Application of cinobufagin in preparation of product for treating temozolomide drug-resistant tumors

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of cinobufagin to preparation of a product for treating temozolomide drug-resistant tumors. The invention provides a novel treatment mode which is safe, exact in curative effect, low in price and simple in administration method and is used for treating temozolomide drug-resistant tumor patients, and particularly, application of cinobufagin to preparation of products for treating temozolomide drug-resistant tumor patients is discovered. Therefore, the compound has a wide application prospect in the aspects of treatment and repair of temozolomide-resistant tumors, glioma, glioblastoma and other diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the use of cinobucoid toxin in preparing a product for treating temozolomide-resistant tumors. Background Art

[0002] Surgical resection and postoperative radiotherapy combined with concurrent adjuvant chemotherapy with temozolomide (TMZ) is the standard treatment for newly diagnosed GBM. However, due to its infiltrative growth pattern and high invasiveness, GBM has a poor prognosis and is prone to recurrence, with a median survival of less than 24 months. Over the past 40 years, there have been few significant advances in GBM prevention, early detection, and treatment. Specifically, over the same period, the 5-year survival rate for GBM patients has only increased from 4% to 7%.

[0003] TMZ was approved by the US Food and Drug Administration (FDA) in 1999 for the treatment of refractory anaplastic astrocytomas in adults, and in 2005 for the treatment of newly diagnosed GBM. It was approved for marketing in China in 2008 and is currently a first-line chemotherapy drug for GBM. As a second-generation oral alkylating agent, TMZ easily crosses the blood-brain barrier and enters the central nervous system. It directly binds to tumor cell DNA through methylation, disrupting its structure and function, inhibiting DNA replication, and causing cell death.

[0004] However, TMZ resistance is a major challenge in GBM treatment and a major cause of chemotherapy failure in GBM patients. At least half of GBM patients develop resistance to TMZ during treatment. The mechanism of TMZ resistance is very complex, with O6-methylguanine-DNA-methyltransferase (MGMT) being the main factor contributing to TMZ resistance in GBM patients. In addition, DNA mismatch repair, base excision repair, abnormal cell signaling pathways, glioma stem cells, tumor microenvironment, epidermal growth factor receptors, microRNAs, etc. can also lead to TMZ resistance.

[0005] To date, the standard of care for GBM has not changed, and TMZ has been the standard drug for GBM. Due to GBM's drug resistance and lack of response to standard therapies, finding new treatment strategies for TMZ-resistant GBM patients is of great significance. Summary of the Invention

[0006] To overcome the defects and shortcomings of the prior art, the present invention provides a new use of cinobucoid venom. The inventors unexpectedly discovered during their research that cinobucoid venom can effectively treat patients with temozolomide-resistant tumors. Based on this discovery, they conducted in-depth research and completed the present invention.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides use of cinobufobagin in preparing a product for treating temozolomide-resistant tumors.

[0009] In another embodiment of the present invention, the temozolomide-resistant tumor includes glioma.

[0010] In another embodiment of the present invention, the temozolomide-resistant tumor comprises glioblastoma.

[0011] In another embodiment of the present invention, the concentration of the cinobucoid toxin in the preparation is 0.0625-0.25 μM.

[0012] Preferably, the concentration of cinobucoid tofu venom in the preparation is 0.25 μM.

[0013] In another embodiment of the present invention, the product is selected from one or more of medicines, kits, health products, and medical devices.

[0014] In another embodiment of the present invention, the medical device includes one or more combinations of medical tape, bandage, gauze, dressing, sponge, and medical suture.

[0015] In another embodiment of the present invention, in the medicine, cinobuccal venom is an active ingredient, and the medicine further comprises a pharmaceutically acceptable carrier or excipient.

[0016] Preferably, in the medicament, the divalent metal lactate is the only active ingredient.

[0017] In another embodiment of the present invention, the dosage form of the drug includes injection or external preparation.

[0018] In another embodiment of the present invention, the external preparation comprises a combination of one or more of ointments, creams, patches, sprays, solutions, and lotions.

[0019] In another embodiment of the present invention, the administration of the product includes one or more of the following: intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or topical application.

[0020] In addition, those skilled in the art will appreciate that the mode of use of the preparation, as well as the dosage and volume of administration, are related to the age, physical condition and disease of the subject, and can be determined by a clinician based on the circumstances.

