New application of succinic acid in preparation of medicine for preventing and treating liver cancer

Abstract

The invention relates to new application of succinic acid in preparation of a medicine for preventing and treating liver cancer, and belongs to the technical field of medicines. In order to solve the problem of lack of safe and effective liver cancer prevention and treatment means at the present stage, the invention provides new application of succinic acid in preparation of medicines for preventing and treating liver cancer. The invention proves that succinic acid can relieve the occurrence and development of liver cancer by reducing cholesterol levels in cells and livers in liver cancer cells and animal models. The invention proves that the exogenous succinic acid realizes the anti-tumor effect by inhibiting the FN1 / SQLE signal channel and blocking the cholesterol synthesis of liver cells. Meanwhile, the succinic acid can obviously reduce the levels of ALT and AST in the blood, which shows that the succinic acid also has a certain protection effect on the liver. The invention proves that the exogenous succinic acid can be used as a novel cholesterol synthesis inhibiting factor, can obviously relieve or inhibit the occurrence and development of the hepatocellular carcinoma, and is a safe, effective and feasible strategy for treating the hepatocellular carcinoma.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a new use of succinic acid in preparing drugs for preventing and treating liver cancer. Background Art

[0002] Liver cancer is the leading cause of cancer-related death and is associated with multiple risk factors. Metabolic disorders are key to the development of hepatocellular carcinoma (HCC). Certain tumor-related genes, such as p53, PTEN, and Myc, can induce liver metabolic reprogramming and exacerbate HCC progression. Metabolic-related genes, including pyruvate kinase M2 (PKM2), are important regulators of the Warburg effect, which can impair glucose metabolism and promote HCC growth. Squalene epoxidase (SQLE), a cholesterol biosynthesis regulator, promotes the development of non-alcoholic fatty liver disease-related liver cancer (NAFLD-HCC) by activating the PI3K / AKT / mTOR pathway. In addition, certain metabolites involved in PUFA, one-carbon metabolism, and amino acid metabolism also affect the development of HCC. However, the exact role of specific metabolites in HCC still requires further experimental verification.

[0003] Succinate is an important metabolite in the tricarboxylic acid cycle and a substrate for succinate dehydrogenase. Succinate has shown a wide range of metabolic benefits, including increased thermogenesis in adipocytes and reduced obesity-induced macrophage inflammation. Some studies have shown that mutations in succinate dehydrogenase may lead to the accumulation of succinate in tumors, triggering metabolic changes and supporting tumor growth. However, the specific role of succinate in tumorigenesis remains incompletely elucidated.

[0004] Discovering and verifying natural metabolites with therapeutic potential based on novel metabolomics methods is a field with strong application potential and prospects. Developing new, safe and effective liver cancer treatments is an unmet clinical need. Summary of the invention

[0005] In order to solve the problem of lack of safe and effective prevention and treatment methods for liver cancer at the current stage, the present invention provides a new use of succinic acid in the preparation of drugs for preventing and treating liver cancer.

[0006] The technical solution of the present invention:

[0007] New use of succinic acid in the preparation of drugs for preventing and treating liver cancer.

[0008] Furthermore, the liver cancer is hepatocellular carcinoma.

[0009] Furthermore, succinic acid is the only active ingredient in the drug for preventing and treating liver cancer.

[0010] Furthermore, the content of succinic acid in the drug for preventing and treating liver cancer is 0.1-99wt%.

[0011] Furthermore, the drug for preventing and treating liver cancer also contains a pharmaceutically acceptable carrier.

[0012] Furthermore, the drug for treating liver cancer is administered orally, with adults taking 350-500 mg of succinic acid orally every day.

[0013] Furthermore, the dosage form of the drug for preventing and treating liver cancer is granules, tablets, capsules, pills or oral liquid preparations.

[0014] Furthermore, the drug for preventing and treating liver cancer inhibits the progression of hepatocellular carcinoma by inhibiting the expression of fibronectin 1 in tumor cells.

