Application of piceatannol in preparation of medicine for treating or preventing esophageal squamous cell carcinoma

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of piceatannol in preparation of a medicine for treating or preventing esophageal squamous cell carcinoma. Experiments prove that piceatannol has good effects of inhibiting proliferation, transfer and invasion of ESCC in both in-vivo and in-vitro experiments, an action target in the ESCC is GMPS, piceatannol can inhibit a GMPS / USP7 axis and inhibit interaction combination of GMPS and USP7, and the piceatannol has remarkable clinical transformation significance for development of targeted therapeutic drugs of ESCC.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to an application of piceatannol in the preparation of a drug for treating or preventing esophageal squamous cell carcinoma. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is highly invasive, with an insidious onset and no obvious early symptoms, but it progresses rapidly and is prone to regional lymph node metastasis. Most patients are already in the middle and late clinical stages when diagnosed, and the prognosis of ESCC patients is poor. At present, in addition to traditional treatments for ESCC such as surgical resection, radiotherapy and chemotherapy, targeted therapy and immunotherapy are new treatment methods that have emerged in recent years, breaking the treatment bottleneck of ESCC and becoming an important part of ESCC treatment.

[0003] Conventional surgery, radiotherapy, chemotherapy and other methods lack targeting, are harmful to the patient, have many side effects, and have poor efficacy. In terms of targeted therapy, due to the unclear pathogenesis of ESCC, there are currently few targeted drugs approved by the FDA for the treatment of ESCC. In addition, some ESCC patients cannot tolerate the adverse reactions related to targeted therapy and are resistant to treatment. The number of ESCC patients who can truly benefit in the long term is actually very limited, and the clinical management of ESCC is still challenging. Therefore, in-depth research and identification of new ESCC treatment target drugs are still needed.

[0004] Piceatannol (PIC) is a natural polyphenol compound and a metabolite of resveratrol. It is found in many natural plants such as grapes, blueberries and tea. Piceatannol has effects in anti-aging, cardiovascular protection, neuroprotection, anti-diabetes, antioxidant, anti-inflammatory and anti-cancer. Piceatannol can selectively inhibit spleen tyrosine kinase (Syk) protein and regulate the immune and inflammatory response of hematopoietic cells. Piceatannol can inhibit COX-2 activity and has significant anti-inflammatory effects. Piceatannol has significant anti-cancer effects in many cancers. In breast cancer and lung cancer, Piceatannol has significant cytotoxic, anti-proliferative and anti-invasive metastatic effects by inhibiting the NF-κB pathway and downstream MMP9. In osteosarcoma, Piceatannol inhibits the proliferation of osteosarcoma cells and induces apoptosis by inhibiting the PI3K / AKT / mTOR pathway. In prostate cancer, Piceatannol inhibits the activity of CDK2 and CDK4, leading to cancer cell cycle arrest and inhibiting cell proliferation. In colorectal cancer, Piceatannol can inhibit the transforming growth factor β1 (TGF-β1) positive feedback autocrine / paracrine loop between M2-like polarized macrophages and cancer cells, affecting the tumor microenvironment and significantly inhibiting tumor growth and metastasis.

[0005] Currently, there is no report on the application of piceatannol in the treatment or prevention of esophageal squamous cell carcinoma. Summary of the invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a use of piceatannol in the preparation of a drug for treating or preventing esophageal squamous cell carcinoma.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] The present application provides the use of piceatannol in preparing a drug for treating or preventing esophageal squamous cell carcinoma.

[0009] Existing studies have revealed that Piceatannol inhibits the malignant phenotype of various cancers. This application first discovered that Piceatannol also plays a significant role in ESCC. Piceatannol can significantly inhibit the proliferation and metastasis of esophageal squamous cell carcinoma (ESCC) in both in vivo and in vitro experiments, promote ESCC apoptosis, and also significantly inhibit the proliferation and metastasis of esophageal squamous cell carcinoma in in vivo animal experiments. There is currently no obvious organ toxicity and biological hazard, so Piceatannol can be used to treat or prevent esophageal squamous cell carcinoma.

