Application of C620-0580 in the preparation of anti-glioma drugs

Abstract

The present invention relates to a kind of application of small molecule compound C620‑0580 in the preparation of antitumor drugs and antitumor drugs, and belongs to the technical field of antitumor drugs. The present invention screens C620‑0580, and has a killing effect on various glioblastoma cell lines. The IC50 test result shows that the concentration of compound C620‑0580 is between 20μM‑60μM and has a significant inhibitory effect on glioblastoma. In vitro experiments have confirmed that small molecule compound C620‑0580 can inhibit the effects such as glioblastoma proliferation, invasion, migration and clone formation, and in vivo experiments have also illustrated that compound C620‑0580 can inhibit tumor growth in glioblastoma, significantly prolonging the survival period of mice. It can be used in the preparation of antitumor drugs, and new options are provided for antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs and relates to the application of a small molecule compound C620-0580 in the preparation of anti-glioma drugs. Background Art

[0002] Gliomas are common central nervous system tumors classified into four grades based on their malignancy, with glioblastoma being the most malignant. Clinical treatment for gliomas primarily relies on surgery combined with chemotherapy and radiotherapy. To date, temozolomide is the only first-line chemotherapy drug for gliomas. Gliomas are highly invasive, with poorly defined borders, prone to recurrence after surgical resection, and face challenges with temozolomide resistance. In 2018, electric field therapy was added to the guidelines. Despite combined treatment with surgery, radiotherapy, and systemic therapies (including chemotherapy and targeted therapies), the overall prognosis for glioblastoma remains poor, with low long-term survival rates. In recent years, molecularly targeted therapies and immunotherapies for tumors have become hot topics in clinical research. While targeted therapies for tumors have yielded significant results, with many targeted drugs for other cancers demonstrating promising clinical efficacy, the only targeted drug for gliomas currently available is the anti-angiogenic drug bevacizumab, which remains a second-line treatment option with minimal benefit. Progress in targeted therapies for gliomas remains slow. Therefore, there is an urgent need to research and develop new targeted therapeutic drugs for gliomas. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a small molecule targeted drug for nuclear transport protein that can target and treat glioblastoma.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] 1. Application of KPNA2 protein inhibitors in the preparation of anti-tumor drugs.

[0006] Furthermore, in the use of the KPNA2 protein inhibitor in the preparation of an anti-tumor drug, the tumor is a glioma.

[0007] Furthermore, in the use of a KPNA2 protein inhibitor in the preparation of an anti-tumor drug, the KPNA2 protein inhibitor is C620-0580.

[0008] Furthermore, the drug usage concentration of C620-0580 is at least 5 mg / Kg.

[0009] Furthermore, in the application of the KPNA2 protein inhibitor in the preparation of anti-tumor drugs, the anti-tumor drug has one of the following functions:

[0010] (1) Inhibit tumor cell proliferation;

[0011] (2) inhibiting tumor cell migration and invasion;

[0012] (3) Inhibit tumor growth.

[0013] 2. Application of C620-0580 in the preparation of reagents for screening anti-tumor drugs.

[0014] Furthermore, the type of the tumor is glioma.

[0015] 3. An anti-tumor drug comprising C620-0580 and pharmaceutically acceptable excipients.

[0016] Furthermore, the anti-tumor drug, the type of tumor is glioma.

[0017] The beneficial effects of the present invention are as follows: C620-0580 was screened by the present invention and had a killing effect on various glioblastoma cell lines. The IC50 test results showed that the compound C620-0580 had a significant inhibitory effect on glioblastoma cells at a concentration between 20 μM and 60 μM. In vitro experiments confirmed that the small molecule compound C620-0580 can inhibit the proliferation, invasion, migration and clone formation of glioblastoma. In vivo experiments also showed that the compound C620-0580 can inhibit the growth of glioblastoma tumors in vivo and significantly prolong the survival of mice (P=0.0005). In addition, toxicity tests showed that the compound C620-0580 had low toxicity, and the effective dose had no significant effect on the heart, liver, spleen, lung, kidney, and brain organs of mice. This fully demonstrates that the small molecule compound C620-0580 can be used to prepare anti-tumor drugs, especially anti-glioblastoma drugs, and can become a potential therapeutic drug for glioma patients, providing a new and effective treatment option for targeted glioma therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0019] Figure 1 This is a flow chart of in vitro and in vivo experiments on the small molecule compound C620-0580 for the treatment of glioblastoma in the present invention.