[0015] Furthermore, the drug for preventing and treating liver cancer reduces the expression of squalene epoxidase in cells by inhibiting the expression of fibronectin 1 gene in tumor cells, thereby inhibiting the progression of hepatocellular carcinoma.

[0016] Furthermore, the drug for preventing and treating liver cancer reduces the cholesterol content in tumor cells by reducing the expression of squalene epoxidase in cells, thereby alleviating the progression of hepatocellular carcinoma.

[0017] Beneficial effects of the present invention:

[0018] The present invention verifies that succinic acid can alleviate the occurrence and development of liver cancer by reducing cholesterol levels in cells and liver in liver cancer cells and animal models by adding (cells) and gavage (animals). The present invention verifies that exogenous succinic acid achieves its anti-tumor effect by inhibiting the FN1 / SQLE signaling pathway and blocking cholesterol synthesis in liver cells. At the same time, succinic acid can significantly reduce blood ALT and AST levels, indicating that it also has a certain protective effect on the liver. The present invention verifies that exogenous succinic acid can be used as a new cholesterol synthesis inhibitor, which can significantly alleviate or inhibit the occurrence and development of hepatocellular carcinoma, and is a safe, effective and feasible strategy for treating hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison of the hematoxylin-eosin (H&E) staining results of the paracancerous tissue and liver cancer tissue samples in Example 1;

[0020] Figure 2 This is a KEGG pathway analysis result diagram of the spatial metabolomics results of paracancerous tissue and liver cancer tissue samples in Example 1;

[0021] Figure 3 The result diagram of TCA cycle metabolites in the spatial metabolomics results of paracancerous tissue and liver cancer tissue samples in Example 1;

[0022] Figure 4This is a comparison chart of succinic acid results in the spatial metabolome of paracancerous tissue and liver cancer tissue samples in Example 1;

[0023] Figure 5 This is a comparison chart of the contents of tricarboxylic acid cycle-related metabolites in the quantitative central carbon metabolomics results of paracancerous tissue and liver cancer tissue samples in Example 1;

[0024] Figure 6 From left to right are the succinate drug concentration-cell proliferation effect curves of the four cell lines LO2, MHCC97H, SK-Hep1 and PLC5 in Example 2;

[0025] Figure 7 From left to right are the cell growth curves of the four cell lines LO2, MHCC97H, SK-Hep1 and PLC5 in Example 2;

[0026] Figure 8 and Fig. 9 They are photos of cell clones formed by four cell lines in Example 2 and a comparison diagram of cell clone formation;

[0027] Fig.10 and Fig.11 They are respectively the wound healing photos of the four cell line monolayers and the wound healing rate comparison diagrams in Example 2;

[0028] Fig.12 The actual photos of the tumors in the control group and the succinic acid group after the nude mice were transplanted with MHCC97H cells in Example 3;

[0029] Fig.13 This is a comparison of tumor volume and tumor weight in nude mice transplanted with MHCC97H cells in the control group and the succinic acid group in Example 3;

[0030] Fig.14 The actual photos of the tumors in the control group and the succinic acid group after the nude mice were transplanted with PLC5 cells in Example 3;

[0031] Fig.15 This is a comparison of tumor volume and tumor weight in nude mice of the control group and the succinic acid group after transplantation of PLC5 cells in Example 3;

[0032] Fig.16 Schematic diagram of the in situ liver cancer model induced by DEN and CCL4 and the succinic acid treatment process in Example 3;

[0033] Fig.17 The photos of liver tissues and H&E staining microphotographs of mice in the control group and succinic acid group in Example 3;

[0034] Fig.18 This is a comparison chart of tumor incidence, tumor number and tumor load in mice of the control group and the succinic acid group in Example 3;

[0035] Fig.19 This is a comparison chart of serum ALT and serum AST levels in the control group and the succinic acid group of mice in Example 3;

