Example 2 Regulatory effect of cupressuflavone on ferroptosis and EMT status in lung cancer (1)To clarify the key pathways by which cupressuflavone regulates the death of lung cancer cells, the effect of cupressuflavone on the gene expression of H1299 cells was analyzed by transcriptome sequencing technology.

[0039] 1. H1299 cells in the logarithmic growth phase were seeded in 10 cm culture dishes and divided into two groups, a control group (0 M hinokiflavone) and an experimental group (80 uM hinokiflavone). After 24 h of drug treatment, total RNA was extracted using TRizol. Then the RNA lysis solution was collected in an EP tube and stored at -80 °C. Sequencing analysis was performed by Shanghai Metware Biotechnology Co., Ltd.

[0040] 2. DESeq2 software (1.20.0) was used to perform differential expression analysis between the two groups. A model based on the negative binomial distribution was used to determine differential expression in digital gene expression data. The Benjamini and Hochberg method was used to adjust the resulting P values (padj) to control the false discovery rate. padj <= 0.05 & |log2(foldchange)| >= 1 was set as the threshold for significantly differentially expressed genes.

[0041] 3. R software (version 4.2.1) was used to perform GO and KEGG enrichment analysis on the differentially expressed genes obtained by the above screening. GO analysis includes three parts: biological process (BP), molecular function (MF), and cellular component (CC). First, the clusterProfiler (4.4.4) software package was used to perform ID conversion and enrichment on the potential targets of hinokiflavone obtained by screening. Subsequently, the ggplot2 (3.3.6) software package was used to visualize the results of the enrichment analysis, and the results obtained were as Figure 9 shown.

[0042] As shown in Figure 9A, compared with the control group, after administration of hinokiflavone, there were a total of 1978 differentially expressed genes in the cells, including 833 up-regulated differentially expressed genes and 1145 down-regulated differentially expressed genes. As shown in Figure 9B, the KEGG enrichment results showed that these differentially expressed genes were enriched in the Ferroptosis signaling pathway. As shown in Figure 9C, the GO enrichment results showed that the cellular components (CC) of these differentially expressed genes were significantly enriched in focal adhesion and cell-substrate junction; while the molecular function (MF) was enriched in cadherin binding, which is closely related to EMT. Therefore, next we will mainly investigate the regulatory effects of hinokiflavone on ferroptosis and EMT in lung cancer cells.

[0043] (2) Detect whether hinokiflavone can induce mitochondrial damage in lung cancer cells Mitochondrial damage is one of the main manifestations of ferroptosis. Therefore, in order to evaluate whether the effect of hinokiflavone on lung cancer cells is related to ferroptosis, we observed the mitochondrial structure of A549 and H1299 cells by transmission electron microscopy, and the results obtained were as Figure 10 shown.

[0044] From Figure 10 Figure 10 The results showed that after treatment with cupressuflavone, the mitochondrial cristae of A549 and H1299 cells were significantly reduced, and the mitochondrial membrane density increased, which are typical morphological changes of ferroptosis. The results indicate that cupressuflavone can induce mitochondrial damage related to ferroptosis in lung cancer cells.

[0045] (3)Detect whether cupressuflavone can induce ferroptosis in lung cancer cells To verify whether cupressuflavone triggers ferroptosis in lung cancer cells, the present invention used Western blot assay to detect the effects of cupressuflavone on the expression levels of ferroptosis markers glutathione peroxidase 4 (GPX4), cystine / glutamate antiporter (SLC7A11), transferrin (TF), ferritin heavy chain 1 (FTH1), arachidonate-15-lipoxygenase (ALOX15) and acyl-CoA synthetase long-chain family member 4 (ACSL4) in lung cancer cells. The results are as Figure 11 shown.

[0046] From Figure 11 Figure 11 the results, it can be seen that cupressuflavone can increase the expression levels of ferroptosis markers TF and ALOX15, and decrease the expression levels of GPX4, SLC7A11 and FTH1. The results indicate that cupressuflavone can induce ferroptosis in lung cancer cells.

[0047] To further clarify whether ferroptosis is the main pathway by which cupressuflavone triggers the death of lung cancer cells, the present invention used the CCK-8 assay to detect the effects of cupressuflavone combined with DFO on the viability of A549 and H1299 cells. The results are as Figure 12 -A shown.

[0048] From Figure 12 Figure 12 the results of Figure 12-A, it can be seen that the inhibitory effect of cupressuflavone on cell growth disappeared after being combined with DFO.

