Application of patinopectin-like in preparation of medicine for relieving and / or treating non-small cell lung cancer

Abstract

The invention relates to the field of biological medicines, and discloses application of patinopectin-like in preparation of a medicine for relieving and / or treating non-small cell lung cancer. Tests find that on the cellular level, the activity of non-small cell lung cancer cells can be reduced, cell migration can be inhibited, and the cell apoptosis process can be activated through treatment with the patinopectin-like; on the protein expression level, apoptosis-related protein can be remarkably activated, non-small cell lung cancer can be effectively treated by adjusting signal channels such as EGFR / PI3K / AKT, the possibility that tumor cells escape in treatment can be reduced, and therefore the clinical treatment efficiency is improved, and the application prospect is extremely good.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of pectenotoxins in the preparation of drugs for relieving and / or treating non-small cell lung cancer. Background Art

[0002] Lung cancer is one of the main cancer types leading to cancer-related deaths globally. According to histological differences, lung cancer is mainly divided into non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC), with NSCLC being the majority, accounting for approximately 85%. Since there are no obvious symptoms in the early stage of NSCLC, the vast majority of patients are not diagnosed until the advanced stage, at which time the cancer cells may have metastasized, making the treatment difficult. Currently, the main treatment methods for advanced NSCLC patients in clinical practice are mainly chemotherapy, targeted therapy, immunotherapy, etc. The main drugs for chemotherapy are cisplatin, paclitaxel, etc. The above-mentioned therapies often easily develop drug resistance and still have relatively large side effects, while other therapies have limitations in use or are relatively expensive. Therefore, developing ideal innovative therapeutic drugs and related methods is of great significance for the clinical treatment of NSCLC.

[0003] Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which includes an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain. EGFR is usually overexpressed in NSCLC and is the main cause of poor prognosis in NSCLC. At present, in addition to the most conventional chemotherapy, EGFR tyrosine kinase inhibitors such as small molecule tyrosine kinase-targeted preparations and EGFR antibody drugs are also widely used in the clinical treatment of NSCLC patients. Related studies have shown that during the development of NSCLC, the kinase activity of EGFR usually increases, and downstream pro-cancer signaling pathways such as extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K) / Akt / mTOR, and interleukin 6 (IL-6) / Janus kinase (JAK) / signal transducer and activator of transcription 3 (STAT3) signaling pathways are activated, affecting biological processes such as cell proliferation, survival, and migration to promote the growth and metastasis of cancer cells. In addition, more and more studies have shown that inducing apoptosis of lung cancer cells by regulating the EGFR / PI3K / AKT signaling pathway may be a promising method to improve the treatment of lung cancer. However, currently, the drug development in this regard is still relatively blank.

[0004] Based on this, there is an urgent need to seek a drug that can effectively treat NSCLC by regulating signaling pathways such as EGFR / PI3K / AKT to enrich the treatment strategies for NSCLC. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0006] An object of the first aspect of the present invention is to provide the use of Yessoenin or a pharmaceutically acceptable salt thereof in the preparation of a drug for relieving and / or treating lung cancer.

[0007] An object of the second aspect of the present invention is to provide an anti-lung cancer drug.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, there is provided the use of Yessoenin or a pharmaceutically acceptable salt thereof in the preparation of a drug for relieving and / or treating lung cancer.

[0009] The application according to the embodiment of the present invention has at least the following beneficial effects: Yessoenin was first isolated from the marine organism Patinopecten yessoensis and belongs to the Yessotoxin class of toxins. Due to the combination of complex cyclic structures in its structure, hYTXs have more possibilities in terms of activity. The present invention found through experiments that after treatment with Yessoenin (hYTXs), the cell viability and migration ability of non-small cell lung cancer cells A549 cells were significantly inhibited, and at the same time, the activation of the apoptosis process and the arrest of the cell cycle in the S phase were induced. At the protein expression level, the apoptosis-related protein Bcl-2 family proteins were significantly activated, and the expression of the cell cycle arrest proteins P53, P21, and P16 increased, suggesting that hYTXs can regulate at the protein expression level to play an anti-non-small cell lung cancer role.

[0010] Furthermore, the present invention found through Western blot and immunofluorescence experiments that hYTXs treatment can significantly inhibit the expression of EGFR, p-PI3K, p-AKT, and p-ERK, indicating that hYTXs can play an inhibitory role in the important molecular mechanism of the development of non-small cell lung cancer, which further proves that hYTXs can play a role in anti-non-small cell lung cancer.

[0011] In addition, the in vivo experimental results of tumor-bearing mice showed that hYTXs can significantly inhibit the development of non-small cell lung cancer, indicating that hYTXs can also play an anti-non-small cell lung cancer activity in vivo and have no effect on the liver, spleen, and kidneys, etc. The above results suggest that hYTXs can be used as a non-small cell lung cancer treatment drug, and at the same time, hYTXs can play an anti-cancer effect by regulating multiple signal pathways, which can effectively reduce the possibility of tumor cells escaping during treatment, thereby improving the clinical treatment efficiency.

