Application of albendazole as DKC1 inhibitor

Abstract

The invention belongs to the technical field of medicines, and provides application of albendazole as a DKC1 inhibitor. Experiments show that albendazole can be combined with DKC1 in vitro and has a relatively strong inhibition effect on pseudouridine synthase activity of DKC1. Albendazole has the effect of inhibiting proliferation of various DKC1 high-expression tumor cells by inhibiting the activity of pseudouridine synthase of DKC1. In addition, albendazole as a marketed drug has the characteristics of low cytotoxicity and low effective concentration. The invention provides a new way for the development of antitumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to use of albendazole as a DKC1 inhibitor. Background Art

[0002] Dyskerin, encoded by the Dyskerin pseudouridine synthase 1 (DKC1) gene, has pseudouridine synthase function and can bind to and catalyze the isomerization of uridine (U) in target RNA to pseudouridine (Ψ). The researchers screened four small molecule compounds, including pyrazofurin (PF), that can inhibit the catalytic activity of DKC1 from the open NCI compound data set, and verified in vitro that they have different degrees of inhibitory effects on the pseudouridine synthase activity of DKC1. The discovered DKC1 inhibitor, PF, inhibits nucleoside synthesis as a pyrimidine nucleoside analog. Although clinical trials have been conducted in a variety of tumors including colorectal cancer, it cannot be used as an effective drug due to its high toxicity and poor clinical efficacy (Moura J AB , et al., 2014.). The remaining three small molecule compounds were only tested for their inhibition of DKC1 pseudouridine synthase activity, but there was no experimental verification that they could directly target DKC1, and it was unclear whether they had anti-tumor activity. Therefore, there are currently no effective DKC1 inhibitors with potential clinical application value.

[0003] Albendazole (ABZ) is an FDA-approved broad-spectrum antiparasitic drug with high efficacy and low toxicity for humans and animals. It has a long-term record of safe and effective use in humans (including school-age children) and has the advantages of low cost and few side effects. Studies have proposed a new strategy for the effective treatment of melanoma and lung cancer with albendazole combined with a novel immune checkpoint CD73 inhibitor (Lin Z, et al., 2022.). In addition, studies have shown that albendazole can inhibit the expression of RING-finger protein 20 (RNF20) by inhibiting the anti-apoptotic protein Bcl-2 family of B cell lymphoma, thereby inhibiting RNF20-Eg5 to block the cell cycle in the G2 / M phase and induce cell apoptosis, ultimately enhancing the chemosensitivity of colon cancer cells to 5-FU (IramF, et al., 2021.). However, it is not clear whether albendazole has other targets. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of albendazole as a DKC1 inhibitor.

[0005] To achieve the above object, the technical solution adopted by the present invention is: First, the present invention provides use of albendazole as a DKC1 inhibitor.

[0006] Furthermore, the present invention provides non-diagnostic and therapeutic uses of albendazole as an in vitro DKC1 inhibitor.

[0007] Secondly, the present invention provides a method for inhibiting the activity of DKC1 pseudouridine synthase, which comprises contacting the DKC1 with a preparation containing albendazole in vitro to inhibit the activity of DKC1 pseudouridine synthase, and the method does not include diagnosis and treatment of the disease.

[0008] Thirdly, the present invention also provides the use of albendazole in preparing a therapeutic drug for cancers associated with high DKC1 expression.

[0009] Furthermore, the cancers associated with high DKC1 expression are gastric adenocarcinoma cell line SGC7901, hepatocellular carcinoma cell line HepG2, colon carcinoma cell line HCT116 and lung adenocarcinoma cell line A549.

