Application of an ART1 inhibitor in improving the efficacy of sorafenib in hepatocellular carcinoma

Abstract

This invention discloses the application of an ART1 inhibitor in enhancing the efficacy of sorafenib against hepatocellular carcinoma, relating to the field of pharmaceutical technology; the application of an ART1 inhibitor in the preparation of a drug for enhancing the efficacy of sorafenib against hepatocellular carcinoma; wherein the hepatocellular carcinoma is a hepatocellular carcinoma with high ART1 expression; the enhanced efficacy of sorafenib against hepatocellular carcinoma is manifested in that, compared with sorafenib alone, the combined use of sorafenib and an ART1 inhibitor can synergistically inhibit the proliferation of hepatocellular carcinoma cells. This invention identifies ART1 as a novel target for enhancing the efficacy of sorafenib; based on the finding that the levels of ART1 and its catalyzed single ADP ribosylation modification are significantly higher in hepatocellular carcinoma tissue than in adjacent normal tissue, a therapeutic approach of sensitizing sorafenib by inhibiting ART1 is proposed; this provides a new and effective combination therapy approach based on existing drugs, in addition to developing entirely new drugs, to address the clinical dilemma of reduced responsiveness after patients use sorafenib.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of an ART1 inhibitor in improving the efficacy of sorafenib in treating hepatocellular carcinoma. Background Technology

[0002] Primary liver cancer is a common malignant tumor of the digestive system worldwide, ranking 6th in incidence and 3rd in mortality among all malignant tumors. In my country, it is the 4th most common malignant tumor and the 2nd leading cause of cancer death. Among them, hepatocellular carcinoma (HCC) is the most common pathological type of primary liver cancer, accounting for 85% to 95% of primary liver cancers.

[0003] Sorafenib is an oral multi-target tyrosine kinase inhibitor and the first molecularly targeted drug approved for first-line treatment of advanced HCC. However, 70% of patients experience a gradual decrease in drug responsiveness after 6 months of sorafenib treatment, severely limiting its clinical efficacy. Studies have shown that activation of the PI3K / AKT / mTOR pathway is the main reason for the reduced efficacy of sorafenib treatment.

[0004] Single ADP ribosylation, catalyzed by single ADP ribosyltransferases, transfers a single ADP ribosome from nicotinamide adenine dinucleotide (NAD+) to a specific amino acid residue on a substrate protein, thereby regulating the structure and function of the substrate protein. In recent years, increasing evidence suggests that abnormal marylation is closely related to tumorigenesis and development.

[0005] ART1 is a key enzyme reported to catalyze arginine-specific single-ADP ribosylation modification in human and mouse tissues. Previous studies have shown that ART1 is highly expressed in colorectal cancer, and inhibiting ART1 can suppress the PI3K / AKT pathway, thereby inhibiting tumor proliferation and migration. ART1 is also highly expressed in hepatocellular carcinoma (HCC) tissues, with higher expression levels in grade III-IV differentiation than in grade I-II. The ART1 inhibitor mI-iodobenzylguanidine (MIBG) can reduce the expression of both ART1 and PI3K, inhibiting HCC cell proliferation. However, it remains unclear whether inhibiting ART1 can suppress the PI3K / AKT / mTOR pathway and thus enhance the responsiveness of HCC cells to sorafenib.

[0006] Based on the aforementioned research background, this invention aims to conduct preliminary research on the effects of ART1 on sorafenib's inhibition of HCC proliferation and its possible molecular mechanisms through in vitro cell experiments and nude mouse xenograft models. This will provide new insights and preliminary experimental evidence for overcoming reduced responsiveness to sorafenib treatment in clinical practice. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing an application of an ART1 inhibitor in improving the efficacy of sorafenib against hepatocellular carcinoma.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The use of an ART1 inhibitor in the preparation of a drug for improving the efficacy of sorafenib in hepatocellular carcinoma.

[0009] Preferably, the hepatocellular carcinoma is a hepatocellular carcinoma with high ART1 expression; the improved efficacy of sorafenib against hepatocellular carcinoma is manifested in that, compared with sorafenib alone, the combined use of sorafenib and ART1 inhibitor can synergistically inhibit the proliferation of hepatocellular carcinoma cells.

[0010] A pharmaceutical composition for treating hepatocellular carcinoma, comprising sorafenib, an ART1 inhibitor, and a pharmaceutically acceptable carrier.