[0010] As a preferred embodiment of the application described in the present application, the target of the piceatannol in esophageal squamous cell carcinoma is guanine mononucleotide, and the piceatannol inhibits the binding of guanine mononucleotide and ubiquitin-specific protease 7.

[0011] The present application uses virtual screening-based molecular docking and drug affinity reaction targeted stability (DARTS) experimental verification to identify that the target of Piceatannol in ESCC is guanine mononucleotide (GMPS). In esophageal squamous cell carcinoma, GMPS in cancer tissue is significantly higher than that in normal tissue. Piceatannol has a stronger effect on tumor cells with high GMPS expression and less effect on normal tissue. Piceatannol can significantly inhibit the malignant phenotype of ESCC by affecting the interaction between GMPS and ubiquitin-specific protease 7 (USP7).

[0012] As a preferred embodiment of the application described in the present application, the piceatannol is prepared into a preparation, and the mass concentration of the piceatannol in the preparation is 25 to 100 μM.

[0013] As a preferred embodiment of the application described in the present application, the mass concentration of piceatannol in the preparation is 100 μM.

[0014] The present application uses Piceatannol at the above mass concentration (25 μM to 100 μM) to treat esophageal squamous cell carcinoma cell lines, and finds that Piceatannol can significantly inhibit the proliferation, stemness and motility of ESCC in vitro and promote the apoptosis of ESCC cells.

[0015] Among them, when the mass concentration of piceatannol was 100 μM, it could better inhibit the proliferation, stemness and motility of ESCC and promote the apoptosis of ESCC cells.

[0016] As a preferred embodiment of the application described in the present application, the esophageal squamous cell carcinoma includes esophageal squamous cell carcinoma cell lines KYSE30 or KYSE180. The esophageal squamous cell carcinoma of the present application includes not only the above cell lines, but also common esophageal squamous cell carcinoma strains in the art.

[0017] In vitro experiments have shown that Piceatannol has a significant inhibitory effect on ESCC cell proliferation. In cell experiments, the proliferation and stemness-forming ability of ESCC cell lines KYSE30 and KYSE180 treated with Piceatannol were significantly weakened in a concentration-dependent manner.

[0018] Moreover, Piceatannol significantly promoted the apoptosis of ESCC cells. In the cell experiment, the apoptosis of ESCC cells KYSE30 and KYSE180 cell lines treated with Piceatannol increased significantly, showing a dose-dependent manner. In the cell experiment, the metastasis and invasion ability of ESCC cells treated with Piceatannol was significantly weakened, and it was concentration-dependent.

[0019] As a preferred embodiment of the application described in the present application, the medicine also includes excipients commonly used in pharmacy.

[0020] The present application also provides the use of piceatannol in preparing a reagent for inhibiting the interaction between guanine mononucleotide and ubiquitin-specific protease 7.

[0021] Molecular docking based on virtual screening and DARTS experimental verification revealed that GMPS is the target of Piceatannol, and Piceatannol inhibits the GMPS / USP7 axis and the interaction between GMPS and USP7, which is of great significance for clinical drug development and targeted treatment of esophageal squamous cell carcinoma.

[0022] As a preferred embodiment of the application described in the present application, the piceatannol is prepared into a preparation, and the mass concentration of the piceatannol in the preparation is 25 to 100 μM.

[0023] The present application also provides the use of piceatannol in preparing an agent for inhibiting plantar-popliteal lymph node metastasis of esophageal squamous cell carcinoma cells in nude mice.

[0024] The nude mouse plantar-popliteal lymph node metastasis model used in this application can ideally simulate the lymph node metastasis process of esophageal cancer in the human body. This technology produces ideal effects in this animal model.

[0025] As a preferred embodiment of the application described in the present application, when the piceatannol is applied to an animal model, the dosage of the piceatannol is 25 to 35 mg / kg. Preferably, the dosage of the piceatannol is 30 mg / kg.

