A pharmaceutical composition for treating drug-resistant non-small cell lung cancer

Abstract

The invention discloses a pharmaceutical composition for treating drug-resistant non-small cell lung cancer (NSCLC). The pharmaceutical composition comprises lulutonic acid and gefitinib, wherein the molar ratio of gefitinib to lulutonic acid is 1:2, 1:1 or 2:1, the inhibition rate of lulutonic acid and gefitinib combined on NSCLC cells is significantly higher than that of single drug use, and the combined use of gefitinib and lulutonic acid can increase the killing effect on cells and the inhibitory effect on cell proliferation and migration, and promote cell apoptosis. The experimental results of the invention provide a theoretical basis for the clinical combined use of lulutonic acid and gefitinib and an effective scheme for treating NSCLC, and have broad application prospects in the field of medicine.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a pharmaceutical composition for treating drug-resistant non-small cell lung cancer. Background Art

[0002] Lung cancer is the malignant tumor with the highest incidence and mortality rate in the world. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) according to its histopathological characteristics, and NSCLC accounts for 85% of all lung cancers.

[0003] Epidermal growth factor receptor (EGFR) is associated with multiple signal transduction pathways such as cell proliferation, metastasis, and apoptosis. It contains a ligand-binding domain outside the cell membrane, a transmembrane region, a proximal nuclear localization signal, and a cytoplasmic tyrosine kinase domain. The discovery of EGFR gene mutations that activate the tyrosine kinase domain has ushered in the era of targeted therapy for NSCLC using small molecule tyrosine kinase inhibitors.

[0004] After being activated by EGF, TGFα, bidirectional regulatory protein, heparin-binding EGF, etc., the epidermal growth factor receptor undergoes conformational changes to form a dimer. Dimerization activates tyrosine kinase activity, thereby activating the downstream Ras pathway, PI3K / AKT pathway, and STAT3 pathway that regulate cell proliferation, growth, and apoptosis, thereby promoting the occurrence and development of NSCLC.

[0005] Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is divided into three generations, and its representative drugs are gefitinib, afatinib and osimertinib. They block EGFR phosphorylation by binding to the ATP binding site of the tyrosine kinase domain of EGFR, thereby preventing the activation of downstream pathways and achieving the effect of inhibiting cell growth and proliferation.

[0006] Patients who benefit from EGFR-TKI treatment will inevitably develop resistance to EGFR-TKI after a period of treatment, so patients cannot achieve complete tumor remission through EGFR-TKI treatment. Activation of the bypass pathway of EGFR downstream pathway signal transduction and transcription activator protein 3 (STAT3) is an important cause of EGFR-TKI resistance. After being phosphorylated and activated by various pathways, STAT3 can form dimers and enter the cell nucleus to bind to specific regulatory elements to induce target gene transcription. High expression of phosphorylated STAT3 (p-STAT3) is a sign of poor prognosis in NSCLC. In clinical cases, it was found that the content of p-STAT3 was strongly correlated with the content of phosphorylated EGFR (p-EGFR), and the expression of p-STAT3 in EGFR mutant NSCLC cells was significantly higher than that in EGFR wild-type cells. STAT3 activation is regulated by a variety of cytokines, growth factors, and intracellular tyrosine kinases, such as epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, IL-6, IL-8, IL-10, JAKs, and Src tyrosine kinase, and has a powerful compensatory activation pathway. The most common cause of compensatory activation of STAT3 is the increased concentration of IL-6 in the tumor microenvironment. Studies have found that lung cancer patients have higher levels of IL-6 in their serum, and NSCLC cell lines with EGFR mutations also secrete more IL-6. Afatinib can also act on fibroblasts to secrete IL-6.

[0007] Due to various reasons, the total amount of IL-6 increases and the activation of complex growth factors, cytokines and other tyrosine kinases in the tumor microenvironment, EGFR-TKI treatment alone cannot effectively inhibit STAT3 activation, leading to EGFR-TKI treatment resistance. Since STAT3 activation has a powerful compensatory pathway, inhibiting only one upstream pathway of STAT3 cannot effectively inhibit STAT3 activation, and inhibiting multiple upstream pathways is difficult to achieve. Inhibitors that directly act on STAT3 are the best choice to inhibit STAT3 activation. There are currently no direct STAT3 inhibitors for clinical use, so research and development of new STAT3 inhibitors is very meaningful for the treatment of NSCLC.

