Application of shikonin combined with osimertinib in reversing osimertinib drug-resistant non-small cell lung cancer

Abstract

The invention discloses application of shikonin combined with osimertinib in reversing osimertinib drug-resistant non-small cell lung cancer, and belongs to the technical field of biological medicine. According to the application of the shikonin combined with the osimertinib in reversing the osimertinib drug-resistant non-small cell lung cancer disclosed by the invention, the shikonin combined with the osimertinib promotes ferroptosis by inhibiting an HES1 / ATF3 / SLC7A11 signal axis, so that the drug resistance of the osimertinib drug-resistant non-small cell lung cancer is reversed; the combined scheme is expected to be a promising treatment means for overcoming the drug resistance of osimertinib drug-resistant non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to an application of shikonin combined with osimertinib in reversing osimertinib-resistant non-small cell lung cancer. Background Art

[0002] Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Among the many treatment strategies, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as osimertinib, have become first-line treatment options due to their significant efficacy in improving progression-free survival and overall survival in patients with EGFR mutant NSCLC. However, the problem of osimertinib resistance inevitably arises, leading to treatment failure, which has become a major challenge in clinical treatment. Therefore, a deep understanding of the mechanism of osimertinib resistance and the exploration of new strategies to overcome this resistance are crucial to improving patients' survival prognosis.

[0003] Ferroptosis is an iron-dependent, regulated form of cell death that typically occurs under conditions of oxidative stress, such as reactive oxygen species (ROS) accumulation and lipid peroxidation. Many diseases, including tumors, have been reported to be associated with ferroptosis, and anti-tumor strategies targeting ferroptosis are increasingly becoming a promising new approach for tumor treatment. In addition, activating ferroptosis may be a potential strategy to overcome resistance mechanisms to traditional cancer therapies, such as chemotherapy, immunotherapy, and targeted therapy. The molecular mechanisms of ferroptosis are complex, involving multiple signaling pathways and cellular responses, which ultimately lead to iron accumulation, lipid peroxidation, and cell death.

[0004] Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) used to treat non-small cell lung cancer (NSCLC). However, like many targeted therapy drugs, patients will develop resistance after a period of time. Overcoming osimertinib resistance is an important topic of current research. The following are some possible approaches and problems in existing technologies:

[0005] Current research directions to overcome osimertinib resistance include:

[0006] (1) Combination with other EGFR-TKIs: Studies have shown that the combined use of different generations of EGFR inhibitors may delay the emergence of drug resistance.

[0007] (2) Combination chemotherapy or immunotherapy: Combining traditional chemotherapy drugs or immune checkpoint inhibitors may provide synergistic effects.

[0008] (3) Targeting drug-resistant mutations: Developing a new generation of EGFR-TKIs targeting osimertinib resistance-associated mutations (such as C797S mutation).

[0009] (4) Bispecific antibodies: Bispecific antibodies targeting EGFR and other related pathways may provide new therapeutic strategies.

[0010] (5) Inhibition of alternative signaling pathways: Drug resistance may be achieved by activating alternative signaling pathways (such as MET, HER2, etc.), and inhibiting these pathways can be used as a strategy.

[0011] (6) Liquid biopsy and personalized treatment: Liquid biopsy technology is used to monitor the emergence of drug-resistant mutations and adjust the treatment plan according to the specific mutations.

[0012] Problems faced in overcoming osimertinib resistance:

[0013] (1) Complex resistance mechanisms: Resistance mechanisms are diverse and complex, and may involve changes in multiple genes and signaling pathways. It is difficult for a single strategy to fully address them. The following is an overview of some of the main mechanisms of osimertinib resistance:

[0014] Secondary gene mutations: In patients treated with osimertinib, tumor cells may develop new EGFR mutations, such as the C797S mutation, which can directly affect the drug's binding ability and lead to drug resistance.

[0015] Activation of bypass signaling pathways: Tumor cells may bypass EGFR inhibition by activating other growth signaling pathways (such as MET, HER2, RAS / RAF / MEK, PI3K / AKT, etc.) to maintain their growth and survival.

[0016] Phenotypic transformation: Some tumor cells may undergo epithelial-mesenchymal transition (EMT) or transform into different phenotypes such as small cell lung cancer (SCLC), and these changes can make cells no longer sensitive to EGFR-TKI treatment.

[0017] Drug efflux and metabolic changes: Drug-resistant cells may increase the expression of drug efflux pumps or alter drug metabolic pathways, thereby reducing the effective concentration of drugs in cells.

[0018] Microenvironmental factors: Changes in the tumor microenvironment, such as hypoxia, acidic environment, or immunosuppressive state, may also promote the occurrence of drug resistance.

[0019] Due to the diversity and complexity of these mechanisms, overcoming osimertinib resistance requires a multi-level comprehensive strategy, including the combined use of multiple targeted drugs, the development of new inhibitors, and personalized treatment plans to effectively address the resistance characteristics of different patients.

[0020] (2) Heterogeneity: Tumor heterogeneity means that different patients may have different resistance mechanisms, making the development of personalized treatment plans challenging. The following are some potential strategies and considerations to address this complex issue: A. Precision treatment: Use genomic sequencing technology to perform detailed molecular characterization of patients' tumors to identify specific resistance mutations and signaling pathway changes. This will help develop targeted treatment plans. B. Dynamic monitoring: Use technologies such as liquid biopsy to monitor patients' resistance mechanisms in real time and adjust treatment plans in a timely manner. This dynamic monitoring can help identify new resistance mutations or pathway activations and take corresponding measures quickly. C. Personalized drug screening: Use patient-derived tumor cells or organoids for in vitro drug screening to evaluate the effectiveness of different drug combinations, so as to tailor treatment plans for patients. D. Combination therapy strategy: Combine the use of multiple targeted drugs to simultaneously inhibit multiple signaling pathways or overcome secondary mutations. For example, osimertinib is used in combination with MET inhibitors or other signaling pathway inhibitors to address bypass activation. E. Combination with immunotherapy: Explore the combination of osimertinib with immune checkpoint inhibitors or other immunotherapies to enhance anti-tumor immune responses and potentially provide additional therapeutic effects.

[0021] (3) Drug toxicity and tolerance: In the process of overcoming osimertinib resistance, drug toxicity and tolerance are important factors that must be considered. Although combination therapy strategies have the potential to improve efficacy, they may also increase drug toxicity, thereby affecting patients' tolerance and quality of life.

[0022] (4) Limitations of clinical trials: New drugs and combination therapies need to undergo rigorous clinical trial verification, which is a long and expensive process.

[0023] (5) Identification of biomarkers: There is a lack of reliable biomarkers to predict and monitor the occurrence and development of resistance. In the process of overcoming osimertinib resistance, the identification of reliable biomarkers is crucial to predicting and monitoring the occurrence and development of resistance. However, this field still faces many challenges. Such as: the diversity of resistance mechanisms, heterogeneity problems, liquid biopsy technology, technical and analytical challenges: Highly sensitive and highly specific detection technologies are the basis for identifying biomarkers, such as circulating tumor DNA (ctDNA) analysis. In short, although the identification and verification of reliable biomarkers face challenges, it is also a key step in achieving precision medicine and personalized treatment. Continuous research and technological advances will help overcome these challenges and improve the ability to predict and manage osimertinib resistance.

[0024] A deeper understanding of resistance mechanisms and the development of more effective treatment strategies can provide better treatment options for patients.

[0025] Therefore, providing the use of shikonin combined with osimertinib in reversing osimertinib-resistant non-small cell lung cancer is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0026] In view of this, the present invention provides the use of shikonin combined with osimertinib in reversing osimertinib-resistant non-small cell lung cancer. The combined use of shikonin and osimertinib can overcome the resistance of non-small cell lung cancer to osimertinib, especially by activating the potential mechanism of ferroptosis, thereby providing new possibilities for improving the treatment effect of patients.

[0027] First, NSCLC osimertinib-resistant cell models (PC9-OR and H1975-OR) were established to evaluate the resistance index and combination index of osimertinib. Secondly, the experimental groups were: control group, shikonin group, osimertinib group, and osimertinib combined with shikonin group. After treatment, the resistant cell lines were collected for DNA and RNA for whole genome sequencing, differentially expressed genes were screened, and subsequent related functions and preliminary molecular mechanism studies were conducted. In addition, a mouse transplant tumor model was established to further verify the results of in vitro cell experiments and preliminarily evaluate the toxic and side effects of combined medication.

[0028] In conclusion, the strategy of combining shikonin with osimertinib provides a new idea for overcoming osimertinib-resistant non-small cell lung cancer. With further research, this combination therapy is expected to become an important treatment for overcoming osimertinib resistance.

[0029] In order to achieve the above object, the present invention adopts the following technical solution:

[0030] Application of shikonin combined with osimertinib in reversing osimertinib-resistant non-small cell lung cancer.

[0031] Furthermore, shikonin is combined with osimertinib in the preparation of drugs for reversing osimertinib-resistant non-small cell lung cancer.

[0032] Furthermore, shikonin is combined with osimertinib in the preparation of drugs for the treatment of non-small cell lung cancer.

[0033] Furthermore, the material for inhibiting HES1 expression is used in the preparation of drugs for treating non-small cell lung cancer.

[0034] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses the use of shikonin combined with osimertinib in reversing osimertinib-resistant non-small cell lung cancer, which has the following beneficial effects:

[0035] 1) Multi-target action: Shikonin, usually refers to the main active ingredient from Lithospermum erythrorhizon, which contains a variety of active ingredients that can work synergistically, target multiple molecules and biological pathways, and exhibit a wide range of biological activities. This multi-target mechanism of action can affect multiple signaling pathways, thereby enhancing the anti-tumor effect: such as inducing cancer cell apoptosis by regulating Bcl-2 family proteins; inhibiting cancer cell proliferation and blocking cell cycle progression; inhibiting angiogenesis and limiting tumor blood supply; regulating PI3K / Akt and MAPK signaling pathways to inhibit cancer cell migration and invasion. Compared with the single target action of targeted drugs, the multi-target characteristics of shikonin may help overcome the development of drug resistance.

[0036] 2) Enhanced efficacy: By regulating multiple signaling pathways simultaneously, shikonin can enhance the efficacy of targeted drugs. For example, shikonin may synergistically increase the anti-cancer effect of targeted drugs through multiple mechanisms such as inhibiting tumor cell proliferation, inducing apoptosis, and inhibiting angiogenesis.

