Application of nevermectin in treatment of malignant tumors

Abstract

The invention provides an application of nevermectin in tumor treatment. According to the present invention, the glucosidase II catalytic subunit GANAB can be subjected to targeted inhibition with the affinity constant KD of 0.312 [mu] M, the effect of the 3-site methoxyl demethylation product Norhistidine of the nehistidine on the GANAB is weak, and the affinity constant KD of the Norhistidine is 97.59 [mu] M; neurotetrastin can inhibit GANAB to induce endoplasmic reticulum stress and unfolded protein reaction of tumor stem cells, promote differentiation of the tumor stem cells, induce death of the tumor stem cells and play a role in inhibiting malignant tumors. In a glioblastoma stem cell model, the cytotoxic activity IC50 of the nevermectin is 7.20 + / -2.10 [mu] M, and a mouse in-situ brain tumor formed by glioblastoma stem cells can be significantly reduced after intratumoral injection, indicating that the nevermectin has good anti-tumor stem cell activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of nidulans in treating malignant tumors. Background Art

[0002] The latest data from the International Agency for Research on Cancer (IARC) indicates that between 2000 and 2018, China's standardized mortality rate for all cancer types decreased by an average of 1.3% per year, while its standardized incidence rate for all cancer types increased by an average of 1.4% per year. In 2022, China ranked first in the world for both new cancer cases and deaths, and cancer remains a major public health issue in China. Currently, cancer treatment primarily encompasses traditional surgery, radiotherapy, chemotherapy, and rapidly developing newer treatment modalities such as targeted therapy and immunotherapy. However, these treatments are often ineffective against advanced malignancies that have recurred, metastasized, or become drug-resistant. The development of drugs to treat these difficult-to-treat malignancies is urgently needed. Cancer stem cells are a cell population within tumors with the potential for self-renewal and multidirectional differentiation. They can initiate and reshape the tumor tissue phenotype and serve as the "seed" cells for tumor initiation, invasion, metastasis, resistance to radiotherapy and chemotherapy, and recurrence. Currently, there are still no targeted therapeutic drugs for cancer stem cells. Although some preclinical studies have shown that drugs targeting stem cell-related signaling pathways such as Notch, Hedgehog, Wnt / β-catenin, etc. have great potential in combating malignant tumors, no related drugs have yet been marketed.

[0003] Therefore, it is urgent to find new therapeutic targets for tumor stem cells and develop new targeted therapeutic drugs. Summary of the Invention

[0004] In view of this, the present invention discloses the use of nidulans in the treatment of malignant tumors, which can inhibit the stemness of tumor stem cells by disrupting their glycosylation modification, thereby providing a promising treatment method for the treatment of targeted tumor stem cells.

[0005] The technical solution provided by the present invention is the use of nidulans in treating tumors. The compound nidulans induces endoplasmic reticulum stress and unfolded protein response in tumor stem cells by targeted inhibition of glucosidase II α subunit GANAB, promotes tumor stem cell differentiation, and induces tumor stem cell death to exert its tumor-suppressing effect.

[0006] Preferably, by inhibiting GANAB, tumor stem cells that highly express GANAB are selectively induced to differentiate and their stemness is reduced.

[0007] The present invention discloses that nidulin can inhibit the catalytic subunit (α-subunit) of glucosidase II, GANAB, by inducing endoplasmic reticulum stress and the unfolded protein response, thereby promoting tumor stem cell differentiation and inducing tumor stem cell death, thereby exerting a tumor-suppressing effect. The present invention discloses that the glucosidase II catalytic subunit, GANAB, is a potential therapeutic target for tumors and that inhibiting GANAB has the effect of inhibiting tumor stem cells.

[0008] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0011] Figure 1 The cytotoxic activities of the compounds provided by the present invention on GSC0617 and LN229 cells, respectively; (A) Relative inhibition rate of 72 compounds treated with GSC0617 cells at a concentration of 10 μM; (B) Relative inhibition rate of 72 compounds treated with LN229 cells at a concentration of 10 μM;

[0012] Figure 2 The cytotoxic activity of nidulan provided by the present invention; (A) chemical structure of nidulan; (B) cytotoxic activity of nidulan;

[0013] Figure 3 The limiting dilution analysis provided by the present invention shows the changes in the frequency of GSC0617 stem cells after nidulans treatment;

[0014] Figure 4 The DARTS analysis provided by the present invention explores the target of nidulans;

[0015] Figure 5 The CETSA analysis provided by the present invention confirms the target of nidulans;

[0016] Figure 6 The SPR analysis provided by the present invention verifies the binding of nidulans and nidulans to GANAB;

[0017] Figure 7The molecular docking results provided by the present invention show the binding of nidulansin to the key amino acids of GANAB;

