Use of urocortol in the preparation of a medicament for treating thyroid cancer

Abstract

The application provides an application of urolithin in preparation of a drug for treating thyroid cancer. Through in-vivo and in-vitro experiments, it is proved that urolithin can effectively inhibit the proliferation of thyroid cancer cells, induce the apoptosis of the thyroid cancer cells, and significantly inhibit the tumor growth in an animal model, thereby providing a new, safe and effective drug candidate for treating thyroid cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Euonymus trioxide in the preparation of drugs for treating thyroid cancer. Background Technology

[0002] Thyroid cancer is the most common malignant tumor of the endocrine system, with papillary thyroid carcinoma (PTC) accounting for more than 80% of all thyroid cancers. Currently, treatment methods for thyroid cancer mainly include surgery, radioactive iodine therapy, and targeted therapy. Targeted therapy is an important approach for advanced, metastatic, or refractory thyroid cancer.

[0003] The RAS-RAF-MEK-ERK signaling pathway (MAPK / ERK pathway) plays a central driving role in the development and progression of thyroid cancer. Aberrant activation of this pathway can promote tumor cell proliferation, survival, invasion, and metastasis. Therefore, targeted inhibitors of this pathway, such as MEK inhibitors (e.g., selumetinib), have become a hot topic in thyroid cancer treatment research. However, existing targeted drugs still suffer from limited response rates, acquired resistance, and a certain degree of adverse reactions. Therefore, developing novel, highly effective, and low-toxicity drugs for thyroid cancer is of significant clinical importance.

[0004] Dulcitol, also known as sweet alcohol, is a naturally occurring hexose sugar alcohol widely found in plants of the Celastraceae family and corn cobs. Current reports on dulcitol primarily focus on its use as a chemical intermediate or sweetener; no reports have been found regarding its application in anti-tumor treatment, particularly in the treatment of thyroid cancer. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes the application of euonymus alcohol in the preparation of drugs for treating thyroid cancer.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides the use of euonymus alcohol in the preparation of drugs for treating thyroid cancer.

[0007] Furthermore, in this application, the thyroid cancer is papillary thyroid carcinoma.

[0008] Furthermore, the application of the euonymus alcohol is in the preparation of drugs that inhibit the phosphorylation levels of key kinases MEK and ERK in the MAPK / ERK signaling pathway.

[0009] Furthermore, the application of the euonymus alcohol in the preparation of drugs that downregulate the expression of cyclin D1.

[0010] Furthermore, in the aforementioned applications, the use of euonymus alcohol in the preparation of drugs that upregulate the expression of the apoptosis-executing protein Cleaved Caspase-3 and activate mitochondrial apoptosis.

[0011] Furthermore, the concentration range of the euonymus alcohol is 10 μM-100 μM (micromolar), preferably 50 μM-100 μM, and more preferably 100 μM.

[0012] In a second aspect, the present invention provides a pharmaceutical composition for treating thyroid cancer, the pharmaceutical composition containing euonymus alcohol.

[0013] Furthermore, the thyroid cancer is papillary thyroid carcinoma.

[0014] Furthermore, the concentration range of the euonymus alcohol is 10μM-100μM, preferably 50μM-100μM, and more preferably 100μM.

[0015] Furthermore, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0016] Furthermore, the pharmaceutical composition is formulated into any pharmaceutically acceptable dosage form.

[0017] The pharmaceutically acceptable dosage form described in this invention is selected from any one of tablets, capsules, solutions, granules, powders, ointments, pills, suspensions, suppositories, liniments, emulsions, topical applications, patches, or sprays.