[0010] The present invention provides a new use of albendazole as a DKC1 inhibitor. Experiments show that albendazole can bind to DKC1 in vitro and has a strong inhibitory effect on the pseudouridine synthase activity of DKC1. Albendazole has the effect of inhibiting the proliferation of multiple DKC1 high-expressing tumor cells by inhibiting the pseudouridine synthase activity of DKC1. In addition, albendazole, as a marketed drug, has the characteristics of low cytotoxicity and low effective concentration. Therefore, the use of albendazole as a novel DKC1 inhibitor will provide a new approach for the development of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Molecular dynamics simulation diagrams of DKC1 and ABZ; Figure A is the stable binding conformation of DKC1 (blue) and albendazole (orange), and the pink area is the most significant conformational change; Figure B is the binding mode of albendazole with the D125, I226 and R227 amino acid residues of DKC1; Figure C is the surface potential of the binding region of DKC1 and albendazole; Figure D is the interaction between albendazole and DKC1.

[0012] Figure 2 The results of the in vitro interaction experiment between ABZ and DKC1; Figure A is the purification of DKC1 prokaryotic protein; Ce represents the whole protein lysate, Chen represents the precipitated sample after centrifugation, S represents the protein supernatant, FL represents the supernatant flow-through sample through the column, W0 represents the protein resuspension buffer flow-through sample, W1-2 represents the elution buffer with different concentrations of imidazole, EL represents the protein sample obtained by the column matrix corresponding to the final elution buffer; Figure B is the ITC titration curve of the interaction between ABZ and DKC1; Figure C is the ITC titration curve of the interaction between ABZ and MBP.

[0013] Figure 3 Schematic diagram of DKC1 expression in tumors and normal tissues in the TCGA database (Figure A) (data source: https: / / www.cancer.gov / ccg / research / genome-sequencing / tcga); COAD: colon cancer; LIHC: hepatocellular carcinoma; LUAD: lung adenocarcinoma; STAD: gastric adenocarcinoma; and Western Blot detection of DKC1 expression in SGC7901, HepG2, HCT116, A549 cells and GES-1 cells (Figure B).

[0014] Figure 4 RNA Dot Blot experimental results and quantitative analysis using Image J software to obtain concentration trend graph and enzyme activity IC 50 Value; wherein, Figure A is the detection result of the effect of PF on the activity of DKC1 pseudouridine synthase in HCT116 cells; Figure B is the detection result of the effect of PF on the activity of DKC1 pseudouridine synthase in A549 cells; Figure C is the detection result of the effect of albendazole on the activity of DKC1 pseudouridine synthase in HCT116 cells; Figure D is the detection result of the effect of albendazole on the activity of DKC1 pseudouridine synthase in A549 cells.

[0015] Figure 5 The results of RNA Dot Blot experiments on the effect of albendazole on the activity of DKC1 pseudouridine synthase in HCT116 cells (Figure A), A549 cells (Figure B) and GES-1 cells (Figure C) and the quantitative analysis using Image J software to obtain the concentration trend graph and enzyme activity IC 50 value.

[0016] Figure 6 The results show the effect of albendazole on the proliferation of SGC7901 cells (Figure A), HepG2 cells (Figure B), HCT116 cells (Figure C), A549 cells (Figure D) and GES-1 cells (Figure E). DETAILED DESCRIPTION

[0017] The following is a further detailed description of this description in conjunction with experiments and experimental data.

