Application of STAT3 dual phosphorylation inhibitors in the preparation of drugs for preventing or treating triple-negative breast cancer

Abstract

The present invention relates to the field of biomedicine and provides the use of a STAT3 dual-phosphorylation inhibitor in the preparation of a drug for preventing or treating triple-negative breast cancer. The STAT3 dual-phosphorylation inhibitor comprises pulsatilla saponin E2 and / or a pharmaceutically acceptable salt thereof. As a STAT3 dual-phosphorylation inhibitor, pulsatilla saponin E2 can effectively inhibit the growth and migration of triple-negative breast cancer cells. Its application in the treatment of triple-negative breast cancer has the advantages of good efficacy and minimal toxic side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of a STAT3 dual phosphorylation inhibitor in the preparation of a drug for preventing or treating triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) is a poorly differentiated subtype of breast cancer characterized by the loss of progesterone receptor, human epidermal growth factor receptor 2, and estrogen receptor expression. High invasiveness and a lack of drug targets are key prognostic factors for TNBC patients. The five-year survival rate after initial surgery is only 40%, and for patients in advanced stages, the five-year survival rate is less than 20%, posing a serious threat to daily life. Internationally, TNBC tends to occur in younger patients, and nearly all deaths are caused by malignant progression. In my country, TNBC is most common in patients between 40 and 70 years old, and most patients present with distant metastases at the time of initial diagnosis, resulting in a very poor prognosis. Currently, chemotherapy, antibody-drug conjugates, immunotherapy, and targeted therapies are commonly used in clinical treatment of TNBC, but patients still experience limited efficacy and severe toxicity. To reduce the malignant progression of TNBC and improve patient outcomes, it is necessary to identify and develop novel, highly effective, low-toxic, and valuable anti-TNBC drugs.

[0003] Targeting signal transducer and activator of transcription 3 (STAT3) has become a hot topic in the development of anti-tumor drugs. Studies have shown that the activation of STAT3 is mainly attributed to the phosphorylation of two key amino acid residues, namely tyrosine 705 (Tyr 705 ) and Serine 727 (Ser 727 ). Normally, STAT3 is activated by JAKs and other tyrosine kinases through its SH2 domain to catalyze the transcription of Tyr 705 STAT3 is activated by phosphorylation, leading to STAT3 dimerization and nuclear translocation, thereby regulating target gene expression and transcription. 727 The site is phosphorylated by several serine kinases, leading to another form of STAT3 activation. Tyr705 Typical role of phosphorylation, STAT3 Ser727 Phosphorylation mainly regulates its mitochondrial function, thereby affecting the metabolic reprogramming of tumor cells. 705 and Ser 727 Therefore, the simultaneous inhibition of Tyr 705 and Ser 727Dual phosphorylation sites can block STAT3 activation, which may be a promising strategy for the development of STAT3 target drugs. Summary of the Invention

[0004] To address the problems of minimal efficacy and severe toxic side effects of existing TNBC drugs, the present invention provides the use of a STAT3 dual-phosphorylation inhibitor in the preparation of a drug for preventing or treating triple-negative breast cancer. The dual-phosphorylation inhibitor is pulsatilla saponin E2, which can effectively inhibit the growth and migration of triple-negative breast cancer cells. Its application in the treatment of triple-negative breast cancer has the advantages of good efficacy and minimal toxic side effects.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides the use of a STAT3 dual phosphorylation inhibitor in the preparation of a drug for preventing or treating triple-negative breast cancer. The STAT3 dual phosphorylation inhibitor comprises pulsatilla saponin E2 and / or a pharmaceutically acceptable salt thereof. The structural formula of pulsatilla saponin E2 is as follows:

[0007]

[0008] Based on the same inventive concept, the present invention provides the use of pulsatilla saponin E2 and / or its pharmaceutically acceptable salt as a STAT3 dual phosphorylation inhibitor in the preparation of a drug for inhibiting the growth or metastasis of triple-negative breast cancer cells.