[0026] After Piceatannol was injected into the nude mouse plantar-popliteal lymph node metastasis model, the volume and weight of the nude mouse plantar in situ tumor in the Piceatannol group were smaller than those in the control group, and the growth of the nude mouse plantar in situ tumor was inhibited. Moreover, the volume and weight of the nude mouse plantar-popliteal lymph nodes in the Piceatannol group were significantly smaller than those in the control group, and the metastasis rate of the popliteal lymph nodes in the Piceatannol group after plantar injection of tumor cells was significantly lower than that in the control group, indicating that Piceatannol inhibits the metastasis of nude mouse plantar-popliteal lymph nodes.

[0027] In animal experiments, no obvious pathological changes were found in the main organs of mice injected with the drug Piceatannol, and the mice did not show any abnormal conditions (such as cyanosis, emaciation, death, etc.) throughout the animal experiment.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] The present application provides a use of piceatannol in the preparation of a drug for treating or preventing esophageal squamous cell carcinoma. The present application proves through experiments that piceatannol has a good effect of inhibiting the proliferation, metastasis and invasion of ESCC both in vivo and in vitro experiments, and the target of action in ESCC is GMPS. Piceatannol can inhibit the GMPS / USP7 axis and inhibit the interaction between GMPS and USP7, which has significant clinical translational significance for the development of targeted therapeutic drugs for ESCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result diagram showing that Piceatannol significantly inhibits the spheroidization ability of ESCC cells;

[0031] Figure 2 This is the result diagram showing that Piceatannol significantly promotes the apoptosis of ESCC cells;

[0032] Figure 3This is the result diagram showing that Piceatannol significantly inhibits the metastasis and invasion ability of ESCC cells;

[0033] Figure 4 2D and 3D results of the docking of Piceatannol and GMPS;

[0034] Figure 5 This is a diagram of the DARTS experimental results in Example 1;

[0035] Figure 6 This is the result diagram of Piceatannol inhibiting the binding between GMPS and USP7;

[0036] Figure 7 This is a graph showing the growth inhibition of plantar in situ tumors in nude mice after Piceatannol treatment;

[0037] Figure 8 The results of plantar-popliteal lymph nodes in nude mice treated with Piceatannol;

[0038] Fig. 9 Representative images of HE staining results of popliteal lymph node metastases in nude mice after Piceatannol treatment and statistical graph of popliteal lymph node metastasis rate. DETAILED DESCRIPTION

[0039] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0040] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0041] In the following examples, the esophageal squamous cell carcinoma cell lines KYSE30 (abbreviated as K30), KYSE180 (abbreviated as K180), and EC109 were donated by Professor Guan Xinyuan of the University of Hong Kong; the EC109 cell line was used to construct the EC109-OE-GMPS overexpression stable strain by lentiviral packaging technology.

[0042] Construction of EC109-OE-GMPS overexpression stable strain:

[0043] The overexpression plasmid was purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. The control plasmid was pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE plasmid, and the GMPS overexpression plasmid was pSLenti-EF1-EGFP-P2A-Puro-CMV-GMPS-3xFLAG-WPRE plasmid. The GMPS gene sequence in the overexpression plasmid was the sequence of the GMPS transcript NM_003875.3. After obtaining the plasmid, lentiviral packaging was performed.

