Application of esulvirin in preparation of hepatocellular carcinoma treatment medicine

Abstract

The invention belongs to the field of medicines for hepatocellular carcinoma, and particularly relates to application of esulvirin in preparation of a medicine for treating hepatocellular carcinoma. Starting from analysis of a mechanism of lipid metabolism negative feedback imbalance in tumor cells, it is found that interaction of ADSL and INSIG1 / 2 protein is an important reason for abnormal activation of lipid synthesis in the tumor cells, and based on the molecular mechanism, computer virtual screening is applied, molecule and cell experiments are combined, and the lipid synthesis activity in the tumor cells is improved. A potential drug esulvirin for treating hepatocellular carcinoma is screened from a compound library approved by FDA. It is found for the first time that the esulvirin can be used for preparing the hepatocellular carcinoma treatment medicine, the esulvirin shows good anti-hepatocellular carcinoma activity in vitro and in vivo, tumor cell proliferation can be inhibited by inhibiting tumor lipid synthesis, and then the purpose of treating hepatocellular carcinoma is achieved.

Description

Technical Field

[0001] This invention belongs to the field of hepatocellular carcinoma medicine, specifically relating to the application of sulfvirine in the preparation of hepatocellular carcinoma treatment drugs. Background Technology

[0002] Hepatocellular carcinoma (HCC) is not only a type of solid tumor with a high mortality rate worldwide, but the five-year survival rate for patients with primary localized HCC is approximately 17%. Most HCC patients are asymptomatic in the early stages and are diagnosed at an advanced stage with local or distant metastases. Traditional methods such as surgery, radiotherapy, and chemotherapy are ineffective for mid-to-late-stage HCC, posing a significant challenge to clinical treatment. In recent years, more than ten targeted drugs targeting multiple kinases or receptors, including lenvatinib (a tyrosine kinase inhibitor), have been used to treat HCC. While these drugs have some therapeutic effect, they all have a high probability of developing drug resistance. Therefore, finding more effective drugs to overcome the drug resistance caused by traditional targeted therapies is an urgent problem to be solved in the treatment of HCC.

[0003] Numerous studies have shown that inhibiting tumor cell growth by affecting tumor cell metabolism, thereby "starving" tumor cells, represents a novel and valuable approach to cancer treatment. Targeting tumor metabolism to treat cancer has become the next major battleground in basic and clinical cancer research, following immunotherapy. In recent years, several anti-tumor drugs targeting nucleic acid synthesis pathways have been approved for marketing in clinical practice, such as methotrexate, cytarabine, and 5-fluorouracil (5-FU). Furthermore, compounds targeting glycolysis (e.g., lonidinamine, TLN-232), glutamine metabolism (e.g., phenylacetate), and fatty acid synthesis metabolism (e.g., orlistat, SB-204990) are currently in clinical trials.

[0004] The phenomenon of tumor cells altering their metabolism to achieve rapid growth is known as tumor metabolic reprogramming. Tumor cell energy metabolism reprogramming, genomic instability and mutations, immune escape, and abnormal infiltration of inflammatory cells are four emerging characteristics of tumor cells that have gained increasing recognition in the last decade. This new understanding of the fundamental characteristics of tumor biology has opened up new avenues for tumor prevention and treatment. Changes in cellular metabolism are inextricably linked to all stages of tumor development. Metabolic reprogramming can lead to a series of growth advantages for tumor cells, such as overcoming telomere replication limitations, obtaining self-sufficient growth signals, reprogramming intracellular gene expression, resisting apoptosis, achieving immune escape, promoting cell migration and invasion, and enhancing angiogenesis. Many metabolic pathways, including glycolysis, the tricarboxylic acid cycle, the urea cycle, uric acid metabolism, nucleic acid metabolism, fatty acid metabolism, cholesterol metabolism, glutamine metabolism, serine metabolism, one-carbon unit metabolism, and choline metabolism, are reprogrammed in tumor cells. In recent years, the theory that abnormal cellular metabolism precedes tumorigenesis has been gradually confirmed in research, and the role and molecular mechanisms of important metabolic abnormalities in malignant transformation of cells have become an international academic frontier. Metabolic reprogramming fulfills three key needs of tumor cells: the energy required for rapid proliferation, sufficient biomolecules for the synthesis of nucleic acids, proteins, and lipids, and the maintenance of redox homeostasis. Therefore, only by fully elucidating the molecular mechanisms of tumor metabolic reprogramming can we uncover the critical weaknesses in tumor metabolic pathways and transform them into novel approaches to cancer treatment.

[0005] Lipid synthesis in normal cells is regulated by negative feedback: when intracellular lipid concentration is within the normal range, the lipid-binding endoplasmic reticulum transmembrane proteins INSIG1 / 2 bind to the lipid-regulating transcription factor SREBP and its escort protein SCAP, preventing SREBP activation and inhibiting cellular lipid synthesis. Conversely, when cellular lipid levels are low, the lipid-free INSIG1 / 2 no longer interacts with SCAP / SREBP, leading to SREBP translocation from the endoplasmic reticulum to the Golgi apparatus. After cleavage by proteases S1P and S2P, the N-terminus of SREBP is translocated to the nucleus, activating the transcription of lipid synthesis-related genes. However, in tumor cells, even with normal intracellular lipid concentration, SREBP-mediated lipid synthesis remains activated. Therefore, the molecular mechanisms underlying lipid metabolism in tumor cells, which differ from those in normal cells, are one of the core issues in tumor metabolism research. This invention will provide new ideas and effective solutions for the development of anti-tumor drugs targeting lipid metabolism reprogramming. Summary of the Invention

[0006] The technical problem this invention aims to solve is to provide the application of anisulin in the preparation of drugs for treating hepatocellular carcinoma. Starting from elucidating the mechanism of negative feedback dysregulation of lipid metabolism in tumor cells, this invention discovers that the interaction between ADSL and INSIG1 / 2 proteins is a crucial cause of abnormal activation of lipid synthesis in tumor cells. Based on this molecular mechanism, using computer-generated virtual screening combined with molecular and cellular experiments, anisulin, a potential drug for treating hepatocellular carcinoma, was screened from an FDA-approved compound library.

[0007] The technical content of this invention is as follows:

[0008] This invention provides the application of elsulfavirine in the preparation of hepatocellular carcinoma treatment drugs.