[0008] According to the Chinese Pharmacopoeia, Lulutong is the dried mature fruit sequence of Liquidambar formosana, a plant of the Hamamelidaceae family. It has the effects of dispelling wind, activating collaterals, promoting diuresis, and promoting menstruation. Lulutong acid is the main component of Lulutong. Studies have shown that Lulutong acid can increase the efficacy of paclitaxel against breast cancer, and also has a certain therapeutic effect on gastric cancer, leukemia, prostate cancer, liver cancer, etc. However, so far, there has been no research report on the combined use of gefitinib and Lulutong acid in the treatment of NSCLC. Summary of the invention

[0009] The purpose of the present invention is to provide a pharmaceutical composition for treating drug-resistant non-small cell lung cancer. The pharmaceutical composition proposes for the first time that gefitinib and lulutonic acid are used in combination to treat non-small cell lung cancer to solve the problem of drug resistance in the treatment of non-small cell lung cancer.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] Combination of lulutonic acid and gefitinib in the treatment of epidermal growth factor tyrosine kinase inhibitor-resistant non-small cell lung cancer.

[0012] The lulutonic acid provided by the present invention can enhance the application of gefitinib in treating NSCLC with acquired resistance to EGFR-TKI. The combination of the two drugs enhances the efficacy of gefitinib, providing a basis for the new use of old drugs.

[0013] The epidermal growth factor tyrosine kinase inhibitor-resistant non-small cell lung cancer is gefitinib-secondary-resistant non-small cell lung cancer.

[0014] Furthermore, the molar ratio of gefitinib to lulutonic acid in the drug is 1:2, 1:1 or 2:1.

[0015] Based on clinical practice, the present invention investigates for the first time the therapeutic effect of gefitinib combined with lulutonic acid on drug-resistant NSCLC. In vitro experiments conducted on H1975 cells show that the combined use of gefitinib and lulutonic acid can increase the killing effect on cells and the inhibitory effect on cell proliferation and migration, while promoting cell apoptosis.

[0016] The present invention explores for the first time the mechanism of synergistic effect of gefitinib and lulutonic acid in combination. Through WB experiments, it is found that the synergistic effect is due to the fact that gefitinib has no inhibitory effect on the phosphorylation of STAT3Tyr705 phosphorylation site, while lulutonic acid can make up for this deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the cytotoxic effect of gefitinib combined with lulutonic acid on H1975 cells; AC is the effect of different ratios of gefitinib combined with lulutonic acid on the survival rate of H1975 cells, and D is the combination index of gefitinib and lulutonic acid.

[0018] Figure 2 The time dependence of the combined use of gefitinib and lulutonic acid.

[0019] Figure 3 This is a picture taken under a microscope for the Edu cell proliferation experiment (200X).

[0020] Figure 4 Results of the Edu cell proliferation experiment; A represents 24h of drug treatment; B represents 48h of drug treatment.

[0021] Figure 5 This is a cell migration experiment; A is a picture taken under a microscope (40X); B is the statistical analysis result of the number of migrated cells (400X).

[0022] Figure 6 It is a cell apoptosis experiment; A is the flow cytometry analysis of cell apoptosis; B is the overall cell apoptosis rate; C is the percentage of cell number in each stage.

[0023] Figure 7 p-STAT3 in H1975 cells Tyr705 A is the statistical result, and B is the example of protein bands.

[0024] Figure 8 p-STAT3 in H1975 cells Ser727 A is the statistical result, and B is the example of protein bands.

[0025] Fig. 9 is the tSTAT3 content in H1975 cells; A is the statistical result, and B is an example of the protein band.

[0026] Fig.10 Tumor growth curve. DETAILED DESCRIPTION

[0027] This example proposes for the first time the therapeutic effect of gefitinib combined with lulutonic acid on drug-resistant NSCLC, and an in vitro experiment was conducted on H1975 cells.

[0028] This example studies whether the combined use of gefitinib and lulutonic acid has a synergistic effect against drug-resistant NSCLC. Taking the gefitinib-resistant NSCLC cell H1975 cell line as the research object, the MTT experiment was first used to investigate the effects of different concentrations of gefitinib and lulutonic acid on cell survival when used alone, and the corresponding IC50 was calculated, and a safe dosing concentration of lulutonic acid was screened at the same time. Then, the MTT experiment was used to investigate the effects of different concentrations of gefitinib combined with a safe concentration of lulutonic acid (cell survival rate>80%) on cell survival rate, and whether the IC50 decreased compared with the use of gefitinib alone. Finally, the combined molar ratio of the two drugs was set according to the ratio of gefitinib to lulutonic acid IC50, and according to the principle of the Chou-Talalay method (median pharmacodynamics method), the combined index (Combination index, CI) of the cell inhibition rate of gefitinib combined with lulutonic acid was investigated by MTT experiment, and the size of CI was judged to evaluate whether the two drugs had a synergistic effect.

