Use of junocin as an inhibitor of the MAU2 sister chromatid cohesion factor

Abstract

The application discloses application of Juno in preparation of a tumor treatment drug. The application first proposes and verifies that Juno can effectively inhibit expression of a MAU2 sister chromatid cohesion factor, achieves the effect of blocking a cell cycle at a G2 phase and / or an M phase, and further inhibits tumor progression, thereby providing a new direction for treatment of non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and more particularly to the application of Junoxin as an inhibitor of MAU2 sister chromatid aggregation factor. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer, accounting for about 85% of all lung cancers. It is a malignant tumor with the highest incidence and mortality rates worldwide. Due to the insidious nature of early symptoms, more than 70% of patients are diagnosed at an intermediate or advanced stage, resulting in a five-year survival rate of less than 5% for patients with advanced disease, making the treatment situation extremely challenging.

[0003] Traditional treatments for NSCLC primarily involve radiotherapy and chemotherapy; however, their efficacy is limited and adverse reactions are significant. Most patients eventually face treatment resistance and disease progression, resulting in an overall unsatisfactory cure rate. In recent years, the application of small-molecule tyrosine kinase inhibitors targeting specific molecules and immunotherapy has brought significant survival benefits to some patients, but the clinical beneficiary population still needs further expansion.

[0004] Junosine is a class of acridinium ketone compounds naturally found in citrus fruits. Its chemical structure is characterized by a ketone group linked to the C9 position of an acridinium ring. Its biological activity has not been reported and requires further investigation. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide the application of Junocin as an inhibitor of MAU2 sister chromatid aggregation factor, and its application in arresting the cell cycle and preparing tumor therapeutic drugs based on this.

[0006] Technical solution: The application of Junoxin as an inhibitor of MAU2 sister chromatid cohesion factor as described in this invention.

[0007] Preferably, the CAS number of the Junoxin is 103956-34-9.

[0008] Preferably, the application is an application that arrests the cell cycle in the G2 phase and / or M phase.

[0009] Preferably, the application is in the preparation of tumor treatment drugs; more preferably, the application is in the preparation of lung cancer treatment drugs; even more preferably, the application is in the preparation of non-small cell lung cancer treatment drugs.

[0010] Preferably, the drug contains Junoxin or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients; more preferably, the drug also contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0011] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that Junocin can effectively inhibit the expression of MAU2 sister chromatid aggregation factor, thereby arresting the cell cycle in the G2 phase and / or M phase, thus inhibiting tumor progression and providing a new direction for the treatment of non-small cell lung cancer. Attached Figure Description

[0013] Figure 1 Figure showing the results of PC9 cell viability and IC50 values ​​after Junoxin treatment; Figure 2 Figure showing the results of A549 cell viability and IC50 values ​​after Junoxin treatment; Figure 3 Figure showing the results of apoptosis level measurement in PC9 cells after Junoxin treatment; Figure 4 Figure showing the results of apoptosis level measurement in A549 cells after Junoxin treatment; Figure 5 Figure showing the results of a scratch assay on PC9 cells after Junoxin treatment; Figure 6 Figure showing the results of scratch assay on A549 cells after Junoxin treatment; Figure 7 Image showing the cell cycle detection results of PC9 cells after Junoxin treatment; Figure 8 Image showing the cell cycle detection results of A549 cells after Junoxin treatment; Figure 9 Figure showing the results of colony-forming ability assay of PC9 cells after Junoxin treatment; Figure 10 Figure showing the results of colony-forming ability assay of A549 cells after Junoxin treatment; Figure 11 Figure showing the results of Junoxin treatment for PC9 cell proliferation. Figure 12 Figure showing the results of A549 cell proliferation assay after Junoxin treatment; Figure 13 Figure 1 shows the results of mitochondrial membrane potential measurement in PC9 cells after Junoxin treatment. Figure 14 Figure 1 shows the results of mitochondrial membrane potential measurement in A549 cells after Junoxin treatment. Figure 15 Figure showing the results of MAU2 expression level detection in PC9 cells after Junoxin treatment; Figure 16 Figure showing the results of MAU2 expression level detection in PC9 cells with MAU2 knockdown; Figure 17 Figure showing the results of viability assays for MAU2 knockdown PC9 cells after Junoxin treatment; Figure 18 Figure 1 shows the colony-forming ability of MAU2 knockdown PC9 cells after Junoxin treatment. Figure 19 Figure showing the proliferation capacity of MAU2 knockdown PC9 cells after Junoxin treatment; Figure 20 The image shows the results of MAU2 expression level detection in PC9 cells overexpressing MAU2. Figure 21 Figure showing the results of viability assays in MAU2-overexpressing PC9 cells after Junoxin treatment; Figure 22 Figure 1 shows the colony-forming ability of MAU2-overexpressing PC9 cells after Junoxin treatment. Figure 23 Figure 1 shows the results of proliferative capacity assay of MAU2-overexpressing PC9 cells after Junoxin treatment. Figure 24 The curve of body weight change in mice after Junoxin treatment; Figure 25 A statistical chart of organ coefficients in mice after Junoxin treatment; Figure 26 Image of tumor tissue in mice after Junoxin treatment; Figure 27 The curves showing the changes in tumor volume in mice after Junoxin treatment; Figure 28 Figure showing the expression of Ki67 and MAU2 in mouse tumor tissues after Junoxin treatment; Figure 29 Figure showing the results of MAU2 expression level detection in mouse tumor tissue after Junoxin treatment. Detailed Implementation

