Application of cinobufagin in preparation of medicine for inhibiting bone metastasis of lung cancer

Abstract

The invention discloses an application of cinobufagin in preparation of a medicine for inhibiting bone metastasis of lung cancer. It is proved for the first time that 1) in-vitro cell experiments prove that cinobufagin can effectively inhibit proliferation and migration ability of lung cancer bone metastasis cells and promote cell apoptosis; 2) a mouse left ventricular injection bone metastasis model and a tibia injection model show that cinobufagin can significantly inhibit the development of lung cancer bone metastasis and osteolytic bone injury; (3) the specific regulation and control mechanism of cinobufagin for inhibiting bone metastasis of lung cancer is explored, and it is found through combined analysis of IP-MS proteomics and LiP-SMap proteomics that cinobufagin may target pyruvate carboxylase to play the function of cinobufagin; and 4) further verifying that cinobufagin can be combined with PC to inhibit bone metastasis of lung cancer through a drug affinity reactive target stability experiment and a heat transfer experiment. The research provides a new target spot and a treatment strategy for inhibiting bone metastasis of lung cancer.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of cinobucinaline in the preparation of a drug for inhibiting bone metastasis of lung cancer. Background Art

[0002] As one of the malignant tumors, lung cancer has the characteristics of rapid development and poor prognosis. According to the information on the number of new cancer cases released by the World Health Organization, the incidence of lung cancer accounts for 11.4% of the new cancer cases in the world, ranking second; it accounts for 22.3% of the new cancer cases in China, ranking first. Because the early symptoms of lung cancer are not obvious and the disease deteriorates rapidly, about 40% of patients have already developed distant metastasis when they seek medical treatment. The occurrence of lung cancer metastasis is the primary cause of lung cancer treatment failure and patient death. The prone sites of lung cancer metastasis include the brain, bones, liver, etc. Among them, the bone metastasis rate accounts for about 40%, which is the second most common site of lung cancer metastasis. Lung cancer is the third most common cancer with bone metastasis after breast cancer and prostate cancer. About 22-59% of patients with lung cancer bone metastasis will experience skeletal related events (SRE), including bone cancer pain, pathological fractures, spinal cord compression, hypercalcemia, etc., which seriously affect the patient's prognosis, quality of life and survival, and rapidly increase the patient's death.

[0003] At present, the treatments for lung cancer bone metastasis include surgery, chemoradiotherapy, bisphosphonate therapy and targeted therapy. Primary tumor resection (PTR) and metastatic tumor resection (MTR) are important treatments for patients with lung cancer bone metastasis. For patients with multiple bone metastases, surgical resection of the lesions is mainly to restore the patient's motor system function to improve the patient's quality of life. Chemotherapy drugs are mostly used to relieve bone cancer pain in the clinical treatment of lung cancer bone metastasis. However, most anti-tumor chemotherapy drugs have side effects, such as toxic reactions and bone marrow suppression. Therefore, anti-tumor chemotherapy drugs should be carefully selected in clinical practice. Commonly used chemotherapy drugs for lung cancer bone metastasis include paclitaxel and Kanglaite. Bisphosphonates are used to treat bone-related diseases because of their high affinity for binding to hydroxyapatite on the surface of bone tissue during bone resorption. However, one of the side effects of bisphosphonate treatment is jaw necrosis. It has been reported that more than 95% of bone metastasis patients develop jaw necrosis in high-dose bisphosphonate treatment. Commonly used targeted drugs for the treatment of lung cancer bone metastasis can be divided into two categories: primary lung cancer lesions and bone metastases. Targeted drugs are very likely to develop drug resistance after use. Therefore, there is still a lack of new and effective targeted drugs for the treatment of lung cancer bone metastasis in clinical practice.

[0004] Cinobufacini is a water-soluble preparation extracted and separated from the dried skin of Bufo bufo or Bufo gargarizans. It has anti-inflammatory, analgesic, myocardial contractility enhancement, anesthesia, edema elimination, and anti-tumor effects. Its main components include bufadienolide, indole alkaloids, sterols, proteins, amino acids, polysaccharides, etc. Among them, bufadienolide and indole alkaloids are considered to be the two most important anti-tumor active ingredients in Cinobufacini. Its anti-tumor effect has been verified in a variety of tumor cells, including liver cancer, gastric cancer, lung cancer, osteosarcoma cells, etc. Cinobufotalin is a diolide isolated from Cinobufacini. Cinobufotalin has been reported to inhibit the occurrence and development of a variety of tumor cells, including lung cancer cells, but its function and mechanism of inhibiting lung cancer bone metastasis have not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide the use of cinobucinaline in the preparation of drugs for inhibiting bone metastasis of lung cancer in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect is to provide the use of cinobucinaline and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates or prodrugs in the preparation of drugs for inhibiting bone metastasis of lung cancer.

[0008] Furthermore, the cinobucinaline and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates or prodrugs inhibit lung cancer bone metastasis by targeting pyruvate carboxylase PC.