[0029] (1) The combination of gefitinib and lulutonic acid has a synergistic effect in enhancing the cytotoxicity of H1975 cells.

[0030] Materials: Fetal bovine serum (FBS) was purchased from ZetaLife, USA; RPMI-1640 medium was purchased from Gibco, USA; dimethyl sulfoxide (DMSO) was purchased from Amresco, USA; trypsin, penicillin-streptomycin double antibody, phosphate buffered saline (PBS), and EDTA were purchased from Solebao Biotechnology Co., Ltd., USA; MTT was purchased from Dalian Meilun Biotechnology Co., Ltd.; passutonic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0031] Methods: H1975 cells in the logarithmic growth phase were digested with trypsin containing EDTA and collected. The cells were counted using a cell counting plate and the number of cells was calculated as 1×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL / well. After the cells adhered to the wall, the culture medium was discarded. 100 μL of drug-containing culture medium containing gefitinib and 100 μL of ...

[0032] Data processing: Microsoft Excel 2019 was used to organize the data, and Graphadprism 8.0 software was used for statistical analysis and plotting. The synergistic effect of gefitinib and lulutonic acid was determined according to the Chou-Talalay principle, and the combination index (CI) of gefitinib and lulutonic acid was calculated using Compusyn software. When CI>1, it indicates antagonism, CI=1 indicates additive effects, CI<1 indicates synergistic / sensitizing effects, and CI<0.3 indicates strong synergistic / sensitizing effects.

[0033] Results: Gefitinib or lulutonic acid alone had a killing effect on H1975 cells, but after reaching a certain concentration (gefitinib 35μM, lulutonic acid 20μM), the killing effect tended to be saturated and the killing effect on H1975 cells no longer increased with the increase of concentration.

[0034] like Figure 1 As shown in the figure, after gefitinib and lulutonic acid were used in combination, the killing effect on H1975 cells was significantly increased, and the cells could be completely killed at high drug concentrations; among them, the cytotoxicity was stronger when the molar ratio of gefitinib to lulutonic acid was 2:1 and 1:1, as shown in the figure. Figure 1 As shown in (B) and (C), gefitinib almost completely killed the cells at a concentration of 40 μM.

[0035] from Figure 1 (D) It can be seen that when the cell inhibition rate is low, the CI of the two drugs combined is greater than 1, which is an antagonistic effect; and as the killing effect on H1975 cells increases, the CI of the two drugs is less than 1, which is a synergistic effect. When the inhibition rate of H1975 cells is greater than 0.8, the combination of gefitinib and lulutonic acid at a molar ratio of 2:1 shows a strong synergistic effect. Among them, when the molar ratio of gefitinib to lulutonic acid is 2:1, the synergistic effect of the two drugs is the best, and the synergistic effect begins to appear when the cell inhibition rate is 0.3.

[0036] (2) The cytotoxicity of gefitinib combined with lulutonic acid on H1975 cells was time-dependent.

[0037] Materials: Fetal bovine serum (FBS) was purchased from ZetaLife, USA; RPMI-1640 medium was purchased from Gibco, USA; dimethyl sulfoxide (DMSO) was purchased from Amresco, USA; trypsin, penicillin-streptomycin double antibody, phosphate buffered saline (PBS), and EDTA were purchased from Solebao Biotechnology Co., Ltd., USA; MTT was purchased from Dalian Meilun Biotechnology Co., Ltd.; passutonic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0038] Methods: H1975 cells in the logarithmic growth phase were digested with trypsin containing EDTA and collected. The cells were counted using a cell counting plate and the number of cells was calculated as 1×10 4The cells were inoculated in a 96-well plate at a density of 100 μL / well. After the cells adhered to the wall, the culture medium was discarded, and 100 μL of drug-containing culture medium containing gefitinib and 100 μL of ...

[0039] Data processing: Microsoft Excel 2019 was used to organize the data, and Graphadprism 8.0 software was used to perform statistical analysis and graphing of the data.

[0040] Results: The molar ratio of gefitinib to lulutonic acid was 2:1, and the cells were treated for 24h, 48h and 72h to observe the effects on the survival rate of H1975 cells. Figure 2 As shown in the figure, as the treatment time increases, the combined use of the two drugs has a stronger killing effect on H1975 cells. The concentration required to completely kill the cells at 24h (in terms of gefitinib concentration) is 35μM, and the concentration required to completely kill the cells at 48h and 72h is only 20μM, which is 15μM less than the concentration required at 24h. At the same drug concentration of 10μM, the cell survival rate is >20% at 48h, while the cell survival rate is <20% at 72h, indicating that the toxicity of 72h treatment to H1975 cells is greater than that of 48h treatment.