[0018] Figure 8 The present invention provides the correlation between the expression of GANAB, PRKCSH, and MOGS in tumor tissues of glioma patients and patient prognosis; (A) Expression of GANAB, PRKCSH, and MOGS in various tumors; (B) Expression of GANAB, PRKCSH, and MOGS in tumor tissues of LGG and GBM patients; (C) Correlation between the mRNA expression of GANAB, PRKCSH, and MOGS; (D) Overall survival and progression-free survival of glioma patients with different expression levels of GANAB;

[0019] Figure 9 Sensitivity of tumor cells and stem cells enriched with nidulans to nidulans provided by the present invention; (A) Expression of GANAB in LN229 and third-generation tumor sphere cells; (B) IC of nidulans on LN229 and third-generation tumor sphere cells 50 ; (C) GANAB expression in MDA-MB-231 and third-generation tumor spheroid cells; (D) IC of nidulans against MDA-MB-231 and third-generation tumor spheroid cells 50 ; (E) Survival rates of HepG2, TE-1, A549 and corresponding second-generation tumor sphere cells under the action of 30 μM nidulans.

[0020] Figure 10 The glioblastoma stem cells provided by the present invention are sensitive to nidulans;

[0021] Figure 11 The present invention provides the morphological changes of glioblastoma stem cells and the expression of stemness-related proteins after knocking down GANAB;

[0022] Figure 12 The RT-PCR provided by the present invention detects the shearing of XBP-1 after nidulans treatment;

[0023] Figure 13 The expression of CHOP and BIP / GRP78 was detected by RT-qPCR provided by the present invention;

[0024] Figure 14 Immunofluorescence detection of glucose and mannose modification levels in glycoproteins provided by the present invention;

[0025] Figure 15 The activity of nidulans provided by the present invention in the glioblastoma stem cell model; (A) IC of nidulans on GSC0617-Luc cells used in the animal model 50; DOXO: doxorubicin, here is the positive control drug; (B) In vivo imaging (IVIS) results of tumor-bearing nude mice; (C) Body weight changes of tumor-bearing nude mice. DETAILED DESCRIPTION

[0026] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. In the following description, when referring to the drawings, like numbers in different figures represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all possible embodiments consistent with the present invention. Rather, they are merely examples of systems consistent with certain aspects of the present invention, as detailed in the appended claims.

[0027] The reagents and consumables mainly used in this embodiment are shown in Table 1.

[0028] Table 1 Reagents and consumables mainly used in this implementation plan

[0029] Reagents and consumables factory Ultra-low attachment 96-well plate Corning Incorporated Phosphate Buffered Saline (PBS) Beijing Solebow Technology Co., Ltd. pancreatic enzymes Thermo Fisher Scientific Accutase digestive solution Thermo Fisher Scientific Gibco GANAB Polyclonal antibody Wuhan Sanying Biotechnology Co., Ltd. β-actin Monoclonal antibody Wuhan Sanying Biotechnology Co., Ltd. Goat anti-rabbit antibody Abcam Goat anti-mouse antibody Abcam Protein molecular weight standards Thermo Fisher Scientific ECL luminescent liquid Sigma-Aldrich Trading Co., Ltd. Recombinant human active GANAB protein Beijing Aorui Dongyuan Biotechnology Co., Ltd. CM5 chip Cytiva 10×PBS-P+ Cytiva PD MiniTrap G-25 Column Cytiva 1.5 ml EP tube without cap Cytiva Rapid Fluorescence Quantitative PCR Premix Reagent Tiangen Biochemical Technology (Beijing) Co., Ltd. ReverTra Ace® qPCR RT Master Mix with gDNA Remover Kit Toyobo (Shanghai) Biotechnology Co., Ltd. D-Luciferin MCE

[0030] The cell lines used in the examples: human glioblastoma LN229 cells and human glioma Hs683 cells were purchased from the American Type Culture Collection (ATCC). Human hepatocellular carcinoma HepG2 cells, human esophageal carcinoma TE-1 cells, human non-small cell lung cancer A549 cells, human glioblastoma U-87 MG cells, human glioblastoma U-251 MG cells, mouse glioma GL261 cells, human immortalized keratinocytes HaCaT cells, human normal lung epithelial cells BEAS-2B, human triple-negative breast cancer cells MDA-MB-231, and human embryonic kidney HEK-293T cells were purchased from the China Center for Type Culture Collection (CCTCC). The first, second and third generation LN229, MDA-MB-231, HepG2, TE-1, and A549 tumor sphere cells LN229-T1, LN229-T2, LN229-T3, MDA-MB-231-T1, MDA-MB-231-T2, MDA-MB-231-T3, HepG2-T1, HepG2-T2, HepG2-T3, TE-1-T1, TE-1-T2, TE-1-T3, A549-T1, A549-T2, and A549-T3 were constructed and preserved by our research group through tumor sphere formation experiments in the early stage. Glioblastoma stem cells GSC0617, GSC0722, GSC0624, GSC0307, ​​and GSC0218 were previously isolated from patient glioblastoma tissue by our research group and characterized in a paper by Professor Ju et al. GSC0617-Luc is a GSC0617 cell stably transfected with the plasmid pLVX-Luc-IRES-mCherry. Normal human astrocytes (NHA) were purchased from CyBio (Shanghai) Biotechnology Co., Ltd.

