Application of AST-487 in medicine for down-regulating SYVN1 expression and inhibiting HCC

Abstract

The invention provides a down-regulation effect of AST-487 in SYVN1 and application of AST-487 in drugs for inhibiting HCC. Wherein the structural formula of the AST-487 is as follows: the small molecule compound AST-487 can obviously down-regulate the expression of SYVN1, and can effectively inhibit the activity, invasion and migration of HCC. More choices are provided for the treatment of HCC patients in exploring new molecular targeting anti-hepatocellular carcinoma drugs, and the application has important significance in developing novel HCC treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical science, and more particularly to the use of AST-487 in drugs that downregulate SYVN1 expression and inhibit HCC. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor. Early-stage HCC often presents with no obvious clinical symptoms, and by the time of diagnosis, it has usually progressed to an intermediate or advanced stage, missing the optimal treatment window. Treatment options for intermediate and advanced-stage HCC are limited, making molecularly targeted therapy a hot research topic in recent years. Currently used targeted drugs in clinical treatment mostly exert their anti-tumor effects through the tyrosine kinase pathway, which has many drawbacks, including significant adverse reactions, low tumor response rates, and easy drug resistance, and does not significantly improve the survival rate of HCC patients. Therefore, developing new targets for HCC drug therapy is of great value. Summary of the Invention

[0003] To address the aforementioned technical challenges, the applicant previously discovered through ubiquitination proteomics analysis that the E3 ubiquitin ligase synoviolin 1 (SYVN1) is highly expressed in HCC and significantly associated with poor prognosis. The research group believes that SYVN1 holds promise as a novel therapeutic target for HCC, and small-molecule inhibitors targeting SYVN1 may offer a new strategy for HCC treatment. Building on this research, the applicant established a monoclonal cell line to screen for inhibitors that negatively regulate the SYVN1 promoter activity. Ultimately, they selected AST-487, a small-molecule compound that significantly inhibits SYVN1 expression and exhibits low cytotoxicity. It is envisioned that AST-487 may provide a novel treatment option for HCC patients.

[0004] Therefore, the applicant conducted a specific study on AST-487 related to HCC, as AST-487 is a potent and selective inhibitor of RET kinase activity. Previous studies have reported that AST-487 inhibits RET kinase-driven TERT expression in bladder cancer. AST-487 inhibits the growth of medullary thyroid carcinoma cells and calcitonin gene expression through different mechanisms. As a novel FMS-like tyrosine kinase 3 (FLT3) inhibitor, studies have reported the anti-leukemic effects of NVP-AST487: potent and selective anti-proliferative effects on primary cells and cell lines expressing FLT3-itd or FLT3 kinase domain point mutations.

[0005] To date, there are few reports on the anti-HCC activity of AST-487. This study aims to explore the role of AST-487 in HCC activity, invasion and migration.

[0006] Based on multiple experiments, the applicant verified that the small molecule compound AST-487 can downregulate SYVN1 expression in vitro and inhibit the activity, invasion, and migration of liver cancer cells. This discovery may reveal a novel pharmaceutical use of AST-487 in downregulating SYVN1 and effectively combating HCC.

[0007] Therefore, this invention proposes the use of AST-487 in downregulating SYVN1 expression and inhibiting HCC;

[0008] The structural formula of AST-487 is as follows:

[0009]

[0010] Furthermore, the use of AST-487 in downregulating SYVN1 expression and inhibiting HCC drugs is proposed.

[0011] Furthermore, the downregulation of SYVN1 expression refers to the downregulation of SYVN1 promoter function, gene expression, protein expression, etc.

[0012] Furthermore, the inhibition of HCC refers to the inhibition of the activity, invasion, and migration levels of HepG2 and Huh7 liver cancer cells.

[0013] The small molecule compound AST-487 involved in this invention is commercially available and can also be synthesized. Beneficial effects: The compound AST-487 involved in this invention has a good downregulation effect on SYVN1 and exhibits excellent inhibitory effects on various HCC indicators, especially HCC activity, invasion, and migration, providing more treatment options for HCC patients and showing promising drug application prospects. Attached Figure Description

[0014] Figure 1 Inhibition curve of AST-487 on HepG2 cell activity.

[0015] Figure 2 Inhibition curve of AST-487 on Huh7 cell activity.