[0041] The experimental results showed that the toxicity of gefitinib combined with lulutonic acid on H1975 cells was time-dependent and suitable for long-term combined use.

[0042] This example studies the effect of gefitinib combined with lulutonic acid on cell proliferation, migration, and apoptosis. The Edu cell proliferation assay was used to investigate whether the combined use of gefitinib and lulutonic acid could synergistically inhibit the proliferation of H1975 cells; the Transwell cell migration assay was used to investigate the effect of gefitinib combined with lulutonic acid on the migration ability of H1975 cells; the Annexin-VAbFluor TM 488 / PI double staining experiment was used to investigate the effect of gefitinib combined with lulutonic acid on apoptosis of H1975 cells.

[0043] (3) Edu cell proliferation assay

[0044] Materials: Fetal bovine serum (FBS) was purchased from ZetaLife, USA; RPMI-1640 medium was purchased from Gibco, USA; dimethyl sulfoxide (DMSO) was purchased from Amresco, USA; trypsin, penicillin-streptomycin double antibody, phosphate buffered saline (PBS), and EDTA were purchased from Solebao Biotechnology Co., Ltd., USA; passutonic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Edu kit and 4% paraformaldehyde were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and TritonX-100 was purchased from Biofroxx.

[0045] Methods: H1975 cells in the logarithmic growth phase were digested with trypsin containing EDTA and the cells were collected. 5 The cells were inoculated at a density of 100 cells / well in a 24-well plate. When the cell confluence reached about 70%, the culture medium was discarded and the drug-containing culture medium was added. A blank control group was set up at the same time. Each group had 3 replicate wells and the culture was continued for 24 hours and 48 hours.

[0046] After culturing for 24h and 48h, remove the cell plate, aspirate half of the culture medium and then add the same volume of 2XEdu working solution (Edu concentration is 20μM), and continue culturing for 2h to allow Edu to mark cells undergoing DNA replication. After Edu labeling is completed, remove the culture medium and wash 3 times with pre-cooled PBS to remove the remaining Edu reagent. Then add 0.4% paraformaldehyde solution to fix at room temperature for 15min. After fixation, remove the fixative and wash with pre-cooled PBS to remove the remaining fixative, then add PBS solution containing 0.3% TritonX-100 and incubate at room temperature for 15min to permeabilize the cells.

[0047] During the permeabilization of cells, prepare the Click reaction solution according to the instructions of the Edu kit. After the permeabilization of cells is completed, remove the permeabilization solution, wash with pre-cooled PBS three times to remove the remaining permeabilization solution, then add 100μL Click reaction solution, gently shake the culture plate to make the reaction solution evenly cover the cells, and incubate at room temperature in the dark for 30 minutes. After the incubation is completed, remove the reaction solution, wash the remaining reaction solution with pre-cooled PBS, then add 1XHoechst3342 solution, and incubate at room temperature for 10 minutes. After the incubation is completed, remove the 1XHoechst3342 solution, wash with pre-cooled PBS three times, and then detect the fluorescence under an inverted fluorescence microscope and take pictures.

[0048] Hoechst3342 solution is a nuclear dye that can bind to the nuclei of all cells and emit blue fluorescence after binding to DNA. Proliferating cells labeled with Edu emit green fluorescence, and the cell proliferation rate = number of green fluorescent cells / number of blue fluorescent cells x 100%.

[0049] Data processing: ImageJ software was used to count cells in the Edu experiment; Microsoft Excel 2019 was used to organize the data; Graphadprism 8.0 software was used to perform statistical analysis and graphing of the data.

[0050] Results: Seven control groups were set up: blank control group, gefitinib 10μM, gefitinib 20μM, lulutonic acid 5μM, lulutonic acid 10μM, gefitinib 10μM + lulutonic acid 5μM, gefitinib 20μM + lulutonic acid 10μM. All six control groups were treated for 24h and 48h to observe the proliferation of Edu cells.

[0051] H1975 cells were treated for 24 h. Figure 3 (A) and Figure 4 As shown in (A), the cell proliferation rate of the blank control group was 20.92%.

[0052] When the concentration of gefitinib was 10 μM and 20 μM, the cell proliferation rate was 4.00% and 2.36%, which was 5.23 times and 8.86 times lower than that of the blank control group with gefitinib concentration of 10 μM and 20 μM.

[0053] The cell proliferation rates were 25.66% and 22.72% when the concentrations of lulutonic acid were 5 μM and 10 μM, which were similar to that of the blank group.