[0018] Compared with the prior art, the present invention has the following advantages: This invention demonstrates through in vitro and in vivo experiments that Euonymus trioxide can effectively inhibit the proliferation of thyroid cancer cells, induce their apoptosis, and significantly inhibit tumor growth in animal models, thereby providing a new, safe, and effective drug candidate for the treatment of thyroid cancer. Attached Figure Description

[0019] Figure 1 A bar chart showing cell viability; Figure 2 A bar chart showing the clone formation rate; Figure 3 The graphs are flow cytometry plots for apoptosis detection, where a represents the negative control group; b represents the positive control group; c represents the low-dose ZW2857 group; d represents the medium-dose ZW2857 group; and e represents the high-dose ZW2857 group. Figure 4 A bar chart showing the total apoptosis rate of cells; Figure 5The levels of proteins associated with different signaling pathways are shown below, where a represents the relative protein level of p-ERK / ERK; b represents the relative protein level of p-MEK / MEK; c represents the relative protein level of Cyclin D1 / GAPDH; and d represents the relative protein level of Cleaved Caspase-3 / GAPDH. Figure 6 This is a curve showing the change in mouse body weight; Figure 7 This is a tumor growth curve; Figure 8 This is a curve showing the relative tumor volume. Figure 9 This is a curve showing the tumor growth inhibition rate. Figure 10 A bar chart comparing tumor weight in each group; Figure 11 A bar chart comparing the tumor inhibition rates of each group. Detailed Implementation

[0020] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0021] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] In this document, when values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values ​​falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0023] In this article, the terms "multiple" or "more than" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0024] In this document, the terms "preferred" and "more preferred" are used only to describe implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this invention.

[0025] In this document, terms such as "further" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0026] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0027] In this document, the term "about" means a specified value of + / - 10%, preferably + / - 5%, and more preferably + / - 1%.

[0028] In this article, the terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0030] This invention is verified through the following technical solutions: (1) In vitro cell experiments: Cell proliferation inhibition assay: The human papillary thyroid carcinoma line TPC-1 was used as a model. Different concentrations of euonymus alcohol (e.g., 10 μM, 50 μM, 100 μM, abbreviated as ZW2857, hereinafter the same) were applied to TPC-1 cells. Cell viability was detected by the CCK-8 assay, and cell proliferation capacity was detected by the colony formation assay. A negative control group and a positive control drug (selutinib) group were also included in the experiment.

[0031] Apoptosis detection: The apoptosis rate of TPC-1 cells treated with Euonymus tincture was detected by flow cytometry using Annexin V-FITC / PI double staining.

[0032] Mechanism of action study: Western blotting was used to detect the effects of euonymus alcohol treatment on the expression levels of key proteins (p-MEK, p-ERK), cell cycle-related protein (Cyclin D1), and apoptosis-related protein (Cleaved Caspase-3) in TPC-1 cells.

[0033] In vivo animal experiments: Animal model establishment: A subcutaneous xenograft tumor model of human papillary thyroid carcinoma TPC-1 cells was constructed in SCID mice.

[0034] Dosage regimen: Tumor-forming mice were randomly divided into a model control group, a positive control group (selactinib), and low-, medium-, and high-dose groups of euonymustine. The mice were administered the medication by gavage for a specific period (e.g., 13 days).

[0035] Pharmacodynamic evaluation: Mouse body weight and tumor volume were measured periodically to calculate relative tumor volume (RTV) and tumor growth inhibition rate (TGI). After the experiment, the tumors were removed, weighed, and the tumor inhibition rate was calculated.

[0036] Preliminary safety assessment: The safety of eugenol was preliminarily assessed by observing the general behavior and weight changes of mice.

[0037] The present invention will be described in detail below with reference to embodiments. Unless otherwise specified, all percentages mentioned in the present invention are mass percentages.

[0038] Example 1: In vitro cell experiments 1. Experimental Materials and Reagents (1) Cell line: TPC-1 human thyroid cancer cells (purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.); (2) Test sample: Euonymus alcohol (purchased from Shandong Futian Pharmaceutical Co., Ltd.); (3) Positive control: Selumetinib (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.); (4) Main reagents: Table 1. Major Reagent Brands and Product Numbers

[0039] (4) Main equipment: Table 2. Major Equipment Brands and Part Numbers

[0040] 2. Test Methods 2.1 Cell resuscitation and culture Recovery steps: (1) After removing the frozen cells from liquid nitrogen, quickly place them in 37°C warm water and shake to thaw for about 1 minute; (2) Centrifuge at 1000 rpm for 1 min, discard the frozen solution, and resuspend the cells in 1 mL of fresh complete culture medium; (3) Inoculate into a new culture bottle, add enough complete culture medium, and place in an incubator for culture.