[0009] Based on the same inventive concept, the present invention provides the effects of Pulsatilla saponin E2 and / or its pharmaceutically acceptable salt on inhibiting STAT3 protein Tyr 705 and / or Ser 727 Applications in phosphorylation.

[0010] Based on the same inventive concept, the present invention provides the use of pulsatilla saponin E2 and / or its pharmaceutically acceptable salt in inhibiting STAT3 nuclear transcription and / or STAT3-dependent mitochondrial oxidative phosphorylation.

[0011] Based on the same inventive concept, the present invention provides the use of pulsatilla saponin E2 and / or its pharmaceutically acceptable salt as and / or in the preparation of a STAT3 dual phosphorylation inhibitor.

[0012] Furthermore, the STAT3 dual phosphorylation inhibitor includes inhibiting STAT3 protein Tyr 705 and Ser 727 Reagents for site phosphorylation.

[0013] Based on the same inventive concept, the present invention provides a drug for preventing or treating triple-negative breast cancer, wherein the active ingredient of the drug includes pulsatilla saponin E2 and / or a pharmaceutically acceptable salt thereof.

[0014] Based on the same inventive concept, the present invention provides a drug for inhibiting the growth or metastasis of triple-negative breast cancer cells, wherein the active ingredient of the drug includes pulsatilla saponin E2 and / or a pharmaceutically acceptable salt thereof.

[0015] Based on the same inventive concept, the present invention provides a STAT3 dual phosphorylation inhibitor, wherein the active ingredient of the STAT3 dual phosphorylation inhibitor includes pulsatilla saponin E2 and / or a pharmaceutically acceptable salt thereof.

[0016] Furthermore, the STAT3 dual phosphorylation inhibitor includes inhibiting STAT3 protein Tyr 705 and Ser 727 Reagents for site phosphorylation.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0018] 1. The use of the STAT3 dual phosphorylation inhibitor of the present invention in the preparation of drugs for preventing or treating triple-negative breast cancer. The present invention first discovered that pulsatilla saponin E2 can act as a STAT3 dual phosphorylation inhibitor, significantly inhibiting the expression of STAT3 protein Tyr 705 Phosphorylation and STAT3 nuclear transcription, significantly inhibiting STAT3 protein Ser 727 Phosphorylation and STAT3-dependent mitochondrial oxidative phosphorylation. PSE2 (Pulchinenoside E2) is a STAT3 dual phosphorylation inhibitor that can use STAT3 as its core target to effectively inhibit the growth and migration of TNBC cells, thereby achieving the prevention or treatment of triple-negative breast cancer. PSE2 can be used as a drug for the prevention and treatment of triple-negative breast cancer, with the advantages of good efficacy and fewer toxic side effects.

[0019] 2. The use of Pulsatilla saponin E2 and / or its pharmaceutically acceptable salt as a STAT3 dual phosphorylation inhibitor in the preparation of a drug for inhibiting the growth or metastasis of triple-negative breast cancer cells. The STAT3 dual phosphorylation inhibitor PSE2 or its pharmaceutically acceptable salt is used as a drug for inhibiting the growth or metastasis of TNBC cells, and can simultaneously act on the STAT3 protein Tyr 705 and Ser 727 phosphorylation site, significantly inhibiting Tyr 705 and Ser 727 It phosphorylates STAT3 and inhibits STAT3 transcriptional activity and mitochondrial STAT3-dependent oxidative phosphorylation, showing the strongest activity in the screening of drug efficacy for inhibiting the growth and migration of triple-negative breast cancer cells, suggesting that PSE2 provides a promising candidate lead compound for the development of drugs to inhibit TNBC growth or metastasis, and has broad development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a diagram showing drug efficacy screening on TNBC cell growth and migration.

[0022] Figure 2 STAT3 is a direct core target of PSE2.