[0020] Figure 2 This is a schematic diagram of sample addition in Example 2.

[0021] Figure 3 This is the SDS-PAGE detection diagram of KPNA2 protein after purification.

[0022] Figure 4 Fitting curve for the affinity experiment between C620-0580 and KPNA2.

[0023] Figure 5These are the results of in vitro cytotoxicity experiments of C620-0580.

[0024] Figure 6 This is a diagram showing the invasion and migration of glioblastoma cells LN229 in transwell chambers after treatment with C620-0580.

[0025] Figure 7 This is a diagram showing the invasion and migration of the primary glioblastoma cell line GBM1 in the transwell chamber after treatment with C620-0580.

[0026] Figure 8 This is a diagram of clone formation after LN229 and GBM1 were treated with C620-0580.

[0027] Figure 9 The effect of intraperitoneal administration of C620-0580 at different concentrations and the DMSO control group for 20 consecutive days on the body weight of BALB / c mice.

[0028] Figure 10 The gross effects of intraperitoneal administration of different concentrations of C620-0580 and the DMSO control group on various organs of BALB / c mice.

[0029] Figure 11 The effects of intraperitoneal administration of different concentrations of C620-0580 and DMSO control group on HE staining of various organs in BALB / c mice.

[0030] Figure 12 These are in vivo fluorescence imaging images of NOD-SCID orthotopic tumor-bearing mice in the C620-0580 treatment group and the DMSO control group.

[0031] Figure 13 This is a comparison of the in vivo fluorescence values ​​of NOD-SCID orthotopic tumor-bearing mice in the C620-0580 treatment group and the DMSO control group.

[0032] Figure 14 This is the effect of C620-0580 treatment group and DMSO control group on the survival of NOD-SCID orthotopic tumor-bearing mice.

[0033] Figure 15 Brain sections and Ki67 immunohistochemical staining of tumor-bearing mice in the C620-0580-treated group and the DMSO control group.

[0034] Figure 16 The IC50 test results of C620-0580 in various glioblastoma cell lines are shown in Figure 5.

[0035] Figure 17 In vitro cytotoxicity test results for the screened compounds.

[0036] Figure 18 This is the WB result of nuclear screening for the targeted drug C620-0580.

[0037] Figure 19 This is the NMR spectrum of C620-0580. DETAILED DESCRIPTION

[0038] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Experimental methods without specific conditions in the examples are generally carried out under conventional conditions or conditions recommended by the manufacturer.

[0039] Figure 1 This is a flow chart for the in vitro and in vivo experimental studies of the small molecule compound C620-0580 for the treatment of glioblastoma. The specific experiments first tested the IC50 value of the small molecule compound in glioblastoma cell lines in vitro, examining the cell killing effect of C620-0580 on glioblastoma cell lines, as well as cell proliferation, invasion, and migration. Secondly, in vivo experiments were conducted, establishing an orthotopic glioblastoma xenograft model. Seven days after tumor implantation, the cells were intraperitoneally administered at a dose of 5 mg / kg daily. A control group received intraperitoneal administration of the same solvent as the experimental group. In vivo fluorescence imaging was performed weekly following treatment to measure tumor size and record survival. Glioblastoma multiforme (GBM) is the most common form of primary malignant brain tumor in adults and the most lethal cancer of the central nervous system. Despite progress in understanding the molecular mechanisms of tumorigenesis, most current treatments remain ineffective, including targeted therapies such as EGFR- and VEGF-targeted drugs. Human GBM cell lines: U87 and T98G; GBM primary cells isolated from fresh tumor tissue: GBM1 and GBM2: GBM1 and U87 are MGMT-deficient GBM cells; GBM2 and T98G are MGMT-positive GBM cells.