[0009] Furthermore, the drug for inhibiting lung cancer bone metastasis also includes a pharmaceutically acceptable excipient.

[0010] The second aspect is to provide a pharmaceutical composition for use in the preparation of a drug for inhibiting bone metastasis of lung cancer, characterized in that it comprises the cinobufolin described in claim 1 and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates or prodrugs.

[0011] Furthermore, the pharmaceutical composition inhibits lung cancer bone metastasis by targeting pyruvate carboxylase PC.

[0012] Furthermore, the drug for inhibiting lung cancer bone metastasis also includes a pharmaceutically acceptable excipient.

[0013] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0014] The present invention proves for the first time that: 1) through in vitro cell experiments, it is proved that cinobucini can effectively inhibit the proliferation and migration ability of lung cancer bone metastasis cells and promote cell apoptosis; 2) the mouse left ventricular injection bone metastasis model and tibia injection model show that cinobucini can significantly inhibit the development of lung cancer bone metastasis and osteolytic bone damage; 3) the specific regulatory mechanism of cinobucini in inhibiting lung cancer bone metastasis is explored, and through the combination of IP-MS proteomics and LiP-SMap proteomics analysis, it is found that cinobucini may target pyruvate carboxylase (Pyruvate Carboxylase, PC) to exert its function; 4) Further verified by drug affinity reactive target stability experiment (DARTS) and thermal transfer experiment (CESTA), cinobucini can bind to PC to inhibit lung cancer bone metastasis; 5) PC plays an important role in the tricarboxylic acid cycle. After inhibiting the function of PC protein by cinobucini and gene level, it was found that the cellular oxygen consumption rate (OCR) was significantly reduced and the content of reactive oxygen (ROS) was significantly increased, suggesting that cinobucini inhibits the intracellular tricarboxylic acid cycle by targeting PC and induces cell death of lung cancer bone metastasis. The above studies provide new targets and therapeutic strategies for inhibiting lung cancer bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The IC of cinobucinaline against A549 and A549-L6 is shown 50 .

[0016] Figure 2 The results of CCK-8 proliferation experiment and plate clone formation experiment are shown; among them, (A) is the CCK-8 proliferation experiment verifying the level of A549L2 and A549-L6 cell proliferation inhibition by cinobucini; (B) is the plate clone formation experiment verifying the level of A549-L2 and A549-L6 cell proliferation inhibition by cinobucini; (C) is the statistical analysis of the cell clone formation level.

[0017] Figure 3 The results of Transwell migration experiment and flat plate scratch experiment are shown; among them, (A) is the Transwell migration experiment to verify that cinobucini inhibits the migration level of A549-L2 and A549-L6 cells; (B) is the statistical analysis of Transwell migration rate; (C) is the flat plate scratch experiment to verify that cinobucini inhibits the migration level of A549-L2 and A549-L6 cells; (D) is the statistical analysis of flat plate scratch migration rate.

[0018] Figure 4 The results of flow cytometry experiments are shown; (A) is flow cytometry verification that cinobufolin significantly promotes apoptosis of A549-L2 and A549-L6 cells; (B) is a statistical analysis of cell apoptosis levels.

[0019] Figure 5 The results of pharmacological evaluation of two mouse models are shown; among them, (A) is IVIS showing the bone metastasis area of ​​lung cancer in mice injected with left ventricle, detecting the development of lung cancer bone metastasis; (B) is IVIS showing the bone metastasis area of ​​lung cancer in mice in situ in the tibia and statistical analysis, detecting the development of lung cancer bone metastasis; (C) is Micro-CT scanning of mice and spine and leg bones, detecting the degree of bone destruction.

[0020] Figure 6 The potential target PCs of cinobucinatolide are shown; among them; (A) is the KEGG analysis of IP-MS differential genes; (B) is the volcano map of Lip-SMap differential genes; (C) is the target PC screened out by the intersection of IP MASS and Lip-SMap results.

[0021] Figure 7 The in vitro validation experimental results of the binding of cinobacillus thiocarbamide to PC are shown; wherein; (A) is the experimental result of IP experiment validating the in vitro binding of cinobacillus thiocarbamide to PC; (B) is the experimental result of DARTS experiment validating the in vitro binding of cinobacillus thiocarbamide to PC; (C) is the experimental result of CESTA experiment validating the in vitro binding of cinobacillus thiocarbamide to PC.

[0022] Figure 8 The results of in vitro proliferation experiments of PC knockdown stable cell lines A549-L2 and A549-L6 are shown; (A) is a WB experiment proving that PC was successfully knocked down in A549-L2 and A549-L6 cells; (B) shows that after knocking down PC, the 96h cell proliferation rate of A549-L2 and A549-L6 cells decreased; (C) shows that after knocking down PC, the 48h cell migration rate of A549-L2 and A549-L6 cells decreased; (D) is a statistical analysis of the migration rate of A549-L2 and A549-L6 cells after knocking down PC.