[0011] Preferably, the ART1 inhibitor is meso-iodobenzylguanidine.

[0012] Preferably, the mass ratio of sorafenib to the ART1 inhibitor is 1:1.

[0013] A kit for improving the efficacy of sorafenib in treating hepatocellular carcinoma, comprising: The first formulation contains sorafenib; The second formulation contains an ART1 inhibitor.

[0014] Preferably, the ART1 inhibitor is meso-iodobenzylguanidine.

[0015] Preferably, the unit dose of sorafenib in the first formulation is 10-100 mg.

[0016] Preferably, the unit dose of the ART1 inhibitor in the second formulation is 10-100 mg.

[0017] A method for screening hepatocellular carcinoma patients suitable for combination therapy with sorafenib and an ART1 inhibitor includes the following steps: S1: Obtain tumor tissue samples from patients with hepatocellular carcinoma; S2: Detect the expression level of ART1 protein in the sample; S3: Patients with ART1 expression levels higher than the control threshold are identified as suitable for receiving the combined treatment.

[0018] The beneficial effects of this invention are as follows: 1. This invention identifies ART1 as a new target for improving the efficacy of sorafenib; based on the finding that the levels of ART1 and its catalyzed single ADP ribosylation modification are significantly higher in hepatocellular carcinoma tissues than in adjacent normal tissues, a therapeutic approach of sensitizing sorafenib by inhibiting ART1 is proposed; this provides a new and effective combination therapy approach based on existing drugs, in addition to developing entirely new drugs, to address the clinical dilemma of reduced responsiveness in patients after using sorafenib.

[0019] 2. At the cellular level, CCK-8 experiments confirmed that the combination of the ART1 inhibitor MIBG and sorafenib significantly inhibited the proliferation of SMMC-7721 liver cancer cells compared to either drug alone, demonstrating a clear synergistic effect. At the animal level, nude mouse xenograft model experiments further demonstrated that, compared to sorafenib monotherapy, the combination therapy with MIBG significantly reduced tumor volume, validating in vivo that this combination regimen effectively inhibits tumor growth and enhances the anti-tumor efficacy of sorafenib.

[0020] 3. Based on the biological characteristic of high ART1 expression in hepatocellular carcinoma, this invention makes the combination therapy regimen particularly suitable for hepatocellular carcinoma patients with high ART1 expression, providing a potential biomarker direction for personalized and precision treatment. In addition, the ART1 inhibitors used in this regimen (such as MIBG) are known compounds, and their combination with sorafenib can enter the clinical development and validation stage more quickly, accelerating the solution to the current drug resistance problem in targeted therapy for hepatocellular carcinoma, and has important clinical application value and market prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of differentially expressed genes (DEGs) between HCC tissue and normal liver tissue in this invention (in the figure: A: volcano diagram, B: heat map); Figure 2 This is a schematic diagram of differentially expressed genes (DEGs) related to single ADP ribosylation between HCC tissue and normal liver tissue in this invention (Figure: A: Venn diagram showing the intersection of differentially expressed genes and genes related to single ADP ribosylation; B: Box plot showing the expression of each differentially expressed gene in HCC tissue (Tumor) and normal liver tissue (Normal). (***: P<0.001)). Figure 3 This is a schematic diagram illustrating the detection of single ADP ribosylation modification expression in hepatocellular carcinoma and adjacent normal tissues using the immunohistochemical method of this invention (in the figure: the upper part is hepatocellular carcinoma tissue, the lower part is adjacent normal tissue; the left side is low magnification, and the right side is high magnification). Figure 4This is a schematic diagram illustrating the immunohistochemical detection of ART1 expression in hepatocellular carcinoma and adjacent normal tissues according to the present invention (in the diagram: the upper part is hepatocellular carcinoma tissue, the lower part is adjacent normal tissue; the left side is low magnification, and the right side is high magnification). Figure 5 This is a graph showing the difference in single ADP ribosylation modification and ART1 expression levels between hepatocellular carcinoma and adjacent normal tissues in this invention (***: P < 0.001) (**: P < 0.01) (In the figure: A: single ADP ribosylation modification level, B: ART1 expression level). Figure 6 The expression diagram of ART1 in normal hepatocytes THLE-3 and various liver cancer cells Huh-7, MHCC97H, and SMMC7721 is shown (****: P < 0.0001). Figure 7 This is a schematic diagram showing the cell proliferation rate of SMMC7721 liver cancer cells as a function of dose and IC50 values ​​for sorafenib and MIBG, respectively, according to the present invention. Figure 8 This is a schematic diagram illustrating the effect of the ART1 inhibitors MIBG, sorafenib alone and in combination on the proliferation of SMMC7721 cells using CCK8 assays (****: P < 0.0001). (In the figure: NC: negative control group; Sorafenib (8 μM): sorafenib monotherapy group, drug concentration 8 μM; MIBG (200 μM): MIBG monotherapy group, drug concentration 200 μM; Sorafenib + MIBG: combination therapy group. Cell proliferation rate was measured after 24 h of cell treatment, with the NC group as 100% normalized). Figure 9 This diagram illustrates the subcutaneous xenografts in SMMC7721 nude mice treated with sorafenib alone and in combination with the ART1 inhibitor MIBG (*: P < 0.05, indicating statistical significance). (Figure A: Group 1 is the sorafenib-only group; Group 2 is the sorafenib + MIBG combined group. Figure B: Xenograft growth curve). Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1: Study on the effect and mechanism of ART1 on the responsiveness of hepatocellular carcinoma to sorafenib 1. Preliminary analysis of marylation level changes in HCC using bioinformatics database data. 1.1 Data Acquisition and Differential Gene Analysis Transcriptome sequencing expression profiles of all TCGA-HCC patients were obtained from the TCGA database. After data integration, 361 HCC tissue samples and 41 normal liver tissue samples were obtained. The transcriptome sequencing expression profiles of 402 samples were imported into R4.0.0 software. The differentially expressed genes between HCC tissues and normal liver tissues were analyzed using the "limma" package. The selection criteria were: |log2FC|>0.585, P-value<0.05. The differential analysis results were visualized using the "ggplot2" and "ggrepel" packages.