[0031] The preparation method of the reagents required in the embodiment of the present invention is as follows:

[0032] EGF and bFGF solutions: Dissolve 1 mg of recombinant human bFGF and recombinant human EGF in 10 mL of sterile PBS, respectively. Aliquot 100 μL / tube and freeze in a -80°C freezer.

[0033] DMEM complete medium: DMEM basic (1×) high glucose medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% non-essential amino acids is used as a culture medium for adherent cells such as LN229, Hs683, U-87 MG, U-251 MG, MDA-MB-231, HepG2, TE-1, A549, HEK-293T, BEAS-2B, and GL261.

[0034] DMEM-F12 complete medium: DMEM / F12 medium supplemented with B-27 supplement (50x), 20 ng / mL human bFGF and 20 ng / mL EGF as suspension cell culture medium.

[0035] Tumor sphere complete medium: DMEM / F12 medium supplemented with B-27 supplement, 20 ng / mL bFGF, 20 ng / mL EGF, 5 μg / mL insulin, 5 μg / mL hydrocortisone and 5 μg / mL heparin.

[0036] NHA medium: DMEM / F12 medium supplemented with B-27 supplement, 1% N-2 supplement, 1% penicillin-streptomycin, 20 ng / mL EGF and 20 ng / mL bFGF.

[0037] HaCaT medium: MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin.

[0038] EDTA solution: 3.1 g EDTA·2Na dihydrate was weighed, completely dissolved in 50 mL PBS and filtered using a 0.22 μm filter membrane. When used, it was diluted to a final concentration of 0.02% and added to 0.25% trypsin.

[0039] In order to enable a more thorough understanding of the present application, the present application will be further described in detail below in conjunction with specific examples.

[0040] Example 1

[0041] CCK-8 (Cell Counting Kit-8) was used to determine the cytotoxic activity of nestin on GSC0617, LN229, NHA and other cells: the principle of CCK-8 detection of cell viability is mainly based on the relationship between cell metabolic activity and cell number, and the level of cell viability is evaluated by measuring the reducing capacity of cells to CCK-8 reagent.

[0042] In this example, glioblastoma LN229 cells and glioblastoma stem cell GSC0617 cells were used to test the cytotoxic activity of 72 compound monomers isolated by the team:

[0043] (1) Cell plating: after the cells were terminated after digestion, 1 mL PBS was added and blown evenly, 20 μL was mixed with 20 μL 0.4% trypan blue, 10 μL was taken out and added to a cell counting plate, a cell counter was used for counting, the cells were diluted to 3000 cells / well, and were evenly added to the adherent or non-adherent 96-well plate, and then returned to the incubator for further culture.

[0044] (2) Drug addition: Add drugs 12-16 hours after plating. Dilute the compound to 100 μM using culture medium, mix thoroughly and dissolve, then transfer 10 μL per well to a 96-well plate to a final concentration of 10 μM for preliminary screening of compound cytotoxic activity. Simultaneously, perform a DMSO negative control group and a DOXO (doxorubicin) positive control group. If activity is observed at a concentration of 10 μM, further determine the half-inhibitory concentration (IC) of cell viability. 50 The compound was diluted to 1 mM using culture medium, and then diluted threefold to seven concentration gradients with the highest concentration as the highest concentration. The mixture was transferred to a 96-well plate, and 10 μL of the test compound was added to each well.

[0045] (3) CCK-8 measurement of cytotoxic activity: 10 μL of CCK-8 was added to each well 72 h after the addition of the compound. After incubation for 4 h, the absorbance at 450 nm and 600 nm (reference) was measured using a microplate reader.

[0046] (4) Calculation of preliminary screening activity of compounds:

[0047] Cell survival rate (%) =

[0048] Relative cell inhibition rate (%) =

[0049] Calculate the half-maximal inhibitory concentration of the compound: Use GraphPad Prism software to import data and calculate IC 50 Select the chart type as XY, set cell viability as Y and concentration as X, click Analyze-Transform-Transformconcentrations to convert the X-axis concentration to Log10, select the converted data table, click Analyze, select Nonliner regression (curve fit) for nonlinear fitting, select log (inhibitor) vs. normalized response -- Variable slope under Dose-response-Inhibition, and obtain IC in Nonlin fit of Transform X of Data. 50 .

[0050] Figure 1 The results showed that the inhibition rate of nidulans on GSC0617 at 10 μM was greater than 50%, and at the same concentration, it had no significant toxicity to glioblastoma LN229 cells (inhibition rate was less than 10%). Figure 2The results showed that nidulan had strong cytotoxic activity against glioblastoma stem cells, and had almost no cytotoxic activity against normal cells, suggesting that nidulan had the effect of selectively inhibiting glioblastoma stem cells.

