Medicine and kit for relieving or treating non-small cell lung cancer

Abstract

The invention discloses a medicine and a kit for relieving or treating non-small cell lung cancer. The effective components of the medicine are PXD and MSAB. According to the application, the WNT inhibitor and the isoflavone derivative are creatively combined to serve as the medicine for relieving or treating the non-small cell lung cancer, an effective medicine combination strategy is provided for treatment of the non-small cell lung cancer, and the application has very remarkable significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a medicine and a kit for alleviating or treating non-small cell lung cancer. Background Art

[0002] Non-small cell lung cancer (NSCLC) has the characteristics of high incidence, high malignancy, and short survival period. Most relapsed and advanced cases do not have the opportunity for surgery. As traditional treatment methods, drug therapy and radiotherapy have entered a plateau period in anti-cancer effects, and the prognosis is still not optimistic. The main reason for the failure of drug therapy is that patients with non-small cell lung cancer develop resistance to drug therapy. Therefore, there is an urgent need to find new treatment options for non-small cell lung cancer to prolong the survival time of patients and improve the cure rate of patients.

[0003] Natural foods such as soybeans and green tea contain a variety of effective antitumor ingredients. Epidemiological studies have shown that the observed differences are attributed to traditional diets rich in soy products and a wide range of dietary and lifestyle factors. Soybeans mainly contain isoflavones, glycines, and glucosamine-polyphenolic plant-derived compounds, which have been shown to be major bioactive antitumor molecules. Therefore, screening natural products and effective metabolites through separation and purification has become a potential solution for the treatment of non-small cell lung cancer. We used an automated drug screening system to test more than 1,500 drugs from the targetmol-natural compound library and selected phenoxodiol (PXD), which has entered phase II / III clinical trials for ovarian cancer, to study its biological effects on NSCLC. Phenoxodiol is a synthetic derivative of genistein. Although Phenoxodiol has good antitumor effects in vitro, it is a drug that exerts antitumor effects through more pathways such as inhibiting topoisomerase II, inhibiting protein tyrosine kinases, inhibiting X-linked inhibitor of apoptosis, and anti-angiogenesis, while the results of in vivo clinical trials are prone to drug resistance. Therefore, rational combination therapy is needed to improve the efficacy of dehydroequol in NSCLC.

[0004] Transcriptomic sequencing revealed that PXD may upregulate Wnt pathway-related binding proteins after treating PC-9 cells in vitro, which may be a way for tumor cells to induce drug resistance. PXD's tumor-suppressing effect may be sensitized by inhibiting Wnt / β-catenin signaling. Studies have shown that MSAB, a specific inhibitor of the Wnt / β-catenin signaling pathway, has a single anti-tumor effect, including head and neck squamous cell carcinoma, bile duct cancer, cervical cancer, breast cancer, colorectal cancer, etc. MSAB can regulate a variety of malignant biological behaviors of tumor cells, including inhibiting tumor cell proliferation and inducing tumor cell apoptosis, but there has been no research on its use in the treatment of NSCLC.

[0005] There are relatively few new and effective drug combination strategies for treating NSCLC disclosed in the prior art, and it is very meaningful to develop more new strategies for treating NSCLC. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a new drug for the preparation of prevention, alleviation or treatment of non-small cell lung cancer.

[0007] To achieve the purpose of the invention, the present invention adopts the following technical solution: a drug for alleviating or treating non-small cell lung cancer, characterized in that the active ingredients of the drug are PXD and MSAB.

[0008] Furthermore, the dosage of PXD is 30-50 mg / kg; the dosage of MSAB is 10-30 mg / kg.

[0009] Furthermore, the dosage of PXD is 40 mg / kg; the dosage of MSAB is 20 mg / kg.

[0010] The present invention also provides a kit for alleviating or treating non-small cell lung cancer, characterized in that it contains the drug.

[0011] The inventors found that a small dose of MSAB combined with PXD can exert a synergistic effect, more strongly inhibiting the growth and proliferation of NSCLC cells, while also having significant therapeutic effects in vivo with low side effects.