[0009] The active ingredient of the drug includes anisulin, which inhibits the growth of hepatocellular carcinoma by inhibiting lipid synthesis in liver cancer cells.

[0010] The drugs also include lenvatinib. The combination of lenvatinib and sulfamethoxazole has a synergistic effect and inhibits the growth of hepatocellular carcinoma. Lenvatinib is a first-line targeted drug for the treatment of liver cancer.

[0011] The drug may also include a pharmaceutically acceptable carrier, or the drug may further contain one or more of the following: disintegrant, humectant, binder, filler, absorption enhancer, solvent, lubricant, surfactant, flavoring agent, sweetener, antioxidant, preservative, pigment, ointment base, and transdermal penetration enhancer.

[0012] The drugs include tablets, capsules, injections, granules, and suspensions.

[0013] Elsulfavirine:

[0014] Aliases: Elipida, R-1206, RO-4970335, RO-5011500, VM-1500

[0015] Molecular formula: C 24 H 17 BrCl2FN3O5S

[0016] The structure is as follows:

[0017]

[0018] Manufacturing company: Viriom, Inc.

[0019] Initial approval date: 2017-06-30

[0020] First country / region to receive approval: Russia

[0021] First approved indication: HIV infection

[0022] InChIKey: ULTDEARCBRNRGR-UHFFFAOYSA-N

[0023] CAS No.: 868046-19-9.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. This invention is the first to discover that anisulin can be used in the preparation of drugs for the treatment of hepatocellular carcinoma, thus expanding the indications for anisulin. Aisulin was approved for marketing in Russia in 2017 for the treatment of HIV infection. This invention has found that anisulin exhibits good anti-hepatocellular carcinoma activity both in vitro and in vivo, and experiments show that anisulin can inhibit tumor cell proliferation by inhibiting tumor lipid synthesis, thereby achieving the goal of treating hepatocellular carcinoma.

[0026] 2. This invention also proposes that the combined use of aniflurin and lenvatinib, a first-line targeted drug for the treatment of liver cancer, has a synergistic effect, resulting in better treatment efficacy for hepatocellular carcinoma.

[0027] 3. Asulfuron-methyl has been approved by the FDA, and its safety is guaranteed. Attached Figure Description

[0028] Figure 1 For the immunoprecipitation and Coomassie Brilliant Blue assay analysis in Example 1, the binding of Flag-INSIG1 to ADSL in Huh7 cells was analyzed with and without glucose treatment. The results of selective peptide hits of proteins binding to Flag-INSIG1, determined by mass spectrometry, are shown on the right.

[0029] Figure 2 For the immunoprecipitation and Coomassie Brilliant Blue assay analysis in Example 1, the binding of Flag-INSIG2 to ADSL in Huh7 cells was determined with and without glucose treatment. The selective peptide hits of proteins binding to Flag-INSIG2, determined by mass spectrometry, are shown on the right.

[0030] Figure 3 For the immunoprecipitation analysis of Example 1, the binding of ADSL with INSIG1 / INSIG2 was detected;

[0031] Figure 4For the immunoprecipitation analysis of Example 2, inhibitors that block the interaction between ADSL and INSIG1 / INSIG2 were identified, wherein U0126, MK-2206, SU6656 and AEB071 were inhibitors of protein kinases ERK1 / 2, AKT, c-SRC and PKC, respectively.

[0032] Figure 5 For the immunoprecipitation analysis of Example 2, the interaction between ADSL and INSIG1 / INSIG2 was detected in Huh7 cells with knockdown of endogenous PKCε and replenishment of PKCεWT and kinase-inactivating mutant (DN) expression, respectively, under glucose stimulation and without.

[0033] Figure 6 For the immunoprecipitation analysis in Example 2, the interaction between ADSL and kinase PKCε was detected in Huh7 and HCCLM3 cells, respectively.

[0034] Figure 7 The mass spectrum of Example 3 shows that ADSL is phosphorylated at the S407 site;

[0035] Figure 8 For the in vitro kinase experiment of Example 3, the phosphorylation of ADSL by PKCε in vitro;

[0036] Figure 9 For the immunofluorescence experiment of Example 3, the colocalization of ADSL and INSIG1 was detected in Huh7 cells with and without glucose stimulation, in the presence and absence of endogenous ADSL and after the expression of ADSL WT and phosphorylation inactivation mutant (S407A) were respectively replenished.

[0037] Figure 10 For the immunoprecipitation analysis in Example 3, the interaction between ADSL and INSIG1 / 2 was detected in Huh7 cells with knockdown of endogenous ADSL and replenishment of ADSL WT and phosphorylation-inactivating mutant (S407A) respectively, under glucose stimulation and without.

[0038] Figure 11 For the immunoprecipitation analysis of Example 4, the interaction between INSIG1 / 2 and SCAP was detected in Huh7 cells with knockdown of endogenous ADSL and replenishment of ADSL WT and phosphorylation-inactivating mutant (S407A) respectively, under glucose stimulation and without.

[0039] Figure 12For the immunoprecipitation analysis in Example 4, in Huh7 cells with knockdown of endogenous ADSL and replenishment of ADSL WT and phosphorylation-inactivating mutant (S407A) respectively, the colocalization of SCAP and endoplasmic reticulum was detected under glucose stimulation and without glucose stimulation. Calnexin: endoplasmic reticulum marker.

[0040] Figure 13 For the immunoprecipitation analysis in Example 4, in Huh7 cells with knockdown of endogenous ADSL and replenishment of ADSL WT and phosphorylation-inactivating mutant (S407A) respectively, the colocalization of SCAP and Golgi apparatus was detected under glucose stimulation and without glucose stimulation. Golgin97: Golgi Marker;

[0041] Figure 14 For the immunoblotting analysis in Example 4, the cleavage of SREBP was detected in Huh7 and HCCLM3 cells with and without glucose stimulation, respectively, after knocking down endogenous ADSL and reintroducing ADSL WT and phosphorylation-inactivating mutant (S407A).

[0042] Figure 15 For the immunoblotting analysis in Example 4, the nuclear translocation of SREBP was detected in Huh7 cells with and without glucose stimulation, in the presence or absence of glucose stimulation, after knocking down endogenous ADSL and reintroducing ADSL WT and phosphorylation-inactivating mutant (S407A), respectively.

