Application of sulfated glycosaminoglycan inhibitor in preparation of antitumor drugs

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a sulfated glycosaminoglycan inhibitor in preparation of antitumor drugs. The invention provides an innovative treatment application of an XYLT1 inhibitor or ChABC in targeted degradation of CSPG, and the XYLT1 inhibitor or ChABC is suitable for lung adenocarcinoma patient groups with obviously increased CSPG expression level. A research result shows that the application has a remarkable accurate treatment potential on the lung adenocarcinoma subtype with the CSPG modification level accumulated abnormally. And by inhibiting the activation of an NF-kappa B signal channel, the invasiveness and metastasis capability of the tumor are effectively reduced. According to the technical scheme, further optimization is carried out on the basis of an existing treatment means, and definite innovativeness and wide clinical application prospects are shown.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of sulfated glycosaminoglycan inhibitors in the preparation of anti-tumor drugs. Background Art

[0002] Lung cancer is a common malignant tumor, and its early detection and timely intervention are crucial to improve the survival rate of patients. However, once lung cancer metastasizes, the complexity and difficulty of treatment will increase significantly. Metastasis is the main cause of death and the main factor limiting the five-year survival rate of patients. Among patients with non-small cell lung cancer (NSCLC), approximately 20% to 50% will have distant metastasis, of which the most common metastatic sites include the brain, bones, liver and adrenal glands. The process of lung cancer metastasis involves cell invasion, angiogenesis and metastatic growth. At present, the treatment of lung cancer includes systemic therapy, radiotherapy, surgery and interventional therapy. Although chemotherapy can kill cancer cells, due to its non-specific killing, it may also damage normal cells, leading to side effects such as nausea, hair loss, and immune system suppression. Radiotherapy may also cause damage to surrounding normal tissues. Although molecular targeted therapy and immunotherapy have good selectivity, they may lead to overactivation of the immune system, trigger a series of side effects, and are costly. The treatment of tumor metastasis currently faces multiple challenges and shortcomings. First, although surgical resection is the preferred treatment for many tumors, it is high-risk, especially for tumors in sensitive areas. Surgery is very traumatic to the human body and may cause the patient's immunity to be reduced and the resistance to disease to be decreased. Radiotherapy can protect uninvolved tissues, but it may cause side effects such as hair loss and loss of sweat gland function. Chemotherapy, as a systemic treatment, has a therapeutic effect on primary lesions, metastatic lesions, and subclinical metastatic lesions, but chemotherapy drugs have poor selectivity. While killing tumor cells, they also kill a large number of normal cells, destroying the body's own immune system function and easily causing drug resistance.

[0003] In addition, the immunosuppressive properties of the tumor microenvironment (TME) also bring additional difficulties to treatment. For example, although immune checkpoint inhibitors (ICIs) have achieved certain results in enhancing anti-tumor immunity, some tumor types do not respond to ICIs due to lack of tumor antigens, antigen presentation defects, T cell activation defects, and rejection of T cells by immunosuppressive TME, and are described as "cold tumors." Moreover, even for tumors that initially respond to ICIs, they may develop resistance due to T cell exhaustion.

[0004] Furthermore, tumor subtypes with certain gene mutation types have stronger tumor invasion, migration and anti-apoptosis capabilities, and conventional drugs have poor therapeutic effects on them. Therefore, it is necessary to develop new anti-tumor drugs for specific tumor subtypes. Summary of the invention

[0005] The first aspect of the present invention aims to provide the use of sulfated glycosaminoglycan inhibitors in the preparation of anti-tumor drugs.

[0006] The second aspect of the present invention is to provide the use of XYLT1 gene as a drug target in drugs for preventing and / or treating lung cancer.

[0007] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides the use of a sulfated glycosaminoglycan inhibitor in the preparation of an anti-tumor drug.

[0009] In some embodiments of the present invention, the sulfated glycosaminoglycan inhibitor includes a substance that reduces the synthesis of the sulfated glycosaminoglycan inhibitor and a substance that promotes the decomposition of sulfated glycosaminoglycan.

[0010] In some embodiments of the present invention, the substance that reduces the synthesis of sulfated glycosaminoglycan inhibitors is a xylosyltransferase 1 (XYLT1) inhibitor, which is an enzyme that plays a key role in the first step of the synthesis of sulfated glycosaminoglycans.

[0011] In some embodiments of the present invention, the substance that promotes the decomposition of sulfated glycosaminoglycans is chondroitinase ABC (ChABC), which can digest the side chains of chondroitin sulfate protein and keratan sulfate.

[0012] In some embodiments of the present invention, the XYLT1 inhibitor comprises at least one of the following:

[0013] (a1) Substances that inhibit the activity of XYLT1 protein;

[0014] (a2) substances that reduce the content of XYLT1 protein;

[0015] (a3) a substance that silences the XYLT1 gene;

[0016] (a4) a substance for knocking out the XYLT1 gene;

[0017] (a5) A substance that inhibits the expression of the XYLT1 gene.

[0018] In some embodiments of the present invention, the nucleotide sequence of the XYLT1 gene is shown in SEQ ID NO:1.

[0019] In some embodiments of the present invention, the XYLT1 inhibitor includes nucleic acid molecules, protein molecules, and small molecule drugs.