[0023] Figure 3 PSE2 can stably bind to STAT3 protein.

[0024] Figure 4 These are the key target screening results for PSE2's anti-TNBC cell invasion and migration.

[0025] Figure 5 PSE2 significantly inhibited p-STAT3 (Tyr 705 ) and STAT3 nuclear transcription.

[0026] Figure 6 PSE2 inhibits p-STAT3 (Ser 727 ) and mitochondrial STAT3-dependent oxidative phosphorylation.

[0027] Figure 7 PSE2 effectively inhibits lung and liver metastasis of TNBC in mice. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0029] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, 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 invention belongs. In the event of any conflict, the present specification shall take precedence.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] The technical principles of the present invention are as follows:

[0032] Pulsatilla chinensis (Bge.) Regel is a perennial herbaceous plant of the Ranunculaceae family, distributed in East Asia, especially in Jilin, Liaoning, Heilongjiang, Hebei, Shandong and other places in China. Pulsatilla saponin E2 (Pulchinenoside E2, PSE2; CAS No.: 244202-36-6; Molecular formula: C 53 H 86 O 21 ) is a saponin compound isolated from Pulsatilla chinensis. This study first discovered: 1) PSE2, the chemical component of Pulsatilla chinensis that exerts its primary pharmacological activity, exhibited the strongest activity in a pharmacological screening to inhibit TNBC cell migration; 2) STAT3 is the core target of PSE2 in its anti-TNBC cell metastasis action; and 3) PSE2 significantly inhibits p-STAT3 Tyr705 and STAT3 nuclear transcription; 4) PSE2 inhibits p-STAT3 Ser727 and mitochondrial oxidative phosphorylation; 5) PSE2 can effectively inhibit the lung and liver metastasis of TNBC in mice.

[0033] Based on this, the present invention proposes for the first time the use of PSE2 as a STAT3 dual phosphorylation inhibitor in the preparation of drugs for the prevention or treatment of triple-negative breast cancer. The present invention proves through experiments that PSE2, as the chemical component of Pulsatilla that exerts the main pharmacological effect, shows the strongest activity in the pharmacodynamic screening of inhibiting TNBC cell growth and migration. Compared with MCF-10A and MCF-7 cells, PSE2 is more selective in inhibiting the migration of MDA-MB-231 cells. Through network pharmacology, molecular docking, molecular dynamics and cellular thermal shift assay (Cellular Thermal Shift Assay, CETSA), the present invention found that STAT3 is a direct core target, and PSE2 directly forms hydrogen bonds with multiple amino acid residues in the STAT3-SH2 domain, and the PSE2-STAT3 complex has good stability. Transwell results showed that PSE2 significantly hindered the invasion and migration of TNBC cells; after knocking down STAT3, the inhibitory effect of PSE2 was weakened. Therefore, STAT3 is likely to be the direct core target of PSE2 to exert its anti-TNBC metastasis effect. Mechanistically, it was found that PSE2 significantly inhibited STAT3 (Tyr 705 ) and STAT3 (Ser 727 ) phosphorylation, inhibiting STAT3 transcriptional activity and mitochondrial STAT3-dependent oxidative phosphorylation. Animal experiments have confirmed that PSE2 significantly inhibits lung and liver metastasis of triple-negative breast cancer in mice. This suggests that PSE2 provides a promising candidate lead compound for the development of anti-triple-negative breast cancer metastasis drugs and has broad development and application prospects.

[0034] The following is a detailed description of the application of the STAT3 dual phosphorylation inhibitor of the present application in the preparation of drugs for preventing or treating triple-negative breast cancer, combined with examples and experimental data.

[0035] Example 1

[0036] In this example, drug efficacy screening on TNBC cell growth and migration was performed.