[0012] In some embodiments of the present invention, the molecular formula of the Yessoenin is C56 H 84 O 21 S2, with a CAS registry number of 196309-94-1.

[0013] In some embodiments of the present invention, the pectenotoxin-like substance can be synthesized by chemical methods or isolated from the marine organism Patinopecten yessoensis.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable salts refer to salts of the free acids or bases of the compounds referred to herein, which are non-toxic, biocompatible or otherwise biologically suitable for administration to a subject. Generally see, S.M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those salts that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation or allergic response. The compounds described herein can have groups that are sufficiently acidic, groups that are sufficiently basic, both types of functional groups, or more than one of each type of functional group, and can thus react with many inorganic or organic bases as well as inorganic and organic acids to form pharmaceutically acceptable salts.

[0015] For compounds containing basic groups (such as amines) described herein, pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, by treatment of the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, etc.) or an organic acid (such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, oxaloacetic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosiduronic acids (such as glucuronic acid or galacturonic acid), α-hydroxy acids (such as mandelic acid, citric acid or tartaric acid), amino acids (such as aspartic acid or glutamic acid), aromatic acids (such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid or cinnamic acid), sulfonic acids (such as laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid or ethanesulfonic acid)), or any compatible mixture of acids (such as those given as examples herein), and any other acids and their mixtures regarded as equivalents or acceptable substitutes according to the ordinary skill level in the art.

[0016] For the compounds containing acidic groups (such as carboxylic acid groups) described herein, the base addition salts can be prepared by any suitable method available in the art, for example, by treating such compounds with a sufficient amount of the desired base (soda ash or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, ammonium, zinc or magnesium salts, or other metal salts; organic amino salts, such as alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts.

[0017] In some preferred embodiments of the present invention, the pharmaceutically acceptable salts are selected from any one of disodium salts, camphorsulfonates, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, oxalates, malonates, succinates, octanedioates, decanedioates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propanesulfonates, benzenesulfonates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates and mandelates.

[0018] In some embodiments of the present invention, the drug for relieving and / or treating lung cancer further comprises a pharmaceutically acceptable excipient.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, sweeteners and flavoring agents.

[0020] In some embodiments of the present invention, the excipient includes water.

[0021] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0022] In some embodiments of the present invention, the binder includes at least one of cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone.

[0023] In some embodiments of the present invention, the wetting agent includes glycerol.

[0024] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate and sodium bicarbonate.

[0025] In some embodiments of the present invention, the absorption promoter comprises a quaternary ammonium compound.

[0026] In some embodiments of the present invention, the surfactant comprises cetyl alcohol.

[0027] In some embodiments of the present invention, the adsorption carrier comprises at least one of kaolin and saponite.

[0028] In some embodiments of the present invention, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0029] In some embodiments of the present invention, the drug for relieving and / or treating lung cancer further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is generally recognized for this purpose and serves as an inactive ingredient of the medicament. A compilation of pharmaceutically acceptable carriers can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd Edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994).

[0030] In some embodiments of the present invention, when the pectenotoxin or its pharmaceutically acceptable salt described in the present invention is used for treating lung cancer, it can be used alone or in the form of a pharmaceutical composition containing the pectenotoxin or its pharmaceutically acceptable salt, that is, the drug further comprises a co-administered drug.

[0031] In some embodiments of the present invention, the drug for relieving and / or treating lung cancer has at least any one of the following effects: A) Reducing the survival rate of lung cancer cells; B) Promoting apoptosis of lung cancer cells; C) Reducing the migration ability of lung cancer cells; D) Inducing cell cycle arrest of lung cancer cells.

[0032] In some embodiments of the present invention, the lung cancer includes small cell carcinoma or non-small cell lung cancer. Preferably, it is non-small cell lung cancer.

[0033] In a second aspect of the present invention, there is provided an anti-lung cancer drug, the active ingredient of which comprises the pectenotoxin or its pharmaceutically acceptable salt.

[0034] In some embodiments of the present invention, the anti-lung cancer drug further comprises a pharmaceutically acceptable excipient.

[0035] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, sweeteners, and flavoring agents.

[0036] In some embodiments of the present invention, the excipient includes water.

[0037] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0038] In some embodiments of the present invention, the binder includes at least one of cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone.

[0039] In some embodiments of the present invention, the wetting agent includes glycerol.

[0040] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0041] In some embodiments of the present invention, the absorption promoter includes quaternary ammonium compounds.

[0042] In some embodiments of the present invention, the surfactant includes cetyl alcohol.

[0043] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and saponite.

[0044] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0045] In some embodiments of the present invention, the anti-lung cancer drug is administered through the gastrointestinal tract or / and non-gastrointestinal administration routes.

[0046] In some embodiments of the present invention, the non-gastrointestinal administration routes are selected from injection administration, respiratory administration, skin administration, mucosal administration, or cavity administration.

[0047] In some embodiments of the present invention, the pharmaceutical dosage forms for non-gastrointestinal administration are selected from injections, sprays, aerosols, patches, etc.

[0048] In some embodiments of the present invention, the pharmaceutical dosage forms for gastrointestinal administration are selected from tablets, capsules, powders, granules, pills, solutions, emulsions, or syrups, etc.