[0021] The beneficial effects of the present invention compared with the prior art are:

[0022] The present invention provides a new treatment for patients with temozolomide-resistant tumors that is safe, effective, inexpensive, and simple to administer. Specifically, the present invention discovers the use of cinobucoid venom in the preparation of a product for treating patients with temozolomide-resistant tumors. Therefore, this product has broad application prospects in the treatment and repair of temozolomide-resistant tumors, gliomas, glioblastomas, and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. 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 any creative work.

[0024] Figure 1 The effects of different concentrations of drugs on cell viability and IC provided in the embodiment of the present invention 50 ;

[0025] Figure A shows the effects of different concentrations of TMZ on the survival rates of U251 and U215 / TMZ cells and the comparison of IC50;

[0026] Figure B shows the effects of different concentrations of Cino on the survival rates of U251 and U215 / TMZ cells and the comparison of IC50 (n=6);

[0027] Figure 2 The effect of Cino provided in the embodiment of the present invention on the clone formation ability of U251 / TMZ cells;

[0028] Figures A, C, and E show the plate clone growth of U251 / TMZ cells in the presence of TMZ alone, Cino alone, and TMZ and Cino combination.

[0029] Panels B, D, and F correspond to the numbers of cell clones in the control and experimental groups of Panels A, C, and E, respectively (n=3, **P<0.01, ****P<0.0001);

[0030] Figure 3 Effects of different concentrations of Cino on U251 / TMZ cell migration provided in the examples of the present invention;

[0031] Figure A is a comparison of scratch distances between the control group and the experimental group at 0h, 24h, and 48h;

[0032] Panel B is a bar graph of scratch migration rates in the control and experimental groups (n=9, ****P<0.0001);

[0033] Figure 4A volcano plot of expression differences between the comparison groups provided in the embodiment of the present invention;

[0034] The horizontal axis represents the fold-change (log(B / A)) value of gene expression between different groups of samples, and the vertical axis represents the statistical significance of the change in gene expression, pValue. The smaller the pValue, the larger the -log(pValue), and the more significant the difference. Each point in the figure represents a gene, with red representing upregulated genes (2989), green representing downregulated genes (3221), and black representing non-differential genes.

[0035] Figure 5 The differential gene expression cluster heat map provided by the embodiment of the present invention;

[0036] Each row in the figure represents a gene, and each column represents a sample. The color indicates the gene's expression level in the sample, with red indicating high expression levels and blue indicating low expression levels. The left side shows a dendrogram of gene clustering. The closer two gene branches are, the closer their expression levels are. The top shows a dendrogram of sample clustering, with the sample names at the bottom. The closer two sample branches are, the closer the expression patterns of all genes in the two samples are, meaning the more similar the gene expression trends are.

[0037] Figure 6 The GO annotation classification histogram of differentially expressed genes provided by the embodiment of the present invention;

[0038] The horizontal axis represents functional classification, with different colors representing different classifications. The light-colored values ​​on the histogram and vertical axis represent differentially expressed genes, while the dark-colored values ​​represent all genes. The right vertical axis represents the number of genes in that classification, and the left vertical axis represents the percentage of genes annotated to that function (differentially expressed genes / all genes).

[0039] Figure 7 A secondary classification summary diagram of the KEGG pathway enrichment results provided in an embodiment of the present invention;

[0040] The vertical axis represents the KEGG signaling pathway name; the horizontal axis represents the number of genes. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0044] TMZ is currently the first-line chemotherapy drug for GBM. After TMZ enters tumor cells, its breakdown products can lead to DNA methylation, which in turn interferes with cellular DNA replication, causes DNA damage, and inhibits tumor cell proliferation. Unfortunately, nearly half of GBM patients develop resistance to TMZ. The mechanisms of TMZ resistance are complex, and the presence of a robust DNA damage repair system and complex repair mechanisms in GBM cells is a key factor mediating TMZ resistance.

[0045] It is generally believed that overexpression of O6-methylguanine DNA methyltransferase (MGMT) is the main cause of TMZ resistance. In addition, the combined effects of mismatch repair, base excision repair, autophagy, and tumor stem cells also lead to TMZ resistance.

[0046] There are many intervention strategies targeting the above targets in the existing technology, such as intervention of MGMT, multidrug resistance protein, GBM stem cells, epidermal growth factor receptor (EGFR) gene and drug therapy, etc., in an attempt to increase chemotherapy sensitivity and reverse TMZ resistance.

[0047] The clinical challenge of treating TMZ-resistant GBM is that, even with multiple treatment options, it remains difficult to significantly extend patient survival. Researching new intervention strategies to address GBM resistance is crucial. Existing treatments for resistant GBM include increasing the TMZ dose, using MGMT inhibitors, and trying new chemotherapy drugs. However, these treatments can increase toxic side effects, treatment costs, and impact patient quality of life.