[0020] In some embodiments of the invention, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide.

[0021] In some embodiments of the present invention, the protein molecule is a specific antibody of XYLT1.

[0022] In some embodiments of the present invention, the small molecule drug is a small molecule that binds to XYLT1.

[0023] In some embodiments of the present invention, the sequence of the shRNA is shown in SEQ ID NO: 2, 3.

[0024] In some embodiments of the present invention, the XYLT1 inhibitor further includes sgRNA used when knocking out the XYLT1 gene in the animal cell or in the animal body using CRISPR / Cas9 technology, or a DNA molecule capable of transcribing the sgRNA, or an expression cassette or expression vector containing the DNA molecule, and of course, may also include Cas9 protein or an expression vector for expressing Cas9 protein. Or shRNA for knocking down XYLT1.

[0025] In some embodiments of the present invention, the vector comprises a viral vector or a non-viral vector.

[0026] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0027] In some embodiments of the present invention, the non-viral vector comprises: at least one of a plasmid vector, a cationic polymer vector, chitosan, a liposome, and a nanoparticle vector.

[0028] In some embodiments of the present invention, the tumor is a tumor in which chondroitin sulfate proteoglycan CSPG is abnormally accumulated.

[0029] In some embodiments of the present invention, the tumor with abnormal CSPG accumulation includes a tumor with high XYLT1 expression.

[0030] The abnormal accumulation of CSPG can be determined preferably but not limited to the following methods: low or high expression of CSPG refers to the conclusion drawn after comparing the amount of CSPG protein between two groups (individuals) of samples. If one group (individuals) of samples has less or more CSPG protein than another group (individuals), the sample is called low or high expression of CSPG. The samples used for protein quantity comparison can be tumor cells and normal cells, tumor tissue and adjacent non-tumor tissue.

[0031] Similarly, the level of XYLT1 expression can be determined by the level of XYLT1 mRNA.

[0032] In some embodiments of the present invention, the tumor is lung cancer, and further is lung adenocarcinoma.

[0033] In some embodiments of the present invention, the medicament comprises a pharmaceutically acceptable excipient.

[0034] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0035] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and as inactive ingredients of medicaments. A compilation of pharmaceutically acceptable excipients can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994); Pharmacopoeia of the People's Republic of China - Catalogue of Pharmaceutical Excipients.

[0036] In some embodiments of the present invention, the dosage form of the drug includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0037] In some embodiments of the present invention, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets;

[0038] In some embodiments of the present invention, the non-gastrointestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0039] In some embodiments of the present invention, the medicament further comprises any one or more other active ingredients.

[0040] The second aspect of the present invention provides the use of XYLT1 gene as a drug target in drugs for preventing and / or treating lung cancer; the use is for non-diagnostic and non-therapeutic purposes.

[0041] In some embodiments of the present invention, the applications include screening of drug targets, screening of drugs, pharmacodynamic evaluation of drugs, and safety evaluation of drugs.

[0042] The beneficial effects of the present invention are:

[0043] The present invention found that high expression of XYLT1 (xylosyltransferase 1) significantly enhanced the abnormal accumulation of sugar chains of chondroitin sulfate proteoglycan (CSPG), promoted the activation of the NF-κB pathway, and thus accelerated the invasion and metastasis of lung adenocarcinoma. Therefore, the regulatory effect of XYLT1 provides a key target for the precise treatment of lung adenocarcinoma with significantly increased CSPG modification levels. Furthermore, the present invention demonstrates unique clinical application potential in lung adenocarcinoma subtypes with significantly increased CSPG modification levels and specific XYLT1 high expression by combining ChABC with traditional anticancer therapy.

[0044] The present invention aims at the role of sulfated glycosaminoglycan (sGAG) in the tumor microenvironment and proposes an enzyme therapy technology for accurately degrading glycosylation modifications on related molecules, thereby effectively inhibiting the invasion, migration and anti-apoptosis ability of tumors. Targeted degradation of sulfated glycosaminoglycan (sGAG), which plays a key role in the process of tumor metastasis, can significantly inhibit the activation of the NF-κB signaling pathway by reducing the modification level of sGAG in tumor cells and on NF-κB molecules, especially the level of CSPG (chondroitin sulfate proteoglycan), thereby weakening the invasion, migration and anti-apoptosis ability of tumor cells, and ultimately effectively preventing the early metastasis of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0046] Figure 1The expression and prognosis of XYLT1 in metastatic lung adenocarcinoma, where (A, B) the expression level of XYLT1 in 18 pairs of early LAD primary tumors and metastases was evaluated by immunohistochemical analysis and quantitative analysis. Two representative paired cases are shown. Scale bar: 50 μm; (C) Kaplan-Meier survival analysis (Log-rank test) evaluates the 5-year overall survival and disease-free survival of the low XYLT1 expression group and the high XYLT1 expression group in the collected LAD patient cohort. (D) Kaplan-Meier survival analysis (Log-rank test) evaluates the 5-year overall survival and disease-free survival of patients with stage I LAD in the TCGALUAD database. Patients were divided into low expression group and high expression group according to the XYLT1 expression level.