[0037] 3) Reduce drug resistance: The heterogeneity and adaptability of cancer cells make them easily resistant to single targeted drugs. The multi-target properties of shikonin can inhibit the growth and survival pathways of cancer cells in many ways, reducing the risk of drug resistance.

[0038] 4) Reduce side effects: The combined use of shikonin may help reduce the side effects of targeted drugs and improve the quality of life of patients; it has good safety and tolerability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0040] Figure 1 The accompanying drawings show that shikonin of the present invention enhances the cytotoxicity of osimertinib to osimertinib-resistant NSCLC cells; (A) Chemical structure of shikonin; (BE) Cells were treated with different drug concentrations for 48 hours, and cell viability was determined by WST-1; (FG) The synergistic effect of combined treatment was evaluated by calculating the CI value using Compuscyn software; (HJ) Cell clone formation after drug treatment for 14 days; (KM) Osimertinib combined with shikonin significantly increased the cell apoptosis rate;

[0041] Figure 2The accompanying drawings show that shikonin combined with osimertinib of the present invention induces ferroptosis in osimertinib-resistant NSCLC cells; (AB) Effects of shikonin and osimertinib combined with other cell death inhibitors on the viability of PC9-OR and H1975-OR cells after 24 hours of treatment; (C) Total ROS levels in PC9-OR and H1975-OR cells were detected by flow cytometry using DCFH-DA fluorescent probe after 24 hours of drug treatment; (D) Quantification of DCFH-DA fluorescence is shown; (E) Lipid peroxidation levels in cells were detected by flow cytometry using C11-BODIPY 581 / 591 probe after 24 hours of drug treatment; (F) Quantification of 581 / 591 fluorescence; (G) After 24 hours of drug treatment, the MDA level in PC9-OR and H1975-OR cells was detected; (H) After 24 hours of drug treatment, the GSH level in PC9-OR and H1975-OR cells was determined; (J) After 24 hours of drug treatment, the Fe 2+ level;

[0042] Figure 3 The accompanying drawings show that HES1 of the present invention is highly expressed in NSCLC and is positively correlated with the poor prognosis of NSCLC patients, and the expression level of HES1 is negatively correlated with the expression level of ATF3;

[0043] (A) Workflow diagram of proteomic and genomic analysis of the combined group; (B) Volcano plot of differentially expressed genes between the control group and the osimertinib combined with PC9-OR shikonin group; Expression analysis of HES1 in unpaired (C) and paired (D) LUAD tissues and adjacent normal tissues in the TCGA-LUAD cohort; (E) Based on TCGA HES1 expression levels obtained from the LUAD cohort were analyzed by Kaplan-Meier analysis for LUAD patients; (F) ROC curves were used to evaluate the diagnostic efficacy of HES1 in LUAD; (G) Venn diagrams depicting the intersection of HES1 target genes and ferroptosis-related genes between different datasets; Relative RNA levels of HES1 and ATF3 were measured in LUAD tissues and adjacent normal tissues (HI) and osimertinib-sensitive and -resistant NSCLC cells (JK); (L) Protein expression levels of HES1, ATF3, SLC7A11, and GPX4 in osimertinib-sensitive and -resistant NSCLC cells; (MN) Cells were treated with the indicated concentrations of osimertinib for 24 h, and cell viability was determined with or without HES1 knockout; (O) Colony formation of cells treated with or without HES1 knockout; (PQ) Quantification of cell clonogenicity; (R) Protein expression levels of HES1, ATF3, and SLC7A11 in cells treated with or without HES1 knockout for 48 h;

[0044] Figure 4 The accompanying drawings show that the combination of shikonin and osimertinib of the present invention induces ferroptosis by weakening the binding of HES-1 to the ATF3 promoter and promoting its transcription and activity; (A) After 48 hours of drug treatment or not, the expression of HES1 and ferroptosis-related proteins in cells was detected by Western blotting; (BC) Colony formation test of cells treated with shikonin and osimertinib in combination with or without erastin or Fer-1; (D) After 48 hours of treatment with shikonin and osimertinib in combination with or without erastin or fer-1, the expression of HES1 and ferroptosis-related proteins in cells was detected; (E) The luciferase activity of the ATF3 promoter in 293T cells was determined using a dual luciferase reporter gene; the luciferase activity was normalized to Renilla; ChIP analysis of HES1 binding to ATF3 promoter in PC9-OR cells by qRT-PCR (F) and agarose gel electrophoresis immunoblotting (G); (H) The HES1 binding site in the ATF3 promoter sequence was predicted by the JASPAR website; (I) Schematic diagram of the predicted HES1 binding sites in the 5' region of ATF3: Mut1 (-910nt to -903nt), Mut2 (-786nt to -779nt); (J) Wild-type (WT) or mutant (Mut) ATF3 luciferase reporter vectors were co-transfected with HES1 or control vector into 293T cells to evaluate luciferase activity; (K) qRT-PCR analysis of ATF mRNA expression in HES1-overexpressing 293T cells and HES1-knockout 293T cells;

[0045] Figure 5 The accompanying drawing shows that osimertinib combined with shikonin of the present invention inhibits xenograft tumor growth in vivo;

[0046] (A) Photos of tumor-bearing mice with different treatments; (B) Representative images of xenograft tumors in mice after 22 days of treatment with or without drugs; (C) Changes in tumor volume of mice during different treatments; (D) Tumor weight at the end of treatment; (E) Changes in body weight during different treatments; ALT (F), AST (G), TP (H), ALB (I), CRE (J), and UREA (K) levels in mice 22 days after different treatments; (L) H&E staining and IHC analysis of xenograft tumor tissues; (MO) qPCR analysis of HES1, ATF3, and SLC7A11 mRNA levels in xenograft tumor tissues; (P) Western blot analysis of HES1, ATF3, and SLC7A11 protein levels in xenograft tumor tissues. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Example 1

[0049] 1) WST-1 detection of cell viability:

[0050] Take cells in the logarithmic growth phase, discard the old culture medium, wash the cells twice with PBS, add 0.5 ml of 0.25% trypsin to the culture bottle to digest the cells, and immediately add an appropriate amount of 1640 culture medium containing 10% FBS to stop digestion when cells fall off under a microscope; transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 minutes; count the cells, add the diluted cells to a 96-well plate according to the cell concentration, add 100 μl of cell suspension to each well, and add PBS 100 μL / well to the wells around the 96-well plate (5000-8000 cells are added to each well for cytotoxicity experiments; 2000-3000 cells are added to each well for siRNA cell transfection experiments). Each concentration gradient has 5 replicate wells, and the culture plate is pre-cultured in an incubator for 24 hours;

[0051] Experiment ① Cultivate to 80% confluence and add drugs: PC9-OR, H1975-OR and other drug-resistant cell lines added Osimertinib (Osi) concentrations are: 0, 4, 8, 16, 32μM; PC9-OR, H1975-OR cell lines added Shikonin (Shi) concentrations are: 0, 0.5, 1, 2, 4, 8μM; PC9-OR, H1975-OR cell lines added 1μM Shikonin combined with different concentrations of Osimertinib, Osimertinib concentration gradients are: 0, 4, 8, 16, 32μM; All the above 0μmol control groups are added with 1640 culture medium containing 10% FBS, without other drugs. The culture was terminated after incubation in the incubator for 48h.

[0052] Experiment ② Cultivate the cells until they reach 80% confluence and add drugs, then set up groups:

[0053] Single-drug group: PC9-OR and H1975-OR cell lines were added with Z-VAD-FMK (apoptosis inhibitor, 10 μM); 3-MA (autophagy inhibitor, 50 μM); Fer-1 (ferroptosis inhibitor, 5 μM); DFO (ferroptosis inhibitor, 5 μM); Erastin (ferroptosis activator, 5 μM); Shikonin combined group: PC9-OR and H1975-OR cell lines were added with Z-VAD-FMK (10 μM), 3-MA (50 μM), Fer-1 (5 μM), DFO (5 μM), Erastin (5 μM) combined with shikonin. Shikonin (1μM); Shikonin + osimertinib combination group: PC9-OR cell line was added with Z-VAD-FMK (10μM), 3-MA (50μM), Fer-1 (5μM), DFO (5μM), Erastin (5μM), each combined with osimertinib (16μM) + shikonin (1μM); H1975-OR cell line was added with Z-VAD-FMK (10μM), 3-MA (50μM), Fer-1 (5μM), DFO (5μM), Erastin (5μM), each combined with osimertinib (12μM) + shikonin (1μM).

[0054] Experiment ③ Culture to a confluence of 30-50% for siRNA cell transfection. Under sterile conditions, dilute siRNA to the desired concentration using serum-free medium. In another tube, dilute the transfection reagent using serum-free medium. Mix the diluted siRNA and transfection reagent solution and mix gently. Let stand for 20 minutes to form the siRNA-transfection reagent complex. Remove the culture medium from the cell culture plate and add fresh serum-free medium. Add the siRNA-transfection reagent complex to the cell culture plate and shake gently for even distribution. After incubation in the incubator for 24 hours, add the drug. The concentrations of osimertinib added to drug-resistant cell lines such as PC9-OR and H1975-OR are: 0, 4, 8, 16, and 32 μM, respectively.

[0055] All the above 0μmol control groups were added with 1640 culture medium containing 10% FBS, without other drugs. Aspirate and discard the old culture medium, replace with 100μl RPMI-1640 culture medium containing 10% WST-1 (without FBS). Incubate the culture plate in the incubator for 0.5-2 hours. For most cases, incubation for 0.5 to 1 hour is sufficient. Place the 96-well plate on a shaker and shake for one minute to fully mix the system to be tested. Use an enzyme marker to measure the absorbance at 450nm, and use a wavelength of 630nm as a reference wavelength for dual wavelength measurement; Viability calculation: cell viability*(%) = [A450(drug addition)-A630(drug addition)] / [A450(0 drug addition)-A630(0 drug addition)]×100%.