[0043] Figure 16 For the luciferase reporter gene experiment in Example 4, the SRE reporter gene activity was detected in Huh7 cells with knockdown of endogenous ADSL and reintroduction of ADSLWT and phosphorylation-inactivating mutant (S407A) under glucose stimulation conditions, respectively.

[0044] Figure 17 For the real-time quantitative PCR experiment of Example 4, the mRNA expression levels of SREBP1 target genes (FASN, SCD, ACACA and GPAM) were detected in Huh7 cells with knockdown of endogenous ADSL and replenishment of ADSLWT and phosphorylation-inactivating mutant (S407A) respectively, with and without glucose stimulation. **P<0.01 (two-tailed t test).

[0045] Figure 18 Example 4 is radioactive. 14 C-glucose incorporation experiment, measured 14 The conversion of C-glucose into triglycerides (TGs) (left) and fatty acids (FAs) (right);

[0046] Figure 19For the immunofluorescence experiment of Example 4, Bodipy and DAPI staining were used for immunofluorescence experiments. The scale bar is 20 μm. The left side is a representative image. The average number of lipid droplets was quantified using ImageJ (right side). ***P<0.001 (two-tailed t-test).

[0047] Figure 20 The results of the mouse liver in situ tumor size detection in Example 5 are shown. The right side shows the tumor volume (n=6). **P<0.01 was verified by a two-tailed Student's t-test.

[0048] Figure 21 For the proliferation experiment in Example 5, the proliferation of Huh7 cells expressing ADSL WT and phosphorylation-inactivating mutant (S407A) was investigated by knocking down endogenous ADSL and reintroducing them, respectively.

[0049] Figure 22 The results of subcutaneous tumor size detection in mice in Example 5 are shown on the left, the middle shows the tumor growth, and the right shows the tumors (n=6). **P<0.01 was verified by a two-tailed Student's t-test.

[0050] Figure 23 For Example 5, immunohistochemical analysis was performed on the tumor shown. The left side shows a representative staining image, and the right side shows the Ki-67 positive cells.

[0051] Figure 24 For Example 5, immunohistochemical analysis was performed on the tumor shown. The left side shows a representative staining image, and the right side shows the TUNEL-positive cells.

[0052] Figure 25 Example 6 shows immunohistochemical staining, where tumor tissue and adjacent normal tissue of hepatocellular carcinoma patients are stained (top side) and IHC staining score is statistically plotted (bottom side). **P<0.01 was determined by a two-tailed Student's t-test.

[0053] Figure 26 Immunohistochemical experiments (left side) and correlation analysis (right side) of human liver cancer samples from Example 6;

[0054] Figure 27 Survival curves for ADSL S407 phosphorylation levels of high expression vs. low expression in Example 6;

[0055] Figure 28 The INSIG2-ADSL complex model of Example 7 is constructed based on a protein-protein complex (INSIG2AlphaFoldDB: Q9Y5U4, ADSL PDB code: 4FLC), with INSIG2 and ADSL displayed as green and cyan, respectively.

[0056] Figure 29 This is a schematic diagram of the virtual screening workflow based on pharmacophores and docking in Example 8, used for screening small molecule inhibitors of the INSIG2-ADSL complex (left side); the binding modes and interaction profiles of the three highest-scoring inhibitors with the INSIG2-ADSL complex are shown on the right side, with hydrogen bonds, metal ion coordination, and salt bridges represented by yellow, purple, and red, respectively; green, violet, and blue represent hydrophobic, positively charged, and polar amino acid residues, respectively.

[0057] Figure 30 For the GST tag pull-down experiment in Example 9, purified His-INSIG2 was first mixed with GST-ADSL, and then incubated for 1 hour with or without the addition of Tezacaftor (5 μM and 50 μM, respectively), Elsulfavirine (2 μM and 25 μM, respectively) or Pyrithioxin (10 μM and 100 μM, respectively), followed by immunoblotting analysis.

[0058] Figure 31 For the immunoprecipitation experiment in Example 9, Huh7 cells expressing Flag-ADSL were pretreated with Tezacaftor (20 μM), Elsulfavirine (10 μM), and Pyrithioxin (100 μM) for 1 hour, and then subjected to high glucose stimulation for 2 hours, respectively, followed by immunoprecipitation and Western blot analysis.

[0059] Figure 32 For the immunofluorescence analysis in Example 9, Huh7 cells were pretreated with Elsulfavirine (10 μM) for 1 hour and then subjected to high glucose treatment, followed by immunofluorescence experiments. The scale bar is 20 μm. The left side is a representative image. ImageJ was used to measure the intensity of the green (ADSL) or red (INSIG1) fluorescence signal along the white diagonal line for colocalization analysis (right side).

[0060] Figure 33 For the immunoblotting analysis in Example 10, Huh7 cells were pretreated with different concentrations of Tezacaftor (5 μM, 20 μM, 50 μM), Elsulfavirine (2 μM, 10 μM, 25 μM), or Pyrithioxin (10 μM, 30 μM, 100 μM) for 1 hour, followed by treatment with or without glucose for 2 hours. Cell lysates were collected, and then immunoblotting analysis was performed using specific antibodies.

[0061] Figure 34For the immunofluorescence analysis in Example 10, Huh7 cells were first treated with Tezacaftor (20 μM), Elsulfavirine (10 μM), or Pyrithioxin (100 μM) for 1 hour, followed by high glucose treatment, and then immunofluorescence experiments were performed. The scale bar is 20 μm. The left side is a representative image. The relative nuclear intensity of at least 30 cells was quantitatively analyzed by ImageJ (right side). ***P<0.001 (two-tailed t-test);

[0062] Figure 35 For the immunofluorescence analysis of Example 10, Huh7 cells were pretreated with Elsulfavirine (10 μM) for 1 hour and then stimulated with high glucose for 4 hours before immunofluorescence experiments were performed. The left side is a representative image, with white arrows indicating SCAPs localized to Calnexin. Image J was used to measure the intensity of green (Calnexin) or red (SCAP) fluorescence signals along the white diagonal lines as colocalization analysis (right side).