[0049] Then, the present invention used a biochemical kit to detect the changes in the intracellular Fe 2+ level of A549 and H1299 cells after treatment with cupressuflavone combined with DFO. The results are as Figure 12 -B shown.

[0050] As can be seen from Figure 12-B, cupressuflavone alone can promote the increase in Fe2+ level, while this promoting effect was significantly reversed after being combined with DFO.

[0051] The above results indicate that cupressuflavone can induce ferroptosis in lung cancer cells.

[0052] (4)Detect the effect of cupressuflavone on the epithelial-mesenchymal transition of lung cancer cells In this invention, the effects of hinokiflavone on the expression levels of transcription factor Snail protein, epithelial marker E-cadherin, tight junction protein (ZO-1), mesenchymal markers vimentin and N-cadherin were detected by WB. Meanwhile, the expression levels of ZO-1, E-cadherin and vimentin in A549 and H1299 cells after treatment with 80 μM hinokiflavone were verified by multicolor immunofluorescence. The results obtained are as Figure 13 and 14 shown.

[0053] From Figure 13 and 14 the results, it can be seen that hinokiflavone can increase the protein expressions of ZO-1 ( Figure 13 Figure 13B and Figure 14B) and E-cadherin ( Figure 13 Figure 13D and Figure 14D) in A549 and H1299 cells, and reduce the protein expressions of N-cadherin (Figure 13C and Figure 14C), vimentin (Figure 13E and Figure 14E) and Snail (Figure 13F and Figure 14F).

[0054] Figure 13G and Figure 14 Figure 14G results show that the effects of hinokiflavone on the expression of EMT-related proteins detected by multicolor immunofluorescence are consistent with the WB results.

[0055] Example 3 Verification of the anti-lung cancer efficacy of hinokiflavone in vivo (1) Detection of whether hinokiflavone can inhibit the growth of xenograft tumors of lung cancer H1299 in nude mice To study whether hinokiflavone also has pharmacological activity in vivo, a xenograft tumor model of H1299 nude mice was constructed to explore the anti-lung cancer efficacy of hinokiflavone in vivo: 1. Drug preparation: An appropriate amount of hinokiflavone was dissolved in DMSO and diluted with 100 μL of normal saline so that the volume of DMSO did not exceed 1%.

[0056] 2. Animal feeding: 5-week-old male BALB / c nude mice were raised in a SPF-class animal house with 12 h light / dark alternation every day. After 1 week of adaptive feeding, the experiment was carried out.

[0057] 3. Take H1299 cells in the logarithmic growth phase, digest and resuspend the cells, and prepare a cell PBS suspension containing 6 cells per nude mouse

[80] . Subsequently, centrifuge at 1000 rpm for 3 min and discard the PBS. Prepare cell-containing saline by mixing with 100 μL of normal saline per nude mouse.

[0058] 4. Subcutaneously inject 100 μL of the cell suspension into the right scapular region of the nude mouse.

[0059] 5. Five days after inoculation, when the tumor volume reaches 100 mm 3 , randomly divide the tumor-bearing nude mice into four groups: model group (0.5% normal saline), biflavone group (160 mg / kg), deferoxamine group (100 mg / kg), and biflavone combined with deferoxamine group (5 mice in each group).

[0060] 6. Administer the drug by gavage once a day for 16 consecutive days. Measure the tumor volume every two days Tumor volume = (length × width 2 ) / 2 7. On the day after the last gavage administration, anesthetize the nude mouse with sodium pentobarbital, then decapitate it by cervical dislocation, immediately remove the tumor, weigh it, take pictures for record, and then fix it with 4% paraformaldehyde.

[0061] 8. Dissect the nude mouse, take the heart, liver, spleen, lungs, and kidneys, and fix them with 4% paraformaldehyde for subsequent H&E staining, EdU staining, immunohistochemistry, and immunofluorescence.

[0062] The results of the growth of transplanted tumors are as Figure 15 shown.

[0063] From Figure 15 it can be seen that the tumor size (Figure 15B), volume (Figure 15C), and mass ( Figure 15 D) of the biflavone group were significantly lower than those of the model group; while the tumor size, volume, and mass of the biflavone combined with deferoxamine group were between those of the model group and the biflavone group. At the same time, as Figure 15 shown in E, the pathological section results showed that compared with the model group, the integrity of the tumor cell membrane and nucleus in the biflavone group was significantly disturbed. After combination with deferoxamine, the degree of interference with the integrity of the tumor cell membrane and nucleus was relatively reduced. The above results indicate that biflavone can inhibit the growth of lung cancer H1299 xenograft tumor-bearing nude mice in vivo by regulating ferroptosis.