[0044] The count is 1×10 6 HEK294T / 293T packaging cells that are in good growth condition and free of mycoplasma contamination are evenly plated in a 10 cm cell culture dish and cultured for 1-2 days. When the 293T packaging cells are cultured to a confluency of 65%-75%, prepare two 1.5 mL EP tubes, label them as tube A and tube B, and then prepare the lentiviral packaging system: Tube A: 2.5 μg GMPS lentiviral plasmid + 5 μL LentiPac TM Mix the packaging plasmid (concentration is 0.5μg / μL), add Opti-MEM to make up to 200μL, and mix well; Tube B: 15μL EndoFectin TM Lenti transfection reagent, fill up to 200μL with OPTI-MEM and mix well. Add the reagent solution in tube B to tube A drop by drop, mix gently and incubate at room temperature for 20-25 minutes to form a transfection complex. Add the transfection complex to 293T cells and shake gently. After culturing in a 37°C, 5% CO2 incubator for 9-13 hours, discard the old culture medium containing the transfection complex, add 10mL containing 400μL fetal bovine serum, 10μL double antibody (penicillin-streptomycin) and 20μL IterBoost TM Freshly prepared DMEM medium containing lentiviral titer enhancer was added. After 48 hours, the cell culture supernatant was collected, centrifuged to remove cell debris, and filtered through a 0.45 μM PES filter to obtain the virus solution. Then, 1.5×10 5The adherent esophageal squamous cell carcinoma cell line EC109 was inoculated in a 6-well plate, and a mixture of 1 mL of virus solution + 1 mL of DMEM complete medium + polybrene (final concentration 5-8 μg / mL) was added to the 6-well plate to infect the cells. After 24 hours of virus infection, the cells were cultured with normal DMEM complete medium. After 48 hours, the DMEM complete medium containing puromycin (concentration of 2.5 μg / mL) was replaced to start screening for resistant cells. Fresh medium containing puromycin was replaced every 2-3 days, and the screening continued for about 7-14 days until all the untransfected control cells died and the transfected cells survived. After detecting the overexpression of GMPS gene level and protein level by RT-qPCR and Western Blot, EC109-OE-GMPS overexpression stable strain was obtained.

[0045] Example 1: Cell spheroidization experiment

[0046] 1. The cell spheroidization experiment was used to verify the effect of the drug Piceatannol in inhibiting the metastasis, proliferation and spheroidization ability of esophageal squamous cell carcinoma cells.

[0047] Take the esophageal squamous cell carcinoma cell lines KYSE30 and KYSE180 in the logarithmic growth phase, digest them with trypsin, collect the cells in a sterile enzyme-free EP tube after terminating the digestion, centrifuge at 1200 rpm for 5 minutes at room temperature, wash the cells once with PBS, centrifuge at room temperature, and discard the upper PBS; add 20 ng of epidermal growth factor (EGF), 20 μL of 50×B27 and 20ng basic fibroblast growth factor (bFGF) were used to prepare stem cell culture medium; 2000 cells were seeded in each well of an ultra-low adsorption six-well plate, and 4mL of stem cell culture medium was added. The 6-well plate was gently shaken to make the cells evenly distributed. The drug Piceatannol was added to the culture medium at concentrations of 0μM, 25μM, 50μM, and 100μM, respectively. The cell spheres were cultured in a 37°C, 5% CO2 incubator for 14-16 days, and the sphere formation of ESCC cells in the 6-well plate and the effect of Piceatannol on the sphere formation ability of ESCC cells were observed under an inverted microscope.

[0048] like Figure 1 The drug Piceatannol significantly inhibited the proliferation and stemness-forming ability of ESCC cells.

[0049] 2. The effect of Piceatannol on apoptosis of esophageal squamous cell carcinoma was verified by apoptosis flow cytometry experiment.

[0050] Esophageal squamous cell carcinoma cell lines KYSE30 and KYSE180 were inoculated in 6-well plates. When the cell confluence reached 50%-60%, Piceatannol at concentrations of 0μM, 25μM, 50μM, and 100μM were added to induce apoptosis. After 2 days, the cells were collected and resuspended in 100μL 1×Annexin V Binding Buffer. 2.5μL AnnexinV-APC Reagent and 2.5μL 7-AAD Reagent were added to the cell suspension, incubated in the dark for 15-20 minutes, and then 400μL 1×AnnexinV Binding Buffer was added. The mixture was mixed and the apoptosis of KYSE30 and KYSE180 after drug treatment was detected by the instrument.

[0051] like Figure 2 As shown, Piceatannol significantly promoted the apoptosis of ESCC cells.

[0052] 3. The effect of the drug Piceatannol in inhibiting the metastasis and invasion of esophageal squamous cell carcinoma cells was determined through transwell experiment.