[0024] 1.2 Acquisition of genes related to single ADP ribosylation All genes related to the "hsa00760 metabolic pathway" were obtained from the KEGG database (https: / / www.genome.jp / kegg / ), and all genes related to the "R-HAS-196807 metabolic pathway" were obtained from the Reactome database (https: / / curator.reactome.org / ). Combined with single ADP ribosylase genes from the literature, a total of 143 single ADP ribosylation-related genes were identified. The chromosomal locations of these genes were located using the "AnnoProbe" package and the "airway" dataset, and visualized using the "RCircos" package.

[0025] 1.3 Screening of Intersecting Genes The "VennDiagram" package was used to obtain the intersection genes of differentially expressed genes and single ADP ribosylation-related genes between the HCC tissue group and the normal liver tissue group, and Venn diagrams were generated. The "ggpubr" package was used to visualize the expression levels of the intersection genes between the HCC tissue group and the normal liver tissue group.

[0026] 2. Immunohistochemical analysis of changes in ART1 and MARylation in HCC tissues Follow the instructions in the kit manual. The specific steps are as follows: (1) Soak the slices in xylene I for 20 min and then in xylene II for 20 min to dewax them; (2) Soak the slices in anhydrous ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 minutes each; (3) Wash with PBS 3 times, 5 min each time; (4) Place the slices in an EDTA antigen retrieval solution and retrieval in a pressure cooker for 15 minutes; (5) After cooling to room temperature, wash three times with PBS for 5 minutes each time; (6) Add reagent 1 and incubate at room temperature in the dark for 20 min; (7) Wash with PBS 3 times, 5 min each time; (8) Add reagent 2, incubate at room temperature for 30 min, and after drying reagent 2, add the diluted primary antibody directly; (9) Dilute the primary antibody with ready-to-use goat serum at the following concentrations: MARylation (1:200) and ART1 (1:150). Incubate overnight at 4°C. (10) Wash with PBS 3 times, 5 min each time; (11) Add reagent 3 and incubate at room temperature for 20 min; (12) Wash with PBS 3 times, 5 min each time; (13) Add reagent 4 drops and incubate at room temperature for 10 min; (14) Wash with PBS 3 times, 5 min each time; (15) Add 50µL LDAB working solution to each slide and stain for 30 seconds. (16) Stain with hematoxylin for 1 min 30 s, then rinse with tap water for 1 min.

