Application of quercetin derivative Q3MG in the preparation of anti-tumor invasion and metastasis drugs

Abstract

The present invention belongs to the field of biomedicine technology, and discloses the use of a quercetin derivative Q3MG in the preparation of anti-tumor invasion and metastasis drugs and drugs for targeted degradation of pro-IL-1β protein in cancer cells. The present invention first discovered that Q3MG can significantly inhibit tumor invasion and metastasis by targeted degradation of pro-IL-1β protein in cancer cells, and has a good therapeutic effect on anti-tumor invasion and metastasis, especially squamous cell carcinoma; it reveals the importance of quercetin derivatives in the preparation of drugs for the treatment of squamous cell carcinoma invasion and metastasis, and even the potential value of flavonoid compounds in various other tumors, which has far-reaching clinical significance and important prospects for promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular to the application of a quercetin derivative, Quercetin 3-O-(6''-O-malonyl)-β-D-glucoside (Chinese name: Quercetin-3-O-(6''-O-malonyl)-β-D-pyranoglucoside, hereinafter abbreviated as Q3MG), in the research and development of drugs for anti-tumor invasion and metastasis, especially anti-squamous cell carcinoma invasion and metastasis. Background Art

[0002] Squamous cell carcinoma is an invasive tumor with high malignancy, easy to metastasize and relapse, and is one of the main causes of cancer death worldwide. Squamous cell carcinoma mainly originates from epithelial tissue and is common in the skin, head and neck, esophagus, lungs and cervix. Exposure to chemical carcinogens, human papillomavirus infection, excessive exposure to ultraviolet radiation, smoking, alcoholism and family inheritance are risk factors for the occurrence of squamous cell carcinoma. The early clinical symptoms of squamous cell carcinoma are not obvious. Most patients are diagnosed in the middle and late stages. They are often accompanied by metastasis and lymph node involvement. They are prone to relapse after treatment and have a poor prognosis. In recent years, although there are treatments for squamous cell carcinoma such as surgery, chemoradiotherapy, targeted therapy and immunotherapy, the US FDA has also approved molecular targeted drugs such as cetuximab and immune checkpoint inhibitors such as nivolumab and pembrolizumab for clinical treatment of squamous cell carcinoma, but the recurrence rate of this cancer is still high and the 5-year overall survival rate is low. Overall, the distant metastasis of squamous cell carcinoma is related to multiple factors, and its treatment is still the biggest challenge currently faced. In-depth research on the biological principles and regulatory mechanisms of squamous cell carcinoma metastasis, the discovery of key metastasis-driving factors and their use as diagnostic and therapeutic targets, such as pro-IL-1β protein, will help to alleviate and eliminate tumors. In order to expand the treatment options for squamous cell carcinoma, it is crucial to find more suitable new therapies and drugs, thereby promoting the improvement of patient efficacy and quality of life.

[0003] Interleukin-1β (IL-1β) is an important inflammatory factor in the tumor microenvironment that can accelerate tumor invasion, progression, and metastasis. IL-1β is produced in a precursor form, called pro-IL-1β. IL1BGene encoding. IL-1β is produced and secreted by immune cells, fibroblasts or tumor cells. Its synthesis and processing respond to infection or other stress stimuli. Subsequently, inflammasomes are assembled, caspase-1 is activated and the precursor is cleaved to form mature IL-1β. In the study of IL-1 family and anti-tumor, studies have shown that IL-1RA and anti-IL-1 monoclonal antibodies can inhibit the incidence to a certain extent. Clinically, under inflammatory conditions, pharmacological targeting of IL-1β has a certain therapeutic effect. However, the development of drugs targeting IL-1β for anti-tumor has not been smooth. The anti-IL-1β monoclonal antibody Canakinumab failed to reach the primary endpoints, namely overall survival and progression-free survival, in a phase III clinical trial for the treatment of lung cancer, and ended in failure, which has raised concerns about the effectiveness of secretory IL-1β as a target for cancer treatment. After the inflammasome is "activated" in bone marrow-derived macrophages, it will feedback and cause IL-1β to be rapidly degraded through the ubiquitin-proteasome pathway to prevent excessive inflammatory factors from causing tissue damage, so IL-1β has a short lifespan. Currently, there are more studies on the role of mature IL-1β as a secretory inflammatory factor, but fewer studies targeting its intracellular precursor protein pro-IL-1β.

[0004] Quercetin is a flavonoid compound found in a variety of plants, fruits, traditional Chinese medicines and vegetables. It is believed to have a variety of biological activities, including antioxidant, anti-inflammatory, anticancer, antibacterial and antiviral effects. However, the bioavailability of quercetin is low, which affects its systemic effects and severely limits its clinical application. Quercetin derivatives refer to natural or synthetic compounds that have undergone specific transformation or modification based on the molecular structure of quercetin. Compared with quercetin, its properties may change, such as enhanced biological activity, improved stability, solubility or improved efficacy. Quercetin derivatives such as 4′-O-methylquercetin, 7-O-methylquercetin, 4′,7-dimethoxyquercetin, quercetin-6-C-β-d-glucopyranoside, etc. have been reported to be more effective than quercetin in inhibiting the growth of lung cancer (A549), melanoma (M14), cervical cancer (Hela), head and neck cancer (M4E), and breast cancer (SKBR) cancer cells.

