Composition for enhancing glioma chemotherapeutic drug sensitivity and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a composition for enhancing sensitivity of glioma chemotherapeutic drugs and application of the composition. The composition comprises a macropinocytosis inhibitor and an autophagy inhibitor, and the macropinocytosis inhibitor comprises any one or more of EIPA and cytochalasin D; the autophagy inhibitor is prepared from any one or more of 3-methyladenine, bajulomycin A1 and hydroxychloroquine. The glioma is glioblastoma, and the chemotherapeutic drug is temozolomide. The invention also provides a pharmaceutical composition for treating glioma. The pharmaceutical composition consists of the macropinocytosis inhibitor, the autophagy inhibitor and temozolomide. Researches prove that the combination of the macropinocytosis inhibitor and the autophagy inhibitor can effectively enhance the chemotherapy effect of glioma temozolomide and prolong the lifetime of glioblastoma patients. The composition provided by the invention has important significance and potential clinical application value for improving GBM chemotherapy resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a composition for enhancing the sensitivity of glioma chemotherapy drugs and an application thereof. Background Art

[0002] Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly invasive and treatment-resistant, and is one of the malignant tumors with the worst survival prognosis in humans. The treatment of glioblastoma is mainly surgical resection, combined with comprehensive treatment methods such as radiotherapy and chemotherapy. However, due to the invasiveness and treatment resistance of the tumor, the survival rate of most patients within 5 years after diagnosis is still very low.

[0003] At present, the resistance of glioblastoma to radiotherapy and chemotherapy is a major challenge in treatment. Due to the resistance of glioma to radiotherapy and chemotherapy, the disease recurrence rate is high and the treatment effect is not ideal. In order to overcome the limitations of these treatments, people have launched multi-faceted explorations. For example, some technicians focus on the study of molecular targeted drugs combined with radiotherapy and chemotherapy. Molecular targeted therapy includes vascular endothelial growth factor (VEGF) inhibitors and epidermal growth factor receptor (EGFR) inhibitors combined with radiotherapy. Molecular targeted drugs can activate specific molecular signal transduction pathways, activate cell apoptosis, inhibit cell cycle or block blood vessel growth, thereby effectively killing tumors. Common molecular targeted drugs include bevacizumab, gefitinib, etc. Among them, bevacizumab is a humanized anti-VEGF monoclonal antibody that can activate VEGF downstream pathways such as RAS-RAF-MAPK through protein phosphorylation. Gefitinib is an effective small molecule inhibitor of EGFR tyrosine kinase. Schwer et al. used SRT combined with gefitinib to treat recurrent high-grade gliomas and reported that the dose of 36 Gy at three sites was well tolerated. Other researchers are exploring the mechanisms of glioblastoma treatment resistance in depth, looking for ways and drugs that can effectively reduce GBM chemotherapy resistance.

[0004] Temozolomide (TMZ) is a first-line chemotherapy drug. When GBM is treated with TMZ, it can activate classical autophagy to produce chemotherapy resistance and lead to treatment failure. Therefore, the combination of autophagy inhibitors may provide a new idea for solving TMZ chemotherapy resistance. However, the latest clinical research results show that even if the maximum loading dose of autophagy inhibitors that GBM patients can tolerate is given, that is, the triple regimen of GBM standard treatment strategy combined with autophagy inhibitors (surgery + TMZ + chloroquine), it does not effectively improve the overall survival of GBM patients. This shows that only focusing on the autophagy mechanism and applying autophagy inhibitors has limited effect on alleviating chemotherapy resistance, and it is necessary to jointly target the biological mechanisms in GBM from multiple pathways to overcome chemotherapy resistance. Therefore, there is an urgent need to find other key mechanisms that mediate GBM chemotherapy resistance.

[0005] In the prior art, some patents disclose technical means for treating gliomas by regulating cell autophagy, such as CN103990126A patent discloses the combined use of cell autophagy inhibitors and beta interferon to treat gliomas; CN116413442A patent discloses that RFC4 inhibitors can effectively reduce temozolomide-induced autophagy activation in glioblastomas and enhance the sensitivity of glioblastoma to temozolomide treatment. In addition, CN117295522A patent discloses the role of macropinocytosis inhibitors in cancer treatment, which confirms that inhibiting macropinocytosis in cancer cells can enhance the anti-cancer efficacy of photodynamic therapy, and the disease types include brain cancer.

[0006] At present, the problem of GBM chemotherapy resistance has not been effectively solved. Analyzing the mechanism of GBM chemotherapy resistance and effectively reducing TMZ resistance have become clinical problems that urgently need to be solved. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a composition for enhancing the sensitivity of glioma chemotherapy drugs, wherein the composition can effectively improve the sensitivity of glioma chemotherapy drugs.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A composition for enhancing the sensitivity of glioma chemotherapy drugs, comprising a macropinocytosis inhibitor and an autophagy inhibitor.