[0049] In some embodiments of the present invention, the drug exists in the form of oral preparations, injections, or topical administration preparations.

[0050] In some embodiments of the present invention, the oral preparations include tablets, capsules, pills, powders, granules, syrups or solutions; the injectables include injection solution dosage forms or freeze-dried powder for injection dosage forms; the topical administration preparations include creams, ointments, sprays, aerosols, gels, cataplasms or patches.

[0051] In some embodiments of the present invention, the carriers that can be selected in the preparation of oral preparations can be conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearates, etc. The freeze-dried powder for injection can be prepared by methods such as aseptic spray drying, low-temperature vacuum drying, freeze drying, etc. The later preparation processes and equipment of each preparation all belong to the conventional technologies in the pharmaceutical field, and the present invention does not make any limitations thereto. Description of the Drawings

[0052] The present invention will be further described below in conjunction with the drawings and examples, where: Figure 1 is the chemical structural formula of the human Yesso scallop toxins (hYTXs) of the present invention.

[0053] Figure 2 is the result of the effect of hYTXs and YTX treatment of the present invention on the survival rate of A549 cells, where * is P <0.05, ** p <0.01 vs Control group; ## is P <0.01 vs YTX group.

[0054] Figure 3 is the result of the effect of hYTXs treatment of the present invention on the colony formation of A549 cells, where A is the monoclonal formation diagram, and B is the statistical result of the monoclonal formation experiment.

[0055] Figure 4 is the result of the effect of hYTXs treatment of the present invention on the apoptosis of A549 cells, where A is the result of the apoptosis test of the flow cytometer, and B is the statistical analysis of the flow cytometer results.

[0056] Figure 5 is the effect of hYTXs treatment of the present invention on the migration ability of A549 cells, where A is the cell migration picture (scale bar is 50 µm), B is the statistical analysis of cell migration, C is the Transwell migration picture (scale bar is 100 µm), and D is the statistical analysis of the Transwell migration results.

[0057] Figure 6Results of the effect of hYTXs treatment of the present invention on the cell cycle progression of A549 cells, where A is the cell cycle distribution of flow cytometry, B is the statistical analysis of the flow cytometry analysis results, C is the Western blot detection results of the cycle-related proteins P53, P21, and P16, D is the statistical analysis of P53, E is the statistical analysis of P21, and F is the statistical analysis of P16.

[0058] Figure 7 Effect of hYTXs treatment of the present invention on the Tubulin cytoskeleton structure of A549 cells.

[0059] Figure 8 Volcano plot of differential proteins between cells treated with hYTXs of the present invention and blank group cells.

[0060] Figure 9 PPI network diagram of differential proteins between cells treated with hYTXs of the present invention and blank group cells.

[0061] Figure 10 KEGG enrichment analysis of corresponding DEPs in cells treated with hYTXs of the present invention.

[0062] Figure 11 GO enrichment analysis of corresponding DEPs in cells treated with hYTXs of the present invention.

[0063] Figure 12 Heat map of differential proteins related to the cell cycle in cells treated with hYTXs of the present invention.

[0064] Figure 13 Heat map of differential proteins related to the transforming growth factor-β signaling pathway in cells treated with hYTXs of the present invention.

[0065] Figure 14 Change diagram of the RMSD of the binding of hYTXs and EGFR protein within 100 ps of the present invention.

[0066] Figure 15 Change diagram of the hydrogen bonds formed between hYTXs and EGFR protein over time during the simulation process of the present invention.

[0067] Figure 16 Three-dimensional free energy topography diagram drawn based on RMSD and radius of gyration (Rg) of the present invention.

[0068] Figure 17 Two-dimensional free energy contour diagram based on RMSD and Rg of the present invention.

[0069] Figure 18This is the result of the effect of hYTXs treatment of the present invention on the expression of EGFR / PI3K / AKT signaling pathway and apoptosis-related proteins in A549 cells. Among them, A is the result of Western blot detection, B is the expression of EGFR protein, C is the expression of PI3K protein, D is the expression of AKT protein, E is the expression of ERK protein, F is the expression of Bax protein, and G is the expression of Bcl-2 protein.

[0070] Figure 19 This is the result of the effect of hYTXs of the present invention on EGFR protein internalization. Among them, A is the effect of hYTXs treatment on EGFR protein internalization in A549 cells, B is the statistical analysis of the internalization effect, and the scale bar is 10 μm.

[0071] Figure 20 This is the result of the effect of hYTXs of the present invention on non-small cell lung cancer in vivo. Among them, A is the liver coefficient of mice treated in different groups, B is the kidney coefficient of mice treated in different groups, C is the spleen coefficient of mice treated in different groups, D is the tumor weight of mice treated in different groups, E is the tumor volume of mice treated in different groups, and F is the tumor pictures of mice treated in different groups. Detailed implementation manners

[0072] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0073] The terms "preferably", "more preferably", etc. in the present invention refer to the embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0074] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0075] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the related listed items.