[0005] Q3MG is a quercetin derivative with a molecular formula of C 24 H 22 O 15, with a molecular weight of 550.425, is composed of quercetin combined with a 6''-O-malonyl group and a β-D-glucoside group, and is a natural flavonol glycoside. Although there have been reports that quercetin inhibits the progression of squamous cell carcinoma, research on the quercetin derivative Q3MG is still scarce, especially its role in tumors has not been clearly reported. In addition, although studies have shown that quercetin can inhibit renal cell pyroptosis by inhibiting the expression of cleaved IL-1β and IL-18, thereby protecting mice from cadmium-induced renal injury, these studies are mainly based on the inflammatory factor IL-1β. However, studies on the specific degradation of the precursor protein pro-IL-1β rather than intervening in IL-1β are still rare, and there are no studies on whether quercetin-related compounds can target the degradation of pro-IL-1β. Summary of the invention

[0006] The present invention aims to solve the problem of distant metastasis and treatment difficulty of squamous cell carcinoma. Based on the discovery and application of a quercetin derivative Q3MG targeting pro-IL-1β protein in the research and development of tumor anti-metastasis drugs, the present invention provides an application of a quercetin derivative Q3MG in the preparation of drugs for anti-tumor invasion and metastasis, which helps to expand the clinical options for the treatment of squamous cell carcinoma and is of great significance for improving the efficacy of patients with squamous cell carcinoma.

[0007] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0008] Application of a quercetin derivative Q3MG in the preparation of anti-tumor invasion and metastasis drugs.

[0009] The use of the quercetin derivative Q3MG in the preparation of anti-tumor invasion and metastasis drugs, preferably, the anti-tumor invasion and metastasis drugs are drugs that inhibit cancer cell invasion and metastasis by targeting and degrading pro-IL-1β protein in cancer cells. The anti-metastasis efficacy of this natural compound Q3MG has a specific mechanism of inhibiting invasion and metastasis by targeting and degrading pro-IL-1β protein in squamous cell carcinoma, thereby having a good therapeutic effect on squamous cell carcinoma, providing a new strategy for the clinical treatment of related tumors.

[0010] Preferably, the anti-tumor invasion and metastasis drug is a drug for anti-squamous cell carcinoma invasion and metastasis.

[0011] Preferably, the squamous cell carcinoma comprises at least one squamous cell carcinoma cell line selected from HK1, FaDu and BxPC3.

[0012] Preferably, the squamous cell carcinoma invasion and metastasis is the squamous cell carcinoma invasion and metastasis simulated by the tail vein-lung metastasis model.

[0013] Based on a general inventive concept, the present invention also provides a use of a quercetin derivative Q3MG in the preparation of a drug for targeted degradation of pro-IL-1β protein in cancer cells.

[0014] The use of the quercetin derivative Q3MG in the preparation of targeted degradation of pro-IL-1β protein in cancer cells, preferably, the cancer cells are squamous cancer cells.

[0015] Preferably, the squamous cancer cells include at least one squamous cell cancer cell line selected from HK1, FaDu, and BxPC3.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Innovation in drug research and development: Existing research still lacks understanding of the quercetin derivative Q3MG. This invention discloses for the first time the anti-metastatic effect of Q3MG, bringing new ideas for drug research and development for the treatment of squamous cell carcinoma and other types of tumors, expanding the research field of anti-tumor drugs, and is expected to provide patients with more treatment options.

[0018] (2) Reduced side effects: As plant-derived compounds, quercetin derivatives generally have better safety and tolerability, and may reduce patients' side effects and toxic reactions compared to traditional chemotherapy drugs or other targeted drugs.

[0019] (3) Innovation in tumor therapy: This invention is based on the constitutively high expression of pro-IL-1β protein in cancer cells instead of the inflammatory factor IL-1β, and discloses that the compound Q3MG accelerates the lysosomal degradation of the precursor protein pro-IL-1β and thus produces a significant therapeutic effect on squamous cell carcinoma. This invention expands the treatment options for squamous cell carcinoma, provides an effective new therapy, and has potential clinical significance and important prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the predicted molecular docking map of Q3MG and IL1B protein (PDB ID: 5R8Q).

[0022] Figure 2It is a Western blot result diagram, which shows that Q3MG has a significant inhibitory effect on pro-IL-1β, where A shows that Q3MG inhibits the expression of pro-IL-1β protein in a dose-dependent manner, and B shows that Q3MG inhibits the expression of pro-IL-1β protein in a time-dependent manner.

[0023] Figure 3 It is a Western blot result chart, comparing the inhibitory effects of Q3MG and other quercetin-related compounds on pro-IL-1β protein, showing that only Q3MG can significantly reduce the protein level of pro-IL-1β, while quercetin-3-O-glucoside and quercetin have no effect on the pro-IL-1β protein level.

[0024] Figure 4 It is a graph of qRT-PCR results, showing that Q3MG does not affect the mRNA expression of IL1B.

[0025] Figure 5 It is the ELISA result graph, which shows that Q3MG does not affect the secretion of IL-1β.

[0026] Figure 6 It is a Western blot result diagram, which shows that Q3MG mediates the degradation of pro-IL-1β protein through the lysosomal pathway rather than the proteasomal pathway, wherein A is the effect of Q3MG on the lysosomal pathway of pro-IL-1β, and B is the effect of Q3MG on the proteasomal pathway of pro-IL-1β.