[0054] The cell proliferation rates of the gefitinib 10 μM + lulutong acid 5 μM and gefitinib 20 μM + lulutong acid 10 μM groups were 4.09% and 2.47%, respectively. Compared with the same concentration of gefitinib, the cell proliferation rates did not change much, while compared with the same concentration of lulutong acid, the cell proliferation rates decreased by 6.27 times and 9.19 times, and the differences were statistically significant.

[0055] H1975 cells were treated for 48 h. Figure 3 (B) and Figure 4 As shown in (B), the cell proliferation rate of the blank control group was 8.74%.

[0056] When the concentration of gefitinib was 10μM and 20μM, the cell proliferation rates were 1.57% and 0.77%, which were 5.57 times and 11.35 times lower than those of the blank control group.

[0057] When the concentration of lulutong acid was 5μM and 10μM, the cell proliferation rate was 4.76% and 7.43%, which was 1.83 and 1.17 times lower than that of the blank group.

[0058] The cell proliferation rates of the gefitinib 10μM + 5μM lulutonic acid and gefitinib 20μM + 10μM lulutonic acid groups were 0.75% and 0.67%, respectively. Compared with the same concentration of gefitinib, the cell proliferation rates decreased by 2.09 times and 1.15 times, respectively. Compared with the same concentration of lulutonic acid, the cell proliferation rates decreased significantly, decreasing by 6.34 times and 11.09 times, respectively. The differences were statistically significant.

[0059] From the above experimental results, it can be seen that gefitinib has a good inhibitory effect on the proliferation of H1975 cells, and the inhibitory effect increases with the increase of drug concentration and the extension of treatment time; the inhibitory effect of lulutonic acid on the proliferation of H1975 cells is weaker than that of gefitinib. Lulutonic acid treatment for 24 hours has no effect on inhibiting cell proliferation, but has a stronger killing effect on cells. Figure 3 As shown in the figure, after treatment with lulutong acid, the cell density was lower than that of other groups. As the treatment time increased, lulutong acid showed an inhibitory effect on cell proliferation. At 48h of treatment, due to the stronger cell killing effect of lulutong acid at a concentration of 10μM, the total number of cells decreased, making the cell proliferation rate higher than when the concentration of lulutong acid was 5μM. At 24h of treatment, the inhibitory effect of the combination of the two drugs on cell proliferation was greater than that of lulutong acid alone, and was not much different from the proliferation inhibitory effect of gefitinib alone. At 48h of treatment, the cell proliferation rate of the combination of the two drugs was lower than that of gefitinib and lulutong acid alone, indicating that the combination of the two has a synergistic inhibitory effect on cell proliferation. The combined use combines the advantages of gefitinib's strong inhibitory effect on cell proliferation and lulutong acid's strong cell killing effect, enhancing the inhibitory effect on tumors.

[0060] (4) Cell migration assay

[0061] Materials: Fetal bovine serum (FBS) was purchased from ZetaLife, USA; RPMI-1640 medium was purchased from Gibco, USA; dimethyl sulfoxide (DMSO) was purchased from Amresco, USA; trypsin, penicillin-streptomycin double antibody, phosphate buffered saline (PBS), and EDTA were purchased from Solebow Biotechnology Co., Ltd., USA; passutonic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 4% paraformaldehyde was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; and crystal violet staining solution was purchased from Beijing Solebow Biotechnology Co., Ltd.

[0062] Methods: This experiment used a 24-well Transwell chamber with a pore size of 8 μM. H1975 cells in the logarithmic growth phase were taken and resuspended in serum-free medium to control the cell density to 5×10 5 / mL. The cell suspension was mixed evenly with a drug-containing medium at a concentration twice the volume. 200μL of drug-containing cell suspension was added to the upper chamber, and 800μL of drug-containing medium containing 10% serum was added to the lower chamber, and cultured for 24h. A blank control group was set up at the same time, with 3 replicates in each group.

[0063] After 24 hours of culture, the culture medium was removed, and the cells were washed three times with pre-cooled PBS to remove the residual culture medium. Then, the chamber was placed in a 4% paraformaldehyde solution for 15 minutes at room temperature to fix the cells. After the cells were fixed, the fixative was removed and the cells were stained with a 0.5% crystal violet solution for 30 minutes. After staining, the excess crystal violet was washed with pre-cooled PBS, and the cells on the inside of the chamber were gently wiped with a cotton swab. The cells were placed under a microscope for observation, and 5 fields of view were randomly selected under a 400x microscope to take pictures and count the cells that passed through the chamber.

[0064] Data processing: ImageJ software was used to count cells in the cell migration experiment; Microsoft Excel 2019 was used to organize the data; Graphadprism 8.0 software was used to perform statistical analysis and graphing of the data.