[0018] Example 1 Molecular dynamics simulation of albendazole and DKC1 Figure 1The molecular dynamics simulation diagram of DKC1 and albendazole. Figure A shows the predicted stable binding conformation of albendazole and DKC1, Figure B shows the binding mode of albendazole with D125, I226 and R227 amino acid residues of DKC1; Figure C shows the surface potential of the binding region of DKC1 and albendazole; Figure D shows the interaction between albendazole and DKC1. The specific simulation process is as follows: The complex structure of the H / ACA ribonucleoprotein complex subunit DKC1 (PDB ID: 7BGB) was set as a receptor for binding to albendazole. The binding mode of DKC1 to albendazole was predicted by AutoDock-vina. The small molecule ligand albendazole was assigned charges using the Acpype-based Gaff force field and the Am1-bcc method. The binding of DKC1-D125 site to albendazole was simulated by molecular dynamics. The system of the binding of DKC1-D125 site to albendazole was established on the CHARMM-GUI web server. The dissolved system was modeled with TIP3P water molecules, and then 150 mM KCl was added to the system. The final simulation box contained 128038 atoms. The simulation of the binding system was based on the GROMACS program and the amberff14sb force field. First, the system was energy minimized. The temperature of the system was raised to 310.15 K using the V-Rescale temperature coupling method in the NVT ensemble. The pressure of the system was stabilized at 1 bar using the Berendsen pressure coupling method in the NPT ensemble. The generation phase of the system was simulated three times with 200 ns molecular dynamics simulations. Then, the RMSD (root mean square deviation) and RMSF (root mean square fluctuation) programs in GROMACS were used for data analysis. Finally, the gmx_MMPBSA tool was used to calculate the contribution of binding energy and residual energy, and PyMOL was used to visualize the binding system.

[0019] Example 2 In vitro binding of albendazole to DKC1 protein In order to detect the in vitro binding of albendazole to DKC1 protein, the prokaryotic protein of DKC1 was extracted, and the DKC1 protein and albendazole were detected by isothermal titration calorimetry to analyze the in vitro binding of albendazole to DKC1 protein.

[0020] 1. Sample collection and processing 1.1 Molecular cloning (1) Clone the target gene into an E. coli expression vector. The vector should be selected based on the protein tag required by the experimental design. (2) After confirming that the recombinant plasmid was successfully sequenced, the plasmid was transformed into Escherichia coli BL21 competent cells for prokaryotic protein expression detection.

[0021] 1.2 Prokaryotic protein expression detection (1) Transfer an appropriate amount of bacterial liquid to 1 mL of resistant liquid LB medium and culture on a shaker at 37°C; (2) After shaking the bacteria for 6 h, take out 200 μL of the bacterial solution and keep the sample (uninduced protein sample); (3) Add 1 μL IPTG to the remaining 800 μL bacterial solution and place on a shaker at 37°C to induce expression; (4) Centrifuge the bacterial solution before and after induction at 4000 rpm for 8 min, retain the bacterial cells and add an appropriate amount of protein loading buffer, denature at 100°C for 5 min, and then perform SDS-PAGE gel electrophoresis; (5) Remove the gel with a gel lifter and place it in a plastic box. Soak the gel in an appropriate amount of staining solution, heat it in a microwave oven to boil, and then stain it in a shaker at room temperature for 30 minutes. (6) Discard the staining solution, add an appropriate amount of decolorizing solution to cover the gel, heat in a microwave oven until boiling, and then decolorize on a shaker at room temperature for 1 h. (7) Place the stained gel under a light to observe whether the amount of the target protein increases significantly after induction. If the expression is successfully induced, subsequent large-scale extraction and purification of the prokaryotic protein can be performed.

[0022] 1.3 Inducible expression of prokaryotic proteins (1) Transfer an appropriate amount of bacterial solution to 20 mL of resistant LB liquid medium and culture at 37°C in a shaking incubator overnight; (2) Inoculate 10-20 mL of bacterial solution per bottle into 1 L of resistant LB liquid medium and culture in a shaking incubator at 37°C and 180 rpm; (3) When the OD value of the bacterial solution reaches about 0.8-1.0, adjust the temperature to 16°C and reduce the speed to 100 rpm; (4) When the temperature dropped to 16°C and stabilized, IPTG (0.2 mM) was added to induce expression for 15 h.

[0023] 1.4 Extraction of prokaryotic proteins (1) Collect the cells using a floor-standing centrifuge and centrifuge at 5000 rpm for 10 min. (2) Add an appropriate amount of resuspension buffer to the bacteria and resuspend them using a vibrating suspension instrument; (3) Use a low-temperature ultra-high-pressure continuous flow cell disruptor to disrupt the bacteria, transfer the protein solution to a low-temperature high-speed centrifuge and centrifuge at 12,000 rpm for 50 min, collect the protein supernatant, and retain the total protein (Ce), precipitate, and protein supernatant (S) samples.