[0047] Figure 2 The results of the effects of XYLT1 on the malignant phenotype of lung adenocarcinoma cells, where A is the result of the wound healing experiment; (B) XYLT1 silencing reverses the promotion of TGF-β1 overexpression on LAD cell migration; (C-F) Transwell invasion assay results show the changes in the invasion ability of LAD cells in different treatment groups. (GJ) Clone formation assay and TUNEL staining results evaluate the survival and anti-apoptosis ability of LAD cells.

[0048] Figure 3The results of the effects of XYLT1 on mice, where (A) wild-type or D745G mutant XYLT1-overexpressing A549 and H1975 cells or corresponding control cells with reporter gene luciferase were injected intraventricularly into nude mice (n=5 per group). Representative bioluminescence imaging showing systemic metastasis; (B) microCT imaging and H&E staining confirmed bone metastasis induced by intraventricular injection of cells. Scale bar: 100 μm; (C) Kaplan-Meier survival analysis (Log-rank test) evaluated the overall survival rate of nude mice after injection of the above cells; (D) TGF-β1-overexpressing A549 and H1975 cells were injected intraventricularly into nude mice, and in vivo imaging showed systemic metastasis; (E) microCT imaging and H&E staining showed representative images of bone metastasis lesions after injection of the above cells. Scale bar: 100 μm; (F) Kaplan-Meier survival analysis to evaluate the overall survival of nude mice after injection of TGF-β1-overexpressing cells; (GI) Low doses (5,000 cells / mouse) of wild-type or D745G mutant XYLT1-overexpressing A549 and H1975 cells or corresponding control cells were inoculated subcutaneously into nude mice (n=5 per group). Representative in vivo imaging (G), bioluminescent growth curves (H), and tumor weights of xenograft tumors (I) are shown; (J) TUNEL staining (green) shows apoptotic cells in paraffin-embedded tumor sections, and DAPI (blue) is nuclear staining. Scale bar: 100 μm; (K) H&E staining and Ki67 immunohistochemical staining show the pathological morphology and proliferating cells of the tumor. Scale bar: 100 μm; Error bars represent mean ± SD, and statistics were performed using two-way ANOVA multiple comparison analysis, P < 0.01.

[0049] Figure 4 The results show that XYLT1 promotes NF-κB pathway activation and promotes P65 nuclear translocation. (A) The sGAG content of LAD cells in the wild-type XYLT1 overexpression group was significantly increased; (BC) TGF-β treatment increased the sGAG content of LAD cells, while XYLT1 silencing significantly reduced the sGAG level; (D) The sGAG level in metastatic LAD tissue was higher than that in primary tumor tissue; (EF) RNA sequencing and enrichment analysis showed that XYLT1 affected RelA / p65 target genes Expression, especially inflammation-related genes; (GH) Wild-type XYLT1 overexpression upregulated RelA / p65 target genes, while XYLT1 silencing inhibited the expression of these genes; (IJ) NF-κB response element luciferase reporter assay showed that wild-type XYLT1 enhanced NF-κB transcriptional activity, while XYLT1 silencing inhibited this signaling activity; (KP) Wild-type XYLT1 promoted the nuclear translocation of p65, while the D745G mutant or XYLT1 silencing inhibited the nuclear translocation of p65.

[0050] Figure 5 ChABC-mediated disruption of sGAG-IκBα modification inhibits XYLT1-mediated LAD progression; (A) Analysis of sGAG levels in whole-cell lysates in A549-XYLT1 or A549-TGF-β cells treated with ChABC; (B) Analysis of sGAG modification levels on purified IκBα protein in A549-XYLT1 or A549-TGF-β cells; (C) Immunoprecipitation showing the effects of ChABC treatment on the levels of polyubiquitinated IκBα, phosphorylated (Ser32 / 36) IκBα, and its interaction with IKKα, IKKβ, or p65 in A549-XYLT1 cells; (D) WB analysis of changes in IκBα protein levels in A549 cells treated with ChABC; (E) Relative Changes in NF-κB response element (NF-κB-RE) luciferase activity in A549-XYLT1 or A549-TGF-β cells; (FG) Transwell invasion assay, showing a significant decrease in the invasion ability of A549-XYLT1 and A549-TGF-β cells, the number of invasive cells in five random fields of view was shown and quantified (scale bar: 300 μm); (HI) Clone formation assay, showing the inhibition of the survival ability of A549-XYLT1 and A549-TGF-β cells by ChABC treatment, the number of clones was shown and quantified; (JK) TUNEL assay showed that ChABC treatment significantly increased the proportion of apoptotic cells, green represents TUNEL-positive cells, and blue represents DAPI-stained nuclei (scale bar: 50 μm). DETAILED DESCRIPTION

[0051] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0052] Example 1 Identification of lung cancer targets

[0053] 1. Experimental methods

[0054] 1) Collect tumor tissue and corresponding normal tissue samples from patients with lung adenocarcinoma (Guangdong Provincial People's Hospital collected LAD tissue in the early stage and paired puncture biopsies to obtain 263 recurrent metastatic specimens, with prior consent from the patients and approval by the Research Ethics Committee of Guangdong Provincial People's Hospital) to ensure the ethical compliance of the samples. The collected tissue samples were fixed, usually using 10% neutral buffered formaldehyde (NBF) for 48 hours. After dehydration and transparency, the tissue samples were immersed in paraffin and waited for complete embedding. Use a microtome to cut the paraffin-embedded tissue into 4-5 micron thick slices and place them on pretreated slides.