[0016] Figure 3 Effects of AST-487 on the in vitro migration ability of HepG2 cells.

[0017] Figure 4 Effect of AST-487 on the in vitro migration ability of Huh7 cells.

[0018] Figure 5 Effects of AST-487 on the in vitro invasion ability of Huh7 cells.

[0019] Figure 6-1Effects of AST-487 on the expression of glycolysis-related genes and proteins;

[0020] Figure 6-2 Effect of AST-487 on LD content in the supernatant of liver cancer cells. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Pharmacological experiment

[0023] (I) Main Experimental Materials

[0024] AST-487 was purchased from MCE; fetal bovine serum was purchased from Gibco, USA; DMEM medium was purchased from Gibco, USA; PBS was purchased from Thermo Fisher Scientific Suzhou Co., Ltd.; double antibiotics were purchased from Beyotime International Co., Ltd.; and Trypsin-EDTA Solution was purchased from Beyotime International Co., Ltd.

[0025] (II) Cell Culture

[0026] This study used three cell lines: human hepatocellular carcinoma cells HepG2 and Huh7; and human normal hepatocytes LO2. Cells were cultured in DMEM medium containing 10% fetal bovine serum at 5% CO2-95% air, saturated humidity, and 37°C.

[0027] (III) CCK-8 cell proliferation experiment

[0028] HepG2 and Huh7 cells in good growth condition were seeded at 2000 mg / well in 96-well plates. Different concentrations of AST-487 were added, with three replicates, a negative control, and a blank well. The plates were incubated for 72 hours. The plates were then removed, the cell supernatant discarded, and 100 μL of fresh culture medium and 10 μL of CCK-8 solution were added to each well. The plates were gently tapped to mix, and the plates were incubated in the dark for 2–4 hours. OD values ​​were measured using a dual-wavelength microplate reader (detection wavelength 450 nm, reference wavelength 630 nm).

[0029] Inhibition curves were fitted using GraphPad Prism 8 (GraphPad software) with drug concentration on the x-axis and cell viability on the y-axis. The inhibition curve of AST-487 on HepG2 cells is shown below. Figure 1 As shown, the inhibition curve of Huh7 cells is as follows: Figure 2 As shown.

[0030] (IV) Detection of the effect of AST-487 on the in vitro migration ability of HepG2 cells

[0031] To further determine the antitumor activity of AST-487, the research group diluted the drug to four concentration gradients: 2 μM, 4 μM, 8 μM, and 16 μM. Human liver cancer cells (HepG2 and Huh7) were treated with a 10 μL pipette tip for 24 hours. 5-FU was used as a positive control, and the culture medium without the drug served as a blank control. The study investigated whether AST-487 affected the in vitro migration ability of human liver cancer cells. The results showed a significant change in migration width after 24 hours of drug treatment compared to the untreated width at 0 h. The migration width gradually increased with increasing drug concentration. The in vitro migration ability of HepG2 cells treated with AST-487 was as follows: Figure 3 As shown, the in vitro migration ability of Huh7 cells is as follows: Figure 4 As shown.

[0032] The specific method for the cell scratch assay is as follows:

[0033] (1) Sterilize the marker pen, ruler and other tools under UV light in advance. Then draw horizontal lines on the back of the 6-well culture plate at intervals of 0.5-1cm, perpendicular to the wells.

[0034] (2) Digested cells were seeded into 6-well cell culture plates, with the cell count per well controlled at approximately 5 × 10⁶ cells / well. 5 Place it in the incubator.

[0035] (3) After overnight, check the cell status under an inverted microscope. Once the cells have covered the culture plate, quickly streak along the bottom of the culture plate with a sterilized 10μL pipette tip.

[0036] (4) Wash the cells twice with medium containing 2% FBS to remove the scratched cells. In the experimental groups, culture medium containing different concentrations of drugs was added. The negative control group used DMEM without FBS, while the positive control group used 5FU. The initial scratch width was recorded by taking pictures under a microscope.

[0037] (5) After treating cells with different drug concentrations (2μM, 4μM, 8μM, 16μM) for 24 h, negative and positive control groups were set up. The scratch width was observed and photographed using an inverted fluorescence microscope after 24 h. The percentage (%) was analyzed using ImageJ software.