[0076] In the description of the present invention, the description of reference terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0077] In the embodiments of the present invention, the biochemical reagents used include the MTT cell proliferation and cytotoxicity assay kit (Nanjing Jiancheng Bioengineering Institute, China), the Cell Cycle and Apoptosis Analysis Kit (Yeasen, 40301ES60), and the Annexin V-FITC / PI Apoptosis Detection Kit (Yeasen, 40302ES60).

[0078] The antibodies used in the present invention include Bax (Cell Signalling Technology, #5023), Bcl-2 (Cell Signalling Technology, #4223), Gapdh (Cell Signalling Technology, #2118), p53 (Cell Signalling Technology, #2527), p21 (Cell Signalling Technology, #2947), p16 (Cell Signalling Technology, #18769), EGFR (Proteintech, 18986-1-AP), Phospho-PI3 Kinase p85 (Cell Signalling Technology, #17366), PI3 Kinase p85 (Cell Signalling Technology, #4257), Phospho-p44 / 42 MAPK (Erk1 / 2) (Cell Signalling Technology, #4370), p44 / 42 MAPK (Erk1 / 2) (Cell Signalling Technology, #4695), Phospho-Akt (Ser473) (Cell Signalling Technology, #4060), Akt (Cell Signalling Technology, #9272), β-Actin (Cell Signalling Technology, #4970), EEA1 (Proteintech, 68065-1-Ig), α-Tubulin (Cell Signalling Technology, #2144).

[0079] The male Balb / c nude mice (3 - 4 weeks old, weighing about 18 g) used were purchased from the Guangdong Provincial Center for Medical Laboratory Animals. Breeding conditions: Experimental Animal Center of Peking University Shenzhen Graduate School (Animal House SPF-level certificate SYXK (Guangdong) 2022 - 0172).

[0080] For those without specific conditions indicated in the examples, they were carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without the manufacturer indicated, they were all conventional products that could be obtained through commercial purchase.

[0081] Example 1: Study on the effect on the survival rate of non-small cell lung cancer cells This example explored the effect of Homoyessotoxin (hYTXs) on the survival rate of non-small cell lung cancer cells. The specific experiments are as follows.

[0082] 1. Experimental materials (1) Compounds: The hYTXs and Yessotoxin (YTX) used in this experiment were purchased from the National Marine Environmental Monitoring Center. The CAS number of hYTXs is 196309-94-1, and its chemical structure is as Figure 1 shown; the CAS number of Yessotoxin is 196309-94-1.

[0083] (2) Cell source: The lung cancer cell line A549 (human non-small cell lung cancer cells) used in this experiment was provided by the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences.

[0084] 2. Experimental methods (1) Cell culture and treatment: Obtain A549 cells and culture them in DMEM medium containing 10% fetal bovine serum and 1% double antibody, and incubate them in an incubator at 5% CO2 and 37°C. When the cells reach 70% - 80% confluence, passage the cells to maintain the cells in the logarithmic growth phase.

[0085] (2) MTT assay: The MTT assay was used to evaluate the effect of different doses of drugs on cell survival rate. The specific method is as follows: Take A549 cells cultured to the logarithmic growth phase, digest them with 0.25% trypsin, centrifuge at 4°C and 800 r / min for 3 min to collect the cells, and resuspend them with DMEM medium. Adjust the cells to 5×10 3 cells / well. At the same time, set up experimental groups, control groups, and blank groups. In the experimental groups, 5, 10, 20, 50, and 100 ng / mL of hYTXs were added for treatment respectively. In the control groups, 5, 10, and 20 ng / mL of YTX were added for treatment respectively. The blank group was not treated with any drugs. After 48 hours of drug treatment, add MTT solution and continue to incubate at 37°C for 2 - 3 hours. Dissolve the formazan crystals with DMSO and measure the absorbance at 570 nm to detect the cell survival rate. There are 5 replicates in each group.

[0086] The above experimental data were expressed as mean ± standard deviation (mean ± SD), and the statistical significance of the data was tested by one-way analysis of variance (ANOVA). P <0.05 was considered to be statistically significant.

[0087] 3. Experimental results The results of the MTT assay were as follows Figure 2 As shown, the viability of A549 cells was significantly reduced after being interfered with by hYTXs at different concentration levels, showing a concentration gradient trend. Meanwhile, the half-maximal inhibitory concentration (IC50) of hYTXs after 48-hour treatment was 30 ng / mL. Subsequently, a fixed concentration of 30 ng / mL or a lower range of 0 - 30 ng / mL was used to explore the effect of hYTXs. In addition, it is worth noting that when the treatment concentrations of both hYTXs and YTX were 20 ng / mL, hYTXs showed a relatively better inhibitory effect on A549 cells.