[0023] Fig. 9 The results show that knocking down PC can inhibit the occurrence of lung cancer bone metastasis in vivo; (A) WB experiment proves that PC was successfully knocked down in LLC1; (B) IVIS shows the area of ​​lung cancer bone metastasis and detects the development of lung cancer bone metastasis; (C) IVIS statistical analysis; (D) X-Ray scans the mouse tibia to detect the degree of bone destruction.

[0024] Fig.10 (A) shows the KEGG enrichment analysis of mass spectrometry differentially expressed genes; (B) shows the enrichment of TCA cycle proteins.

[0025] Fig.11(A) shows that after knocking down PC, the OCR levels of A549-L2 and A549-L6 cells decreased; (B) shows that after knocking down PC, the ROS levels of A549-L2 and A549-L6 cells increased.

[0026] Fig.12 (A) shows the successful knockdown of PC by WB experiment; (B) shows the IC of cinobucini to A549-L6 sh PC cells 50 Higher than A549-L6 sh NC cells; (C) shows that after knocking down PC, the proliferation level of A549-L6 cells inhibited by cinonabin is weakened; (D) shows that after knocking down PC, the migration level of A549-L6 cells inhibited by cinonabin is weakened, which is verified by the plate scratch experiment. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0028] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0029] The main experimental materials used in the following examples include:

[0030] Experimental cells: Human non-small cell lung cancer A549, lung cancer bone metastasis cell lines A549-L2 and A549-L6, epithelial cells 293T, and mouse lung cancer cells LLC1 were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0031] Experimental animals: BALB / c nude and C57 BCL6 mice were ordered by the school animal center and raised in the SPF-level experimental animal center. All operations involving mice during the experiment strictly followed the regulations of the school's Laboratory Animal Welfare Ethics Committee, and the animal ethics review approval number is ARXM2024083.

[0032] Example 1 Drug half-inhibitory concentration experiment

[0033] 1.1 Experimental Purpose

[0034] Primary non-small cell lung adenocarcinoma cells A549 and lung cancer bone metastasis cells A549-L6 were selected for half inhibitory concentration (IC 50 ) detection to explore the inhibitory efficiency of cinobucini on the growth of lung cancer bone metastasis cancer cells.

[0035] 1.2 Specific experimental methods include

[0036] (1) Cell plating

[0037] 1) Digest the cells, centrifuge and resuspend.

[0038] 2) Take 20 μL of the digested cell suspension and mix it evenly with 20 μL of trypan blue solution;

[0039] 3) Pipette 20 μL of the mixture, add it to a counting plate and count, calculate the required volume of the cell suspension based on the concentration of the obtained cell suspension, and ensure that every 100 μL of solution contains 3000 cells;

[0040] 4) Add DMEM medium containing serum to the centrifuge tube according to the total number of wells plated, ensuring that each well contains 100 μL;

[0041] 5) Invert the centrifuge tube upside down to mix the cell solution;

[0042] 6) Transfer the cell solution to a 96-well plate, with 100 μL of cell suspension per well;

[0043] 7) Add 100 μL of PBS buffer solution to each well around the plated cell wells;

[0044] 8) Place the cells in a 37°C cell incubator for culture.

[0045] (2) Prescription and Dosage

[0046] 1) Dissolve the drug in DMSO to prepare a 50 mM stock solution;

[0047] 2) Dilute 50 mM drug stock solution with DMEM medium containing serum to dilutions of different concentrations: DMSO, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, ensuring that each well of the 96-well plate contains 100 μL of drug solution, and each drug concentration treatment group has 3 replicate wells;

[0048] 3) Discard the original culture medium and add culture medium solutions containing different concentrations of drugs to the 96-well plate;

[0049] 4) Place the cells back into a 37°C cell incubator and culture for 48 hours.

[0050] (3) Cell IC50 determination

[0051] 1) Observe the degree of cell death after drug killing cells under an optical microscope;

[0052] 2) Prepare a sufficient amount of CCK-8 solution using serum-free DMEM medium at a ratio of CCK-8:DMEM = 1:10, ensuring that each well contains 100 μL of solution, and protect from light throughout the process;

[0053] 3) Discard the original drug-containing culture medium in the 96-well plate, add the prepared CCK-8 solution, and place it in a 37°C cell incubator for 1 hour incubation, protecting from light throughout the process;

[0054] 4) Take out the 96-well plate, put it into the microplate reader, and measure the absorbance value at 450 nM, keeping it away from light;

[0055] 5) The obtained absorbance values ​​were normalized and the IC value of the drug was fitted using Graphpad software. 50 The value is observed to see whether it is consistent with the cell death observed under an optical microscope. If it is consistent, the value is the IC50 value of the drug. Otherwise, the algorithm is changed until they are consistent.

[0056] 1.3 Experimental Results

[0057] like Figure 1 The results showed that cinobucini has a killing effect on both primary non-small cell lung adenocarcinoma cells A549 and lung cancer bone metastasis cells A549-L6. 50 The value is between 200-300nM.