[0027] Figure 7 The figure is the result of the scratch healing experiment, which shows that Q3MG significantly inhibits the migration of tumor cells.

[0028] Figure 8 This is the result of the Transwell migration experiment, showing that Q3MG significantly inhibits the migration of tumor cells.

[0029] Fig. 9 This is the result of the Transwell invasion experiment, which shows that Q3MG significantly inhibits the invasion of tumor cells.

[0030] Fig.10 This is a graph of CCK8 experimental results, showing that Q3MG does not affect the survival of tumor cells in vitro.

[0031] Fig.11 This is a colony formation experiment result diagram, which shows that Q3MG does not affect the proliferation of tumor cells in vitro.

[0032] Fig.12The figure is the result of the scratch healing experiment, which shows that the addition of Q3MG to tumor cells with stable knockout of pro-IL-1β has no effect on the migration of tumor cells.

[0033] Fig.13 The figure is the result of Transwell migration experiment, which shows that the addition of Q3MG to tumor cells with stable knockout of pro-IL-1β has no effect on the migration of tumor cells.

[0034] Fig.14 The figure is the result of Transwell invasion assay, showing that the addition of Q3MG to tumor cells with stable knockout of pro-IL-1β has no effect on the invasion of tumor cells.

[0035] Fig.15 This is a curve chart of body weight changes in nude mice in the tail vein lung metastasis model.

[0036] Fig.16 It is a scatter plot of the general picture of lung tissue and the statistical number of metastatic nodules obtained from the nude mouse tail vein lung metastasis model.

[0037] Fig.17 These are representative images of H&E staining of major organs of nude mice in the tail vein-lung metastasis model.

[0038] Fig.18 The following are representative H&E staining and immunohistochemical staining results of nude mouse lung metastases. The right side is a scatter plot of the number of nude mouse lung metastases in 15-20 tissue sections in each group under a microscope (n = 5). DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0040] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0042] The quercetin derivative used in the present invention is quercetin-3-O-(6''-O-malonyl)-β-D-pyranoglucoside (English name: Quercetin 3-O-(6''-O-malonyl)-β-D-glucoside, hereinafter abbreviated as Q3MG, chemical formula: C24 H 22 O 15 , CAS number: 96862-01-0).

[0043] The previous study of the present invention observed that pro-IL-1β is highly expressed in squamous cell carcinoma. Targeted drug screening targeting pro-IL-1β constitutively highly expressed in squamous cell carcinoma will provide a new direction for anti-tumor treatment. Therefore, one of the purposes of the present invention is to screen specific inhibitors / degradation drugs for pro-IL-1β protein highly expressed in squamous cell carcinoma.

[0044] Step 1: High-throughput virtual screening of molecular compounds targeting Human pro-IL-1β protein.

[0045] Schrödinger Maestro 12.8 software was used to perform structure-based high-throughput virtual screening of the binding pocket of human pro-IL-1β protein and its ligand Z2643472210. The X-ray crystal structure of Human IL1B (PDB ID: 5R8Q) was obtained from the RCSB protein database and further processed by the Protein Preparation Wizard module of Schrödinger software. The MCE Bioactive Compound Library Plus, which contains 19.1K compounds, was processed by the LigPrep Module of Schrödinger software for energy optimization and other processing before virtual screening. These optimized compounds then entered the virtual screening workflow for molecular docking. First, all compounds were screened in Glide HTVS mode, and the top 15% of the compounds with the highest scores were docked in Glide standard precision (SP). Subsequently, the top 15% of the molecules in Glide SP were docked in Glide high precision (XP) mode. The higher the absolute value of the predicted docking score, the stronger the binding affinity. 2D and 3D docking models were created by PyMol. in, In silico Docking showed that Q3MG binds to the allosteric pocket on the surface of pro-IL-1β protein through hydrogen bonding, and the key residues of the pocket are L80 / L134 / K74 / V132 ( Figure 1 ).

[0046] Step 2: Verify that Q3MG works by targeting the precursor protein pro-IL-1β rather than interfering with the secretion of mature IL-1β.

[0047] Next, in squamous cell carcinoma cell lines HK1, FaDu, and BxPC3, the compound Q3MG was added at a working concentration of 0 / 10 / 20 / 30 / 40 / 50 μM, DMSO-PBS was added as a control, the action time was 24 h, and the cell extract protein was collected. Western blot experiments showed that Q3MG had a significant inhibitory effect on the pro-IL-1β protein level. With the increase of Q3MG dose, the inhibitory effect became more obvious. When Q3MG was added at a working concentration of 50 μM, the reduction of pro-IL-1β protein level was most significant. In addition, in HK1, FaDu, and BxPC3 cells, the compound Q3MG was added at a working concentration of 50 μM, DMSO-PBS was added as a control, and the action time was 0 / 12 / 24 / 48 h, respectively. The effect on the pro-IL-1β protein level was also detected by Western blot. As the duration of Q3MG action increases, the inhibitory effect becomes more pronounced. When the duration of Q3MG action exceeds 24 hours, a significant effect on the degradation of pro-IL-1β protein has already appeared. Therefore, the present invention clarifies that Q3MG significantly inhibits the level of pro-IL-1β protein in a dose- and time-dependent manner ( Figure 2 ).

