Use of asct2 small molecule inhibitors and pharmaceutical compositions

Abstract

The application of an ASCT2 small molecule inhibitor in the preparation of a drug for enhancing the chemotherapy sensitivity of tumor doxorubicin-resistant cells, wherein the inhibitor is ZR001, the molecular formula of which is C 35 H 39 N3O3, and the structural formula is as follows: The combination of ZR001 and doxorubicin in the present application can significantly improve the sensitivity of liver cancer cell drug-resistant strains to doxorubicin, thereby improving the inhibition of cell proliferation by doxorubicin and achieving excellent therapeutic effect, and the inhibition rate of liver cancer tumor growth reaches 70%, which is significantly higher than 20% of single ADM single drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to the application of ASCT2 small molecule inhibitors and pharmaceutical compositions. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a common and highly lethal malignant tumor in my country. Early symptoms are often insidious, and more than 70% of patients are diagnosed at an advanced stage, where they can only receive systemic treatment such as chemotherapy. Doxorubicin (ADM) is a classic anthracycline broad-spectrum chemotherapy drug. Although it has good anti-tumor activity, its efficacy is often limited by the development of chemotherapy resistance.

[0003] Chemotherapy resistance in liver cancer cells is closely related to metabolic reprogramming, with glutamine metabolism playing a crucial role in maintaining cellular energy supply and antioxidant homeostasis. ASCT2, a major protein mediating transmembrane glutamine transport, is overexpressed in various tumors. High ASCT2 activity can enhance intracellular glutathione (GSH) synthesis and improve oxidative stress tolerance, thereby leading to tumor cell resistance to chemotherapeutic drugs such as doxorubicin. Previous studies have confirmed that ASCT2 is highly expressed in various drug-resistant strains, but research on its targeted inhibition to improve chemosensitivity is still limited.

[0004] Regarding doxorubicin, the development of doxorubicin resistance in cancer cells is the result of multiple pathways and mechanisms, such as overexpression of drug efflux pumps like P-gp, hyperglutamine metabolism, Topo II abnormalities at the target and gene levels, enhanced DNA repair, p53 mutations, and microenvironmental influences. Furthermore, because different tumors have completely different driver genes, ABC transporter expression profiles, DNA repair subtypes, metabolic phenotypes, and microenvironments, the resistance mechanisms to doxorubicin do not differ among different drug-resistant cancer cell lines. This is the core reason why some methods for improving the sensitivity of drug-resistant cancer cell lines to doxorubicin are effective in some drug-resistant tumor cell lines but ineffective in others. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the ASCT2 small molecule inhibitor ZR001 in enhancing the chemosensitivity of doxorubicin-resistant liver cancer cells.

[0006] Based on the above effects, a combination drug composition of ZR001 and doxorubicin is provided to improve the therapeutic effect on liver cancer.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The application of an ASCT2 small molecule inhibitor in the preparation of a drug that enhances the chemosensitivity of doxorubicin-resistant tumor cells, wherein the small molecule inhibitor is ZR001, and its molecular formula is C35 H 39 N3O3 has the following structural formula: .

[0009] Furthermore, the tumor-resistant cells are drug-resistant liver cancer cells.

[0010] A pharmaceutical composition containing the ASCT2 small molecule inhibitor ZR001, characterized in that: the pharmaceutical composition is composed of the above-mentioned small molecule inhibitor ZR001 and doxorubicin.

[0011] The above-mentioned drug composition can improve the sensitivity of drug-resistant liver cancer cell lines to doxorubicin.

[0012] Furthermore, in the pharmaceutical composition, the concentration of doxorubicin is 1 μM and the concentration of ZR001 is 5 μM.

[0013] The present invention has the following technical effects: In this invention, by combining ZR001 with doxorubicin, the sensitivity of drug-resistant liver cancer cell lines to doxorubicin can be significantly improved, thereby enhancing the inhibitory effect of doxorubicin on cell proliferation and achieving excellent therapeutic effects. The tumor growth inhibition rate of liver cancer reaches 70%, which is significantly higher than the 20% of ADM monotherapy. Attached Figure Description

[0014] Figure 1 This invention describes the process of investigating glutamine metabolism and redox levels in SMMC-7721 doxorubicin-resistant cells.

[0015] Figure 2 This invention explores how glutamine affects the redox balance and thus the sensitivity of drug-resistant cells to doxorubicin.

[0016] Figure 3 This invention explores the effect of the glutamine transporter ASCT2 on the drug resistance of doxorubicin strains in liver cancer.

[0017] Figure 4 The ASCT inhibitor ZR001 in this invention enhances the sensitivity of drug-resistant cells to doxorubicin.

[0018] Figure 5 In this invention, ZR001, in combination with doxorubicin, disrupts the redox balance of drug-resistant cells, leading to DNA damage.

[0019] Figure 6 In this invention, ZR001, in combination with doxorubicin, inhibits the mTOR signaling pathway and cell cycle in drug-resistant cells.

[0020] Figure 7In this invention, NAC and GSH partially reverse the cell damage caused by the combined treatment of ZR001 and doxorubicin.

[0021] Figure 8 In this invention, the combined treatment of ZR001 and doxorubicin inhibits the proliferation of doxorubicin-resistant cells in a nude mouse xenograft tumor model. Detailed Implementation

[0022] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0023] Example 1 Cell protein extraction and quantification (1) Discard the culture medium in the six-well plate, add 1 mL of 1×PBS solution to each well for washing, repeat twice, add freshly prepared RIPA high-efficiency lysis buffer (with appropriate amounts of protease inhibitor and phosphatase inhibitor), and place on ice for lysis for 15 min. After lysis, carefully scrape the protein from the dish with a protein scraper, collect it into a centrifuge tube with a pipette, and then sonicate it (conditions: 30% power, run for 3 s, pause for 3 s, sonicate for 1 min). After sonication, place at 4 ℃, centrifuge at 12000 rpm for 15 min, and transfer the supernatant to another clean 1.5 mL centrifuge tube, place on ice to allow protein degradation.

[0024] (2) Protein quantification: Take 7 mL of protein stock solution, dilute it 10 times with ultrapure water, and then use the BCA protein concentration assay kit to determine the protein concentration. The specific experimental steps are as follows: a. Dilute the protein standard with ultrapure water at a 10-fold dilution ratio. Add the standard and sample to a 96-well microplate, adding 20 μL of liquid to each well. Set up three replicates for each sample.

[0025] b. Prepare the BCA working solution according to the ratio of BCA reagent A solution to BCA reagent B solution = 50:1. Prepare and use immediately. After mixing thoroughly, add 180 μL of BCA working solution to each well.

[0026] c. Place the microplate in a 60 ℃ oven and heat for 15 min.

[0027] d. Load the processed 96-well plate into the detection chamber of a multi-functional microplate reader and perform absorbance scanning at a monochromatic wavelength of 562 nm. Establish a standard curve (R²≥0.99) based on the optical density values ​​of the serially diluted standard wells. Substitute the average OD value of three repeated measurements of the sample wells into the equation to calculate the actual protein concentration (μg / mL).

[0028] e. First, adjust the protein concentration of each sample to 5 μg / μL using RIPA high-efficiency lysis buffer. Then, add 0.2 times the sample volume of 5× SDS-PAGE loading buffer to the sample, vortex thoroughly for 30 s, and then transfer to a preheated 100 ℃ dry incubator for 10 min of heat denaturation. Centrifuge to remove condensate from the tube wall, aliquot into low-adsorption centrifuge tubes, label with sample number, and temporarily store at -20 ℃ for short-term use. For long-term storage, transfer to -80 ℃.