[0056] 2) Plate colony formation experiment:

[0057] Cells in the logarithmic growth phase were obtained, digested, centrifuged, and inoculated into 6-well plates. The plates were pre-cultured in an incubator for 24 hours before adding drugs;

[0058] Experiment ①: The control group, shikonin group, osimertinib group, and osimertinib combined with shikonin group were set up; PC9-OR cell line osimertinib (16μM), shikonin (1μM); H1975-OR cell line osimertinib (12μM), shikonin (1μM); the culture was terminated after incubation in the incubator for 48h;

[0059] Experiment ②: Perform siRNA cell transfection. Under sterile conditions, dilute siRNA to the desired concentration using serum-free medium. In another tube, dilute the transfection reagent using serum-free medium. Mix the diluted siRNA and transfection reagent solution and mix gently. Let stand for 20 minutes to form the siRNA-transfection reagent complex. Remove the medium from the cell culture plate and add fresh serum-free medium. Add the siRNA-transfection reagent complex to the cell culture plate and shake gently to distribute evenly.

[0060] Experiment ③: The drug addition settings were osimertinib + shikonin group, osimertinib + shikonin group + Erastin (5μM), osimertinib + shikonin group + Fer-1 (5μM), PC9-OR cell line osimertinib (16μM), shikonin (1μM); H1975-OR cell line osimertinib (12μM), shikonin (1μM); the culture was terminated after incubation in the incubator for 48h;

[0061] Digestion, centrifugation, cell counting, inoculate 200-400 cells in each well, inoculate 2-3 duplicate wells in each treatment group, and continue to culture in an incubator for 10-14 days; observe once every 2-3 days, replace the culture medium every 3 days or replace the culture medium according to the situation of dead cells in the culture medium, and terminate the culture when clones visible to the naked eye appear in the culture dish; discard the supernatant and carefully rinse with PBS twice; add methanol to fix the cells 1mL / well and fix for 15min; discard the fixative and dry; add an appropriate amount of 1% crystal violet staining solution to stain for 20 minutes; recover the crystal violet staining solution, slowly wash off the staining solution with tap water, air dry, and take pictures; count the clones of each group under a microscope, and the clones with more than 50 cells are counted as effective clones; clone formation rate % = number of clones / number of inoculated cells x 100%;

[0062] 3) Detection of cell apoptosis by flow cytometry (AnnexinV-FITC cell apoptosis detection kit, C1062L, Bio-Tech)

[0063] Take cells in the logarithmic growth phase, digest, centrifuge, count the cells, and add 15-18x 10 4 Cells / well, 2 ml of 1640 culture medium containing 10% FBS was added to each well and placed in an incubator for pre-culture for 24 hours. Add drugs. The concentration of osimertinib added to PC9-OR resistant cell lines is: 16μM; the concentration of osimertinib added to H1975-OR resistant cell lines is: 12μM; the concentration of shikonin added to PC9-OR, H1975-OR and other resistant cell lines is: 1μM; PC9-OR resistant cell lines added osimertinib combined with 1μM shikonin, the concentration of osimertinib is: 16μM; H1975-OR resistant cell lines added osimertinib combined with 1μM shikonin, the concentration of osimertinib is: 12μM; all the above 0μmol control groups are added with 1640 culture medium containing 10% FBS, without other drugs. After incubation in the incubator for 48 hours, the culture was terminated; the culture medium was poured off, the cells were moderately digested with EDTA-free trypsin, the cells were gently blown, centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded; the cells were washed twice with PBS and centrifuged at 1000 r / min for 5 minutes; the cells were suspended with 100 μl of Binding Buffer; 5 μl of AnnexinV-EGFP was added and mixed, and then 5 μl of PI was added and mixed; incubated at room temperature, away from light, for 5-15 minutes; the cells were suspended with 400 μl of Binding Buffer; and the cells were analyzed by flow cytometry.

[0064] 4) Detection of total intracellular ROS level by flow cytometry (Reactive Oxygen Species Detection Kit, S0033S, Bio-Tech)

[0065] Take cells in the logarithmic growth phase, digest, centrifuge, count the cells, and add 15-18x 10 4Cells / well, 2 ml of 1640 culture medium containing 10% FBS was added to each well and placed in an incubator for pre-culture for 24 hours. Add drugs and group them: control group: add 1640 culture medium containing 10% FBS, without other drugs; osimertinib group: PC9-OR and H1975-OR resistant cell lines added osimertinib concentrations were: 16μM and 12μM respectively; shikonin group: PC9-OR and H1975-OR resistant cell lines added shikonin concentrations were: 1μM; combined group: PC9-OR resistant cell lines added osimertinib (16μM) combined with shikonin (1μM); combined group: H1975-OR resistant cell lines added osimertinib (16μM) and shikonin (1μM); The cell line was added with osimertinib (12 μM) and shikonin (1 μM); the culture was terminated after incubation in the incubator for 24 hours; 24 hours after the addition of the drug, the culture medium was poured out, the cells were moderately digested with trypsin, gently blown, centrifuged at 1000 rpm, and the supernatant was discarded for 3 minutes; the cells were washed twice with serum-free culture medium RPMI1640 and centrifuged at 1000 r / min for 3 minutes; DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 μM; 1 ml of diluted 10 μM DCFH-DA was added to each sample, and the cells were suspended in the diluted DCFH-DA (cell concentration was 1 million to 20 million / ml), and incubated in a cell culture incubator at 37°C for 20 minutes; inverted and mixed every 3-5 minutes to allow the probe and cells to fully contact; the cells were washed two to three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells; the results were detected and analyzed by flow cytometry.

[0066] 5) Detection of intracellular lipid ROS levels by flow cytometry (BODIPY 581 / 591C11 kit, HY-D1301, MCE)

[0067] Take cells in the logarithmic growth phase, digest, centrifuge, count the cells, and add 15-18x 10 4cells / well, add 2 ml of 1640 culture medium containing 10% FBS to each well and place in an incubator for pre-culture for 24 hours. Add drugs and group them: control group: add 1640 culture medium containing 10% FBS, without other drugs; osimertinib group: PC9-OR and H1975-OR resistant cell lines added osimertinib concentrations were: 16μm and 12μM, respectively; shikonin group: PC9-OR and H1975-OR resistant cell lines added shikonin concentrations were: 1μM; combination group: PC9-OR resistant cell line added osimertinib (16μM) combined with shikonin (1μM); H1975-OR resistant cell line added osimertinib (12μM) combined with shikonin (1μM); incubate in the incubator for 24h and then terminate the culture; pour out the culture medium, digest the cells moderately with trypsin containing EDTA, blow gently, centrifuge at 1000rpm, and discard the supernatant for 3min; wash twice with PBS and centrifuge at 1000r / min for 5min; add 1mL (5μM) BODIPY 581 / 591C11 working solution, incubate at room temperature for 15 minutes. Usually add 100μl to each well of a 96-well plate, 250μl to each well of a 24-well plate, 500μl to each well of a 12-well plate, and 1ml to each well of a 6-well plate. Incubate at room temperature in the dark for 10-20 minutes. Wash the cells twice with PBS, centrifuge at 1000rpm for 5min; resuspend the cells with 1mL of serum-free medium or PBS, and detect and analyze the results under a flow cytometer.

[0068] 6) Detection of intracellular MDA content by ELISA

[0069] Cells in logarithmic growth phase were inoculated in 6 cm culture dishes, 15-20×10 4The cells were divided into control group, osimertinib group, shikonin group, osimertinib + shikonin group, osimertinib + shikonin + Erastin group, and osimertinib + shikonin + Fer-1 group. The cells were cultured in an incubator for 24 hours. Drugs were added. Grouping: Control group: 1640 culture medium containing 10% FBS was added without other drugs; Osimertinib group: PC9-OR and H1975-OR resistant cell lines were added with osimertinib at concentrations of 16μM and 12μM, respectively; Shikonin group: PC9-OR and H1975-OR resistant cell lines were added with shikonin at concentrations of 1μM; Osimertinib + shikonin group: PC9-OR resistant cell lines were added with osimertinib (16μM) and 12μM, respectively. μM) combined with shikonin (1μM); osimertinib + shikonin group: H1975-OR resistant cell line plus osimertinib (12μM) combined with shikonin (1μM); osimertinib + shikonin + Erastin group: PC9-OR resistant cell line plus osimertinib (16μM) + shikonin (1μM) + Erastin (5μM); osimertinib + shikonin + Erastin group: H1975-OR resistant cell line Drug-resistant cell line plus osimertinib (12μM) + shikonin (1μM) + Erastin (5μM); osimertinib + shikonin + Fer-1 group: PC9-OR resistant cell line plus osimertinib (16μM) + shikonin (1μM) + Fer-1 (5μM); osimertinib + shikonin + Erastin group: H1975-OR resistant cell line plus osimertinib (12μM) + shikonin (1μM) + Fer-1 (5μM); incubate in the incubator for 24h and then terminate the culture; pour out the culture medium, digest the cells moderately with trypsin containing EDTA, blow gently, centrifuge at 1000rpm, discard the supernatant for 3min; wash twice with PBS and centrifuge at 1000r / min for 5min; lyse with Western cell lysis buffer, place the cells on ice, use 0.1ml lysis buffer for every 1 million cells, and lyse for 15min. After cell lysis, centrifuge at 10,000g-12,000g for 10 minutes to obtain the supernatant for subsequent determination. After sample preparation, the protein concentration was determined using a BCA protein concentration assay kit to facilitate the subsequent calculation of the MDA content per unit protein weight in the cell.

[0070] Preparation of lipid oxidation (MDA) detection kit (S0131S, Bio-Tech):

[0071] Preparation of TBA storage solution: Weigh an appropriate amount of TBA and use TBA preparation solution to prepare a TBA storage solution with a concentration of 0.37%. That is, 25 mg of TBA is prepared with 6.76 ml of TBA preparation solution, and the final concentration is 0.37%. The prepared TBA storage solution is stored at room temperature and away from light.

[0072] Preparation of MDA working solution: According to the number of samples to be tested (including controls), refer to Table 1 to freshly prepare an appropriate amount of MDA working solution just before the test (the prepared MDA working solution must be used on the same day).

[0073] Table 1

[0074] Number of detections 1 time 10 times 20 times 50 times TBA diluent 150μl 1500μl 3000μl 7500μl TBA storage solution 50μl 500μl 1000μl 2500μl Antioxidants 3μl 30μl 60μl 150μl

[0075] Dilution of standard: Take an appropriate amount of standard and dilute it with distilled water to 1, 2, 5, 10, 20, 50 μM for subsequent preparation of standard curve.

[0076] Sample determination: Add 0.1 ml of lysate as a blank control, add 0.1 ml of the above-mentioned standard of different concentrations to prepare the standard curve, add 0.1 ml of sample for determination; then add 0.2 ml of MDA detection working solution. Refer to Table 2 to set up the detection reaction system.