[0012] The present invention creatively combines two drugs, WNT inhibitors and isoflavone derivatives, as drugs for alleviating or treating non-small cell lung cancer. The present invention proves in non-small cell lung cancer cell lines that the combined use of WNT inhibitors and isoflavone derivatives can synergistically inhibit the proliferation of non-small cell lung cancer cells and induce apoptosis thereof, and has a more significant effect of inhibiting the proliferation of non-small cell lung cancer cells and inducing apoptosis of non-small cell lung cancer cells than single therapy. In animal experiments, the PDX mouse model also proves that the combination of the two drugs can synergistically inhibit the tumor formation process of non-small cell lung cancer in mice. The present invention provides an effective drug combination strategy for the treatment of non-small cell lung cancer, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :Flow chart of high-throughput drug screening system for selecting drug dehydroequol for the treatment of non-small cell lung cancer;

[0014] Figure 2 :The results of cell proliferation inhibition of lung cancer PC-9 cells after treatment with different doses of dehydroequol for 24h, 48h, and 72h;

[0015] Figure 3:The results of cell proliferation inhibition after different doses of dehydroequol treated lung cancer H1650 cells for 24h, 48h, and 72h;

[0016] Figure 4 : The results of colony formation of lung cancer PC-9 cells treated with different doses of dehydroequol;

[0017] Figure 5 :The results of colony formation of lung cancer H1650 cells treated with different doses of dehydroequol;

[0018] Figure 6 : Flow cytometry results of cell cycle detection of lung cancer PC-9 cells after treatment with different doses of dehydroequol for 48 hours;

[0019] Figure 7 :Statistical results of cell cycle distribution of lung cancer PC-9 cells after treatment with different doses of dehydroequol for 48 hours;

[0020] Figure 8 : Flow cytometry results of cell cycle detection of lung cancer H1650 cells after treatment with different doses of dehydroequol for 48 hours;

[0021] Fig. 9 : Statistical results of cell cycle distribution of lung cancer H1650 cells after treatment with different doses of dehydroequol for 48 hours;

[0022] Fig.10 : Western blot analysis results of cell cycle-related genes in lung cancer PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0023] Fig.11 : Western blot analysis results of cell cycle-related genes in lung cancer H1650 cells treated with different doses of dehydroequol for 48 hours;

[0024] Fig.12 : The results of P21 and P27 immunofluorescence detection in lung cancer PC-9 cells after treatment with different doses of dehydroequol for 48 hours;

[0025] Fig.13 : The results of P21 and P27 immunofluorescence detection in lung cancer H1650 cells after treatment with different doses of dehydroequol for 48 hours;

[0026] Fig.14 : Flow cytometry results of apoptosis in lung cancer PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0027] Fig.15 : Statistical results of cell apoptosis rate of lung cancer PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0028] Fig.16 :Statistical results of apoptosis rate of lung cancer H1650 cells after treatment with different doses of dehydroequol for 48 hours;

[0029] Fig.17 : Flow cytometry results of apoptosis in lung cancer H1650 cells treated with different doses of dehydroequol for 48 hours;

[0030] Fig.18 : Western blot analysis results of apoptosis-related genes in lung cancer PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0031] Fig.19 : Western blot analysis results of apoptosis-related genes in lung cancer H1650 cells treated with different doses of dehydroequol for 48 hours;

[0032] Fig. 20 : Immunofluorescence detection results of apoptosis-related proteins in lung cancer PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0033] Fig.21 : Immunofluorescence detection results of apoptosis-related proteins in lung cancer H1650 cells after treatment with different doses of dehydroequol for 48 hours;

[0034] Fig. 22 : The results of cell migration ability test of lung cancer PC-9 cells treated with different doses of dehydroequol within 48 hours;

[0035] Fig.23 : Statistical results of cell migration rate of lung cancer PC-9 cells treated with different doses of dehydroequol within 48 hours;