[0065] Results: Four control groups were set up: blank control group, gefitinib 20 μM group, lulutonic acid 10 μM group and gefitinib 20 μM + lulutonic acid 10 μM combined administration group.

[0066] like Figure 5 As shown in A, the cell density of the gefitinib group, the lulutong acid group and the combined drug group was significantly lower than that of the blank control group, among which the combined drug group had the lowest cell density. The cells were counted in the pictures taken under a 400x microscope, and the number of cells passing through each field of view in the blank group was about 84. The number of cells passing through each field of view in the gefitinib group was about 32, which was 2.63 times lower than that in the blank group; the number of cells passing through each field of view in the lulutong acid group was about 60, which was 1.38 times lower than that in the blank control group; the number of cells passing through each field of view in the combined drug group was about 14, which was 6 times lower than that in the blank control group. The number of cells passing through each field of view in the combined drug group was 2.29 times and 4.29 times lower than that in the gefitinib group and the lulutong acid group, respectively, and the difference was statistically significant.

[0067] The above experimental results indicate that both gefitinib and lulutonic acid have the ability to inhibit the migration of H1975 cells. When the two drugs are used in combination, the inhibitory effect on the migration of H1975 cells is stronger than when one drug is used alone. The combination of the two drugs can synergistically enhance the inhibitory effect on the migration of H1975 cells.

[0068] (5) Annexin-VAbFluor TM 488 / PI double staining cell apoptosis experiment

[0069] Materials: Fetal bovine serum (FBS) was purchased from ZetaLife, USA; RPMI-1640 medium was purchased from Gibco, USA; dimethyl sulfoxide (DMSO) was purchased from Amresco, USA; trypsin, penicillin-streptomycin double antibody, phosphate buffered saline (PBS), and EDTA were purchased from Solebo Biotechnology Co., Ltd., USA; pamoic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Annexin-VAbFluor TM The 488 / PI kit was purchased from Abbkine.

[0070] Method: H1975 cells in the logarithmic growth phase were taken and the cells were cultured at 5×10 5 The cells were inoculated into a 12-well plate at a density of 100 cells / well. When the cell confluence reached 80-90%, the culture medium was discarded, and different drug-containing culture media were added and cultured for 24 hours. A blank control group was set up, and 3 replicates were set up for each group.

[0071] After 24 hours of culture, the culture medium was discarded, the cells were collected by digestion with EDTA-free trypsin, and washed twice with pre-cooled PBS. After centrifugation, the cells were resuspended in 100 μL of 1xAnnexin-VBindingBuffer (obtained by diluting 5xAnnexin-VBindingBuffer with deionized water), and 5 μL of Annexin-VAbFluor was added to each 100 μL of cell suspension. TM 488 and 2μLPI, gently mix. Incubate at room temperature in the dark for 15 minutes. After the staining incubation, add 400μL 1xAnnexin-VBindingBuffer, gently mix and place on ice. Use flow cytometer for detection within 30 minutes.

[0072] Data processing: FlowJo was used to analyze and plot the flow cytometry data of cell apoptosis; Microsoft Excel 2019 was used to organize the data; Graphadprism 8.0 software was used to perform statistical analysis and plot the data.

[0073] Results: Four drug-treated groups were set up: blank control group, gefitinib 20 μM group, lulutonic acid 10 μM group, and gefitinib 20 μM + lulutonic acid 10 μM combined administration group. TM After 488 / PI double staining, the cells were detected using a flow cytometer.

[0074] The cells were divided into four phases: Q4 for live cells, Q2 for late apoptotic cells, Q3 for early apoptotic cells, and Q1 for mechanically damaged cells. The total amount of apoptotic cells was the sum of early apoptotic cells and late apoptotic cells, so the apoptotic rate was Q2%+Q3%.

[0075] like Figure 6 As shown, the apoptosis rate of gefitinib group and lulutong acid group was significantly higher than that of blank control group. The apoptosis rate of gefitinib group was 7.69%, which was 2.61 times higher than that of 2.94% in blank control group, and the apoptosis rate of lulutong acid was 13.43%, which was 4.57 times higher than that of blank control group. The apoptosis rate of combined drug group was 51.47%, which was significantly higher than that of single drug group, and the difference was statistically significant.

[0076] The apoptosis rate in the combined drug group was 6.69 times and 3.83 times higher than that in the gefitinib group and lulutonic acid group, respectively. Figure 6 As shown in C, the combination of drugs can increase the early and late apoptosis rates of H1975 cells, among which the effect on the late apoptosis rate is greater.

