Application of irbesin in overcoming drug resistance of tumor cells

Abstract

The invention discloses application of irbesin in overcoming drug resistance of tumor cells. The invention proposes and verifies that the irbesin can significantly reduce the activity of osimertinib drug-resistant tumor cells by inducing cancer cell ferroptosis for the first time, further enhances the treatment effect of osimertinib, inhibits the progress of drug-resistant non-small cell lung cancer, and provides a new direction for treatment of osimertinib drug-resistant lung cancer.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and more particularly to the application of ibecin in overcoming drug resistance in tumor cells. Background Technology

[0002] Lung cancer is the leading cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent pathological type, accounting for approximately 85% of all lung cancers. In Asian patients with lung adenocarcinoma, activating mutations in the epidermal growth factor receptor (EGFR) gene are particularly common, with a positive rate of 40-60%. For EGFR-mutant advanced NSCLC, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the standard treatment. Among them, third-generation EGFR-TKIs, represented by osimertinib, have become an important first-line treatment option due to their ability to simultaneously inhibit EGFR-sensitive mutations and T790M resistance mutations, significantly improving overall survival. However, osimertinib often faces the serious challenge of acquired resistance in clinical application.

[0003] Ibexin is a steroidal alkaloid isolated from the bulb of Fritillaria thunbergii, and its specific biological activities are not yet clear. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the use of ibexin in the preparation of a treatment or adjuvant therapy for non-small cell lung cancer, especially osimertinib-resistant non-small cell lung cancer, whether used alone or in combination with osimertinib.

[0005] Technical solution: The application of ibecin as described in this invention in overcoming drug resistance in tumor cells.

[0006] Preferably, the CAS number of the ibecin is 143502-51-6.

[0007] Preferably, the application is in the preparation of drugs for the treatment or adjuvant treatment of drug-resistant non-small cell lung cancer.

[0008] Preferably, the drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0009] Preferably, the drug contains ibexin or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0010] Preferably, the drug further contains osimertinib as an active ingredient; more preferably, the mass ratio of ibexin to osimertinib in the drug is 1.5~2.5:1.

[0011] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0012] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that ibexin can significantly reduce the activity of osimertinib-resistant tumor cells by inducing ferroptosis in cancer cells, and further enhance the therapeutic effect of osimertinib, inhibit the progression of drug-resistant non-small cell lung cancer, and provide a new direction for the treatment of osimertinib-resistant lung cancer. Attached Figure Description

[0014] Figure 1 Figure showing the IC50 values ​​of PC9 / OR cells after ibexin treatment; Figure 2 Figure 1 shows the flow cytometry results of apoptosis levels in PC9 / OR cells after treatment. Figure 3 This is a quantitative analysis diagram of the flow cytometry results of PC9 / OR cell apoptosis levels after treatment; Figure 4 The image shows the results of the colony-forming ability assay for PC9 / OR cells after treatment. Figure 5 This is a graph showing the quantitative analysis of the colony-forming ability of PC9 / OR cells after treatment. Figure 6 The graph shows the results of the PC9 / OR cell proliferation assay after treatment. Figure 7 This is a graph showing the quantitative analysis results of the PC9 / OR cell proliferation assay after treatment; Figure 8 The figure shows the flow cytometry results of mitochondrial membrane potential measurement in PC9 / OR cells after treatment. Figure 9 This is a quantitative analysis graph of the flow cytometry results of mitochondrial membrane potential measurement in PC9 / OR cells after treatment. Figure 10 Fluorescence images of ROS levels in PC9 / OR cells after treatment; Figure 11 This is a quantitative analysis of fluorescence images used to measure ROS levels in PC9 / OR cells after treatment. Figure 12 Figure 1 shows the flow cytometry results of ROS level determination in PC9 / OR cells after treatment. Figure 13 The graph shows the results of ferroptosis marker measurement in PC9 / OR cells after treatment; Figure 14 The graph shows the statistical results of mouse body weight in different treatment groups; Figure 15 Representative images of H&E staining in lung tissues of mice from different treatment groups; Figure 16 Representative images of Masson staining in lung tissues of mice from different treatment groups; Figure 17 Statistical results of tumor volume growth curves in mice of different treatment groups; Figure 18 Representative images of Ki67 protein immunohistochemical staining in tumor tissues of mice in different treatment groups; Figure 19 A statistical chart showing the results of ferroptosis-related indicators in tumor tissues of mice in different treatment groups. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: In vitro efficacy validation of yibeissine (YIBE) in the treatment of drug-resistant non-small cell lung cancer 1. Cell viability assay PC9 / OR cells (purchased from Zhejiang Meisen Cell Technology Co., Ltd., catalog number CTCC-0550-NY) were used at a rate of 2×10⁻⁶ cells / year. 3 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 h. Then, the original medium was replaced with DMEM complete medium containing YIBE (purchased from MedChemExpress LLC., catalog number HY-121631) at final concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, and 64 µM. After 24 h of treatment, 100 μL of DMEM basal medium containing 10% CCK8 reagent (purchased from Novizan, catalog number A311-01) was added to each well and the plates were incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0017] The results are as follows Figure 1 As shown, with the continuous increase of YIBE treatment concentration, the viability of PC9 / OR cells decreased significantly, exhibiting a YIBE dose-dependent effect. The IC50 value after 24 h of treatment was [data missing]. 50 The value was 10.069 µM. Therefore, the YIBE treatment concentration was set to 10 µM in subsequent experiments.