[0036] Fig.24 : Statistical results of cell migration rate of lung cancer H1650 cells treated with different doses of dehydroequol within 48 hours;

[0037] Fig.25 : The results of cell migration ability test of lung cancer H1650 cells treated with different doses of dehydroequol within 48 hours;

[0038] Fig.26 : Results of cell invasion ability test of lung cancer PC-9 cells treated with different doses of dehydroequol within 48 hours;

[0039] Fig. 27 : Statistical results of the number of invasive cells per visual field in lung cancer PC-9 cells treated with different doses of dehydroequol within 48 hours;

[0040] Fig.28: Statistical results of the number of invasive cells per visual field in lung cancer H1650 cells treated with different doses of dehydroequol within 48 hours;

[0041] Fig.29 : The results of cell invasion ability test of lung cancer H1650 cells treated with different doses of dehydroequol within 48 hours;

[0042] Fig.30 : Western blot analysis of cell migration and invasion-related genes in lung cancer PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0043] Fig.31 : Western blot analysis of cell migration and invasion-related genes in lung cancer H1650 cells treated with different doses of dehydroequol for 48 hours;

[0044] Fig.32 : The results of immunofluorescence detection of E-cadherin and N-cadherin in lung cancer PC-9 cells after treatment with different doses of dehydroequol for 48 hours;

[0045] Fig.33 : The results of immunofluorescence detection of E-cadherin and N-cadherin in lung cancer H1650 cells after treatment with different doses of dehydroequol for 48 hours;

[0046] Fig.34 :Representative results of lung cancer tissue proliferation capacity after 14 days of administration in PDX mouse model;

[0047] Fig.35 : PDX mouse model lung cancer tissue volume and mass changes and mouse body weight changes after 14 days of administration;

[0048] Fig.36 : Pathological test results of normal and tumor tissues of PDX mouse model 14 days after administration;

[0049] Fig.37 : Western blot analysis results of cell cycle-related genes in PDX mouse model 14 days after administration;

[0050] Fig.38 : Western blot analysis results of apoptosis-related genes in PDX mouse model 14 days after administration;

[0051] Fig.39 : Western blot analysis results of migration and invasion-related genes in PDX mouse model 14 days after administration;

[0052] Fig.40: Transcriptomic sequencing suggests that Wnt pathway-related binding genes are significantly upregulated in GO analysis bubble chart and bar chart;

[0053] Fig.41 :The qPCR analysis results verifying that PXD upregulates Wnt pathway-related genes;

[0054] Fig.42 : Western blot analysis results verifying that PXD upregulates Wnt pathway-related genes;

[0055] Fig.43 :Changes in the morphology of autophagosomes in PC-9 cells treated with different doses of dehydroequol for 48 hours under transmission electron microscopy;

[0056] Fig.44 :The expression changes of autophagy-related genes in PC-9 cells treated with different doses of dehydroequol for 48 h;

[0057] Fig.45 :Changes of LC3B and P62 immunofluorescence in PC-9 cells after treatment with different doses of dehydroequol for 48 h;

[0058] Fig.46 :Changes in autophagosomes in PC-9 cells treated with different doses of dehydroequol for 48 h;

[0059] Fig.47 :Changes in autophagosomes in PC-9 cells treated with different doses of dehydroequol for 48 hours;

[0060] Fig.48 :The results of cell proliferation inhibition after treating lung cancer PC-9 cells with different doses of Wnt inhibitor MSAB for 48 hours

[0061] Fig.49 :The results of qPCR analysis of low-dose MSAB inhibiting Wnt pathway-related genes;

[0062] Fig.50 :The results of cell proliferation inhibition after different doses of dehydroequol combined with low dose of MSAB treated lung cancer PC-9 cells for 48 hours;

[0063] Fig.51 : Statistical results of cell cycle distribution of PC-9 cells after 48h of combined treatment with two drugs;

[0064] Fig.52 : Statistical results of apoptosis rate of PC-9 cells after 48 hours of combined treatment with two drugs;