[0055] 2) Immunohistochemical staining

[0056] Antibody selection: Select a highly specific anti-XYLT1 monoclonal antibody and verify its applicability in lung adenocarcinoma tissue.

[0057] Antibody dilution: Dilute the anti-XYLT1 antibody to an appropriate concentration according to the product instructions.

[0058] Staining steps:

[0059] a. Dry the sections in an oven at 60°C for 1 hour, then dewax and hydrate.

[0060] b. Rinse the sections with phosphate-buffered saline (PBS) to remove excess water.

[0061] c. Add diluted anti-XYLT1 antibody and incubate overnight at 4°C.

[0062] d. On the next day, rinse the sections three times with PBS, 5 minutes each time.

[0063] e. Add appropriate secondary antibody (such as HRP-labeled anti-rabbit IgG) and incubate, usually for 1 hour at room temperature.

[0064] f. Rinse the sections again with PBS.

[0065] 3) Color reaction

[0066] Color development agent preparation: Use DAB (3,3'-diaminobenzidine) as the color development substrate and prepare it according to the instructions. Immerse the slices in the DAB color development solution and observe the color development reaction, which usually reacts for 5-10 minutes at room temperature. After the reaction is completed, rinse the slices with tap water to terminate the reaction.

[0067] 4) Observation and analysis

[0068] Microscope observation:

[0069] Observe the sections using an optical microscope and select an appropriate magnification (such as 400 times) for observation.

[0070] The staining intensity and number of positive cells were recorded to evaluate their distribution.

[0071] Calculation of immunohistochemistry score (SI value):

[0072] Staining intensity scoring: Scoring was performed based on the staining intensity (0-3 points), with 0 for no staining and 3 for strong staining.

[0073] Positive cell ratio scoring: Scoring was performed based on the ratio of positive cells (0-4 points), with 0 being 0% and 4 being >75%.

[0074] Comprehensive score: The staining intensity score and the positive cell ratio score were added to obtain the final immunohistochemistry score (SI value).

[0075] 5) Data statistics and analysis

[0076] Statistical analysis: Statistical software (such as SPSS or GraphPad Prism) was used for data analysis to compare the differences in XYLT1 expression among different groups.

[0077] 2. Experimental results

[0078] Interpretation of results: Based on the above data, the correlation between XYLT1 expression and patient prognosis, tumor stage and other clinical characteristics was analyzed.

[0079] Through the above experimental steps, the expression level of XYLT1 in clinical samples of lung adenocarcinoma patients can be effectively evaluated, thereby identifying targets related to CSPG accumulation in metastatic early lung adenocarcinoma.

[0080] 2. Upregulation of XYLT1 in metastatic LAD and its correlation with poor prognosis

[0081] 1. High expression of XYLT1 in metastatic LAD

[0082] This study analyzed tissue samples from 263 patients with early-stage lung adenocarcinoma (LAD), including surgically resected primary LAD and paired metastatic LAD tissues ( Figure 1 Middle A). The expression level of XYLT1 was detected by immunohistochemistry (IHC) staining, and the staining index (SI) was calculated.

[0083] Quantitative analysis results showed that XYLT1 protein expression was significantly upregulated in metastatic tissues compared with primary LAD tumors ( Figure 1 (B).

[0084] 2. Correlation between XYLT1 expression and patient prognosis

[0085] Kaplan–Meier survival analysis

[0086] Kaplan-Meier survival curve analysis showed that patients with higher XYLT1 expression in primary LAD had a significantly poor prognosis:

[0087] Overall survival (OS): The 5-year survival rate of the high XYLT1 expression group was only 28.5%, while that of the low expression group was 80.0%.

[0088] Metastasis-free survival (MFS): The 5-year MFS in the high XYLT1 expression group was 0%, while that in the low expression group was 20%.

[0089] 2.2.TCGA Dataset Validation

[0090] We further analyzed the mRNA expression level of XYLT1 in the TCGA lung adenocarcinoma (LUAD) dataset and verified its correlation with poor prognosis:

[0091] Overall survival (OS): The 5-year OS of the high XYLT1 expression group was 48.6%, which was significantly lower than that of the low expression group (60.9%).

[0092] Relapse-free survival (RFS): The 5-year RFS of the high expression group was 54.3%, while that of the low expression group was 63.5%.

[0093] 2.3. Stage analysis

[0094] In patients with stage I LAD, high XYLT1 expression was significantly associated with poor OS and RFS, whereas in patients with advanced LAD (stage II), the expression level of XYLT1 had no significant difference in predicting prognosis and RFS ( Figure 1 (middle D).

[0095] Example 2 XYLT1 enhances the malignant phenotype of lung adenocarcinoma cells

[0096] 1. Experimental methods

[0097] 1) Cell culture A549 and H1975 cells were cultured in a medium containing 10% fetal bovine serum until the logarithmic growth phase, and lentivirus infection was used to construct stable expression and knockdown cell lines.

[0098] Lentivirus preparation and infection

[0099] Virus preparation: Lentivirus is packaged by packaging cells (293T cells). The target gene plasmid (overexpression XYLT1, shXYLT1 plasmid) and packaging plasmid (such as psPAX2 and pMD2.G) are transfected into packaging cells at a certain ratio, and the viral supernatant is collected after 48-72 hours of culture.