[0027] (17) Add hydrochloric acid alcohol for 3 seconds, lithium carbonate for 3 seconds; (18) Soak in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 5 min each, and then soak in xylene for 15 min. (19) Mount the slides with neutral resin, observe them under a microscope and score them; (20) Immunohistochemical results: The expression of ART1 and the level of MARylation in tumor cells were graded by a comprehensive score of the percentage of positive cells and staining intensity; I. Number of positive cells: Observe the proportion of positive cells in every 100 tumor cells. 5%~25% of positive cells is 1 point, 26%~50% of positive cells is 2 points, 51%~75% of positive cells is 3 points, and 76%~100% of positive cells is 4 points. II. Positive staining intensity: No staining is 0 points, pale yellow is 1 point, brownish-yellow is 2 points, and dark brown is 3 points. The product of the above two is the positive grade: 0 points is negative (-), 1-4 points is weakly positive (1+), 5-8 points is moderately positive (2+), and 9-12 points is strongly positive (3+). This process is performed by two pathologists in a double-blind manner, and the average value is used for statistical analysis. (21) Data analysis was performed using GraphpadPrism 9.0.0 software. The statistical method selected was Kruskal-Wallis univariate analysis. P < 0.05 was considered statistically significant.

[0028] 3. Cell Culture THLE-3, Huh-7, MHCC97H, and SMMC7721 cells were cultured in DMEM complete medium. The complete medium was prepared as follows: 10% fetal bovine serum + 1% triple antibiotics + DMEM basal medium.

[0029] 3.1 Cell resuscitation (1) Remove the cells to be revived from liquid nitrogen and heat them in a water bath at 37°C until the cryopreservation solution is completely thawed.

[0030] (2) Add 1 mL of complete culture medium to the centrifuge tube, then add the cell suspension, mix well, and centrifuge at 1000 rpm for 3 min.

[0031] (3) Discard the supernatant and add 1 mL of complete culture medium to the precipitate to resuspend the cells.

[0032] (4) Take 5 mL of complete culture medium into a culture dish, add cell suspension, and mix well.

[0033] (5) Culture the cells in a constant temperature incubator containing 5% CO2 at 37°C.

[0034] 3.2 Cell passage (1) Discard the old culture medium and wash 3 times with PBS.

[0035] (2) Add 2 mL of 0.25% trypsin to the culture dish; (3) Add 3 mL of complete culture medium to the culture dish to stop digestion, gently pipette until the cells are completely detached, collect the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm for 3 min; (4) Discard the supernatant and resuspend the cells in 2 mL of complete culture medium; (5) Prepare two clean culture dishes, add 5 mL of complete culture medium and 1 mL of cell suspension to each dish, and mix well; (6) Culture the cells in a constant temperature incubator containing 5% CO2 at 37°C.

[0036] 3.3 Cell cryopreservation (1) Discard the old culture medium and wash 3 times with PBS.

[0037] (2) Add 2 mL of 0.25% trypsin to the culture dish.

[0038] (3) Add 3 mL of complete culture medium to the culture dish to stop digestion, gently pipette until the cells are completely detached, collect the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm for 3 min.

[0039] (4) Discard the supernatant and resuspend the cells in 1 mL of serum-free cell cryopreservation solution.

[0040] (5) Aspirate the cell suspension into cryovials and place them in an ultra-low temperature freezer at -80°C. After 24 hours, transfer them to liquid nitrogen.

[0041] 4. Western Blot detection of ART1 expression in liver cancer cell lines THLE-3 was used as a control, and Huh-7, SMMC7721 and MHCC97H cells were used as research subjects.

[0042] 4.1 Cell culture is the same as in method 3.

[0043] 4.2 Extraction and concentration determination of total cell protein (1) Remove the culture dish and wash it three times with sterile PBS solution.

[0044] (2) Add 2 mL of 0.25% trypsin for digestion, then add 3 mL of complete culture medium. After the cells are completely detached, transfer the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 3 min.

[0045] (3) Discard the supernatant, add 1 mL of sterile PBS solution to wash once, and centrifuge at 1000 rpm for 3 min.

[0046] (4) Discard the supernatant and add 100µL of cell lysis buffer. The cell lysis buffer is prepared in the following ratio: RIPA lysis buffer: phosphatase inhibitor: protease inhibitor: PMSF = 100: 1: 1: 1.

[0047] (5) After lysing on ice for 1 hour, centrifuge at 12,500 rpm and 4°C for 20 minutes using a low-temperature high-speed centrifuge and take the supernatant.