[0048] The present invention verified the effects of other quercetin-related compounds (such as quercetin-3-O-glucoside and quercetin) on pro-IL-1β protein levels in squamous cell carcinoma cells. The results showed that only Q3MG could significantly reduce the protein level of pro-IL-1β, while quercetin-3-O-glucoside and quercetin had no effect on pro-IL-1β protein expression ( Figure 3 ).

[0049] The compound Q3MG was added to the squamous cell carcinoma cell line at a working concentration of 50 μM. After 24 h of action, the cells and supernatant were collected, and the cell RNA was extracted. qRT-PCR showed that Q3MG did not affect the mRNA expression of IL1B ( Figure 4 ), the supernatant was collected and ELISA experiments showed that Q3MG did not affect the secretion of mature IL-1β ( Figure 5 ).

[0050] Step 3: Provide the degradation mode of the target compound Q3MG on pro-IL-1β protein.

[0051] The present invention uses lysosomal inhibitor chloroquine (CQ) and proteasome inhibitor MG132 to treat squamous cell carcinoma cells respectively to further clarify the specific way in which compound Q3MG degrades pro-IL-1β protein. After treating HK1, FaDu and BxPC3 cells with lysosomal inhibitor CQ, it was found that Q3MG no longer regulated the expression of pro-IL-1β protein. After treatment with MG132, Western blot experiments showed that Q3MG can still degrade pro-IL-1β protein. This indicates that Q3MG mediates pro-IL-1β protein degradation through the lysosomal pathway, rather than through the proteasome pathway ( Figure 6 ).

[0052] Based on the constitutively highly expressed pro-IL-1β protein in squamous cell carcinoma, a compound Q3MG that can accelerate its specific degradation was discovered. This natural quercetin derivative can produce effective anti-metastatic therapeutic effects in tumors, showing potential clinical value and important prospects for promotion and application. Therefore, the second purpose of the present invention is to provide verification of the application effect of the quercetin derivative Q3MG based on pro-IL-1β protein targeting in the development of anti-metastatic drugs for squamous cell carcinoma.

[0053] Step 1: Verify that Q3MG inhibits the invasion and migration of squamous cell carcinoma cells in vitro.

[0054] In squamous cell carcinoma cells HK1, FaDu, and BxPC3, the scratch healing assay showed that the addition of Q3MG could inhibit the migration of squamous cell carcinoma cells compared with the control ( Figure 7 ). Through Transwell migration and invasion experiments, the results also showed that Q3MG can significantly inhibit the migration and invasion ability of squamous cell carcinoma cells ( Figure 8 , Fig. 9 ). Through CCK-8 experiments and colony formation experiments, it was found that the proliferation and survival of squamous cell carcinoma cells in vitro were not affected by the addition of Q3MG ( Fig.10 , Fig.11 ).

[0055] Step 2: Verify that Q3MG inhibits the invasion and migration of squamous cell carcinoma cells by targeting pro-IL-1β.

[0056] Q3MG was added to the squamous cell carcinoma cell lines HK1 and FaDu in which pro-IL-1β was stably knocked out, and the migration and invasion of squamous cell carcinoma cells were tested by scratch healing assay and Transwell migration and invasion assay to determine whether there were changes. The results showed that Q3MG no longer affected the migration and invasion of tumor cells after pro-IL-1β was knocked out ( Fig.12 , Fig.13 , Fig.14 ), indicating that Q3MG's regulation of tumor cell motility and invasion is mediated by pro-IL-1β.

[0057] Step 3: Verify the ability of Q3MG to inhibit the metastasis of squamous cell carcinoma cells in vivo.

[0058] The present invention established a nude mouse tail vein-lung metastasis model. Female BALB / c nude mice aged 4-5 weeks were prepared, and each nude mouse was injected with HK1 and FaDu cells several times through the tail vein (total cell injection volume 4×10 6 The mice were randomly divided into a control group and a drug treatment group. Drug treatment was performed 2-3 weeks after tumor cell inoculation. The drug was dissolved in 2% DMSO, 40% PEG300, 5% Tween-80, and 53% Saline. The dosage was 15 mg / kg / 2 days, and 100 μL / mouse was intravenously administered for 7 times. The body weight of mice was monitored weekly during the experiment. Lung tissue and major organs were removed by dissection after 8 weeks for analysis.

[0059] The experimental results show that compared with the control group, the body weight of nude mice in the Q3MG treatment group increased significantly ( Fig.15 ), the number of metastatic nodules on the lung surface was significantly reduced ( Fig.16 No obvious toxic side effects of the drug were observed in the hematoxylin and eosin (H&E) staining results of major organs ( Fig.17 ). The results of H&E and immunohistochemical staining of lung tissue paraffin sections showed that the pro-IL-1β expression signal in the Q3MG drug-treated group was significantly weakened, and the number and area of ​​lung metastatic lesions were significantly lower than those in the control group ( Fig.18 ). This indicates that Q3MG has a significant inhibitory effect on the metastasis of squamous cell carcinoma cells in vivo.

[0060] The present invention uses squamous cell carcinoma as an example to illustrate the technical solution, but the present invention is not limited to this, and embodies the application of the quercetin derivative Q3MG in the research and development of tumor anti-metastasis drugs.