[0029] Western Blot (1) Gel preparation: Determine the molecular weight of the target protein and select different concentrations of separating gels. Accurately measure the volume ratio of 30% acrylamide / methylenebisacrylamide mixture (29:1), Tris-HCl buffer (1.5 M, pH = 8.8), and deionized water to prepare sufficient gel preparation solution. The glass plate assembly was treated with an ultrasonic cleaner (40 kHz) for 15 min and then rinsed with ultrapure water. The clean glass plate was vertically installed on the gel preparation frame, and ultrapure water was injected for a seal test (no leakage after standing for 5 minutes) to confirm that the integrity of the device met the requirements of the electrophoresis experiment. The pre-cooled separating gel solution was slowly injected, with the injection volume being 80% of the gel preparation mold volume. The separating gel was flattened using ultrapure water and solidified at a constant temperature of 25 ℃ for 30 min. After complete solidification, the ultrapure water was removed, and residual liquid at the interface was absorbed using double-layer dust-free filter paper. Prepare a stacking gel solution (4% acrylamide, Tris-HCl pH = 6.8 buffer system) and slowly inject it into the upper space of the separating gel until it is 5 mm from the top. Quickly insert a 15-well gel casting comb (1.5 mm pore size) and use a micropipette to remove air bubbles between the teeth. After polymerization at 25 °C in the dark for 15 minutes, store the gel system in a humidified sealed box for later use. Before use, check the integrity of the comb pores. The optimal separation range for different concentrations of separating gel is shown in Table 1.

[0030] Table 1:

[0031] The preparation methods for stacking gel and non-separating gel are shown in Table 2.

[0032] Table 2:

[0033] (2) SDS-PAGE electrophoresis: Place the glass plate with the prepared gel into the electrophoresis clamp, and after ensuring that there is no leakage, add 1× electrophoresis buffer into the electrophoresis clamp; take out the protein sample, melt it at room temperature, vortex to mix and centrifuge; carefully remove the comb teeth on the gel, use a pipette to blow away the residual gel strands, add protein sample with the same total protein amount to each sample well, put it into the electrophoresis tank, add an appropriate amount of 1× electrophoresis buffer, and electrophoresis under constant pressure (80 V, 40 min, 120 V, 1 h).

[0034] (3) Wet transfer: Activate the polyvinylidene fluoride (PVDF) membrane by soaking it in methanol for 1 min. In pre-cooled 1× transfer buffer, prepare a "sandwich clip" in the following order: negative electrode (black clip) - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - positive electrode (white clip). Vertically insert the assembled transfer assembly into the transfer tank and inject sufficient transfer working solution until the device is completely submerged. The transfer system is placed in a low-temperature environment maintained by crushed ice, and a circulating cooling device is connected to maintain the buffer temperature ≤10 ℃. Set the electroporator to constant current mode (300mA) according to the target protein molecular weight range. (10-180kDa) Determine the transfer time (15-120 minutes).

[0035] (4) Blocking: After the PVDF membrane has been transferred, it is briefly rinsed with TBST and the position of the pre-stained protein molecular weight standard reference is marked with a waterproof marker. The membrane is completely immersed in freshly prepared blocking buffer (5% skim milk powder dissolved in TBST buffer, containing 0.1% Tween-20) and placed on a horizontal shaker (25 ℃, 60 rpm) for continuous shaking for 60 min.

[0036] (5) Primary antibody incubation: After removing the blocking solution, wash three times independently with TBST buffer (5 min / wash). Prepare the primary antibody working solution with antibody dilution buffer according to the manufacturer's recommended ratio (1:1000). Transfer the membrane to the antibody incubation box and inject sufficient primary antibody solution. Remove air bubbles and seal. Incubate on a shaker at 4 ℃ for 14-16 h.

[0037] (6) Secondary antibody incubation: After recovering the primary antibody solution, wash three times with TBST (5 min / wash). Prepare a 1:5000 dilution working solution with TBST according to the species of the secondary antibody (e.g., HRP-labeled goat anti-rabbit IgG). Immerse the membrane in the secondary antibody solution and incubate at 25 °C in the dark for 60 min.

[0038] (7) Chemiluminescence imaging: After discarding the secondary antibody solution, wash three times with TBST (5 min / time). Transfer the membrane to the imaging platform and evenly drop ECL chemiluminescence substrate (Pierce™ Enhanced CL Substrate) to completely cover the membrane surface. Use a chemiluminescence imaging system for exposure acquisition (10s-300s).

[0039] Cellular RNA extraction and reverse transcription (1) Cellular RNA extraction: a. Discard the culture medium in the six-well plate. Add 1 mL of 1×PBS solution to each well and wash twice. Add 500 μL of FreeZol Reagent to each well and incubate at room temperature for 5 min. Use a pipette to gently pipette the bottom of the plate to fully lyse the cells, then transfer them to an enzyme-free centrifuge tube. Add 150 μL of Dilution Buffer, vortex to mix, and incubate at room temperature for 5 min. Centrifuge at 12000g for 15 min at 4°C, and aspirate 500 μL of the clear supernatant into a clean enzyme-free centrifuge tube.

[0040] b. Add 500 μL of isopropanol to the centrifuge tube, invert repeatedly to mix, and let stand at room temperature for 10 min to precipitate RNA. Centrifuge at 12000 g for 15 min at 4°C, then carefully discard the liquid, being careful not to discard the precipitate.

[0041] c. Add 1 mL of 75% ethanol (prepared with DEPC water) to the tube, gently pipette the precipitate to suspend it in the ethanol, wash the precipitate thoroughly, and then centrifuge at 8000 g for 3 min at 4°C. Repeat three times.

[0042] d. Carefully discard the liquid, invert the centrifuge tube and place it on filter paper, let it stand for 30 minutes to allow the precipitate to dry.

[0043] e. Add 50-100 μL of DEPC water according to the amount of precipitate, vortex to mix the precipitate, and incubate in a 60℃ incubator for 15 min to fully dissolve the precipitate. After the precipitate has fully dissolved and homogenized, determine the RNA concentration using a NanaDrop 2000 micro spectrophotometer.

[0044] f. Perform reverse transcription or store at -80°C.

[0045] (2) Cellular RNA reverse transcription a. Removal of genomic DNA The genomic DNA removal reaction system consisted of 4 μL of 4×DNA wiper Mix, 2 μL of RNA (0.5 μg / μL), and 10 μL of RNase-free ddH2O. After preparation, the mixture was placed in a gene amplification instrument and reacted at 42℃ for 2 min.

[0046] b. Reverse transcription reaction Constructing the reverse transcription reaction system: 16 μL of the product from the previous step + 4 μL of 5×qRT SuperMix were placed in a gene amplification instrument for reaction: 25℃, 10 min → 42℃, 45 min → 85℃, 5 min. After the instrument cooled to 4℃, cDNA samples were obtained. The obtained cDNA samples were diluted with 150 μL of RNase-free ddH2O and stored at -20℃.

[0047] Real-time quantitative PCR (qPCR) (1) Primer design Gene sequences were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and primer sequences were designed using Primer Premier 5.0 software and compared online in the Blast database. Sequences for qPCR and below are shown in Table 3.

[0048] Table 3:

[0049] Cell IC50 Calculation (1) Cell plating: Wash the healthy cell lines in logarithmic growth phase with sterile PBS, add 2.5% trypsin (containing EDTA) for digestion, stop digestion with complete culture medium, blow off the cells and transfer the cell suspension to a clean, sterile centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, add 1 mL of complete culture medium to resuspend the cell pellet, and count the cells. Add 180 mL of cell suspension to each well of a 96-well plate to control the cell count to 3000 cells per well. Add an appropriate amount of 1×PBS to the wells around the perimeter of the 96-well plate for sealing, and incubate in a 37℃ CO2 incubator.