[0048] (6) Take the lysis supernatant and add it to a 96-well plate for BCA determination in the following proportion: 200 µL BCA working solution + 16 µL double-distilled water + 4 µL lysis supernatant per well (note that no bubbles should be generated); incubate at 37°C for 30 min.

[0049] (7) Measure the OD value at 570 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the protein concentration and loading amount using the standard curve.

[0050] (8) Add 5× protein loading buffer (4:1 ratio) to the lysis buffer supernatant and incubate at 100°C for 5 min.

[0051] 4.3 SDS-PAGE Gel Preparation (1) Clean the glass slide and put it in an oven to dry.

[0052] (2) Assemble the glass slide with the adhesive frame, and prepare the upper and lower adhesive layers as shown below:

[0053] (3) Add 5 mL of the prepared lower layer adhesive to the grooves of the two glass slides (be gentle and avoid creating small air bubbles), then inject 1 mL of anhydrous ethanol to press it flat. When a visible dividing line appears, discard the anhydrous ethanol.

[0054] (4) Add 1.5 mL of the prepared top layer adhesive to the grooves of the two glass slides, insert a clean adhesive comb, and let it stand for about 15 minutes to solidify before use.

[0055] 4.4 Electrophoresis (1) Assemble the electrophoresis rack, SDS-PAGE gel and electrophoresis apparatus according to the instructions. Add the newly prepared electrophoresis solution to the inner tank and the recycled electrophoresis solution to the outer tank.

[0056] (2) Slowly pull out the gel comb and add the colored pre-stained protein marker and protein sample in sequence.

[0057] (3) Connect the power supply, first set the voltage to 60V for 30 minutes, then set it to 120V for 90 minutes, and stop electrophoresis when the protein sample reaches the bottom of the lower gel.

[0058] 4.5 Transfer (1) Cut PVDF membranes of appropriate size, mark them, soak them in methanol for 1 minute, and then soak them in electroporation solution.

[0059] (2) Take out the SDS-PAGE gel, place it in the pre-cooled electroporation solution, and cut out the area where the target protein and internal control are located.

[0060] (3) Assemble the film in the order of black glue and white film, which is: white side - filter paper - PVDF membrane - glue - filter paper - black side.

[0061] (4) Place the assembled sandwich clip into the electrophoresis apparatus, add electrophoresis solution to soak, and soak the periphery of the electrophoresis tank with a mixture of ice and water.

[0062] (5) Connect the power supply, set the current to a constant current of 250mA, and the film transfer time to 60min.

[0063] 4.6 Antibody incubation (1) After the transfer is completed, take out the film and place it in 5% skim milk powder and seal it at room temperature for 1 hour.

[0064] (2) Wash the PVDF membrane in TBST solution for 5 min, 3 times.

[0065] (3) Prepare primary antibodies using primary and secondary antibody dilution buffers at the following dilution ratios: mouse ART1 monoclonal antibody (1:8000) and mouse β-actin monoclonal antibody (1:10000).

[0066] (4) Place the PVDF membrane in the diluted primary antibody and incubate overnight at 4°C. The next day, wash the membrane three times with TBST solution, 5 min each time.

[0067] (5) Dilute the secondary antibody with TBST, incubate at room temperature for 1 hour, and then wash with TBST solution 3 times, 5 min each time.

[0068] 4.7 Exposure Imaging Analysis (1) Prepare ECL luminescent solution according to the instructions, with equal volumes of reagent A and reagent B.

[0069] (2) Remove the TBST solution from the membrane surface, then place it in a wax plate and add luminescent liquid to make it evenly cover the membrane surface.

[0070] (3) Use a chemiluminescence imaging system to take photos.

[0071] (4) Gray values ​​were detected using Image J software and data analysis was performed using Graphpad Prism 9.0.0 software. One-way ANOVA was selected as the statistical method. P < 0.05 was considered statistically significant. All experiments were repeated three times or more.

[0072] 5. CCK-8 cell proliferation assay to detect the IC50 values ​​of sorafenib and the ART1 inhibitor MIBG in SMMC7721 cells. 5.1 Cell Culture The specific steps are the same as in method 3. 5.2 Cell Plating in 96-Well Plates (1) Take SMMC-7721 cells in the logarithmic growth phase, remove the old culture medium, wash once with PBS, and then add an appropriate amount of trypsin for digestion.