[0058] Example 2

[0059] 2.1 Experimental Purpose

[0060] The inhibitory effect of cinobuczalin on the proliferation of lung cancer bone metastasis cell lines A549-L2 and A549-L6 was further verified by CCK-8 proliferation assay and plate clone formation assay.

[0061] 2.2 Specific experimental methods include

[0062] 2.2.1CCK-8 cell proliferation assay

[0063] (1) Cell plating

[0064] 1) Digest and count the cells to ensure that each well of a 96-well plate contains 2,000 cells in 100 μL of cell solution;

[0065] 2) Add 100 μL of PBS buffer solution to each well around the plated cell wells;

[0066] 3) Place the cells in a 37°C cell incubator for culture.

[0067] (2) CCK-8 assay

[0068] 1) After the cells adhere, prepare CCK-8 solution and keep it away from light;

[0069] 2) Discard the original culture medium in the 96-well plate, add the prepared CCK-8 solution, place it in a 37°C cell incubator for 1 hour, then take it out and measure the absorbance value at 450 nM using an ELISA reader;

[0070] 3) Measure once every 24 hours, and measure the absorbance values ​​at 0h, 24h, 48h, 72h, and 96h respectively;

[0071] 4) Use Graphpad software to fit the cell proliferation curve.

[0072] 2.2.2 Plate colony formation experiment

[0073] (1) Cell plating

[0074] 1) Digest and count the cells to ensure that each well of a 12-well plate contains 2,000 cells, and calculate the volume of cell suspension required for each well;

[0075] 2) Add 1 mL of DMEM medium containing serum to each well of a 12-well plate, add the cell suspension, and shake the 12-well plate to ensure uniform cell distribution;

[0076] 3) Place the cells in a 37°C cell incubator for culture.

[0077] (2) Cell proliferation

[0078] 1) After the cells adhere to the wall, discard the original culture medium;

[0079] 2) Add 500 μL PBS buffer solution to each well and wash once;

[0080] 3) Discard the PBS buffer solution and add 1 mL of drug-added culture medium of different concentrations to each well;

[0081] 4) Change the medium every two days until visible cell clones are formed.

[0082] (3) Plate staining

[0083] 1) Discard the original culture medium, add 500 μL PBS buffer solution to each well, let stand and wash for 3 minutes, and repeat the washing 3 times;

[0084] 2) Discard the PBS buffer solution, add 500 μL 4% paraformaldehyde to each well, and let it stand for 20 minutes;

[0085] 3) Discard the 4% paraformaldehyde solution, add 500 μL PBS buffer solution to each well, let stand and wash for 3 minutes, and repeat the washing 3 times;

[0086] 4) Discard the PBS buffer solution, add 500 μL of 0.9% crystal violet solution to each well, and let it stand for staining for 20 minutes;

[0087] 5) Discard the 0.9% crystal violet solution, add 500 μL PBS buffer solution to each well, let stand and wash for 3 minutes, and repeat the washing 3 times;

[0088] 6) After leaving it to dry, take photos and take statistics.

[0089] 2.3 Experimental Results

[0090] like Figure 2 The results showed that the cell proliferation rate and cell clone formation ability of the cinobucini treatment group were significantly reduced, and the ability to form cell clones gradually weakened with the increase in concentration, indicating that cinobucini can significantly inhibit the proliferation level of lung cancer bone metastasis cells in a concentration gradient-dependent manner.

[0091] Example 3

[0092] 3.1 Experimental Purpose

[0093] Transwell migration experiment verified that cinobucini inhibited the migration level of A549-L2 and A549-L6 cells; plate scratch experiment verified that after cinobucini treatment, the migration ability of tumor cells at the scratch point in the plate gradually decreased.

[0094] 3.2 Specific experimental methods include

[0095] 3.2.1 Transwell cell migration assay

[0096] (1) Cell digestion and plating

[0097] 1) Add 800 μL of DMEM medium containing serum to a 12-well plate;

[0098] 2) Slowly add the Transwell chamber into the well to avoid bubbles;

[0099] 3) Add 200 μL of serum-free DMEM medium into the Transwell chamber;

[0100] 4) After cell digestion, count the cells to ensure that each well of the 12-well plate contains 200,000 cells;

[0101] 5) Add the cell suspension to serum-free DMEM medium;

[0102] 6) Place the cells in a 37°C cell incubator and culture for 24 hours.