[0063] Figure 36 For the immunofluorescence analysis in Example 10, Huh7 cells were pretreated with Elsulfavirine (10 μM) for 1 hour and then stimulated with high glucose for 2 hours before immunofluorescence experiments were performed. The left side shows a representative image with a scale bar of 20 μm. The white arrows indicate SCAPs located in the Golgi apparatus. Image J was used to measure the intensity of the green (Golgin97) or red (SCAP) fluorescence signal along the white diagonal line as colocalization analysis (right side).

[0064] Figure 37 For the real-time quantitative PCR analysis in Example 10, Huh7 cells were pretreated with Elsulfavirine and stimulated with high glucose for 8 hours. The mRNA expression levels of SREBP-1 target genes (FASN, SCD, ACACA and GPAM) were measured using real-time quantitative PCR. **P<0.01 (two-tailed t-test).

[0065] Figure 38 For the immunoblotting analysis in Example 10, Huh7 and HCCLM3 cells were pretreated with Elsulfavirine (10 μM) for 1 hour before high glucose stimulation, followed by immunoblotting analysis using a specific antibody.

[0066] Figure 39 The radioactivity of Example 10 14 C-glucose incorporation experiment, measured 14The conversion of C-glucose into triglycerides (TGs) (left side) and fatty acids (FAs) (right side) is shown in mean ± standard deviation. *P<0.05 (two-tailed t-test) (n=6 biological replicates).

[0067] Figure 40 For the immunofluorescence experiment of Example 10, Huh7 cells were pretreated with Elsulfavirine (10 μM) for 1 hour and then stimulated with high glucose. Immunofluorescence experiments were performed using Bodipy and DAPI staining. The scale bar is 20 μm. The top image is a representative image. The average number of lipid droplets was quantified using ImageJ (bottom). ***P<0.001 (two-tailed t-test).

[0068] Figure 41 In Example 11, tumor growth was observed in nude mice treated with lenvatinib, esulvirine, or a combination of both. On day 7 after inoculation, mice were injected intraperitoneally with the drugs daily. On day 28 after injection, the mice were euthanized and the tumor growth was examined. The tumor volume was measured. The data are expressed as mean ± standard deviation. **Compared with the control group, P < 0.01 (two-tailed t-test).

[0069] Figure 42 In Example 11, tumor growth was observed in nude mice treated with lenvatinib, esulvirine, or a combination of both. On day 7 after inoculation, mice were injected intraperitoneally with the drugs daily. On day 28 after injection, the mice were euthanized and the tumor weight was measured. Data are expressed as mean ± standard deviation. **P<0.01 compared with the control group (two-tailed t-test).

[0070] Figure 43 Immunohistochemical experiments of mouse tumor samples from Example 12;

[0071] Figure 44 Immunohistochemical analysis of the tumor shown in Example 13 after treatment was performed. The left side shows a representative staining image, and the right side shows the Ki-67 positive cells.

[0072] Figure 45 Immunohistochemical analysis of the tumor shown in Example 13 after treatment was performed. The left side shows a representative staining image, and the right side shows the TUNEL-positive cells. Detailed Implementation

[0073] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials are commercially available.

[0074] I. Detection methods involved in the embodiments

[0075] 1. Protein level detection

[0076] First, proteins are extracted from cells. Then, corresponding antibodies are used to react with the proteins to form an antigen-antibody reaction, and the expression and function of specific target proteins are determined by substrate color development.

[0077] The protein level detection system used in this invention includes: immunoprecipitation and immunoblotting assay.

[0078] The antibodies used were ADSL (ab154872), INSIG1 (ab70784), PKCεS729 (ab63387), SRC (pY418)(ab4816), Calnexin (ab22595), Ki-67 (ab21700), SREBP1 (ab28481), SREBP2 (ab28482), SCAP (ab125186), SCD1 (ab19862), and FASN (ab22759), which were purchased from Abcam. PKCε (#2683), AKT (#9272), p44 / 42MAPK (ERK1 / 2) (#9102), AKT (pS473) (#4060), c-SRC (#2108), HA (#50297) (mouse), and HA (#3724) (rabbit) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027), mouse ADSL (sc-365623), PKCθ (sc-81534), ERK1 / 2 (sc-514302), SREBP-1 (sc-13551), His (sc-8036), GST (sc-138), and tubulin (sc-8035) antibodies were purchased from Santa Cruz Biotechnology. Elsulfavirine, Tezacaftor, and Pyrithioxin were purchased from MedChemExpress. Lenvatinib was purchased from Selleck.

[0079] 2. Immunofluorescence detection and analysis

[0080] After treatment, the cells were fixed and incubated with a 1:100 dilution of primary antibody, a fluorescent dye-conjugated secondary antibody, and DAPI. Immunofluorescence microscopy images of the cells were obtained and observed and photographed using an IX81 confocal microscope system (Olympus America). Finally, the acquired images were analyzed for fluorescence intensity using ImageJ (National Institutes of Health).

[0081] Antibodies used:

[0082] INSIG2(PA5-41707), Golgin97(CDF4)(14-9767-82), BODIPY(D3922), Calnexin(MA3-027), 6-diamidino-2-phenylindole(DAPI), Alexa Fluor 488goat anti-rabbit(A11008), Alexa Fluor 594goat anti-rabbit (A11012), Alexa Fluor 488goat anti-mouse (A11029) and Alexa Fluor 594goat anti-mouse antibodies (A11005) were purchased from ThermoFisher Scientific.

[0083] 3. Real-time quantitative PCR

[0084] Cellular RNA was extracted using the TRIzol method, and 1 μg of RNA from each sample was used for cDNA synthesis using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems). In Light... Real-time quantitative PCR (Takara) was performed using the SYBR Premix Ex Taq kit on a 480PCR system (Bio-Rad). In the experiments, α-Tubulin protein was used as a reference gene for normalization.