[0061] Example:

[0062] A quercetin derivative Q3MG is used in the preparation of drugs for preventing squamous cell carcinoma or for targeted degradation of pro-IL-1β protein in cancer cells.

[0063] In order to better illustrate the technical solution of the present invention, the following embodiments are given, but the present invention is not limited thereto.

[0064] 1. Targeting the pro-IL-1β protein that is highly expressed in squamous cell carcinoma, a specific inhibitor / degradation drug was screened out: Q3MG.

[0065] 1. High-throughput virtual screening of molecular compounds targeting Human pro-IL-1β protein.

[0066] (1) Protein preparation: Download the three-dimensional structure of Human IL1B from the RCSB PDB database (PDB ID: 5R8Q). Use the Protein Preparation Wizard module to hydrogenate the protein and remove water molecules and SO4 2- , retaining the A chain and the small molecule JGY (i.e., Z2643472210), followed by energy optimization (OPLS2005 force field, RMSD of 0.30 Å). The processed protein was used to generate a grid file using the Receptor Grid Generation module, with the pocket where the small molecule Z2643472210 was located as the center, and the box size was set to 20 Å×20 Å×20 Å.

[0067] (2) Compound preparation: The 2D format of the compound library HY-L001 (MCE Bioactive Compound Library Plus, containing 19.1K compounds) was processed by the Schrödinger software LigPrep Module for hydrogenation, energy optimization, etc., and the 3D structure was output for virtual screening.

[0068] (3) Virtual screening: Use the Virtual Screening Workflow module for virtual screening, import the prepared compounds, and use the Glide module for molecular docking, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching.

[0069] (4) Result output: First, the prepared molecules were screened using the high-throughput screening (HTVS) mode in the Glide module, and the molecules with the top 15% of the scores were selected for the second round of screening using the standard (SP) mode; then the molecules with the top 15% of the scores were selected for the third round of screening using the high-precision (XP) mode, and all screening results were output. The binding force between the target and the compound, the compound structure, etc. were manually reviewed, and the top 50 compounds in the Bioactive Compound Library Plus library were selected for output (the results are shown in Table 1 below).

[0070] Table 1: Top 50 compounds by molecular docking scores

[0071]

[0072] (5) Molecular docking: In silico Docking showed that IL1B protein binds to Q3MG through an allosteric pocket (see results in Figure 1 ). The docked compounds were mapped to the IL1B protein crystal structure (PDB ID: 5R8Q) using PyMol software. Potential allosteric sites of IL1B protein were predicted using Allosite (http: / / mdl.shsmu.edu.cn / AST / ). Q3MG can form 7 hydrogen bonds with the target protein Human IL1B: the carbonyl group forms 2 hydrogen bonds with LEU80 and LEU134, and the hydroxyl group can form 5 hydrogen bonds with LYS74, VAL132, and LEU134.

[0073] 2. Experiments have verified that Q3MG works by targeting the precursor protein pro-IL-1β rather than interfering with the secretion of mature IL-1β.

[0074] (1) Taking squamous cell carcinoma cells HK1, FaDu, and BxPC3 as examples, the cell lines used in the present invention are all preserved in the Molecular Genetics Laboratory of the Institute of Oncology, Central South University. Human nasopharyngeal carcinoma cell line: HK1, squamous epithelial-like, cell adherent growth; human hypopharyngeal carcinoma cell line: FaDu cell line, squamous epithelial-like, cell adherent growth; human pancreatic squamous cell carcinoma cell line: BxPC3 cell line, epithelial-like, cell adherent growth. The cell culture conditions are: RPMI1640 liquid culture medium with 10% fetal bovine serum (FBS) and 1% double antibody (penicillin, streptomycin), adherent growth in a constant temperature incubator at 37°C, 95% humidity, and 5% CO2 concentration.

[0075] Compound Q3MG was purchased from MCE Biotechnology, catalog number HY-N9397, chemical formula C 24 H 22 O 15 , solid powder, solubility in DMSO is 33.33 mg / mL.

[0076] In HK1, FaDu, and BxPC3 cells, the experimental groups were added with the compound Q3MG at working concentrations of 0 / 10 / 20 / 30 / 40 / 50 μM in the cell culture medium, and DMSO-PBS was added as the control group. After 24 h of action, the cells were collected to extract total protein and the pro-IL-1β protein level was detected by Western blot (results in Figure 2 As shown in Figure A, Q3MG can significantly inhibit the protein expression of pro-IL-1β in a dose-dependent manner. As the drug dose increases from 0 to 50 μM, the decrease in pro-IL-1β protein level becomes more and more significant.

[0077] In HK1, FaDu, and BxPC3 cells, the experimental group added the compound Q3MG at a working concentration of 50 μM in the cell culture medium, and the control group added DMSO-PBS. After the action time was 0 / 12 / 24 / 48 h, the cells were collected to extract total protein and the pro-IL-1β protein level was detected by Western blot experiment (results in Figure 2 As shown in Figure B, Q3MG can significantly inhibit the protein expression of pro-IL-1β in a time-dependent manner. As the drug action time increases from 0 to 48 h, the decrease in pro-IL-1β protein level becomes more and more significant.