[0103] (2) Chamber dyeing

[0104] 1) Discard the original culture medium, add 500 μL PBS buffer solution to the upper and lower parts of each Transwell chamber, let it stand for 3 minutes, and repeat the washing 3 times;

[0105] 2) Discard the PBS buffer solution, add 500 μL of 4% paraformaldehyde solution to the upper and lower sides of each Transwell chamber and let it stand for 20 minutes;

[0106] 3) Discard the 4% paraformaldehyde solution, add 500 μL PBS solution to the upper and lower parts of each Transwell chamber, let stand and wash for 3 minutes, and repeat the washing 3 times;

[0107] 4) Discard the PBS buffer solution, add 0.9% crystal violet solution to the upper and lower parts of each Transwell chamber, and let it stand for staining for 20 minutes;

[0108] 5) Discard the 0.9% crystal violet solution, add 500 μL PBS buffer solution to the upper and lower parts of each Transwell chamber, let stand and wash for 3 minutes, and repeat the washing 3 times;

[0109] 6) Use a cotton swab to wipe off the cells on the upper layer of the Transwell chamber, leaving only the cells that have migrated through the chamber;

[0110] 7) After standing to dry, take photos of cell migration.

[0111] 3.2.2 Flat plate scratch test

[0112] (1) Cell digestion and plating

[0113] 1) Draw a horizontal line on the bottom of the 6-well plate with a black pen to facilitate later positioning;

[0114] 2) Digest and plate cells to ensure that the cell density in each well of a 6-well plate is 100%;

[0115] 3) Place the cells in a 37°C cell incubator for culture.

[0116] (2) Cell streaking

[0117] 1) After the cells have attached, use a 20 μL pipette tip to draw a vertical line in the well plate;

[0118] 2) Discard the original culture medium and add 1 mL of PBS buffer solution to each well for washing once;

[0119] 3) Discard the PBS buffer solution and add 1 mL of drug-added culture medium of different concentrations to each well.

[0120] (3) Scratch photography

[0121] 1) Photograph the scratch under a white light background of an optical microscope;

[0122] 2) Take photos every 24 hours to see the cell migration at the same location at 0 hours, 24 hours, 48 ​​hours, and 72 hours.

[0123] 3.3 Experimental Results

[0124] like Figure 3 The results of the Transwell migration experiment showed that after the addition of cinobucoid, the number of tumor cells passing through the Transwell chamber was significantly reduced; the results of the plate scratch experiment showed that after treatment with cinobucoid, the migration ability of tumor cells at the scratch point in the plate gradually decreased.

[0125] Example 4 Flow cytometry

[0126] 4.1 Specific experimental methods include

[0127] (1) Cell digestion and plating

[0128] 1) After cell digestion, spread the cells in a 6-well plate, ensuring that each well has 3×10 6 cells;

[0129] 2) Place the cells in a 37°C cell incubator and wait for them to adhere to the cell wall;

[0130] 3) The cells were grouped and treated differently and cultured for 48 hours.

[0131] (2) Cell apoptosis detection

[0132] 1) Digest the cells and collect the original culture medium, PBS buffer solution, and digested cell suspension into a 15 mL centrifuge tube;

[0133] 2) Centrifuge, 1000 rpm, 5 min;

[0134] 3) Discard the supernatant and resuspend the cells in PBS buffer solution for washing;

[0135] 4) Centrifuge, 1000 rpm, 5 min;

[0136] 5) Repeat the above washing operation once;

[0137] 6) Dilute the apoptosis buffer to 1× buffer solution with PBS buffer solution;

[0138] 7) Resuspend the cells with 1× Buffer solution;

[0139] 8) The cells in each treatment group were divided into four groups: Blank group, PI single staining group, APC single staining group, and double staining group;

[0140] 9) Stain the cells according to the above grouping, APC:PI=1:2, stain for 5 minutes, and protect from light throughout the process;

[0141] 10) Detect cell apoptosis levels using flow cytometry.

[0142] 4.2 Experimental Results

[0143] like Figure 4 The results showed that the apoptosis level of tumor cells in the cinobucini-treated group was significantly increased, indicating that cinobucini-treated group could significantly promote the apoptosis of lung cancer bone metastasis cells in a concentration gradient-dependent manner.

[0144] Example 5

[0145] Two mouse models were constructed for pharmacological evaluation:

[0146] The first is the left ventricle injection of lung cancer bone metastasis mouse model, which is a common model for studying the ability of tumor cells to metastasize to bone and the degree of bone tissue damage. First, the A549-L6-luc stable cell line was successfully constructed. Then, 1×10 6 A549-L6-luc cells were used to successfully construct a mouse model of lung cancer bone metastasis. After the tumor grew for one week, the mice were randomly divided into 2 groups, with at least 3 mice with lung cancer bone metastasis in each group. One group was the control group (Control), and the other group was the Cinobufotalin treatment group (Cinobufotalin). The treatment was continued for 21 days by daily intraperitoneal injection, and the development of lung cancer bone metastasis was recorded by IVIS shooting every week.

[0147] The results show that Figure 5 A), compared with the control group, the area of ​​lung cancer bone metastasis in mice treated with cinobuccal sulfamethoxazole was significantly reduced, indicating that cinobuccal sulfamethoxazole can significantly inhibit the development of lung cancer bone metastasis in mice. After the experiment, the mice were killed and the hind limb bones and spine of the mice were removed. Micro-CT scanning was used to observe the bone destruction ( Figure 5 C). We found that compared with the control group, the bone tissue in the Cinobufacini-treated group was more intact, and Cinobufacini-treated group could significantly inhibit the occurrence of osteolysis during lung cancer bone metastasis.