[0048] 1. General experimental materials: Induction of U251 / TMZ cell line

[0049] U251 cells (human glioma cell line) were revived, and logarithmically growing cells were added with 100 μM TMZ. The cells were cultured for 72 h as one cycle. After one cycle of culture, the cells were passaged according to the cell density and induced with 100 μM TMZ for 12 weeks. Cell clones were collected and CCK-8 resistance index was determined. Figure 1 As shown in A, the IC of the original strain and the resistant strain 50 They were 16.89μM and 166.2μM respectively, with RI>5, indicating that the drug-resistant strain U251 / TMZ was successfully constructed.

[0050] For cell-based experiments, Cino was used at a final drug concentration of 1 μM. The stock was prepared at 1 mM according to the protocol in Table 1, and the solvent was DMSO. The cells were added at a 1:1000 dilution in complete culture medium.

[0051] Table 1 Prepared drug stock

[0052]

[0053] 2. Effect of Cinobufacini on the Proliferation of Drug-Resistant U251 / TMZ Cells

[0054] 1. Test method

[0055] The drug-resistant U251 / TMZ cell strain obtained according to the "General Experimental Materials" was seeded into 96-well plates and arranged into 10 groups as shown in Table 2. After culturing in a CO2 incubator for 24 hours, the cell adhesion was observed under a microscope. The old culture medium was discarded, and 100 μL of pre-prepared drug-containing culture medium was added to each well at the concentration of cinona tofu venom base shown in Table 2. After drug addition, the cells were cultured in a CO2 incubator for 72 hours.

[0056] Table 2 CCK-8 experimental grouping table

[0057]

[0058]

[0059] CCK-8 experimental detection: Under sterile and light-proof conditions, CCK-8 detection reagent and complete culture medium were mixed in a volume ratio of 1:9 to prepare a working solution (final concentration 10%). Use 15mL light-proof centrifuge tubes as containers. After the aliquoting is completed, wrap them in aluminum foil and store in the dark. After 72 hours of drug intervention, use a multichannel pipette to slowly remove the culture medium in the well plate at a 30° tilt angle. Be careful to avoid touching the adherent cells at the bottom of the well during the operation. Accurately add 100μL of freshly prepared CCK-8 working solution to each well. During the addition process, keep the pipette tip 2mm above the liquid surface and drain the liquid at a constant speed. Transfer the 96-well cell culture plate to a 37°C, 5% CO2 saturated humidity incubator and incubate in the dark for 60 minutes. Use a multifunctional microplate reader to measure the absorbance, set the detection wavelength to 450nm, record the optical density value of each well, and calculate the cell inhibition rate.

[0060] 2. Experimental results

[0061] like Figure 1As shown in B, the CCK-8 assay results showed that the viability of U251 and U251 / TMZ cells treated with different concentrations of Cino was dose-dependent, and the IC values ​​of the original strain and the resistant strain were significantly different. 50 0.08027 μM and 0.08874 μM respectively.

[0062] 3. Cinobufacini venom inhibits the formation of drug-resistant U251 / TMZ cell clones

[0063] 1. Test method

[0064] U251 / TMZ cells obtained according to the "General Experimental Materials" protocol were seeded into 6-well plates and arranged into 12 groups as shown in Table 3. Each group consisted of three replicate wells, with 1000 cells seeded per well in 2 mL of complete medium. The seeded cells were cultured in a CO2 incubator for 24 hours. After 24 hours of incubation, cell attachment was observed microscopically. The old medium was discarded, and 2 mL of pre-prepared drug-containing medium was added to each well at the concentrations shown in Table 3. After drug addition, the cells were cultured in a CO2 incubator for 12 days.

[0065] Table 3 Grouping table of plate cloning experiment

[0066]

[0067]

[0068] Plate cloning assay: Observe cell morphology every 48 hours using an inverted microscope from the start of culture. After cloning is complete, discard the old culture medium, wash once with PBS, add 1 mL of 4% tissue fixative to each well, fix for 60 minutes, and wash once with PBS. Add 1 mL of 2.5% crystal violet to each well and stain the cells for 20 minutes. Wash several times with PBS, air dry, photograph, and count the number of colonies.