[0065] Fig.53 :Changes in the morphology of autophagosomes in PC-9 cells under transmission electron microscopy after the two drugs were combined for 48 hours;

[0066] Fig.54 :The expression changes of apoptosis and autophagy related genes in PC-9 cells after 48h of combined treatment with the two drugs;

[0067] Fig.55 :Changes of LC3B and P62 immunofluorescence in PC-9 cells after 48h of combined treatment with the two drugs;

[0068] Fig.56 : Changes of autophagosomes in PC-9 cells after 48h of combined treatment with the two drugs;

[0069] Fig.57 :Changes in autophagic lysosomes in PC-9 cells after 48h of combined treatment with the two drugs;

[0070] Fig.58 :Representative results of lung cancer tissue proliferation capacity in PDX mouse model after 14 days of combined administration of two drugs;

[0071] Fig.59 : The results of changes in lung cancer tissue volume and mass in the PDX mouse model after 14 days of combined administration of the two drugs. DETAILED DESCRIPTION

[0072] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0073] The drug dehydroequol powder and MSAB powder involved in the following examples were purchased from MedChemExpress Biological Company.

[0074] Lung cancer cell lines (including PC-9 and H1650) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0075] Example 1 Flowchart of high-throughput automated drug screening system

[0076] The operation method is: take 5×10 3PC-9 lung cancer cell line in logarithmic growth phase was inoculated in 96-well cell culture plates. The control group was treated with DMSO, and the experimental group was treated with more than 1500 drugs (such as Sanguinarine chloride; Escin; Digitoxin; Periplocin) in the TargetMol-natural compound library at a concentration of 20μM for 48h (a 20μM drug was added to each well of the 96-well plate for 48h, and more than 1500 drugs required about 20 96-well plates in total). Dehydroequol PXD, which has an inhibition rate of more than 70% on PC-9 cell viability and is approved by the FDA for II / III clinical trials, was selected for research. The flow chart is as follows Figure 1 shown.

[0077] Example 2 Inhibitory effect of dehydroequol PXD on proliferation of lung cancer cell lines

[0078] The operation method is: take 5×10 3 Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated in 96-well cell culture plates, respectively. The control group was treated with DMSO, and the experimental group was treated with different concentrations of dehydroequol. After 24h, 48h, and 72h of treatment, the cell proliferation level was detected using CCK8 kit (Lambolide, Beijing). The statistical results of cell proliferation level are shown in Figure 2 and Figure 3 shown.

[0079] Take 5×10 2 Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated into six-well plates, treated with different concentrations of dehydroequol for 48 hours, and then the medium was changed. Two weeks later, the cells were fixed with paraformaldehyde for 30 minutes, stained with crystal violet for 30 minutes, and photographed. Figure 4 and Figure 5 shown.

[0080] The above results showed that compared with the control group, dehydroequol had a dose-dependent inhibitory effect on the growth and proliferation of lung cancer cells. 50 were 86.2, 57.2, and 33.1 μM, respectively, while the IC 50 After 24, 48, and 72 hours of treatment, the concentrations of PXD were 146.7, 46.1, and 15.0 μM, respectively. Moreover, PXD could inhibit the colony formation ability of lung cancer cells in a dose-dependent manner.

[0081] Example 3 PXD induces G0 / G1 phase cell cycle arrest in lung adenocarcinoma cells

[0082] The operation method is: take 4×10 5Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated in 6-well cell culture plates. The control group was treated with DMSO, and the experimental group was treated with PXD at 20μM, 40μM, and 60μM for H1650 cells and 30μM, 60μM, and 90μM for PC-9 cells. After 48h of treatment, half of the cells were taken to detect cell cycle distribution by PI flow cytometry and statistically analyze the changes in cell distribution. The results are shown in Figure 6-9 .

[0083] The other half of the cells were lysed with 200 μL RIPA lysis buffer on ice for 30 min, and total protein was extracted for Western blot to detect the expression of cell cycle-related gene protein levels. The results are shown in Fig.10 and Fig.11 .