[0085] The following primers are used for qRT-PCR:

[0086] FASN, 5′-CACAGGGACAACCTGGAGTT-3′ and 5′-ACTCCACAGGTGGGAACAAG-3′; SCD, 5′-CGACGTGGCTTTTCTTCTC-3′ and 5′-CCTTCTCTTTGACAGCTGGG-3′; ACACA, 5′-AGTGGGTCACCCATTGTT-3′ and 5′-TTCTAACAGGAGCTGGAGCC-3′;

[0087] GPAM, 5′-TTGTGGCTTGCCTGCTCCTCTA-3′ and 5′-AATCACGAGCCAGGACTTCCTC-3′; HMGCR, 5′-TCTGGCAGTCAGTGGGAACTATT- L DLR, 5′-AACGGTCATTCACCCAGGTC-3′ and 5′-GGCTGAAGAATAGGAGTTGCC-3′; FDFT1, 5′-CGATAGCTGTGTGCAAAGTAACT-3′ and 5 ′-CCATCTGCTGAGTGCTTTCTG-3′; α-Tubulin, 5′-TATCGAGCGCCCAACCTACAC-3′ and 5′-CACCAGGTTGGTCTGGAATTCTGT-3′.

[0088] 4. Luciferase assay analysis

[0089] SRE-driven luciferase activity in cell lysates was detected using a luciferase detection system. First, Huh7 cells in 24-well plates were transfected with 0.1 μg of SRE-driven luciferase reporter protein and 0.075 μg of β-galactosidase. Twenty-four hours after transfection, the cells were cultured in a high-glucose medium for 6 hours, and the activities of luciferase and β-galactosidase in the cell lysates were measured.

[0090] 5. Acquisition and analysis of mouse tumor samples

[0091] Huh7 cells (1×10) 6Subcutaneous injections were administered to 6-week-old male BALB / c nude mice. Each experimental group consisted of six mice. They were then randomly assigned to four groups: a control group, a lenvatinib group (10 mg / kg daily), an elsulfavirine group (10 mg / kg daily), and a combination therapy group (10 mg / kg lenvatinib daily plus 10 mg / kg elsulfavirine daily). Treatment of mice consisted of daily gavage with 0.5% methylcellulose (control group) or daily gavage with lenvatinib diluted in 0.5% methylcellulose for 20 days, or daily gavage with elsulfavirine diluted in 0.5% methylcellulose for 20 days. Twenty-eight days after injection of Huh7 cells, tumors were removed, fixed in 4% formaldehyde, and embedded in paraffin. Tumor volume was calculated using the formula V = 1 / 2a. 2 The measurements were performed using the formula b, where V represents volume, a represents the shortest diameter, and b represents the longest diameter. Animal handling complied with relevant institutional and national guidelines and regulations. The use of the animals was approved by the Institutional Review Committees of the First Affiliated Hospital of Zhejiang University School of Medicine and the Qingdao Cancer Institute, China.

[0092] II. Materials Used in the Examples

[0093] 1. Cell types:

[0094] Huh7 and HCCLM3 cells (human liver cancer cell lines) were obtained from the Cell Bank of the Chinese Academy of Sciences;

[0095] The athymic nude mouse is the BALB / c athymic nude mouse.

[0096] 2. The shRNA sequence used for gene knockdown is as follows:

[0097] ADSL-shRNA:5'-GCAGAACATTTCTGAAGGATT-3'.

[0098] Example 1: High glucose conditions can induce the interaction between ADSL and INSIG1 / INSIG2.

[0099] Huh7 cells overexpressing Flag-INSIG1 / Flag-INSIG2 were treated with and without high glucose for 2 hours. Immunoprecipitation experiments were performed using Flag antibodies. The samples obtained from the immunoprecipitation were further separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Figure 1 and Figure 2 As shown, after high glucose treatment, immunoprecipitation yielded a distinct differential protein band, which was subsequently identified as ADSL by mass spectrometry.

[0100] To further demonstrate the interaction between ADSL and INSIG1 / INSIG2 under high glucose conditions, we treated Huh7 cells with and without high glucose for 2 hours and performed immunoprecipitation experiments using endogenous ADSL antibodies. Figure 3 As shown, high sugar levels promote the interaction between ADSL and INSIG1 / 2.

[0101] Conclusion: In hepatocellular carcinoma cells, high glucose treatment promotes the interaction between ADSL and INSIG1 / 2.

[0102] Example 2: Under high glucose conditions, kinase PKCε mediates the interaction between ADSL and INSIG1 / INSIG2.

[0103] Huh7 cells were pretreated for 30 min with or without the ERK1 / 2 inhibitor U0126 (20 μM), the AKT inhibitor MK-2206 (10 μM), the c-SRC inhibitor SU6656 (4 μM), and the PKC inhibitor AEB0071 (5 μM). Following this, they were treated for 2 hours with or without high glucose, and immunoprecipitation experiments were performed. Figure 4 As shown, GSK2656157 inhibited the interaction between ADSL and INSIG1 / 2 under high glucose stimulation.

[0104] To further verify that PKCε activity is crucial for the binding of ADSL and INSIG1 / 2, we constructed cell lines with knockdown of endogenous PKCε and re-expression of PKCε-WT and PKCε-DN (kinase-inactivating point mutations). Immunoprecipitation experiments were performed after high glucose treatment. Similarly, as... Figure 5 As shown, ADSL interacts with INSIG1 / 2 only in cells expressing PKCε-WT, while it does not in cells expressing PKCε-DN. Furthermore, Figure 6 Immunoprecipitation experiments showed that PKCε can indeed interact with ADSL in response to high glucose stimulation.

[0105] Conclusion: Under high glucose stimulation, PKCε can mediate the interaction between ADSL and INSIG1 / 2.

[0106] Example 3: PKCε phosphorylates the ADSL S407 site and promotes its binding to INSIG1 / 2.

[0107] Since PKCε typically functions as a kinase, we incubated kinase-active PKCε with prokaryotically purified His-ADSL in vitro in the presence of ATP, and then analyzed the incubation product by phosphorylation mass spectrometry. Figure 7 As shown, ADSL is phosphorylated at the S407 site.

[0108] PKCε, possessing kinase activity, was incubated in vitro with prokaryotically purified His-ADSL WT and His-ADSL S407A in the presence of ATP-γ-S, and then subjected to Western blotting experiments, such as... Figure 8 As shown, phosphorylation signals only occur when both PKCε and His-ADSL WT are present, while no phosphorylation was detected in the ADSL S407A mutation.

[0109] Next Figure 9 Immunofluorescence experiments showed that ADSL colocalized with INSIG1 in response to high glucose stimulation only in Huh7 cells expressing ADSL WT, while this colocalization was lost in cells expressing ADSL S407.