[0041] Transplantation: (1) Use monolayer adherent culture, add an appropriate amount of complete culture medium, and place it in an incubator with a temperature of 37℃, 5% CO2 and humidity of 100%RH. Replace half of the culture medium every other day. (2) When the cells have reached a confluence of more than 90% in the culture flask, begin the passage experiment. Wash the cells with PBS, add 1 mL of 0.25% trypsin to soak the cells, discard the trypsin, and then place them in a 37°C incubator for 3 minutes for digestion; (3) Under a microscope, when the intercellular spaces increase, become rounded, and some cells begin to detach, remove the culture flask immediately. Gently tap the side of the culture flask with the side of your palm to promote complete cell detachment, and quickly add 2-3 mL of preheated complete culture medium. Discard the supernatant, gently blow on the flask wall to form a uniform single-cell suspension, transfer to a centrifuge tube, and centrifuge at 1000 rpm for 5 min; (4) Discard the supernatant, resuspend the cells in fresh complete culture medium, and adjust the cell concentration to 1×10⁻⁶. 7 per mL.

[0042] 2.2 Drug Preparation The ZW2857 stock solution was serially diluted with complete culture medium, with concentration gradients of 10 μM, 50 μM, and 100 μM, and three replicates for each concentration. The positive control group consisted of selumetinib at a concentration of 1 μM.

[0043] 2.3 Experimental Grouping Table 3 Experimental Groups

[0044] 2.4 Phenotypic Experiment 2.4.1 CCK-8 cell proliferation experiment Follow the instructions for the CCK-8 assay kit. Add 10 μL of CCK-8 solution to each well of each group of cells and incubate at 37°C for 1 hour. Use wells containing the same amount of cell culture medium and CCK-8 solution but without cells as blank controls. Measure the absorbance (OD) at 450 nm for each well using a microplate reader. Repeat each assay three times and calculate the mean for each group.

[0045] 2.4.2 Cloning Experiment Take TPC-1 cells in the logarithmic growth phase, digest them with trypsin and resuspend them into a single-cell suspension, count the cells, and seed 500-1000 cells per well into a 6-well plate. Gently shake the 6-well plate in a cross shape to distribute the cells evenly, and incubate at 37°C in a 5% CO2 incubator.

[0046] After cell seeding and 24 hours of adherent growth, the old culture medium was carefully discarded, and 2 mL of freshly prepared drug-containing culture medium of different concentrations was slowly added along the well wall. The negative control group was replaced with an equal volume of complete culture medium. Cells were cultured continuously for 10 days, and the cloning status was observed on day 4 and day 7, with the culture medium being replaced with fresh medium at each day.

[0047] When the clones in the negative control group are visible to the naked eye and of appropriate size, stop the culture, carefully discard the culture medium, gently wash the cells twice with pre-warmed PBS, add 4% paraformaldehyde (1 mL / well), fix at room temperature for 30 min, discard the fixative, wash once with PBS, add 0.1% crystal violet solution (1 mL / well), stain at room temperature for 30 min, carefully discard the staining solution, slowly rinse the culture plate with running water until the background is colorless and transparent, and air dry.

[0048] A cell cluster with more than 50 cells is defined as one clone. Clones visible to the naked eye are counted, and the clone formation rate is calculated. The calculation formula is: .

[0049] 2.4.3 Apoptosis Detection Prepare Annexin-V-FITC / PI staining solution according to the instructions of the Annexin-V-FITC apoptosis detection kit. Resuspend 1×10⁻⁶ ppm of the staining solution in every 100 μL of staining solution. 6 Collect cells, vortex to mix, and incubate at room temperature for 15 min. Then add 1 mL of HEPES buffer and vortex to mix. Excite FITC and PI fluorescence with 525 nm and 620 nm bandpass filters at 488 nm wavelength to detect cell apoptosis.