[0014] The technical solution of the present invention will be further described below.

[0015] Example 1: In vitro efficacy validation of Junosine (JUSE) in non-small cell lung cancer 1. JUSE for non-small cell lung cancer (NSCLC) cells 1.1 Cell viability and IC50 value determination PC9 cells or A549 cells at 2×10 3 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 h. Then, the cells were treated with JUSE (purchased from MedChemExpress LLC., catalog number HY-N13665) at final concentrations of 0, 1, 2, 5, 10, 15, 20, and 30 µM in DMEM complete medium containing 10% fetal bovine serum for 24 h. After treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0016] The results are as follows Figure 1 , 2 As shown, the higher the concentration of JUSE, the lower the cell viability, and both types of NSCLC cell showed a dose-dependent effect; the IC50 value of JUSE for PC9 cells was 9.36 µM, while the IC50 value for A549 cells was 10.15 µM.

[0017] 1.2 Measurement of Apoptosis Level PC9 cells or A549 cells at 2 × 10⁶ cells per well 4 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight until adherence. Then, cells were treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE 10), and 15 µM (JUSE 15) for 24 h in DMEM complete medium containing 10% fetal bovine serum. Cells were collected after treatment and stained using the Novizan Annexin V-PE / 7AAD apoptosis detection kit (catalog number A213-01). The stained cells were analyzed using a Beckman CytoFLEX flow cytometer to quantify apoptosis levels.

[0018] The results are as follows Figure 3 , 4 As shown, for PC9 cells, the late apoptosis levels in cells treated with 0, 10, and 15 µM JUSE were 6.39%, 14.3%, and 22.2%, respectively; while for A549 cells, the late apoptosis levels in cells treated with 0, 10, and 15 µM JUSE were 7.39%, 17.1%, and 22.5%, respectively. The results indicate that the apoptosis levels in both cell types gradually increased with increasing JUSE concentration, exhibiting a dose-dependent effect.

[0019] 1.3 Cell Scratch Test PC9 cells or A549 cells at 2 × 10⁶ cells per well4 Cells were seeded at a density of 70 ± 5% per well in 6-well plates. Once the cell density reached 70 ± 5%, cells were scratched in each well using a 10 µL pipette tip. Cells were treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE10), and 15 µM (JUSE15) for 24 h, followed by 24 h of further culture. Cells were observed and images were acquired at 0 h and 48 h after scratching under an optical microscope.

[0020] The results are as follows Figure 5 , 6 As shown, after 24 h of treatment with JUSE, the scratch healing ability of PC9 and A549 cells was significantly inhibited, and the inhibitory effect on the scratch healing ability became more pronounced with increasing JUSE concentration.