[0069] 2. Experimental results

[0070] like Figure 2 As shown in the figure, the results of the plate cloning experiment show that after TMZ administration, the clone formation ability of U251 / TMZ cells was not significantly different from that of the control group; compared with the control group, the Cino administration group, TMZ and Cino combined administration groups all significantly inhibited the clone formation of U251 / TMZ cells, and the results were significantly different, proving that Cino has a significant inhibitory effect on the clone formation of U251 / TMZ cells, and shows a good dose-dependency.

[0071] IV. Cinobufacini toxin base inhibits the lateral migration of drug-resistant U251 / TMZ cells

[0072] 1. Test method

[0073] Use a marker to draw a horizontal line and three vertical lines on the bottom of each well of a 6-well plate for subsequent scratch photography. The U251 / TMZ cells obtained according to the "General Experimental Materials" were inoculated into the 6-well plate. As shown in Table 4, 4 groups were set up. Each group had 3 replicate wells, and each well was inoculated with 1×10 6 cells, a total of 2mL complete medium. The inoculated cells were placed in a carbon dioxide incubator and cultured for 24 hours. After culturing in the carbon dioxide incubator for 24 hours, the cell adhesion state was observed under a microscope. A 200μL pipette tip was used to draw a parallel straight line at the diameter of the well plate perpendicular to the well plate. The old medium was aspirated and discarded. 1mL of complete medium was added and photographed under a fluorescence microscope. Three sites on each straight line were selected for photography and the initial scratch width was measured. The old medium was discarded and 2mL of pre-prepared drug-containing medium was added to each well. The concentrations are shown in Table 4. After drug addition, the cells were placed in a carbon dioxide incubator and cultured for 24 hours. After culturing for 24 hours with drug addition, the old medium was discarded, 1mL of PBS was washed once, 1mL of complete medium was added, and photographed under a fluorescence microscope. The sites were the same as the three sites photographed at 0 hours. The scratch widths at 24 hours and 48 hours were measured.

[0074] Table 4 Scratch test grouping table

[0075] Grouping Cino concentration (μM) 1 0 2 0.0625 3 0.125 4 0.25

[0076] 2. Experimental results

[0077] like Figure 3 As shown, Figure 3 The results of the scratch test show that after treatment with Cino, the lateral migration ability of U251 / TMZ cells was significantly reduced compared with the control group, proving that Cino inhibits the lateral migration ability of cells in a time- and dose-dependent manner.

[0078] 5. RNA-Seq Sequencing

[0079] 1. Test method

[0080] The U251 / TMZ cells obtained according to the “General Experimental Materials” were inoculated into 75T culture flasks. Two groups were set up: Control group and Cino-1μM group. Each group had 4 parallel controls. Each 75T culture flask was inoculated with 3×10 6 cells, 15 mL complete medium. Place the inoculated cells in a CO2 incubator and culture for 24 hours.

[0081] Drug-added culture: Observe the cell adhesion status under a microscope, discard the old culture medium, add 15 mL of pre-prepared drug-containing culture medium to each 75T culture flask, and culture the cells in a carbon dioxide incubator for 48 hours after drug addition.

[0082] Total RNA extraction from cells: Discard the old culture medium and wash the cells twice with 5 mL of ice-cold PBS. Discard the PBS and aspirate any remaining PBS with a pipette. Add 1 mL of TriZol lysis buffer to the bottom of the culture flask. Shake repeatedly on ice to allow the TriZol lysis buffer to fully contact the cells at the bottom of the flask. Shake for six minutes to fully lyse the cells at the bottom of the flask (complete lysis is defined as the liquid being non-viscous and fluid when shaken). Remove the cells using a cell scraper. After scraping, shake evenly until they are gathered at the bottom of the flask. Collect the cells with a pipette and transfer them to a 1.5 mL centrifuge tube. Quickly freeze the sample on dry ice. Once all samples are collected, transport them along with the dry ice. Testing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0083] 2. Experimental results

[0084] like Figure 4 As shown, after Cino-1 μM intervention in U251 / TMZ cells for 48 h, transcriptomic sequencing obtained a total of 6210 differentially expressed genes, including 2989 upregulated genes and 3221 downregulated genes.

[0085] 6. Cino target gene selection

[0086] 1. Test method

[0087] Figure 5 For the clustering heat map of differential gene expression, three genes upregulated by Cino (BMP3, FGF23, and MRO) and three genes downregulated by Cino (SPANXN4, IL20, and RNASE7) in the test samples were used as candidate validation indicators.