[0053] In the present application, esophageal squamous cell carcinoma cell lines KYSE30 and KYSE180 were treated with Piceatannol at concentrations of 0 μM, 25 μM, 50 μM, and 100 μM, and then a transwell experiment was performed.

[0054] like Figure 3 As shown in the figure, the drug Piceatannol significantly inhibited the metastasis and invasion ability of KYSE30 and KYSE180 esophageal squamous cell carcinoma cell lines.

[0055] 4. Docking experiment of Piceatannol and GMPS:

[0056] Download the protein crystal structure of GMPS from the RCSB PDB database and download small molecule compounds from 8 compound libraries (① Selleck·FDA-Approved Drug Library, catalog number: L1300; ② TargetMol·Natural Compound Library, catalog number: L6000; ③ MedChemExpress·Natural Product Library, catalog number: HY-L021; ④ Pharmacodia (Yaodu)·Natural Product Library; ⑤ BioBioPha BBP (Xili)·Natural Product Library; ⑥ APExBIO·Anti-cancer-Compound-Library Plus, catalog number: L1023; ⑦ Selleck·Preclinical / Clinical Compound Library catalog number: L2000; ⑧ MedChemExpress·Immunology / Inflammation Compound Library, catalog number: HY-L007).

[0057] Open the "Protein Preparation Wizard" module of Schrödinger software to perform preprocessing of protein preprocess, regenerate states of native ligand, H-bond assignment optimization, protein energy minimization, and remove waters on the obtained GMPS protein crystals respectively. Then, open the "LigPrep" module to preprocess small molecule compounds. After that, open the "QikProp" module to predict all ADME / T parameter scores of these small molecule compounds. Then, open the "Receptor Grid Generation" module of Schrödinger software to identify the active pocket of GMPS. Next, open the "Glide docking" module of Schrödinger software to perform HTVS rigid docking, SP semi-flexible docking, and XP flexible docking of the processed ligands in the 8 compound libraries with the active site of GMPS protein in sequence, gradually improving the accuracy of molecular docking. After outputting the docking scores, perform MM-GBSA analysis using Schrödinger software, and screen out potential drug candidate molecules according to the MM-GBSA scores and molecular docking binding situations. According to the docking results, piceatannol is found to be a potential targeted small molecule drug for GMPS.

[0058] like Figure 4 Shown is the result of docking Piceatannol with GMPS.

[0059] 5. DARTS experiments were performed in KYSE30 and KYSE180 esophageal squamous cell carcinoma cell lines to verify that the target of Piceatannol is GMPS, and the two can stably bind.

[0060] Esophageal squamous cell carcinoma cell lines KYSE30 and KYSE180 were grown in 10 cm culture dishes to 80-85% confluence, the old culture medium was discarded, and after washing with PBS, 1 mL of M-PER was added. TM Mammalian protein extraction reagent, scrape the cells with a cell scraper, collect the lysate in a sterile enzyme-free EP tube. After lysing on ice for 20 minutes, centrifuge in a precooled centrifuge at 4°C; transfer 900 μL of supernatant (cell lysate) to a new 1.5 mL EP, add 100 μL 10X TNC buffer, mix evenly to obtain a cell sample solution, and measure the protein concentration of the cell sample solution by BCA method. Then take 495 μL of cell sample solution and divide it into 5 groups, each with 99 μL, of which 1 group is the control group, adding 1 μL 1×TNC buffer, and the remaining 4 experimental groups are added with 1 μL DMSO and 3 different volumes of small molecule Piceatannol drug solutions, so that the concentrations of small molecule Piceatannol drugs in the 4 experimental groups are 0, 1, 10, and 100 μM, respectively, and incubate at 4°C overnight. Then, in the four experimental groups, according to the ratio of total protein in the cell sample solution to PronaseE of 1:1000, an appropriate volume of 0.1% PronaseE (mother solution is 10 mg / mL) was added to each EP tube, and the control group added the same volume of 1×TNC buffer, incubated at room temperature for 20 minutes, and then added an appropriate volume of protease phosphatase inhibitor mixture (5×), incubated on ice for 10 minutes, and terminated the protease digestion reaction of each group. Then, an appropriate volume of 5× loading buffer was added, and after heating and denaturation at 98°C for 10 minutes, the Western Blot experiment was performed to observe the results.