[0077] This example preliminarily explores the mechanism of synergistic effect of lulutonic acid combined with gefitinib. Western blotting (WB) was used to investigate the effects of gefitinib and lulutonic acid alone and in combination on STAT3 protein in H1975 cells.

[0078] (6) Western blotting

[0079] Materials: BCA protein quantification kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd., Anti-Phospho-STAT3 (Tyr705) Recombinant Rabbit Monoclonal Antibody and Anti-Phospho-STAT3 (Ser727) Recombinant Rabbit Monoclonal Antibody were purchased from Hangzhou Huaan Biotechnology Co., Ltd., STAT3 antibody (F-2) was purchased from Santa Cruz Biotechnology, ECL colorimetric solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., GAPDH Rabbit pAb primary antibody and Goat Anti-Rabbit IgG-HRP were purchased from Wuhan Aibote PMSF and TEMED were purchased from Sigma-Aldrich, USA; RIPA lysis buffer was purchased from Beijing Solebow Biotechnology Co., Ltd.; Trisbase was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.; glycine was purchased from Beijing Bailingwei Technology Co., Ltd.; NaCl was purchased from Tianjin Ruijinte Chemical Co., Ltd.; Tween 20 was purchased from Tianjin Komio Chemical Reagent Co., Ltd.; acrylamide was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; skim milk powder was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.; protein marker was purchased from Shanghai Yishen Biotechnology Co., Ltd.; PVDF membrane was purchased from Bio-Rad Laboratories, USA; pleuronate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0080] Methods: (1) Cell plating and treatment: H1975 cells in the logarithmic growth phase were taken and plated at 1.5×10 6 Each well was inoculated into a sterile 6-well plate. When the cells adhered to the wall and grew to 80% confluence, the culture medium was discarded and different drug-containing culture media were added respectively, and the culture was continued for 24 hours. A blank control group was set up at the same time, and 3 replicates were set up for each group.

[0081] (2) Cell protein extraction: After drug treatment, remove the 6-well plate, discard the culture medium, and wash 2-3 times with pre-cooled PBS. Add RIPA lysis buffer containing 1mMPMSF and 1x phosphatase inhibitor mixture to each well, and vortex intermittently on ice for 30 minutes to fully lyse the cells. Transfer the cell lysate to a centrifuge tube, centrifuge at 12000rpm at 4℃ for 10 minutes, and take the supernatant. Use the BCA kit to determine the protein concentration of the cell lysate. Mix the extracted cell lysate with 5× loading buffer in a ratio of 4:1, then boil it in boiling water for 10 minutes and store it at -20℃.

[0082] (3) Immunoblotting experiment: Prepare SDS-PAGE gel, add protein sample to the loading well, ensure that the amount of protein loaded in each well is 20-40μg, set up a hole for each gel to add marker, run at 80V constant voltage until the protein is compressed into a straight line, adjust the voltage to 120V, and run at constant voltage until the protein is completely separated (by observing the separation of the marker color). PVDF membrane is activated with methanol and covered on the gel, and the membrane is transferred at a constant current of 300A in the transfer solution for 120min. After the transfer is completed, the PVDF membrane is washed 3 times with TBST solution, and then the PVDF membrane is placed in TBST containing 5% skim milk powder for 2 hours. After the blocking is completed, the PVDF membrane is washed 3 times with TBST solution, and then placed in the primary antibody solution and incubated overnight at 4℃ in a shaker. After the primary antibody incubation is completed, the PVDF membrane is washed 3 times with TBST solution, and then the PVDF membrane is placed in the secondary antibody solution and incubated at room temperature for 1h in a shaker. After the secondary antibody incubation, the PVDF membrane was washed 3 times with TBST solution and 2 times with TBS solution. The ELC chemiluminescence kit was used for development and SageCapture software was used for observation and image acquisition.

[0083] Data processing: ImageJ software was used to analyze the grayscale values ​​of the protein bands obtained from the protein immunoblotting experiment, Microsoft Excel 2019 was used to organize the data, and Graphadprism 8.0 software was used to perform statistical analysis and graphing of the data.

[0084] Results: STAT3 has two phosphorylation sites, Tyr705 and Ser727, which activate STAT3 and promote cell proliferation and survival. The phosphorylated STAT3 (p-STAT3Tyr705, p-STAT3Ser727) content and total STAT3 (tSTAT3) content in H1975 cells were detected after 24h treatment with 10μM and 20μM gefitinib, 5μM and 10μM gefitinib + 5μM gefitinib, and 20μM gefitinib + 10μM gefitinib.