[0050] (2) Drug treatment: The drug was serially diluted using serum-free medium. Three replicates were set for each concentration. 20 μL of drug solution was added to each well, and the mixture was gently shaken and then placed in a 37℃ CO2 incubator for incubation.

[0051] (3) Cell viability assay: After 72 h of drug treatment, CCK-8 was diluted with serum-free medium to prepare 10% CCK-8 working solution. The medium in the 96-well plate was discarded, and 100 μL of CCK-8 working solution was added to each well. The plate was then placed in a 37℃ CO2 incubator for 1.5 h. After incubation, the plate was shaken on a plate shaker for 5 min, and the absorbance of each well was measured at an excitation wavelength of 450 nm using a microplate reader. The cell growth inhibition rate of each drug concentration treatment group was calculated using the solvent well as a control. The calculation formula is as follows:

[0052] ROS detection Discard the culture medium in the dish. Load the DCFH-DA probe at a ratio of DCFH-DA:serum-free medium = 1:1000. Add 1 mL of DCFH-DA working solution to each well, gently shake to mix, and incubate in a 37℃ CO2 incubator for 20 min. Discard the liquid in the dish, wash the cells with 1×PBS, then add 0.25% trypsin (EDTA-free) for digestion. After the cells have digested to a suitable level, add 100 μL of FBS to stop the digestion. Gently blow the cells off the bottom of the dish and transfer them to a 1.5 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. Resuspend and wash the cells with 1 mL of 1×PBS, centrifuge at 1000 rpm for 5 min, and repeat twice. Resuspend the cells with 500 μL of 1×PBS and detect fluorescence using flow cytometry at an excitation wavelength of 488 nm.

[0053] Detection of intracellular GSH levels (1) Cell sample processing: Discard the culture medium, gently wash the cells twice with pre-chilled 1×PBS, and aspirate any residual liquid. Add an appropriate amount of trypsin to digest the cells, centrifuge (600 xg, 4℃, 5 min) to collect the cell pellet, and record the volume of the cell pellet. Add pre-chilled protein removal reagent solution (e.g., 30 μL solution for 10 μL of cell pellet) at 3 times the volume of the cell pellet, vortex to mix for 30 s, and lyse on ice for 10 min. After lysis, centrifuge at 10,000 g for 10 min at 4℃, and collect the supernatant as the sample to be tested. The sample can be temporarily stored at 4℃. If long-term storage is required, it should be aliquoted and frozen at -70℃. Before detection, dilute with protein removal reagent solution 5-20 times according to the concentration.

[0054] (2) Preparation of standard curve: Take 10 mM GSSG stock solution and dilute it sequentially with protein removal reagent solution to prepare standard solutions of 15, 10, 2, 1, and 0.5 μM (freshly prepared, avoid repeated freeze-thaw cycles). To detect GSSG content, add 20 μL of diluted GSH removal auxiliary solution and 4 μL of GSH removal reagent working solution to every 100 μL of standard solution, vortex to mix, and react at 25°C in the dark for 60 minutes.

[0055] (3) Total GSH detection: For each sample, prepare 6.6 μL of glutathione reductase diluted 5-fold, 6.6 μL of DTNB stock solution, and 150 μL of total glutathione detection buffer. Mix well to prepare the total glutathione detection working solution. Prepare a 96-well microplate, add 10 μL of the prepared standard solution / sample solution to each well, and add 150 μL of the total glutathione detection working solution. React at room temperature for 25 min. After the reaction is complete, add 50 μL of 0.5 mg / ml NADPH to each well, mix well, and immediately use a microplate reader to detect the absorbance at a wavelength of 412 nm. Calculate the intracellular total GSH content based on the standard equation fitted to the standard curve.

[0056] (4) GSSG content detection: Add 20 μL LSH removal auxiliary solution and 4 μL LSH removal working solution to the prepared sample, vortex quickly to mix, and react at 25℃ for 60 min to remove GSH from the sample. Perform absorbance detection according to step (3), and then calculate the intracellular GSSG content based on the standard equation fitted by the standard curve.

[0057] Glutamine uptake test (1) Cell preparation: After digestion and treatment of cells, an appropriate amount of cell suspension is evenly spotted into 24-well plates. (2) Isotope uptake: First, prepare an isotope solution of 0.5 μCi / mL [3H]-L-glutamine using MEM medium. Then, add 500 μL of the prepared isotope solution to each well of a 24-well plate and incubate at 37°C for 15 min. After incubation, discard the isotope solution from the 24-well plate. During this process, the time for adding and removing the isotope in each well must be strictly controlled to ensure that the incubation time for cells in each well is precisely 15 min. Then, wash the cells in the 24-well plate three times with 1×PBS solution. When washing with 1×PBS solution for the first time, the washing time for each well should be strictly controlled to be the same. The washing time does not need to be strictly controlled during the second and third washes. If the next experimental operation cannot be performed immediately, the 24-well plate should be stored at -20°C.

[0058] (3) Detection of radioactivity: Add 220 μL of 1 N NaOH solution to each well of a 24-well plate and treat for 30 min to ensure complete cell lysis. After lysis, take 20 μL of liquid from each well for Nanodrop protein quantification. Then add 200 μL of 1 N HCl solution to each well for neutralization. After neutralization, add 2 mL of scintillation solution to each well, mix thoroughly, and transfer to a 96-well plate for radioactivity detection. The absolute value of glutamine uptake can then be obtained.

[0059] (4) NanoDrop protein quantification: Protein concentration was determined using a NanoDrop 2000 micro-volume spectrophotometer.

[0060] Detection of intracellular α-kg content (1) Cell preparation: After washing the cells thoroughly with 1×PBS, add the lysis buffer in the kit, lyse the cells on ice for 10 min, then centrifuge at 12000 rpm at 4°C for 5 min, and take the supernatant for subsequent detection.

[0061] (2) Kit preparation: Thaw the relevant reagents and prepare the relevant solutions according to the kit instructions.

[0062] (3) Sample determination: Take 10 μL of α-kg Standard, add 90 μL of Buffer A for Metabolic Assay, mix well, and prepare an α-kg standard solution with a concentration of 1000 μM. Take 0, 0.4, 1, 2, 4, 10, and 20 μL of 1000 μM α-kg standard solution and add them to the standard wells of a 96-well plate, respectively, and make up to 20 μL with Buffer A for Metabolic Assay to obtain the corresponding standard curves with concentrations of 0, 20, 50, 100, 200, 500, and 1000 μM.

[0063] Add 20 μL of sample to each well of a 96-well plate, and set up a blank control containing only Buffer A for Metabolic Assay. Add 80 µL of Amplex Red working solution to each well, mix well, and incubate at 37ºC in the dark for 30 minutes. Measure the absorbance at 570 nm and calculate the α-kg concentration per well using the calculated standard curve.

[0064] Preparation of E. coli culture reagent (1) LB liquid culture medium: Accurately weigh 10.00 g of tryptone, 10.00 g of sodium chloride, and 5.00 g of yeast extract using an analytical balance, and transfer them to a 2 L glass beaker. Add 800 mL of ultrapure water and place the beaker on a constant temperature magnetic stirrer (45 ℃, 500 rpm) and stir continuously until the solutes are completely dissolved. Quantitatively transfer the solution to a volumetric flask and dilute to the 1000 mL mark with deionized water. Dispense the prepared solution into heat-resistant glass bottles and sterilize them in an autoclave (121 ℃, 103.4 kPa) for 30 min. After sterilization, allow the solution to cool naturally to 25 ℃ in a biosafety cabinet, and add ampicillin sodium salt (working concentration 100 μg / mL, added as 1% by volume of the stock solution) under aseptic conditions. Mix thoroughly using a vortex mixer (3000 rpm, 10 s), dispense into sterile storage bottles, label with batch number and expiration date, store at 4 ℃ protected from light, shelf life 30 days.