[0021] 1.4 Cell cycle detection PC9 cells or A549 cells at 2 × 10⁶ cells per well 4 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adherence, cells were treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE 10), and 15 µM (JUSE 15) for 24 h in DMEM complete medium containing 10% fetal bovine serum. Cells were then collected and fixed overnight with 70% ethanol at -20°C. The fixed cells were centrifuged at 300×g for 5 min, and the cell pellet was recovered. The cells were resuspended in PBS buffer containing 50 μg / mL propidium iodide (MedChemExpress LLC., catalog number HY-D0815) and 100 μg / mL RNase A (Beyotime, catalog number ST579) and incubated at 37°C in the dark for 30 min. Cell cycle analysis was performed using a Beckman CytoFLEX flow cytometer and ModFitLT software.

[0022] The results are as follows Figure 7 , 8 As shown, for PC9 cells, the proportions of cells in the G2 / M phase treated with JUSE at final concentrations of 0, 10, and 15 µM were 11.7%, 11.9%, and 17.2%, respectively; for A549 cells, the proportions of cells in the G2 / M phase treated with JUSE at final concentrations of 0, 10, and 15 µM were 9.02%, 11.6%, and 15.6%, respectively. This indicates that JUSE can arrest NSCLC cells in the G2 / M phase, thereby affecting the cell viability of PC9 and A549 cells.

[0023] 1.5. Cell clone formation ability assay PC9 or A549 cells were seeded at a density of 500 cells / well in 6-well plates and cultured adherently for 24 h. Cells were then treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE 10), and 15 µM (JUSE 15) in DMEM complete medium containing 10% fetal bovine serum for 24 h. After treatment, the medium was replaced with fresh DMEM complete medium without JUSE every 2 days. Cells were stained with Beyotime crystal violet reagent (catalog number C0121-100ml), and observed and images were acquired under a microscope.

[0024] The results are as follows Figure 9 , 10 As shown, after treatment with 15 µM JUSE, the number of cell clones formed in PC9 and A549 cells was significantly lower than that in the DMSO control group that was not treated with JUSE after 24 h.

[0025] 1.6. Cell proliferation capacity assay PC9 cells or A549 cells at 5 × 10⁶ cells per well 3 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured adherently for 24 h. Cells were then treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE 10), and 15 µM (JUSE 15) in DMEM complete medium containing 10% fetal bovine serum for 24 h. After treatment, 20 μL of EdU reagent (from Novizan, catalog number A413-01) was added to each well, and the cells were incubated at 37°C for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 10 min, the cells were permeabilized with Beyotime immunostaining permeabilization buffer (catalog number P0096) at room temperature for 15 min. Click reaction solution (from Novizan, catalog number A413-01) was then added, and the cells were incubated in the dark for 30 min. The cells were then mounted with Beyotime anti-fluorescence quenching mounting buffer (catalog number P0131), and the images were observed and acquired using a fluorescence microscope.

[0026] The results are as follows Figure 11 , 12 As shown, JUSE treatment significantly reduced EdU red fluorescence signal in PC9 and A549 cells, indicating a decrease in the proliferation capacity of both cell types.

[0027] 1.7 Mitochondrial membrane potential measurement PC9 cells or A549 cells at 2 × 10⁶ cells per well 4 Cells were seeded at a density of 1 cell per well in 6-well plates and cultured overnight. After adherence, the cells were treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE 10), and 15 µM (JUSE 15) in DMEM complete medium containing 10% fetal bovine serum for 24 h.

[0028] Cells were collected after treatment and incubated in PBS buffer containing 10 µg / mL JC-1 (purchased from MedChemExpress LLC., catalog number: HY-15534) at 37°C in the dark for 30 min. Fluorescence signals were detected using a Beckman CytoFLEX flow cytometer. The excitation wavelength for JC-1 monomer (green fluorescence) was set to 488 nm, and the emission wavelengths were detected at 525 / 50 nm. The emission wavelengths for JC-1 multimer (red fluorescence) were detected at 590 / 40 nm.