[0110] Similarly, the following Figure 10 Immunoprecipitation also showed that in Huh7 cells expressing ADSL WT, ADSL interacts with INSIG1 in response to high glucose stimulation, while in cells expressing ADSL S407, ADSL no longer interacts with INSIG.

[0111] Conclusion: Phosphorylation of the ADSL S407 site under high glucose stimulation is crucial for the interaction between ADSL and INSIG1 / 2.

[0112] Example 4: Phosphorylation of the ADSL S407 site and its interaction with INSIG1 / 2 promoted the activation of the SREBP pathway and lipid synthesis.

[0113] The separation of SCAP from INSIG1 / 2 is the first step in cellular lipid synthesis in response to high glucose stimulation. Immunoprecipitation experiments show that, as Figure 11 As shown, in cells expressing ADSL WT, SCAP dissociates from INSIG1 / 2 in response to high glucose stimulation, while in cells expressing ADSL S407, SCAP no longer dissociates from INSIG1 / 2 even under high glucose conditions.

[0114] Similarly, immunofluorescence assay ( Figure 12 and Figure 13 This indicates that in cells expressing ADSL WT, SCAP translocates from the endoplasmic reticulum to the Golgi apparatus in response to high glucose stimulation, while in cells expressing ADSL S407, this translocation of SCAP does not occur. Here, Calnexin and Golgin97 serve as markers for the endoplasmic reticulum and Golgi apparatus, respectively.

[0115] The following immunoblotting experiment ( Figure 14This indicates that cells expressing ADSL WT underwent 8 hours of high glucose treatment, which promoted SREBP1 cleavage, while this promotion was inhibited in ADSL S407A cells. Similarly, immunofluorescence ( Figure 15 The experiment showed that SREBP entered the nucleus in cells expressing ADSL WT in response to high glucose stimulation, while SREBP entry into the nucleus was significantly inhibited in cells expressing ADSL S407A.

[0116] In Huh7 cells, after knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and the Flag-rADSL S407A mutant were reintroduced. Simultaneously, a plasmid containing β-galactosidase and an SRE-driven luciferase reporter group was expressed, followed by high glucose treatment for 8 hours. The activity of the SRE-driven luciferase was measured 24 hours after transfection. Figure 16 As shown, high glucose treatment increases SRE-driven luciferase activity, while the ADSL S407A mutation can no longer respond to high glucose stimulation and increase luciferase activity.

[0117] After knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and Flag-rADSLS407A mutant Huh7 cell lines were reintroduced and treated with high glucose for 8 hours. The mRNA expression levels of FASN, SCD, ACACA, and GPAM genes were detected using qPCR. Figure 17 As shown, high glucose treatment increases the mRNA expression level of the above genes, while the ADSL S407A mutation does not increase the mRNA expression level of the above genes.

[0118] After knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and Flag-rADSLS407A mutant Huh7 cell lines were reintroduced and used... 14 C-glucose labeling and treatment for 8 hours. For example... Figure 18 As shown, compared with Flag-rADSLWT cells, cells expressing the ADSL S407A mutation had a significantly weakened ability to synthesize TG (triglycerides) and FAs (fatty acids).

[0119] After knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and Flag-rADSLS407A mutant Huh7 cell lines were reintroduced. Following 12 hours of high glucose stimulation, Bodipy staining was used to reflect lipid droplet accumulation. Figure 19 As shown, high sugar treatment increased the number of lipid droplets, while the number of lipid droplets in the ADSL S407A mutant was significantly reduced under high sugar stimulation.

[0120] Conclusion: Phosphorylation at the ADSL S407 site promotes its interaction with INSIG1 / 2. This interaction is essential for promoting downstream SREBP activation and lipid synthesis.

[0121] Example 5: PKCε-mediated phosphorylation of the ADSL S407 site promotes liver cancer growth.

[0122] After knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and Flag-rADSLS407A mutant Huh7 cells were reintroduced and injected intraperitoneally into the livers of 6-week-old male athymic BALB / c nude mice. Tumor size was assessed 28 days later (e.g., ...). Figure 20 The right side shows the statistical results of tumor volume. Compared with tumors expressing Flag-rADSL WT, tumors expressing S407A mutations are significantly smaller.

[0123] After knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and Flag-rADSLS407A mutant Huh7 cells were reintroduced at a rate of 5 x 10⁻⁶. 3 The cells were seeded at different densities onto 10cm dishes and cultured for 3 days, after which they were collected and counted. Figure 21 As shown, compared with Huh7 cells expressing Flag-rADSL WT, cells expressing the S407A mutation showed a significantly slower growth rate.

[0124] After knocking down endogenous ADSL with ADSL shRNA, wild-type Flag-rADSL and Flag-rADSLS407A mutant Huh7 cells were reintroduced and subcutaneously injected into 6-week-old male athymic BALB / c nude mice. Tumor size was measured 28 days later (e.g., ...). Figure 22 The middle and right sides show the statistical results of tumor volume and tumor weight, respectively. Compared with tumors expressing Flag-rADSL WT, tumors expressing S407A mutations have significantly smaller volume and weight.

[0125] like Figure 23 As shown, the corresponding mouse tumor tissue was cut into 5-micrometer-thick slices, and the proliferation ability of the tumor cells was detected and quantitatively analyzed using Ki-67.

[0126] like Figure 24 As shown, the corresponding mouse tumor tissue was cut into 5-micrometer-thick slices, and apoptotic cells were stained and quantitatively analyzed using the DeadEndColorimetric TUNEL System (Promega).

[0127] Conclusion: Phosphorylation at the S407 site of ADSL promotes the development and progression of hepatocellular carcinoma.

[0128] Example 6: S407 phosphorylation of ADSL is associated with prognosis in patients with liver cancer.

[0129] The protein levels of PKCεpS729, ADSLpS407, and SREBP1 in clinical liver cancer tissues and adjacent non-cancerous tissues were compared using immunohistochemistry. Figure 25 The results showed that, compared with normal liver tissue, the protein levels of PKCεpS729, ADSL pS407, and SREBP1 were low in adjacent normal liver tissue, while these indicators were high in cancerous tissue.