[0065] (2) LB solid culture medium: Using an analytical balance, accurately weigh agarose powder (30.00 g), tryptone (20.00 g), sodium chloride (20.00 g), and yeast extract (10.00 g) sequentially, and transfer them to a 2 L glass Erlenmeyer flask. Add 800 mL of ultrapure water and place on a constant-temperature magnetic stirrer (50 ℃, 600 rpm) and stir continuously until the solutes are completely dissolved. After pre-filtration through a 0.22 μm filter membrane, transfer the solution to a volumetric flask and dilute to the 1 L mark. Dispense the mixture into high-temperature resistant glass containers. The bottles were sterilized in an autoclave (121 ℃, 103.4 kPa) for 30 min. After sterilization, the containers were transferred to a constant temperature water bath. 100 mg / mL ampicillin stock solution (final concentration 100 μg / mL) was aseptically injected at a 1:1000 volume ratio, and homogenized using a vortex mixer for 10 s. The antibiotic-containing culture medium was aliquoted into 90 mm culture dishes in a sterile workbench, 20 mL per dish. After gelation at room temperature, the dishes were sealed with a sealing film. The batch number, preparation date, and expiration date of the culture medium were labeled. The medium was stored upright at 4 ℃ protected from light for 14 days. Sterility testing was required before use.

[0066] ASCT2 plasmid construction (1) Primer design for overexpression of human ASCT2 sequence Agel-Forward 5'-ATAACCGGTGCCACCATGGGCGGCGGCGGCG-3' Sacl-Rervese 5'-ATAGAGCTCTCAGGCGCCGGCG-3' PCR reaction system: 25 μL 2×Phanta Max Master Mix + 1 μL dNTP (10 mM) + 5 μL cDNA template (50 ng / μL) + 2.5 μL each of forward and reverse primers (10 μM) + ddH2O to a final volume of 50 μL. Follow the procedure below. Amplification: Pre-denaturation (98℃, 3 min, 1 cycle) → Denaturation (98℃, 10 s, 30 cycles) → Annealing (64℃, 30 s, 30 cycles) → Extension (72℃, 2 min, 30 cycles) → Complete extension (72℃, 10 min, 1 cycle) (2) Enzyme digestion and enzyme chain a. Linearization vector preparation: 2 μg Flag plasmid + 5 μL 10×CutSmart buffer + 1 μL AgeI + 1 μL SacI + ddH2O to make up to 50 μL, mix well and incubate at 37℃ for 2 h for enzyme digestion.

[0067] b. Enzyme-linked The PCR product obtained in step (1) was recovered according to the instructions of the gel recovery / DNA purification kit. It was then digested with restriction endonucleases AgeI and SacI. The digested products were verified by nucleic acid electrophoresis and then recovered by gel extraction. The ligation system was prepared as follows: linearized vector (AgeI / SacI) X μL (50 ng) + ASCT2 insert fragment (AgeI / SacI) Y μL (molar ratio X:Y = 1:5) + 5×T4 Ligase Buffer 2 μL + T4 DNA Ligase 1 μL + ddH2O to a final volume of 10 μL. The mixture was incubated overnight at room temperature. The ligation product was then transformed into Stbl3 competent cells, and positive clones were screened by plate plating.

[0068] Plasmid transformation, amplification and extraction (1) Plasmid transformation: Thaw DH5α competent cells (selected according to plasmid resistance) on ice, add 20 μL to a 1.5 mL EP tube, and add 3-5 μg of linearized plasmid. Gently mix, incubate on ice for 20 min → heat shock in a 42℃ water bath for 45 s → incubate on ice for 5 min → add 500 μL of antibiotic-free LB liquid medium, and culture at 37℃ with shaking at 200 rpm for 30 min.

[0069] (2) Plate preparation: 30 min in advance, place the LB solid medium containing ampicillin (100 μg / mL) at room temperature. Centrifuge the transformed bacterial culture at 1000 rpm for 3 min, discard 400 μL of supernatant, resuspend and spread evenly on the plate, incubate at 7℃ upright for 15 min, then invert overnight.

[0070] (3) Plasmid amplification: Pick 3-5 single colonies and inoculate them into 4 mL of LB liquid medium containing ampicillin. Incubate at 37℃ and 200 rpm for 12-16 h.

[0071] (4) Sequencing identification: Take 200 μL of bacterial culture and send it to the sequencing company. Store the remaining bacterial culture at 4℃ for later use.

[0072] (5) Plasmid extraction: Extract plasmids according to the instructions of the plasmid extraction kit to ensure that there is no endotoxin contamination.

[0073] (6) Concentration determination: The plasmid concentration was determined using NanoDrop 2000 and diluted with TE buffer to a working concentration of 1 μg / μL.

[0074] plasmid transfection (1) Cell plating: Take an appropriate amount of cell suspension (2 mL) and add it to a 6 / 96-well plate. After adding, gently shake the plate to ensure that the cells are evenly dispersed at the bottom. Then, incubate the plate in a 37℃ CO2 incubator for 12 h to ensure that the cells are in good condition when the plasmid is added. The density is around 40%.

[0075] (2) Preparation of plasmid transfection working solution: Prepare two sterile 1.5 mL centrifuge tubes, label them A (transfection reagent tube) and B (plasmid tube), and add 125 μL of serum-free culture medium to each. Add 2 μL of transfection reagent Lipofectamine 3000 to tube A and gently mix by pipetting. Add 2 μg of target plasmid and 4 μL of P3000 transfection enhancer to tube B, gently mix by pipetting, and let stand at room temperature for 5 min. Then add the liquid from tube A to tube B, gently mix by pipetting, and let stand at room temperature for 15 min.

[0076] (3) Cell transfection: Discard the original culture medium in the well, add 1.75 mL of serum-free culture medium, and then add 250 μL of the above transfection working solution to each well. Gently shake to mix, and then incubate in a 37℃ CO2 incubator. After 12 h, replace with serum-containing complete culture medium.

[0077] Cellular siRNA interference (1) siRNA sequence: Negative control (NC): 5'-AACUUAUTAUTTTGGCACAUTC-3' ASCT2 siRNA-1: 5'-CCUGUAUCAUGGUGCUCAATT-3' ASCT2 siRNA-2: 5'-GGAUCAAGCUCAUCGUGUATT-3' (2) Cell transfection with siRNA steps a. Cell Plating: Observe the cells under a microscope. When the cell density reaches 80%-90%, perform cell plating. In a clean bench, use a pipette to discard the culture medium. Add 2 mL of sterile PBS buffer to wash the cells twice. After discarding the PBS, add 2 mL of 0.25% trypsin (containing EDTA) along the wall of the culture dish. Gently shake the dish to ensure the trypsin completely covers the cells at the bottom. Digest at room temperature for 5 min. Observe under a microscope. Once the cells become rounded, immediately add cells containing 10% FBS. Digestion was stopped by adding 1 mL of complete culture medium. Cells were carefully transferred from the bottom of the culture dish to a centrifuge tube and centrifuged at 800 rpm for 5 min at room temperature. The supernatant was carefully discarded, and an appropriate amount of complete culture medium was added. Cells were gently pipetted until completely dispersed. 20 μL of cell suspension was added to a cell counting plate, and cell counting was performed using a cell counter. 3 μL of the cell suspension was added to each well of a six-well plate. 10 5 3000 cells were added to each well of a 96-well plate. After adding the cells, the plate was gently shaken to ensure that the cells were evenly distributed on the bottom. The plate was then incubated in a 37°C CO2 incubator for 12 hours to ensure that the cell density was about 40% when minimal disturbance was introduced.