[0010] The present invention found that autophagy inhibition is significantly correlated with macropinocytosis activation. When autophagy inhibitors are used alone, the classical autophagy process is inhibited, the expression of related proteins and mRNA decreases, but the expression of macropinocytosis-related proteins and mRNA increases significantly. Autophagy-related proteins include LC3B, SEC22B, and ATG5; macropinocytosis-related proteins include RAC1, NRF2, SDC1, NHE1, SNX5, CDC42, Rab5a, and Rab7a. Further studies have found that macropinocytosis activation plays an important role in glioblastoma chemotherapy resistance, and GBM chemotherapy-resistant cell lines have stronger macropinocytosis activity, and inhibiting macropinocytosis can significantly alleviate its chemotherapy resistance. In addition, macropinocytosis core protein has an adverse effect on GBM survival prognosis and is negatively correlated with autophagy activity. The present invention confirms that the combined use of macropinocytosis inhibitors and autophagy inhibitors can effectively alleviate temozolomide chemotherapy resistance and improve its chemotherapy sensitivity; thereby effectively inhibiting glioblastoma growth and prolonging patient survival.

[0011] Furthermore, the chemotherapy drug includes temozolomide.

[0012] Furthermore, the macropinocytosis inhibitor includes any one or more of EIPA and cytochalasin D.

[0013] Furthermore, the autophagy inhibitor includes any one or more of 3-methyladenine, bafilomycin A1, and hydroxychloroquine.

[0014] Furthermore, the glioma includes glioblastoma.

[0015] A second object of the present invention is to provide a pharmaceutical composition for treating glioma.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

[0017] A pharmaceutical composition for treating glioma, comprising the above composition and temozolomide.

[0018] The third object of the present invention is to provide an application of the aforementioned composition in the preparation of a sensitizer for enhancing the efficacy of glioma chemotherapy.

[0019] To achieve the above object, the present invention adopts the following technical solutions:

[0020] Application of the above composition in the preparation of a sensitizer for improving the sensitivity of glioma chemotherapy drugs.

[0021] Furthermore, the chemotherapy drug includes temozolomide.

[0022] Furthermore, the glioma includes glioblastoma.

[0023] Furthermore, the composition alleviates temozolomide chemotherapy resistance and enhances the chemotherapy efficacy of glioma by inhibiting the expression of autophagy-related proteins and macropinocytosis-related proteins; the autophagy-related proteins include any one or more of LC3B, SEC22B, and ATG5; the macropinocytosis-related proteins include any one or more of RAC1, NRF2, SDC1, NHE1, SNX5, CDC42, Rab5a, and Rab7a.

[0024] A fourth object of the present invention is to provide an application of the aforementioned pharmaceutical composition in the preparation of a drug for treating glioma.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] Use of the aforementioned pharmaceutical composition in the preparation of a drug for treating glioma.

[0027] Furthermore, the glioma includes glioblastoma.

[0028] A fifth object of the present invention is to provide a use of the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting glioma growth and / or prolonging the survival of glioma patients.

[0029] To achieve the above object, the present invention adopts the following technical solutions:

[0030] The use of the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting the growth of glioma and / or prolonging the survival of glioma patients.

[0031] Furthermore, the glioma includes glioblastoma.

[0032] The beneficial effects of the present invention are:

[0033] The present invention found that not only the autophagy pathway is involved in the chemotherapy resistance of glioma, but macropinocytosis also plays an important role in the chemotherapy resistance of glioma. The application of autophagy inhibitors in glioma can significantly activate the macropinocytosis process, and macropinocytosis activation can promote the chemotherapy resistance of glioblastoma. Inhibiting the macropinocytosis process has a significant effect on improving the treatment resistance of glioma. Further studies have confirmed that the dual blocking therapy of the macropinocytosis inhibitor EIPA combined with the autophagy inhibitor HCQ can effectively alleviate chemotherapy resistance and enhance the therapeutic effect of the chemotherapy drug temozolomide, which has important application prospects for prolonging patient survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of Western blotting to detect the changes in the expression of intracellular autophagy and macropinocytosis-related proteins in GBM cells treated with temozolomide, hydroxychloroquine and their combination;

[0035] Figure 2 The results of qPCR detection of the expression levels of autophagy and macropinocytosis-related genes in GBM cells treated with temozolomide, hydroxychloroquine and their combination;

[0036] Figure 3 This is the result of immunoblotting to detect the changes in autophagy activity after ATG5 knockdown in GBM1 cells, and the results of detecting ATG5 protein expression;