[0064] The results of EdU staining are as Figure 16 shown.

[0065] From Figure 16As a result, it can be seen that the EdU fluorescence intensity of the hinokiflavone group was significantly lower than that of the model group, while the fluorescence intensity of the hinokiflavone combined with deferoxamine group was enhanced compared with the hinokiflavone group. The results indicate that hinokiflavone can inhibit the proliferation activity of lung cancer H1299 xenograft tumors by inducing ferroptosis.

[0066] (2)Examine whether hinokiflavone has obvious organ toxicity To investigate the safety of hinokiflavone in vivo, we examined the damage of the hearts, livers, spleens, lungs and kidneys of nude mice in each group by H&E staining, and the results obtained are as Figure 17 shown.

[0067] From Figure 17 the results, it can be seen that there was no obvious damage in the tissues of the hearts, livers, spleens, lungs and kidneys of nude mice in each group, suggesting that hinokiflavone has good safety and no obvious organ toxicity.

[0068] (3)Examine whether the anti-tumor effect of hinokiflavone in vivo is related to ferroptosis and EMT To further verify the relationship between the anti-tumor activity of hinokiflavone in vivo and EMT and ferroptosis, the present invention detected the expression of ferroptosis-related regulatory proteins (GPX4, SLC7A11, TF, FTH1 and ALOX15) by immunohistochemical staining, and the results obtained are as Figure 18 shown.

[0069] From Figure 18 the results, it can be seen that TF and ALOX15 in the hinokiflavone group showed positive expression in tumor tissues, while GPX4, SLC7A11 and FTH1 showed negative or weak positive expression, and the above behaviors were reversed after combination with deferoxamine. The results indicate that the tumor inhibitory effect of hinokiflavone in vivo is closely related to ferroptosis.

[0070] Meanwhile, the present invention detected the expression of EMT-related proteins (E-cadherin and Vimentin) by immunofluorescence, and the results obtained are as Figure 19 shown.

[0071] From Figure 19 the results, it can be seen that the fluorescence intensity of E-cadherin in the hinokiflavone group was significantly higher than that of the model group, and the fluorescence intensity of Vimentin was significantly lower than that of the model group, while the fluorescence intensity of the combined deferoxamine group was between the hinokiflavone group and the deferoxamine group. The above results suggest that hinokiflavone can inhibit the EMT process of lung cancer cells by inducing ferroptosis.

[0072] Example 4 Discovery of potential anti-lung cancer targets of hinokiflavone and verification of its mechanism Erlotinib: Molecular weight, 393.40, CAS No. 183321-74-6, purchased from MedChemExpress, catalog number: HY-50896, purity ≥99.98%. Epidermal growth factor (EGF) was purchased from MedChemExpress, catalog number: HY-P1960A, purity ≥96.75%.

[0073] (1) Screening and acquisition of potential targets of cupressuflavone To obtain the potential targets of cupressuflavone against lung cancer, the SwissTarget Prediction database was used to screen the potential targets of cupressuflavone. First, visit the official website of SwissTarget Prediction (http: / / www.swisstargetprediction.ch), and upload the SMILES format string of cupressuflavone on the main page. After confirmation, click the "Predict Targets" button to predict its possible action targets, and the results are as Figure 20 shown.

[0074] From Figure 20 the results, it can be seen that the present invention finally obtained 105 potential targets against lung cancer. Intersect these 105 targets with the differential targets screened by previous RNA-seq sequencing, and finally obtained 6 potential intersection targets, as Figure 20 shown in B, namely TNKS2, TNKS, PTPRS, IGF1R, AXL and EGFR. Through KEGG enrichment analysis of 105 cupressuflavone targets against lung cancer, as shown in Figure 20C, we found that these targets were mainly enriched in signal pathways such as EGFR tyrosine kinase inhibitor resistance.

[0075] (2) Screening of core targets and differential expression analysis Intersect the screened cupressuflavone targets with the differential genes screened by RNA-seq sequencing to obtain the core targets of cupressuflavone against lung cancer for subsequent analysis.