[0078] (2) Quercetin-related compounds were added to HK1, FaDu, and BxPC3 cells at a working concentration of 50 μM: quercetin-3-O-glucoside, quercetin, and Q3MG, respectively, in the cell culture medium. DMSO-PBS was added as a control group. After 24 h of exposure, the cells were collected to extract total protein and the pro-IL-1β protein level was detected by Western blot (results are shown in Figure 3 ). As shown in the figure, only Q3MG can significantly reduce the protein level of pro-IL-1β, while quercetin-3-O-glucoside and quercetin have no effect on the protein level of pro-IL-1β.

[0079] (3) In HK1, FaDu, and BxPC3 cells, the experimental group added the compound Q3MG at a working concentration of 50 μM in the cell culture medium, and the control group added DMSO-PBS. After 24 h of exposure, the cells were collected and RNA was extracted for qRT-PCR experiments to detect changes in IL1B mRNA expression (results are shown in Figure 4 ). As shown in the figure, Q3MG does not affect the mRNA expression level of IL1B.

[0080] (4) In HK1, FaDu, and BxPC3 cells, the experimental group added the compound Q3MG at a working concentration of 50 μM in the cell culture medium, and the control group added DMSO-PBS. After 24 h of exposure, the cell supernatant was collected for ELISA to detect the secretion of mature IL-1β (results are shown in Figure 5 ). As shown in the figure, Q3MG does not affect the secretion of mature IL-1β.

[0081] 3. Provide the degradation mode of the target compound Q3MG on pro-IL-1β protein.

[0082] (1) Prepare the following reagents: lysosome inhibitor: CQ, proteasome inhibitor: MG132, squamous cell carcinoma cells, compound: Q3MG, pro-IL-1β specific antibody, culture medium and buffer required for other experiments, etc.

[0083] (2) Cell culture and treatment: Squamous cell carcinoma cells were cultured to an appropriate density and divided into different groups: control group and Q3MG treatment group; CQ and MG132 were added or not added to the culture medium to make the final concentration reach the required experimental concentration (20 μM). After a certain period of treatment, the cells were collected.

[0084] (3) Western blot and result analysis: Cell proteins were extracted and protein samples were prepared. Western blot analysis was performed to detect the expression level of pro-IL-1β protein. The expression levels of pro-IL-1β protein in different treatment groups were compared, and the effects of CQ and MG132 on the degradation of pro-IL-1β protein by Q3MG were analyzed (see the results). Figure 6 ). As shown in the figure, after adding CQ, Q3MG no longer inhibited the protein expression of pro-IL-1β; while adding MG132 did not affect the degradation of pro-IL-1β protein by Q3MG, indicating that Q3MG mediated the degradation of pro-IL-1β protein through the lysosomal pathway rather than the proteasome pathway.

[0085] 2. Provide verification of the application effect of the quercetin derivative Q3MG based on pro-IL-1β protein targeting in the development of anti-metastatic drugs for squamous cell carcinoma.

[0086] 1. Verify that Q3MG inhibits the invasion and migration of squamous cell carcinoma cells in vitro.

[0087] (1) Prepare HK1, FaDu, and BxPC3 cells, and set up a control group and a Q3MG treatment group (working concentration 50 μM), respectively.

[0088] (2) Cell migration assay: Using the scratch healing assay and Transwell chamber migration assay, scratches were made on the Q3MG-treated group and the control group or cells were added to the Transwell chamber. After a certain period of observation, the scratch healing status was measured (see the results). Figure 7 ) or count the number of migrated cells at the bottom of the Transwell chamber (see Figure 8 ), the migration ability of cells in the Q3MG treatment group and the control group was compared. As shown in the figure, Q3MG inhibits the migration of squamous cell carcinoma cells.

[0089] (3) Cell invasion assay: Matrigel was pre-coated on the bottom of the Transwell chamber, serum-free cell suspension was added to the upper chamber of the Transwell well of the Q3MG treatment group and the control group, and the chamber was placed in a cell plate containing 20% ​​FBS culture medium and incubated for a certain period of time. The chamber was removed, the non-invaded cells on the upper surface were removed, fixed and stained, counted and analyzed, and the invasion ability of the cells in the Q3MG treatment group and the control group was compared (the results are shown in Fig. 9 ). As shown in the figure, Q3MG significantly inhibited the invasion of squamous cell carcinoma cells.

[0090] (4) Cell viability detection: Cells from different treatment groups were inoculated in a 96-well plate at a quantity of 1000 cells / well, and 5 wells were set for each cell type, for a total of 6 groups of replicates. CCK-8 was added at regular intervals at selected time points (Day 0, 1, 2, 3, 4, 5) for cell viability detection. 10 μL of CCK-8 reagent was added to each well, shaken well, and returned to the incubator for incubation for 3 h. The absorbance of each well was detected at a wavelength of 450 nm using a multifunctional microplate reader, and a growth curve was drawn to compare the survival of cells in the Q3MG treatment group and the control group (results are shown in Fig.10 ). As can be seen from the figure, Q3MG does not affect the survival of squamous cell carcinoma cells.

[0091] (5) Colony formation experiment: 1000 cells / well were taken from different treatment groups, and three biological replicates were set up. They were scattered and seeded in 6-well plates and placed in an incubator. After 2-3 weeks of culture, when macroscopic clones were observed (size between 0.3-1.0 mm), the culture was terminated; fixed and stained, the number of clones was counted for statistical analysis, and the proliferation capacity of cells in the Q3MG treatment group and the control group was compared (results are shown in Fig.11 ). As can be seen from the figure, Q3MG does not affect the proliferation ability of squamous cell carcinoma cells.