[0037] Figure 4 Representative images of TMR-DEX uptake in GBM1 and LN229 cells after different treatments, scale bar = 10 μm;

[0038] Figure 5 This is a diagram for statistical analysis of the relative activity of macropinocytosis of GBM1 and LN229 cells after different treatments; Figure 5 -A is the relative macropinocytosis activity of LN229 cells after different treatments, Figure 5 -B is the relative macropinocytosis activity of GBM1 cells after different treatments;

[0039] Figure 6Representative images and statistical analysis of immunofluorescence detection of autophagy core protein LC3B and macropinocytosis uptake of TMR-DEX in GBM1 cells under different treatment groups, among which, Figure 6 -A is a representative image of TMR-DEX for immunofluorescence detection of autophagy core protein LC3B and macropinocytosis uptake in GBM1 cells of different treatment groups, scale bar = 10 μm; Figure 6 -B is the distribution of LC3B in GBM1 cells in different treatment groups (LC3B represents the level of cell autophagy). Figure 6 -C is the relative macropinocytosis activity of GBM1 cells in different treatment groups;

[0040] Figure 7 This is the result of detecting the interaction between SEC22B and RAC1 in GBM1 cells by co-immunoprecipitation (Co-IP), where: Figure 7 -A is a diagram showing the interaction between SEC22B and RAC1 in GBM1 cells detected by immunoprecipitation using SEC22B as a capture antibody; Figure 7 -B is a diagram showing the interaction between SEC22B and RAC1 in GBM1 cells detected by immunoprecipitation using RAC1 as a capture antibody;

[0041] Figure 8 This is a diagram showing the co-localization of SEC22B and RAC1 in GBM1 cells treated with HCQ (10 μM, 2 h) by immunofluorescence detection and statistical analysis; Figure 8 -A is a graph showing the co-localization of SEC22B and RAC1 in GBM1 cells treated with HCQ by immunofluorescence detection. Figure 8 -B is Figure 8 -Statistical results of A; scale bar = 10 μm;

[0042] Fig. 9 This is a graph showing the IC50 values ​​of GBM1 and its resistant cell line GBM1-R cell line detected by CCK8 method after iterative induction of temozolomide, among which the IC50 value of GBM1 is 722.1μM, and the IC50 value of GBM1-R is 1995.3μM;

[0043] Fig.10 The representative images and statistical analysis results of immunofluorescence detection of TMR-DEX uptake in GBM1 and GBM1-R cells treated with temozolomide and HCQ, among which, Fig.10 -A is a representative image of TMR-DEX uptake detected by immunofluorescence in GBM1 and GBM1-R cells treated with temozolomide and HCQ. Fig.10 -B is Fig.10 -Statistical results of A; scale bar = 10 μm;

[0044] Fig.11 This is a graph showing the IC50 of GBM1 cells iteratively induced by temozolomide and its resistant cell line GBM1-R cell line treated with the macropinocytosis inhibitor EIPA using the CCK8 method;

[0045] Fig.12 The representative images and statistical analysis results of immunofluorescence detection of TMR-DEX uptake in GBM1-R cells treated with temozolomide, HCQ, and EIPA, among which, Fig.12 -A is a representative image of immunofluorescence detection of TMR-DEX uptake in GBM1-R cells treated with temozolomide, HCQ, and EIPA, scale bar = 10 μm; Fig.12 -B is the relative macropinocytosis activity of GBM1-R cells in different treatment groups; Fig.12 -C is the relative macropinocytosis activity of LN299-R cells in different treatment groups;

[0046] Fig.13 This is the growth curve of GBM1-R and LN229-R cells detected by CCK8 method after TMZ, HCQ, EIPA treatment alone and in combination, among which, Fig.13 -A is the growth curve of GBM1-R cells detected by CCK8 method after TMZ, HCQ, EIPA treatment alone and in combination, Fig.13 -B is the growth curve of LN229-R cells detected by CCK8 method after TMZ, HCQ, EIPA treatment alone and in combination;

[0047] Fig.14 The following is the analysis of RAC1 expression in the TCGA-GBM dataset and the survival analysis results of the relationship between expression level and survival prognosis in the TCGA-GBM and CGGA databases. Fig.14 -A is the analysis result of RAC1 expression in TCGA-GBM dataset; Fig.14 -B is the survival analysis result diagram of the relationship between the expression level of RAC1 in the TCGA-GBM database and the survival prognosis; Fig.14 -C is the survival analysis result diagram of the relationship between the expression level of RAC1 in the CGGA database and the survival prognosis;