[0084] Take 1×10 5 Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated in 24-well cell culture plates and fixed with 4% paraformaldehyde for 10 min at room temperature after drug treatment for 48 h. The results of immunofluorescence detection of cell cycle-related gene protein levels are shown in Fig.12 and Fig.13 .

[0085] Depend on Figure 6-9 It can be seen that compared with the control group, the proportion of cells in the G0 / G1 phase in the experimental group increased, and the proportion in the S phase showed a downward trend, indicating that the cells were blocked in the G0 / G1 phase. Fig.10 and 11 The results showed that the expression of P21, P27, Cyclin D1, and Cyclin E1 proteins were up-regulated, while the expression of Cyclin B1, CDK4, CDK2, and CDK1 were down-regulated. Fig.12 and 13 Immunofluorescence results also showed that PXD could upregulate the expression levels of P21 and P27 in a dose-dependent manner. These results suggest that PXD arrests H1650 and PC-9 cells in the G0 / G1 phase.

[0086] Example 4 PXD induces apoptosis in lung adenocarcinoma cells

[0087] The operation method is: take 4×10 5 Lung cancer cell lines PC-9 and H1650 in the logarithmic growth phase were inoculated in 6-well cell culture plates, respectively. The control group was treated with DMSO, and the experimental group was treated with PXD at 20μM, 40μM, and 60μM for H1650 cells; the PXD concentration for PC-9 cells was set at 30μM, 60μM, and 90μM. After 48h of treatment, half of the cells were taken to detect the level of cell apoptosis using Annexin V / PI flow cytometry and the cell apoptosis rate was calculated. The results are shown in Figure 14-17 .

[0088] The other half of the cells were lysed with 200 μL RIPA lysis buffer on ice for 30 min, and the total protein was extracted for Western blot to detect the expression of apoptosis-related gene protein levels. The results are shown in Fig.18 and Fig.19 .

[0089] Take 1×10 5 Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated in 24-well cell culture plates and fixed with 4% paraformaldehyde for 10 min at room temperature after drug treatment for 48 h. The results of immunofluorescence detection of apoptosis-related gene protein levels are shown in Figure 20-21 .

[0090] As shown in Figures 14-17, PXD increased the apoptosis rate of H1650 and PC-9 cells in a dose-dependent manner. Fig.18 and 19 The results showed that PXD could induce apoptosis by downregulating the apoptosis inhibitor XIAP. PXD could upregulate the expression of Bax in a concentration-dependent manner, while the expression of the anti-apoptotic factor Bcl-2 gradually decreased. The ratio of the two increased, and the accumulation of cleaved casp9, cleaved casp3, and cleaved PARP increased, indicating that PXD could induce apoptosis through endogenous pathways. The expression of FLIP decreased, and the activation cleavage band of cleaved casp8 appeared, indicating that PXD could also induce apoptosis of lung adenocarcinoma cells through the death receptor pathway. Fig. 20 and 21 Immunofluorescence results also showed that PXD could increase the fluorescence intensity of Cleaved casp9, Cleaved casp8, and Cleaved casp3 in a dose-dependent manner. These results suggest that PXD induces endogenous and exogenous apoptosis in lung adenocarcinoma cells.

[0091] Example 5 PXD inhibits the migration and invasion of lung adenocarcinoma cells

[0092] The operation method is: take 1×10 6 Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated in 6-well cell culture plates. After the cells were fully grown, scratches were made and cultured with low serum medium. The control group treated the cells with DMSO, and the experimental group treated the cells with PXD at 20μM, 40μM, and 60μM for H1650 cells and 30μM, 60μM, and 90μM for PC-9 cells. The healing of the scratches was statistically analyzed at 0, 24, and 48 hours after treatment. The results are shown in Figure 2. Figure 22-25 shown.