[0130] Immunohistochemical staining of PKCεpS729, ADSL pS407, and SREBP1 was performed on 30 liver cancer tissue samples. Figure 26 The results showed that correlation analysis indicated that ADSL S407 phosphorylation was positively correlated with the protein levels of PKCεpS729 and SREBP1.

[0131] In a sample of 84 liver cancer patients, ADSL S407 phosphorylation levels were used to classify them into high-expression and low-expression groups. Kaplan-Meier plots of overall survival were then constructed, and the results are as follows: Figure 27 As shown, the phosphorylation level of ADSL S407 is negatively correlated with a good prognosis in patients with liver cancer.

[0132] Conclusion: S407 phosphorylation in ADSL is positively correlated with poor prognosis in patients with liver cancer.

[0133] Example 7: Schematic diagram of ADSL-INSIG2 interaction

[0134] like Figure 28 As shown, a binding complex of the INSIG2-ADSL complex was constructed based on the protein-protein complex (INSIG2 AlphaFoldDB:Q9Y5U4, ADSL PDBcode:4FLC). INSIG2 and ADSL are shown in green and blue, respectively.

[0135] Example 8: Screening for drugs that can inhibit the ADSL-INSIG interaction from an FDA-approved molecular library using computer-generated virtual screening.

[0136] like Figure 29 As shown, a virtual screening workflow based on pharmacophores and docking is used to find small molecule inhibitors for INSIG2-ADSL inhibitors (left); the binding modes and interaction profiles of the three best-performing inhibitors with the INSIG2-ADSL complex are shown on the right.

[0137] For the preparation of drug compounds, FDA-approved compounds were first downloaded from the MCE database in SDF format. Ligand preparation was performed using the "LigPrep" tool in Maestro within the Schrodinger software suite. The following criteria were used during ligand preparation: (1) OPLS3 force field, (2) generation of all possible ionization states at pH 7.0 ± 2.0, (3) desalting option, (4) generation of tautomers for all conformations, and (5) generation of a low-energy conformation for each ligand. Subsequently, a structure-based virtual screening process was performed on the prepared ligands using the Glide module in the Schrodinger suite. The HTVS mode was used to filter chemical entities proposed from the molecular library, the SP mode was used for further screening, and the XP mode was used to obtain more accurate docking calculations.

[0138] Example 9: Isovirine can effectively inhibit the interaction between ADSL and INSIG.

[0139] Purified His-INSIG2 and GST-ADSL were mixed in vitro, and then incubated for 1 hour with or without the addition of Tezacaftor (5 μM and 50 μM, respectively), Elsulfavirine (2 μM and 25 μM, respectively), or Pyrithioxin (10 μM and 100 μM, respectively). GST pull-down and immunoblotting analysis were then performed. Figure 30 As shown, elsulfavirine blocked the binding of ADSL to INSIG in a dose-dependent manner.

[0140] In Huh7 cells, the Flag-ADSL plasmid was first transfected and overexpressed for 18 hours. Then, the cells were pretreated for 1 hour with Tezacaftor (20 μM), Elsulfavirine (10 μM), and Pyrithioxin (100 μM), followed by 2 hours of treatment with and without high glucose stimulation. Immunoprecipitation and Western blot analysis were then performed. Figure 31 As shown, elsulfavirine can significantly inhibit the interaction between ADSL and INSIG.

[0141] Huh7 cells were pretreated with Elsulfavirine (10 μM) for 1 hour, followed by high glucose treatment, and then immunofluorescence assays were performed using ADSL and INSIG1 antibodies. Figure 32As shown, high glucose stimulation enables colocalization of ADSL and INSIG, while elsulfavirine treatment prevents colocalization. The left image shows representative images, with colocalization analysis performed using ImageJ to measure the intensity of green (ADSL) or red (INSIG1) fluorescence signals along the white diagonal line (right image).

[0142] Conclusion: Elsulfavirine can effectively inhibit the interaction between ADSL and INSIG.

[0143] Example 10: Asulfvirine can effectively inhibit lipid synthesis in liver cancer cells.

[0144] Huh7 cells were pretreated for 1 hour with different concentrations of Tezacaftor (5 μM, 20 μM, 50 μM), Elsulfavirine (2 μM, 10 μM, 25 μM), or Pyrithioxin (10 μM, 30 μM, 100 μM). Then, cells were treated with and without high glucose for 2 hours. Cell lysates were collected for Western blotting experiments. Figure 33 As shown, the immunoblotting results indicate that elsulfavirine inhibits glucose-induced SREBP1 cleavage in liver cancer cells in a dose-dependent manner.

[0145] Huh7 cells were first treated with Tezacaftor (20 μM), Elsulfavirine (10 μM), or Pyrithioxin (100 μM) for 1 hour, followed by high glucose treatment, and then immunofluorescence assays were performed. The scale bar is 20 μm; the left side shows a representative image. Quantitative analysis of the relative nuclear intensity of at least 30 cells was performed using ImageJ (right side), ***P < 0.001 (two-tailed t-test). Figure 34 As shown, the immunofluorescence results indicate that elsulfavirine inhibits the nuclear translocation of SREBP1 induced by high glucose in liver cancer cells.

[0146] In response to high glucose stimulation, SCAPs translocate from the endoplasmic reticulum to the Golgi apparatus. Huh7 cells were pretreated with DMSO or Elsulfavirine (10 μM) for 1 hour, followed by high glucose stimulation for 4 hours, and then immunofluorescence experiments were performed. The left image shows a representative image; the white arrows indicate SCAPs localized to Calnexin. Image J was used to measure the intensity of the green (Calnexin) or red (SCAP) fluorescence signal along the white diagonal line as colocalization analysis (right image). Figure 35As shown, in response to high glucose stimulation, SCAP no longer colocalizes with the endoplasmic reticulum, while SCAP in cells treated with elsulfavirine remains localized to the endoplasmic reticulum.

[0147] Huh7 cells were pretreated with Elsulfavirine (10 μM) for 1 hour, followed by high glucose stimulation for 2 hours before immunofluorescence experiments. The left image is representative, with a scale bar of 20 μm. White arrows indicate SCAPs localized to the Golgi apparatus. Image J was used to measure the intensity of green (Golgin97) or red (SCAP) fluorescence signals along the white diagonal lines for colocalization analysis (right image). Figure 36 As shown, SCAP translocates to the Golgi apparatus in response to high glucose stimulation, while SCAP does not translocate in cells treated with elsulfavirine.