[0088] Example 2: Effects on the proliferation and apoptosis of non-small cell lung cancer cells In this example, the effects of hYTXs on the proliferation and apoptosis progression of non-small cell lung cancer cells were detected. Specifically, in this example, the effects of hYTXs on the proliferation and apoptosis progression of non-small cell lung cancer cells were explored through colony formation assay, cell apoptosis assay, scratch migration assay, and Transwell migration assay. The specific experimental procedures are as follows: 1. Experimental method The source of the compound yessotoxin used in this experiment and the culture process of A549 cells refer to Example 1 above. The specific methods of colony formation assay, cell apoptosis assay, scratch migration assay, and Transwell migration assay are as follows: (1) Colony formation assay: A549 cells were seeded into 6-well plates at a density of 500 cells / well, and then treated with different concentrations of hYTXs (0, 1, 3.75, 7.5 ng / mL). The cells were cultured for 14 days, and the culture medium was changed every 3 days. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min, stained with crystal violet solution, and then photographed for monoclonal colonies.

[0089] (2) Cell apoptosis assay: A549 cells were seeded into 6-well plates and then treated with different concentrations of hYTXs (0, 15, 30 ng / mL) for 48 h. The cells were collected into 1.5 mL centrifuge tubes and stained with Annexin V-FITC / PI for detecting cell apoptosis, and a flow cytometer was used to detect and reflect the cell apoptosis situation.

[0090] (3) Scratch migration assay: A549 cells were seeded into 6-well plates. When the cell density reached 80% confluence, a scratch was made using a 200 μL pipette tip, and then the cells were treated with different concentrations of hYTXs (0, 30 ng / mL) for 72 h. The scratch was photographed at 0, 12, 24, 48, and 72 h using an inverted microscope, and the scratch area was analyzed.

[0091] (4)Transwell migration assay: Tumor migration is an important process in systemic development. Exploring the migratory ability of cancer cells is of great significance for controlling tumor development. Based on this, this experiment investigated the effect of hYTXs treatment on the migratory ability of A549 cells. The specific experimental method is as follows: A549 cells were seeded into Transwell chambers at a density of 1×10 5 / well, and then transferred to a 24-well plate containing 600 μL / well DMEM (containing 20% FBS). Drugs were added at 0, 15, and 30 ng / mL hYTXs and cultured for 48 h. Subsequently, the chambers were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with crystal violet, washed and dried, and pictures of different fields were taken using an inverted microscope to count the number of migrated cells.

[0092] The above experimental data were expressed as mean ± standard deviation (mean ± SD). The statistical significance of the data was tested by one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.

[0093] 2. Experimental results The results of the colony formation assay are as Figure 3 shown. Figure A is the monoclonal formation diagram, and Figure B is the statistical result of the monoclonal formation assay. Through the monoclonal formation assay, it was found that hYTXs also showed a significant concentration gradient inhibitory effect. As the concentration of hYTXs increased, the growth and proliferation ability of A549 cells was inhibited, and the cells could not grow when the concentration reached 7.5 ng / mL.

[0094] The results of cell apoptosis are as Figure 4 shown. The detection results showed that compared with the control group, the A549 cells treated with hYTXs for 48 h showed a trend of migrating towards early / late apoptosis. At the same time, as the concentration increased (30 ng / mL), late apoptotic / necrotic cells were predominant, and the number of apoptotic cells reached 34.5%, indicating that hYTXs could inhibit cancer cell development by inducing apoptosis.

[0095] The results of the scratch wound healing assay and Transwell assay are as Figure 5As shown, where A is a picture of cell migration, B is the statistical analysis of cell migration, C is a picture of Transwell migration, and D is the statistical analysis of Transwell migration results, showing that the number of metastatic cells of A549 cells treated with hYTXs is significantly reduced compared with the control group after 48 h or even 72 h of migration. Further, the Transwell results also show that hYTXs can dose-dependently inhibit the migration of A549 cells. After treatment with 30 ng / mL hYTXs for 48 h, the number of migrating cells is reduced to 51.4%.

[0096] The above results show that hYTXs has excellent inhibitory effects on the proliferation of non-small cell lung cancer cells. At the same time, hYTXs can effectively inhibit the development of cancer cells by inducing apoptosis and inhibiting cell migration.

[0097] Example 3: Study on the effect of hYTXs on the cell cycle progression of A549 cells The cell cycle progression is an important physiological process of cell growth. Disorders in the cell cycle can lead to inhibited cell growth and even cell death. In this experiment, by detecting the effect of hYTXs on the cell cycle progression of A549 cells, the specific role of hYTXs in inhibiting the development of non-small cell lung cancer was further explored. The specific experimental procedures are as follows: 1. Experimental method The source of the compound Yesso scallopin used in this experiment and the culture process of A549 cells refer to Example 1 above. The specific methods for the cell cycle experiment, immunofluorescence experiment, and Western blot experiment are as follows: (1) Cell cycle experiment: A549 cells were seeded in 6-well plates and then treated with different concentrations of hYTXs (0, 15, 30 ng / mL) for 48 h. After collecting the cells in 1.5 mL centrifuge tubes, the cells were stained with PI, and the DNA content and sub-G1 peak were analyzed using a flow cytometer to explore the cell cycle progression.

[0098] (2) Western blot experiment: A549 cells were seeded in 6-well plates and then treated with different concentrations of hYTXs (0, 15, 30 ng / mL) for 48 h. The cells were lysed with cell lysis buffer, and the proteins were collected by centrifugation. SDS-PAGE electrophoresis was performed, and after transfer to the membrane, the target antibody was used for detection, specifically referring to the well-known methods in the art.