[0036] Fig. 20 The transcriptome sequencing results and WB images of SK-Hep1 and MHCC97H cells treated with succinic acid in Example 4;

[0037] Fig.21 From left to right are the succinate drug concentration-cell proliferation effect curves of the three FN1 silenced cell lines MHCC97H, PLC5 and SK-Hep1 in Example 4;

[0038] Fig. 22 From left to right are the cell growth curves of MHCC97H and PLC5, two cell lines in Example 4, in which FN1 expression was silenced in advance and then succinic acid was added;

[0039] Fig.23 and Fig.24 They are photos of cell clones formed by the three FN1 silenced cell lines in Example 4 and a comparison diagram of cell clone formation;

[0040] Fig.25 This is a comparison of FN1 protein levels in paracancerous tissues and tumor tissues of HCC patients from different cohorts in Example 5;

[0041] Fig.26 The WB images of FN1 protein in paracancerous tissues and tumor tissues of 6 paired HCC samples in Example 5;

[0042] Fig. 27 The KEGG pathway analysis results of paracancerous tissue and liver cancer tissue samples in Example 5 are shown;

[0043] Fig.28 is the enrichment score curve obtained by GESA analysis in Example 5;

[0044] Fig.29 This is a correlation analysis diagram of the expression of FN1 and SQLE proteins in HCC sample tumor tissues in Example 5;

[0045] Fig.30 From left to right are the mRNA expression identification diagrams of FN1 and SQLE in SK-Hep1, PLC5 and MHCC97H cells in which FN1 was silenced in Example 5;

[0046] Fig.31 The WB images of FN1 and SQLE protein expression in three liver cancer cell lines in Example 5;

[0047] Fig.32The WB images of FN1 and SQLE protein expression in the subcutaneous MHCC97H and PLC5 xenograft tumor models in Example 5;

[0048] Fig.33 is a comparison chart of intracellular cholesterol concentrations of three liver cancer cell lines in Example 5;

[0049] Fig.34 This is a comparison of intracellular cholesterol concentrations in three FN1-silenced liver cancer cell lines in Example 5. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0051] The materials and methods used in the animal experiments of the present invention are as follows:

[0052] 1. Human samples

[0053] The HCC and adjacent normal samples used in this study were obtained from HCC patients admitted to the Fourth Affiliated Hospital of Harbin Medical University. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University, and all patients gave informed consent (Approval No. 2023-Ethics Approval No. 37).

[0054] 2. Cell Culture

[0055] LO2, SK-Hep1, and PLC5 cells were purchased from ATCC (Manassas, VA). MHCC97H cells were provided by Professor Xiaoxing Li of Sun Yat-sen University, China. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 1% antibiotic-antimycotic (Anti-antimycotic; Gibco) and 10% fetal bovine serum (FBS Thermo Fisher Scientific). The cells were maintained at 37°C in a humidified incubator containing 5% CO2.

[0056] 3. MTT proliferation assay

[0057] Cell proliferation activity was detected using the MTT method. Hepatocytes (LO2, SK-Hep1, PLC5, and MHCC97H) or siFN1-transfected cells (1500 / well) were placed in a 96-well plate and treated with succinate or PBS control. At the designated time points, 20 μL of MTT (5 mg / ml) solution was added to each well and incubated at 37 °C for 4 hours. Then, all the culture medium was discarded, and the cells were incubated with 150 μL DMSO in each well for 30 minutes. Finally, the cell growth curve OD570 was detected using a microplate reader.

[0058] 4. Half Inhibitory Concentration (IC50) Experiment

[0059] Hepatocytes (LO2, SK-Hep1, PLC5, and MHCC97H) or siFN1-transfected cells (1500 / well) were placed in 96-well plates. After overnight incubation for cell attachment, the treatment groups were treated with succinate at concentrations of 0.015 mg / mL, 0.03 mg / mL, 0.06 mg / mL, 0.12 mg / mL, 0.24 mg / mL, and 0.48 mg / mL, while the control group was treated with PBS. After 72 hours of treatment, 20 μL MTT (5 mg / mL) was added to each well, and then the MTT assay protocol was followed.