[0018] 2. Measurement of apoptosis level PC9 / OR cells at 2×10 4Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. After adherence, the cells were treated for 24 h with DMEM complete medium containing 1‰ DMSO, or osimertinib (OSI, MedChemExpress LLC., catalog number HY-15772) at a final concentration of 5 µM, or YIBE at a final concentration of 10 µM, or both OSI at a final concentration of 5 µM and YIBE at a final concentration of 10 µM. Cells were then collected and stained using the Annexin V-PE / 7AAD apoptosis detection kit (catalog number A213-01). The stained cells were analyzed using a Beckman CytoFLEX flow cytometer to quantify apoptosis levels.

[0019] The results are as follows Figure 2 , 3 As shown, for PC9 / OR cells, the level of apoptosis induced by OSI was relatively low, only about 8.8%, which was not significantly different from the DMSO treatment group. However, after treatment with 10 µM YIBE, the apoptosis level of PC9 / OR cells increased to about 25%. In the combined treatment group of 5 µM OSI and 10 µM YIBE, the apoptosis level of PC9 / OR cells was about 36%. These experimental results show that YIBE can not only induce a high level of apoptosis on its own, but also significantly increase the apoptosis induced by OSI in PC9 / OR cells. That is, YIBE can effectively overcome the OSI resistance problem that occurs in the treatment of non-small cell lung cancer.

[0020] 3. Cell clone formation ability assay PC9 / OR cells were seeded at a density of 500 cells / well in 6-well plates and cultured for 24 h. Then, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or OSI at a final concentration of 5 µM, or YIBE at a final concentration of 10 µM, or both OSI at a final concentration of 5 µM and YIBE at a final concentration of 10 µM, for another 24 h. After treatment, the medium was replaced with fresh, drug-free DMEM complete medium, and the medium was changed every 2 days for a total of 14 days. Cells were then washed with PBS, fixed with methanol, stained with Beyotime crystal violet reagent (C0121-100ml), and observed under a microscope for quantitative analysis.

[0021] The results are as follows Figure 4 , 5 As shown, compared with the DMSO-treated group of PC9 / OR cells, there was no significant change in cell clonogenic ability before and after OSI treatment. However, YIBE treatment significantly inhibited the clonogenic ability of PC9 / OR cells and significantly enhanced the inhibitory effect of OSI drug on the clonogenic ability of PC9 / OR cells, showing a significant difference.

[0022] 4. Cell proliferation capacity assay PC9 / OR cells were used at a rate of 5 × 10 3 The wells were seeded at a density of 1‰ in 96-well plates. After 24 h of incubation, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or OSI with a final concentration of 5 µM, or YIBE with a final concentration of 10 µM, or both OSI with a final concentration of 5 µM and YIBE with a final concentration of 10 µM, for another 24 h. After treatment, 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well, and the plates were incubated at 37℃ for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 15 min, the plates were permeated with Beyotime immunostaining permeabilization solution (catalog number P0096) at room temperature for 20 min. Then, Click reaction solution (purchased from Novizan, catalog number A413-01) was added, and the plates were incubated in the dark for 30 min. The plates were then mounted with Beyotime anti-fluorescence quenching mounting solution (catalog number P0131), and the images were observed and quantitatively analyzed using a fluorescence microscope.