[0093] 5×10 4The PC-9 and H1650 lung cancer cell lines in logarithmic growth phase were resuspended in low serum medium containing 5 and 10 μM PXD and inoculated in the upper chamber of the Transwell chamber covered with Matrigel, and the complete medium containing 5 and 10 μM PXD was added to the lower chamber. After 48 hours of treatment, the cells were fixed with formaldehyde and stained with crystal violet. The number of cells transferred to the lower layer of Matrigel was counted to reflect the changes in cell invasion ability. The results are shown in Figure 26-29 shown.

[0094] Total protein was extracted from cells treated in the same way and Western blot was performed to detect the expression of gene proteins related to cell migration and invasion. The results are shown in Fig.30 and Fig.31 .

[0095] Take 1×10 5 Lung cancer cell lines PC-9 and H1650 in logarithmic growth phase were inoculated in 24-well cell culture plates, and fixed with 4% paraformaldehyde for 10 minutes at room temperature after drug treatment for 48 hours. The results of immunofluorescence detection of cell migration and invasion-related gene protein levels are shown in Fig.32 and Fig.33 .

[0096] The above results showed that PXD inhibited the migration and invasion ability of H1650 and PC-9 cells in a dose-dependent manner. Fig.30 and 31 The results showed that the expression of E-cadherin protein was up-regulated, while the expression of N-cadherin protein, matrix metalloproteinase family MMP2 and MMP9 was down-regulated. Fig.32 and 33 Immunofluorescence results also showed that PXD could downregulate the expression level of N-cadherin in a dose-dependent manner.

[0097] Example 6 Evaluation of the killing effect of PXD on lung cancer at the animal level

[0098] The operation method is: the lung adenocarcinoma tissue from the patient is inoculated subcutaneously into the right forelimb of the nude mouse. 3 The mice were randomly divided into a control group and a PXD-treated group, and then administrated. PXD was administered intraperitoneally at a dose of 40 mg / kg. The administration date was taken as the 0th day, and the drug was administered once a day for 14 days. There were 6 mice in each group. After the administration, the nude mice were euthanized. Fig.34 The tumor volume and mass of the control and treatment groups are shown in Fig.35 The tumor tissues and heart, liver, spleen, lung, and kidney tissues of nude mice were taken for HE staining. The results are shown in Fig.36 The tumor tissues of nude mice were taken for Western blot detection, and the results were as follows. Figure 37-39 shown.

[0099] Depend on Fig.34 and 35 It can be seen that PXD can reduce the weight and volume of tumor nodules in lung adenocarcinoma PDX, and there is no significant difference in the body weight of the two groups of nude mice. Fig.36 It can be seen that PXD has no obvious effect on the normal organs of the two groups of animals, including the heart, liver, spleen, lungs, and kidneys, indicating that PXD has little side effects on animals. Figure 37-39 As shown, the expression of cell cycle-related P21, P27, and Cyclin D1 proteins was upregulated, while Cyclin B1, CDK1, CDK4, and phosphorylated Rb (i.e., p-Rb) were downregulated; the apoptosis-related Bax / Bcl2 ratio was upregulated, C-casp8, C-caspa9, C-casp3, and PARP cleavage increased, and FLIP and XIAP expression was downregulated. The above results indicate that PXD significantly inhibited the process of lung cancer tumorigenesis in the PDX mouse model.

[0100] Example 7 Transcriptomic sequencing revealed that PXD upregulates Wnt pathway-related proteins

[0101] The operation method is: 3 groups of 6cm dishes were repeated, the control group was DMSO, and the drug group was 60μM PXD. After RNA extraction, purification, and library construction, the next-generation sequencing technology (Next-Generation Sequencing, NGS) was used to perform paired-end (PE) sequencing on these libraries based on the Illumina sequencing platform. Cluster analysis (bidirectional clustering analysis, PCA analysis, sample correlation analysis) and enrichment analysis (GO analysis, KEGG analysis, GSEA analysis) were performed on the differentially expressed genes. Fig.40 The bubble chart and bar chart of GO analysis enrichment for up-regulated differentially expressed genes indicate that Wnt-related genes are significantly up-regulated after PXD treatment. qPCR and WB experiments were used to verify the effect of PXD on the mRNA and protein levels of these Wnt-related binding genes. The results are shown in Fig.41 and 42 shown.