[0100]

[0101] XYLT1-shRNA2:GGGAGAATGTCTACGATGA (SEQ ID NO: 2)

[0102] XYLT1-shRNA2:CAAGCATCATGCTACCAAT (SEQ ID NO: 3)

[0103] Lentivirus infection: The virus solution was mixed with A549 and H1975 cells, and polybrene (5 μg / mL) was added to enhance the infection efficiency. The cells were incubated at 37°C and 5% CO 2 Incubate for 6-12 hours under the same conditions and then replace with fresh medium.

[0104] Target gene expression analysis: RNA or protein was extracted, and qRT-PCR and Western Blot were performed to detect the expression of the target gene.

[0105] 2) Scratch operation: Use a cell scraper to gently scratch a straight line on the cell monolayer to form a scratch, and wash away the detached cells with PBS to ensure that only attached cells are retained. Take images of the scratch area at different time points (such as 0 hours, 24 hours, and 48 hours). Measure the scratch width using image analysis software and calculate cell migration rate.

[0106] 3) Clone formation experiment

[0107] Objective: To investigate the effects of overexpression and knockdown of XYLT1 on cell proliferation and clone formation.

[0108] Experimental steps:

[0109] The cells were divided into a high-expression XYLT1 group and a knockdown XYLT1 group, and the cells were collected after 48 hours of culture.

[0110] a. Inoculate the treated cells in a 6-well plate at an appropriate density (e.g., 500-1000 cells / well) and culture them in a medium containing 10% fetal bovine serum. Culture the cells for about 10-14 days until obvious clones are formed.

[0111] b. Collect the samples and fix the clones with 4% paraformaldehyde at room temperature for 10 minutes.

[0112] c. Stain with crystal violet staining solution at room temperature for 30 minutes.

[0113] d. Rinse with PBS to remove excess dye, and then observe and count the number of colonies under a microscope after drying.

[0114] 4) TUNEL assay

[0115] Objective: To evaluate the effects of high expression of XYLT1 and mutation of XYLT1 enzyme activity site (base 2234 in SEQ ID NO: 1 mutated from A to G, and the corresponding amino acid sequence 745 mutated from D to G) on cell apoptosis.

[0116] Experimental steps:

[0117] a. The cells were divided into a high-expression XYLT1 group and a mutated XYLT1 enzyme activity site group, and the cells were collected after 48 hours of treatment.

[0118] b. Fix the cells in 6-well plates with 4% paraformaldehyde at room temperature for 20 minutes.

[0119] c. Treat cells with 0.1% Triton X-100 in PBS and permeabilize for 5-10 minutes at room temperature.

[0120] d. According to the TUNEL kit instructions, add the reaction solution (containing TdT enzyme and labeled nucleoside) to the cells and incubate at 37°C for 1 hour.

[0121] e. Observe the stained cells under a fluorescence microscope and record the number of positive cells.

[0122] f. Calculate the cell apoptosis rate and evaluate the effect of XYLT1 on cell apoptosis.

[0123] g. Data statistics: Use statistical software (such as SPSS or GraphPad Prism) to compare the cell migration rate, clone formation ability and apoptosis rate between the XYLT1 high expression group and the XYLT1 knockdown group.

[0124] 2. Experimental results

[0125] 2.1 Effect of XYLT1 overexpression on the malignant phenotype of LAD cells

[0126] Migration and invasion ability: Ectopic overexpression of wild-type XYLT1 significantly enhanced the migration and invasion ability of LAD cells. This phenomenon was confirmed by wound healing assay and Matrigel-coated Transwell invasion assay ( Figure 2 Specifically, cells overexpressing wild-type XYLT1 significantly accelerated the healing speed in the wound healing assay, and the number of invasive cells in the Transwell assay increased significantly.

[0127] Effect of D745G mutant XYLT1: Compared with the wild type, overexpression of D745G mutant XYLT1 had little effect on the migration and invasion ability of LAD cells ( Figure 2 A, C, D, and F), indicating that its enzymatic activity is essential for the malignant-promoting effects of XYLT1.

[0128] 2.2 Reversal of the promoting effect of XYLT1 silencing on TGF-β signaling

[0129] Silencing XYLT1 in TGF-β1-overexpressing LAD cells significantly reversed the promoting effect of TGF-β signaling on cell migration and invasion ( Figure 2 This further demonstrates the key role of XYLT1 in TGF-β-mediated malignant progression of LAD cells.

[0130] 2.3 Effects of XYLT1 on survival and anti-apoptosis ability of LAD cells

[0131] Further research found that:

[0132] Survival: Ectopic overexpression of wild-type XYLT1 significantly enhanced the viability of LAD cells under serum-deficient conditions, as evidenced by increased clonogenicity ( Figure 2 Middle G).

[0133] Anti-apoptotic ability: Cells overexpressing wild-type XYLT1 showed a significantly reduced number of apoptotic cells in TUNEL staining ( Figure 2 HJ), indicating that it has an anti-apoptotic effect.

[0134] Effect of the D745G mutant: Unlike the wild type, overexpression of the D745G mutant XYLT1 failed to significantly enhance the survival and anti-apoptotic ability of LAD cells ( Figure 2 Chinese GJ).