[0076] Differential expression analysis using TCGA data. Briefly, RNA-seq data from the TCGA-LUAD and TCGA-LUSC projects were downloaded and curated from the TCGA database (https: / / portal.gdc.cancer.gov). These data included 1149 samples, of which 108 were adjacent tissue samples. RNA-seq data were extracted in FPKM format and processed as log2(value + 1). The analysis was performed using R software (version 4.2.1), and the statistical methods were selected and analyzed using the stats (4.2.1) and car (3.1-0) packages. In addition, the ggplot2 (3.3.6) package was used for data visualization; (3) Survival analysis The data used for survival analysis were from the TCGA database and relevant literature. Supplementary survival analysis was performed using R software (version 4.2.1). The proportional hazards hypothesis test and survival regression fitting were performed using the survival (3.3.1) package. The survminer and ggplot2 (3.3.6) packages were used to visualize the results. The statistical significance was set at p < 0.05. To ensure the continuity of the results, a data filtering strategy was adopted to exclude normal samples and clinically meaningless data points, and the prognostic type was overall survival (OS).

[0077] (4) Molecular docking The prognosis-related targets confirmed according to the results were analyzed and docked with cupressuflavone. The receptor protein was the EGFR (PDBID: 6JXT) protein, and the protein 3D structure file was downloaded from the PDB database. The PyMOL 2.3.0 software was used to check the protein structure for docking. The ligand small molecule was Cupressuflavone (Pubchem CID: 5281609), and its 3D structure file was downloaded from the PubChem database and the MMFF94 force field of the OpenBabel software was used to optimize the small molecule structure, and finally the optimal molecular structure in the lowest energy state was obtained. The AutoDock Tools 1.5.6 was used to hydrogenate the protein, hydrogenate the small molecule and determine the rotatable bonds, and save it as a pdbqt file. The Grid section was used to set the molecular docking parameters, Center (X,Y,Z) = (-6, 54, 1), Size (X×Y×Z) = (30.0×23.0×25.0); the docking mode was set as semi-flexible docking, the docking accuracy exhaustiveness was 25, and the docking algorithm was the Lamarckian genetic algorithm. The AutoDock Vina 1.2.0 software was run for molecular docking to obtain the docking binding free energy and the docking result file. To verify the reliability of molecular docking, in this study, the protein and ligand were docked 10 times under the same conditions, and the differences in the ten molecular docking results were compared. In addition, the co-crystallized ligand in the EGFR protein crystal structure was used for reproducibility docking with the EGFR protein to further verify the reliability of the molecular docking method. If the RMSD value of the co-crystallized ligand before and after reproducibility docking was less than 2 Å, it represented that the molecular docking methodology verification passed.

[0078] (5)Molecular dynamics simulation The Gromacs 2022 software was used to perform molecular dynamics simulation on the cupressuflavone-EGFR complex. The Amber14sb force field was used for the protein and the Gaff2 force field was used for the ligand. The system was immersed in the SPC / E water model in a 1.2-nanometer periodic box, and sodium ions and chloride ions were added to neutralize the charges by the Monte Carlo method. The long-range electrostatic interaction was treated by the particle mesh Ewald (PME) method. Before simulation, the system was energy-minimized for 50,000 steps by the steepest descent algorithm until the maximum force was below 1000 kJ / mol. Subsequently, the system was sequentially equilibrated at 310K under constant volume (NVT) and constant pressure (NPT), with each stage running for 50,000 steps (time step 2 femtoseconds). The formal simulation ran for 100 nanoseconds (time step 2 femtoseconds), and the coordinates were saved every 10 picoseconds. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), hydrogen bonds, and solvent accessible surface area (SASA) of the trajectory were analyzed at the 0, 25, 50, 75, and 100 nanosecond time points, respectively.

[0079] (6)Surface Plasmon Resonance (SPR) Experiment The SPR experiment was carried out using a Biacore T200 system. The chip surface was activated for 240 seconds by injecting a mixture of 400 mM EDC and 100 mM NHS at a flow rate of 20 μL / min. EGFR (purchased from AntibodySystem, catalog number EHB86901) was dissolved in the immobilization buffer at a concentration of 50 μg / mL and immobilized on the chip surface at a flow rate of 20 μL / min. Subsequently, it was blocked with 1 M ethanolamine hydrochloride for 240 seconds. Six concentration gradients (10, 5, 2.5, 1.25, 0.625, and 0 μM) of hinokiflavone analyte were set, and each concentration was injected at a flow rate of 20 μL / min for 240 seconds for the binding phase, followed by a 360-second dissociation phase. All concentrations were detected in 6 cycles in ascending order, and between each cycle, the chip surface was regenerated with 10 mM glycine-HCl (pH not indicated) at a flow rate of 150 μL / min for 10 seconds.