[0037] The experimental method is as follows:

[0038] First, a network pharmacology approach was used to screen the active ingredients of Pulsatilla chinensis. Targets corresponding to the active ingredients were collected from a drug target database. Targets corresponding to TNBC metastasis were screened from a disease database. A "Pulsatilla chinensis-active ingredient-target-TNBC metastasis" interaction network was constructed to predict the potential role of chemical components of Pulsatilla chinensis that exert the primary antitumor effect in TNBC metastasis. Subsequently, the cell viability and migration abilities of the putative active compounds of Pulsatilla chinensis were verified using MTT and Transwell assays, respectively. Finally, the Transwell assay was used to examine the effects of PSE2 on the migration of TNBC cells (MDA-MB-231), breast cancer cells (MCF-7), and normal breast epithelial cells (MCF-10A), highlighting the selectivity of PSE2 for TNBC cell migration.

[0039] 1. Network pharmacology prediction of the chemical components of Pulsatilla scabra that exert the main anti-tumor effects:

[0040] A network pharmacology approach was used to collect as many Pulsatilla components as possible from the TCMSP database (https: / / old.tcmsp-e.com / tcmsp.php) and literature. Active ingredients from Pulsatilla were screened using a drug-likeness (DL) ≥ 0.18 as a criterion. Active ingredients were then merged based on those reported in the literature. Subsequently, drug active ingredient targets were collected from the TCMSP, Super-PRED (https: / / prediction.charite.de / subpages / target_prediction.php), Swiss Target Prediction (STP, http: / / swisstargetprediction.ch / ), and PharmMapper (http: / / www.lilab-ecust.cn / pharmmapper / ) online databases. TNBC metastasis targets were screened using the GeneCards database. Cytoscape 3.10.1 software was used to construct a "Pulsatilla-active ingredient-target-TNBC metastasis" interaction network to predict the potential role of Pulsatilla active ingredients in TNBC metastasis.

[0041] 2. MTT method to verify the cell activity of effective compounds of Pulsatilla dasyphylla:

[0042] MDA-MB-231 cells were seeded in a 96-well plate and incubated overnight at 37°C in a cell culture incubator. 0-4 μM of the active compound described above was added to each well for 48 hours. 10 μL of MTT solution was then added and incubated for another 4 hours. The supernatant was discarded, and 100 μL of DMSO was added. The plate was shaken to mix thoroughly, and the OD value of each well was measured at 570 nm using a microplate reader.

[0043] 3. Transwell assay to verify cell migration of effective compounds of Pulsatilla dasyphylla:

[0044] MDA-MB-231 cells were seeded in the upper chamber of a Transwell nested tube, and 0-0.5 μM of the above-mentioned effective compound of Pulsatilla dasyphylla was added to the lower chamber for treatment for 12 h. The chamber was removed, fixed with 4% paraformaldehyde, and stained with crystal violet. The migrated cells were counted and photographed under a light microscope.

[0045] 4. Transwell assay to detect the selectivity of PSE2 on TNBC cell migration:

[0046] MDA-MB-231 cells were seeded in the upper chamber of a Transwell nested tube, and 0-1.0 μM PSE2 was added to the lower chamber for treatment for 24 h. The chamber was removed, fixed, and stained with crystal violet. The number of migrated cells was counted under a light microscope and photographed.

[0047] The experimental results are as follows:

[0048] Using network pharmacology methods, 58 Pulsatilla components were collected from the TCMSP database and published literature. The active ingredients of Pulsatilla were screened with a DL ≥ 0.18 standard. Combined with the active ingredients in the literature, 25 active ingredients of Pulsatilla were finally obtained ( Figure 1 A). Targets corresponding to drug active ingredients were collected from the TCMSP database and SwissTargetPrediction platform, and 5877 TNBC metastasis-related genes were obtained from the GeneCards disease database. The "Pulsatilla-active ingredient-target-TNBC metastasis" network was constructed using Cytoscape software ( Figure 1 A) to predict the potential role of the active ingredients of Pulsatilla in TNBC metastasis. At the cellular level, the inhibitory effects of these 25 active ingredients of Pulsatilla on MDA-MB-231 cell proliferation and migration were further explored. The results showed that saponin active ingredients (PSE2, PSE4, etc.) are the main chemical component group of Pulsatilla that exerts anti-proliferative and migration effects ( Figure 1 B-1C). PSE2 is the main chemical component of Pulsatilla truncatula that plays a major role in inhibiting cell growth (IC 50 =1.05μM 48h) and migration (inhibition rate 11.35%, 0.5μM PSE2, 12h) showed the strongest activity ( Figure 1 B-1C). In addition, compared with MCF-10A and MCF-7 cells, PSE2 inhibited the migration of MDA-MB-231 cells more selectively (P<0.05) ( Figure 1 D).