[0048] Fig.15 The analysis of NRF2 expression in the TCGA-GBMLGG dataset and the relationship between expression and survival prognosis in the CGGA and TCGA-GBMLGG databases were performed. Fig.15 -A is the analysis result of NRF2 expression in TCGA-GBMLGG dataset, Fig.15 -B is the survival analysis result diagram of the relationship between the expression level of NRF2 in the CGGA database and the survival prognosis. Fig.15-C is the survival analysis result diagram of the relationship between the expression level of NRF2 in the TCGA-GBMLGG database and the survival prognosis;

[0049] Fig.16 The results of comparative analysis of immunohistochemical staining of autophagy core protein (LC3B) and macropinocytosis core protein (RAC1, NRF2, SDC1) in serial sections of the same clinical sample are shown in the figure. Fig.16 -A is the immunohistochemical staining result of autophagy core protein and macropinocytosis core protein in clinical sample GBM0329. Fig.16 -B is the immunohistochemical staining result of autophagy core protein and macropinocytosis core protein in clinical sample GBM2767;

[0050] Fig.17 This is a picture showing the effects of HCQ, EIPA, and HCQ combined with EIPA treatment on GBM1-derived NOD-SCID transplanted tumors detected by in vivo imaging of small animals;

[0051] Fig.18 Representative images of HE staining to detect the effects of HCQ, EIPA, and HCQ combined with EIPA on GBM1-derived NOD-SCID transplanted tumors;

[0052] Fig.19 Statistical analysis of the growth of transplanted tumors in the control group, HCQ group, EIPA group, and HCQ+EIPA group, n=8;

[0053] Fig. 20 This is the result of Kaplan Meier survival analysis of the effects of the control group, HCQ group, EIPA group and HCQ+EIPA group on the survival of GBM1-derived NOD-SCID transplanted tumors, n=8.

[0054] In the above figures, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no statistical difference. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0056] At present, the main hypotheses about the mechanism of TMZ chemotherapy resistance include the following: 1) various DNA repair mechanisms based on MGMT repair enzymes; 2) glioma stem cell theory; 3) GBM multidrug resistance mechanism; 4) abnormal tumor microenvironment and signaling pathways; 5) classic autophagy and macropinocytosis mechanisms. Regarding the role of autophagy in GBM, previous studies have shown that autophagy promotes chemotherapy resistance, but the results of phase II / III clinical studies on autophagy inhibitors have shown that the addition of autophagy inhibitors did not improve the overall survival of GBM; and the results of preliminary experiments have shown that after the application of autophagy inhibitors, the number of macropinocytic bodies in GBM cells has increased significantly, and inhibiting macropinocytosis can alleviate chemotherapy resistance, which seems to indicate that macropinocytosis is related to GBM chemotherapy resistance.

[0057] Recent studies have reported that when tumor cells are under metabolic stress (such as radiotherapy, chemotherapy, and starvation), in addition to activating autophagy to maintain survival, they can also survive by taking up extracellular macromolecular nutrients through macropinocytosis. This indicates that macropinocytosis is a supply pathway for many nutrients, especially amino acids, to support the growth and proliferation of tumor cells. Many tumors have the ability of macropinocytosis. Tumor cells take up extracellular macromolecular nutrients through macropinocytosis to synthesize carbohydrates, lipids, proteins, and nucleotides required for their own metabolism, and resist treatment targeting anabolic pathways. In GBM, tumor cells with RAS, PI3K, or PTEN mutations have significantly activated macropinocytosis, which internalizes extracellular proteins by absorbing extracellular fluid, degrading and releasing amino acids and glutamine, thereby supporting the metabolic adaptation of cells and promoting tumor growth. In addition, recent studies have shown that in proliferating tumor cells, extracellular amino acids and lipids contribute most of the bioenergy required for rapid proliferation of tumor cells, rather than glucose, so this further emphasizes the important role of macropinocytosis based on the intake of liquid substances in tumor growth and survival. Therefore, macropinocytosis may be one of the key mechanisms of GBM chemotherapy resistance, but the way and mechanism by which the macropinocytosis process is activated in GBM and the regulatory network involved still need further study.

[0058] The present invention found in the early stage of studying TMZ chemotherapy resistance caused by autophagy that when autophagy is inhibited, a large number of macropinocytosis bodies are formed in the cell, and current research believes that macropinocytosis (MP) is an evolutionarily highly conserved cell biological process that promotes the growth and survival of cells in extreme environments by non-selectively taking up extracellular nutrients and liquid macromolecular organic matter. The preliminary experimental results of the present invention show that: 1) after the application of autophagy inhibitors, the macropinocytosis bodies marked by the specific dye TMR-DEX increased significantly; 2) there is a stronger macropinocytosis activity in TMZ-resistant GBM cell lines; 3) the application of macropinocytosis inhibitors can significantly alleviate GBM's resistance to TMZ chemotherapy. This shows that inhibiting autophagy can promote macropinocytosis activation, and macropinocytosis activation may be one of the key mechanisms of GBM chemotherapy resistance. Therefore, in-depth research on the mechanism behind the inhibition of autophagy leading to macropinocytosis activation and its role in the formation of TMZ treatment resistance may be of great significance and potential clinical application value for improving GBM chemotherapy resistance.