[0099] (3) Immunofluorescence experiment: A549 cells were seeded on 14-mm coverslips and then treated with hYTXs (treatment concentrations were 0 and 30 ng / mL) for 48 h. The cells were fixed with 4% paraformaldehyde and permeabilized. They were blocked with 5% BSA solution at room temperature for 1 h, stained with anti-α-Tubulin antibody, incubated with fluorescent secondary antibody and DAPI, and images were taken by confocal microscopy after mounting.

[0100] 2. Experimental results The results of flow cytometry for the cell cycle changes of A549 cells treated with different concentrations of hYTXs are as Figure 6 shown. The results showed that after treatment with hYTXs for 48 h, the cells were all aggregated in the S phase, and the cell DNA replication process was blocked, resulting in the arrest of the cell cycle process and the accumulation of DNA damage.

[0101] Furthermore, the key proteins in the cell cycle process were detected by Western blot (as shown in C-F Figure 6 ). The expression levels of cell cycle inhibitory proteins p53, p21, and p16 in A549 cells after treatment with hYTXs for 48 h were all significantly increased and were correlated with the concentration of hYTXs.

[0102] In addition, relevant studies have shown that inhibiting the polymerization of tubulin usually leads to cell cycle arrest. In this experiment, immunofluorescence was used to observe the changes in the intracellular tubulin network structure. The results are as Figure 7 shown, indicating that the microtubules of the cells treated with hYTXs were significantly depolymerized, the cytoskeleton structure was damaged, and multinuclear aggregation was difficult to separate into independent cells, while the microtubule structures of untreated A549 cells were filamentous and intact and clear. All these indicate that hYTXs can induce cell cycle arrest by activating the p53 signaling pathway and inhibiting tubulin polymerization, affecting the occurrence of normal cell processes, and thus promoting cell death.

[0103] The above results show that hYTXs can induce cell cycle arrest and accelerate the cell death process by promoting the expression of cell cycle inhibitory proteins p53, p21, and p16 and inhibiting tubulin polymerization.

[0104] Example 4: Protein level study In this example, the specific mechanism of action of hYTXs in inhibiting the development of A549 cells at the protein level was studied. The specific experimental procedure is as follows: 1. Experimental method The source of the compound yessotoxin used in this experiment and the process of culturing A549 cells refer to Example 1 above.

[0105] A549 cells were seeded in 6-well plates and then treated with hYTXs (treatment concentration: 30 ng / mL) for 48 h. After treatment, the cells were scraped off, washed 3 times with PBS, centrifuged at 1000×g for 10 min at 4°C, the precipitate was collected, snap-frozen in liquid nitrogen and stored at -80°C. The blank control group (Con group) was not treated with hYTXs.

[0106] The protein was quantified by the Bicinchoninic acid (BCA) method. The concentration was adjusted to be consistent. 5 mM DTT was added and incubated at 55°C for 30 min, then 10 mM iodoacetamide was added and incubated in the dark for 15 min. The protein was precipitated with acetone and treated at -20°C for 4 h. The protein was collected by centrifugation at 8000×g. The protein was digested with trypsin and desalted and freeze-dried by SPE. The peptide segments were separated by EASY-nLC 1200 and analyzed in the DIA mode by timsTOF Pro mass spectrometer. The data was processed by Spectronaut software, and functional pathway enrichment analysis was performed in combination with GO, KEGG and PPI networks.

[0107] 2. Experimental results The results of proteomic analysis are as Figure 8 shown, showing that a total of 842 differentially expressed proteins (DEPs) were detected in the hYTXs group compared with the Con group, among which 531 proteins were up-regulated and 311 proteins were down-regulated. The PPI network further demonstrated multiple major proteins and their interactions (specifically as Figure 9 shown), among which multiple proteins involved in regulating apoptosis, cell cycle and signal transduction such as CDKN1A, APP, SERPINE1, PLAU and TGFBR1, etc. revealed that hYTXs could regulate these signal pathways at the molecular level and play related roles.

[0108] The results of KEGG enrichment of differentially expressed proteins are as Figure 10 shown, indicating that hYTXs regulate the activation of PI3K-Akt signaling pathway, P53 signaling pathway and apoptosis pathway. GO analysis (as Figure 11 shown) further revealed pathways related to DNA replication, DNA damage and ATP binding.

[0109] In addition, compared with the Con group, the expression of protein markers in the cell cycle and transforming growth factor-β pathways (as Figures 12 to 13 shown) increased or decreased, including p53, p21, TGFBR2 family. These results further indicated that hYTXs could inhibit the growth of A549 cells by regulating apoptosis. In addition, the cell cycle and transforming growth factor signaling pathways also played certain roles.

[0110] The above results indicate that hYTXs can regulate these signaling pathways by affecting apoptosis, cell cycle, and signal transduction-related proteins at the cellular level, thereby exerting tumor-suppressive effects.