[0073] (2) After observing under a microscope that the cells have become rounded and the gaps between them have widened, add serum-containing complete culture medium to stop digestion, and gently pipette to prepare a single-cell suspension. Use a cell counting chamber to count the cells, and dilute the cell suspension to 5×10⁻⁶. 4 Density of cells / mL. (3) Seed the cells into 96-well plates, adding 100 μL of cell suspension to each well. That is, the number of cells per well is 5000. Divide the cells into experimental wells (containing cells, culture medium, and different concentrations of drugs), negative control wells (containing cells, culture medium, and no drugs), and blank wells (containing only culture medium and no cells). In addition, add 100-200 μL of PBS to each well in the outermost ring of the 96-well plate.

[0074] (4) Place the inoculated 96-well plate in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere completely and return to the logarithmic growth phase.

[0075] 5.3 Drug administration (1) Sorafenib and MIBG were diluted to (4 μM, 8 μM, 12 μM, 16 μM, 20 μM) and (80 μM, 160 μM, 240 μM, 320 μM, 400 μM, 480 μM) in sterile EP tubes using complete culture medium.

[0076] (2) Remove the 96-well plate that has been pre-cultured for 24 hours from the incubator, aspirate the old culture medium from each well, and add 100 μL of fresh complete culture medium containing different concentrations of the drug to each well. Five replicates should be set for each concentration to ensure statistical validity. The negative control group is added with complete culture medium containing an equal amount of solvent, and the blank control group is added with complete culture medium without cells.

[0077] (3) Place the 96-well plate with the added drug back into the incubator and continue culturing for 24 hours.

[0078] 5.4 CCK8 testing (operation in the dark) (1) Prepare the required total volume of working solution according to the ratio of complete culture medium: CCK-8 reagent = 10:1.

[0079] (2) Take out the 96-well plate that has been pre-cultured for 24 hours from the incubator, carefully remove the old culture medium from each well, add 110 μl of working solution to each well, and put it back into the incubator for 2 hours.

[0080] (3) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0081] 5.5 Data Processing and IC50 Calculation (1) Calculate the average OD value of replicates for different concentration groups. Then calculate the cell proliferation rate using the following formula:

[0082] (2) Using the logarithm of drug concentration as the X-axis and cell proliferation rate as the Y-axis, the dose-response curve was plotted using GraphPadPrism software. The curve was fitted and the half-inhibitory concentration (IC50) was calculated using the nonlinear regression analysis function in the software.

[0083] 6. CCK-8 cell proliferation assay to detect changes in HCC proliferation after sorafenib combined with ART1 inhibitor MIBG Drug dosage and grouping are as follows: ① Control group: containing cells + solvent (DMSO + PBS) ②MIBG monotherapy group: containing cells + 200 μM MIBG ③Sorafenib monotherapy group: containing cells + 8μM sorafenib ④ Combination therapy group: containing cells + 8μM sorafenib + 200μM MIBG 7. Effects of sorafenib combined with the ART1 inhibitor MIBG on HCC xenograft growth in a nude mouse subcutaneous xenograft model. 7.1 Establishment of a subcutaneous xenograft model in nude mice (1) Collect SMMC-7721 cells in the logarithmic growth phase by trypsin digestion and centrifugation. Wash twice with PBS, resuspend the cells and count them. Adjust the cell density to 1×10⁻⁶. 7 cells / mL (i.e., 1×10⁻⁶ cells / mL) 6 (cells).

[0084] (2) Using a 1mL insulin syringe, draw up the cell suspension and subcutaneously inject 0.1mL of the cell suspension (containing 1×10⁻⁶ cells) into the right axilla of each nude mouse. 6 (Each cell). Small wheals can be seen under the skin after injection.

[0085] (3) After vaccination, observe 3 times a week. Once the tumor can be clearly felt after 1 week, medication can be started.

[0086] 7.2 Animal grouping and drug administration (1) The tumor-bearing nude mice were randomly divided into two groups of six each.

[0087] ① Sorafenib monotherapy group (Sorafenib, 30 mg / kg); ② Combination therapy group (Sorafenib 30mg / kg + MIBG 30mg / kg) (2) Administration method and frequency: intraperitoneal injection, once every other day, for a total of 7 times.

[0088] (3) Observe the mental state, activity level, appetite and skin condition of nude mice daily.