[0135] Based on the above experimental results, this embodiment found that:

[0136] XYLT1 plays a pro-malignant role in LAD cells through its enzymatic activity, promoting cell migration, invasion, survival and anti-apoptosis ability.

[0137] The D745G mutant XYLT1, which lacks enzyme activity, is unable to achieve the malignant effects of wild-type XYLT1, emphasizing its key role in the biosynthesis of sulfated glycosaminoglycans (sGAGs).

[0138] High expression of XYLT1 not only promotes the malignant phenotype of LAD cells, but is also closely associated with the poor clinical prognosis of metastatic LAD.

[0139] Example 3 XYLT1 promotes the metastasis and survival of LAD cells

[0140] 1. Experimental methods

[0141] 1) Construction of vector

[0142] Plasmid vectors expressing wild-type XYLT1 and mutant XYLT1 were constructed (same as in Example 2).

[0143] 2) Transfect cells

[0144] Cells were transfected with Lipofectamine 3000 to obtain cells with high expression of wild-type and mutant XYLT1.

[0145] 3) Ventricular injection

[0146] The BALB / c nude mouse model was selected to ensure compliance with ethical requirements. The transfected cells (1×10^6 cells / 100μL PBS / 1 mouse) were injected into the mouse heart by ventricular injection. The health status of the mice was observed regularly after injection, and the survival rate and any adverse reactions were recorded.

[0147] 4) Metastasis detection: Perform regular in vivo imaging of animals after injection to detect metastasis. 4-6 weeks after injection, dissect the mice and remove major organs (lungs, liver, lymph nodes) for subsequent analysis.

[0148] 5) Slice preparation: The removed tissue was fixed with 10% neutral buffered formaldehyde for 24 hours. Dehydration, clearing and paraffin embedding were performed according to conventional methods. The tissue was cut into 4-5 micron thick slices using a microtome and placed on pre-treated slides.

[0149] 6) Ki67 staining: Select a specific anti-Ki67 antibody (Anti-Ki67 antibody [SP6] ab16667), dewax and hydrate the sections, add the anti-Ki67 antibody, and incubate overnight at 4°C. Use a secondary antibody for incubation, and observe under a microscope after color development.

[0150] 7) TUNEL staining: Use 4% paraformaldehyde to fix the slices and permeabilize them. Follow the instructions of the TUNEL kit (DeadEnd TM Fluorometric TUNEL System Technical Bulletin. G3250), add the reaction solution to the slices and incubate at 37°C for 1 hour. Observe the stained cells under a fluorescence microscope, record the number of positive cells, and calculate the apoptosis rate.

[0151] 2. Experimental results

[0152] 2.1XYLT1 promotes distant metastasis of LAD cells

[0153] In the nude mouse intracardiac injection model, LAD cells overexpressing wild-type XYLT1 showed stronger distant metastasis ability, and their bioluminescent signals were significantly enhanced compared with the control group and the D745G mutant XYLT1 group ( Figure 3 (A).

[0154] Micro-CT imaging and histological analysis showed that nude mice in the wild-type XYLT1 overexpression group developed obvious cancerous lesions in the bone tissue, while the control group and the D745G mutant XYLT1 group had weak metastatic ability ( Figure 3 (middle B).

[0155] 2.2 Effect of XYLT1 on survival

[0156] The metastasis-free survival and overall survival of nude mice in the wild-type XYLT1 overexpression group were significantly shortened ( Figure 3 Middle C).

[0157] XYLT1 silencing significantly reversed the adverse effects of TGF-β1 on metastasis and survival ( Figure 3 (in DF).

[0158] In the allogeneic transplantation model, all mice in the wild-type XYLT1 overexpression group (5 / 5) developed distant metastases, while only 1 / 5 mice in the control group and the D745G mutant XYLT1 group developed metastases ( Figure 3 ).

[0159] 2.3XYLT1 promotes tumor growth and recurrence

[0160] In the low-dose subcutaneous inoculation model, the tumor growth rate of the wild-type XYLT1 overexpression group was significantly accelerated, the bioluminescence signal was enhanced, and the tumor volume and weight were significantly increased ( Figure 3 Medium GI).

[0161] TUNEL and Ki67 staining results showed that wild-type XYLT1 overexpressing tumor cells had stronger cell proliferation ability and higher anti-apoptosis ability ( Figure 3 (in JK).

[0162] In the model of subcutaneous transplantation followed by surgical resection, the recurrence rate in the wild-type XYLT1 overexpression group was significantly higher than that in the control group and the D745G mutant XYLT1 group ( Figure 3 ).

[0163] Example 4 XYLT1 promotes NF-κB pathway activation and P65 nuclear translocation

[0164] 1. Experimental methods

[0165] 1) Nuclear-cytoplasmic separation experiment

[0166] Objective: To separate the nuclei and cytoplasms of XYLT1-WT, XYLT1-MT A549 and H1975 cells to detect the localization changes of NF-κB, so as to clarify the mechanism by which XYLT1 causes the malignant progression of lung adenocarcinoma.

[0167] Experimental steps:

[0168] a. Grow A549 and H1975 cells in medium containing 10% fetal bovine serum and ensure that the cells are in the logarithmic growth phase.