[0078] b. siRNA preparation: Take 1 OD siRNA powder, add 125 μL of sterile DEPC water, vortex for 10 s, and centrifuge for later use.

[0079] c. Complex preparation: Prepare two sterile 1.5 mL centrifuge tubes, labeling them A (transfection reagent tube) and B (siRNA tube), respectively. Add 125 μL of serum-free culture medium to each tube. Add 5 μL of Lipofectamine 3000 transfection reagent to tube A, gently pipette to mix, and incubate at room temperature for 5 min. Add 5 μL of fully dissolved siRNA solution to tube B, then add all the liquid from tube A to tube B, gently pipette to mix, and incubate at room temperature for 15 min.

[0080] d. Cell transfection: Discard the original culture medium in the wells, add 1.75 mL of serum-free culture medium, then add 250 μL of the above transfection working solution to each well, gently shake to mix, and incubate in a 37℃ CO2 incubator. After 6 h, replace with serum-containing complete culture medium.

[0081] Experimental results Figure 1 A represents the IC50 of the doxorubicin-sensitive strain SMMC-7721 in hepatocellular carcinoma. 50 Line graph; Figure 1B represents the IC50 of the doxorubicin-resistant strain SMMC-7721 in hepatocellular carcinoma. 50 Line graph. Figure 1 The results of AB showed that the resistance fold of SMMC-7721A was 48, indicating a significant resistance phenotype. Figure 1 C is a comparison of glutamine uptake experiments between the SMMC-7721-sensitive and SMMC-7721A-resistant liver cancer strains; Figure 1 D is a comparison of the relative mRNA expression levels of glutamine metabolism-related genes (ASCT2, GLS, GLUL) in the SMMC-7721 sensitive strain and the SMMC-7721A resistant strain of liver cancer. Figure 1 E is a Western blotting plot and quantitative analysis of ASCT2 protein expression levels in the SMMC-7721 sensitive and SMMC-7721A resistant liver cancer strains. Figure 1 F is a Western blotting plot and quantitative analysis of GLS protein expression levels in the SMMC-7721-sensitive and SMMC-7721A-resistant liver cancer strains; Figure 1 G is a comparison of α-ketoglutarate (α-KG) levels in hepatocellular carcinoma strains sensitive to SMMC-7721 and resistant strains of SMMC-7721A. Figure 1 The results of CG showed that the SMMC-7721A drug-resistant strain exhibited upregulated glutamine uptake and metabolism. Furthermore, since the α-KG content in the drug-resistant strain did not change significantly, but considering its active glutamine metabolism, it is suggested that the glutamine ingested in the drug-resistant strain may not primarily enter the tricarboxylic acid cycle for synthesis, but rather maintains the drug-resistant phenotype by altering the intracellular oxidative stress state. Figure 1 H is a flow cytometry diagram and statistical graph showing the intracellular reactive oxygen species (ROS) levels in the SMMC-7721-sensitive and SMMC-7721A-resistant liver cancer strains. Figure 1 Figure I shows a comparison of the ratio of reduced glutathione to oxidized glutathione (GSH / GSSG) in the SMMC-7721-sensitive and SMMC-7721A-resistant liver cancer strains. Figure 1 The results of HI showed that, compared with the susceptible strain, the drug-resistant strain SMMC-7721A had significantly lower basal ROS levels and a higher GSH / GSSG ratio, suggesting that its antioxidant capacity was significantly enhanced. Figure 1 J is a flow cytometry diagram and statistical graph showing the changes in ROS levels in the SMMC-7721-sensitive and SMMC-7721A-resistant liver cancer strains after treatment with doxorubicin (ADM). Figure 1 The results showed that under ADM induction, the ROS level of the sensitive strain increased significantly, while the ROS level of the resistant strain did not change significantly, indicating that the SMMC-7721A resistant strain can effectively resist drug-induced oxidative stress by maintaining redox homeostasis.

[0082] Figure 2 A represents the IC50 of the SMMC-7721A hepatocellular carcinoma strain resistant to ADM before and after glutamine (Gln) deprivation. 50 Curve comparison chart; Figure 2 B is a bar chart showing the effect of glutamine deprivation on the inhibition rate of SMMC-7721A resistant strains under different concentrations of doxorubicin (ADM) treatment; Figure 2 C is a comparison of the effects of simple glutamine deprivation (-Gln) on the inhibition rate of SMMC-7721A resistant cells; Figure 2 AC results showed that depriving glutamine would reduce the IC50 of drug-resistant strains. 50 The decrease from 2.41±0.744 μM to 0.67±0.070 μM significantly enhanced the sensitivity of the resistant strain to ADM, while deprivation of glutamine alone had no significant effect on the cell viability of the resistant strain, indicating that glutamine is directly related to ADM resistance. Figure 2 D is a graph showing the trend of the GSH / GSSG ratio in the SMMC-7721A resistant strain after glutamine deprivation and combined ADM treatment; Figure 2 E is a flow cytometry diagram and statistical graph showing the changes in ROS levels in the SMMC-7721A resistant strain after glutamine deprivation and combined ADM treatment. Figure 2 The DE results showed that ADM treatment of cells while depriving them of glutamine significantly reduced intracellular GSH levels and increased intracellular ROS accumulation, thus disrupting intracellular redox balance.

[0083] Figure 3 A shows the mRNA level detection in the SMMC-7721A drug-resistant strain after interfering with ASCT2 expression using siRNA; Figure 3 B is a Western Blot diagram showing the protein level detection in the SMMC-7721A drug-resistant strain after interfering with ASCT2 expression using siRNA; Figure 3 C is a comparison of the effects of interfering with ASCT2 expression on the survival rate of SMMC-7721A resistant cell lines; Figure 3 D is a bar chart showing the effect of ASCT2 interference on the inhibition rate of SMMC-7721A resistant cells under different concentrations of doxorubicin (ADM) treatment. Figure 3 AD results showed that knockdown of ASCT2 alone had no killing effect on cells, while knockdown of ASCT2 significantly enhanced the sensitivity of drug-resistant strains to ADM. Figure 3 E is a Western Blot graph showing the protein level detection after ASCT2 overexpression (OE-ASCT2) in the SMMC-7721 sensitive strain; Figure 3F is a bar chart showing the effect of ASCT2 overexpression on the inhibition rate of SMMC-7721 sensitive cells under different concentrations of ADM treatment. Figure 3 EF results showed that the resistance of susceptible strains to ADM increased after treatment with ASCT2.

[0084] Example 2 ZR001 Enhanced Sensitivity Test for Drug-Resistant Cells to Doxorubicin Growth curve experiment Hepatocellular carcinoma cells in good condition and in the logarithmic growth phase were digested, pipetted, and transferred to sterile centrifuge tubes. After centrifugation, the cells were resuspended and counted. The required cell volume was resuspended in complete culture medium. After homogenization, 3000 cells per well were added to a 96-well plate containing 180 μL of complete culture medium, ensuring uniform distribution of cells. The plates were incubated at 37 ℃ CO2 for 12 h. After cell attachment, the cells were treated with relevant drugs. Three replicates were set up for each concentration, and seven experimental groups were set up for each concentration. Subsequently, one group of culture medium was discarded each day, and 100 μL of prepared 10% CCK-8 solution was added. The plates were incubated at 37 ℃ CO2 for 1.5 h. The CCK-8 solution after the reaction was completed was aspirated and added to an ELISA plate. The absorbance at 450 nm was measured using an ELISA reader. The reaction was repeated for 7 days, and a cell growth curve was plotted.