[0148] The second is a mouse model of lung cancer bone metastasis by orthotopic injection of tibia. First, LLC1-luc stable cell line was successfully constructed, and then 8×10 5 LLC1-luc cells were used to successfully construct a mouse model of lung cancer bone metastasis in the tibia. After the tumor grew for one week, the mice were divided into the control group and the Cinobufotalin group. The drugs were administered in situ in the tibia for 21 days, and the development of lung cancer bone metastasis was recorded by IVIS every week. Figure 5 B) The results were consistent with previous results, that is, the area of ​​bone metastasis of lung cancer in mice treated with cinobufolin was significantly reduced.

[0149] Example 6

[0150] IP-MS analysis and Lip-SMap were used to predict target proteins. Then, the targets obtained from the two sequencing results were intersected to screen out the potential target PC of cinobucoidin ( Figure 6 ).

[0151] Next, in vitro experimental verification was performed. The specific experimental methods included:

[0152] 6.1 Protein Immunoprecipitation (IP)

[0153] (1) Protein extraction

[0154] 1) Collect the cell pellet from a 10 cm culture dish and add protein lysis buffer, phosphatase inhibitors, and protease inhibitors;

[0155] 2) Ultrasonication in an ice water bath for 10 min;

[0156] 3) Turn the EP tube upside down and sonicate again in an ice water bath for 10 minutes;

[0157] 4) Centrifuge at 4°C, 12000 rpm, 15 min;

[0158] 5) Aspirate the supernatant protein solution and transfer it to a new EP tube.

[0159] (2) Protein binding

[0160] 1) Divide the total protein solution into 3 groups;

[0161] 2) One group added biotin, one group added 100nM biotin-Cinobufotalin, and one group added 300nM biotin-Cinobufotalin

[0162] 3) Aspirate 10% of the volume of solution from each of the two treatment groups and add 6× loading buffer to lyse the protein for use as input;

[0163] 4) Incubate on a rotating shaker at 4°C for 3 h.

[0164] (3) Incubate beads

[0165] 1) Washing beads: Add 700 μL of washing buffer to the beads, mix by inverting, centrifuge, centrifuge at 4°C, 1000 rcf, 1 min, discard the supernatant, and repeat washing three times;

[0166] 2) Divide the beads into 3 groups and add them into 3 treatment groups respectively;

[0167] 3) Incubate on a rotating shaker at 4°C for 1 hour;

[0168] (4) Sample collection

[0169] 1) Discard the supernatant and add Washing buffer to wash the beads three times after incubation. The washing process is the same as above.

[0170] 2) Add 1× loading buffer to lyse the protein;

[0171] 3) Place the cleaved protein in a metal bath and cook for 15 minutes;

[0172] 4) Verify the interaction between drug and protein by Western blot.

[0173] 6.2 Drug affinity responsive targetstability (DARTS)

[0174] (1) Protein cleavage

[0175] 1) Collect the cell pellet and add cell lysis buffer to resuspend the cell pellet;

[0176] 2) Ultrasonicate in an ice bath for 10 minutes, then turn the EP tube upside down and ultrasonicate in an ice bath for another 10 minutes;

[0177] 3) Centrifugation at 4°C, 12,000 rpm, 15 min;

[0178] 4) Pipette the supernatant into a new EP tube.

[0179] (2) Protein quantification

[0180] 1) Prepare the reaction solution;

[0181] 2) Add the reaction solution to a 96-well plate, 100 μL per well;

[0182] 3) Add standard and protein supernatant;

[0183] 4) Place in a 37°C incubator for 30 min;

[0184] 5) Measure the absorbance at 562 nm using an enzyme-labeled instrument;

[0185] 6) Calculate the concentration of the protein supernatant.

[0186] (3) Group processing

[0187] 1) The protein supernatant was equally divided into two groups, one group was a control group, and the other group was a drug-added group;

[0188] 2) Place on a rotary shaker and incubate at room temperature for 30 minutes.

[0189] (4) Protein degradation

[0190] 1) Calculate the concentration of protease based on the measured concentration of protein supernatant, with the ratios being 0, 1:900, and 1:600 ​​respectively;

[0191] 2) Add protease and incubate in a 37°C water bath for 5 min;

[0192] 3) Add 6× loading buffer to terminate degradation;

[0193] 4) Boil the sample in a metal bath for 15 minutes;

[0194] 5) Verify the binding of protein and drug by Western blot.

[0195] 6.3 Cellular Thermal Shift Assay (CESTA)

[0196] (1) Protein cleavage

[0197] 1) Collect the cell pellet, add PBS buffer solution, proteasome inhibitor, phosphatase inhibitor, and resuspend the cell pellet;

[0198] 2) Freeze and thaw twice in liquid nitrogen and then in a 37°C water bath.