[0029] The results are as follows Figure 13 , 14 As shown, for the PC9 cell group, the ratio of JC-1 polymers to JC-1 monomers (Q2 / Q3) in cells treated with JUSE at final concentrations of 0, 10, and 15 µM were 5.9, 2.0, and 1.1, respectively; for the A549 cells, the ratio of JC-1 polymers to JC-1 monomers in cells treated with JUSE at final concentrations of 0, 10, and 15 µM were 4.6, 2.5, and 1.3, respectively. This indicates that the mitochondrial activity of PC9 and A549 cells significantly decreased after JUSE treatment, leading to a dose-dependent decrease in cell viability.

[0030] 1.8. Measurement of MAU2 sister chromatid cohesion factor (MAU2 expression level) PC9 cells were used at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1 cell per well in 6-well plates and cultured overnight. After adherence, the cells were treated with JUSE at final concentrations of 0 µM (1‰ DMSO), 10 µM (JUSE 10), and 15 µM (JUSE 15) in DMEM complete medium containing 10% fetal bovine serum for 24 h. Cells were then collected, and lysis was performed on ice with shaking for 30 min using RIPA lysis buffer (Catalog No. WB3100). The cells were then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was collected, and RIPA 5× Loading buffer (Catalog No. WB2001) was added. The cells were heated to boiling in a metal bath for 10 min, followed by SDS-PAGE electrophoresis. After transfer and blocking, MAU2 primary antibody (purchased from Abcam, Catalog No. ab183033) or GAPDH primary antibody diluted 1:1000 was added, and the cells were incubated overnight at 4°C. After rinsing, secondary antibody (purchased from Proteintech, Catalog No. SA00001-2) diluted 1:5000 was added, and the cells were incubated at room temperature for 2 h. Finally, the cells were developed using the RIPA ultrasensitive ECL chemiluminescence kit (Catalog No. P10100), and the developed images were acquired.

[0031] The results are as follows Figure 15As shown, JUSE treatment significantly reduced MAU2 protein expression, and the higher the JUSE concentration, the lower the MAU2 protein level.

[0032] 2. JUSE for non-small cell lung cancer cells regulated by MAU2 expression 2.1 JUSE for MAU2 knockdown of non-small cell lung cancer cells 2.1.1 Construction and validation of MAU2 knockdown non-small cell lung cancer cells Based on the MAU2 gene mRNA sequence (NCBI Reference Sequence: NM_015329.4), a siRNA with the sequence 5'-AAAUUUAACAUCUUCGAACUG-3' (siRNA-MAU2) was designed and synthesized by Suzhou Genewise Biotechnology Co., Ltd., while a random control siRNA (si-NC) was synthesized at the same time.

[0033] PC9 cells were fed at a rate of 2 × 10 4 The cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, siRNA-MAU2 and si-NC were transfected into PC9 cells using the Novizan Lipomaster 2000 Transfection Reagent Kit (catalog number TL201-01) to obtain si-MAU2 PC9 cells and si-NC PC9 cells, respectively.

[0034] Cells transfected for 24 h were collected, and the MAU2 expression level was determined based on the aforementioned experimental steps.

[0035] The results are as follows Figure 16 As shown, the expression level of MAU2 in PC9 cells transfected with siRNA-MAU2 was significantly reduced, indicating that the construction of PC9 cells with MAU2 knockdown was successful.

[0036] 2.1.2 Cell viability assay si-MAU2 PC9 cells or si-NC PC9 cells at 2×10 3 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured for 24 h. Then, the cells were treated with 10 µM (JUSE 10) of DMSO (DMSO) for 24 h in DMEM complete medium containing 10% fetal bovine serum. After treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0037] The results are as follows Figure 17As shown, the decrease in absorbance of si-NC cells after JUSE treatment was much greater than the change in absorbance of si-MAU2 cells, thus preliminarily confirming that the effect of JUSE on PC9 cell activity depends on MAU2.

[0038] 2.1.3. Cell clone formation ability assay si-MAU2 PC9 cells or si-NC PC9 cells were seeded at a density of 500 cells / well in 6-well plates and cultured adherently for 24 h. Cell colony formation ability was then determined based on the aforementioned experimental steps.

[0039] The results are as follows Figure 18 As shown, the clonogenic ability of si-MAU2 cells was significantly lower than that of si-NC cells, meaning that JUSE had a significantly greater effect on the clonogenic ability of si-NC cells than on si-MAU2 cells.