[0077] Table 2

[0078] Blank control Standards sample Homogenate, lysis buffer or PBS 0.1ml － － Standards － 0.1ml － Samples to be tested － － 0.1ml MDA detection working solution 0.2ml 0.2ml 0.2ml

[0079] After mixing, heat the tube at 100°C for 15 minutes. (When heating, be sure to avoid violent boiling and splashing of the liquid. Use a heavy object to press the centrifuge tube cap tightly). Cool to room temperature in a water bath and centrifuge at 1000g for 10 minutes at room temperature. Take 200 microliters of supernatant and add it to a 96-well plate. Then use an ELISA reader to measure the absorbance at 532nm. (450nm can be set as the reference wavelength for dual-wavelength measurement). Calculate the MDA content and analyze the results.

[0080] 7) Detection of intracellular GSH content by ELISA

[0081] Cells in logarithmic growth phase were inoculated in 6 cm culture dishes, 15-20×10 4There were 3 replicate wells per well, including control group, osimertinib group, shikonin group, osimertinib + shikonin group, osimertinib + shikonin + Erastin group and osimertinib + shikonin + Fer-1 group. Each group had 2 replicate wells and cultured in the incubator for 24 hours; then drug was added. Grouping: Control group: added 1640 culture medium containing 10% FBS, without other drugs; Osimertinib group: PC9-OR and H1975-OR resistant cell lines added osimertinib concentrations were: 16μM and 12μM, respectively; Shikonin group: PC9-OR and H1975-OR resistant cell lines added shikonin concentrations were: 1μM; Osimertinib + Shikonin group: PC9-OR resistant cell line plus osimertinib (16μM) combined with shikonin (1μM); Osimertinib + Shikonin group: H1975-OR resistant cell line plus osimertinib (12μM) combined with shikonin (1μM); Osimertinib + Shikonin + Erastin group: PC9-OR resistant cell line plus osimertinib (16μM) + Shikonin (1μM) + Erastin (5μM); Osimertinib + Shikonin + Erastin Tin group: H1975-OR resistant cell line plus osimertinib (12μM) + shikonin (1μM) + Erastin (5μM); Osimertinib + shikonin + Fer-1 group: PC9-OR resistant cell line plus osimertinib (16μM) + shikonin (1μM) + Fer-1 (5μM); Osimertinib + shikonin + Erastin group: H1975-OR resistant cell line plus osimertinib (12μM) + shikonin (1μM) + Fer-1 (5μM); Incubate in the incubator for 24h and then terminate the culture; Pour away the culture medium, moderately digest the cells with trypsin containing EDTA, gently blow and centrifuge at 1000rpm for 3min to discard the supernatant; Label the centrifuge tubes, weigh and record the weight of the centrifuge tubes before collecting the cells, wash the cells once with PBS, collect the cells by centrifugation, and aspirate the supernatant. Add 3 times the volume of the cell pellet, that is, if the cell pellet is 10 μl, add 30 μl of the protein removal reagent M solution, and fully vortex. (The volume of the cell pellet can be estimated based on the weight of the cell pellet. Weigh the centrifuge tube before and after collecting the cells, so that the weight of the cell pellet can be calculated. The volume of 10 mg of cell pellet can be roughly regarded as 10 μl.) Then use -80℃ and 37℃ water baths to quickly freeze and thaw the sample twice. Place it in 4℃ or ice bath for 5 minutes. Centrifuge at 4℃, 10,000g for 10 minutes. Take the supernatant for the determination of total glutathione GSH (samples that are not measured immediately can be stored at -70℃, but should not exceed 10 days. Try to use fresh cells for determination instead of frozen cells). For the treated cell samples, it is usually necessary to dilute them appropriately with the protein removal reagent M solution before determination, and the dilution multiple can be up to 20 times.Another duplicate well of the sample was lysed with Western cell lysis buffer, the cells were placed on ice, 0.1 ml of lysis buffer was used for every 1 million cells, and the lysis was performed for 15 minutes. After the cells were lysed, they were centrifuged at 10,000g-12,000g for 10 minutes and the supernatant was taken to measure the protein concentration using the BCA protein concentration assay kit, so as to facilitate the subsequent calculation of the total glutathione GSH content in the cells per unit protein weight.

[0082] Preparation of GSH and GSSG detection kit (S0053, Bio-Tech):

[0083] Preparation of GSSG stock solution: Add 816 microliters of Milli-Q grade pure water to 5 mg of GSSG provided in this kit, dissolve and mix well to obtain GSSG stock solution with a concentration of 10 mM. Except for the portion to be used immediately, the remaining GSSG stock solution should be appropriately divided and stored at -20°C.

[0084] Preparation of DTNB stock solution: Add 1.5 ml of DMSO provided in this kit to 4.5 mg of DTNB provided in this kit, dissolve and mix well to obtain the DTNB stock solution. Except for the portion to be used immediately, the remaining DTNB stock solution should be appropriately divided and stored at -20°C.

[0085] Preparation of Protein Removal Reagent M Solution: Weigh 0.2 g of Protein Removal Reagent M, add 4 ml of Total Glutathione Assay Buffer, and prepare 4 ml of 5% aqueous solution. According to this ratio: Weigh 0.05 g of Protein Removal Reagent M each time, add 1 ml of Total Glutathione Assay Buffer, and prepare 1 ml of 5% aqueous solution. Protein Removal Reagent M solution must be freshly prepared and used only on the same day.

[0086] Preparation of NADPH stock solution (40 mg / ml): Add 100 microliters of Milli-Q grade pure water to the 4 mg NADPH provided in this kit, dissolve and mix well to obtain the NADPH stock solution. Except for the portion to be used immediately, the remaining NADPH stock solution should be appropriately divided and stored at -70°C.

[0087] Preparation of 5-fold diluted glutathione reductase: Take 50 μl of glutathione reductase, add 200 μl of total glutathione detection buffer, mix well, and you will get 5-fold diluted glutathione reductase.

[0088] Preparation of total glutathione detection working solution: According to the number of samples to be detected, refer to the table below to prepare an appropriate amount of total glutathione detection working solution. The total glutathione detection working solution is obtained by mixing the three reagents in Table 3 in proportion.

[0089] Table 3

[0090] 1 sample 10 samples 20 samples 5-fold dilution of glutathione reductase 6.6μl 66μl 132μl DTNB stock solution 6.6μl 66μl 132μl Total Glutathione Assay Buffer 150μl 1.5ml 3ml

[0091] Preparation of 0.5mg / ml NADPH: Take 10 microliters of NADPH stock solution, add 790 microliters of total glutathione detection buffer, and mix well to obtain 0.5mg / ml NADPH. 50 microliters of 0.5mg / ml NADPH are required for each sample tested.

[0092] Preparation of standard: dilute 10mM GSSG stock solution with Protein Removal Reagent M solution to 15μM GSSG solution. Then dilute to 10, 5, 2, 1, 0.5μM GSSG solution in sequence. Take 15, 10, 5, 2, 1, 0.5μM GSSG solution as six points for standard curve. Note: Since GSSG is not very stable in Protein Removal Reagent M solution, the GSSG solution prepared with Protein Removal Reagent M solution must be freshly prepared before use and cannot be frozen for reuse.

[0093] Determination of samples and standards: Refer to Table 4, use a 96-well plate, add samples or standards in sequence, mix well. Add 150 μl of total glutathione detection working solution, mix well, and incubate at 25°C or room temperature for 5 minutes.

[0094] Table 4

[0095] Blank Standard curve Sample Sample or standard 0μl 10μl xμl Protein Removal Reagent M Solution 10μl 0μl 10-xμl Total glutathione assay working solution 150μl 150μl 150μl Incubate at 25°C or room temperature 5min 5min 5min 0.5mg / ml NADPH 50μl 50μl 50μl

[0096] Add 50 microliters of 0.5 mg / ml NADPH solution and mix well. Immediately measure A412 with an enzyme-labeled instrument. 25 minutes after adding the NADPH solution and mixing well, measure A412 only once. If the instrument can set the temperature, set it to 25°C, otherwise measure it at room temperature. If the enzyme-labeled instrument cannot measure A412, you can measure the absorbance in the range of 405-414 nm. If the standard curve is good, but the absorbance of the sample is relatively low, you can extend the incubation time to 30-60 minutes. The absorbance of the standard and sample will increase linearly with time within a certain range. Calculation of total glutathione content in the sample: Single-point determination method: that is, the absorbance is measured only once after 25 minutes (or 30-60 minutes) of reaction. Make a standard curve based on the different absorbances measured by standards of different concentrations. The total glutathione can be calculated by comparing the sample with the standard curve (multiply the GSSG concentration calculated by the standard curve by 2). The actual calculated total glutathione content is equivalent to multiplying the content of oxidized glutathione (GSSG) by 2 and adding the content of reduced glutathione.

[0097] 8) Detection of intracellular Fe by fluorescence microscopy 2+ level:

[0098] Cells in logarithmic growth phase were inoculated in 12-well plates at 5×-8×10 4The cells were cultured in an incubator for 24 h, and drugs were added. Grouping: Control group: added 1640 culture medium containing 10% FBS, without other drugs; Osimertinib group: PC9-OR and H1975-OR resistant cell lines added osimertinib concentrations were: 16μM, 12μM, respectively; Shikonin group: PC9-OR and H1975-OR resistant cell lines added shikonin concentrations were: 1μM; Combination group: PC9-OR resistant cell line plus osimertinib (16μM) combined with shikonin (1μM); Combination group: H1975-OR resistant cell line plus osimertinib (12μM) combined with shikonin (1μM); Incubate in the incubator for 24h and then terminate the culture; Wash with serum-free medium 3 times and replace with fresh serum-free medium; Solution preparation: Add 35μl DMSO to the tube containing 24μg FerroOrange, mix with a pipette, and prepare 1μmmol / l FerroOrange solution. Dilute 1μmmol / l FerroOrange solution with ddH2O to prepare 1μmol / l FerroOrange working solution. Add 500μL of 1μmol / l FerroOrange working solution and culture in a 37℃, 5% CO2 incubator for 30 minutes. No need to wash. After culture, observe the cells and take pictures directly under the BioTek Cytation 5 Cell Imaging Multifunctional Microplate Detection System. FerroOrange detection conditions use green excitation light (Ex: 532nm) to excite FerroOrange. Data analysis: Use the BioTek Cytation 5 Cell Imaging Multifunctional Microplate Detection System to quantitatively analyze the fluorescence intensity and compare the differences between different groups.