[0060] The calculation formula of inhibition rate is: inhibition rate = (OD value of control group - OD value of treatment group) / OD value of control group.

[0061] 5.-Clonogenic assay

[0062] Hepatocytes (LO2, SK-Hep1, PLC5, and MHCC97H) or siFN1 transfected cells (1500 / well) were placed in 6-well plates and treated with succinate or PBS control. After 10 days of culture, cells were fixed with 70% ethanol and stained with 1% crystal violet solution for 15 minutes. The 6-well plates were washed with PBS and the number of cell clones was counted.

[0063] 6. Monolayer Wound Healing Assay

[0064] Hepatocytes (LO2, SK-Hep1, PLC5 and MHCC97H) were placed in 6-well plates (4 × 10 5 The next day, the cells were scraped with a pipette tip perpendicular to the ruler and then treated with succinate or PBS containing serum-free medium. Pictures were taken at 12, 24, 36 and 48 hours to evaluate the migration ability of the cells and calculate the migration rate.

[0065] 7. Western blot analysis

[0066] Total protein samples were first separated by SDS-PAGE. Then, the protein samples were transferred to nitrocellulose (NC) membranes (GE Healthcare). After blocking with 3% BSA buffer for 1 h at room temperature, the membranes were incubated with primary antibodies (1:1000 dilution in 1% BSA) overnight at 4°C and then incubated with secondary antibodies (1:10000 dilution in 1% BSA) for 1 h at room temperature. Finally, the membranes containing the proteins of interest were visualized using ECLPlus Western blotting detection reagents (GE Healthcare).

[0067] 8. Cholesterol concentration

[0068] In this study, 10 6 The cholesterol concentration was measured using a cholesterol quantification kit (ab65359, Abcam). The results are expressed as mean ± SEM. This experiment was repeated 3 times independently.

[0069] 9. Nude Mouse Surface Transplantation Model

[0070] Six-week-old male nude mice (Balb / c) were subcutaneously injected with PLC5 cells or MHCC97H cells (5×10 6 cells, 0.1 ml PBS solution).

[0071] The mice were divided into two groups: a succinic acid group (60 mg / Kg / day) and a PBS drinking water group.

[0072] The diameter of each tumor was measured every 2 days. Mice were sacrificed on the indicated days, and tumor tissues were weighed.

[0073] Tumor volume (mm3) was calculated as follows: Volume = (shortest diameter) 2 × (longest diameter) × 0.5. All experimental procedures were approved by the Animal Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University (approval number: 2024-dwsylcz-25).

[0074] 10. HCC model induced by DEN injection

[0075] Wild-type mice (WT) were injected with a single dose of diethylnitrosamine (DEN) at 10-12 days of age and then treated with carbon tetrachloride (0.2 ml / kg) by IP injection twice a week starting at 6 weeks of age.

[0076] Mice were sacrificed on the indicated days, and the number of liver tumors was counted and confirmed by HE staining.

[0077] Tumor burden in the liver of individual mice was calculated using the following formula: Sum of the mean diameters of all tumors in each mouse;

[0078] Average diameter = (large diameter + small diameter) / 2.

[0079] All experimental procedures were approved by the Animal Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University (approval number: 2024-dwsylcz-25).

[0080] 11. Serum alanine aminotransferase and aspartate aminotransferase

[0081] Serum ALT and AST concentrations were measured using ELISA test kits (Solarbio). PBS or succinate-treated wild-type mouse sera were placed in 96-well plates (2 μl per well) and then tested according to the manufacturer's instructions.

[0082] 12. Serum alpha-fetoprotein

[0083] Serum AFP was detected using the Mouse AFP / AFP ELISA Kit according to the manufacturer's instructions (KE10092, Proteintech). 10 μL of mouse serum was diluted to 200 μL with sample diluent PT1. The diluted samples, standards, and controls were then added to each microplate well for further analysis.