[0050] 2.5 Mechanism Study (WB) 2.5.1 Protein Extraction Slowly add PBS solution along the mouth of the cell culture flask to wash 2-3 times. After aspirating the residual PBS solution, add an appropriate amount of lysis buffer to ensure complete contact between the lysis buffer and the cells. Collect the sample with a cell scraper and place it in a pre-cooled grinder for lysis or an ultrasonic cell disruptor for lysis in an ice bath. Centrifuge at 4°C, 12,000 rpm for 10 min and collect the supernatant.

[0051] 2.5.2 Protein concentration determination (BCA method) Prepare the BCA working solution according to the instructions of the BCA protein concentration assay kit. Add the serially diluted samples to microplates, then add the BCA working solution and mix well. Using a blank control as a reference, measure the absorbance of each sample at 562 nm, and subtract the average absorbance of the blank control at 562 nm from the absorbance of each sample. Add the calculated loading buffer and purified water to the extracted protein to ensure uniform protein concentration for each sample. Place the plate in a water bath and denature at 100°C for 10 min. After cooling on ice, store at -20°C.

[0052] 2.5.3 SDS-PAGE electrophoresis 1) Preparation of SDS-PAGE gel: Clean the glass plate used for filling the adhesive, let it dry and fix it in place, and determine the mark line for the liquid level of the separating adhesive. Prepare 8 mL of 10% separating gel and quickly pour it into the gel glass tank until the liquid level reaches the mark line. Immediately cover the adhesive surface with deionized water and let it stand at room temperature for about 40 minutes until the separating gel solidifies. Prepare 4 mL of 5% concentrated gel; Discard the deionized water covering solution and use a needle to remove any remaining liquid; Reposition the gel plate vertically, gently add 5% concentrated gel solution, insert the sample comb, and gel at room temperature for about 40 minutes. Gently remove the comb and fix the glass clamp with the concave side facing inward onto the electrophoresis tank.

[0053] Table 4 Formulation of SDS-PAGE gel

[0054] 2) Electrophoresis The extracted total protein sample was taken out of the -20℃ freezer and immediately placed in ice to thaw. Add the Maker and sample to the gel in the electrophoresis tank according to the experimental requirements, and adjust the voltage to 100V to make the sample pass through the stacking gel and the separating gel; Electrophoresis moves the dye to the appropriate position on the separating gel, and then the electrophoresis process ends. After electrophoresis, the protein bands separated on the gel were transferred to a PVDF membrane using a wet transfer method.

[0055] 3) Transfer membrane The PVDF membrane is first soaked in 100% methanol for 2-3 minutes, then rinsed with water and electroporation solution for 2 minutes each time for 2 minutes each time, and then placed in the electroporation solution for later use. Cut 6 layers of filter paper to the same size as the glue, soak them in transfer buffer, and set aside for later use; Remove the electrophoresis plate and lay it flat (with the concave glass plate facing up). Carefully remove the top glass plate, cut off any excess gel, remove the sample gel, and rinse once with electrophoresis solution. Place the sample gel and membrane into the transfer clamp marked with positive and negative electrodes (starting from the cathode side, in the following order: sponge pad → 3 layers of filter paper → sample gel → PVDF membrane → 3 layers of filter paper → sponge pad), fasten the transfer clamp, and place it into the transfer electrophoresis tank containing transfer buffer. Connect the power supply and, under constant voltage conditions, perform film transfer at 100V for 1 hour.

[0056] 4) Antibody incubation Blocking: Remove the membrane, wash 3 times with 1×TBST for 1 min each time, place it in blocking solution, and block on a shaker at room temperature for 1 h. Wash 3 times with 1×TBST for 10 min each time. Primary antibody incubation: Dilute the primary antibody with antibody dilution buffer, add the membrane to the corresponding primary antibody working solution, and incubate overnight at 4°C; Secondary antibody incubation: After primary antibody incubation, wash three times with 1×TBST, 10 min each time. Dilute the secondary antibody with 1×TBST in 3% skim milk powder, place the membrane in the secondary antibody working solution, and incubate on a shaker at room temperature for 1 h. Wash three times with 1×TBST, 10 min each time, to remove free secondary antibody.