[0038] (V) Effects of AST-487 on the in vitro invasion ability of Huh7 cells

[0039] The research group used Transwell assays to investigate whether AST-487 inhibited the invasion of human hepatocellular carcinoma cells (Huh7). After treating cells with different drug concentrations for 24 hours, the number of cells invading the lower chamber was observed under a fluorescence microscope. It was observed that the number of cells invading the lower chamber decreased significantly with increasing drug concentration. Cells penetrating the lower chamber were counted using ImageJ software. Results are as follows: Figure 5 As shown.

[0040] The specific method for the cell scratch assay is as follows:

[0041] (1) Place the Matrigel at 4°C one day in advance to allow it to fully melt. Pre-cool the Transwell chamber and 200 μL pipette tip at -20°C, dissolve the Matrigel in FBS-free culture medium, and mix well.

[0042] (2) Add 100 μL of diluted matrix gel to the upper layer of the chamber, place it in the incubator for 2 hours, and then gently aspirate the liquid from the upper layer.

[0043] (3) Prepare a cell suspension by adding 200 μL of cell suspension to the upper layer, containing approximately 5 × 10⁻⁶ cells. 4 Add 800 μL of complete culture medium to the bottom layer of the cells. Incubate in an incubator for 24 hours.

[0044] (4) Use tweezers to pick up the upper chamber, absorb the original culture medium, gently wipe away the cells that have not penetrated the upper chamber with cotton swabs, wash with PBS and air dry.

[0045] (5) Then, fix the chamber in paraformaldehyde for 20 min. After fixation, wash three times with PBS. Next, stain the chamber in 0.1% crystal violet solution for 30 min. After staining, wash with PBS and air dry. Observe and record under a microscope, and calculate the number of cells in the lower chamber. The results are as follows: Figure 6-1 , Figure 6-2 As shown. Figure 6-1 and Figure 6-2 Together, they form a diagram showing the effect of AST-487 on glycolysis-related genes and proteins in Huh7 cells and the LD content in the culture supernatant of liver cancer cells.

[0046] (vi) Effects of AST-487 on glycolysis-related genes and proteins in Huh7 cells and the LD content in the culture supernatant of hepatocellular carcinoma cells.

[0047] The expression levels of key glycolytic enzymes, lactate dehydrogenase (LDHA), PKM2, and HK2, in Huh7 cells were detected using RT-qPCR and Western blot. Results showed that the expression of LDHA, PKM2, and HK2 gradually decreased with increasing drug concentration. The LD content in the cell culture supernatant was measured using a kit. Results showed that, compared to the untreated control group, lactate content decreased continuously with increasing drug concentration.

[0048] The specific methods for extracting total RNA from cells are as follows:

[0049] Trizol extraction was performed, and all necessary reagents were prepared in advance. All reagents and consumables were enzyme-free.

[0050] (1) Aspirate the waste liquid in the 6-well plate, wash the cells with PBS 1-2 times, add 1 ml Trizol / well, wait for the cells to be completely digested and lysed for 15 min, then pipette up and down to aspirate the liquid into the corresponding EP tubes and label each EP tube (operate on ice throughout).

[0051] (2) Add 200 μL of chloroform (20% of Trizol) to each EP tube, vortex and mix, let stand on ice for 5 min, and then centrifuge for 15 min (4℃, 12000 rpm).

[0052] (3) Prepare a new EP tube (1.5ml) in advance, and slowly aspirate 400μL of supernatant into the tube. Add 500μL of isopropanol to the EP tube containing the supernatant, invert it to mix thoroughly, let it stand on ice for 10min, and then centrifuge for 10min (4℃, 12000rpm).

[0053] (4) During this period, prepare 75% ethanol (1 part DEPC water + 3 parts anhydrous ethanol), 1 ml for each sample, discard the supernatant, and wipe the tube opening dry with paper. Add 1 ml of 75% ethanol and centrifuge for 5 min (4℃, 7500 rpm).

[0054] (5) Gently remove the remaining ethanol with a 10μL pipette tip, open the biosafety cabinet, let it air dry for 10-30 minutes, add 10-50μL of DEPC water to dissolve the white precipitate, mix with a shaker and then centrifuge.