[0099] 9) Collect cell samples for RNA transcriptome sequencing:

[0100] Sample collection and processing: Cells in the logarithmic growth phase were inoculated in a 10 cm culture dish. After 24 hours of culture, drugs were added. The control group, osimertinib group, shikonin group, and osimertinib + shikonin group were set up. The culture was terminated after incubation in the incubator for 48 hours. RNA extraction: Total RNA was extracted using a commercial RNA extraction kit (Sevenfast total RNA extraction kit, SM130-02, Seven Innovation Biotechnology Co., Ltd.). DNase was added during the RNA extraction process to remove possible genomic DNA. RNA quality detection: RNA integrity detection: The integrity and purity (A260 / A280 ratio) and concentration of RNA were detected using a bioanalyzer. Library construction: mRNA enrichment, poly A tail capture: For eukaryotic mRNA, poly A tail capture method (such as oligo (dT) magnetic beads) was used to enrich mRNA; rRNA removal: rRNA removal kit was used to remove the abundant rRNA in the cells. cDNA synthesis: Reverse transcriptase was used to reverse transcribe the enriched mRNA into cDNA. Random primers or oligo(dT) primers are usually used in this step. End repair and A-tailing: The synthesized cDNA is end-repaired and A-tailed. Adapters are connected for subsequent PCR amplification and sequencing. Library amplification: PCR amplification is performed to increase the amount of the library to ensure sufficient DNA for sequencing. Select the Illumina high-throughput sequencing platform for sequencing. Data analysis, data cleaning: remove low-quality reads and adapter contamination; alignment: align the cleaned sequence to the reference genome or transcriptome, using alignment tools (such as STAR, HISAT2, etc.); expression calculation: use tools (such as HTSeq, featureCounts, etc.) to calculate the expression of each gene; differential expression analysis: use statistical methods (such as DESeq2, edgeR, etc.) to perform differential expression analysis to identify genes with significant changes in expression under different conditions; functional enrichment analysis: perform GO and KEGG enrichment analysis on differentially expressed genes, etc.

[0101] 10) Download and analyze the data of lung adenocarcinoma patients from public databases

[0102] Data download: Open the official website of TCGA (https: / / www.cancer.gov / tcga). Select "Lung Adenocarcinoma (LUAD)" as the cancer type of interest to download and obtain genomic data. Download the required clinical and gene expression data on the GDC website (https: / / gdc.cancer.gov / ). Use R to read the downloaded data. Process missing values ​​and outliers to ensure data quality. According to clinical information, paired samples of cancer tissue and adjacent tissue were screened out. All cancer tissue samples can be selected for unpaired samples; differential expression analysis was performed using the "DESeq2" or "edgeR" package of R to compare the HS1 gene expression levels between cancer tissue and adjacent tissue. Based on the results of the differential analysis, samples with high expression of HS1 in cancer tissue were screened out. Survival analysis: Survival time and survival status (such as survival / death) were extracted from clinical data, and Kaplan-Meier survival curves were drawn using the "survival" and "survminer" packages of R. ROC analysis was performed using the "pROC" package of R to evaluate the performance of HS1 in predicting survival status and calculate the AUC value.

[0103] 11) Public databases predict downstream target genes of transcriptional repressor HES1

[0104] The following three public databases were used to predict the downstream target genes of HES1: Gene Transcription Regulation Database (https: / / gtrd20-06.biouml.org / ); hTFtarget Database (https: / / guolab.wchscu.cn / hTFtarget#! / ); Cistrome DB Database (http: / / cistrome.org / db); and then, the Ferroptosis Database (http: / / www.zhounan.org / ferrdb / current / ) was used to download ferroptosis-related genes. The target genes predicted by the above three databases were intersected with the ferroptosis-related genes downloaded from the Ferroptosis Database to screen out the HES1 target genes.

[0105] 12) RT-qPCR detection of HES1 and related ferroptosis gene mRNA levels

[0106] (1) Total RNA extraction: Sample collection: For cells cultured in suspension or adherent cells that have been digested and detached, centrifuge and discard the supernatant to keep the cell pellet (recommended cell amount: 1–2 × 10 6 ); flick the tube wall to completely loosen the cell pellet and resuspend it, add 500 μL Lysis solution, mix by pipetting, then shake vigorously by hand for 20 seconds until there are no cell clumps, and let stand for 1 minute to fully lyse. Collect RNA: Add the mixture to the adsorption column (the adsorption column is placed in the collection tube), centrifuge at 12,000rpm (13,400×g) for 30 seconds, and discard the filtrate as much as possible. Rinse: Add 500μL of rinsing solution to the adsorption column (please check whether anhydrous ethanol has been added first!), centrifuge at 12,000rpm (13,400×g) for 30 seconds, and discard the filtrate as much as possible. Repeat this step once. Remove ethanol: Put the adsorption column back into the empty collection tube, centrifuge at 12,000rpm (13,400×g) for 2 minutes to dry the residual ethanol in the membrane matrix. Elution: Take out the adsorption column and put it into a clean RNase Free centrifuge tube. Add 25-50 μL RNase-Free H2O to the middle of the adsorption membrane according to the expected RNA yield (preheating in a 70-90°C water bath can increase the yield). Let it stand at room temperature for 1 minute and centrifuge at 10,000 rpm (9,600 × g) for 1 minute. To increase the yield, you can use the first elution solution to add back to the adsorption column and repeat the elution.

[0107] (2) RNA concentration detection: Sample preparation: dilute the RNA sample to an appropriate concentration to ensure that it is within the instrument's detection range; usually use RNase-free DEPC water for dilution. Turn on the machine, turn on the spectrophotometer, and preheat to a stable state; enter the system and select RNA detection; blank correction: wipe the detection hole on the machine and drip 1.5μl of DEPC water into the small hole, press blank, then drip 1.5ul of DEPC water into the small hole, press measure to see Data (Table), 260 / 280 is below 0.004. Measure the sample, take 1.5μl of RNA liquid sample and add it to the cuvette, put it into the spectrophotometer, and press measure to detect (measure the absorbance at 260nm (A260), the concentration of RNA is proportional to A260). Save and name the test sample and record the data, record the A260 value and calculated concentration of each sample.

[0108] (3) Reverse transcription RT: RNA sample preparation: Ensure that the RNA sample is free of DNA contamination. Genomic DNA can be removed by DNase treatment. The RNA concentration is usually diluted to 5 μg / μL total RNA. The preparation of the DNA removal reaction solution is shown in Table 5.

[0109] Table 5

[0110]

[0111] The reverse transcription reaction is shown in Table 6.

[0112] Table 6

[0113]

[0114] Place in RT machine for amplification for 15 min. After the reaction program is completed, dilute cDNA with 180 μl DEPC water to make the final concentration of synthesized cDNA 5 ng / μl and store in a -20°C refrigerator.

[0115] (4) Steps of RT-qPCR experiment:

[0116] Prepare the reaction solution on ice, see Table 7.

[0117] Table 7

[0118]

[0119] Primers: HES1, ATF3, SLC7A11. The specific primer sequences are as follows:

[0120] HES1-F: 5'-TTCCCCCAAGGGAAAAATAC-3'; SEQ ID NO.1;

[0121] HES1-R: 5'-AGACAGAGCCTTGCGATGTT-3'; SEQ ID NO.2;

[0122] ATF3-F: 5'-AGAGCTAAGCAGTCGTGGTATGG-3'; SEQ ID NO.3;

[0123] ATF3-R: 5'-GCCCTCCCGCCTTGATGG-3'; SEQ ID NO.4;

[0124] SLC7A11-F: 5'-CTCCAGGACAGCAGGACTTC-3'; SEQ ID NO.5;

[0125] SLC7A11-R: 5'-CAGCCAGGTCTGCATCAGTA-3'; SEQ ID NO.6;

[0126] GAPDH-F: 5'-CAGTGATGGCATGGACTGTG-3'; SEQ ID NO.7;

[0127] GAPDH-R: 5'-GGGTGTGAACCATGAGAAGT-3'; SEQ ID NO. 8.

[0128] First add 2μl cDNA to the PCR plate, then add 8μl reaction liquid, stick a transparent film (to prevent cDNA from volatilizing due to high temperature during the loading process), and centrifuge the PCR plate at 2000rpm for 2min. qPCR program settings: initial denaturation: 95℃30s; denaturation: 95℃30s; annealing / extension: 60℃30s; cycle steps, usually 40 cycles;

[0129] (5) Data collection and analysis: Use real-time PCR instrument software to record fluorescence signals; analyze Ct values ​​(cycle threshold values) and calculate relative or absolute expression levels.

[0130] 13) Western blot detection of HES1 and related ferroptosis gene mRNA levels

[0131] (1) Extraction of total protein: 48 hours after drug addition or 72 hours after transfection, digest and centrifuge the cells, and wash twice with pre-cooled PBS. Add 100 μl of Lysis Buffer to each well (cell lysis buffer: prepared according to the ratio of RIPA: PMSF = 100:1). Lyse on ice for 20 minutes, scrape off the biological cells with a clean biological cell scraper, and transfer the cell solution to a 1.5 ml EP tube with a pipette. The cells are fully lysed under the ultrasonic lysator. The basic conditions for the working operation control are: amplitude 8.0%, interval treatment time 4s, and lysis treatment time 4s. Centrifuge: 4℃, 12000rpm, 20min. Transfer the supernatant to a new EP tube to avoid aspirating the precipitate, measure the protein concentration, and store in a -80℃ refrigerator.