[0040] In vitro cell IC50 assay of small molecule compound C620-0580

[0041] 1. Wash the glioblastoma cell lines LN229, T98G, U87, GBM1, GBM2, and GBM3 twice with PBS, discard the PBS, add 1 ml of trypsin for digestion, blow off the cells to form a suspension and transfer to a 15 ml centrifuge tube. Centrifuge at 800 rpm for 5 minutes, then resuspend in DMEM complete medium and count.

[0042] 2. Take the cell suspension and add DMEM complete medium to a 96-well plate, inoculating 1000 cells / 100 μl per well. Incubate at 37°C in a 5% CO2 incubator.

[0043] 3. After the cells have attached, discard the supernatant and add DMEM complete medium containing different concentrations of C620-0580. Continue culturing in the incubator.

[0044] 4. After 72 hours of drug treatment, discard the drug-containing culture medium and add DMEM with 10% CCK8 concentration. Continue incubation at 37°C for 2 hours, measure the OD value at a wavelength of 450nm, and calculate the drug IC50.

[0045] Figure 16 The IC50 values ​​of C620-0580 in various glioblastoma cell lines are shown. The small molecule compound has a killing effect on various glioblastoma cell lines, with IC50 values ​​ranging from 20μM to 60μM. The results show that compound C620-0580 has a significant inhibitory effect on glioblastoma cells at concentrations between 20μM and 60μM.

[0046] The present invention targets the nucleocytoplasmic transporter protein that is specifically and highly expressed in gliomas. 35 compounds were screened from the drug-like compound library chemdivV2017 (specs~1469931) and further tested for in vitro cytotoxicity (such as Figure 17 As shown), small molecule compounds were used to inhibit the cargo protein transported by KPNA2 into the nucleus to screen the targeted drug C620-0580 (as shown Figure 18 shown).

[0047] The present invention first demonstrated in vitro that the small molecule compound C620-0580 can inhibit glioblastoma proliferation, invasion, migration, and cloning. Further in vivo in situ tumor model construction and intraperitoneal administration experiments also demonstrated that compound C620-0580 can inhibit glioblastoma tumor growth in vivo and significantly prolong mouse survival. Compound C620-0580 also has a strong affinity for the KPNA2 protein, making KPNA2 an effective target for C620-0580. Toxicity tests demonstrated that compound C620-0580 has low toxicity, with an effective dose having no significant effects on the heart, liver, spleen, lungs, kidneys, and brain of mice. It could be a potential therapeutic drug for patients with gliomas that overexpress KPNA2, providing a new treatment option for targeted glioma therapy.

[0048] The experimental process should be:

[0049] 1. Compound C620-0580 was screened;

[0050] 2. Purify KPNA2 protein and perform affinity testing with the compound, confirming that the compound has strong affinity for KPNA2 protein;

[0051] 3. IC50 test of the compound in different cell lines;

[0052] 4. Compounds kill glioblastoma cell lines, in vitro cytotoxicity experiments, invasion and migration experiments, etc.

[0053] 5. In vivo animal experiments: acute toxicity test - long-term toxicity test - drug treatment experiment after intracranial tumor formation.

[0054] Example 1 KPNA2 protein purification:

[0055] 1. Take 100 μl of Rosstta BL21 competent cells (Kamed Biotech, China), thaw on ice, take a 1.5 ml sterile centrifuge tube in the clean bench, and divide the competent cells into 50 μl.

[0056] 2. Add 20 μl of KPNA2 plasmid (Tianjin KAMD Biotechnology Co., Ltd., ABX12606-2 COA) to the competent cells in a clean bench, mix gently, and incubate on ice for 30 minutes.

[0057] 3. Heat shock at 42°C for 90 seconds and immediately place on ice for 5 minutes. Take 30 μl and spread on LB (containing 50 μg / ml kanamycin or ampicillin) plate. Incubate at 37°C for 14 hours (overnight culture) and pick out single colonies for activation culture.