[0088] A search of the PubMed database revealed that BMP3 primarily exerts its tumor suppressor effects by regulating cell differentiation (e.g., inhibiting myofibroblast transformation) and the SMAD signaling pathway, and may have a protective function, particularly within the fibrosis-associated tumor microenvironment. FGF23 plays a dual role in tumor progression: on the one hand, it participates in tumor malignancy by directly promoting cell proliferation and invasion; on the other hand, it affects systemic metabolism by mediating hypophosphatemia. MRO genes play a complex role in tumors, participating in both maintaining genomic stability (tumor suppression) and driving tumor progression (oncogenicity) under specific conditions. SPANXN4 is a multifunctional X-linked gene that may promote tumor progression by regulating the immune microenvironment. IL20 has pleiotropic effects, supporting tumor growth through a pro-inflammatory microenvironment and promoting metastasis through mechanisms such as EMT. RNASE7 primarily participates in tumor microenvironment remodeling by regulating immune and inflammatory responses, and its downregulation may promote tumor immune escape.

[0089] 2. Experimental results

[0090] according to Figure 5The results showed that BMP3, FGF23, and MRO were lowly expressed in U251 / TMZ tissues and could play the role of tumor suppressor genes; SPANXN4, IL20, and RNASE7 were highly expressed in U251 / TMZ tissues and could play the role of tumor suppressor genes.

[0091] 7. GO analysis of Cino target genes

[0092] 1. Test method

[0093] GO analysis was performed on 6210 genes in U251 / TMZ cells. GO annotation cluster analysis mainly divided genes into three categories: biological process (BP), cellular component (CC), and molecular function (MF). BP classification involved cellular process, biological regulation, metabolic process, regulation of biological process, and response to stimulus, while CC classification involved cell, cell part, organelle, organelle part, and membrane, and MF classification involved binding, catalytic activity, molecular function regulator, signal transducer activity, and transporter activity.

[0094] 2. Experimental results

[0095] GO analysis results are as follows Figure 6 As shown, Figure 6 This is a histogram of differentially expressed genes (DOPs) by GO annotation. The horizontal axis represents functional categories, with different colors representing different categories. Lighter colors on the histogram and vertical axis represent differentially expressed genes, while darker colors represent all genes. The right vertical axis represents the number of genes in that category, and the left vertical axis represents the percentage of genes annotated with that function (DOPs / Total Genes).

[0096] 8. KEGG signaling pathway enrichment analysis

[0097] Experimental results

[0098] KEGG signaling pathway enrichment analysis structure Figure 7 As shown, Figure 7 This is a secondary classification summary of the KEGG pathway enrichment results. The threshold for KEGG pathway enrichment was set at P < 0.05. The results showed that the differentially expressed genes were primarily involved in six signaling pathways: signal transduction, cellular community-eukaryotes, cell growth and death, development and regeneration, endocrine system, and nervous system.

[0099] Based on the above experimental results, Cino can inhibit U251 / TMZ cell viability, cell cloning, and migration in a time- and dose-dependent manner. Therefore, it is expected to be used for the treatment and repair of temozolomide-resistant tumors, gliomas, glioblastomas, and other diseases. RNA-Seq sequencing, target gene selection, GO analysis, and KEGG signaling pathway enrichment analysis indicate that Cino's inhibition of U251 / TMZ cell proliferation may be achieved by reducing the expression of BMP3, FGF23, and MRO, and increasing the expression of SPANXN4, IL20, and RNASE7.

[0100] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. Use of cinobucoid venom in the preparation of a product for treating temozolomide-resistant tumors.

2. The use according to claim 1, characterized in that The temozolomide-resistant tumor includes glioma.

3. The use according to claim 1, characterized in that The temozolomide-resistant tumor includes glioblastoma.

4. The use according to claim 1, characterized in that The concentration of the cinobucoid toxin in the product is 0.0625-0.25 μM.

5. The use according to claim 1, characterized in that The product is selected from one or more of medicines, test kits, health products, and medical devices.

6. The use according to claim 5, characterized in that The medical device includes one or more combinations of medical adhesive tape, bandage, gauze, dressing, sponge, and medical suture.

7. The use according to claim 5, characterized in that In the medicine, cinobucoid toxin is an active ingredient, and the medicine further comprises a pharmaceutically acceptable carrier or excipient.

8. The use according to claim 5, characterized in that The dosage form of the drug includes injection or external preparation.

9. The use according to claim 8, characterized in that The external preparation includes one or more combinations of ointments, creams, patches, sprays, solutions, and lotions.

10. The use according to claim 1, characterized in that The administration of the product includes one or more of the following: intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or topical application.

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