[0023] The results are as follows Figure 6 , 7 As shown, there was no significant difference in cell proliferation ability before and after OSI treatment in PC9 / OR cells, indicating that PC9 / OR cells are insensitive to OSI drugs. However, YIBE treatment significantly enhanced the inhibitory effect of OSI on the proliferation ability of PC9 / OR cells.

[0024] 5. Mitochondrial membrane potential measurement PC9 / OR cells at 2×10 4 Inoculate the cells at a density of cells / well in 6-well plates, incubate overnight, and then replace the original medium with DMEM complete medium containing 1‰ DMSO, or OSI with a final concentration of 5 µM, or YIBE with a final concentration of 10 µM, or both OSI with a final concentration of 5 µM and YIBE with a final concentration of 10 µM for 24 h.

[0025] Cells were collected after treatment and incubated in PBS buffer containing 10 µg / mL JC-1 (purchased from MedChemExpress LLC., catalog number: HY-15534) at 37°C in the dark for 30 min. Fluorescence signals were detected using a Beckman CytoFLEX flow cytometer. The excitation wavelength for JC-1 monomer (green fluorescence) was set to 488 nm, and the emission wavelengths were detected at 525 / 50 nm. The emission wavelengths for JC-1 multimer (red fluorescence) were detected at 590 / 40 nm.

[0026] The results are as follows Figure 8 , 9As shown, the mitochondrial membrane potential of PC9 / OR cells in the DMSO treatment group was higher (Q1 / Q2=3.08), and the mitochondrial membrane potential of PC9 / OR cells in the OSI group was also at a high level (Q1 / Q2=2.91). This indicates that OSI has no significant effect on the mitochondrial membrane potential of PC9 / OR cells and does not alter cell function. However, YIBE treatment significantly reduced the mitochondrial membrane potential of PC9 / OR cells (Q1 / Q2=1.37), and the mitochondrial membrane potential was the lowest in the YIBE+OSI treatment group (Q1 / Q2=0.88). This indicates that YIBE can induce a decrease in mitochondrial activity in PC9 / OR cells, thereby leading to a decrease in cell viability and achieving the goal of treating non-small cell lung cancer.

[0027] 6. Measurement of reactive oxygen species (ROS) levels PC9 / OR cells were used at a rate of 1×10 4 Cells were seeded at a density of 1 cell / well in 24-well plates pre-placed with glass slides. After overnight culture and adhesion, the cells were treated for 24 h with DMEM complete medium containing 1‰ DMSO, or OSI at a final concentration of 5 µM, or YIBE at a final concentration of 10 µM, or both OSI at a final concentration of 5 µM and YIBE at a final concentration of 10 µM. The treated cells were then fixed with 4% paraformaldehyde at 37°C for 15 min, permeabilized with Beyotime immunostaining and permeabilization solution at room temperature for 20 min, and finally stained with a 5 μM DCFH-DA probe (purchased from MedChemExpress LLC., catalog number HY-D0940) at 37°C for 30 min. The slides were then mounted with Beyotime anti-fluorescence quenching mounting solution, and the images were observed and quantitatively analyzed using a fluorescence microscope.

[0028] The results are as follows Figure 10 , 11 As shown, the ROS fluorescence intensity of the DMSO treatment group and the OSI treatment group was almost the same, with no significant difference. However, the ROS fluorescence intensity of the YIBE treatment group and the YIBE+OSI combined treatment group increased significantly, and the fluorescence intensity of the YIBE+OSI combined treatment group was the highest.

[0029] PC9 / OR cells were used at a rate of 5 × 10 3 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. After adhesion, the cells were treated for 24 h with DMEM complete medium containing 1‰ DMSO, or OSI at a final concentration of 5 µM, or YIBE at a final concentration of 10 µM, or both OSI at a final concentration of 5 µM and YIBE at a final concentration of 10 µM. The treated cells were then stained with a DCFH-DA probe at a final concentration of 5 μM at 37°C for 30 min. Quantitative analysis was performed by flow cytometry.

[0030] The results are as follows Figure 12 As shown, for PC9 / OR cells, the ROS levels in the DMSO control group and OSI-induced cells were low and showed no significant difference. However, after YIBE treatment, it was found that YIBE not only induced higher ROS levels on its own, but also significantly increased the ROS levels in OSI-induced PC9 / OR cells.