[0051] Example 2

[0052] Nidulin reduces glioblastoma stem cell frequency:

[0053] The limiting dilution method was used to detect the sphere formation frequency of GSC0617 cells after the addition of the compound. The specific steps are as follows:

[0054] (1) Cell plating: GSC0617 cells were digested and counted, then diluted to 50, 25, 10, 5, and 1 cells per well, and plated onto ultra-low adsorption round-bottom 96-well plates to allow sphere formation. After stabilization, the wells with 1 cell per well were counted to confirm that each well contained only 1 cell.

[0055] (2) Drug treatment: On the second day, nidulans and negative control DMSO were added to the cells at final concentrations of 10 μM and 20 μM, respectively. Nidulans and DMSO were added every three days.

[0056] (3) Calculation of sphere formation frequency: After 14 days, each well was observed and the cell clusters appearing in the well were measured or counted. If there were cell spheres larger than 50 μm, spheres were considered to have formed. The frequency of cell sphere formation was calculated for different initial cell numbers. The data were analyzed using the ELDA analysis tool (https: / / bioinf.wehi.edu.au / software / elda / index.html). The effect of nidulans on the frequency of GSC0617 stem cells was analyzed by comparing the sphere formation frequencies of the DMSO group and the nidulans-treated group.

[0057] Figure 3 The results showed that after treatment with nidulin, the stem cell frequency in glioblastoma stem cells GSC0617 decreased from 1 / 11 to 1 / 100 and even failed to form spheres, indicating that nidulin significantly reduced the stem cell frequency of glioblastoma GSC0617, suggesting the selective anti-glioblastoma stem cell potential of nidulin.

[0058] Example 3

[0059] SPR experiments were used to detect the interaction between Nidulin and GANAB protein.

[0060] (1) Protein pretreatment

[0061] Calculate the protein isoelectric point, protein-to-small molecule molecular weight ratio, and estimated Rmax value. Desalt the protein. Remove the top cap of the PD MiniTrap G-25 column and pour out the solution inside. Remove the inner cap and fill the column with equilibration buffer (deionized water), allowing the equilibration buffer to completely enter the packed bed. Repeat twice, using a total of 8 mL of equilibration buffer. Discard the filtrate and add 200 μL of sample. If the sample volume is less than 0.5 mL, add equilibration buffer after the sample has completely entered the packed bed, adjust the sample volume to 0.5 mL, and discard the filtrate. Elute with 1.0 mL of buffer and collect the eluate containing the GANAB protein. Spin down the liquid to 100 μL using a freeze-drying centrifuge and store at 4°C until needed.

[0062] (2) Ligand coupling

[0063] Add 10% DMSO to the prepared 1.05×PBS-P+ solution. Be sure to prepare it immediately before use. Ultrasonication is required before use. Place it in the A solution position and check that the B solution is sufficient deionized water. Select Insert Chip-New Chip in the software, select the CM5 chip, and place the chip in the designated position. Hold the chip with the written side facing up. According to the direction of the arrow on the chip, slowly move the chip into the card slot and finally close the door of the chip compartment. Click Dock Chip. After the end, select Tools → Prime command. After clicking Start, the instrument will control the flow of running buffer to the entire chip. After the end, click Close and the instrument system will enter the standby state. Click Open / New wizard template under File in the software, select immobilization, and double-click. Select CM5 in Chip type and 1 in Flow cells per cycle. Select Flowcell 2, select "amine" for "method," enter the ligand name for "Ligand," select "specify contact time and flowrate" for high coupling, enter the contact time (in this experiment, enter 420 s), and typically set the flow rate to 10 μL / min. Click "Next" twice. Prepare the ligand protein GANAB by diluting it to 10 μg / mL using sodium acetate (pH 4.0). Click "Eject Rack," remove the sample rack, and place the prepared ligand protein GANAB and the amino coupling kit sample in the corresponding positions. Run the program and wait for the coupling results. For this experiment, the coupled protein amount was 15,000 RU.

[0064] (3) Sample preparation

[0065] Dilute the 5 mM small molecule stock solution 10-fold with 1.05× PBS-P to obtain a 500 μM concentration of the small molecule in 10% DMSO. Then, dilute the nidulans solution in 10% DMSO to 200 μM as the highest injection concentration using the prepared 10% DMSO running buffer. Continue diluting downward through at least five concentration steps. Always prepare samples immediately before use and load them onto the instrument immediately after preparation to prevent DMSO evaporation and inaccurate concentrations.

[0066] (4) On-machine testing

[0067] Set up the multi-cycle kinetic detection program, adjust the concentration unit and specific concentration value, set the dissociation parameters, etc., adjust the sample loading position, set the save parameters, place the sample in the designated position, and run the program.