[0102] The above results indicate that PXD treatment of PC-9 cells causes upregulation of Wnt pathway-related proteins. The Wnt pathway is related to the biological behaviors of tumor cells such as growth, proliferation, and drug resistance. This upregulation may be a mechanism for tumor cells to resist drug killing and induce drug resistance.

[0103] Example 8 PXD inhibits autophagy in lung adenocarcinoma PC-9 cells

[0104] The operation method is as follows: PC-9 cells were inoculated into 6-well plates and starved for 24 hours to induce autophagy. The control group treated the cells with DMSO, and the PXD concentrations of the experimental group were set to 30μM, 60μM, and 90μM. After 48 hours of treatment, the autophagosomes and autolysosomes were examined and photographed under a transmission electron microscope. The results are as follows Fig.43 After PC-9 cells were treated for 48 hours (same method as above), Western blot was used to detect the changes in autophagy-related proteins, and the results are shown in 44. After PC-9 cells were treated for 48 hours (same method as above), immunofluorescence was used to detect the changes in autophagy-related proteins, and the results are shown in 45. After PC-9 cells were treated for 48 hours (same method as above), The changes of autophagosomes were detected by staining with the Autophagy Detection Kit 2.0, and the results are shown in 46. After PC-9 cells were treated for 48 hours (same method as above), the changes of autophagolysosomes were detected by acridine orange (AO) staining, and the results are shown in 47.

[0105] Depend on Fig.43 It can be seen that as the concentration of PXD increases, the number of autophagosomes and autolysosomes decreases under the electron microscope. Fig.44 It can be seen that the LC3Ⅱ / Ⅰ ratio is downregulated (LC3B has two bands, and the ratio of Ⅱ / Ⅰ band is the result of observation), and the expression of P62 is increased, indicating autophagy inhibition. ATG5 and Beclin1 involved in the initiation of autophagy are downregulated, and PXD inhibits the initiation of autophagy in PC-9 cells. Fig.45 The results of immunofluorescence detection showed that LC3B in the control group was punctately aggregated and diffusely distributed in the cytoplasm with a low fluorescence level, while the fluorescence intensity of P62 increased in a dose-dependent manner. Fig.46 It can be seen that PXD inhibits the formation of autophagosomes in the early stage of autophagy. Fig.47 It can be seen that the formation of late autophagic lysosomes is also inhibited. The above results show that PXD can inhibit autophagy in lung cancer cells.

[0106] Example 9: MSAB, a specific inhibitor of the Wnt / β-catenin pathway, can inhibit the growth and proliferation of lung adenocarcinoma cells

[0107] The operation method is: take 5×10 3 Lung cancer cell lines PC-9 in logarithmic growth phase were inoculated in 96-well cell culture plates. The control group was treated with DMSO, and the experimental group was treated with different concentrations of MSAB. After 48 hours of treatment, the absorbance value was detected by CCK8 kit and a cell viability bar graph was drawn. Fig.48 PC-9 cells were treated with 1 μM MSAB, which did not affect the cell viability of PC-9 cells, to verify the effect on the mRNA level of Wnt binding proteins. The results are shown in Fig.49 shown.

[0108] The above results showed that MSAB inhibited the growth and proliferation ability of lung adenocarcinoma cell PC-9 in a dose-dependent manner, and low-dose MSAB could inhibit the Wnt / β-catenin pathway.

[0109] Example 10: The two drugs combined to synergistically inhibit the growth and proliferation of lung cancer cell lines

[0110] The operation method is: take 5×10 3 PC-9 lung cancer cell lines in logarithmic growth phase were inoculated in 96-well cell culture plates. The control group was treated with DMSO, and the experimental group was treated with different concentrations of dehydroequol and 1μM MSAB. After 48h, the cell proliferation level was detected by CCK8 kit, and the King's formula Q = Ea + b / (Ea + Eb-Ea * Eb) was used to evaluate the efficacy of combined drug treatment. When the concentration of PXD exceeded 20μM, Q> was 1.15, indicating a synergistic effect. The results are shown in Fig.50 The synergistic use of 60 μM PXD and 1 μM MSAB was selected for subsequent experiments.