[0031] 7. Measurement of ferromorigenesis-related indicators PC9 / OR cells were loaded at 2×10 4 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, the culture medium was replaced with DMEM complete medium containing 1‰ DMSO, or OSI with a final concentration of 5 µM, or YIBE with a final concentration of 10 µM, or both OSI with a final concentration of 5 µM and YIBE with a final concentration of 10 µM, for 24 h. After treatment, the cell culture supernatant was collected, and the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and glutathione (GSH) in the supernatant were detected using the Beyotime Lipid Oxidation (MDA) Detection Kit (Catalog No. S0131S), the Sangon Biotech 4-Hydroxynonenal (4-HNE) ELISA Kit (Catalog No. D751041), and the Beyotime GSH and GSSG Detection Kit (Catalog No. S0053).

[0032] The results are as follows Figure 13 As shown, there were no significant differences in ferroptosis-related indicators between the DMSO control group and the OSI treatment group for PC9 / OR cells. However, after YIBE treatment, for indicators positively correlated with ferroptosis, YIBE not only induced higher levels of MDA, 4-HNE, and Fe alone, but also significantly increased the levels of MDA, 4-HNE, and Fe in OSI-induced PC9 / OR cells. For indicators negatively correlated with ferroptosis, YIBE pretreatment showed that YIBE not only induced lower levels of GSH alone, but also significantly reduced the GSH level in OSI-induced PC9 / OR cells.

[0033] The above experiments show that YIBE can not only effectively induce ferroptosis in non-small cell lung cancer, but also greatly solve the OSI resistance problem that occurs during its treatment.

[0034] Example 2: Validation of the therapeutic effect of ibexin in a mouse model of drug-resistant non-small cell lung cancer Female Balb / c nude mice aged 6-8 weeks and weighing 18-20 g were purchased from the Experimental Animal Center of Nantong University and housed in an SPF-grade animal room at a temperature of 22±2℃ and a humidity of 50±10%, with a 12-hour day-night cycle. They were acclimatized for one week before the experiment.

[0035] PC9 / OR cells were distributed at a rate of 5 × 10⁵ cells per mouse. 5 One cell was inoculated subcutaneously under the left axilla to create a tumor. The tumor was induced when it reached approximately 100 mm in size. 3 At that time, tumor-bearing mice were randomly divided into 4 groups, including: saline group, OSI treatment group (dose of 10 mg / kg), YIBE treatment group (dose of 20 mg / kg), and YIBE+OSI combined treatment group (YIBE dose of 20 mg / kg and OSI dose of 10 mg / kg), and drug treatment was started at the same time.

[0036] The drug was administered intraperitoneally every 3 days, while the saline group received the same volume of saline. Treatment continued until day 18. Upon reaching the experimental endpoint, the mice were euthanized, and tumor and lung tissues were collected from each group for subsequent analysis of relevant indicators.

[0037] 1. Weight monitoring Starting from day 0 of the drug administration, the body weight of mice in each experimental group was measured every 3 days.

[0038] The results are as follows Figure 14 As shown, there was no significant difference in body weight among the four groups of mice in the experiment, indicating that the drug dosage in the experiment did not cause biological toxicity.

[0039] 2. H&E staining Mouse lung tissue was immersed in 4% paraformaldehyde solution for 48 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 3 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0040] The results are as follows Figure 15 As shown, the H&E staining results of lung tissue from mice in all treatment groups were almost identical: the cell nuclei were dark blue, indicating normal cell activity; and the cytoplasm was pink, indicating a healthy cytoplasm.

[0041] 3. Masson staining Masson staining of liver tissue The preliminary steps were the same as those for H&E staining. In the staining section, the lung tissue was stained using the Beyotime Masson Tricolor Staining Kit (catalog number C0189S). After mounting with neutral resin, the tissue was observed and images were acquired under a microscope.

[0042] The results are as follows Figure 16 As shown, the Masson staining results of lung tissue from all treatment groups were almost identical, indicating that the drug treatment did not induce lesions in the lung tissue of the mice.

[0043] 4. Tumor volume monitoring Starting from day 0 of the drug administration, the tumor volume of mice in each experimental group was measured every 3 days. The length and width of the tumor were recorded, and then the tumor volume was calculated according to the following formula and statistically analyzed: Tumor volume (mm) 3 = length × width × width / 2.