[0169] b. Stimulation (TNF-α, concentration of 10 ng / mL) was applied to the cells in the treatment group for 30 minutes to 1 hour.

[0170] c. Collect the cells and pellet them by centrifugation (1000 rpm, 5 minutes).

[0171] d. Prepare nuclear plasma separation lysis buffer containing: HEPES (pH 7.9): 10 mM, KCl: 10 mM, MgCl2: 1.5 mM, NP-40: 0.4%, and protease inhibitor (PMSF).

[0172] The cells were resuspended in lysis buffer, gently pipetted to mix, and placed on ice for 10 minutes. The nuclei and cytoplasm were separated by centrifugation (3000 rpm, 5 minutes).

[0173] The supernatant was the cytoplasm, and the precipitate was the cell nucleus, which were collected and stored at -80°C and subsequently used for western blot experiments.

[0174] 2) Immunofluorescence detection of NF-κB nuclear translocation

[0175] Experimental steps:

[0176] a. A549 and H1975 cells were seeded on glass slides and cultured until they were 70%-80% confluent.

[0177] b. The control group was not treated, and the experimental group was stimulated with TNF-α (10 ng / mL, treatment time was 30 minutes).

[0178] c. Fix the cells with 4% paraformaldehyde and incubate at room temperature for 10-15 minutes. Wash with PBS three times, 5 minutes each time.

[0179] d. Permeabilize cells with 0.1% Triton X-100 in PBS, incubate at room temperature for 5-10 minutes, and wash again with PBS three times.

[0180] e. Block the cells with PBS containing 5% bovine serum albumin (BSA) and incubate at room temperature for 1 hour.

[0181] f. Dilute NF-κB antibody (such as p65 antibody) to an appropriate concentration (usually 1:100), add to the cells, and incubate at 4°C overnight.

[0182] g. On the next day, wash three times with PBS.

[0183] h. Add fluorescently labeled secondary antibody (such as FITC or Cy3 labeled anti-rabbit IgG) and incubate at room temperature for 1 hour. Wash with PBS 3 times.

[0184] i. DAPI staining: Treat cells with DAPI staining solution and incubate at room temperature for 5-10 minutes to mark cell nuclei. Wash again with PBS for 3 times.

[0185] j. Observe the cells with a fluorescence microscope, record the localization of NF-κB, and analyze the fluorescence intensity in the nucleus and cytoplasm.

[0186] k. Comparison of NF-κB nuclear translocation between XYLT1-WT and XYLT1-MT groups using GraphPad Prism.

[0187] 2. Experimental results

[0188] By analyzing clinical samples from patients with lung adenocarcinoma, we found that XYLT1 was highly expressed in metastatic recurrence lesions, and that it could activate the NF-κB signaling pathway by increasing the modification level of CSPG in the metastatic microenvironment and enhancing the modification of IκBα sugar chains, thereby promoting the nuclear translocation of NF-κB and ultimately accelerating tumor metastasis.

[0189] The sGAG level in LAD cells overexpressing wild-type XYLT1 was significantly higher than that in the control group and the D745G mutant XYLT1 group ( Figure 4 After TGF-β treatment, the sGAG content in A549 and H1975 cells increased gradually with the extension of treatment time, while XYLT1 silencing could significantly reduce the sGAG level ( Figure 4 The sGAG content in metastatic tissues was significantly higher than that in primary tumor tissues ( Figure 4 (middle D).

[0190] Relationship between XYLT1 and TGF-β signaling:

[0191] XYLT1 silencing did not affect the SMAD binding element (SBE)-luciferase activity after TGF-β treatment, suggesting that XYLT1 may act through a non-classical TGF-β signaling pathway ( Figure 4 Medium EF).

[0192] XYLT1 regulates the expression of RelA / p65 target genes:

[0193] RNA sequencing showed that in TGF-β-treated LAD cells, genes regulated by RelA / p65 were significantly enriched among XYLT1-downregulated genes, including inflammation-related genes such as IL1B, IL6, and MMP9 ( Figure 4 Medium EF).

[0194] Overexpression of wild-type XYLT1 significantly upregulated the expression of RelA / p65 target genes, whereas silencing of XYLT1 suppressed the expression of these genes in cells activated by TGF-β signaling ( Figure 4 in GH).

[0195] XYLT1 enhances NF-κB signaling pathway activity:

[0196] NF-κB response element luciferase reporter assay showed that wild-type XYLT1 overexpression significantly enhanced NF-κB transcriptional activity, while XYLT1 silencing significantly inhibited TGF-β signaling-induced NF-κB activity ( Figure 4 (in Chinese).

[0197] Immunoblotting and immunofluorescence staining showed that wild-type XYLT1 promoted the nuclear translocation of p65, while XYLT1 silencing significantly blocked TGF-β-induced p65 nuclear localization ( Figure 4 (in KP).

[0198] Example 5 ChABC inhibits the malignant phenotype of lung adenocarcinoma cells

[0199] 1. Experimental methods

[0200] 1) Cell culture

[0201] A549 and A549-XYLT1 overexpressing cells were seeded in 6-well plates and cultured to 70%-80% confluence.