[0049] Figure 1Diagram showing drug efficacy screening for TNBC cell growth and migration. A: Pulsatilla-active ingredient-target-TNBC metastasis network constructed using Cytoscape 3.10.1 software: triangular nodes represent active ingredients in Pulsatilla; elliptical nodes represent putative targets of Pulsatilla for TNBC. B: MTT assay for the activity of the 25 putative Pulsatilla-active compounds at 0-4 μM in MDA-MB-231 cells: *P < 0.05, **P < 0.01, compared to the control group (0 μM, untreated). C: Transwell assay for the migration of the 25 putative Pulsatilla-active compounds at 0-0.5 μM in MDA-MB-231 cells: *P < 0.05, **P < 0.01, compared to the control group (0 μM, untreated). D: Transwell assay to detect the effect of 0-3 μM PSE2 on the migration of MDA-MB-231, MCF-7 and MCF-10A cells: **P<0.01, compared with the MDA-MB-231 group. Among them: 1. Pinoresinol, 2. Sophora flavescensine, 3. Korean scutellaria acetoside, 4. Antirrhinol, 5. Lanosterol, 6. Podophyllotoxin, 7. β-Sitosterol, 8. Isobehenolide, 9. Betulinic acid, 10. Tricosanoic acid, 11. Oleanolic acid, 12. Ergosterol, 13. Isorhamnetin, 14. Sitoside, 15. Stigmasterol, 16. Ursolic acid, 17. Tetracosanoic acid, 18. Scutellaria acetoside B4, 19. Scutellaria acetoside A, 20. Scutellaria acetoside E2, 21. Scutellaria acetoside E4, 22. Hydroxybetulinic acid, 23. Hederagenoside A, 24. β-Sitosterol acetate, 25. Hederagenoside.

[0050] Example 2

[0051] This example explores the core target of PSE2 in combating TNBC cell metastasis.

[0052] The experimental method is as follows:

[0053] Network pharmacology was used to identify PSE2 targets and TNBC metastasis-associated targets. These targets were then intersected to identify shared targets. Core targets were further identified using the Centiscape 2.2 plugin in Cytoscape software, with thresholds set. Molecular docking was performed to compare and predict the most direct core target of PSE2 (STAT3). Molecular dynamics simulations were performed to examine the binding pattern between PSE2 and STAT3. CETSA assays were used to determine the binding stability of PSE2 and STAT3 proteins. After knockdown of STAT3, a Transwell assay was used to verify that STAT3 is a key target of PSE2 in inhibiting TNBC cell invasion and migration.

[0054] 1. Network pharmacology prediction of PSE2 as a core target for the treatment of TNBC metastasis:

[0055] Network pharmacology was used to identify PSE2 targets in the Super-PRED, STP, and PharmMapper online databases. TNBC metastasis targets were identified using the GeneCards database. The two databases were intersected to identify shared targets. Core targets were further identified using the Centiscape 2.2 plugin in Cytoscape software, with thresholds set.