[0040] 2.1.4. Cell proliferation capacity assay si-MAU2 PC9 cells or si-NC PC9 cells at 5 × 10⁶ cells per well 3 Cells were seeded at a density of 1 cell per well in 96-well plates and cultured adherently for 24 hours. Cell proliferation was then measured based on the aforementioned experimental procedures.

[0041] The results are as follows Figure 19 As shown, JUSE inhibits the proliferation of si-MAU2 cells much less than it does si-NC cells.

[0042] 2.2 JUSE for MAU2-overexpressing non-small cell lung cancer cells 2.2.1 Construction and validation of MAU2-overexpressing non-small cell lung cancer cells Based on the MAU2 gene mRNA sequence (NCBI Reference Sequence: NM_015329.4), Suzhou Genewise Biotechnology Co., Ltd. was commissioned to construct the MAU2 gene overexpression plasmid pCDNA3.1 / MAU2 based on the pCDNA3.1 vector.

[0043] PC9 cells were fed at a rate of 2 × 10 4 The cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, the synthesized pCDNA3.1 / MAU2 or pCDNA3.1 vector was transfected into PC9 cells using the Novizan Lipomaster 2000 Transfection Reagent Kit (catalog number TL201-01) to obtain MAU2-OE PC9 cells and CTRL PC9 cells.

[0044] Cells transfected for 24 h were collected, and the MAU2 expression level was determined based on the aforementioned experimental steps.

[0045] The results are as follows Figure 20 As shown, the expression level of MAU2 in PC9 cells transfected with pCDNA3.1 / MAU2 was significantly increased, indicating that PC9 cells overexpressing MAU2 were successfully constructed. 2.2.2 Cell viability assay MAU2-OE PC9 cells or CTRL PC9 cells at 2×10 3 Cells were seeded at a density of 1 cell per well in 96-well plates and cultured for 24 h. Cell viability was then measured based on the aforementioned experimental procedures.

[0046] The results are as follows Figure 21 As shown, the absorbance of MAU2-OE PC9 cells was significantly higher than that of control cells, further indicating that MAU2 is crucial for the activity of PC9 cells. After JUSE treatment, the decrease in absorbance of MAU2-OE cells was much greater than that of CTRL cells, meaning that JUSE had a significantly greater effect on the activity of MAU2-overexpressing cells than on CTRL cells.

[0047] 2.2.3. Cell clone formation ability assay MAU2-OE PC9 cells or CTRL PC9 cells were seeded at a density of 500 cells / well in 6-well plates and cultured adherently for 24 h. Cell colony formation capacity was then determined based on the aforementioned experimental procedures.

[0048] The results are as follows Figure 22 As shown, the clonogenic ability of MAU2-overexpressing cells was significantly higher than that of CTRL cells, meaning that JUSE had a significantly greater effect on the clonogenic ability of MAU2-overexpressing cells than on CTRL cells.

[0049] 2.2.4. Cell proliferation capacity assay MAU2-OE PC9 cells or CTRL PC9 cells were used at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1 cell per well in 96-well plates and cultured adherently for 24 hours. Cell proliferation was then measured based on the aforementioned experimental procedures.

[0050] The results are as follows Figure 23 As shown, JUSE has a much greater inhibitory effect on the proliferation of MAU2-overexpressing cells than on CTRL cells.

[0051] Example 2: In vivo efficacy validation of Junoxin in a mouse model of non-small cell lung cancer Female C57BL / 6 mice, aged 6-8 weeks and weighing 18-20 g, were purchased from the Experimental Animal Center of Nantong University. Mouse lung cancer cells LLC were added at a rate of 5 × 10⁻⁶ cells per mouse. 5 One cell was subcutaneously inoculated into the left axilla to create a tumor. The tumor was allowed to grow to approximately 100 mm². 3Tumor-bearing mice were randomly divided into two groups: a saline group and a JUSE treatment group (dose of 20 mg / kg), and drug administration was initiated simultaneously. The first administration was recorded as day 0. The drug was administered by gavage every two days, while the saline group received the same volume of saline. Treatment continued until day 18. Upon reaching the experimental endpoint, the mice were euthanized, and tumor tissue, as well as heart, liver, spleen, lungs, and kidneys, were collected from each group for subsequent analysis of relevant indicators.