[0085] Plate colony formation experiment Hepatocellular carcinoma cells in good condition and in the logarithmic growth phase were digested, pipetted, and transferred to sterile centrifuge tubes. After centrifugation, the cells were resuspended and counted. The resulting hepatocellular carcinoma cells were added at a rate of 4000 cells per well to 6-well plates containing 2 mL of complete culture medium. The plates were shaken to ensure that the cells were evenly distributed in the plates. The plates were then incubated at 37 ℃ in a CO2 incubator for 12 h. The culture medium was discarded, and 2 mL of complete culture medium was added again. The cells were then treated according to different experimental purposes. After treatment, the cells were placed in a 37℃ CO2 incubator and cultured. Cell colony formation was observed under a microscope daily, and the culture medium was replaced with fresh complete medium every 4 days. When the cell colony reached 200 cells and was visible to the naked eye under the microscope, the original culture medium was discarded, and the cells were carefully washed 3 times with 1×PBS buffer. 1 mL of paraformaldehyde was added to each well for fixation for 30 min. After fixation, the cells were carefully washed 3 times with 1×PBS buffer, stained with crystal violet solution for 20 min, and the crystal violet solution was recovered. Excess staining solution was carefully washed away along the well wall with tap water, taking care not to wash away the cell clumps. The cells were then air-dried at room temperature before taking pictures for analysis.

[0086] Experimental results Figure 4Figure A shows the detection of glutamine uptake capacity of the hepatocellular carcinoma SMMC-7721A drug-resistant strain under different concentrations of ZR001 treatment. Figure 4 The results of A indicate that as the concentration of the ASCT2 inhibitor ZR001 increases, the glutamine uptake capacity of drug-resistant cells gradually decreases. Figure 4 Figure B shows the comparative effects of ZR001, V-9302, and BPTES combined with doxorubicin (ADM) on the cell viability of the SMMC-7721A drug-resistant strain. Figure 4 Results B showed that ZR001 alone had no cytotoxic effect, but when used in combination with ADM, it significantly enhanced the sensitivity of resistant strains to ADM, which was more significant compared to V-9302 and BPTES. ZR001 had almost no effect on the activity of resistant cells (~98%), indicating that it has low intrinsic toxicity and causes little damage to non-chemosensitive cells. In contrast, V-9302 alone showed some inhibitory effect (~76%), indicating that it has higher intrinsic toxicity. The comparison between the two suggests that ZR001 can be used as a chemosensitizer without increasing the risk of single-drug toxicity, which is very important in clinical combination therapy. Figure 4 C represents the IC50 of the SMMC-7721A resistant strain after treatment with different concentrations of ZR001 combined with ADM. 50 Curve and numerical comparison chart. Figure 4 The results of C indicate that as the concentration of ZR001 increases, the IC50 of resistant cells to ADM decreases. 50 It shows a significant downward trend. Figure 4 D is a heatmap of the synergistic index (ZIP) of the killing effect of ZR001 and ADM in combination on the drug-resistant SMMC-7721A strain. Figure 4 The results of D showed that the synergistic index (ZIP) of ZR001 and doxorubicin was >10, suggesting a synergistic effect between ZR001 and ADM. Furthermore, dose matrix analysis determined the optimal combination concentration to be 1 μM ADM and 5 μM ZR001, providing a drug concentration reference for subsequent experiments. Figure 4 E represents the cell proliferation activity curves of the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 4 Figure F shows the results of the colony formation experiment of the SMMC-7721A drug-resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 4 The results from EF showed that ZR001 combined with ADM could significantly inhibit the proliferation and colony formation ability of drug-resistant strains.

[0087] Example 3 ZR001 combined with doxorubicin: ELISA method for the detection of 8-hydroxydeoxyguanosine 8-Hydroxyguanosine is a deoxyguanine that directly attacks nuclear DNA caused by reactive oxygen species (ROS). Therefore, the level of intracellular DNA oxidative damage can be quantitatively assessed by measuring 8-hydroxyguanosine. The specific steps are as follows: (1) Cell sample processing: Cell digestion and treatment were as described above. A certain concentration of cell suspension was added to a six-well plate and cultured for 12 h. After the cells adhered, the drug was added to treat the cells for 72 h. Cell samples were then collected for 8-hydroxydeoxyguanosine detection. The culture medium in the six-well plate was discarded, and the cells were gently washed twice with 1×PBS. 1 mL of 0.25% trypsin (containing EDTA) was added to each well. After cell digestion, the cells were blown off and the cells were disrupted using an ultrasonic disruptor (conditions: 30% power, run for 3 s, pause for 3 s, disrupt for 5 min). After disruption, the cells were centrifuged at 12000 rpm for 15 min, and the supernatant was collected for subsequent detection.

[0088] (2) Standard curve preparation: Add 1 mL of the standard dilution solution to the standard, let it stand at room temperature for 10 min, repeatedly invert it to dissolve it completely, shake it gently to mix, avoid the formation of air bubbles, and prepare a 100 ng / mL standard working solution. Dilute it according to the gradient to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0 ng / mL.

[0089] (3) Sample detection: Prepare biotinylated antibody working solution (freshly prepared) and HRP enzyme conjugation working solution (freshly prepared) according to the ELISA kit instructions; prepare the ELISA plate provided by the kit, add 50 μL of standard / sample to each well, then add 50 μL of biotinylated antibody working solution, cover the ELISA plate with a membrane, and incubate at 37 ℃ for 45 min. Note that when adding the sample / standard, try to add it to the bottom of the ELISA plate and avoid generating air bubbles; after the reaction is complete, shake off the liquid in the well, pat dry on clean absorbent paper, add 350 μL of washing buffer to each well, soak for 1 min, then pat dry the liquid in the ELISA plate, repeat 3 times; then add 100 μL of HRP enzyme conjugation working solution, cover the ELISA plate with a membrane, and incubate at 37 ℃ for 30 min, shake off the liquid in the well again, and wash 5 times; add 90 μL of substrate solution to each well, cover the ELISA plate with a membrane, and incubate at 37 ℃ in the dark for 15 min. min; after the reaction is complete, add 50 μL of stop solution to each well and quickly measure the absorbance at 450 nm using a microplate reader. Calculate the concentration of 8-hydroxydeoxyguanosine for each sample based on the standard equation fitted to the standard curve.

[0090] Comet Experiment The comet assay is the gold standard for directly, intuitively, and quantitatively detecting DNA strand breaks at the single-cell level. The specific steps are as follows: (1) Reagent preparation: Neutral buffer: Weigh an appropriate amount of Tris powder and prepare a 0.4 M neutral buffer; then add an appropriate amount of concentrated hydrochloric acid to adjust the pH to 7.5. Electrophoresis buffer: Weigh 4 g NaOH + 186.12 mg EDTA powder, add ultrapure water to make up to 500 mL, stir until fully dissolved at room temperature, and place in a 4 ℃ refrigerator for pre-cooling. (2) Preparation of comet electrophoresis slides: Prepare transparent slides, prepare a 1% agarose solution, spread it evenly on the transparent slides, and allow the agarose gel to completely solidify at room temperature. Digest the cells, collect the cell suspension, and adjust the concentration of the cell suspension to 1×10⁻⁶. 6 Cells / mL. Prepare a 0.7% low-melting-point agarose solution using ultrapure water and place it in a 37 ℃ water bath to prevent solidification. Add 20 μL of cell suspension to 150 μL of 0.7% low-melting-point agarose solution and gently pipette to disperse the cells evenly. Quickly drop the solution onto a 1% agarose gel slide, being careful to avoid air bubbles. Use tweezers to pick up a coverslip and quickly place it over the added agarose cell suspension to form a "sandwich" structure. Allow the agarose to solidify at room temperature to form a comet electrophoresis slide.