[0132] (2) Protein concentration was determined using the BCA protein concentration assay kit (P0010, Biotech): Preparation of protein standard: Add 0.8 ml of BSA Preparation Solution to a tube of 20 mg of Protein Standard (BSA) and prepare a 25 mg / ml protein standard solution after fully dissolving. Take an appropriate amount of 25 mg / ml Protein Standard (BSA) and dilute to a final concentration of 2 mg / ml. Prepare 0, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000, and 2000 μg / ml Protein Standard (BSA). Mix thoroughly each time you dilute. Preparation of BCA Plus working solution: Each reaction requires 180 μl of BCA Plus working solution. According to the number of samples to be tested (including the standard), prepare BCA Plus working solution (the volume ratio of A solution to B solution is 50:1) and mix thoroughly. Take 20μl of protein standards of different concentrations and add them to the standard wells of a 96-well plate. Add 2μl of protein sample to the sample wells of a 96-well plate and add 18μl of PBS to make up to 20μl. Add 180μl of BCA Plus working solution to each well, mix with a microplate oscillator for 30 seconds, and place at room temperature (about 25°C) for 30 minutes. Use an enzyme marker to measure the absorbance at A480 wavelength. The absorbance measurements of the standard and sample must be subtracted from the absorbance measurement of the 0μg / ml blank standard. The absorbance of the standard and sample after blank correction is calculated and used to draw the standard curve and calculate the sample concentration. Calculate the protein concentration B (μg / ml) of the sample well according to the standard curve. Measure the OD value corresponding to the protein standard sample, calculate the effective concentration of the protein based on the reference standard distribution action curve, and finally multiply by 10, which is the proportion of the diluted treatment. Balance of protein: Combine 4X loading buffer and Lysis buffer in an appropriate ratio to balance the protein to equal mass and effective concentration (final concentration is 0.75μg / μl). After balancing, heat in a 100℃ water bath for about 8-10 minutes to completely denature the protein. Cool on ice and store in a -20℃ refrigerator.

[0133] (3) Western Blotting: Gel preparation, separation gel preparation: Select the separation gel concentration: Select the appropriate separation gel concentration according to the molecular weight of the target protein. For proteins with larger molecular weight, choose a low-concentration separation gel (such as 8-10%), and for proteins with smaller molecular weight, choose a high-concentration separation gel (such as 12-15%). Prepare separation gel: Mix acrylamide, Tris-HCl, SDS, water, ammonium persulfate and TEMED according to the specific gravity on the instructions. Inject gel: Pour the separation gel into the gel preparation equipment, leaving space for adding concentrated gel. Add ethanol: Add 1 mL of anhydrous ethanol to the separation gel to flatten the liquid surface, and let it stand for 30 minutes until a clear dividing line appears on the upper edge of the glass plate, indicating that the separation gel has polymerized. Prepare concentrated gel: Mix acrylamide, Tris-HCl, SDS, water, ammonium persulfate and TEMED. Inject gel: Pour the concentrated gel on the polymerized separation gel until it is full. Insert the sample comb: Insert the sample comb immediately to avoid bubbles, and let it stand for 20 minutes until the concentrated gel polymerizes. Loading, preparing electrophoresis equipment: Place the glass plate symmetrically in the electrophoresis clamp and clamp it, check for leakage. Sample preparation: Take out the protein sample from the refrigerator, thaw and centrifuge for 3 minutes. Loading: Add the balanced protein sample to the sample well, 20μL per well, and add 3μL protein marker on both sides as a mark. Electrophoresis, setting up the electrophoresis equipment: Place the electrophoresis clamp in the electrophoresis tank, making sure the electrode is in the correct direction. Inject electrophoresis buffer: Inject electrophoresis buffer to the "2Gels" mark. Run electrophoresis: Run at 80V voltage to compress the protein sample in the concentrated gel. After the Marker bands are separated, the voltage can be adjusted to a maximum of 120V until the small molecule protein reaches the bottom of the electrophoresis plate and the electrophoresis is stopped. Transfer, select the membrane pore size: Select the pore size of the PVDF membrane according to the protein size, 0.2μm for small molecule proteins, and 0.45μm for large molecule proteins. Membrane treatment: Soak the PVDF membrane in methanol (5min) to activate it, and then balance it in transfer buffer. Assemble the transfer cassette: Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer cassette in order, making sure there are no bubbles. Perform the transfer: Transfer the membrane at an appropriate current (100-120mA) for 90 minutes. Antibody incubation, membrane blocking: Place the membrane in a blocking solution (such as 5% skim milk powder) and incubate at room temperature for 1 hour. Primary antibody incubation: Incubate the membrane with a primary antibody solution (1:1000) of HES1, ATF3, SLC7A11, and GPX4, usually overnight at 4°C. Wash: Wash the membrane 3 times with wash buffer (TBS containing Tween-20), 5-10 minutes each time. Secondary antibody incubation: Incubate the membrane with HRP-labeled secondary antibody solution at room temperature for 1 hour. Re-wash: Wash the membrane 3 times with wash buffer again. Color development and imaging, color development: Immerse the membrane in ECL color development reagent and incubate for a few minutes. Imaging: Capture the signal in a chemiluminescent imaging system.

[0134] 14) CHIP experiment

[0135] Grow cells to the desired density in a 10 cm dish under appropriate culture conditions. Crosslinking: Add formaldehyde to the cell culture medium, usually at a final concentration of 1%. Incubate at room temperature for 10-20 minutes to fix the interaction between protein and DNA. Add glycine to terminate the reaction, usually at a final concentration of 125 mM, and incubate for 5 minutes. Cell collection: Collect cells and wash twice with cold PBS. Lysis and ultrasonic disruption: Resuspend the cell pellet with lysis buffer, incubate on ice for 10 minutes, and use a sonicator to fragment the chromatin, usually with a fragment size between 200-1000 bp. Keep the sample cool during this period to prevent overheating. Co-immunoprecipitation: Incubate the lysate with non-specific IgG or beads without antibody to remove non-specific binding. Antibody incubation: Add specific antibodies and incubate with rotation, usually overnight at 4°C. Bind beads: Add magnetic beads and incubate for 1-2 hours to bind antibody-chromatin complexes. Wash and elute: Wash the beads multiple times with wash buffer to remove non-specific binding.

[0136] Elute the immune complexes with elution buffer. Reverse crosslinking and DNA purification: Add anti-crosslinking buffer and heat (usually overnight at 65°C) to reverse the crosslinks. Add proteinase K to digest the proteins. Analyze the purified DNA using conventional DNA extraction methods. Use RT-qPCR and gel agarose to detect the enrichment of specific DNA sequences.

[0137] 15) Dual luciferase reporter gene assay

[0138] (1) Plasmid transfection

[0139] Cell Culture: Grow cells to logarithmic growth phase under appropriate culture conditions; cell density is typically 70-90% confluence for transfection. Cell Seeding: Depending on the cell growth rate, seed the cells into appropriate culture plates so that they reach the desired confluence on the day of transfection. Transfection Mix Preparation, Plasmid DNA Dilution: Dilute the plasmid DNA in serum-free medium (e.g., Opti-MEM). Typically, 0.5-5 μg of DNA is used per well, and the specific amount needs to be optimized based on cell type and plate size. Transfection Reagent Dilution: In a separate tube, dilute the transfection reagent in serum-free medium. The amount to be used is determined according to the manufacturer's instructions and experimental optimization results. Mix and Incubate: Mix the diluted plasmid DNA with the diluted transfection reagent. Incubate at room temperature for 15-30 minutes to form the DNA-transfection reagent complex. Transfection, Cell Treatment: Remove the culture medium from the cell culture plate. Wash the cells once with serum-free medium (optional step). Add Transfection Mix: Add the DNA-transfection reagent complex to the cells. Add an appropriate amount of serum-free medium to completely cover the cells. Incubate: Incubate in a 37°C, 5% CO2 incubator for 4-6 hours. Replace medium: Replace the transfection mixture with regular medium containing serum. Continue to incubate the cells for the required time (usually 24-72 hours) for subsequent analysis. Depending on the purpose of the experiment, perform fluorescence microscopy, Western blot, qPCR and other analyses to verify transfection efficiency and gene expression.

[0140] (2) Cell lysis: After transfection for a certain period of time, discard the culture medium, wash once with PBS, completely cover the cells with cell lysis solution and mix thoroughly, add cell lysis solution according to the method in Table 8, and fully lyse the cells for about 30 minutes to 1 hour.

[0141] Table 8

[0142] Culture Plate 96-well plate 48-well plate 24-well plate 12-well plate 6-well plate Cell lysis buffer dosage 100μL 150μL 250μL 500μL 600μL

[0143] (3) Preparation of reagents: Briefly centrifuge the firefly / renilla luciferase substrate (200X) before use; prepare it according to the needs of each experiment:

[0144] Cell lysis buffer (5X): 5X cell lysis buffer was diluted with ddH2O at a ratio of 1:5 to a working solution (1X) before use.

[0145] Firefly luciferase reaction solution: Firefly luciferase substrate (200×) was diluted with firefly luciferase buffer at a ratio of 1:200 to prepare the reaction solution before use.

[0146] Renilla luciferase reaction solution: Renilla luciferase substrate (200×) was diluted 1:200 with Renilla luciferase buffer before use.

[0147] (4) Detection steps: Take 50 μL of cell lysate and add it to the ELISA plate. According to the experimental needs, 3-5 wells can be set up for replication; add 100 μL of firefly luciferase reaction solution, shake the plate to mix, and detect the activity of firefly luciferase. The detection should be completed within 30 minutes; add 100 μL of Renilla luciferase reaction solution, shake the plate to mix, and detect the activity of Renilla luciferase. The detection should be completed within 30 minutes; analyze the data.

[0148] 16) Use interfering RNA technology to construct siHES1: Determine the mRNA sequence of the HES1 gene, select a suitable target sequence, and use online tools or software (such as siRNA Target Finder, BLOCK-iT RNAi Designer) to design the siHES1 sequence. Follow the design principles, such as avoiding consecutive identical bases, selecting a GC content between 30% and 50%, etc. You can choose to chemically synthesize or in vitro transcribe siHES1 for subsequent experiments, and use transfection reagents (such as Lipofectamine 2000, RNAiMAX) to introduce the siHES1 expression vector into cells. The siHES1 sequence is as follows:

[0149]

[0150]

[0151] Construction of HES1-OE overexpression plasmid: GV658 (pcDNA3.1(-)) was digested with restriction endonucleases (KpnI, PacI) to obtain a linearized vector;

[0152] Construction of ATF3-OE overexpression plasmid: GV534 (pcDNA3.1(-)) was digested with restriction endonucleases (NheI, HindIII) to obtain a linearized vector;

[0153] Prepare the target gene fragment by PCR amplification; when designing the amplification primers, a homologous recombination sequence must be added to the 5' end. The target gene fragment is amplified using this primer, and the sequences at the 5' and 3' ends of the amplified product are completely consistent with the sequences at both ends of the linearized cloning vector. Prepare a reaction system with the linearized vector and the target gene amplification product, perform a recombination reaction, and realize the in vitro circularization of the linearized vector and the target gene fragment. The recombinant product is directly transformed, and the single clone on the plate is picked for PCR identification, and the positive clones are sequenced and the results are analyzed. The correct cloned bacterial solution is expanded and extracted to obtain a high-purity plasmid for subsequent experiments. The specific primer sequences are as follows:

[0154] HES1 (NM_005524.4)

[0155] HES1-F:

[0156] 5'-GTGGATCCGAGCTCGGTACCCGCCACCATGCCAGCTGATATAATG-3'; SEQ ID NO.15;

[0157] HES1-R:

[0158] 5'-ATATTTTATTACCGGTTTAATTAATCAGTTCCGCCACGGCCTCCACATGG-3'; SEQ ID NO.16;

[0159] ATF3 (NM_001674.4-promoter)

[0160] ATF3-F: 5'-CGGTACCTGAGCTCGCTAGCGAGATAACAAATAACTTCATTC-3'; SEQ ID NO.17;

[0161] ATF3-R: 5'-AGTACCGGATTGCCAAGCTTGCCGGCGAGAAGAGAGCTGTGC-3'; SEQ ID NO. 18.