[0111] Take 4×10 5 PC-9 lung cancer cell line in logarithmic growth phase was inoculated in 6-well cell culture plates. The control group was treated with DMSO, and the experimental group was treated with PXD single drug concentration of 60μM, MSAB single drug concentration of 1μM and P+M (i.e. PXD+MSAB, the same below) combined drug treatment PC-9 cells. After 48h of treatment, the cell cycle distribution was detected by PI staining after alcohol fixation, and the cell apoptosis rate was detected by AnnexinV / PI staining. The results are shown in Figure 51-52 .

[0112] The above results show that the combined drug group can significantly inhibit the proliferation of lung adenocarcinoma and has a synergistic effect. The G0 / G1 phase arrest and apoptosis induction are higher than those of the single drug group. This shows that the combined drug group can synergistically inhibit the proliferation of lung cancer cells.

[0113] Example 11 The effect of the combination of two drugs on inducing autophagy in lung cancer cell lines

[0114] The operation method is as follows: PC-9 cells were inoculated into a 6 cm culture dish and starved for 24 h to induce autophagy. The control group treated the cells with DMSO, the experimental group PXD concentration was set to 60 μM, MSAB concentration was set to 1 μM, and the combined drug group. After 48 h of treatment, the autophagosomes and autolysosomes were examined and photographed under a transmission electron microscope. The results are as follows Fig.53 Western blot was used to detect changes in autophagy-related proteins. The results are shown in Fig.54 As shown. Immunofluorescence was used to detect changes in autophagy-related proteins. The results are shown in Fig.55 shown. The changes of autophagosomes were detected by staining with the Autophagy Detection Kit 2.0. The results are as follows Fig.56 AO staining was used to detect changes in autophagy and lysosomes. The results are shown in Fig.57 shown.

[0115] Depend on Figure 55-59 It can be seen that: in the combined drug group, more autophagosomes and autophagolysosomes can be observed than in the PXD single drug group, the LC3B-II / I ratio is upregulated, and the expression of autophagy-related protein P62 is reduced. Compared with the PXD group, the combined drug group promotes the generation of autophagic vesicles and presents a stronger red fluorescence symbolizing the acidic organelle lysosome. The above results show that the combined use of PXD and MSAB involved in the present invention can induce autophagy.

[0116] Example 12 PXD combined with MSAB further inhibits the growth of lung adenocarcinoma in vivo

[0117] The operation method is: inoculate lung adenocarcinoma tissue into the subcutaneous part of the right forelimb of nude mice. When the tumor volume reaches 100mm 3 The mice were randomly divided into a control group, a PXD-administered group, an MSAB-administered group, and a combined drug group. The PXD concentration was set at 40 mg / kg / day, the MSAB concentration was set at 20 mg / kg / day, and the combined drug group. The mice were intraperitoneally injected once a day according to the prescribed dose. The starting date of the drug administration was set as day 0, and the drug was administered once a day for 14 days, with a total of 6 mice in each group. After the drug administration, the nude mice were euthanized. The experimental results are as follows Figure 58-59 shown.

[0118] The results showed that the PXD combined with MSAB treatment group significantly inhibited the weight and volume of the tumor compared with the control group, PXD or MSAB group, and did not produce obvious side effects.

[0119] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A drug for alleviating or treating non-small cell lung cancer, characterized in that: The active ingredients of the medicine are PXD and MSAB.

2. The drug according to claim 1, characterized in that The dosage of PXD is 30-50 mg / kg; the dosage of MSAB is 10-30 mg / kg.

3. The drug according to claim 1, characterized in that The dosage of PXD is 40 mg / kg; the dosage of MSAB is 20 mg / kg.

4. A kit for alleviating or treating non-small cell lung cancer, characterized in that: Contains the drug according to any one of claims 1 to 3.

Images