[0024] 1.5 Purification of prokaryotic proteins (1) Rinse and incubate the column with first-grade water and resuspension buffer in advance; (2) Pour out the beads in the column and incubate with the protein supernatant at low temperature; (3) Pass the incubated system through the column and collect the flow-through protein solution (FL); (4) Rinse the column with a large amount of resuspension buffer to wash out the impurities and collect the effluent protein solution (W0); (5) Prepare the elution buffer according to the target protein tag, flush the column several times with a small amount of elution buffer until the universal dye does not turn blue, and collect the protein sample; (6) Perform SDS-PAGE gel electrophoresis on the protein samples collected each time (Ce, sediment, S, FL, W0, EL, etc.) and stain and decolorize to observe whether the purity and amount of the eluted protein meet the experimental requirements; (7) The protein solution that meets the requirements is dialyzed to reduce salt, and then centrifuged and concentrated to obtain a high concentration of the target protein. The protein sample is packaged and quickly frozen in liquid nitrogen and stored at -80°C.

[0025] like Figure 2 As shown in A, in this example, a prokaryotic expression vector of DKC1 was constructed, and a DKC1-MBP protein with high purity was obtained by prokaryotic protein extraction and purification.

[0026] 2. Experimental Methods 2.1 ITC Experiment There are two cells in the microcalorimeter, one contains water, which serves as a reference cell, and the other contains the sample. The microcalorimeter must keep these two cells at exactly the same temperature. A thermal device detects the temperature difference between the two cells when binding occurs and feeds back to the heater, which compensates for the temperature difference and returns the two cells to the same temperature. Prepare the sample cell and syringe, add the buffer to the sample cell, and add the reference substance and the substance to be tested to the syringe. Place the sample cell and syringe in the ITC instrument and control the temperature at a constant temperature. Inject the reference substance in the sample cell and record the baseline value of the thermal effect signal. Add the substance to be tested to the sample cell and record the change in the thermal effect signal. Calculate the thermodynamic parameters such as the thermodynamic equilibrium constant, enthalpy change, and entropy change based on the measurement results.

[0027] 2.2 Experimental Results and Analysis like Figure 2As shown in B and 2C, based on the titration curve of albendazole and DKC1-MBP protein, it can be found that albendazole can interact with DKC1 protein but not with MBP empty vector.

[0028] The ITC experimental data were further analyzed and fitted using the analysis software MicroCal PEAQ-ITC Analysis. Through concentration gradient exploration, the final experimental results showed that when the working concentrations of ABZ and DKC1 proteins were 400 μM and 20 μM, respectively, that is, when the concentration ratio of the two was 20 times, the mutual binding effect was optimal, and the binding constant KD at this time was 4.66e -6 M.

[0029] Example 3 Analysis of the expression of DKC1 in some cancers using the TCGA database and detection of the expression of DKC1 in different cells using Western blot experiments ( Figure 3 ) 1. Experimental methods, results and analysis of changes in DKC1 expression after albendazole treatment We found through TCGA database analysis that DKC1 is highly expressed in a variety of cancers ( Figure 3 A). COAD is colon cancer; LIHC is hepatocellular carcinoma; LUAD is lung adenocarcinoma; STAD is gastric adenocarcinoma.

[0030] 2. Experimental methods, results and analysis of detecting DKC1 expression in different cells Experimental groups: Normal SGC7901, HepG2, HCT116, A549 cells and common gastric epithelial cells GES-1 were obtained and preserved in this laboratory.

[0031] Medication treatment: The cells in the experimental group were treated with albendazole, and then cell samples were collected at the corresponding treatment time points.