[0089] 7.3 Tumor Measurement and Data Collection (1) Starting from the first administration, measure the long diameter (L) and short diameter (W) of the tumor before each administration, and calculate the tumor volume according to the following formula: (Record the data and plot the tumor growth curve)

[0090] (2) Tumor removal: After the administration of the drug (2 days after the last administration), nude mice were euthanized by cervical dislocation. The subcutaneous tumor was completely dissected under sterile conditions, the connective tissue was removed, and the tumor was removed.

[0091] (3) Photograph preservation: Arrange the dissected tumors of each group neatly and take photos for comparison.

[0092] (4) Preservation: The tumor tissue was aliquoted, and a portion was flash-frozen in liquid nitrogen and stored at -80°C (for subsequent Western Blot molecular mechanism detection), while another portion was fixed in 10% buffered paraformaldehyde solution (for HE staining and immunohistochemistry).

[0093] Experimental results: 1. Preliminary analysis of bioinformatics database data on changes in marylation levels in HCC Differential expression analysis was performed on transcriptome data from hepatocellular carcinoma tissues and normal liver tissues using the TCGA database. The results showed that a total of 5424 differentially expressed genes were identified, of which 1395 were upregulated and 3029 were downregulated. Figure 1 ).

[0094] The intersection of differentially expressed genes in HCC with 143 single ADP ribosylation-related genes was used to screen out 18 single ADP ribosylation-related genes that were differentially expressed in HCC tissues. Figure 2 ).

[0095] 2. Immunohistochemical analysis of changes in ART1 and MARylation in HCC tissues The results showed that MARylation modification was mainly located in the cytoplasm and cell membrane, presenting as brownish-yellow granular distribution. Nuclear staining was observed in some cells. In hepatocellular carcinoma tissue, the positive staining signal was significantly stronger than that in adjacent normal tissue. Figure 3 ART1 expression is mainly found in the cytoplasm and cell membrane. Figure 4 ).

[0096] In 30 HCC tissue samples, single ADP ribosylation modification was observed, with 10 showing weak positive expression, 17 showing moderate positive expression, and 3 showing strong positive expression. In 10 adjacent normal control tissue samples, single ADP ribosylation modification was also observed, with all 10 showing weak positive expression. The level of single ADP ribosylation modification was significantly higher in HCC tissues than in adjacent normal tissues (P < 0.001). ART1 expression levels were also observed, with 9 showing weak positive expression, 18 showing moderate positive expression, and 3 showing strong positive expression in the 30 HCC tissue samples; and 8 showing weak positive expression and 2 showing moderate positive expression in the 10 adjacent normal control tissues. The ART1 expression level was significantly higher in HCC tissues than in adjacent normal tissues (P < 0.01).

[0097] The levels of single ADP ribosylation modification in hepatocellular carcinoma and adjacent normal tissues (controls) are as follows:

[0098] ART1 expression levels in hepatocellular carcinoma and adjacent normal tissues (control):

[0099] 3. Western Blot detection of ART1 expression in hepatocellular carcinoma cell lines The results showed that ART1 expression levels were increased to varying degrees in all three hepatocellular carcinoma cell lines. ART1 expression was significantly higher in SMMC7721, Huh-7, and MHCC97H cells than in THLE-3 cells (P < 0.0001, P < 0.001, and P < 0.001, respectively). Significant differences in ART1 protein expression levels were observed among the different hepatocellular carcinoma cell lines (Huh-7, MHCC97H, and SMMC7721). Compared with normal hepatocellular carcinoma cells (THLE-3), SMMC7721 cells showed the highest ART1 expression level, followed by Huh-7 cells, while MHCC97H cells showed relatively lower expression levels. Figure 6 Therefore, SMMC7721 cells were used as the subject of subsequent experiments.

[0100] 4. CCK-8 cell proliferation assay to detect the IC50 values ​​of sorafenib and the ART1 inhibitor MIBG in SMMC7721 cells. SMMC7721 cells were treated with different concentrations (0, 4, 8, 12, 16, 20 μM) of sorafenib for 24 h, and the cell proliferation rate was detected by CCK-8 assay. Figure 7 As shown in the left image, the IC50 value of sorafenib treatment for SMMC7721 cells for 24 hours, calculated using GraphPadPrism software, is approximately 8.05 μM.

[0101] The inhibitory effect of MIBG on the proliferation of SMMC7721 cells was detected using the same method. MIBG concentration gradients of 0, 80, 160, 240, 320, 400, and 480 μM were set, and cell proliferation rate was measured after 24 h of treatment. Figure 7 (Right) shows the proliferation of SMMC7721 cells. According to the fitted curve, the IC50 value of SMMC7721 cells treated with MIBG for 24 h was approximately 218.2 μM.