[0068] (5) Data analysis

[0069] After the run is complete, open the Biacore S200 Evaluation Software and click "Open" in the "Data" folder to locate the file. First, click "Solvent Correction" to perform solvent correction analysis. The solvent correction curve is generally required to fall between -500 and +1000 RU, with a Chi² value less than 2 to indicate reliable values ​​and correct calibration. For samples with rapid dissociation and adsorption, perform affinity fitting. Select the appropriate concentration and click "Fit" in the upper left corner to display the fitted results.

[0070] like Figure 4 As shown in the figure, the target of nidulans was identified as glucosidase II α subunit GANAB by drug affinity reaction target stability experiment (DARTS), and the cell thermal shift experiment ( Figure 5 ) confirmed that nidulans can increase the thermal stability of GANAB;

[0071] like Figure 6 As shown in the figure, SPR experiments detected that nidulin can directly bind to GANAB with an affinity constant KD of 0.312 μM. As a control, the affinity constant KD of nidulin's 3-methoxy demethylation product, nornidulin, with GANAB is 97.59 μM, indicating that GANAB is the target of nidulin and that nidulin can directly bind to the GANAB protein. Figure 7 Molecular docking results showed that nidulans directly bound to the key amino acids of GANAB, aspartic acid at position 283, methionine at position 543, and histidine at position 678, demonstrating the key functional amino acids of nidulans.

[0072] Example 4

[0073] The expression level of GANAB protein in cells is positively correlated with the activity of nidulans. When the level of GANAB protein increases, the cytotoxic activity of nidulans on cells increases. 50 When the level of GANAB protein decreases, the cytotoxic activity of nidulans on cells increases. 50 Increased, activity decreased.

[0074] The expression level of GANAB in three consecutive generations of tumor sphere cells, including LN229 cells, enriched for stemness in tumor sphere formation experiments, was determined by immunoblotting. The cytotoxic activity of nidulans was also tested. The specific steps are as follows:

[0075] (1) Protein cleavage

[0076] Resuspend the cells at the time of passage in 1 mL of PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of RIPA lysis buffer, mix thoroughly, and place on ice for more than 15 min. Vortex mix and add to a pre-cooled centrifuge, centrifuge at 13000 rpm for 15 min, remove the supernatant, and transfer to a new EP tube.

[0077] (2) Protein quantification

[0078] After mixing the supernatant, take out 2 μL and mix BCA reagent solution B: solution A at a ratio of 50:1. Then add 100 μL to each well of a 96-well plate. Add 2 μL of sample and standard protein solution to each well respectively. After incubation in a 37°C incubator for 20 min, use a microplate reader to measure the absorbance at 562 nm, calculate the protein concentration and sample volume, and the sample volume is 10 μg.

[0079] (3) SDS-PAGE electrophoresis

[0080] Add the remaining protein sample to a solution of 5-mercaptoethanol supplemented with β-mercaptoethanol. Heat in a 100°C metal bath for 10 minutes. Once cooled, the sample can be loaded. Add electrophoresis buffer to the well and prepare for sample injection by slowly pulling the comb vertically away from the gel. Add a desired amount of sample to the well. Set a constant voltage of 90 V. Once the sample forms a thin line at the interface between the stacking gel and the separating gel, switch the voltage to 120 V. Stop running when the bromophenol blue line reaches the green bottom plate.

[0081] (4) Wet transfer

[0082] Remove the glass plate and tilt the two sides off. Cut off the unused portions of the stacking and separating gels on the top side and place them on a metal plate with transfer buffer. Activate the polyvinylidene fluoride (PVDF) membrane with methanol for at least 1 minute. Place a sponge pad and three sheets of filter paper on each side of the tweezers. Remove any bubbles and place the PVDF membrane. Place the gel on the black side of the plate and the PVDF membrane on the transparent side. Remove any bubbles and close the tweezers. Transfer the gel to the black side of the tweezers, and the transparent side to the red side. Place the black side of the tweezers on the black side of the tweezers, and the transparent side on the red side of the tweezers. Place the tweezers in a 4°C refrigerator and add sufficient pre-chilled transfer buffer for constant current electroporation.

[0083] (5) Closed

[0084] Rinse the membrane once in an incubator pre-filled with 1×TBST. Add blocking solution to completely cover the PVDF membrane and block the membrane on a shaker at 30 rpm for 1 hour at room temperature.

[0085] (6) Incubation with primary antibody

[0086] Prepare the primary antibody. Dilute the target protein antibody to the specified concentration using blocking buffer and incubate overnight in a 4°C refrigerator on a shaker at 30 rpm.

[0087] (7) Membrane washing

[0088] Add 1×TBST to completely cover the PVDF membrane and wash at 50 rpm at room temperature for 10 min. Repeat three times.