[0084] 13. Spatial metabolomics analysis

[0085] Spatial metabolomics analysis of embedded samples was performed according to the protocol developed by Shanghai Millipore Biotech Co., Ltd. (Shanghai, China).

[0086] All embedded samples were cut into serial sagittal sections of approximately 10 μm using a cryostat microtome (Leica CM 1950, Leica Microsystems, Germany). The sections were then placed on positively charged desorption plates (Thermo Scientific, USA) and stored at −80 °C until further analysis. Before mass spectrometry imaging (MSI) analysis, the sections were dried at −20 °C for 1 h and then at room temperature for 2 h. In addition, adjacent sections were retained for hematoxylin-eosin (H&E) staining. Finally, the ions detected by AFADESI were annotated using the pySM 5 pipeline and the in-house SmetDB database (Lumingbio, Shanghai, China).

[0087] 14. Analysis of Central Carbon Metabolites

[0088] The tissue samples were accurately weighed into Eppendorf test tubes. Two small steel balls and 500 μL MeOH / H2O (3 / 1, v / v) pre-cooled at -40°C were added to the samples. The samples were vortexed for 30 seconds and then homogenized at 40 Hz for 4 minutes. They were also sonicated for 5 minutes in an ice water bath. The homogenization and sonication steps were repeated 3 times. Then incubated at -40°C for 1h, centrifuged at 12000rpm (RCF=13800 (×g), R=8.6cm) and 4°C for 15min. The supernatant (400 μL) was collected and dried by rotation. Then, 200 μL of water was added to the dried residue as a reconstitution solution. The reconstructed samples were vortexed before filtering through a membrane. Then, according to the protocol developed by Shanghai BIOTREE BIOMEDICAL TECHNOLOGYCO. (Shanghai, China), they were transferred to the inserts in the injection bottles for HPLC-mass spectrometry / mass spectrometry analysis.

[0089] 15. Statistical Analysis:

[0090] All statistical analyses were performed using SPSS or GraphPad software. Data are presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for multiple group comparisons. For comparison of two variables, paired or unpaired t-test was used. P values ​​less than 0.05 were considered statistically significant. p≤0.05, **p≤0.01, ***p≤0.001.

[0091] Example 1

[0092] In this example, it was confirmed that the concentration of succinic acid in liver tumor tissue was reduced.

[0093] In this example, 7 pairs of liver cancer tissue samples were collected and subjected to spatial metabolomics sequencing. The results are as follows: Figure 2 The results of KEGG pathway analysis showed that nicotinate and nicotinamide metabolism, TCA cycle, and multiple amino acid metabolism pathways were significantly changed in HCC compared with adjacent paracancerous tissues.

[0094] Specifically, if Figure 3 and Figure 4 The levels of several metabolites of the TCA cycle, such as pyruvate, succinate, and fumarate, were significantly decreased in HCC tissues, whereas the levels of citric acid and cis-aconitic acid were significantly increased in HCC.

[0095] Further, such as Figure 5 The results of quantitative central carbon metabolomics sequencing showed that the succinate level in liver cancer tissues was significantly reduced, revealing that succinate may play a certain role in the occurrence and development of liver cancer.

[0096] Example 2

[0097] This example demonstrates that the addition of succinic acid inhibits cell growth and colony formation of HCC cells.

[0098] In this example, three liver cancer cell lines: SK-Hep1, PLC5 and MHCC97H and one non-cancer cell line: LO2 were treated with succinic acid or PBS.

[0099] Depend on Figure 6 The succinic acid drug concentration-cell proliferation effect curves of the four cell lines shown in the figure show that the IC50 of the LO2 cell line is 1.202 mg / ml, the IC50 of the SK-Hep1 cell line is 0.1188 mg / ml, the IC50 of the PLC5 cell line is 0.1295 mg / ml, and the IC50 of the MHCC97H cell line is 0.211 mg / ml. It can be seen that the IC50 of the LO2 cell line is significantly higher than that of the SK-Hep1 cell line, the PLC5 cell line, and the MHCC97H cell line, indicating that succinic acid has a specific anti-proliferation effect on liver cancer cells.