[0148] Huh7 cells were pretreated with DMSO or Elsulfavirine (10 μM) for 1 hour, followed by high glucose stimulation for 8 hours. The mRNA expression levels of FASN, SCD, ACACA, and GPAM genes were detected using qPCR. Figure 37 As shown, high glucose treatment increased the mRNA expression level of the above genes, while elsulfavirine effectively inhibited the increase in the mRNA level of the above genes under high glucose conditions.

[0149] Huh7 cells were pretreated with DMSO or Elsulfavirine (10 μM) for 1 hour, followed by high glucose stimulation for 24 hours. The protein expression levels of FASN, SCD1, etc., were detected by Western blotting. Figure 38 As shown, high glucose treatment increased the expression levels of the aforementioned proteins, while elsulfavirine effectively inhibited the increase in FASN and SCD1 protein levels under high glucose conditions.

[0150] Huh7 cells were pretreated with DMSO or Elsulfavirine (10 μM) for 1 hour before being used. 14 C-glucose labeling and treatment for 8 hours. For example... Figure 39 As shown, compared with the DMSO expression group, the ability to synthesize TG (triglycerides) and FAs (fatty acids) was significantly weakened in the elsulfavirine treatment group.

[0151] Huh7 cells were pretreated with DMSO or Elsulfavirine (10 μM) for 1 hour, and after high glucose stimulation for 12 hours, Bodipy staining was used to reflect lipid droplet accumulation. Figure 40As shown, high sugar treatment increases the number of lipid droplets, while the number of lipid droplets in the elsulfavirine treatment group is significantly reduced under high sugar stimulation.

[0152] Example 11: Asulfvirine can effectively inhibit the growth of hepatocellular carcinoma and has a sensitizing effect when used in combination with lenvatinib.

[0153] Huh7 cells (1×10) 6 Subcutaneous injection was administered to 6-week-old male BALB / c nude mice. Each experimental group consisted of six mice, who were then randomly assigned to four groups: a control group (0.5% methylcellulose), a lenvatinib group (10 mg / kg daily), an elsulfavirine group (10 mg / kg daily), and a combination therapy group (10 mg / kg lenvatinib plus 10 mg / kg elsulfavirine daily), administered by gavage for 20 consecutive days. Tumor volume was recorded every three days starting from day 10 after Huh7 cell injection. On day 28 after Huh7 cell injection, the mice were euthanized, the tumors were removed, and tumor weight was measured. Figure 41 and Figure 42 As shown, sifluvirine can effectively slow tumor growth, and its effect is even better when used in combination with lenvatinib.

[0154] Conclusion: Asulfvirine can effectively slow the growth of hepatocellular carcinoma, and its effect is even better when used in combination with lenvatinib.

[0155] Example 12: Isovirine inhibited lipid synthesis in mouse hepatocellular carcinoma.

[0156] Huh7 cells (1×10) 6 Six-week-old male BALB / c nude mice were injected subcutaneously with lenvatinib (10 mg / kg daily), with each experimental group consisting of six mice. These mice were then randomly assigned to four groups: a control group (0.5% methylcellulose), a lenvatinib group (10 mg / kg daily), an elsulfavirine group (10 mg / kg daily), and a combination therapy group (10 mg / kg lenvatinib plus 10 mg / kg elsulfavirine daily). Administration was by gavage for 20 consecutive days. On day 28 after Huh7 cell injection, the mice were euthanized, tumors were removed, fixed in 4% formaldehyde, embedded in paraffin, and subsequently sectioned and stained. Figure 43 As shown, lipid synthesis indicators (SREBP1, ACLY, and FASN) were significantly reduced in the asulfonvirine-treated group, and these indicators were further reduced when used in combination with lenvatinib.

[0157] Conclusion: Asulfvirine can effectively inhibit lipid synthesis in hepatocellular carcinoma.

[0158] Example 13: Asulfuron-Viline inhibits the proliferation of hepatocellular carcinoma and promotes its apoptosis.

[0159] Huh7 cells (1×10) 6 Six-week-old male BALB / c nude mice were injected subcutaneously with lenvatinib (10 mg / kg daily), with each experimental group consisting of six mice. These mice were then randomly assigned to four groups: a control group (0.5% methylcellulose), a lenvatinib group (10 mg / kg daily), an elsulfavirine group (10 mg / kg daily), and a combination therapy group (10 mg / kg lenvatinib plus 10 mg / kg elsulfavirine daily). Administration was by gavage for 20 consecutive days. On day 28 after Huh7 cell injection, the mice were euthanized. Tumors were removed, fixed in 4% formaldehyde, and embedded in paraffin. The corresponding mouse tumor tissue was then sliced ​​into 5-micrometer-thick sections. Figure 44 As shown, Ki-67 was used to detect and quantitatively analyze the proliferation ability of tumor cells.

[0160] like Figure 45 As shown, apoptotic cells were stained and quantitatively analyzed using the DeadEnd Colorimetric TUNEL System (Promega).

[0161] Conclusion: Asulfvirine can inhibit the proliferation of hepatocellular carcinoma and promote its apoptosis.

[0162] Of course, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of anisuvirine in the preparation of drugs for the treatment of hepatocellular carcinoma.

2. The application as described in claim 1, characterized in that: The active ingredient in the drug includes sifluvirin.

3. The application as described in claim 2, characterized in that: The drugs also include lenvatinib.

4. The application as described in claim 3, characterized in that: The anisulin inhibits the growth of hepatocellular carcinoma by suppressing lipid synthesis in liver cancer cells.

5. The application as described in claim 4, characterized in that: The combination of anisuline and lenvatinib has a synergistic effect, inhibiting the growth of hepatocellular carcinoma.

6. The application as described in claim 5, characterized in that: The drug may also include a pharmaceutically acceptable carrier, or the drug may contain one or more of the following: disintegrant, humectant, binder, filler, absorption enhancer, solvent, lubricant, surfactant, flavoring agent, sweetener, antioxidant, preservative, pigment, ointment base, and transdermal penetration enhancer.

7. The application as described in claim 6, characterized in that: The drugs include tablets, capsules, injections, granules, and suspensions.

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