[0058] 4. Pick a single clone from the LB plate, add 2 ml LB liquid medium (containing 50 μg / ml kanamycin) to activate the bacteria until the OD600 is 0.6-0.8, then transfer to 5 ml LB liquid medium (containing 50 μg / ml kanamycin) and culture until the OD600 of the bacteria is 0.6-0.8. Then add 1 mM IPTG to induce expression at 37°C for 4 hours, and then induction at 16°C for 30 hours, and then collect the bacteria.

[0059] 5. Take the bacteria before and after induction, perform ultrasonic disruption, and take the supernatant and precipitate to prepare samples for SDS-PAGE analysis.

[0060] 6. Scale up expression: Select the optimal clone from the expression identification in step 4, culture it in 300 ml at 37°C, add 150 μl of IPTG and induce at 37°C for 4 hours. Collect the supernatant. Centrifuge, filter the supernatant through a 0.22 μg filter, and then perform Ni affinity enrichment and purification to obtain purified KPNA2 protein. Figure 3 This is the SDS-PAGE detection diagram after KPNA2 protein purification, from left to right: M: Marker; Lane 1: flow-through; Lanes 2-7: elution products at different imidazole concentrations.

[0061] Example 2

[0062] Affinity test of small molecule compound C620-0580 and KPNA2 protein

[0063] Detection instrument: Octet RE96E, fortebio, NTA (manufacturer: Fortebio, catalog number: 18-5101), which can specifically capture HIS-tagged proteins for kinetic detection.

[0064] 1. Protein dilution: Dilute KPNA2 protein to 20 μg / mL with PBS for subsequent solidification.

[0065] 2. Compound dilution: Take the purchased C620-0580 powder (chemdiv, Inc. Shanghai Taosu Biochemical Co., Ltd., the molecular structure and QC test results are as follows Figure 19 The 50 mM C620-0580 compound was diluted 100-fold with PBST as the highest concentration, and then serially diluted 2-fold with 1% DMSO+PBST for a total of 6 concentrations for subsequent analysis.

[0066] C620-0580, molecular formula: C 23 H 26 N4O4, structural formula:

[0067] 3. NTA solidified protein detection compound: Mix the above samples and reagents according to Figure 2 The sample plate was added in the order shown, wherein 200 μL of 1% DMSO + PBST was added to wells C1-C6 and D1-D12; 200 μL of different concentrations of C620-0580 were added to wells C7-C12, with the concentrations being 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM, respectively. The program was set and signal detection was performed. The detection results are shown in FIG. Figure 4 As shown in the figure, the SSG R^2 values ​​were all above 0.99. The results showed that C620-0580 at different concentrations had strong affinity with KPNA2. Figure 4 Fitting curve for affinity experiment between purified protein and compound C620-0580.

[0068] Example 3

[0069] In vitro cytotoxicity study of small molecule compound C620-0580

[0070] 1. Wash LN229 and GBM1 cells twice with PBS, discard the PBS, add 1 ml of trypsin for digestion, blow off the cells into a suspension and transfer to a 15 ml centrifuge tube. Centrifuge at 800 rpm for 5 minutes, resuspend in DMEM complete medium, and count.

[0071] 2. Take the cell suspension and add DMEM complete medium to a 96-well plate, seeding 1000 cells / 100 μl. Incubate at 37°C in a 5% CO2 incubator.

[0072] 3. After the cells have attached, discard the supernatant and add DMEM complete medium containing 20 μM C620-0580. Continue culturing in the incubator.

[0073] 4. After drug treatment for 3, 6, 12, 24, 48, 72, and 92 hours, the drug-containing culture medium was discarded, and DMEM with 10% CCK8 was added. The cells were incubated at 37°C for 2 hours, and the OD value was measured at a wavelength of 450 nm.

[0074] The experimental results are as follows Figure 5 As shown by Figure 5 It can be seen that the inhibitory effect of C620-0580 treatment on the glioblastoma cell line LN229 and the primary glioblastoma cell line GBM1 increases with time.

[0075] Transwell invasion and migration assay

[0076] 1. Melt the Matrigel stored at -20℃ at 4℃, prepare a working solution with serum-free DMED at a ratio of 1:1, and store at 4℃ for later use.