[0055] (6) Prepare 2.5μL and 10μL pipettes, 10μL pipette tips, paper towels, and Diethyl pyrocarbonate in advance. Turn on the NanoDrop2000 spectrophotometer, calibrate the instrument to ensure the concentration error is within ±0.2, then test the concentration of the extracted sample and adjust the sample concentration to about 500 ng / μL. Then store at -80℃ for later use.

[0056] RNA reverse transcription

[0057] (1) Based on the 10μL system, RNA→cDNA was prepared on ice with the following reaction solution (500 / sample concentration + 2μL reverse transcriptase + DEPC water to make up to 10μL).

[0058] (2) After thoroughly mixing the above test, transfer it into the reverse transcription instrument and set the program to (37℃ for 15 min, 80℃ for 5 s, 4℃ forever).

[0059] (3) After the above procedure is completed, dilute the cDNA and store it at -20℃.

[0060] PCR amplification

[0061] Prepare PCR reaction solutions using TAKARA 420A, and mix the reaction solutions per well according to the following ratios.

[0062] Table 1. PCR amplification reaction system

[0063]

[0064] Take 8 μL of the above liquid (9 μL), add 8 μL of the above liquid and 2 μL of the previously reversed cDNA to each well of the PCR plate, mix well, centrifuge, and then perform the PCR test. The PCR program settings are as follows:

[0065]

[0066] Copy the data, and based on the obtained CT values, use 2 -ΔΔCT Data is processed using relative quantification methods.

[0067] The specific method for Western blot experiments is as follows:

[0068] protein extraction

[0069] (1) After the cells were transfected with the overexpression plasmid for 24 hours, they were washed with pre-cooled PBS, the culture plate was rotated and shaken to wash the cells, and then the washing solution was aspirated. After washing 3 times, the cell culture plate was placed on ice.

[0070] (2) Add 80 μL of RIPA lysis buffer (RIPA: protease inhibitor = 100:1) to each well. After lysing for 30 min, use a clean scraper to scrape the cells to one side of the plate (one direction). Transfer the liquid to an EP tube (1.5 ml) to collect the suspension. Centrifuge in a refrigerated high-speed centrifuge (4℃, 12000 rpm, 15 min).

[0071] BCA method for detecting protein concentration

[0072] (1) Preparation of protein standards: Protein standard preparation solution (1200μL) + protein standard (30mgBSA) are thoroughly mixed → 25mg / ml.

[0073] (2) Dilute the above protein standard with PBS to → 0.5 mg / ml.

[0074] (3) Preparation of BCA working solution: Prepare an appropriate amount of BCA working solution according to (50 volumes of BCA reagent A + 1 volume of BCA reagent B). Use within 24 hours.

[0075] (4) Protein concentration determination: Add 0, 1, 2, 4, 8, 12, 16 and 20 μL of standard to the standard wells of the 96-well plate, respectively, and then add standard diluent to make up to 20 μL, which are equivalent to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg / ml, respectively.

[0076] (5) Add 200 μL of the prepared (3) to the above well using a pipette, set the shaker temperature to 37°C, and incubate for 30 min.

[0077] (6) Set the iMark microplate reader to 560 nm and detect the absorbance. Finally, calculate the protein concentration of the sample based on the obtained standard curve and the sample volume used.

[0078] protein denaturation

[0079] (1) Add 5×SDS-PAGE loading buffer according to the volume ratio of supernatant to 5×SDS-PAGE loading buffer = 4:1 and mix by blowing.

[0080] (2) Place the EP tube containing protein lysis buffer and loading buffer in a 95°C metal bath and boil for 10 min to denature the protein. Cool on ice, vortex and shake, then place in a -80°C freezer for subsequent experiments.

[0081] Rubber Mixing

[0082] (1) Preparation of the lower gel: 15 ml solution, 15 ml buffer, 300 μL coagulant (4 pieces). Add the mixed lower gel solution to the cleaned glass plates one by one, then add methanol and press the liquid surface flat. After about 30 minutes, the lower gel will solidify naturally, then pour off the methanol.

[0083] (2) Prepare the top layer gel: 4 ml solution, 4 ml buffer solution, 80 μL coagulant (4 pieces). Add the prepared top layer gel mixture to the solidified bottom layer gel, immediately insert the comb, and wait for the top layer gel to solidify naturally.

[0084] Sample loading: Load the sample according to the measured protein concentration. Place the solidified gel into the electrophoresis tank, tighten it, fill with liquid, and check for leaks. Vertically pull the comb out of the electrophoresis solution to prevent air bubbles from forming. Add markers and the extracted protein sample to the wells.