[0061] like Figure 5 As shown, GMPS protein was resistant to proteolytic enzyme hydrolysis after incubation with Piceatannol in a concentration-dependent manner, demonstrating that GMPS is the target of Piceatannol in ESCC cell lines and the two can be stably bound.

[0062] 6. Previous studies have shown that GMPS and USP7 bind to each other (references 1-2). Through co-immunoprecipitation experiments, we tested whether Piceatannol can inhibit the interaction between GMPS and USP7 and inhibit the malignant phenotype of ESCC by inhibiting the GMPS / USP7 axis.

[0063] The adherent cells EC109-OE-GMPS were digested and resuspended, evenly divided into 5 groups, and cultured in 5 10 cm culture dishes. DMEM complete medium containing 0, 25, 50, 75, and 100 μM Piceatannol was added respectively. After 48 hours of culture, the medium was discarded, washed with pre-cooled PBS, 1.1 mL of IP lysis buffer was added, the cells were scraped off with a cell scraper, and the lysis buffer was collected in a 1.5 mL sterile enzyme-free EP tube. The sample was placed on ice for lysis for 30 minutes. During this period, the EP tube was turned upside down every 10 minutes to fully lyse. After the lysis was completed, centrifuged at 12000 rpm for 15-20 minutes in a pre-cooled centrifuge at 4°C, the precipitate was discarded, and the supernatant was collected to obtain the cell lysate. The protein concentration of the cell lysate was then determined by the BCA method.

[0064] Next, pre-treat the Protein A+G magnetic beads: fully resuspend the Protein A+G magnetic beads, then transfer 20μL of Protein A+G magnetic beads to a 1.5mL EP tube, add 500μL IP lysis buffer, fully resuspend the magnetic beads, place the EP tube on a magnetic rack, and perform magnetic separation. Discard the supernatant. Repeat this step twice. After pre-treating the magnetic beads, place them on ice for later use. Then, take 80μL of cell lysate and add SDS-PAGE protein loading buffer, mix thoroughly, and boil at 99℃ for 10 minutes as the input group. Take 500μL cell lysate (total protein amount is 500-1000μg), add 10μL Flag antibody according to the instruction manual of Flag antibody, as IP group, mix and incubate overnight at 4℃, then take 500μL cell lysate (total protein amount is 500-1000μg), add Mouse IgG, which is homologous and isotyped with IP antibody, as IgG group, mix and incubate overnight at 4℃. Add the incubated sample to the magnetic beads prepared in the pretreatment, and incubate at room temperature for 1 hour. After incubation, place the EP tube on the magnetic stand, and after magnetic separation, discard the supernatant. Add 500μL IP lysate and invert the EP tube several times. Place the EP tube on the magnetic stand, and after all the magnetic beads are adsorbed, discard the supernatant, and repeat washing three times. Add 50-80μL SDS-PAGE protein loading buffer to the washed magnetic beads for elution, place the sample in a metal bath, and heat at 99℃ for 10 minutes. Western Blot detection was performed to observe the experimental results.

[0065] like Figure 6As shown, Piceatannol can inhibit the interaction between GMPS and USP7, thereby suppressing the malignant phenotype of ESCC by inhibiting the GMPS / USP7 axis.

[0066] References:

[0067] 1. van der Knaap JA, Kozhevnikova E, Langenberg K, Moshkin YM, VerrijzerCP: Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-SpecificProtease 7in Transcriptional Regulation of Ecdysteroid Target Genes. Molecular and Cellular Biology 2010, 30(3): 736-744.

[0068] 2.Reddy BA, van der Knaap JA, Bot AG, Mohd-Sarip A, Dekkers DH, TimmermansMA, Martens JW, Demmers JA, Verrijzer CP: Nucleotide biosynthetic enzyme GMPsynthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell 2014,53(3):458-470.