[0077] 2. Invasion gel: Take 20 μl of Matrigel working solution and evenly spread it in the Transwell chamber, place the chamber in a 24-well plate, and dry it in a 37°C incubator;

[0078] 3. Wash glioblastoma cells LN229 and GBM1 twice with warm PBS, add 1 ml of Accutase enzyme for digestion, blow off the cells to form a suspension and transfer to a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 minutes, resuspend in serum-free DMEM, and count.

[0079] 4. Take 400 μl of DMED containing 10% FBS and add it to a new 24-well plate.

[0080] 5. Prepare a 40,000 cell suspension in 200 μl of counted cells and add it to the upper layer of the Transwell chamber (with gel for invasion and no gel for migration). Then gently place the chamber into the culture medium in step 4 to avoid bubbles at the bottom.

[0081] 6. After 24 hours of invasion and 16 hours of migration in a 37°C incubator, remove the chamber and wash three times with PBS.

[0082] 7. Fix with 4% paraformaldehyde solution at room temperature for 15 minutes and wash three times with PBS.

[0083] Crystal violet staining was performed for 20 minutes, and cells were gently rinsed with running water. The cells on the upper layer of the chamber were gently wiped clean with a cotton swab. The cells were photographed under a microscope and counted. Figure 6 and Figure 7 As shown, Figure 8 This is a diagram of the clone formation experiment.

[0084] Figure 6-8 Figures show the inhibitory effect of C620-0580 treatment on the glioblastoma cell line LN229 and the primary glioblastoma cell line GBM1, as well as transwell assays of glioma cell invasion, migration, and colony formation in the treated and DMSO control groups. The results demonstrate that C620-0580 significantly inhibits glioblastoma proliferation, which increases over time, and also inhibits glioblastoma cell invasion, migration, and colony formation in vitro.

[0085] Example 4

[0086] C620-0580 acute toxicity test

[0087] 1. Dosage setting and grouping: solvent control group, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, a total of nine groups, ten animals in each group.

[0088] 2. Intraperitoneal administration, 10 ml / kg, single dose.

[0089] 3. The solvent control group was injected with 10% dimethylacetamide at a volume of 10 ml / kg. The mice in the drug-treated groups were observed as follows: The drug was administered by intraperitoneal injection. The mental state, activity, diet, water intake, and urination and defecation of each group of mice were recorded daily after administration. The observation period was 14 days. The number of deaths in each group was counted, and the median lethal dose (LD50) of mice was calculated using the Bliss method. LD50 = 414.7 ± 70.2 (342.9 ~ 483.3) mg / kg.

[0090] Table 1 Death of mice after intraperitoneal injection of different doses of C620-0580 (n=10)

[0091]

[0092] Table 2 Calculation results of median lethal dose LD50 (Bliss method)

[0093]

[0094] Example 5

[0095] C620-0580 Low-dose, long-term toxicity test

[0096] 1. According to the LD50 of the acute toxicity test, set a dosage concentration gradient of less than 1 / 10 and administer the drug continuously every day.

[0097] 2. Record the weight, mental state, etc. of the mice ( Figure 9 ).

[0098] 3. After 20 days, the heart, liver, spleen, lung, kidney and brain of the mice were taken out to observe the gross morphological changes of each organ ( Figure 10 ).

[0099] 4. HE staining of paraffin sections of each organ, and comparison of the differences between the drug-treated group and the control group ( Figure 11 ).

[0100] HE staining.

[0101] 1. Dewaxing: Bake the sample under a baking lamp for 30 minutes, and dewax in the order of xylene I (15 minutes) → xylene II (15 minutes);

[0102] 2. Hydration: Absolute alcohol I (10 min) → Absolute alcohol II (10 min) → 95% alcohol (5 min) → 85% alcohol (5 min) → 75% alcohol (5 min) in this order. Rinse the slides under tap water for 3 min and then wash the sections with ultrapure water 3 times, 5 min each time.

[0103] 3. Nuclear staining: Place the slide in hematoxylin solution for 20 seconds, then rinse with tap water. Place it in hydrochloric acid alcohol for 2 seconds, then rinse with tap water for 10 minutes to reverse blue.