[0091] (3) Comet electrophoresis: The prepared comet electrophoresis slide gel was placed in lysis buffer and lysed overnight at 4 °C. The next day, the slide was removed and rinsed three times with 1×PBS. The slide was placed in the electrophoresis tank, and electrophoresis buffer was added so that the liquid level was 2-3 cm above the slide. The slide was placed on ice in the dark for 60 min to allow the DNA to unwind. Electrophoresis was performed on ice at 25 V for 30 min in the dark. After electrophoresis, the slide was removed and neutralized with neutral buffer at 4 °C for 10 min. The slide was rinsed with 1×PBS and then 4 sgreen DNA dye was added. The slide was stained at room temperature in the dark for 30 min. The slide was removed, washed three times with ultrapure water, dried, and placed in a 37 °C oven to dry. After drying, the slide was observed and photographed using a fluorescence inverted microscope.

[0092] Cell cycle detection By using flow cytometry to detect the cell cycle, the distribution of different phases of the cell cycle and the degree of cell cycle arrest are analyzed to determine the regulatory mode of drug effects on cell proliferation. The specific steps are as follows: Cell digestion was performed as described above. A certain concentration of cell suspension was added to a six-well plate, and the cells were cultured for 12 h. After cell attachment, the cells were treated with the drug for 72 h, and then cell samples were collected for cell cycle analysis. The culture medium in the wells was discarded, and the cells were washed with 2 mL of 1×PBS, repeated twice. 0.25% trypsin (without EDTA) was added for digestion. After adequate digestion, 100 μL of FBS was added to stop the digestion. The cells were gently blown off the bottom of the plate and transferred to a 1.5 mL centrifuge tube. The plate was centrifuged at 1000 rpm for 5 min. Resuspend the cell pellet in 1×PBS, wash the cells thoroughly, centrifuge at 1000 rpm for 5 min, and repeat the reading twice. Discard the supernatant, add 200 μL of ultrapure water and gently mix the cell pellet. Use a pipette to aspirate the cell suspension and add it dropwise to 800 μL of pre-chilled 90% ethanol at 4℃. Fix overnight at 4℃. The next day, centrifuge at 1000 rpm for 5 min, discard the fixative, add 1 mL of pre-chilled 1×PBS to resuspend the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 0.5 mL of prepared PI staining solution to resuspend the cells, and incubate at room temperature in the dark for 30 min. After staining, detect red fluorescence at an excitation wavelength of 488 nm using a flow cytometer.

[0093] Experimental results Figure 5 A is a flow cytometry diagram and statistical graph showing the changes in ROS levels in the drug-resistant liver cancer SMMC-7721A strain after treatment with ZR001 alone and in combination with doxorubicin (ADM). Figure 5 B is a comparison of the GSH / GSSG ratio in the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 5 The results from AB showed that, consistent with glutamine deprivation, the combination of ZR001 and ADM significantly increased intracellular ROS levels and significantly reduced intracellular GSH accumulation. Figure 5 C is a graph showing the detection levels of the DNA oxidative damage marker 8-OHdG in the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 5 The results showed that the combination of ZR001 and ADM significantly increased the intracellular 8-OHdG level, indicating that the oxidative stress caused by the combination of drugs further affected DNA damage. Figure 5 D is an immunofluorescence staining image of γ-H2AX, a marker of DNA double-strand break, in the SMMC-7721A drug-resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 5 E is a Western blotting image showing the expression level of γ-H2AX protein in the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 5 The results of DE showed that combined administration of ZR001 (5 μM) and ADM (1 μM) significantly increased the fluorescence intensity of γ-H2AX in the cell nucleus and the protein content of γ-H2AX in the cells, indicating that ZR001 enhanced ADM-induced DNA damage. Figure 5 F is a Western Blot diagram showing the expression levels of DNA damage response pathway-related proteins (p-ATM, ATM, p-BRCA, p-CHK2, CHK2) in the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 5 The results of F showed that the combined administration of ZR001 and doxorubicin led to a significant increase in the expression of p-ATM, p-BRCA, and p-CHK2, indicating activation of the DDR pathway. Figure 5 G is a fluorescence microscope image of DNA breakage detected by comet assay in the SMMC-7721A drug-resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 5 H is a statistical graph of Olive tail moments (OTM) of cells in each group during the comet experiment; Figure 5 I is a statistical graph showing the tail DNA content of cells in each group during the comet experiment. Figure 5 The results from GI showed that when ZR001 and ADM were administered in combination, the cells exhibited significant tailing, with both the tailed DNA and Olive tail moments being significantly higher than those in the single-drug group and the control group, indicating that the cells had significant DNA double-strand breaks.

[0094] Figure 6 A is a Western Blot diagram showing the expression levels of p-MTOR and mTOR proteins in the drug-resistant liver cancer SMMC-7721A strain after treatment with ZR001 alone and in combination with doxorubicin (ADM). Figure 6 B is a Western blotting image showing the expression levels of PI3K / AKT signaling pathway-related proteins (p-PI3K, PI3K, p-AKT, AKT) in the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 6 The results of the AB study showed that the combination of ZR001 and ADM significantly downregulated proteins related to the mTOR-AKT-PI3K pathway (such as p-mTOR, p-PI3K, and p-AKT), thereby inhibiting the proliferation of drug-resistant cells. Figure 6 C is a Western Blot diagram showing the expression levels of cell cycle regulation-related proteins (cyclin-B, p-CDC25C, CDC25C, CDC2, p-CDC2) in the SMMC-7721A drug-resistant strain after treatment with ZR001 alone and in combination with ADM. Figure 6The results showed that Cyclin B expression was downregulated in the ZR001 and ADM co-treatment group, while the expression of the active form of phosphorylated CDC2 was reduced, which exhibited cycle arrest characteristics. Figure 6 D is a flow cytometry diagram and bar chart showing the cell cycle distribution of the SMMC-7721A resistant strain after treatment with ZR001 alone and in combination with ADM, and the statistical bar chart of the proportion of each cell cycle. Figure 6 The results showed that the proportion of cells in the G2 / M phase was significantly increased in the group treated with the combined ZR001 and ADM, further clarifying that the combination of drugs induced cell cycle arrest.