[0032] Sample collection and processing: The cells were washed twice with pre-cooled 1× PBS buffer and then collected. After the cells were fully lysed with NETN 420 buffer, they were centrifuged at 13,000 rpm for 10 min and the supernatant was taken as the total cell protein extract. The protein was quantified using the BCA method and quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0033] Experimental methods: Western blot was used to detect the internal reference protein β-actin and DKC1 protein in the samples: take an equal amount of sample for SDS-PAGE gel electrophoresis → 0.3A, constant flow transfer for 60 min → 5% skim milk blocking solution: 30 min → 1× TBST buffer washing → DKC1 detection antibody (Proteintech) incubated at room temperature for 1h → 1× TBST buffer washing 3 times, 10 min each time → horseradish peroxidase (HRP) labeled secondary antibody incubated at room temperature for 45 min → 1× TBST buffer washing 3 times, 10 min each time → developer for development.

[0034] Experimental results and analysis: like Figure 3 As shown in A, DKC1 is highly expressed in colon cancer, hepatocellular carcinoma, lung adenocarcinoma, and gastric adenocarcinoma, so we used HCT116 (colon cancer), A549 (lung adenocarcinoma), SGC-7901 (gastric cancer), and HepG2 (liver cancer) cells for subsequent experiments. Figure 3 As shown in B, Western blot experiments showed that DKC1 protein was highly expressed in the four tumor cells used compared with normal cells, which was consistent with the expectations of TCGA database analysis.

[0035] Example 4 Albendazole inhibits the enzyme activity of tumor cells DKC1 In order to detect the changes in DKC1 enzyme activity levels after albendazole treatment, this example detected the pseudouridine modification level of cells treated with concentration gradient albendazole, analyzed the changes in DKC1 enzyme activity levels in cells after albendazole concentration gradient treatment, and used PF treatment on two types of tumor cells to compare the effects of albendazole and existing DKC1 enzyme activity inhibitors.

[0036] 1. Experimental methods, results and analysis of the detection of pseudouridine modification levels in cells after albendazole and PF treatment ( Figure 4 ) 1.1 Experimental Grouping Normal HCT116 and A549 cells (preserved in this laboratory) were obtained, of which HCT116 is a colon cancer cell line and A549 is a lung adenocarcinoma cell line. The cells were randomly divided into 6 groups: 1 control group and 5 experimental groups. Three parallel experiments were set up for each test sample.

[0037] 1.2 Sample collection and processing Centrifuge the cells at 1000 rpm for 5 minutes at room temperature (15-25 ºC) and discard the supernatant or remove the medium from the monolayer and wash once with pre-chilled PBS. 7Add 1 mL TRIzol reagent to the cells. Precool the centrifuge at 4°C and transfer the tissue or cell lysate to a 1.5 mL RNase-free EP tube. Place on ice and let stand for 5 minutes. Add 200 μL of chloroform to each tube, mix thoroughly and let stand on ice for 10 minutes to completely dissociate the nucleoprotein complex. Centrifuge at 13,000 rpm at 4°C for 15 minutes. During this time, take a new EP tube, add 500 μL of isopropanol and precool it on ice. After centrifugation, transfer the upper aqueous phase (about 500 μL) to the new EP tube. Let stand on ice and precipitate with alcohol for 10 min. Centrifuge at 13,000 rpm for 10 minutes. Remove the supernatant. Wash the RNA precipitate once with 1 mL 75% ethanol. Centrifuge at 12,000 rpm for 5 minutes. Remove the supernatant and air-dry or vacuum-dry the RNA precipitate for 5-10 minutes. Dissolve the RNA in 30-50 μL of DEPC-treated deionized water (depending on the amount of RNA precipitate). Perform spectrophotometric analysis to determine sample concentration and purity.