[0199] (2) Group processing

[0200] 1) The protein supernatant was equally divided into two groups, one group was a control group, and the other group was a drug-added group;

[0201] 2) Place on a rotary shaker and incubate at 4°C for 3 h.

[0202] (3) High temperature degradation

[0203] 1) The two groups of samples were treated at different temperatures: 45°C, 48°C, 51°C, 54°C, and 57°C;

[0204] 2) Place each sample in a PCR instrument and let it stand for 3 minutes, then let it stand at room temperature for 3 minutes;

[0205] (4) Protein cleavage

[0206] 1) Centrifugation at 4°C, 12000 rpm, 5 min;

[0207] 2) Transfer the supernatant to a new EP tube;

[0208] 3) Add 6× loading buffer;

[0209] 4) Boil the sample in a metal bath for 15 minutes;

[0210] 5) Verify the binding of protein and drug by Western blot.

[0211] 6.4 Western Blot (WB)

[0212] (1) Glue making

[0213] 1) Clean the rubber sheet, clamp it on the rubber rack, add double distilled water, and check for leakage;

[0214] 2) Prepare the lower layer of separation gel according to the recipe and pour it into the gel plate;

[0215] 3) Quickly add 1 mL of anhydrous ethanol to seal the solution and let it stand for 1 hour to gel;

[0216] 4) Discard the anhydrous ethanol, prepare the upper separation gel according to the recipe, and pour it into the gel plate;

[0217] 5) Quickly insert the rinsed comb and let it sit for 1 hour to gel;

[0218] 6) Remove the comb and take the polyacrylamide gel off the gel stand for electrophoresis;

[0219] 7) Wrap the remaining protein gel with paper towels, wet it with dd H2O, and store it in a 4°C refrigerator.

[0220] (2)SDS-PAGE

[0221] 1) Prepare 1L 1× Running Buffer with dd H2O;

[0222] 2) Install the polyacrylamide gel in the running gel tank and pour in 1× Running Buffer;

[0223] 3) Add the protein samples to the loading wells in sequence;

[0224] 4) Add 1 μL of protein marker on both sides of the protein sample;

[0225] 5) Fill with 1× loading buffer;

[0226] 6) Add 1× Running Buffer to the sample;

[0227] 7) Connect the electrophoresis instrument, and make sure the positive and negative poles of the power supply are connected in the same way;

[0228] 8) Perform electrophoresis at a constant voltage of 80V. If bubbles are observed, it is normal.

[0229] 9) After the protein markers are separated, increase the voltage to 120V and continue running the gel until the bromophenol blue indicator electrophoreses to the bottom of the gel tank, then stop running the gel.

[0230] (3) Transfer

[0231] 1) Prepare filter paper and NC membrane, and prepare 1L 1×Transfer Buffer with dd H2O;

[0232] 2) Remove the protein gel from the gel tank and cut it;

[0233] 3) Add the following materials to the transfer clip in order: cotton, filter paper, protein glue, NC membrane, filter paper, cotton;

[0234] 4) Install the transfer clip in the transfer tank, put it in an ice box, and pour in 1× Transfer Buffer;

[0235] 5) Connect the electrophoresis instrument, and make sure the positive and negative poles of the power supply are connected correctly;

[0236] 6) Transfer the membrane in an ice water bath at a constant current of 200 mA. The transfer time should be slightly longer than the molecular weight of the target protein.

[0237] (4) Closed

[0238] 1) Prepare blocking solution: 7% skim milk, the solvent is PBS buffer solution;

[0239] 2) Remove the NC membrane from the transfer tank and transfer clip, add PBS buffer solution, place it on a shaker and wash for 5 minutes, and repeat the washing three times;

[0240] 3) Discard the PBS buffer solution, add blocking solution, place on a shaker, and block for 1 hour.

[0241] (5) Primary antibody incubation

[0242] 1) Prepare antibody diluent: 3% BSA, the solvent is sterile PBS solution, filter with a 0.45 μM filter and syringe;

[0243] 2) Dilute the primary antibody with antibody diluent at a ratio of 1:1000;

[0244] 3) Discard the blocking solution, add PBS buffer solution, wash on a shaker for 5 minutes, and repeat the washing three times;

[0245] 4) Cut the membrane according to the molecular weight of the target protein and the protein marker, and retain the NC membrane containing the target protein;

[0246] 5) Place the NC membrane in a dark box and add the corresponding diluted primary antibody;

[0247] 6) Place on a shaker at 4°C and incubate overnight.

[0248] (6) Secondary antibody incubation

[0249] 1) Dilute the secondary antibody with antibody diluent at a ratio of 1:10000;

[0250] 2) Recover the primary antibody, add PBST buffer solution, wash on a shaker for 5 minutes, and repeat the washing three times;

[0251] 3) Discard the PBST buffer solution and add the corresponding secondary antibody;

[0252] 4) Incubate on a shaker at 4°C for 1 h;

[0253] 5) Recover the secondary antibody, add PBST buffer solution, wash on a shaker for 5 minutes, and repeat the washing three times;

[0254] 6) Discard the PBST buffer solution and add PBS buffer solution;

[0255] 7) Scan the membrane for analysis using a membrane scanner.