[0111] Example 5: Signaling Pathway Research In this example, the pathways by which hYTXs inhibit the proliferation and migration of A549 cells and promote apoptosis were studied. The EGFR / PI3K / Akt signaling pathway is one of the main pathways affecting the growth and reproduction of cancer cells. Relevant studies have shown that inhibiting the occurrence of this pathway can promote cancer cell apoptosis and inhibit its growth ability, thereby achieving the effect of inhibiting cancer development. In addition, epidermal growth factor (EGFR) mutations are important mutation sites in non-small cell lung cancer. In-depth research on the EGFR pathway is of great significance for inhibiting the occurrence of lung cancer. The above KEGG enrichment pathway results showed that hYTXs may have a role similar to that of EGFR kinase inhibitors and are related to the expression of the PI3K / Akt pathway. Based on this, in this example, experiments were carried out on the molecular interaction mechanism between hYTXs and EGFR. The specific experiments are as follows.

[0112] 1. Experimental Method The culture and drug administration of A549 cells in this experiment were carried out according to the method of Example 1.

[0113] To deeply explore the mechanism pathway by which hYTXs inhibit the development of non-small cell lung cancer, molecular dynamics simulation was used to explore the binding of hYTXs to EGFR protein at the molecular level. The molecular dynamics simulation was performed using GROMACS 2022.1 software, combined with the charmm36-jul2022 force field. The protein structure was derived from the PDB database, and the ligand parameters were generated by CGENFF. The system was dissolved using the SPC216 water model and neutralized by adding Na + and Cl - ions. After energy minimization and NVT and NPT equilibration, a 100 ns production simulation was carried out. The Leap-Frog algorithm was used during the simulation, and the analysis included RMSD, RMSF calculations, and the MM / PBSA method to evaluate the protein-ligand interaction energy.

[0114] During the Western blot experiment, the treatment concentrations of hYTXs were 0, 15, and 30 ng / mL. Cells were lysed with cell lysis buffer, and proteins were collected by centrifugation. SDS-PAGE was used for electrophoresis, and after transferring the membrane, the target antibody was used for detection, which was carried out specifically according to the well-known methods in the art; the immunofluorescence experiment was carried out according to the method of Example 3 above, and the relevant antibodies were EGFR and EEA1.

[0115] 2. Experimental Results The results of the molecular interaction simulation analysis of hYTXs and EGFR are as follows Figure 14 shown, indicating that during the 100 ns binding simulation of hYTXs and EGFR protein, a low and stable root mean square deviation (RMSD) change (0.2 nm - 0.5 nm) was maintained, suggesting that the binding structure of hYTXs and EGFR protein reached thermodynamic equilibrium, with small conformational changes in the protein-ligand complex and a tendency towards stable binding. Hydrogen bond binding gradually stabilized after 30 - 40 ns (as shown in Figure 15 ), also indicating that the complex structure tended to be stable.

[0116] Figure 16 and Figure 17 respectively show the three-dimensional free energy and two-dimensional plan view based on the change of RMSD over time. The lowest free energy region (blue) with an RMSD value of 0.2 nm - 0.5 nm shows that the binding conformation is the most stable at this time. In summary, the results of the molecular interaction simulation of hYTXs and EGFR protein show that they can bind stably, indicating that hYTXs may be able to affect the normal function of EGFR protein to a certain extent and interfere with the normal operation of downstream pathways.

[0117] As Figure 18 shows the results of the effect of hYTXs treatment on the expression of EGFR / PI3K / AKT signaling pathway and apoptosis-related proteins in A549 cells. Among them, A is the Western blot detection results of EGFR, PI3K, AKT, ERK, Bax, and Bcl-2 proteins, B is the expression of EGFR protein, C is the expression of PI3K protein, D is the expression of AKT protein, E is the expression of ERK protein, F is the expression of Bax protein, and G is the expression of Bcl-2 protein. The results show that after treatment with 15 and 30 ng / mL hYTXs for 48 h, the expression of EGFR in A549 cells was significantly inhibited, and the inhibitory effect increased with the increase of concentration. Since EGFR usually regulates multiple signaling pathways by activating downstream kinases such as PI3K, Akt, and erk, the effect of hYTXs on downstream signaling pathways was further evaluated. The results show that the phosphorylation process of downstream PI3K was also inhibited, and similar results were observed for the phosphorylation of Akt and erk, indicating that hYTXs treatment significantly inhibited the role of EGFR pathway and its downstream tyrosine kinases, blocked the transmission of non-small cell carcinogenic signals, and inhibited the process of cancer occurrence. In addition, the apoptotic family proteins Bcl-2 and Bax were also activated, which is also consistent with the previous research conclusion that inhibiting the PI3K / AKT pathway can prevent the further development of lung cancer.

[0118] Furthermore, to deeply explore how hYTXs treatment affects the expression of the EGFR pathway, the EGFR internalization ability was evaluated by immunofluorescence. EGFR internalization is a key step in its intracellular transmission, and the internalization process greatly affects the activation of EGFR and its downstream signaling pathways. The results of the effect of hYTXs on EGFR protein internalization are as Figure 19 shown. Compared with the control group, the co-localization of EGFR and the endosomal protein (EEA1) in A549 cells treated with hYTXs was significantly reduced, indicating that the process of EGFR entering early endosomes was inhibited, and the process of translocation to the nucleus and activation of the signaling pathway was disrupted.