[0059] In the implementation of the present invention, 3-methyladenine, bafilomycin A1, hydroxychloroquine, EIPA and other autophagy inhibitors were purchased from Selleck, with the item numbers S2767, S1413, E4824 and S9849, respectively. Temozolomide and cytochalasin D were purchased from Sigma, with the item numbers NO.85622-93-1 and NO.22144-77-0, respectively.

[0060] In the implementation of the present invention, the cell lines LN229 and U87 involved were purchased from the ATCC Biological Standard Resource Center in the United States, GBM1 was isolated and constructed by this laboratory, and the glioma tissue samples were derived from the biological sample bank of this unit, with the ethics approval number: KY2020294.

[0061] Example 1. Study on the correlation between activation of macropinocytosis and secretory autophagy

[0062] The present invention has found that in addition to playing a degradation function as one of the important mechanisms of chemotherapy resistance, autophagy also has a new form of autophagy that mediates secretory function, namely secretory autophagy. Secretory autophagy can extend the direct influence of key intracellular signal factors to the extracellular microenvironment, and plays an important role in the remodeling of the microenvironment and intercellular communication. At the same time, it is interconnected with other biological processes and affects the fate of tumor cells. Therefore, the present invention uses immunoblotting, qPCR and other means to verify the correlation between the activation of macropinocytosis and secretory autophagy. The specific method is as follows:

[0063] (1) Clarify the detection method of macropinocytosis activation: Rhodamine B isothiocyanate-labeled dextran (TMR-DEX) with a molecular weight of 70KD can specifically label the macropinocytosis process. When intracellular macropinocytosis is activated, it is taken up by the cells and red fluorescent macropinosomes can be observed under a fluorescence microscope or confocal microscope, which can be used for statistical counting analysis.

[0064] (2) Grouping: Glioma cell lines and primary cell lines were treated with autophagy inhibitors: 3-methyladenine (3-MA, 1 mM), bafilomycin A1 (BafA1, 1 nM), and hydroxychloroquine (HCQ, 10 μM). The experimental group was the TMZ+autophagy inhibitor group, and the control group was the TMZ+placebo group.

[0065] (3) Cell transfection: Lentiviral transfection was used to construct GBM-shLC3B and GBM-shATG5 cell models with low autophagy activity or even no autophagy by knocking down LC3B and ATG5 as a supplement to autophagy inhibitor drugs.

[0066] (4) In the above steps (2) and (3), cells were collected from each treatment group and control group 24 hours after transfection and sent to the BGI gene detection platform for transcriptome sequencing. After sequencing, GSEA, GO, KEGG and other pathway enrichment analyses were performed, focusing on the activation of genes in macropinocytosis-related pathways, and absolute quantitative analysis was performed to analyze the expression of macropinocytosis core transcription factors RAC1, NRF2, SDC1, NHE1, SNX5, CDC42, Rab5a, and Rab7a.

[0067] (5) After the treatment in step (2) and step (3), each group of cells was washed twice with PBS and placed in serum-free cell culture medium, and TMR-DEX was added so that the concentration in the culture medium reached 1 mg / ml. After 1 hour, the cells were fixed and stained, observed and photographed under a confocal microscope, and the cell uptake of each group was statistically analyzed.

[0068] (6) Collect the cell samples from each group processed in step (2) and step (3), and use RT-PCR and Western blot to detect the core genes and proteins related to the autophagy and macropinocytosis process for in vitro verification.