[0057] 5) Color development and imaging Mix ECL solution A and solution B in a 1:1 (V / V) ratio. Apply the ECL mixture evenly to the PVDF membrane, ensuring that the ECL mixture completely covers the entire PVDF membrane. Take care to avoid light during the process.

[0058] After the reaction is complete, the PVDF film is placed in a chemiluminescence imager, and appropriate exposure parameters are selected for color imaging.

[0059] Table 5 Formulation of separating gel

[0060] 3. Experimental Results 3.1 CCK-8 test Table 6 CCK-8 Test Results

[0061] The results are shown in Table 6 and Figure 1 As shown, ZW2857 can inhibit the proliferation of TPC-1 thyroid cancer cells in a dose-dependent manner. 10 μM: produced a mild but significant inhibitory effect (survival rate 86%). This indicates that this concentration is the effective concentration; 50 μM: produced a strong inhibitory effect (survival rate 63%), indicating that this concentration has reached near the half-maximal inhibitory concentration (MCC); 100 μM: produced a strong inhibitory effect (survival rate 35%), indicating that most cell proliferation was inhibited at this concentration.

[0062] 3.2 Cloning experiment Table 7 Results of the Clonal Formation Experiment

[0063] The results are shown in Table 7 and Figure 2 As shown, ZW2857 can inhibit the clonogenic ability of TPC-1 thyroid cancer cells in a dose-dependent manner.

[0064] 3.3 Apoptosis Detection Table 8. Results of apoptosis detection

[0065] The results are shown in Table 8. Figure 3 and Figure 4 As shown, ZW2857 can induce apoptosis in a dose-dependent manner. 10 μM: produced an apoptosis-inducing effect (total apoptosis rate of 12.1%), indicating that ZW2857 can initiate the apoptosis program at a low concentration; 50 μM: produced a strong apoptosis effect (total apoptosis rate of 27.6%), with a significant increase in the proportion of late apoptosis (Q2), indicating a strong drug effect and rapid cell apoptosis process; 100 μM: produced a strong apoptosis effect (total apoptosis rate of 54.1%), with most cells in an apoptotic state.

[0066] 3.4 WB Table 9 WB Detection Results

[0067] Conclusions: (1) p-ERK / ERK & p-MEK / MEK: as shown in Table 9 and Figure 5 a, Figure 5 As shown in b, both selumetinib and ZW2857 can significantly inhibit the phosphorylation of key proteins in the MAPK signaling pathway; the inhibitory effect of ZW2857 is dose-dependent, and the inhibitory effect of the high-dose group is comparable to that of selumetinib, with no statistically significant difference. (2) Cyclin D1: as shown in Table 9 and Figure 5 As shown in c, Cyclin D1 is a key downstream protein of the MAPK pathway. Its expression level decreases with the inhibition of the MAPK pathway, and the trend is consistent with p-ERK, indicating that the cell cycle is arrested in the G1 phase. (3) Cleaved Caspase-3: as shown in Table 9 and Figure 5 As shown in Figure d, cleaved caspase-3 is key to apoptosis, and its elevated level is a marker of irreversible apoptosis. Its expression level significantly increased with increasing ZW2857 dosage, demonstrating that ZW2857 can induce cell death by activating the caspase-3-dependent apoptosis pathway.

[0068] In summary, the multi-target kinase inhibitor ZW2857 can effectively inhibit the proliferation of human papillary thyroid carcinoma cell line TPC-1 by dose-dependently inhibiting the RAS-MAPK-ERK signaling pathway. Its molecular mechanism lies in inducing cell cycle G1 phase arrest and triggering mitochondrial apoptosis.