[0080] (7)Microscale Thermophoresis (MST) Analysis The MST technique was used to study the interaction between EGFR and hinokiflavone. EGFR was fluorescently labeled using the Monolith NT Protein Labeling Kit RED-NHS. CPF was diluted to a gradient concentration from 25 μM to 762.94 pM in MST buffer (1×PBST pH 7.4 containing 0.05% Tween 20 and 1% DMSO) containing 1% DMSO and mixed with the labeled EGFR in equal volume (final volume 10 μL / sample). The mixture was added to a standard-treated capillary, and detected at 25 °C using a Nanotemper MonolithNT.115 device with the parameters set to medium MST power and 100% LED light source intensity. The dissociation constant (Kd) of the EGFR-CPF interaction was calculated using MO.Affinity Analysis software v2.3.

[0081] The results obtained are as Figure 21 shown.

[0082] As can be seen from Figure 21 it, in Figure 21 A, the expression levels of 6 potential targets (i.e., TNKS2, TNKS, PTPRS, IGF1R, EGFR, and AXL) in human lung cancer tissues and normal lung tissue samples were detected using the TCGA database, and significant differences were found in all the above 6 targets. As Figure 21 shown in B to Figure 21As shown in Figure G, survival analysis revealed that only EGFR (Figure 21F) showed significant differences, indicating that its high expression was associated with poor prognosis. Then, molecular docking was performed. Generally, a binding energy less than 0 kcal·mol -1 represents that the receptor and ligand can bind spontaneously without external energy, and a binding energy less than -7.2 kcal·mol -1 indicates strong binding. As Figure 21 shown in Figure H, the docking binding energy of EGFR protein and Cupressuflavone in this docking was -8.7 kcal·mol -1 , indicating a high degree of binding between EGFR protein and Cupressuflavone. The reproducibility docking of the co-crystal ligand with the EGFR protein showed that the RMSD value of the co-crystal ligand before and after docking was 1.954 Å, representing the successful verification of the molecular docking methodology. As Figure 21 shown in Figure I, molecular dynamics simulation showed that the Cupressuflavone-EGFR complex exhibited good stability in terms of RMSD, RMSF, Rg, number of hydrogen bonds, and SASA: the RMSD value fluctuated slightly, the curves of RMSF and Rg were smooth, the number of hydrogen bonds was stable, and the SASA curve was steady. In addition, the binding ability of Cupressuflavone to EGFR was verified by surface plasmon resonance (SPR) and microscale thermophoresis (MST) experiments, and the experimental results were highly consistent with the computational predictions, further corroborating the targeting mechanism of CPF ( Figure 21 Figure J-K).

[0083] Example 5 Regulatory effect of Cupressuflavone on EGFR protein phosphorylation in lung cancer cells As a member of the epidermal growth factor receptor (HER) family, EGFR plays an important role in physiological processes such as cell growth, proliferation, and differentiation. From the results of survival analysis and molecular docking, it can be seen that EGFR may be the key core target for Cupressuflavone to exert its anti-lung cancer activity. Therefore, we used WB, in combination with the epidermal growth factor receptor tyrosine kinase inhibitor Erlotinib and epidermal growth factor (EGF), to detect the effects of Cupressuflavone on the expression of total EGFR protein and phosphorylated protein in A549 and H1299 cells, and the results are as Figure 22 shown.

[0084] From Figure 22 it can be seen that Cupressuflavone had no effect on the expression of total EGFR protein, but could reduce the expression of p-EGFR. After co-treatment with EGF, the expression of both total EGFR protein and p-EGFR increased. After co-treatment with Erlotinib, the expression of p-EGFR was still inhibited. The results showed that Cupressuflavone could regulate the expression of EGFR and inhibit the phosphorylation of EGFR.

[0085] Example 6 Cupressuflavone mediates lung cancer cell growth and lipid peroxidation by regulating EGFR To clarify the regulatory role of EGFR in the anti-lung cancer activity of cupressuflavone, we further combined Erlotinib or EGF and detected the cell viability of A549 and H1299 by CCK-8 assay. The results obtained are as Figure 23 shown

[0086] As Figure 23 can be seen, compared with the use of cupressuflavone alone, the cytotoxic effect of cupressuflavone was reversed when combined with EGF; but when combined with Erlotinib, the growth inhibitory effect of cupressuflavone on A549 (Figure 23A) and H1299 ( Figure 23 B) cells was partially enhanced