[0256] The experimental results are as follows Figure 7 As shown in Figure 2, IP experiments proved that cinobucini can directly bind to PC, and the binding ability gradually increases with the increase of drug concentration. DARTS and CESTA also proved that under the action of protease and high temperature protein degradation, the PC content in the cinobucini treatment group was higher, proving that cinobucini can bind to PC and protect PC from degradation by protease and high temperature.

[0257] Example 7

[0258] Two different PC knockdown sequences were designed, and the corresponding knockdown stable cell lines were successfully constructed in two lung cancer bone metastasis cells, A549-L2 and A549-L6. Subsequently, we detected the proliferation and migration levels of the six cell lines. We measured the cell proliferation levels at 0h and 96h before and after PC knockdown by CCK-8 proliferation assay, and calculated the cell proliferation rate.

[0259] turn out( Figure 8 ), compared with the shNC group, the cell proliferation rate of the sh PC-1 group and the sh PC-2 group was significantly decreased in both lung cancer bone metastasis cells. Similarly, the cell scratch assay found that in both lung cancer bone metastasis cells, the cell migration rate of the two sh PC groups after 48 hours was significantly lower than that of the shNC group. The above results show that knocking down PC inhibits lung cancer bone metastasis in vitro.

[0260] In this example, two stable cell lines, LLC1-shNC-luc and LLC1-sh PC-luc, were successfully constructed. Subsequently, 8×10 5 LLC1-sh NC-luc and LLC1-sh PC-luc cells were used to successfully establish a mouse model of orthotopic lung cancer metastasis in the tibia, ensuring that each group had at least 3 mice with successful modeling. During the growth of the tumor, the development of the tumor was recorded by IVIS every week. The results showed that ( Fig. 9 ), compared with shNC, the area of ​​lung cancer bone metastasis in mice in the sh PC group was significantly reduced, indicating that lung cancer bone metastasis was significantly inhibited. After the 21-day experiment, the mice were killed and the tibia was dissected out, and the bone destruction was observed by X-Ray. The results showed that sh PC cells destroyed less bone in the tibia of mice than shNC cells. Based on the above results, it can be concluded that knocking down PC can inhibit the occurrence of lung cancer bone metastasis in vitro and in vivo.

[0261] Example 8

[0262] By performing mass spectrometry analysis on A549-L6 cells before and after treatment with cinobucini, and then further enriching the differentially expressed genes through KEGG pathway analysis, it was found that cinobucini may affect the TCA cycle of lung cancer bone metastasis cells ( Fig.10 ).

[0263] The metabolic levels of A549-L2 and A549-L6 cells were detected before and after the addition of cinobucini. The results showed that ( Fig.11 ), Cinobufacini can significantly reduce the OCR level of A549-L2 and A549-L6 cells in a concentration gradient-dependent manner and increase their ROS level. Therefore, it can be concluded that Cinobufacini can inhibit the TCA cycle of lung cancer bone metastasis cells.

[0264] Example 9

[0265] This example detects the inhibitory effect of cinobucini on PC-deficient lung cancer bone metastasis cells. First, we treated A549-L6 sh NC and A549-L6 sh PC cells with cinobucini, respectively. Fig.12 B) We found that cinobufolin kills the IC of A549-L6 sh PC cells 50 The value was higher than that of A549-L6 sh NC cells, indicating that after knocking down PC, the killing ability of cinobufolin on A549-L6 cells was weakened. Plate clone formation experiment ( Fig.12 C) and flat plate scratch test ( Fig.12 D) showed that cinobucini can significantly reduce the cell proliferation and migration rate of shNC cells, but has no obvious inhibitory effect on sh PC cells, indicating that the ability of cinobucini to inhibit the proliferation and migration of lung cancer bone metastasis is reduced after knocking down PC. The above results indicate that cinobucini may inhibit lung cancer bone metastasis by targeting PC (Pyruvate Carboxylase).

[0266] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of cinobucinatolide and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates or prodrugs in the preparation of drugs for inhibiting bone metastasis of lung cancer.

2. The use according to claim 1, characterized in that: The cinobucinaline and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates or prodrugs inhibit lung cancer bone metastasis by targeting pyruvate carboxylase PC.

3. The use according to claim 1, characterized in that: The drug for inhibiting lung cancer bone metastasis also includes pharmaceutically acceptable excipients.

4. Use of a pharmaceutical composition in the preparation of a drug for inhibiting lung cancer bone metastasis, characterized in that: The invention comprises the cinobufolin as claimed in claim 1 and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates or prodrugs.

5. The use according to claim 4, characterized in that: The pharmaceutical composition inhibits lung cancer bone metastasis by targeting pyruvate carboxylase PC.

6. The use according to claim 4, characterized in that: The drug for inhibiting lung cancer bone metastasis also includes pharmaceutically acceptable excipients.

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