[0044] The results are as follows Figure 17 As shown, the tumor volume change curves in the saline treatment group and the OSI treatment group were almost identical, indicating that the OSI drug did not induce a significant tumor inhibition effect. However, after YIBE treatment or YIBE+OSI treatment, the tumor volume in nude mice was much smaller than that in the saline treatment group, and the tumor volume in the YIBE+OSI treatment group was the smallest. This indicates that YIBE is effective in treating non-small cell lung cancer and can significantly enhance the therapeutic effect of OSI drugs.

[0045] 5. Immunohistochemical detection of Ki67 expression in tumor tissue Tumor tissue samples were fixed overnight with 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, and cut into 4 μm sections. Antigen retrieval was performed at 95°C for 20 min with citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide solution for 10 min, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were then incubated overnight at 4°C with Ki67 primary antibody (1:10000, Proteintech, catalog number 27309-1-AP) at 1:10000 dilution. After washing, the sections were incubated with horseradish peroxidase (HRP) conjugated with secondary antibody (Proteintech, catalog number SA00004-2) at 1:500 dilution for 1 h at room temperature. Signal detection was performed using the Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. After mounting with neutral resin, the sections were observed and images were acquired under a microscope.

[0046] The results are as follows Figure 18 As shown, there was no significant difference in the positive rate of Ki67 between the OSI treatment group and the saline treatment group. However, YIBE and YIBE+OSI treatments significantly reduced Ki67 expression, with a greater reduction in the YIBE+OSI group. This preliminarily suggests that YIBE can significantly reduce Ki67 protein expression levels in vivo, thereby inhibiting the growth of OSI-resistant tumors.

[0047] 6. ELISA method for detecting ferroptosis-related indicators 100 mg of tumor tissue was collected, ground in liquid nitrogen using a mortar and pestle, and then 1 mL of Beyotime Western blotting and IP cell lysis buffer (product number P0013) was added to completely lyse the tissue. After lysis on ice for 30 min, the tissue was centrifuged at 12000 rpm for 30 min to obtain the supernatant. The supernatant was then analyzed using the Beyotime Lipid Oxidation (MDA) Detection Kit (product number S0131S), the Sangon Biotech 4-Hydroxynonenoic Acid (4-HNE) ELISA Kit (product number D751041), the Beyotime GSH and GSSG Detection Kit (product number S0053), and the Elite Cell Ferrous Ion Fluorescence Assay Kit (product number E-BC-F101). The results showed the presence of malondialdehyde (MDA), 4-hydroxynonenoic acid (4-HNE), glutathione (GSH), and ferrous ions (Fe2+) in the supernatant. 2+ ) level.

[0048] The results are as follows Figure 19 As shown, there were no significant changes in ferroptosis markers between the OSI-treated group and the control group, indicating that OSI drugs alone cannot induce ferroptosis to achieve the goal of tumor treatment. Meanwhile, after YIBE treatment, the levels of MDA, Fe, and 4-HNE in tumor tissue significantly increased, while the level of GSH significantly decreased. This suggests that YIBE can significantly induce ferroptosis in OSI-resistant tumors, meaning that YIBE can significantly increase the effectiveness of OSI treatment for OSI-resistant non-small cell lung cancer.

Claims

1. Use of ibesin in overcoming tumor cell drug resistance.

2. Use according to claim 1, characterized in that, The CAS number of the ibesin is 143502-51-6.

3. Use according to claim 1, characterized in that, The use is the use in preparing a drug for treating or adjuvant treating drug-resistant non-small cell lung cancer.

4. Use according to claim 3, characterized in that, The drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

5. Use according to claim 3, characterized in that, The drug contains ibesin or a pharmaceutically acceptable salt, solvate, hydrate thereof as an active ingredient.

6. Use according to claim 5, characterized in that, The drug also contains osimertinib as an active ingredient.

7. Use according to claim 6, characterized in that, The mass ratio of ibesin to osimertinib in the drug is 1.5-2.5:

1.

8. Use according to claim 3, characterized in that, The drug also contains a pharmaceutically acceptable excipient.

9. Use according to claim 8, characterized in that, The pharmaceutically acceptable excipient includes any one or more of excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffering substances, solubilizers, cosolvents or solvents.

10. Use according to claim 3, characterized in that, The dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, pellets, injection solutions, infusion solutions, suspensions, patches, suppositories, transdermal patches, microemulsion liposomes, nanoparticles.

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