[0162] After transfection of cells, ATF3 mRNA levels were detected by RT-qPCR (the operation steps were the same as before);

[0163] 17) Animal experiments

[0164] Female Balb / c-nude mice aged 4-6 weeks were raised, a total of 40 mice, and rested in an SPF environment for 1 week. Method for establishing a tumor model: PC-9OR cells in the logarithmic growth period were digested with trypsin and then resuspended and washed once with PBS, and finally resuspended in 0.2ml 1:1PBS / Matrigel, and the cell density was adjusted to 106 / ml and stored on ice. After the left armpit of the mouse was disinfected with 75% alcohol, the needle was inserted at a 45-degree angle, being careful not to break through the peritoneum, and 200μl / point of cell suspension was injected. After the injection, the needle was slowly withdrawn to prevent leakage of the injection solution. Tumor volume and body weight were monitored twice a week. The tumor volume reached 80-100mm 3 The mice were divided into groups.

[0165] Experimental animals and grouping: The mice were randomly divided into the following groups according to their body weight: control group: 10 mice; shikonin group: 10 mice; osimertinib group: 10 mice; osimertinib combined with shikonin treatment group: 10 mice; Mouse treatment: Control group: intraperitoneal injection of normal saline every 3 days after tumor formation; Shikonin treatment group: intraperitoneal injection of shikonin (0.8 mg / kg) every 3 days after tumor formation; Osimertinib treatment group: intraperitoneal injection of osimertinib (5 mg / kg) every 3 days after tumor formation; Shikonin combined with osimertinib treatment group: Shikonin (0.8 mg / kg) combined with osimertinib (5 mg / kg) every 3 days after tumor formation. Transplant tumor analysis data: Body weight: Before each treatment and during the experiment, record the weight of mice and tumor size; Tumor solid photos: After the experiment ended for 21 days (expected experimental time, until the largest tumor reaches 2 cm), the mice were euthanized by decapitation and dislocation. After the death was determined, the eyeballs were immediately enucleated and blood was collected. The mice were dissected, the tumor weight and maximum diameter were weighed and recorded, and tumor comparison photos were taken (at least 6 samples per group); After the tumor was photographed, the tumor tissue was quickly cut into two halves, and one half was placed in formalin fixative for subsequent HE and IHC detection: At least 6 mice in each group were taken for HE, ki-67, HES1, ATF3, and SLC7A11 immunohistochemical staining of tumor tissue. It would be best if the HE and IHC results can be scanned in full, otherwise at least 40X photos (3 photos per slide) are provided. The other half was frozen in a -80℃ refrigerator and waited for grinding to collect protein and RNA for WB / PCR detection; if tumor metastasis was found in the adjacent tissues of the tumor after the mouse was dissected, the corresponding data of the metastatic tumor should be recorded. For example, comparison photos of lung metastatic nodules.

[0166] 18) Blood biochemical index detection

[0167] Eyeball blood collection steps, fixation: Place the euthanized nude mouse on the dissection table. Tilt its head slightly for easy operation. Blood collection: Use the thumb and index finger to gently squeeze the skin around the eye socket to make the eyeball protrude slightly. Use a sterile blood collection needle or capillary to insert into the posterior ocular venous plexus and collect the required amount of blood (usually 100-200 microliters). Transfer the collected blood quickly to a blood collection tube containing an anticoagulant (such as EDTA or heparin). Blood sample processing, centrifugation: Centrifuge the collected blood sample at 3000rpm for 10 minutes at 4°C to separate the serum. Serum collection: Carefully transfer the supernatant serum to a new sterile centrifuge tube to avoid disturbing the precipitate. Biochemical index detection: Use an automatic biochemical analyzer to detect the following indicators: Liver function test: AST (aspartate aminotransferase) and ALT (alanine aminotransferase): According to the instructions of the kit, add appropriate amount of serum and reagents, incubate and measure the absorbance change. Calculate enzyme activity, usually expressed in U / L. Renal function test: ALB (albumin) and TP (total protein): Use colorimetric method according to the instructions of the kit. CRE (creatinine) and UREA (urea): Use the corresponding colorimetric kit to measure the absorbance and calculate the concentration, which is usually expressed in mg / dL or mmol / L.

[0168] 19) Immunohistochemistry (IHC)

[0169] Sample preparation: Fixation: The transplanted tumor tissue samples are fixed in formalin to preserve tissue structure and protein antigenicity. Embedding: The fixed tissue samples are embedded in paraffin for sectioning. Sectioning: The paraffin-embedded tissue is cut into thin slices, usually 4-6 μm thick, using a microtome, and the slices are mounted on slides. Dewaxing and hydration: The paraffin sections on the slides are treated in a series of gradient concentrations of xylene and ethanol to remove the paraffin and rehydrate the tissue sections. Antigen retrieval: According to the characteristics of the antibody and antigen, an appropriate antigen retrieval method (such as heat retrieval or enzyme retrieval) is selected to expose the antigen epitope. Blocking: A blocking solution (such as a buffer containing normal serum or BSA) is applied to the sections to reduce nonspecific binding. Primary antibody incubation: The sections are incubated with an appropriately diluted primary antibody (specific antibody against the target protein), usually at room temperature for 1-2 hours or at 4°C overnight. Washing: The sections are washed with PBS or TBS buffer to remove unbound primary antibody. Secondary antibody incubation: Incubate the sections with an appropriate secondary antibody (usually a biotinylated or enzyme-labeled antibody corresponding to the species from which the primary antibody is derived). Washing: Wash the sections again to remove unbound secondary antibody. Color development: Apply a colorimetric reagent (such as DAB or AEC) to detect the activity of the enzyme-labeled secondary antibody to produce a visual signal on the section. Counterstaining: Counterstain the cell nucleus with dyes such as hematoxylin to better observe the tissue structure. Dehydration and mounting: Dehydrate the sections through a series of gradient concentrations of ethanol and xylene, and then mount them with mounting media. Microscope observation: Observe and record the results under a microscope to analyze the expression and localization of the target protein.

[0170] Experimental Results

[0171] 1) Shikonin enhances the cytotoxicity of osimertinib against osimertinib-resistant NSCLC cells

[0172] Experiment ①: The WST-1 method was used for the first time to detect the cytotoxicity of shikonin against osimertinib-resistant non-small cell lung cancer cells (including PC9-OR and H1975-OR cells). Figure 1 As shown in BC, shikonin inhibited the proliferation of PC9-OR and H1975-OR cells in a dose-dependent manner. Then, the antiproliferative effect of shikonin combined with osimertinib was evaluated. Shikonin significantly enhanced the proliferation inhibition of PC9-OR and H1975-OR cells by osimertinib ( Figure 1 DE). In addition, the combination index (CI) value of the combination treatment was calculated using CompuSyn software to evaluate whether the combination of shikonin and osimertinib showed a synergistic enhancement of cytotoxicity. Figure 1As shown in FG, the CI values ​​of osimertinib and shikonin in PC9-OR and H1975-OR were both less than 1.0, which means that shikonin combined with osimertinib has a synergistic effect in PC9-OR and H1975-OR cells. In addition, compared with the use of osimertinib or shikonin alone, the combination of the two drugs significantly reduced the colony formation rate ( Figure 1 HJ). Annexin VFITC / PI staining and flow cytometry were used to detect the apoptosis rate after 48 hours of different treatments. It was found that compared with osimertinib or shikonin alone, osimertinib combined with shikonin significantly increased the apoptosis rate ( Figure 1 KM). In conclusion, shikonin enhanced the cytotoxicity of osimertinib against PC9-OR and H1975-OR cells.

[0173] 2) Shikonin combined with osimertinib induced ferroptosis in osimertinib-resistant NSCLC cells.

[0174] Experiment ②: To further determine whether the synergistic effect of shikonin combined with osimertinib is related to the ferroptosis pathway, various cell death inhibitors or activators [Z-VAD-FMK (apoptosis inhibitor), 3-methyladenine (3-MA, autophagy inhibitor), Ferrostatin-1 (Fer-1, ferroptosis inhibitor), Deferoxamine (DFO, ferroptosis inhibitor) and Erastin (ferroptosis activator)] were used to treat cells with or without osimertinib combined with shikonin. Figure 2 As shown in AB, Z-VAD-FMK and 3-MA slightly increased cell survival compared with the shikonin group or the osimertinib combined with shikonin group, but the effects of ferroptosis inhibitors DFO and Fer-1 were more significant than those of apoptosis and autophagy inhibitors. In contrast, compared with Erastin alone, the ferroptosis activator Erastin combined with shikonin or osimertinib combined with shikonin significantly inhibited cell proliferation. The results showed that shikonin combined with osimertinib promoted ferroptosis. Subsequently, the related indicators of ferroptosis, including intracellular reactive oxygen species (ROS), lipid ROS, malondialdehyde (MDA), glutathione (GSH), and intracellular Fe 2+ First, the peroxide-sensitive fluorescent probe DCFH-DA was used to detect the level of intracellular ROS. As expected, after combined treatment with osimertinib and shikonin, the accumulation of ROS in PC9-OR and H1975-OR cells was significantly higher than that with osimertinib or shikonin alone ( Figure 2 CD). Next, the lipid ROS level was detected using the C11-BODIPY kit. The results showed that in PC9-OR and H1975-OR cells, the lipid ROS level in the osimertinib combined with shikonin group was significantly higher than that in the osimertinib or shikonin group ( Figure 2EF). In addition, malondialdehyde (MDA), a marker of oxidative stress, also showed a significant increase in PC9-OR and H1975-OR cells after osimertinib combined with shikonin treatment, an effect that could be reversed by ferrostatin-1 and promoted by erastin ( Figure 2 G). In contrast, after osimertinib combined with shikonin treatment, GSH levels in PC9-OR and H1975-OR cells were significantly decreased ( Figure 2 H), this effect can also be reversed by Ferrostatin-1, while Erastin further depletes GSH. In addition, compared with the osimertinib or shikonin group, the fluorescence intensity in PC9-OR and H1975-OR cells treated with osimertinib and shikonin increased, indicating that the combination of osimertinib and shikonin promoted the Fe 2+ The accumulation of Figure 2 IJ). Collectively, these findings strongly suggest that shikonin combined with osimertinib can induce ferroptosis in osimertinib-resistant NSCLC cells.