[0092] 2. Verify that Q3MG inhibits the in vitro invasion and migration of squamous cell carcinoma cells by targeting pro-IL-1β.

[0093] HK1 and FaDu cells with stable knockout of pro-IL-1β were prepared, and the control group and Q3MG treatment group (working concentration 50 μM) were set up in the same way, and the scratch healing experiment was performed (the results are shown in Fig.12 ), Transwell migration assay (results see Fig.13 ) and Transwell invasion assay (results see Fig.14 ), to detect whether the effect of Q3MG on the invasion and migration ability of tumor cells is mediated by pro-IL-1β. Figure 12-14It can be seen that the addition of Q3MG to pro-IL-1β knockout squamous cell carcinoma cells has no effect on the migration and invasion of tumor cells, indicating that Q3MG inhibits invasion and migration through pro-IL-1β.

[0094] The process of constructing HK1 and FaDu cells with stable knockout of pro-IL-1β is as follows:

[0095] (1) Design sgRNA: Use the Synthego website to design sgRNA. The end of the sgRNA must be a protospacer adjacent motif (PAM) sequence. PAM is usually composed of three bases: NGG (N is an arbitrary base). Change the U base in the sgRNA given by the website to a T base. When using Bpil (BbsI) enzyme for enzyme digestion, it is necessary to add the homologous sequence after Bpil digestion at both ends of the sequence. Add the base CACCG before the forward sequence (i.e., the sequence immediately following NGG). If the first base of the forward sequence is G, then G does not need to be added. Add the base AAAC before the reverse sequence and add the base C at the end. If the first base of the forward sequence is G, that is, the last base of the reverse sequence is C, then C can be omitted. Verify the effectiveness of the sgRNA: Consider Tm (56-62℃), GC% (45-60%) and secondary structure, and finally determine the appropriate sgRNA sequence (see Table 2).

[0096] Table 2: sgRNA sequence information (as shown in SEQ ID No. 1-12)

[0097]

[0098] (2) Enzyme digestion: The designed enzyme digestion system is 50 μL, and a total of 2 systems are required. The whole process is performed on ice. The enzyme digestion reaction system is shown in Table 3:

[0099] Table 3: Enzyme digestion reaction system

[0100]

[0101] After instant centrifugation at 3000 rpm, the system was placed in a 37°C water bath for 3 h and then stored at 4°C.

[0102] (3) Verify the product by agarose gel electrophoresis.

[0103] (4) Recover the target product from the gel (refer to the instructions of the gel recovery kit). The reaction system is shown in Table 4.

[0104] Table 4: Adhesive recovery reaction system

[0105]

[0106] (5) Phosphorylation and annealing: The system is 20 μL and the whole process is performed on ice. The reaction system is shown in Table 5:

[0107] Table 5: Phosphorylation and annealing reaction system

[0108]

[0109] The PCR program was set as follows: phosphorylation: 37°C, 20 min; 75°C, 10 min; annealing: 95°C, 5 min; the sample was taken out immediately after the program was completed and placed at room temperature for 30 min-1 h to allow the sample temperature to drop to 25°C; then 180 μL of water was added to the product to dilute the product 10 times.

[0110] (6) Ligation: The ligation system is 10 μL and the whole process is performed on ice. The reaction system is shown in Table 6:

[0111] Table 6: Ligation reaction system

[0112]

[0113] After instant centrifugation at 3000 rpm, the reaction was performed using a PCR instrument at 25°C for 1 h and then stored at 4°C.

[0114] (7) Plasmid transformation: 14-16 hours later, 3-5 monoclonal colonies were picked from each group and sequenced after overnight shaking.

[0115] (8) After successful identification, preserve the bacterial strain and perform plasmid extraction (refer to the instructions of the endotoxin-free plasmid extraction kit: Kangwei Century CW2105).

[0116] (9) Transfect cells by electroporation.

[0117] Electroporation transfection method:

[0118] (1) Preparation of cell suspension: digest and centrifuge the cells, remove the supernatant, add electroporation buffer (Opti-MEM or serum-free medium) preheated at 37°C to wash the cells 2-3 times, add serum-free medium, pipette and mix to make a cell suspension, and count. Prepare 1×10 cells per group. 6 cells.

[0119] (2) Preparation of cell and DNA mixture: Prepare a mixture with a DNA content of 10 μg and a total volume of 100 μL. After mixing evenly, transfer the mixture to an electroporation cup and place it on ice for 5 min. Be careful to avoid generating bubbles.

[0120] (3) Electroporation transfection: Electroporation was performed using Super Electroporator NEPA21 Type Ⅱ electroporator; the electroporation parameters were set to 275 V, 0.5 ms (pulse voltage: 200 V, 230 V, 260 V, 300 V, 350 V; pulse time: 5 ms, 10 ms, 15 ms, 20 ms, 25 ms, 30 ms, 60 ms, resistance value between 30-50 Ω).

[0121] (4) Cell seeding: Remove the electroporation cup and immediately add preheated complete culture medium. Use the provided pipette to aspirate the cell suspension and seed the cells into a 6-well plate.