[0089] (8) Incubation with secondary antibody

[0090] Prepare secondary antibody. Dilute the secondary antibody corresponding to the primary antibody species in blocking buffer to the specified concentration and incubate at room temperature at 30 rpm for 1 hour.

[0091] (9) Membrane washing

[0092] Recover the secondary antibody, add 1×TBST to completely cover the PVDF membrane, and wash at 50 rpm for 10 min at room temperature, repeat three times.

[0093] (10) Development

[0094] The excess TBST solution was removed with dust-free paper, ECL substrate was added for reaction, and the target band was exposed using the ultra-sensitive multifunctional imager AI680RGB.

[0095] Antibody incubation method: GANAB and β-actin were prepared at a dilution of 1:2000 in 5% skim milk powder; anti-rabbit secondary antibody and anti-mouse secondary antibody were prepared at a dilution of 1:20000 in 5% skim milk powder.

[0096] like Figure 8As shown in the results, glucosidase I MOGS, glucosidase II α subunit GANAB, and β subunit PRKCSH are highly expressed in multiple cancers, and their expression is significantly increased in glioblastoma. The expression of glucosidase II α subunit GANAB is correlated with that of β subunit PRKCSH and glucosidase I MOGS in glioblastoma, and high expression of GANAB significantly predicts poor overall survival and progression-free survival in patients.

[0097] like Figure 9 As shown in the results, with the increase of the generation number, the expression of GANAB in the stemness-enriched LN229 and MDA-MB-231 tumor sphere cells gradually increased, and after the increase of GANAB expression, the cytotoxic activity of nidulans was significantly enhanced, from about 92.31 μM and >100 μM in the parent cells to the lowest 19.74 μM and 51.88 μM, respectively. In addition, the sensitivity of HepG2, TE-1, and A549 tumor sphere cells to nidulans was enhanced, indicating that nidulans has the activity of selectively inhibiting tumor stem cells.

[0098] like Figure 10 As shown in Figure 2, after knocking down GANAB in cancer stem cells GSC0617 and GSC0722, the activity of nidulans was significantly weakened, and the cytotoxic activity IC 50 The highest concentrations increased to 51.64 μM and 180.30 μM, respectively, both increasing by approximately 3 times, suggesting that the tumor stem cell cytotoxicity of nidulans depends on the expression level of GANAB.

[0099] like Figure 11 As shown in the results, after GANAB knockdown, cancer stem cells showed a differentiated morphology, and the expression of stemness markers such as CD44, SOX2, and CD133 in cancer stem cells was reduced, suggesting that GANAB affects the stemness of cancer stem cells.

[0100] Example 5

[0101] Nidulin induced increased expression of endoplasmic reticulum stress markers XBP-1 cleavage, CHOP, and BIP / GRP78.

[0102] Endoplasmic reticulum stress activates ATF6, leading to increased levels of BIP / GRP78 and PERK, which in turn induces increased CHOP levels, while RPL19 expression remains unchanged. Therefore, RNA was collected from tumor spheres LN229-T2 cells treated with nidulans for different periods of time. RNA was then analyzed for XBP-1 cleavage using RT-PCR, and for CHOP, BIP / GRP78, and RPL19 expression using RT-qPCR.

[0103] The specific steps are as follows:

[0104] (1) Sample lysis

[0105] Collect the cells and discard the supernatant. Wash the pellet with an appropriate amount of PBS to remove the residual culture medium. Add 1 mL of Trizol solution to the cell pellet, fully resuspend the cell pellet until there is no white floc, and then transfer it to an EP tube.

[0106] (2) RNA extraction

[0107] Add 200 μL of chloroform to the EP tube and mix vigorously for 30 seconds. After standing at 4°C for 10 minutes, centrifuge at 12,000 rpm for 15 minutes at 4°C. Carefully transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, and gently shake to mix. Place in a -20°C refrigerator to allow overnight precipitation for 12–16 hours. Centrifuge at 12,000 rpm for 15 minutes at 4°C, and discard the supernatant. Add 1 mL of 75% ethanol to wash and precipitate the RNA. Centrifuge at 12,000 rpm for 10 minutes. Repeat the ethanol wash twice, discard the supernatant, and air-dry the tube in a clean bench for approximately 10 minutes. Dissolve the RNA in enzyme-free water. Determine the concentration using a nanodrop instrument, and perform nucleic acid electrophoresis to verify the integrity of the extracted RNA.

[0108] (3) RNA reverse transcription

[0109] Prepare the reverse transcription system according to Table 2 and keep on ice throughout the process according to the RNA reverse transcription kit instructions. Before use, add 1 / 50 volume of gDNA removal reagent to the 4× DN Master Mix. Incubate the RNA solution at 65°C for 5 minutes.