[0069] Results: The present invention treated the primary glioblastoma cells GBM1 with temozolomide and the classic autophagy inhibitor hydroxychloroquine alone or in combination, and then detected the autophagy-related proteins and their mRNAs, as well as the core proteins related to macropinocytosis. Figure 1-Figure 2As shown, when autophagy inhibitors are applied, the classical autophagy process is inhibited, the expression of related proteins and mRNA is reduced, but the expression of macropinocytosis-related proteins and mRNA is significantly increased. The study found that when autophagy inhibitors are used to inhibit the classical autophagy of GBM, the autophagic vesicles are blocked from degradation and then enter the secretory process. The corresponding secretory autophagy activity is significantly increased, and the expression of the vesicle transport protein SEC22B specifically related to secretory autophagy is significantly increased. And with the increase in secretory autophagy activity, the uptake capacity of GBM cells is also significantly enhanced, and a large number of macropinosomes are formed in the cells. Therefore, the present invention further silences the autophagy core gene ATG5 in GBM cells to inhibit autophagy, and at the same time uses rhodamine B isothiocyanate-labeled dextran (TMR-DEX) to mark the macropinocytosis process. The results also show that inhibiting autophagy enhances macropinocytosis activity. As Figure 3-Figure 6 As shown in the figure, compared with the control group, the cells in the autophagy inhibitor group contained more abundant macropinocytic bodies; and the activation degree of GBM macropinocytosis was positively correlated with the intensity of secretory autophagy; at the same time, the experiment showed that the changes in the SEC22B protein specifically related to secretory autophagy were consistent with the changes in the small GTPase RAC1 that initiated the macropinocytosis process. These results show that the expression of macropinocytosis-related genes and proteins was upregulated after the application of autophagy inhibitors, and was positively correlated with the activity of secretory autophagy, suggesting that secretory autophagy plays a key role in the process of inhibiting the activation of macropinocytosis after classical autophagy.

[0070] In order to explore the specific mechanism of macropinocytosis initiation, the present invention further verified the relationship between the secretory autophagy specific transporter SEC22B and the macropinocytosis initiation protein RAC1. First, immunoprecipitation experiments were performed in GBM1 and LN229 cells, and it was found that they had obvious interactions when inhibiting classical autophagy. Figure 7 The results of immunofluorescence colocalization also suggest that the two have obvious colocalization and inhibiting autophagy can promote their colocalization. Figure 8 The secretory autophagic vesicle transporter SEC22B can form a complex with the RAC1 protein that initiates macropinocytosis.

[0071] The above results indicate that the activation of macropinocytosis is significantly correlated with secretory autophagy, and inhibition of classical degradative autophagy can significantly activate the macropinocytosis process.

[0072] Example 2. Study on the role of macropinocytosis in glioblastoma chemotherapy resistance and its correlation with patient survival prognosis

[0073] 1. Study on the role of macropinocytosis activation in glioblastoma chemotherapy resistance

[0074] The present invention clarifies the important role of macropinocytosis in glioma chemotherapy resistance by detecting the macropinocytosis core genes and proteins of glioma samples in the biological sample library. The specific method is as follows:

[0075] (1) Tissue samples of patients who were resistant to temozolomide (poor efficacy and short survival after temozolomide chemotherapy) and those who were sensitive to temozolomide (good efficacy and long survival after temozolomide chemotherapy) were retrieved through the clinical information system of the sample library and prepared into frozen sections and paraffin sections.

[0076] (2) The macropinocytosis-related proteins RAC1, NRF2, SDC1, NHE1, SNX5, and CDC42 were stained by immunohistochemistry and their expression intensity was quantified by immunohistochemical H-score, and the relative expression levels were compared between the treatment-resistant group and the treatment-sensitive group.

[0077] (3) Use "GlioVis" in the TCGA and CGGA databases to search for the effects of macropinocytosis-related core proteins on the survival prognosis of glioblastoma, and evaluate the specific role of macropinocytosis in glioblastoma temozolomide chemotherapy resistance.

[0078] (4) The region with abundant expression of macropinocytosis core protein in step (2) is selected and cut by microdissection, and protein spectrum analysis and proteome sequencing are performed to find the key protein with increased macropinocytosis.

[0079] (5) Detecting the expression level of the macropinocytosis-related protein obtained in step (4) in the constructed temozolomide treatment-resistant cell line model to clarify its relationship with glioblastoma temozolomide chemotherapy resistance.

[0080] (6) Using lentivirus or adenovirus transfection experimental methods, the core gene SEC22B for initiating macropinocytosis obtained in step (4) was knocked down in the temozolomide treatment-resistant cell line to observe the changes in the expression level of RAC1 and whether its IC50 value decreases under the treatment of temozolomide.

[0081] Results: A cell model of glioblastoma temozolomide resistance was established. Our self-isolated primary cell lines were exposed to TMZ until their IC 50 The value reached more than 2 times, and the chemotherapy-resistant cell line GBM1-R was successfully constructed. Fig. 9 ; TMR-DEX (1 mg / ml, 1 h) was added, and the results showed that the treatment-resistant strain had higher macropinocytosis activity, see Fig.10 The results are shown in Table 2. Fig.11 As shown, its IC 50 The values ​​were significantly reduced, indicating that inhibiting the macropinocytosis process can increase the sensitivity of temozolomide treatment. Fig.12The results also confirmed that EIPA significantly reduced macropinocytosis activity. The chemotherapy-resistant cell lines were placed in different treatment groups. The results showed that the group with combined inhibition of macropinocytosis and autophagy had a better therapeutic effect and a stronger killing effect than the single combination of autophagy inhibitors. For details, see Fig.13 The results showed that GBM chemotherapy-resistant cell lines have stronger macropinocytosis activity, and inhibiting macropinocytosis significantly alleviates their chemotherapy resistance.