[0102] 5CCK-8 cell proliferation assay to detect changes in HCC proliferation after sorafenib combined with the ART1 inhibitor MIBG Based on the established IC50 values ​​of sorafenib and MIBG on SMMC7721 cells, this invention further explores whether the combined application of the two drugs has a synergistic inhibitory effect on SMMC7721 cell proliferation. The CCK-8 assay was used to detect the effects of sorafenib (8 μM) alone, MIBG (200 μM) alone, and the combined treatment of both drugs on the proliferation rate of SMMC7721 cells after 24 hours. Figure 8As shown, compared with the control group, the sorafenib combined with MIBG group had the most significant inhibitory effect on cell proliferation, which was statistically significant (P < 0.0001).

[0103] 6. Effect of Sorafenib Combined with ART1 Inhibitor MIBG on HCC Xenograft Growth in Nude Mice Subcutaneous Xenograft Model SMMC7721 cells were inoculated subcutaneously into the right axilla of nude mice, and the tumor-bearing nude mice were randomly divided into two groups (n=6 per group). ① Sorafenib monotherapy group: Sorafenib 30 mg / kg was administered intraperitoneally every other day for a total of 7 doses.

[0104] ② Combination therapy group (Sorafenib+MIBG): Sorafenib 30mg / kg was administered intraperitoneally in combination with MIBG 30mg / kg, every other day, for a total of 7 doses.

[0105] like Figure 9 As shown in Figure B, the tumor growth rate in the combination therapy group was significantly slower than that in the sorafenib monotherapy group. Starting from the fourth dose, the difference in tumor volume between the two groups gradually became apparent, with the tumor volume in the combination therapy group being smaller than that in the sorafenib monotherapy group.

[0106] The mean tumor volume at each time point is shown in the table below.

[0107] like Figure 9 As shown in Figure C, the tumor volume in the combination therapy group was significantly smaller than that in the sorafenib monotherapy group. Measurements showed that the average tumor volume in the sorafenib monotherapy group was 1.092 cm³. 3 The average tumor volume in the combination therapy group was 0.644 cm. 3 The tumor volume in the combination therapy group was reduced by approximately 41.0% compared to the sorafenib monotherapy group, and the difference was statistically significant (P < 0.05).

[0108] The average volume of xenografts in the two groups of nude mice under different administration frequencies is as follows:

[0109] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of an ART1 inhibitor in the preparation of a drug for improving the efficacy of sorafenib in hepatocellular carcinoma treatment.

2. The application according to claim 1, characterized in that, The hepatocellular carcinoma mentioned is hepatocellular carcinoma with high ART1 expression; the improved efficacy of sorafenib against hepatocellular carcinoma is demonstrated by the fact that, compared with sorafenib alone, the combined use of sorafenib and ART1 inhibitors can synergistically inhibit the proliferation of hepatocellular carcinoma cells.

3. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that, It includes sorafenib, ART1 inhibitors, and pharmaceutically acceptable carriers.

4. The pharmaceutical composition according to claim 3, characterized in that, The ART1 inhibitor is meso-iodobenzylguanidine.

5. The pharmaceutical composition according to claim 4, characterized in that, The mass ratio of sorafenib to the ART1 inhibitor is 1:

1.

6. A kit for improving the efficacy of sorafenib in treating hepatocellular carcinoma, characterized in that, Include: The first formulation contains sorafenib; The second formulation contains an ART1 inhibitor.

7. The reagent kit according to claim 6, characterized in that, The ART1 inhibitor is meso-iodobenzylguanidine.

8. The reagent kit according to claim 7, characterized in that, The unit dose of sorafenib in the first formulation is 10-100 mg.

9. The reagent kit according to claim 7, characterized in that, The unit dose of the ART1 inhibitor in the second formulation is 10-100 mg.

10. A method for screening hepatocellular carcinoma patients suitable for combination therapy with sorafenib and an ART1 inhibitor, characterized in that, Including the following steps: S1: Obtain tumor tissue samples from patients with hepatocellular carcinoma; S2: Detect the expression level of ART1 protein in the sample; S3: Identify patients whose ART1 expression levels are higher than the control threshold as suitable for receiving the combined treatment.

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