[0085] Electrophoresis: First, run the target protein at a constant voltage of 80V until it reaches the boundary between the upper and lower gel layers, then adjust the power supply to 120V.

[0086] Transfer: Cut the PVDF membrane into pieces (8cm long, 5cm wide) beforehand and immerse it in methanol for activation (2 min). Pour the prepared TBST (pre-cooled) into the transfer tank. Wet the filter paper and sponge with transfer buffer. Place the filter paper → gel → PVDF membrane (sandwich) into the transfer clamp in the following order, avoiding air bubbles during stacking. Clamp the transfer clamp tightly. The entire process is carried out in the transfer buffer. Then place the membrane in the transfer tank, pour in fresh transfer buffer, and set the constant current to 250mA for 2 hours for transfer.

[0087] Blocking: After the transfer is complete, gently pick up the PVDF membrane and place it in TBST. Rinse it on a shaker for about 5 minutes to remove any residual transfer solution. Then, use tweezers to pick up the PVDF membrane and place it in the prepared blocking solution. Incubate at room temperature for 2 hours.

[0088] Incubate antibodies:

[0089] (1) Primary antibody incubation: Dilute according to the corresponding antibody dilution, then put the blocked PVDF membrane into the diluted antibody, ensuring that the antibody and PVDF membrane can be in full contact, and then incubate overnight on a shaker at 4°C.

[0090] (2) Washing the membrane: Recover the primary antibody, place the PVDF membrane in TBST solution, wash 2-3 times, and shake on a shaker for 10 minutes each time.

[0091] (3) Secondary antibody incubation: Hold the PVDF in the TBST with tweezers and incubate it in the prepared secondary antibody dilution solution at room temperature for 2 hours.

[0092] (4) Membrane washing: Same as (2).

[0093] Exposure: Prepare ECL luminescent solution (A:B = 1:1), place the strip on the luminescence analyzer plate, add an appropriate amount of the prepared luminescent solution and ensure it evenly wets the PVDF film, expose using the Tianneng gel imaging analysis system, and save the image. Grayscale analysis: Perform grayscale analysis on the exposed strip using ImageJ software.

[0094] The specific method for determining lactic acid content is as follows:

[0095] (1) Preparing cells

[0096] (2) Drug pretreatment: Prepare the drug at the required concentrations of 2μM, 4μM, 8μM and 16μM, set up a blank control group, and incubate in an incubator for 24h.

[0097] (3) Preparation of working solution (enzyme): Before the experiment, mix the stock solution (reagent 2) and the diluent (reagent 1) in a ratio of 1:100. Prepare before each experiment and store in a refrigerator at 4°C. The shelf life is 24 hours.

[0098] (4) Preparation of colorimetric solution: Take reagent 3 and reagent 4 from the kit, shake and invert repeatedly to fully dissolve and mix them. This is the colorimetric solution. Store at 4°C away from light. It is valid for 2 weeks.

[0099] (5) Sample pretreatment: Dilute the cell culture medium with physiological saline to different concentration gradients for preliminary experiments. This ensures that the measured OD value is between 0.05 and 0.35 (sample-blank). If the OD value is <0.05, the sample concentration needs to be increased and the measurement repeated; if the OD value is >0.35, the sample needs to be diluted again and the test repeated. Add reagents according to the order specified in the instructions.

[0100] (6) Mix the centrifuge tubes containing the samples thoroughly. Under specific conditions of a wavelength of 530 nm, a light path of 1 cm, and zero distilled water, measure the absorbance (A) of each tube. Determine the lactic acid content using the formula provided in the kit. Results are as follows: Figure 6-1 , Figure 6-2 As shown.

Claims

1. The use of AST-487 in drugs that downregulate SYVN1 expression and inhibit HCC.

2. The use according to claim 1, characterized in that: The structural formula of AST-487 is as follows: 。 3. The use according to claim 1, characterized in that: The downregulation of SYVN1 expression refers to the downregulation of SYVN1 promoter function, gene, and protein expression.

4. The use according to claim 1, characterized in that: The inhibition of HCC refers to the suppression of the activity, invasion, and migration of HepG2 or Huh7 liver cancer cells.

Images