[0087] Meanwhile, in this invention, the intracellular reactive oxygen species levels of A549 and H1299 cells were quantitatively detected by flow cytometry using the BODIPY 581 / 591 C11 lipid peroxidation fluorescent probe. The results obtained are as Figure 24 shown

[0088] As Figure 24 can be seen, after combined with EGF, the ROS levels in A549 and H1299 cells were significantly lower than those when using cupressuflavone alone; after combined with Erlotinib, the ROS levels in A549 and H1299 cells were significantly higher than those when using cupressuflavone alone. The above results indicate that the growth inhibitory effect and the promotion of lipid peroxide accumulation of cupressuflavone on lung cancer cells are closely related to the activation of EGFR

[0089] Example 7 Cupressuflavone mediates ferroptosis and EMT of lung cancer cells by regulating EGFR To verify the specific regulatory role of EGFR in cupressuflavone-induced ferroptosis and inhibition of the EMT process of lung cancer cells, we first detected the expression of ferroptosis-related proteins GPX4 and FTH1 by immunofluorescence staining after cupressuflavone was combined with Erlotinib or EGF. The results obtained are as Figure 25 shown

[0090] As Figure 25It can be seen that compared with the sole use of hinokiflavone, the inhibitory effects of hinokiflavone on the protein expressions of GPX4 and FTH1 in A549 and H1299 cells were significantly reversed after the combination of hinokiflavone and EGF. After the combination of hinokiflavone and Erlotinib, the inhibitory effect of hinokiflavone on the protein expression of FTH1 was only enhanced, and there was no difference in the inhibitory effect on the expression of GPX4 compared with the sole use.

[0091] Meanwhile, we also detected the expressions of EMT-related proteins E-cadherin and Vimentin by immunofluorescence staining after the combination of hinokiflavone and Erlotinib or EGF, and the results are as Figure 26 shown.

[0092] From Figure 26 it can be seen that compared with the sole use of hinokiflavone, after the combination of hinokiflavone and EGF, the inhibitory effect of hinokiflavone on the protein expression of Vimentin and the promoting effect on the protein expression of E-cadherin in A549 and H1299 cells were both significantly reversed. After the combination of hinokiflavone and Erlotinib, the promoting effect of hinokiflavone on the protein expression of E-cadherin was enhanced. The above results indicate that hinokiflavone can further mediate ferroptosis and EMT of lung cancer cells by regulating the activation of EGFR.

Claims

1. Use of hinokiflavone in the preparation of an inhibitor for targeted inhibition of EGFR phosphorylation, characterized in that, The CAS number of the cupressuflavone is 3952-18-9.

2. An inhibitor for targeted inhibition of EGFR phosphorylation, characterized in that, The inhibitor consists of cupressuflavone and a pharmaceutically acceptable carrier, and the CAS number of the cupressuflavone is 3952-18-9.

3. The inhibitor according to claim 2, wherein In the inhibitor, the concentration of the cupressuflavone is 20-80 μM.

4. Use of hinokiflavone in the preparation of a drug for inhibiting lung cancer metastasis, characterized in that, The CAS number of the cupressuflavone is 3952-18-9.

5. The application according to claim 4, characterized in that, The cupressuflavone inhibits lung cancer metastasis by targeting and inhibiting EGFR phosphorylation.

6. A drug for inhibiting lung cancer metastasis, characterized in that, The drug consists of cupressuflavone and a pharmaceutically acceptable carrier, and the CAS number of the cupressuflavone is 3952-18-9; The concentration of the cupressuflavone is 20-80 μM.

7. Use of hinokiflavone in the preparation of a drug for promoting ferroptosis of lung cancer cells, characterized in that, The CAS number of the cupressuflavone is 3952-18-9, and the cupressuflavone promotes ferroptosis of lung cancer cells by targeting and inhibiting EGFR phosphorylation.

8. A drug for promoting ferroptosis of lung cancer cells, characterized in that, The drug consists of cupressuflavone and a pharmaceutically acceptable carrier, and the CAS number of the cupressuflavone is 3952-18-9; The concentration of the cupressuflavone is 20-80 μM.

9. Use of a composition in the preparation of a drug for inhibiting lung cancer metastasis and ferroptosis of lung cancer cells, characterized in that, The composition consists of cupressuflavone and erlotinib, the CAS number of the cupressuflavone is 3952-18-9, and the CAS number of the erlotinib is 183321-74-6.

10. The application according to claim 9, wherein, In the composition, the concentration of the cupressuflavone is 80 μM, and the concentration of the erlotinib is 10 μM.

Images