[0038] 1.3 Experimental methods RNA Dot Blot experiment: Take out the RNA sample to be tested, measure the RNA concentration after thawing, and operate on ice throughout the process. The sample concentration is unified, and the sample is continuously diluted and mixed in a new enzyme-free tube to a final concentration of 500 ng / μL. After centrifugation, the RNA sample is heated at 95℃ for 3 minutes to destroy the secondary structure, and immediately cooled on ice after the end to prevent the secondary structure from re-forming. Use the thick end of a 1 mL pipette tip to leave a circular mark on the NC membrane to guide the sample addition, and then cut the NC membrane into a suitable size and transfer it to a clean plastic box. Drop 2 μL of RNA sample on the NC membrane in sequence. Be careful not to touch the membrane with the pipette tip. The sample should diffuse naturally on the membrane. The pipette tip needs to be replaced each time the sample is added. After the NC membrane is slightly dried at room temperature, the RNA and membrane can be cross-linked. Method 1: Oven at 37℃ for 30 min. Method 2: Irradiate under a UV lamp with a wavelength of 254 nm for 30 min-1 h. Wash the membrane with 10 mL TBST for 5 min to wash away unbound RNA. Discard TBST, add 10 mL of blocking solution, and incubate at room temperature with shaking for 1 hour. Discard blocking solution, add blocking solution containing primary antibody and incubate overnight at 4°C. Wash the membrane 3 times with 10 mL TBST, 10 min each time. Incubate with secondary antibody at room temperature for 1 hour. Wash the membrane 3 times with 10 mL TBST, 10 min each time. Incubate with ECL substrate for 5 min, take pictures under the developer, and after taking pictures, place the membrane in 10 mL of methylene blue staining buffer and incubate at room temperature with shaking for 30 min. Wash with ddH2O until the background is roughly clean, about 30-60 s. White light imaging collects the image after methylene blue staining as a loading control.

[0039] 1.4 Experimental Results and Analysis like Figure 4As shown in the figure, RNA Dot Blot experiments showed that albendazole treatment inhibited the activity of pseudouridine synthase in tumor cells. With the increase of drug treatment concentration, the activity level of pseudouridine synthase in cells gradually decreased. The blotting results were quantitatively analyzed using ImageJ software to obtain the concentration change trend graph and enzyme activity IC 50 The results showed that PF had an IC value of 1. 50 The value was 2.458 μM (Figure A), and the IC value of albendazole on the enzyme activity of HCT116 cells was 50 The value was 1.221 μM (Figure B), and the IC value of PF on the enzyme activity of HCT116 cells 50 The value was 1.467 μM (Figure C), and the IC value of albendazole on the enzyme activity of A549 cells was 50 The value was 1.228 μM (Figure D). This result shows that the concentration of albendazole is better than that of the existing DKC1 inhibitor PF.

[0040] 2. Experimental methods, results and analysis of intracellular pseudouridine modification levels in normal cells and other tumor cells with high expression of DKC1 after albendazole treatment 2.1 Experimental Grouping: Normal SGC7901, HepG2, and GES-1 cells (preserved in this laboratory) were obtained, among which SGC7901 is a gastric adenocarcinoma cell line, HepG2 is a hepatocellular carcinoma cell line, and GES-1 is a human gastric mucosal epithelial cell line. The cells were randomly divided into 6 groups in equal amounts: 1 control group and 5 experimental groups. Three parallel experiments were set up for each test sample.

[0041] 2.2 Experimental method is as in 1.

[0042] 2.3 Experimental Results and Analysis like Figure 5 As shown in the figure, the other two tumor cells SGC7901 and HepG2 were treated with albendazole, and normal human gastric mucosal epithelial cells GES-1 were treated at the same time. The blotting results were quantitatively analyzed using Image J software to obtain the concentration change trend graph and enzyme activity IC 50 The results showed that the IC value of albendazole on the enzyme activity of SGC7901 cells 50 The value of albendazole was 4.423 μM (Figure A), and that of HepG2 was 2.836 μM (Figure B), while there was no significant inhibitory effect on GES-1 cells (Figure C) at low concentrations. This result shows that albendazole has a more significant inhibitory effect on DKC1 enzyme activity in tumor cells with higher DKC1 expression levels.