[0069] Example 2 1. Experimental Materials (1) Test sample: Euonymus alcohol (ZW2857, purchased from Shandong Futian Pharmaceutical Co., Ltd.); (2) Positive control: Selmetinib (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0070] 2. Laboratory animals and tumor cell lines 2.1 Laboratory Animals: Thirty-six female SPF-grade SCID mice weighing 16-18g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They underwent a 7-day quarantine acclimatization period. Cage type: breathable plastic cages; stocking density: 6 mice / cage; environmental conditions: light intensity: 15-20 LX; temperature: 22±1.5℃; relative humidity: 50±10%RH. Feed type: normal breeding diet; normal group rats: complete nutritional solid diet. Storage conditions: solid diet was stored in a dedicated feed room, kept at low temperature, dry, and hygienic. Feeding method: fed once daily, with free access to food, using sterilized water as drinking water.

[0071] SCID mice were chosen for thyroid animal experiments because their severe T and B lymphocyte immune deficiencies ensured that human thyroid cancer cells or tissues could be successfully xenografted and grow in vivo.

[0072] 2.2 Tumor cell line: TPC1 human thyroid cancer cells within three generations (Shenzhen Haodi Huatuo Biotechnology Co., Ltd.).

[0073] 3. Experimental Methods 3.1 Establishment of a mouse model of thyroid cancer 3.1.1 Laboratory animals: Thirty-six female SCID mice, 6 weeks old and weighing 16-18g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All animals were housed in plastic cages in a stable environment with 12 / 12h light / dark cycles and free access to water and food. All mice were acclimatized for one week at 22℃ and 40%-60% RH.

[0074] 3.1.2 TPC1 cell resuscitation and passage culture Recovery steps: (1) After removing the frozen cells from liquid nitrogen, quickly place them in 37°C warm water and shake to thaw for about 1 minute; (2) Centrifuge at 1000 rpm for 1 min, discard the frozen solution, and resuspend the cells in 1 mL of fresh complete culture medium; (3) Inoculate into a new culture bottle, add enough complete culture medium, and place in an incubator for culture.

[0075] Transplantation: (1) Use monolayer adherent culture, add an appropriate amount of complete culture medium, and place it in an incubator with a temperature of 37℃, 5% CO2 and humidity of 100%RH. Replace half of the culture medium every other day. (2) When the cells have reached a confluence of more than 90% in the culture flask, begin the passage experiment. Wash the cells with PBS, add 1 mL of 0.25% trypsin to soak the cells, discard the trypsin, and then place them in a 37°C incubator for 3 minutes for digestion; (3) Under a microscope, when the intercellular spaces increase, become rounded, and some cells begin to detach, remove the culture flask immediately. Gently tap the side of the culture flask with the side of your palm to promote complete cell detachment, and quickly add 2-3 mL of preheated complete culture medium. Discard the supernatant, gently blow on the flask wall to form a uniform single-cell suspension, transfer to a centrifuge tube, and centrifuge at 1000 rpm for 5 min; (4) Discard the supernatant, resuspend the cells in fresh complete culture medium, and adjust the cell concentration to 1×10⁻⁶. 7 per mL.

[0076] 3.1.3 Model Construction and Grouping Take 30 mice from 5 cages. Draw cell suspension into a 1mL syringe, gently pinch the skin on the mouse's back, insert the needle horizontally subcutaneously, and slowly inject 100μL of cell suspension. After withdrawing the needle, press the puncture site briefly to prevent leakage. Return the mice to their cages and feed them normally. Observe the mice's consciousness, diet, and spontaneous activity daily, and measure their weight and tumor volume (V=a×b). 2 / 2). In another cage, three animals with similar weights were selected and marked as the blank control group.

[0077] When the tumor grows to 75mm 3 When the tumor-bearing animals were randomly divided into 5 groups (n=6), it was ensured that there was no significant difference in the initial tumor volume among the groups.

[0078] The groups are as follows: Table 10 Mouse Grouping

[0079] Dosage frequency and cycle: Administer once daily for 13 consecutive days, followed by euthanasia and pathological sampling on the 14th day.

[0080] 3.1.4 Detection of Pharmacodynamic Indicators 1) Measurement of body weight and tumor volume (1) Weight: Measured once a day; (2) Tumor volume: Measured every two days. The longest diameter (a) and shortest diameter (b) of the tumor were measured using calipers. The formula for calculating tumor volume (TV) is: ; (3) Relative tumor volume (RTV): The calculation formula is as follows Where V0 is the tumor volume measured on the day of drug administration (d0), and V t This represents the tumor volume at each measurement.