[0175] 3) HES1 is highly expressed in NSCLC and positively correlated with the poor prognosis of NSCLC patients.

[0176] To explore the molecular mechanism of ferroptosis induced by combined treatment, high-throughput sequencing experiments were performed to determine the mRNA expression profile in PC9-OR cells. Figure 3 As shown in A. Differentially expressed genes were detected by comparing the control group and the combination group (adjusted P value < 0.001, fold change > 1.0). The volcano plot shows the top 10 down-regulated genes and 10 up-regulated genes in the combination group. Among these differentially expressed proteins, the protein level of HES1 in the combination group was significantly decreased ( Figure 3 B). The mRNA expression of HES1 in LUAD patient tissue samples was investigated using the TCGA dataset. The results showed that in the TCGA-LUAD cohort, in the unpaired ( Figure 3 C) and pairing ( Figure 3 D) In ​​cancer tissues and adjacent normal tissues, the expression of HES1 in LUAD cancer tissues was higher than that in adjacent normal tissues. Kaplan-Meier survival curve analysis showed that patients with high HES1 expression had a poor prognosis ( Figure 3 E) To determine the diagnostic value of HES1 in LUAD, a diagnostic ROC analysis was performed. Figure 3As shown in F, the results showed that HES1 (P < 0.05, AUC = 0.699) could be used as an independent diagnostic predictor for LUAD. Taken together, these findings suggest that HES1 may be a target gene for ferroptosis in osimertinib-induced NSCLC cells.

[0177] 4) Knockout of HES1 inhibits cell proliferation and induces ferroptosis

[0178] Experiment ③: HES1 is a well-known transcriptional repressor. Public databases such as the Gene Transcription Regulation Database (https: / / gtrd20-06.biouml.org / ), hTFtarget Database (https: / / guolab.wchscu.cn / hTFtarget#! / ) and Cistrome DB Database (http: / / cistrome.org / db) were used to predict downstream target genes, and the predicted target genes and ferroptosis-related genes were selected as the intersection genes, and ATF3 was selected as the target gene for subsequent research ( Figure 3 G). Then, the expression levels of HES1 and ATF3 in lung adenocarcinoma tissues and adjacent paracancerous tissues were detected. It was found that HES1 expression was higher in LUAD tissues compared with adjacent paracancerous tissues ( Figure 3 H), while the expression of ATF3 was lower ( Figure 3 I). In addition, the expression levels of HES1 and ATF3 in osimertinib-sensitive and osimertinib-resistant NSCLC cell lines were analyzed. The results showed that the expression of HES1 was upregulated in osimertinib-resistant cell lines PC9-OR and H1975-OR compared with sensitive cell lines PC9 and H1975 ( Figure 3 J), in contrast, ATF3 expression was downregulated in osimertinib-resistant cell lines ( Figure 3 K). The expression of HES1 and ferroptosis-related genes (ATF3, SLC7A11, GPX4) in these cell lines was also evaluated, and it was found that the protein levels of HES1, SLC7A1, and GPX4 were upregulated in PC9-OR and H1975-OR cells, whereas the protein level of ATF3 was downregulated in PC9-OR cells and H1977-OR cells compared with PC9 and H1975 cells ( Figure 3 L).

[0179] To investigate whether knocking out HES1 can induce ferroptosis, two non-overlapping siRNAs targeting HES1 (siHES1-1 and siHES1-2) were used to silence the expression of HES1 in PC9-OR and H1975-OR cells. After transfection, the cells were treated with or without osimertinib, and the cell proliferation rate was evaluated. The results showed that knocking out HES significantly inhibited cell proliferation ( Figure 3MN). In addition, when HES1 expression was reduced, the colony formation rate was significantly reduced ( Figure 3 OQ). Figure 3 As shown in Figure R, siHES1 mediated the reduction of HES1 and SLC7A11 expression and promoted ATF3 expression in PC9-OR and H1975-OR cells. Taken together, these findings indicate that knockdown of HES1 significantly inhibited the proliferation of osimertinib-resistant NSCLC cells and induced ferroptosis.

[0180] 5) Combination treatment induces ferroptosis by attenuating HES1 binding to the ATF3 promoter and promoting ATF3 transcription and activity

[0181] The levels of HES1 and ferroptosis-related ATF3, SLC7A11, and GPX4 proteins were detected after cells were treated with different treatment groups. Figure 4 As shown in A, the levels of HES1, SLC7A11, and GPX4 in the combined group were decreased, while the level of ATF3 was increased. In addition, the combination of osimertinib and shikonin with erastin significantly inhibited colony formation, while the combination with Fer-1 significantly increased colony formation ( Figure 4 BC). In addition, the combination of osimertinib and shikonin with Erastin further downregulated the expression of HE1, SLC7A11 and GPX4 proteins, and also upregulated the expression of ATF3 protein, while the combination with Fer-1 upregulated the expression levels of HE1, SLC7A11, GPX4 and downregulated the expression level of ATF3 ( Figure 4 D). The results showed that the combination group induced ferroptosis by downregulating HES1 and upregulating ATF3 expression. In addition, at the same time, silencing of HES1 in PC9-OR and H1975-OR cells significantly upregulated the expression of ATF3 ( Figure 3 R). Therefore, the hypothesis was proposed whether the combination group could overcome osimertinib-resistant NSCLC via the HES1 / ATF3 / SLC7A11 signaling pathway.

[0182] To further verify this hypothesis, we investigated whether HES1 directly binds to ATF3 as a transcriptional repressor and negatively regulates ATF3 transcription. Dual-luciferase reporter gene assays were performed to verify the predicted direct interaction between HES1 and ATF3. First, the dual-luciferase reporter gene assay showed that OE-HES1 significantly reduced the fluorescence intensity of the OE-ATF3 group ( Figure 4 E). In addition, chromatin immunoprecipitation (ChIP) analysis revealed that HES1 binds to sequences within the ATF3 promoter in PC9-OR cells ( Figure 4 FG).

[0183] Collectively, these results demonstrate a role for HES1 in binding to the ATF3 promoter and acting as a negative regulator of ATF3 transcription. To investigate the binding sites within the ATF3 promoter in non-small cell lung cancer, the JASPAR database was used to explore the transcriptional repressor binding sites of HES1 ( Figure 4 H), and found that there are two HES1 binding sites in the promoter region of the ATF3 gene ( Figure 4 I). Dual luciferase reporter gene assays showed that HES1 has the ability to bind to and downregulate the wild-type (WT) ATF3 promoter, mutant binding sites 1 (MUT1) and 2 (MUT2). However, there was no significant difference in fluorescence intensity between WT and MUT1, while there was a significant statistical difference in luminescence intensity between the WT and MUT2 groups. These results indicate that HES1 binds to the ATF3 promoter of MUT2, but its effect is not as strong as that of the wild type, indicating that mutant site 2 is not the only binding site and that there may be other binding sites ( Figure 4 J). In addition, the mRNA expression level of ATF3 was detected after overexpression and knockdown of HES1 in 293T cells (the specific experimental steps were the same as above). Figure 4 As shown in Figure K, in 293T cells, the mRNA expression level of ATF3 was decreased after HES1 overexpression compared with the control group, while the mRNA expression level of ATF3 was increased after HES1 knockdown compared with the control group. The above results indicate that the combination group induces ferroptosis by weakening the binding of HES1 to the ATF3 promoter and promoting ATF3 transcription and activity.

[0184] 6) The combination group inhibited xenograft tumors in vivo

[0185] The effect of the combination group on the growth of PC9-OR xenografts was evaluated in vivo. Mice inoculated with xenografts were intraperitoneally injected with osimertinib (5.0 mg / kg) and / or shikonin (0.8 mg / kg) or an equal amount of saline (as a control) every 3 days. Compared with the control group and the use of a single drug alone, the combination group significantly inhibited tumor growth and reduced tumor volume ( Figure 5 AD). In addition, the combination group did not produce observable toxic effects based on the body weight of the mice ( Figure 5 E). There were no significant differences in serum ALT, AST, TP, ALB, CRE and BUN levels among the groups ( Figure 5 FK), indicating that the drug doses used in this study had no significant effect on the liver and kidney function of mice. H&E, Ki-67, HES1, ATF3, and SLC7A11 staining were performed on isolated tumor tissues from all treatment groups. Figure 5As shown in LP, compared with the use of drugs alone, the combination group showed the highest degree of tumor tissue damage, manifested as reduced cell density and nuclear shrinkage; compared with the use of drugs alone, the combination group significantly downregulated the expression of Ki-67, HES1 and SLC7A11, and significantly upregulated the expression of ATF3 protein. These results indicate that the combined treatment has a synergistic effect on PC9-OR cells resistant to osimertinib in vivo without significantly increasing toxic side effects.

[0186] in conclusion:

[0187] Shikonin combined with osimertinib promotes ferroptosis by inhibiting the HES1 / ATF3 / SLC7A11 signaling axis, thereby reversing the resistance of osimertinib-resistant non-small cell lung cancer. This combination regimen is expected to be a promising treatment to overcome the resistance of osimertinib-resistant non-small cell lung cancer.

[0188] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of shikonin combined with osimertinib in reversing osimertinib-resistant non-small cell lung cancer.

2. Application of shikonin combined with osimertinib in the preparation of drugs for reversing osimertinib-resistant non-small cell lung cancer.

3. Application of shikonin combined with osimertinib in the preparation of drugs for the treatment of non-small cell lung cancer.

4. Application of materials that inhibit HES1 expression in the preparation of drugs for the treatment of non-small cell lung cancer.

Images