[0052] 1. Weight monitoring Starting from day 0 of the drug treatment, the body weight of mice in each group was measured every 2 days.

[0053] The results are as follows Figure 24 As shown, there was no significant difference in body weight between the saline group and the JUSE treatment group, indicating that the concentration of JUSE used in the experiment did not cause biological toxicity.

[0054] 2. Organ coefficient detection The heart, liver, spleen, lungs, and kidneys of each mouse were weighed separately. The organ coefficient was calculated using the following formula: Organ coefficient (%) = Organ wet weight ÷ Mouse body weight × 100.

[0055] The results are as follows Figure 25 As shown, there was no significant difference in organ coefficients between the saline group and the JUSE treatment group, further indicating that the concentration of JUSE used in the experiment did not cause biological toxicity.

[0056] 3. Tumor volume monitoring Starting from day 0 of the drug administration, the tumor volume of mice in each experimental group was measured every two days. The length and width of the tumor were recorded, and the tumor volume was then calculated and statistically analyzed according to the following formula: Tumor volume (mm) 3 = length × width × width / 2.

[0057] Tumor images at the experimental endpoint are as follows Figure 26 As shown, the tumor volume monitoring results are as follows: Figure 27 As shown, the tumor volume in the JUSE treatment group was much smaller than that in the control group, indicating that JUSE is effective in treating non-small cell lung cancer.

[0058] 4. Immunohistochemical detection of Ki67 and MAU2 protein expression in tumor tissues Tumor tissues from the saline group and the JUSE treatment group were fixed overnight with 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, and cut into 4 μm sections. Antigen retrieval was performed for 20 min at 95°C with citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited for 10 min with 3% hydrogen peroxide solution, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were incubated overnight at 4°C with Ki67 primary antibody diluted 1:10000 (Proteintech, catalog number 27309-1-AP) or MAU2 primary antibody diluted 1:300. After washing, the sections were incubated with horseradish peroxidase (HRP) conjugated secondary antibody diluted 1:500 (Proteintech, catalog number SA00004-2) at room temperature for 1 h. Signal detection was performed using the Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope.

[0059] The results are as follows Figure 28 As shown, Ki67 and MAU2 in the tumor tissue of the JUSE treatment group were significantly lower than those in the control group, which further illustrates that JUSE has a significant inhibitory effect on non-small cell lung cancer.

[0060] 5. Western blot method to determine the protein expression level of MAU2 100 mg of tumor tissue was taken, ground in liquid nitrogen using a mortar and pestle, and then 1 mL of Beyotime Western chemiluminescence buffer containing protease inhibitors and phosphatase inhibitors and IP cell lysis buffer (catalog number P0013) was added to completely lyse the tissue. After lysis at 4°C for 30 min, the tissue was centrifuged at 12000 rpm for 30 min to obtain the supernatant, which was used for SDS-PAGE electrophoresis. After transfer and blocking, the tissue was incubated overnight at 4°C with MAU2 primary antibody or GAPDH primary antibody. After rinsing, the tissue was incubated at room temperature with secondary antibody for 2 h. The tissue was then developed and images were acquired using the NewSyne Ultrasensitive ECL Chemiluminescence Kit (catalog number P10100).

[0061] The results are as follows Figure 29 As shown, the MAU2 protein level in the JUSE-treated group was significantly lower than that in the saline group, consistent with the results of in vitro experiments, indicating that JUSE inhibits NSCLC by targeting and suppressing the expression level of MAU2 protein.

Claims

1. An application of Junoxin as an inhibitor of MAU2 sister chromatid aggregation factor, characterized in that, Junoxin has the CAS number 103956-34-9, and its application is in the preparation of drugs for the treatment of non-small cell lung cancer.

2. The application according to claim 1, characterized in that, The drug contains Junoxin or a pharmaceutically acceptable salt thereof as its active ingredient.

3. The application according to claim 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: excipients, lubricants, glidants, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, powders, microcapsules, injections, suspensions, patches, suppositories, microemulsions, liposomes, and nanoparticles.

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