[0081] (4) Tumor growth inhibition rate (TGI): The calculation formula is as follows TRTV represents the RTV in the treatment group, and CRTV represents the RTV in the model control group.

[0082] 2) Endpoint sample collection Twenty-four hours after the last administration, the mice were euthanized. Tumor tissue was dissected, tumor weight was measured, and the tumor inhibition rate (IR) was calculated using the following formula: .

[0083] The excised tumor fragments were divided into three parts: one part was fixed with 4% paraformaldehyde for pathological sectioning; one part was rapidly frozen in liquid nitrogen and then transferred to -80°C for protein extraction; and one part was placed in RNA preservation solution for RNA extraction. Simultaneously, samples were collected from the heart, liver, spleen, lungs, and kidneys for preliminary toxicity observation. This fragment, along with Western blotting and qRT-PCR samples, was sent for external testing.

[0084] 4. Test Results 4.1 General clinical symptoms and weight Throughout the experiment, all animals were in good spirits, and no significant abnormalities were observed in their behavior, respiration, excretion, eating, or drinking. No animals in any group died.

[0085] The average weight fluctuation range of each group was as follows: normal control group: 18.0~21.9g, model control group: 18.0~18.6g, positive control group: 18.1~19.2g, low-dose euonymus alcohol group: 18.0~20.6g, medium-dose euonymus alcohol group: 18.0~20.5g, and high-dose euonymus alcohol group: 18.0~19.3g.

[0086] The results are as follows Figure 6 As shown: (1) Extremely significant difference (p<0.001): Blank control group VS model control group, proving that the tumor model was successfully established; all ZW2857 groups VS model group, proving the anti-tumor effect of ZW2857; positive control group VS low-dose ZW2857 group, showing the safety advantage of ZW2857; (2) Significant difference (p<0.05): Blank control group VS medium-dose ZW2857 group, showing that medium dose has a slight effect; positive control group VS high-dose ZW2857 group, indicating that high-dose safety is better than positive drug; among the different dose groups of ZW2857, dose dependence is shown; (3) No significant difference (p>0.05): Blank control group VS high-dose ZW2857 group, proving that high-dose ZW2857 has high safety.

[0087] Weight statistics results as follows Figure 6 As shown, the data is presented in the table below: Table 11 Weight Statistics Table

[0088] 4.2 Tumor volume Tumor volume variation range among groups: blank control group (NA); model control group (75.2–1250.5 mm). 3 The positive control group ranged from 76.8 to 650.3 mm. 3 The low-dose group of Euonymus alcohol had a concentration of 74.5–980.6 mm. 3 The mid-dose group of Euonymus alcohol ranged from 77.1 to 510.4 mm. 3 The high-dose group of Euonymus alcohol had a serum concentration of 75.9–380.2 mm. 3 .

[0089] The tumor volume statistics are shown in the table below. The results are as follows: Figure 7 As shown.

[0090] Table 12 Tumor Volume Statistics

[0091] The statistical analysis of each group compared with the model group is shown in the table below. Table 13 Statistical analysis of each group compared with the model group

[0092] 4.3 Relative tumor volume (RTV) Calculation formula:

[0093] The calculation results are as follows Figure 8 Specific values ​​are shown in Table 14: Table 14 Relative Tumor Volume Table

[0094] Based on relative tumor volume (RTV) analysis, the tumor in the model control group grew the fastest, reaching an RTV of 16.63 on day 14; all dose groups of ZW2857 showed significant tumor-suppressing effects. Furthermore, the tumor-suppressing effect showed a dose-dependent pattern: high-dose group > medium-dose group > low-dose group.

[0095] 4.4 Tumor Growth Inhibition Rate (TGI) Calculation formula: .

[0096] The calculation results are as follows Figure 9 The specific values ​​are shown in Table 15.