[0104] 4. Stain with 0.5% eosin solution for 20 seconds; / 5. Dehydration and sealing: Dehydrate and transparentize the slides in the following order: 75% alcohol (3 minutes) → 85% alcohol (3 minutes) → 95% alcohol (3 minutes) → 100% alcohol (5 minutes) → 100% alcohol (5 minutes) → xylene I (20 minutes) → xylene II (20 minutes). Place in a ventilated place overnight and seal with neutral resin and take pictures ( Figure 11 ).

[0105] Figure 9 The results show that intraperitoneal administration of C620-0580 at different concentrations and a DMSO control group for 20 consecutive days had no significant effect on the body weight of BALB / c mice. Figure 10 The gross effects of intraperitoneal administration of different concentrations of C620-0580 and the DMSO control group on various organs of BALB / c mice. Figure 11The effects of intraperitoneal administration of different concentrations of C620-0580 and DMSO control group on HE staining of various organs in BALB / c mice. Figures 9-11 The results show that the C620-0580 group had no significant effect on the organs of mice at concentrations of 20 mg / kg and below.

[0106] Example 6

[0107] Prepare C620-0580 solution for intraperitoneal administration in mice.

[0108] Prepare a 20 mg / ml stock solution of purchased C620-0580 powder in DMSO. Add 240 μl of polyethylene glycol (PEG300) to 40 μl of the stock solution, mix thoroughly, and once clarified, add 80 μl of Tween 80. Once clarified, add 440 μl of distilled water to achieve a 10 mg / kg dosing concentration. For a 5 mg / kg dosing concentration, dilute accordingly.

[0109] Orthotopic transplant tumor model construction and C620-0580 in vivo administration:

[0110] 1. Glioblastoma cell line LN229 was transfected with luciferase virus.

[0111] 2. Collect LN229 cells and seed 2×10 cells per NOD-SCID mouse. 5 pcs / 5μl.

[0112] 3. Purchase 4-6 week old female NOD / SCID mice (purchased from Jicui Yaokang Biological Company), anesthetize the mice with sodium pentobarbital anesthetic, and take 5 μl of LN229 cell suspension using a microinjector.

[0113] 4. LN229 cells were injected into the right cerebral hemisphere, 4 mm posterior to the intersection of the anterior midline of the mouse brain and the line connecting the lateral canthus, and 3 mm to the right. The needle was advanced 4 mm. Slowly insert the needle and slowly inject the cells. After the cells are injected, wait 30 seconds before slowly withdrawing the needle.

[0114] 5. Seven days after tumor implantation, the drug was administered intraperitoneally at a dose of 5 mg / kg daily. The control group (same DMSO concentration as the experimental group) was administered intraperitoneally with the same solvent as the experimental group at a dose of 5 mg / kg daily. The control group was administered intraperitoneally with the same solvent as the experimental group. In situ in vivo fluorescence imaging was performed to detect the size of the transplanted tumors on day 0, seven days after tumor implantation, and weekly after treatment, and survival was recorded. Brain sections of tumor-bearing mice in the C620-0580-treated group and the DMSO control group were sliced ​​and Ki67 immunohistochemically stained.

[0115] In situ intravital fluorescence imaging to detect transplanted tumor size:

[0116] After the intracranial transplanted tumors were established, the tumor formation of mice was detected by live imaging. The mice were randomly divided into control group and treatment group and the drug was administered for 7 days, 14 days, and 21 days. Each mouse was intraperitoneally injected with 200uL of Luciferin working solution each time. The tumor formation of the experimental group and the control group mice was then observed using IVIS live animal imaging. Figure 12 ), statistical fluorescence intensity ( Figure 13 ).

[0117] Survival statistics

[0118] The death time of tumor-bearing mice in the drug-treated group and the control group was recorded, and the survival time of the two groups was counted and calculated ( Figure 14 The results showed that C620-0580 could prolong the survival of tumor-bearing mice.

[0119] Immunohistochemical staining.

[0120] 1. Take the brains of mice in the control group and the treatment group, fix them in formalin for 1 day, embed them in wax blocks and perform paraffin sections.