[0095] Figure 7 A is a flow cytometry diagram and statistical graph showing the ROS level in the drug-resistant liver cancer SMMC-7721A strain treated with ZR001 in combination with doxorubicin (ADM) after the addition of antioxidants (NAC, GSH). Figure 7 The results from A indicate that exogenous supplementation of NAC and GSH restored the intracellular redox balance in drug-resistant cells. Figure 7 B is a fluorescence microscopy image of the ROS levels in each group of SMMC-7721A drug-resistant strains after the addition of antioxidants; Figure 7 C is a statistical graph showing the compensatory effect of adding antioxidants on DNA damage (Olive tail moment in comet experiment) induced by ZR001 combined with ADM; Figure 7 D is a statistical graph showing the compensatory effect of adding antioxidants on DNA damage (DNA content in the tail of the comet experiment) induced by ZR001 combined with ADM. Figure 7 E is a Western blot diagram showing the expression levels of DNA damage-related proteins (p-CHK2, γ-H2AX) in the SMMC-7721A drug-resistant strain after the addition of antioxidants. Figure 7 The BE results showed that supplementation with NAC and GSH rescued DNA damage caused by oxidative stress. Figure 7 F is a comparison of the effects of ZR001 combined with ADM on the recovery of cell viability of SMMC-7721A resistant strains after the addition of antioxidants. Figure 7 The results showed that supplementation with NAC and GSH rescued the activity inhibition caused by the combined administration of ZR001 and ADM. Figure 7 G is a Western blot diagram showing the expression levels of p-MTOR and p-AKT signaling pathway proteins in the SMMC-7721A drug-resistant strain after the addition of antioxidants. Figure 7 H represents a flow cytometry diagram and statistical bar chart of the cell cycle distribution of the SMMC-7721A drug-resistant strain after the addition of antioxidants. Figure 7 The GH results showed that supplementation with NAC and GSH salvaged the periodic arrest induced by the combination therapy.

[0096] Example 4 ZR001, in combination with doxorubicin, inhibited the proliferation of hepatocellular carcinoma cells in a nude mouse xenograft tumor model. Constructing a subcutaneous xenograft model in nude mice Collect SMMC-7721A cells in the logarithmic growth phase, resuspend the cells in pre-chilled PBS, and dilute the cells to 5 × 10⁻⁶. 7 Cells / mL. Before inoculation, the nude mice were disinfected at the inoculation site with 75% alcohol swabs. 200 μL of a homogeneous cell suspension was drawn up using a 1.0 mL syringe and inoculated subcutaneously into the left and right axillae of the mice. Inoculation continued until the tumor volume reached 500 mm in length. 3 The mice were then euthanized, the tumor was removed, and the pieces were cut into 1.5 mm pieces. 3 The tumor was inoculated into the right axilla of nude mice using a cannula. When the average tumor volume was approximately 40 mm3, the tumor-bearing mice were randomly divided into different groups and administered different drugs, as shown in Table 4.

[0097] Table 4:

[0098] The drug preparations for the above animal experiments are as follows: (1) ZR001 Stock solution preparation: 160 mg ZR001 was dissolved in 800 μL of dimethyl sulfoxide (DMSO).

[0099] Preparation of working solution: 250 μL stock solution + 1.5 mL PEG400 + 0.25 mL Tween 80 + 3 mL ultrapure water.

[0100] (2) ADM Stock solution preparation: 36 mg ADM + 1.5 mL DMSO.

[0101] Working solution preparation: 150 μL stock solution + 2850 μL ultrapure water.

[0102] (3) VER Stock solution preparation: 10 mg + 250 μL DMSO.

[0103] Working solution preparation: 25 μL stock solution + 475 μL ultrapure water.

[0104] Animal experimental test indicators and calculation methods a. General observation: Observe the growth of mice; observe the skin and fur of mice; observe feces and urine.

[0105] b. Weight changes: The weight of mice was measured and recorded every other day during the administration process.

[0106] c. Tumor volume (TV), calculated using the following formula: TV = a b2 / 2 (Measure the longest diameter a and shortest diameter b of the tumor with calipers every other day during the administration process).

[0107] d. Relative tumor volume (RTV), calculated using the following formula: RTV = Vt / V0 (Vt is the tumor volume obtained from each tumor measurement, and V0 is the tumor volume before drug administration).

[0108] e. Relative tumor proliferation rate (T / C)%, calculated using the following formula: (T / C)% = TRTV / CRTV 100% TRTV: RTV in the treatment group; CRTV: RTV in the negative control group. f. Tumor inhibition rate, calculated using the formula: Tumor inhibition rate = (1 - T / C)% Experimental results Figure 8 A is a schematic diagram of the drug administration regimen for the SMMC-7721A liver cancer mouse model; Figure 8 B shows the gross anatomical diagram of tumor tissue in each group of tumor-bearing mice after the drug administration was completed; Figure 8 C is a graph showing the change in body weight of mice in each group during the drug administration period; Figure 8 D is a growth curve of tumor volume in each group of mice as a function of drug administration time; Figure 8 E is a scatter plot of tumor weight statistics for each group of mice at the end of drug administration. Figure 8 The results of the AE showed that the tumor volume and relative tumor volume in the ZR001 medium / high dose combination group were significantly lower than those in the ADM and ZR001 monotherapy groups. Based on the tumor growth inhibition rate formula, the tumor growth inhibition rates in the medium / high dose combination group were 70% and 65%, respectively, which were significantly higher than the 20% in the ADM monotherapy group and the 35% and 41% in the medium / high dose ZR001 monotherapy group. Figure 8 F represents the immunohistochemical staining and quantitative statistical graph of the expression level of the proliferation marker Ki67 in tumor tissues of each group. Figure 8 The results showed that the ratio of Ki67 positive cells in the tumor tissue of mice treated with the combination of ZR001 and doxorubicin was significantly reduced, indicating that the combination of ZR-001 and doxorubicin can also significantly inhibit the proliferation of drug-resistant liver cancer cells in vivo. Figure 8 G shows HE-stained pathological sections of tumor tissue from each group of mice. Figure 8The results showed that the combination of ZR001 and doxorubicin led to nuclear condensation and tissue disintegration.

[0109] Regarding the selection of cell types for action, this invention initially considered the possibility that ASCT2 is highly expressed in various tumors (including breast cancer and liver cancer), and whether it could significantly enhance the sensitivity of ADM in tumor cells with high ASCT2 expression. However, the experimental results of this invention show that the effects of ZR001 are not entirely consistent in different tumor cells. Based on existing research and previous experiments, ZR001 mainly exhibits a good direct inhibitory effect in breast cancer cells, while in doxorubicin-resistant liver cancer cells, ZR001 alone has minimal effect on cell viability, with almost no inhibitory effect. However, when combined with doxorubicin, it can significantly enhance chemosensitivity, exhibiting a different synergistic effect pattern (see [link to relevant documentation]). Figure 4 (B) We tried using the same ZR001 in combination with ADM in breast cancer cells, but did not obtain the expected more significant effect of improving ADM sensitivity, which is why we ultimately chose liver cancer cells.

[0110] The above-mentioned differences indicate that although ASCT2 is highly expressed in various tumors, the functional effect of ZR001 on ADM sensitivity is significantly cell type dependent, and it does not achieve the same chemosensitizing effect in all ASCT2-highly-expressing tumors. Especially in hepatocellular carcinoma drug-resistant models, it significantly enhances the effect of doxorubicin through metabolic regulation, demonstrating a more prominent application value. Therefore, this invention selects doxorubicin-resistant hepatocellular carcinoma cells as the research object, based on the specific chemosensitizing effect exhibited by ZR001 in this system, rather than simply deducing from ASCT2 expression levels.

Claims

1. The application of an ASCT2 small molecule inhibitor in the preparation of a drug that enhances the chemosensitivity of doxorubicin-resistant tumor cells, wherein the inhibitor is ZR001, and its molecular formula is C 35 H 39 N3O3 has the following structural formula: 。 2. The application of ZR001 as described in claim 1, characterized in that: The drug-resistant tumor cells mentioned are drug-resistant liver cancer cells.

3. A pharmaceutical composition containing the ASCT2 small molecule inhibitor ZR001, characterized in that: The pharmaceutical composition consists of the aforementioned small molecule inhibitor ZR001 and doxorubicin.

4. The pharmaceutical composition according to claim 3, characterized in that: In the pharmaceutical composition, the concentration of doxorubicin is 1 μM and the concentration of ZR001 is 5 μM.

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