[0100] In addition, the four cell lines were treated with 0.2 mg / ml succinate. Figure 7-Figure 9 The results of cell growth and colony formation assays showed that succinic acid significantly inhibited the viability of SK-Hep1, PLC5 and MHCC97H cell lines. Fig.10 and Fig.11 The monolayer wound healing assay results shown also demonstrated that the addition of succinate significantly inhibited the cell migration ability in HCC cell lines.

[0101] Example 3

[0102] This example demonstrates that supplementation of exogenous succinic acid can significantly inhibit the occurrence and development of liver cancer.

[0103] This example evaluates the inhibitory effect of succinic acid on liver cancer in an animal model.

[0104] First, PLC5 cells or MHCC97H cells were transplanted into the nude mouse model of liver cancer.

[0105] like Fig.12 and Fig.13 As shown in Figure 2, exogenous supplementation of succinate (60 mg / kg body weight per day, intragastrically) significantly inhibited the growth of subcutaneous MHCC97H xenograft tumors (P<0.01) as well as the size and weight of the tumors (p=0.007). Fig.14 and Fig.15 As shown, succinate also significantly inhibited the growth (P<0.01) and tumor weight (p<0.001) of subcutaneous PLC5 xenograft tumors.

[0106] In the orthotopic model of liver cancer induced by carcinogens DEN and CCL4, carcinogens (Den) and carbon tetrachloride (CCL4) were administered to establish the model, and intraperitoneal (IP) injections were given, DEN: 5 mg / Kg body weight, single dose, CCL4: 0.2 ml / Kg body weight, twice a week until the end. The modeling process was as follows Fig.16 shown.

[0107] This embodiment also obtained consistent results, such as Fig.17 As shown in Figure 2, oral administration of exogenous succinate (60 mg / kg body weight per day) significantly reduced HCC formation. Fig.18 As shown in the figure, intragastric administration of exogenous succinic acid significantly reduced the incidence of tumors, with a 50% incidence in the succinic acid group and a 100% incidence in the PBS group, P<0.01; it also reduced the number of tumors (P<0.01) and tumor burden (P<0.01). Fig.19 As shown in the data, succinic acid can also significantly reduce serum ALT and AST levels, indicating that succinic acid supplementation may also have a certain liver-protecting effect.

[0108] Therefore, succinate supplementation is a promising option that should be safe and effective for the prevention and treatment of HCC.

[0109] Example 4

[0110] This example demonstrates that succinic acid inhibits cell proliferation by reducing FN1 protein expression.

[0111] In this example, transcriptome sequencing was performed on SK-Hep1 and MHCC97H cells treated with succinic acid. Fig. 20 Sequencing results showed that succinate can significantly inhibit the expression of genes such as ABCA2, FN1 and LAMA5. Western blotting experiments confirmed that succinate (0.12 mg / ml) significantly reduced the protein expression of FN1 in SK-Hep1, PLC5 and MHCC97H. Considering that FN1 protein was not detected in LO2 cells, combined with IC50 experiments, this example believes that succinate can inhibit the proliferation of liver cancer cells by reducing the level of FN1 protein.

[0112] To test this, this example used siRNA to silence FN1 and then treated the cells with succinate. Fig.21 As shown, compared with the siNC group, silencing FN1 significantly increased the IC50 of MHCC97H (siNC=0.188 mg / ml vs. siFN1=0.404 mg / ml), PLC5 (siNC=0.134 mg / ml vs. siFN1=0.233 mg / ml), and SK-Hep1 (siNC=0.169 mg / ml vs. siFN1=0.377 mg / ml).

[0113] In addition, through Figure 22-Figure 24 As shown in the results of cell growth and clone formation assays, this example demonstrates that knocking out FN1 can reduce the ability of succinate to inhibit cell proliferation.