[0110] Table 2 RNA reverse transcription system

[0111] reactants volume 4×DN Master Mix 2 μL Total RNA template 0.5 µg Enzyme-free water Fill to 8 μL

[0112] After thorough mixing, incubate at 37°C for 5 min, then add 2 μL of 5× RT Master Mix II to a total volume of 10 μL. Incubate at 37°C for 15 min, incubate at 50°C for 5 min, and terminate the reaction by heating at 98°C for 5 min to obtain sample cDNA. The concentration was determined using a nanodrop instrument, followed by dilution and storage in a -20°C freezer.

[0113] (4) Prepare fluorescence quantitative PCR reaction solution

[0114] Prepare the fluorescence quantitative PCR system according to Table 3, add ddH2O to a final volume of 20 μL, mix thoroughly, and then perform the fluorescence quantitative PCR reaction. Set the reaction program according to Table 4 for fluorescence quantitative PCR.

[0115] Table 3 Fluorescence quantitative PCR reaction system

[0116] reactants volume 2×FastReal qPCR PreMix 10 μL Primer Forward (10 µM) 0.6 μL Primer Reverse (10 µM) 0.6 μL cDNA 100 ng

[0117] Table 4 Fluorescence quantitative PCR reaction procedure

[0118] step Reaction temperature time 1 50 ℃ 2 min (hot start) 2 95 ℃ 2 min (preliminary denaturation) 3 95 ℃ 15 s (denaturation) 4 58 ℃ 15 s (annealing) 5 72 ℃ 1 min (extension) 6 16 ℃ ∞

[0119] Place the sample into the fluorescent quantitative PCR instrument LC480 and repeat steps 3-5 for 40 cycles.

[0120] GANAB can hydrolyze N-glycosylated α-1,3-linked glucose and is a key protein for protein quality control in the endoplasmic reticulum. Its inhibition will lead to the unfolding / misfolding of glycoproteins and their accumulation in the endoplasmic reticulum, inducing endoplasmic reticulum stress, triggering the expression of a series of downstream effector molecules, and leading to the activation of the unfolded protein response (UPR). If the UPR effect fails to alleviate endoplasmic reticulum stress, it will eventually induce cell death. Figure 12-14 As shown, RT-PCR was used to detect changes in XBP-1 cleavage in cells after nidulin treatment, revealing that nidulin treatment began to induce XBP-1 cleavage in the UPR pathway 24 hours after treatment. Amplification using corresponding primers revealed that CHOP levels began to increase significantly 12 hours after nidulin treatment, and BIP / GRP78 levels increased significantly after 24 hours. Meanwhile, expression of the reference gene RPL19 remained unchanged. PC, the positive control drug Tunicamycin, indicates that nidulin treatment induces endoplasmic reticulum stress and an unfolded protein response in tumor stem cells. Furthermore, the present invention observed, through lectin ConA staining and fluorescence microscopy, that nidulin inhibited GANAB, leading to increased levels of glucose and mannose modification of glycoproteins in cells. These results demonstrate that nidulin can target GANAB, leading to abnormal glycosylation of glycoproteins, inducing changes in endoplasmic reticulum stress markers and glycosylation modification levels, confirming the role of nidulin in inducing endoplasmic reticulum stress in tumor stem cells by inhibiting the glucosidase GANAB.

[0121] Example 6

[0122] In vivo activity of nidulans in tumor-bearing nude mice.

[0123] Glioblastoma stem cells GSC0617-Luc were stereotactically injected according to the location of intracranial glioma. After tumor formation was confirmed, different doses of nidulans and a solvent control group were injected into the tumor in situ through pre-buried positioning pins. At the same time, changes in body weight and fluorescence intensity of brain tumors in nude mice were continuously observed to explore the safety and efficacy of nidulans in the in situ nude mouse model of xenotransplanted glioblastoma stem cells.

[0124] The specific steps are as follows:

[0125] (1) Nail embedding

[0126] Purchase SPF grade nude mice from a model animal center and raise for one week. Anesthetize the nude mice and place them in a small animal stereotaxic instrument. Fix the head using ear bars and incisor bar adapters. Apply ophthalmic ointment to the eyes to prevent dehydration. Check whether the animal is anesthetized by a toe pinch pain reflex and confirm anesthesia before proceeding to the next step. Disinfect the head skin of the nude mice with 75% ethanol and iodophor solution, ensure the surgical site is sterile, and make a midline incision with a small scalpel to separate the skin and connective tissue. Locate the bregma by observing the skull and use a micromanipulator to position a Hamilton syringe at the bregma. Adjust the positioning frame to move 1.0 mm up and 2.5 mm left. Mark the position with a drill bit. Carefully drill a hole at the position without damaging the arachnoid membrane and brain parenchyma, and bury a positioning nail at the position. Use a screwdriver to rotate the nail and suture the scalp with a surgical suture needle. Subcutaneously administer meloxicam for analgesia. Transfer the animal to an observation room and monitor the body temperature, respiratory rate, and heart rate until full recovery. Remove the suture needle after the head heals for one week. Measure the body weight every week.