[0085] from Figure 7It can be seen that 10μM and 20μM gefitinib treatment of H1975 cells for 24h had little effect on the intracellular p-STAT3Tyr705 content, and there was no statistically significant difference compared with the blank group. After 5μM and 10μM lulutong acid treatment of H1975 cells for 24h, the intracellular p-STAT3Tyr705 content decreased to 58% and 49% respectively compared with the blank control group, and the difference was statistically significant. The p-STAT3Tyr705 content of the two combined drug groups decreased to 34% and 14% respectively compared with the blank group; compared with gefitinib alone, the intracellular p-STAT3Tyr705 content decreased significantly, and the difference was statistically significant. Compared with lulutong acid alone, the p-STAT3Tyr705 content had a downward trend, and the combined drug group with a higher concentration had a more obvious downward trend, and the difference was statistically significant.

[0086] from Figure 8 It can be seen that after H1975 cells were treated with gefitinib and lulutonic acid, the intracellular p-STAT3Ser727 content was significantly lower than that of the blank control group, and the difference was statistically significant.

[0087] Depend on Fig. 9 It can be seen that gefitinib and lumefantrine alone treated H1975 cells, which had little effect on the intracellular tSTAT3 content, while the high-concentration combined drug group significantly reduced the intracellular tSTAT3 content.

[0088] The above experimental results show that gefitinib and lulutong acid alone do not affect the intracellular tSTAT3 content, and both can effectively inhibit the phosphorylation of STAT3Ser727 phosphorylation site. The lack of inhibitory effect of gefitinib on the phosphorylation of intracellular STAT3Tyr705 phosphorylation site may be the reason for its drug resistance. Lulutong acid can effectively inhibit the phosphorylation of STAT3Tyr705 phosphorylation site, which just makes up for the deficiency of gefitinib. When the two are used together at a higher concentration, the intracellular tSTAT3 content can be reduced, further enhancing the inhibitory effect on cell proliferation and survival caused by STAT3 activation. Therefore, the synergistic anti-NSCLC effect of gefitinib and lulutong acid may be achieved by inhibiting the phosphorylation of STAT3Tyr705 phosphorylation site.

[0089] (7) This example investigates the in vivo efficacy of lulutonic acid combined with gefitinib.

[0090] Materials: Four-week-old male Balb / c-Nude mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., pamoic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and gefitinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0091] Methods: First, a tumor-bearing mouse model was established. This study used 4-week-old male Balb / c-Nude mice weighing approximately 18-20 g. H1975 cells in the logarithmic growth phase were collected by trypsin digestion and resuspended in PBS to adjust the cell concentration to 2.5×10 7 200 μL of H1975 cell suspension was inoculated into the left armpit of mice, and the tumor growth of mice was observed every other day.

[0092] When the mouse tumor grew to 80 mm 3 The mice were randomly divided into 4 groups, 6 mice in each group: ① blank control group (control); ② gefitinib group (GEF); ③ BA group (BA); ④ gefitinib and BA group (GEF+BA). Dosage: poqd; dosing cycle: 14 days; dosing dose: 50 mg / kg gefitinib and 25 mg / kg BA. The growth of mice and the change of tumor volume were recorded during the treatment. The effectiveness of the treatment was investigated by the change of tumor volume and the final weight of tumor tissue.

[0093] Data processing: This experiment used Microsoft Excel 2019 to organize the data, and Graphadprism 8.0 software was used to perform statistical analysis and graphing of the data.

[0094] Results: The therapeutic effects of different drug administration groups were determined by monitoring the changes in tumor volume during treatment. Fig.10 As shown, the tumor growth rates of the four groups are arranged in order from fast to slow: control group, BA group, GEF group, GEF+BA group, and thus the tumor growth inhibition effects are arranged in order from strong to weak: GEF+BA group, GEF group, BA group.

[0095] The above experimental results provide a theoretical basis for the clinical combination of gefitinib and lumefantrine and an effective regimen for the treatment of EGFR-TKI-resistant NSCLC, and have broad application prospects in the field of medicine.

[0096] The above is only a preferred implementation of the present invention, but the protection scope of the present invention is not limited thereto, and any modification and replacement based on the technical solution and inventive concept provided by the present invention should be covered within the protection scope of the present invention.

Claims

1. The use of lulutonic acid and gefitinib in combination for the preparation of a drug for the treatment of epidermal growth factor tyrosine kinase inhibitor-resistant non-small cell lung cancer; The epidermal growth factor tyrosine kinase inhibitor-resistant non-small cell lung cancer is gefitinib-resistant non-small cell lung cancer; The molar ratio of gefitinib to lulutonic acid in the drug is 2:1.

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