[0069] Example 2: Animal Experiment

[0070] 1. Sixteen 4-week-old male nude mice were randomly divided into two groups: control group and Piceatannol group. 2×10 6 KYSE30 in esophageal squamous cell carcinoma cells.

[0071] 2. After 10 days, the nude mice in the Piceatannol group were intraperitoneally injected with Piceatannol at a dose of 30 mg / kg, and the control group was intraperitoneally injected with an equal amount of solvent (4% DMSO + 30% PEG300 + 5% Tween80 + 61% ddH2O), and the drugs were administered every day.

[0072] After 3 and 4 weeks, the mice were killed and the plantar tumor in situ and popliteal lymph nodes were removed. The size, volume and weight of the plantar primary tumor were used to determine the effect of Piceatannol in inhibiting the proliferation of esophageal squamous cell carcinoma in vivo. The plantar-popliteal lymph node metastasis rate of the tumor was calculated by HE staining of the mouse popliteal lymph nodes to determine the effect of Piceatannol in inhibiting the metastasis of esophageal squamous cell carcinoma in vivo. Figure 7 to Figure 9 ).

[0073] like Figure 7 As shown in the figure, after injection of Piceatannol, the volume and weight of the nude mouse plantar tumor in situ group were smaller than those in the control group, and the growth of the nude mouse plantar tumor in situ was inhibited, suggesting that Piceatannol inhibits the proliferation of esophageal squamous cell carcinoma. Figure 8 As shown, after the injection of Piceatannol, the volume and weight of the plantar-popliteal lymph nodes of nude mice in the Piceatannol group were significantly smaller than those in the control group.

[0074] like Fig. 9 As shown, according to the results of HE staining of popliteal lymph nodes, 7 out of 8 nude mice injected with esophageal squamous cell carcinoma cells in the control group developed plantar-popliteal lymph node metastasis, while only 2 out of 8 nude mice injected with the same number of esophageal squamous cell carcinoma cells in the Piceatannol group developed plantar-popliteal lymph node metastasis. There was a statistical difference in the popliteal lymph node metastasis rate between the control group and the Piceatannol group, indicating that Piceatannol inhibits plantar-popliteal lymph node metastasis in nude mice.

[0075] In animal experiments, no obvious pathological changes were found in the main organs of mice injected with the drug Piceatannol, and the mice did not show any abnormal conditions (such as cyanosis, emaciation, death, etc.) throughout the animal experiment.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. The use of piceatannol in the preparation of drugs for treating or preventing esophageal squamous cell carcinoma.

2. The use according to claim 1, characterized in that The target of the piceatannol in esophageal squamous cell carcinoma is guanine mononucleotide, and the piceatannol inhibits the combination of guanine mononucleotide and ubiquitin-specific protease 7.

3. The use according to claim 1, characterized in that The piceatannol is prepared into a preparation, and the mass concentration of the piceatannol in the preparation is 25-100 μM.

4. The use according to claim 3, characterized in that The mass concentration of piceatannol in the preparation is 100 μM.

5. The use according to claim 1, characterized in that The esophageal squamous cell carcinoma includes esophageal squamous cell carcinoma cell lines KYSE30 or KYSE180.

6. The use according to claim 1, characterized in that The medicine also includes auxiliary materials commonly used in pharmacy.

7. Use of piceatannol in the preparation of reagents for inhibiting the interaction between guanine mononucleotide and ubiquitin-specific protease 7.

8. The use according to claim 7, characterized in that The piceatannol is prepared into a preparation, and the mass concentration of the piceatannol in the preparation is 25-100 μM.

9. Application of piceatannol in the preparation of reagents for inhibiting plantar-popliteal lymph node metastasis of esophageal squamous cell carcinoma cells in nude mice.

10. The use according to claim 9, characterized in that When the piceatannol is applied to an animal model, the dosage of the piceatannol is 25-35 mg / kg.

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