[0043] Example 5 Detection of the effect of albendazole on tumor cell proliferation In order to detect the effect of albendazole treatment on tumor cell proliferation, this example treated different tumor cells with drugs, and detected the cell proliferation level of cells treated with gradient concentrations to analyze the effect of concentration gradient treatment on cell proliferation.

[0044] 1. Experimental methods, results and analysis of cell proliferation levels after albendazole treatment 1.1 Experimental Grouping Normal SGC7901, HepG2, HCT116, A549, and GES-1 cells were obtained and preserved in this laboratory. SGC7901 is a gastric adenocarcinoma cell line, HepG2 is a hepatocellular carcinoma cell line, HCT116 is a colon cancer cell line, A549 is a lung adenocarcinoma cell line, and GES-1 is a human gastric mucosal epithelial cell line. The cells were randomly divided into a control group and five experimental groups in a ratio of 6:1. Three parallel experiments were set up for each test sample.

[0045] 1.2 Experimental methods Day 1: (1) Digest, centrifuge, and count the cells cultured the day before.

[0046] (2) Inoculate 100 μL of 3 k-7 k cells / well on a 96-well plate and culture at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0047] the next day: (1) Observe the cell growth status and density under a microscope, and select wells with good growth status and uniform cell distribution and density for the experiment.

[0048] (2) Prepare sample solutions with different concentration gradients. Add three replicates of each concentration to a 96-well plate and incubate at 37°C, 5% CO2, and 90% humidity for an appropriate length of time, such as 6, 12, 24, or 48 hours.

[0049] Day 3 / Day 4: (1) CCK8 was thawed at room temperature and centrifuged before use.

[0050] (2) Use an inverted microscope to monitor the cell growth status and take photos of wells with good growth status and uniform cell distribution and density.

[0051] (3) Add 10 μL of CCK-8 solution to each well.

[0052] (4) Incubate at 37°C, 5% CO2, and 90% humidity for 0.5-4 hours.

[0053] (5) Measure the absorbance at 450 nm using an enzyme reader.

[0054] (6) Process and analyze the results using Excel and Graphpad Prism.

[0055] 1.3 Experimental Results and Analysis like Figure 6 As shown in the CCK-8 experiment, albendazole inhibited the proliferation of the four different types of tumor cells tested in a concentration-dependent manner. It is worth noting that albendazole had no significant inhibitory effect on the growth of normal gastric epithelial cells GES-1.

[0056] The experimental data were processed and analyzed using Graphpad Prism9 to obtain concentration trend graphs and proliferation IC 50 The results showed that albendazole had an IC value of 1. 50 The IC value for the proliferation of HepG2 cells was 2.118 μM (Figure A). 50 The IC value of HCT116 cell proliferation was 1.612 μM (Figure B). 50 The IC value for the proliferation of A549 cells was 0.452 μM (Figure C). 50 The concentration of albendazole was 0.238 μM (Figure D), while it had no significant inhibitory effect on the proliferation of GES-1 cells (Figure E) within the above concentration range. This result further shows that albendazole can selectively inhibit the proliferation of tumor cells while having no effect on normal cells.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of albendazole as a DKC1 inhibitor.

2. The use of albendazole as a DKC1 inhibitor according to claim 1, characterized in that: The use of albendazole as a DKC1 inhibitor is a non-diagnostic and non-therapeutic use of albendazole as an in vitro DKC1 inhibitor.

3. A method for inhibiting DKC1 pseudouridine synthase activity, characterized in that: The method comprises contacting the DKC1 with a preparation containing albendazole in vitro to inhibit the pseudouridine synthase activity of DKC1, and the method does not include the diagnosis and treatment of the disease.

4. Application of albendazole in the preparation of therapeutic drugs for cancers associated with overexpression of DKC1.

5. The use according to claim 4, characterized in that: The cancers associated with high DKC1 expression are gastric adenocarcinoma cell line SGC7901, hepatocellular carcinoma cell line HepG2, colon carcinoma cell line HCT116 and lung adenocarcinoma cell line A549.

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