[0121] 2. Dewaxing and hydration of paraffin sections: bake in a 65-degree oven for 30 minutes, dewax in the order of xylene I (15 minutes) → xylene II (15 minutes) → anhydrous ethanol I (10 minutes) → anhydrous ethanol II (10 minutes) → 95% ethanol (5 minutes) → 85% ethanol (5 minutes) → 75% ethanol (5 minutes), and wash the sections in distilled water three times, 5 minutes each time;

[0122] 3. Antigen repair: Add citric acid repair solution to the pressure cooker on the induction cooker for acid repair. Place the slide upside down until it is completely submerged in the repair solution. After the pressure is increased, continue heating for 2.5 minutes before stopping the repair and let it cool to room temperature.

[0123] 4. Block endogenous peroxidase: Use freshly prepared 2% H2O2 blocking solution, add it to the repair box so that it covers the slides, and then block at 37°C for 30 minutes. Then wash with PBS three times, each time for 5 minutes;

[0124] 5. Blocking: Shake off the moisture around the slices, circle the tissue sample with a brush, add ready-to-use goat blocking serum and block at 37°C for 30 minutes;

[0125] 6. Primary antibody incubation: Shake off the serum on the slides, add Ki67 primary antibody (#9449, CST, USA) on the surface of the sections, dilute the primary antibody at a ratio of 1:1000 with antibody diluent, and incubate in a humidified chamber at 4°C overnight;

[0126] 7. Incubation with secondary antibody: Wash with PBS 4 times, 5 min each time, adding Tween-20 for the last wash; add universal secondary antibody DAKO (DAKO EnVision FLEX, High pH, ​​USA) on the surface of the slice and incubate in a humidified chamber at 37°C for 30 min;

[0127] 8. Color development: Mix DAB color development solution A and B in a ratio of 1:50, add DAB working solution to the glass slide, 40uL per slide, and rinse with tap water after color development is completed;

[0128] 9. Nuclear staining: Place the slide in hematoxylin solution for 2 minutes, then rinse with tap water, place in hydrochloric acid alcohol for 2 seconds, then rinse with tap water for anti-blueing;

[0129] 10. Dehydration and sealing: Dehydrate and transparentize the slides in the following order: 75% alcohol (3 min) → 85% alcohol (3 min) → 95% alcohol (3 min) → 100% alcohol (5 min) → 100% alcohol (5 min) → xylene I (20 min) → xylene II (20 min). Place in a ventilated place overnight and seal with neutral resin.

[0130] Figure 12 In vivo fluorescence imaging of NOD-SCID orthotopic tumor-bearing mice in the C620-0580-treated group and the DMSO control group showed that C620-0580 significantly inhibited the growth of orthotopic glioblastoma xenografts.

[0131] Figure 13 Comparison of in vivo fluorescence values ​​of NOD-SCID orthotopic tumor-bearing mice between the C620-0580-treated group and the DMSO control group. Figure 12 and Figure 13 The results showed that C620-0580 could significantly inhibit the growth of glioblastoma orthotopic transplanted tumors.

[0132] Figure 14 The results show that C620-0580 can prolong the survival of tumor-bearing mice.

[0133] Figure 15 Brain sections of tumor-bearing mice were sliced ​​and Ki67 immunohistochemically stained for C620-0580-treated and DMSO-controlled groups. The results showed that C620-0580 significantly inhibited tumor size and reduced Ki67 expression in glioblastoma cells, thereby inhibiting glioblastoma proliferation.

[0134] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Use of C620-0580 as an inhibitor of KPNA2 protein in the preparation of an anti-glioblastoma drug, wherein the structural formula of C620-0580 is: 。 2. The use according to claim 1, characterized in that The drug concentration of C620-0580 is at least 5 mg / Kg.

3. The use according to claim 1 or 2, characterized in that The anti-glioblastoma cell has one of the following functions: (1) Inhibit the proliferation of glioblastoma cells; (2) inhibiting glioblastoma cell migration and invasion; (3) Inhibit the growth of glioblastoma.

Images