[0122] (5) Screening: Change the medium 24 hours after electroporation, when the transfection peak is reached; 72 hours later, treat with complete medium containing puromycin (Puro, 0-10 μg / mL) for 2 days.

[0123] (6) Cell culture: After visible cell colonies are formed, the independent colonies are repeatedly blown off with digestion solution and transferred to a 12-well plate for expansion culture.

[0124] (7) Cell identification: Extract gDNA for sequencing to detect IL1B The expression of pro-IL-1β protein was detected by Western blot, and the identified successful cells were expanded and cryopreserved.

[0125] 3. Verify the ability of Q3MG to inhibit the metastasis of squamous cell carcinoma cells in vivo.

[0126] (1) Experimental animal preparation: 4-5 week-old BALB / c nude mice were adaptively raised in an SPF animal laboratory for one week. A total of 20 mice were randomly divided into two groups inoculated with HK1 / FaDu tumor cells. The two groups were further randomly divided into a PBS control group and a Q3MG drug treatment group (n=5).

[0127] (2) Cell preparation: The total amount of cells injected into the tail vein of nude mice depends on the metastatic ability of the injected tumor cells. Taking HK1 / FaDu cells as an example, the total cell injection volume for a single nude mouse is 4×10 6 The cells can be injected multiple times within a few weeks. Since cells tend to aggregate and cause venous thrombosis, the single cell injection volume should not be too large. A single tail vein injection of 1×10 6 The cells of each group grown at the same time were digested and washed, and then supplemented with sterilized 1× PBS to make a cell suspension. After counting, the final concentration was 1×10 6 / 100 μL of cell suspension.

[0128] (3) Drug preparation: PBS / Q3MG drug was dissolved in 2% DMSO, 40% PEG300, 5% Tween-80, and 53% Saline. The dosage was 15 mg / kg / 2 days, and the intravenous administration was 100 μL / mouse for a total of 7 times.

[0129] (4) Tail vein injection: Place the nude mouse in a nude mouse holder. After wiping the tail with alcohol cotton, press and hold the injection point to dilate the blood vessels at the tip of the tail. Select the dorsal vein or the veins on both sides and insert the needle parallel to the vein from the distal 1 / 4. The needle should enter the mouse tail about 3 mm. The injection should be smooth and without resistance. The track of liquid injection can be seen in the vein, indicating a successful injection. The first day of tumor cell injection in nude mice is counted as day 1. Drug treatment will begin 2-3 weeks later. Tail vein injection is used for all injections. The body weight is measured every week after tumor cell injection to observe the condition of the nude mice.

[0130] (5) Termination of the experiment: After 8 weeks, the nude mice were killed by cervical dislocation, and the lung tissue and other major organs (heart, liver, spleen, and kidney) of the nude mice were completely dissected and removed; the tissues were washed with saline and fixed.

[0131] (6) Result analysis: The weight growth changes of mice in each group were statistically analyzed (see Fig.15 ); After the lung tissue was fixed, the water was aspirated and photographed, and the number of nodules on the surface of the lung tissue was counted (the results are shown in Fig.16 ), to compare the effect of Q3MG drug treatment on the metastasis of squamous cell carcinoma cells in vivo; the main organs of the mice were fixed, dehydrated, paraffin-embedded and sectioned, and then H&E stained to observe whether the drug had obvious side effects or systemic toxicity (results see Fig.17 ); H&E and immunohistochemical staining of mouse lung tissues were performed, and the number of nude mouse lung metastatic lesions in 15-20 tissue sections in each group was counted to determine whether the pro-IL-1β protein level in nude mouse lung metastases was clearly changed after Q3MG drug treatment (see the results). Fig.18 ).

[0132] In summary, the present invention discovered for the first time that Q3MG can significantly inhibit tumor invasion and metastasis by targeted degradation of pro-IL-1β protein in cancer cells, and has a good therapeutic effect on squamous cell carcinoma; it reveals the importance of quercetin derivatives in the preparation of drugs for the treatment of squamous cell carcinoma invasion and metastasis, and even the potential value of flavonoids in a variety of other tumors, which has far-reaching clinical significance and important prospects for promotion and application.

Claims

1. A use of a quercetin derivative Q3MG in the preparation of an anti-tumor invasion and metastasis drug, characterized in that: The quercetin derivative Q3MG is quercetin-3-O-(6''-O-malonyl)-β-D-pyranoglucoside, and the anti-tumor invasion and metastasis drug is a drug for preventing squamous cell carcinoma invasion and metastasis.

2. The use of the quercetin derivative Q3MG according to claim 1 in the preparation of anti-tumor invasion and metastasis drugs, characterized in that: The anti-tumor invasion and metastasis drug is a drug that inhibits the invasion and metastasis of cancer cells by targeting and degrading pro-IL-1β protein in cancer cells.

3. The use of the quercetin derivative Q3MG according to claim 1 in the preparation of anti-tumor invasion and metastasis drugs, characterized in that: The squamous cell carcinoma comprises at least one squamous cell carcinoma cell line selected from HK1 and FaDu.

4. The use of the quercetin derivative Q3MG according to claim 1 in the preparation of anti-tumor invasion and metastasis drugs, characterized in that: The invasion and metastasis of the squamous cell carcinoma is lung metastasis.

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