[0082] 2. Study on the correlation between macropinocytosis activation and survival prognosis in human glioblastoma

[0083] The correlation between macropinocytosis activation and the survival prognosis of glioblastoma patients was clarified through the glioma database and bioinformatics analysis tools. The specific methods are as follows:

[0084] (1) Specimens diagnosed with glioblastoma from 2014 to 2022 were collected from the biobank of this platform, including 50 paraffin-embedded specimens and 30 fresh surgical frozen specimens, including clinical information and follow-up information of all specimens.

[0085] (2) Based on the clinical paraffin specimens of glioblastoma collected in the biological sample library of this experimental platform, the activation of macropinocytosis in the samples treated with temozolomide was detected, and the macropinocytosis-related core proteins SEC22B, RAC1, NRF2, SDC1, NHE1, SNX5, and CDC42 were immunohistochemically stained.

[0086] (3) Cut the collected fresh and chemotherapy-treated glioma tissue into 1mm (length * width * height = 1mm * 1mm * 1mm) tissue blocks. If the tissue block is too large, the fixative, dehydrating agent, dye, etc. cannot penetrate into the middle part, which will seriously affect the dehydration, staining, and sectioning of the tissue. Because the specimens will be lost to a certain extent during the sample preparation process, 3-6 pieces of tissue from the same part (the same observation target) are placed in a specimen bottle (containing electron microscope fixative) to ensure that there is a piece of tissue that can be placed on the electron microscope in the end to detect the formation of macropinosomes in the tissue and conduct statistical analysis.

[0087] (4) The fresh and chemotherapy-treated glioma tissues were embedded in an embedding agent and frozen into sections with a thickness of 10 μm. After being fixed with ice-acetone, immunofluorescence staining was performed. The macropinosome core protein was labeled using the double antibody sandwich method, and ImageJ was used to semi-quantitatively analyze the relative content changes.

[0088] (5) Glioblastoma LN229 and U87 cell lines and primary cell lines 090116 (GBM1) and 0901214 (GBM2) isolated and constructed by our research group were used as in vitro experimental models. The above cells were treated with different concentration gradients (0-1000 μm / l) of temozolomide for 24 h, and the expression of genes related to macropinocytosis activation was detected by RT-PCR and Western blot.

[0089] Results: By analyzing the expression of macropinocytosis core proteins NRF2 and RAC1 in the TCGA and CGGA databases, it was found that they were highly expressed in GBM, and survival analysis showed that their expression levels were negatively correlated with prognosis. For details, see Figure 14-15 Immunohistochemical staining of GBM tissue samples found that the higher the autophagy activity, the weaker the expression of macropinocytosis protein, and the lower the autophagy activity, the higher the macropinocytosis activity. Fig.16 The results suggest that macropinocytosis core protein has an adverse effect on GBM survival prognosis and is negatively correlated with autophagy activity. In general, macropinocytosis activation plays an important role in glioblastoma chemotherapy resistance.

[0090] Example 3. Study on improving chemotherapy sensitivity of glioblastoma by combining autophagy inhibition with macropinocytosis inhibition

[0091] Inhibiting macropinocytosis and autophagy may have potential therapeutic significance for alleviating chemotherapy resistance. Therefore, the present invention further established an orthotopic transplant tumor model in mice to detect the sensitization effect of the macropinocytosis inhibitor EIPA combined with the autophagy inhibitor. The specific method is as follows:

[0092] (1) In vitro cell experiments, glioma cells were treated with autophagy inhibitors 3-methyladenine (3-MA, 1 mM), bafilomycin A1 (BafA1, 1 nM), hydroxychloroquine (HCQ, 10 μM) and macropinocytosis-specific inhibitors amiloride (EIPA, 600 nM) and cytochalasin D (100 nM).

[0093] (2) Grouping: control group, autophagy inhibitor group, TMZ group, macropinocytosis inhibitor group, TMZ + autophagy inhibitor group, TMZ + macropinocytosis inhibitor group, TMZ + autophagy inhibitor + macropinocytosis inhibitor group.

[0094] (3) Glioma cell lines and chemotherapy-resistant cell lines were plated in 96-well plates, with 3,000 cells plated in each well. The two treatment groups were given and CCK-8 assay was performed daily to detect cell proliferation.