[0119] The above results show that hYTXs can inhibit the development of lung cancer by blocking the EGFR / PI3K / AKT signaling pathway conduction and promoting the activation of the apoptosis-related protein family.

[0120] Example 6: In vivo experiment To evaluate the effect of hYTXs on tumor growth in vivo, this example conducted a detailed analysis of the tumor growth in different treatment groups (G1 control group, G2 hYTXs group). The specific experiment is as follows: 1. Experimental method Twelve male Balb / c nude mice aged 3 - 4 weeks and weighing about 18 g were purchased and randomly divided into 2 groups (G1 control group, G2 hYTXs group), with 6 mice in each group. They were treated with PBS and hYTXs (1 mg / kg) by in situ tumor injection for 10 days. Then the mice were sacrificed, the tumor tissues were isolated, photographed, weighed and fixed with paraformaldehyde for further immunohistochemical evaluation.

[0121] 2. Experimental results The results of the effect of hYTXs on non-small cell lung cancer in vivo are as Figure 20 shown. Among them, A is the liver coefficient of the mice treated in different groups, B is the kidney coefficient of the mice treated in different groups, C is the spleen coefficient of the mice treated in different groups, D is the tumor weight of the mice treated in different groups, E is the tumor volume of the mice treated in different groups, and F is the tumor picture of the mice treated in different groups. The results show that the tumor development in mice treated with hYTXs was significantly inhibited. In addition, there was no difference in the liver, spleen and kidney coefficients of the mice compared with those of the control group mice, indicating that hYTXs treatment had no effect on the normal life of the mice.

[0122] In summary, the present invention provides the application of haliotoxins in the preparation of drugs for relieving and / or treating non-small cell lung cancer. Through experiments, the present invention found that after treatment with haliotoxins (hYTXs), the cell viability and migration ability of non-small cell lung cancer cell line A549 were significantly inhibited. Meanwhile, the apoptosis process was activated, the cell cycle was arrested at the S phase, and at the protein expression level, the apoptosis-related protein Bcl-2 family protein was significantly activated, and the expression levels of cell cycle arrest proteins P53, P21, and P16 were increased, suggesting that hYTXs can regulate at the protein expression level to exert its anti-non-small cell lung cancer effect.

[0123] Furthermore, through Western blot and immunofluorescence experiments, the present invention found that hYTXs treatment can significantly inhibit the expression of EGFR, p-PI3K, p-AKT, and p-ERK, indicating that hYTXs can inhibit the important molecular mechanisms of non-small cell lung cancer development, which further proves that hYTXs can play a role in anti-non-small cell lung cancer.

[0124] In addition, the in vivo experimental results of tumor-bearing mice showed that hYTXs can significantly inhibit the development of non-small cell lung cancer, indicating that hYTXs can also exert anti-non-small cell lung cancer activity in vivo and has no effect on the liver, spleen, kidney, etc. The above results suggest that hYTXs can be used as a therapeutic drug for non-small cell lung cancer. At the same time, hYTXs can exert its anti-cancer effect by regulating multiple signaling pathways, which can effectively reduce the possibility of tumor cells escaping during treatment, thereby improving the clinical treatment efficiency.

[0125] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the above embodiments. Within the knowledge scope of those of ordinary skill in the art to which the present invention pertains, various changes can be made without departing from the purpose of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. Use of scallopacin or a pharmaceutically acceptable salt thereof in the preparation of a drug for relieving and / or treating lung cancer.

2. The application according to claim 1, wherein, The molecular formula of said pectenotoxin is C 56 H 84 O 21 S2, and the CAS registration number is 196309-94-1.

3. The application according to claim 1, wherein The pharmaceutically acceptable salt includes a salt of the free acid or base of scallopacin.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable salt is selected from any one of disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, oxalate, malonate, succinate, octanedioate, decanedioate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

5. The application according to claim 1, characterized in that The drug for relieving and / or treating lung cancer has at least any one of the following effects: A) Reducing the survival rate of lung cancer cells; B) Promoting apoptosis of lung cancer cells; C) Reducing the migration ability of lung cancer cells; D) Inducing cell cycle arrest of lung cancer cells.

6. The application according to any one of claims 1 to 5, characterized in that, The lung cancer includes small cell carcinoma or non-small cell lung cancer.

7. An anti-lung cancer drug, characterized in that, The active ingredient contains scallopacin or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 6.

8. The anti-lung cancer drug according to claim 7, wherein It also contains a pharmaceutically acceptable excipient.

9. The anti-lung cancer drug according to claim 8, wherein, The pharmaceutically acceptable excipient includes at least one of diluent, excipient, filler, binder, disintegrant, absorption promoter, surfactant, adsorption carrier, lubricant, sweetener and flavoring agent.

10. The anti-lung cancer drug according to claim 8, characterized in that, The anti-lung cancer drug is administered by gastrointestinal administration or / and non-gastrointestinal administration routes.

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