[0202] ChABC treatment: Wash the cells twice with PBS and remove the culture medium. Prepare a ChABC solution of appropriate concentration (ChondroitinaseABC (MedChemexpress, HY-P2727, 0.1 IU / mL) and add it to the cells, incubate at 37°C for 1 hour to degrade CSPG. After treatment, wash the cells three times with PBS to remove ChABC.

[0203] 2) Transwell invasion assay

[0204] a. Add an appropriate amount of matrix glue (such as Matrigel) to the upper chamber of the Transwell to simulate the extracellular matrix and solidify at 37°C.

[0205] b. The ChABC-treated cells (A549 and A549-XYLT1) were seeded into the upper chamber of the Transwell at an appropriate concentration (eg, 1×10^5 cells / well).

[0206] c. Add culture medium containing 10% FBS as a chemoattractant to the lower chamber.

[0207] d. Place the Transwell at 37°C and 5% CO 2 Incubate in an incubator for 24 hours.

[0208] e. Take out the Transwell, remove the unmigrated cells in the upper chamber, and wash with PBS.

[0209] f. Cells were fixed with methanol and subsequently stained with crystal violet.

[0210] g. Observe and count the number of cells migrating into the lower chamber under a microscope, and compare the cell migration ability before and after ChABC treatment.

[0211] 3) Scratch test

[0212] a. A549 and A549-CSPG cells were seeded in 6-well plates and cultured until fully confluent.

[0213] b. Use a sterile scraper to make even scratches in the cell monolayer.

[0214] c. The floating cells were washed with PBS and then treated with medium containing ChABC (0.1 U / mL ChABC).

[0215] d. Set up a control group (no ChABC treatment) and an experimental group (ChABC treatment).

[0216] e. Take photos at different time points (such as 0 hours, 12 hours, and 24 hours) to record the wound healing status.

[0217] f. Use image analysis software (such as ImageJ) to measure the wound width and calculate the healing rate.

[0218] 3) Data analysis

[0219] Use appropriate statistical software (such as SPSS or GraphPad Prism) to perform statistical analysis on the data of the Transwell assay and the scratch assay to compare the differences between the ChABC-treated group and the control group.

[0220] Through the above experimental steps, the inhibitory effect of ChABC in lung adenocarcinoma cell lines with significantly increased CSPG modification levels can be verified, providing an experimental basis for further studying the role of CSPG in tumor metastasis.

[0221] 2. Experimental results

[0222] 2.1 Modification, regulation and degradation of IκBα by sGAG

[0223] ChABC treatment significantly reduced sGAG levels in whole-cell lysates of wild-type XYLT1-overexpressing or TGF-β-pretreated LAD cells ( Figure 5 (A).

[0224] The level of sGAG modification on IκBα protein was significantly reduced, further confirming the key role of sGAG in regulating IκBα function ( Figure 5 (middle B).

[0225] 2.2 Protein interactions and activity regulation of IκBα and NF-κB signaling

[0226] ChABC treatment reduced the polyubiquitination and degradation of IκBα in XYLT1-overexpressing or TGF-β-activated LAD cells, while enhancing the interaction of IκBα with p65 and inhibiting its binding to IKKα / IKKβ ( Figure 5 The transcriptional activity of NF-κB and the expression of RelA / p65 target genes were significantly decreased after ChABC treatment ( Figure 5 Middle E).

[0227] 2.3 Functional inhibition experiment

[0228] ChABC treatment significantly inhibited the invasion, survival and anti-apoptosis ability of XYLT1-overexpressing or TGF-β-activated LAD cells ( Figure 5 FK), indicating that inhibition of NF-κB nuclear translocation can weaken XYLT1-related tumor invasion and anti-apoptosis ability, indicating that inhibition of XYLT1-mediated NF-κB signaling has potential therapeutic significance.

Claims

1. Application of sulfated glycosaminoglycan inhibitors in the preparation of anti-tumor drugs; The sulfated glycosaminoglycan inhibitors include XYLT1 inhibitors and / or chondroitinase ABC.

2. The use according to claim 1, characterized in that: The XYLT1 inhibitor includes at least one of the following: (a1) Substances that inhibit the activity of XYLT1 protein; (a2) substances that reduce the content of XYLT1 protein; (a3) a substance that silences the XYLT1 gene; (a4) a substance for knocking out the XYLT1 gene; (a5) A substance that inhibits the expression of the XYLT1 gene.

3. The use according to claim 2, characterized in that: The XYLT1 inhibitors include nucleic acid molecules, protein molecules, and small molecule drugs; Preferably, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide; Preferably, the protein molecule is a specific antibody of XYLT1; Preferably, the small molecule drug is a small molecule that binds to XYLT1.

4. The use according to claim 3, characterized in that: The sequences of the shRNA are shown in SEQ ID NOs: 2 and 3.

5. The use according to claim 1, characterized in that: The tumor is a tumor with high expression of XYLT1.

6. The use according to claim 1, characterized in that: The tumor is lung cancer.

7. The use according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

9. Application of XYLT1 gene as a drug target in the prevention and / or treatment of lung cancer; The use is for non-diagnostic, non-therapeutic purposes.

10. The use according to claim 9, characterized in that: The applications include screening of drug targets, screening of drugs, pharmacodynamic evaluation of drugs and safety evaluation of drugs.

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