[0095] (4) Glioma cell lines and chemotherapy-resistant cell lines were plated in 96-well plates, with 6,000 cells per well. Each group was treated for 48 h, and the CCK-8 cytotoxicity assay was performed to detect the killing effect.

[0096] (5) A subcutaneous tumor transplant mouse model was constructed. Each group was treated with EIPA (7.5 mg / kg), TMZ (5 mg / kg), and HCQ (10 mg / kg) for 7 days. All the above drugs were injected intraperitoneally. The mice were killed after 2 weeks, and the tumor weight and volume were measured, and statistical analysis was performed.

[0097] (6) Orthotopic xenograft mouse models (PDX) were constructed and each group was treated with EIPA (7.5 mg / kg), TMZ (5 mg / kg), and HCQ (10 mg / kg) for 7 days. All the above drugs were injected intraperitoneally. The tumor growth of each group of mice was detected every 7 days using the luciferase chemiluminescence method and statistical analysis was performed.

[0098] Results: This example firstly carried out an in vivo experiment on NOD-SCID transplanted tumor mice according to the in vitro experimental grouping in Example 2. All treatment groups were treated with TMZ (5 mg / kg) for 5 consecutive days. Fig.17 As shown in the figure, the double blocking group using macropinocytosis inhibitor EIPA and autophagy inhibitor hydroxychloroquine significantly inhibited the growth of transplanted tumors compared with the control group using TMZ alone. And HE staining of the transplanted tumors in each group showed that the tumor area in the HCQ+EIPA group was significantly smaller than that in the other groups. Fig.18 Statistical analysis of the tumor growth of each group of mice showed that the HCQ+EIPA group had a statistically significant reduction compared with the control group, and prolonged the overall survival of the mice. Figure 19-20 That is, the combination of macropinocytosis inhibitors and autophagy inhibitors significantly prolonged the survival of mice with orthotopic GBM transplanted tumors. These results indicate that the combined use of macropinocytosis inhibitors and autophagy inhibitors plays an important role in improving the chemosensitivity of the chemotherapy drug temozolomide.

[0099] In summary, the present invention deeply explores the sensitization effect of temozolomide chemotherapy by macropinocytosis inhibitor EIPA under the state of glioma autophagy inhibition. By detecting the core genes and proteins of macropinocytosis of glioma samples in the biological sample library, the important role of macropinocytosis process in glioma chemotherapy resistance is clarified. At the same time, the correlation between macropinocytosis activation and the survival prognosis of glioblastoma patients is clarified through the glioma database and bioinformatics analysis tools. RT-PCR, Western blotting, TMR-DEX uptake experiment, immunofluorescence, immunohistochemistry and other conventional methods are used to detect related components. Finally, a mouse orthotopic transplant tumor model is established to detect the sensitization effect of macropinocytosis inhibitor EIPA combined with autophagy inhibitor. The results show that the application of autophagy inhibitors in glioma can significantly activate the macropinocytosis process, and macropinocytosis activation can promote glioblastoma chemotherapy resistance. Therefore, the macropinocytosis inhibitor EIPA combined with autophagy inhibitors can sensitize the temozolomide chemotherapy effect of glioma, which has important application prospects for prolonging patient survival.

Claims

1. A composition for enhancing the sensitivity of glioma chemotherapy drugs, characterized in that: The composition includes a macropinocytosis inhibitor and an autophagy inhibitor.

2. The composition according to claim 1, characterized in that The chemotherapy drug includes temozolomide.

3. The composition according to claim 1, characterized in that The macropinocytosis inhibitor includes any one or more of EIPA and cytochalasin D.

4. The composition according to claim 1, characterized in that The autophagy inhibitor includes any one or more of 3-methyladenine, bafilomycin A1, and hydroxychloroquine.

5. The composition according to claim 1, characterized in that The glioma includes glioblastoma.

6. A pharmaceutical composition for treating glioma, characterized in that: The pharmaceutical composition comprises the composition according to any one of claims 1 to 5 and temozolomide.

7. Use of the composition according to any one of claims 1 to 5 in the preparation of a sensitizer for improving the sensitivity of glioma chemotherapy drugs.

8. The use according to claim 7, characterized in that: The composition alleviates temozolomide chemotherapy resistance and enhances the chemotherapy efficacy of glioma by inhibiting the expression of autophagy-related proteins and macropinocytosis-related proteins; the autophagy-related proteins include any one or more of LC3B, SEC22B, and ATG5; the macropinocytosis-related proteins include any one or more of RAC1, NRF2, SDC1, NHE1, SNX5, CDC42, Rab5a, and Rab7a.

9. Use of the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating glioma.

10. Use of the pharmaceutical composition according to claim 6 in the preparation of a drug for inhibiting the growth of glioma and / or prolonging the survival of glioma patients.

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