[0127] (2) Cell preparation

[0128] Collect and disperse GSC0617-Luc cells into a single cell suspension and resuspend 1 x 10 5 cells in 10 μL of PBS per injection per animal. Move the cell suspension into a 1.5 mL sterile centrifuge tube and place on ice for in situ injection.

[0129] (3) Intracranial injection

[0130] After anesthetizing and fixing the nude mice, mix the collected cell suspension thoroughly and load into a Hamilton syringe and install on the positioning frame. Introduce the needle into the small hole of the positioning nail and advance to 3.0 mm below the cortical surface. Set the injection volume and flow rate (1 μL / min) using an electric stereotactic injector. After injection, gradually remove the needle at a rate of 1.0 mm every 1 min.

[0131] (4) Postoperative recovery

[0132] Transfer the animal to an observation room and monitor the body temperature, respiratory rate, and heart rate until full recovery. Administer meloxicam for analgesia.

[0133] (5) In situ administration of animals

[0134] Intravital imaging (IVIS) was performed within 24 hours of cell transplantation and every three days thereafter. The appropriate time for intervention was determined and randomized into groups. After nidulin was fully dissolved, different doses of nidulin and solvent were administered. In situ administration was performed using intracranial positioning pins every other day for seven consecutive doses, followed by discontinuation. After the start of treatment, intravital imaging was performed every four days until the end of the experiment.

[0135] The specific steps for small animal in vivo imaging are as follows:

[0136] (1) Instrument preparation

[0137] Turn on the Revvity IVIS Imaging System and launch the accompanying Living Image software. Click Initialize to pre-cool the instrument to the desired temperature and maintain a stable imaging environment with appropriate ambient temperature and humidity.

[0138] (2) Reagent preparation

[0139] Prepare the luciferase substrate D-luciferin by dissolving it in PBS to 15 mg / mL, sterilize it by 0.22 μm filtration, and aliquot it. Store it at -20°C in the dark and return it to room temperature before use.

[0140] (3) Substrate injection

[0141] D-Luciferin solution was injected intraperitoneally at a dose of 150 mg / kg. After injection, wait for 10-15 minutes to allow the substrate to be evenly distributed in the body and reach the peak signal.

[0142] (4) Animal anesthesia

[0143] Place the animal in an anesthesia induction chamber and anesthetize it with isoflurane. Once fully anesthetized, transfer it to the imaging chamber and place it prone on the imaging platform. Maintain anesthesia using a nasal ventilation port to ensure brain exposure.

[0144] (5) Imaging settings

[0145] After initialization is complete, select the bioluminescence imaging mode (Bioluminescence), set the exposure time, select the appropriate field of view and focal length, and click the "Acquire" button to start acquiring images.

[0146] (6) Data analysis

[0147] Use software tools (e.g., the ROI tool) to select the region of interest and measure the optical signal intensity (in photons / second / cm² / solid angle, p / s / cm² / sr). Export the image and data to the desired format (TIFF).

[0148] (7) Animal recovery

[0149] After imaging, turn off the anesthetic gas, move the animal to a warm environment, and observe the animal until it fully awakens.

[0150] On the 17th day after cell injection, the tumor was injected with nidulans via pre-embedded nails and treated 7 times with nidulans at two doses of 1.5 mg / kg and 0.16 mg / kg. Figure 15 As shown, IVIS results showed that the growth rate of brain tumors in nude mice treated with 1.5 mg / kg nidulans was significantly slowed, while high- and low-dose nidulans treatment had no effect on the body weight of tumor-bearing nude mice. These results demonstrate that nidulans has anti-glioblastoma stem cell activity in vivo and has no significant effect on the body weight of tumor-bearing nude mice.

[0151] In summary, the results of the target stability experiment of drug affinity reaction, cell thermal shift experiment, surface plasmon resonance and other experiments show that nidulans targets and inhibits the catalytic subunit GANAB of glucosidase II; the results of immunoblotting show that knocking down GANAB promotes the differentiation of glioblastoma stem cells GSC0617 and reduces the expression of stemness-related proteins such as CD44, that is, inhibiting GANAB can selectively induce the differentiation of tumor stem cells and reduce the degree of stemness; RT-PCR, qPCR experiments and lectin experiments verify that nidulans can induce endoplasmic reticulum stress of tumor stem cells by inhibiting GANAB, thereby inducing cell death.

[0152] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These changes and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of nidulans in treating malignant tumors, characterized in that: The compound nidulans targets and inhibits the catalytic subunit (α subunit) of glucosidase II, GANAB, thereby inducing endoplasmic reticulum stress and unfolded protein response in cancer stem cells and inducing their death.

2. The use of nidulans in treating malignant tumors according to claim 1, characterized in that: By inhibiting the glucosidase II α subunit GANAB, which is highly expressed in tumor stem cells, it selectively induces tumor stem cell differentiation and reduces the degree of stemness.

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