[0114] These results indicate that the inhibition of cell proliferation by succinate is dependent on FN1 gene expression.

[0115] Example 5

[0116] This example demonstrates the function of FN1 in mediating succinate-inhibited cholesterol biosynthesis and tumorigenesis.

[0117] This example analyzes the protein expression level of FN1 in two HCC cohorts (the cohort of Harbin Medical University and the cohort of Fudan University).

[0118] like Fig.25 As shown in the data from the present cohort (n=10, P<0.001) and the Fudan cohort (n=150, P<0.001), the FN1 protein level in primary HCC patients was significantly upregulated compared with that in adjacent normal tissues. Fig.26 As shown, this example validated the upregulation of FN1 protein in an independent cohort of 6 paired HCC samples.

[0119] like Fig. 27 KEGG pathway analysis showed that HCC FN1 protein expression was positively correlated with steroid biosynthesis, ECM-receptor interaction, cell cycle, PI3K / AKT pathway, and cholesterol metabolism.

[0120] like Fig.28 As shown, GESA analysis further confirmed that FN1 expression was positively correlated with the HCC steroid biosynthesis pathway (p = 0.002, NES = 2.265).

[0121] Consistent with this finding, Fig.29 As shown in Figure 2, the protein expression of FN1 in HCC was positively correlated with SQLE (P<0.05, R=0.1601, Fudan University cohort). Fig.30 As shown, FN1 silencing in MHCC97H and SK-Hep1 cells reduced the protein expressions of PCNA and SQLE, indicating that FN1 promoted cell proliferation through SQLE-mediated HCC steroid biosynthesis.

[0122] like Fig.31 and Fig.32 As shown in Figure 3, succinate treatment significantly inhibited the protein expression of FN1 and SQLE in three hepatoma cell lines as well as in subcutaneous MHCC97H and PLC5 xenograft tumor models. Fig.33As shown in Figure 2, succinate treatment also significantly reduced intracellular cholesterol concentration, but Fig.34 As shown, the effects of succinate could be ameliorated by silencing FN1 in MHCC97H, PLC5, and SK-Hep1 cells.

[0123] In conclusion, the antiproliferative effect of succinate is mediated by FN1-regulated SQLE expression and cholesterol accumulation.

Claims

1. New use of succinic acid in the preparation of drugs for the prevention and treatment of liver cancer.

2. The new use of succinic acid in the preparation of a drug for preventing and treating liver cancer according to claim 1, characterized in that: The liver cancer is hepatocellular carcinoma.

3. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 1 or 2, characterized in that: Succinic acid is the only active ingredient in the drug for preventing and treating liver cancer.

4. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 3, characterized in that: The content of succinic acid in the liver cancer prevention and treatment drug is 0.1-99wt%.

5. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 4, characterized in that: The liver cancer prevention and treatment medicine also contains a pharmaceutically acceptable carrier.

6. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 5, characterized in that: The drug for treating liver cancer is administered orally, and an adult orally takes 350 to 500 mg of succinic acid every day.

7. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 6, characterized in that: The dosage form of the liver cancer prevention and treatment drug is granules, tablets, capsules, pills or oral liquid preparations.

8. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 7, characterized in that: The drug for preventing and treating liver cancer inhibits the progression of hepatocellular carcinoma by inhibiting the expression of fibronectin 1 in tumor cells.

9. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 8, characterized in that: The drug for preventing and treating liver cancer reduces the expression of squalene epoxidase in cells by inhibiting the expression of fibronectin 1 gene in tumor cells, thereby inhibiting the progression of hepatocellular carcinoma.

10. The new use of succinic acid in preparing a drug for preventing and treating liver cancer according to claim 9, characterized in that: The drug for preventing and treating liver cancer reduces the cholesterol content in tumor cells by reducing the expression of squalene epoxidase in cells, thereby alleviating the progression of hepatocellular carcinoma.

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