[0097] Table 15 Tumor Growth Inhibition Rate

[0098] According to the tumor growth inhibition rate (TGI) analysis, on day 14, the TGI of the positive control group was 49.09%, while the TGI of the high-dose ZW2857 group reached 69.88%, showing the best tumor inhibition effect. The tumor inhibition effect gradually increased over time, and all treatment groups showed statistically significant tumor inhibition effects.

[0099] 4.5 tumor weight Twenty-four hours after the final drug administration, mice were euthanized, tumors were removed, and tumor weight was measured. Results showed... Figure 10 The tumor weight in the model control group was 1.32±0.18g, the tumor weight in the positive control group was 0.68±0.11g, the tumor weight in the low-dose ZW2857 group was 1.01±0.15g, the tumor weight in the medium-dose ZW2857 group was 0.55±0.09g, and the tumor weight in the high-dose ZW2857 group was 0.42±0.07g.

[0100] The results of the statistical analysis are shown in Table 16. Figure 10 As shown.

[0101] Table 16 Results of statistical analysis of tumor weight

[0102] The analysis results show that: (1) Successful modeling: The tumor growth in the model group and the absence in the blank control group indicates that the tumor model has been successfully established.

[0103] (2) Positive drug efficacy: The positive control group showed a highly significant difference compared with the model control group (p<0.001), indicating that the positive drug had a significant anti-tumor effect.

[0104] (3) Dose-dependent effect of ZW2857: There were significant differences between each dose group and the model group: low dose group p < 0.05 (weak but significant effect); medium dose group p < 0.001 (significant effect); high dose group p < 0.001 (strongest effect). (4) The high-dose group showed the best effect: the high-dose group had a highly significant difference compared with the positive control group (p<0.001), indicating that the high-dose group had a better tumor-suppressing effect than the positive control.

[0105] (5) The medium-dose group was comparable to the positive control group: There was no significant difference between the medium-dose group and the positive control group (p=0.087), indicating that the effect of the medium-dose group was comparable to that of the positive control group.

[0106] 4.6 Tumor inhibition rate The formula for calculating the tumor inhibition rate (IR) based on tumor weight is as follows: .

[0107] The calculation results are as follows Figure 11The positive control group was 48.5%, the low-dose ZW2857 group was 23.5%, the medium-dose ZW2857 group was 58.3%, and the high-dose ZW2857 group was 68.2%.

[0108] Conclusion: ZW2857 demonstrated good safety and significant antitumor effects in this SCID mouse thyroid cancer model experiment. Compared with the positive control selactinib, ZW2857 significantly improved treatment safety while maintaining efficacy. Significant differences existed among the dose groups, showing a clear dose-response relationship, with the high-dose group significantly superior to the positive control, medium-dose, and low-dose groups, indicating potential clinical application value.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of euonymus alcohol in the preparation of drugs for treating thyroid cancer.

2. The application according to claim 1, characterized in that: In this application, thyroid cancer refers to papillary thyroid carcinoma.

3. The application according to claim 1, characterized in that: The application of the euonymus alcohol is in the preparation of drugs that inhibit the phosphorylation levels of key kinases MEK and ERK in the MAPK / ERK signaling pathway.

4. The application according to claim 1, characterized in that: The application of the aforementioned application is the use of euonymus alcohol in the preparation of drugs that downregulate the expression of cyclin D1.

5. The application according to claim 1, characterized in that: In the aforementioned applications, the use of euonymus alcohol in the preparation of drugs that upregulate the expression of the apoptosis-executing protein Cleaved Caspase-3 and activate mitochondrial apoptosis.

6. The application according to claim 1, characterized in that: The concentration range of the euonymus alcohol is 10 μM-100 μM.

7. A pharmaceutical composition for treating thyroid cancer, said pharmaceutical composition comprising euonymus alcohol.

8. The pharmaceutical composition according to claim 7, characterized in that: The thyroid cancer mentioned is papillary thyroid carcinoma.

9. The pharmaceutical composition according to claim 7, characterized in that: The concentration range of the euonymus alcohol is 10 μM-100 μM.

10. The pharmaceutical composition according to claim 7, characterized in that: The concentration range of the euonymus alcohol is 50 μM-100 μM.

Images