[0056] 2. Molecular docking to screen the targets of PSE2:

[0057] Crystal structures corresponding to 11 core targets were obtained using the AlphaFold2 or RCSB PDB databases. The resulting protein crystals were processed using the Protein Preparation Wizard module of Schrödinger software for protein preprocessing, regenerate states of native ligand, H-bond assignment optimization, protein energy minimization, and water removal. The optimal binding site was predicted using the SiteMap module of Schrödinger software, and the Receptor Grid Generation module of Schrödinger software was used to set the optimal enclosing box to perfectly enclose the predicted binding site. Based on this, the active sites of the 11 proteins were obtained. The processed ligand compound PSE2 was molecularly docked with the active sites of the 11 proteins, and the MM-GBSA binding free energy was calculated and analyzed.

[0058] 3. Molecular dynamics simulation to detect the binding mode of PSE2 and STAT3:

[0059] Molecular dynamics simulations were performed using the OPLS4 force field to parameterize the protein and small molecule, and the SPCE model to parameterize the aqueous solvent. The docked complexes were simulated unrestricted for 100 ns. Maestro 2023 was used to analyze interactions and generate dynamic trajectory animations. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) plots of PSE2 and STAT3 proteins were obtained, along with stable binding modes and temporal changes in the interaction between specific amino acids in the PSE2 and STAT3 proteins.

[0060] 4. Detection of the binding of PSE2 and STAT3 protein by CETSA method:

[0061] When TNBC cells reached 80% growth, they were treated with 0.75 μM PSE2 or DMSO for 1 hour. The cells were harvested and suspended in PBS containing PMSF and phosphatase inhibitors. The cells were aliquoted into 0.2 mL centrifuge tubes, 100 μL per tube. Each tube was heated to the designated temperature for 2 minutes, immediately cooled to room temperature, and frozen in liquid nitrogen. To lyse the cells, each tube was subjected to three freeze-thaw cycles in liquid nitrogen. The cell lysate was centrifuged at 20,000 g for 20 minutes at 4°C. STAT3 protein levels in the cell lysate were analyzed by Western blot.

[0062] 5. Transwell assay for cell invasion and migration:

[0063] TNBC cells with stable STAT3 knockdown were seeded in the upper chamber of a Transwell insert containing Matrigel. The lower chamber was treated with or without 0.75 μM PSE2 for 24 hours. The chamber was then removed, fixed with 4% paraformaldehyde, and stained with crystal violet. Invading cells were counted and photographed under a light microscope. Cell migration assays were performed without Matrigel, using the same method as above.

[0064] The experimental results are as follows:

[0065] In order to find the direct target of PSE2 in treating TNBC metastasis, the 97 potential PSE2 targets collected were intersected with 2000 TNBC metastasis-related targets to obtain 41 common targets ( Figure 2 A). Using the network pharmacology method, the Centiscape2.2 plug-in of Cytoscape software was used to set the Closeness unDir, Betweenness unDir and Degree unDir thresholds to identify 11 key target genes (STAT3, JUN, MMP9, HSP90AA1, FGF2, KDR, IL2, ACE, PTPRC, ITGAV, NR3C1) ( Figure 2 B) The crystal structures corresponding to these 11 proteins were molecularly docked with PSE2, and it was found that STAT3 had the lowest binding free energy (kcal / mol = -28.62) ( Figure 2 C), indicating the highest binding stability. In addition, PSE2 penetrates deep into the active pocket of STAT3, and the residue MET of STAT3 660 、ILE 659 etc. form hydrophobic interactions with PSE2, and PSE2 and residue GLU 625 LYS 626 Each forms a hydrogen bond, suggesting that STAT3 is likely to be the direct core target ( Figure 2D). Molecular dynamics simulations revealed that the PSE2-STAT3 complex has small RMSF fluctuations and stable RMSD values, indicating that the PSE2-STAT3 complex has good stability ( Figure 3 A) The interaction trajectory between PSE2 and specific amino acids of STAT3 protein shows that the amino acid residue GLU in the STAT3-SH2 domain 625 LYS 626 GLN 635 SER 636 , GLU 638 TYR 657 LYS 658 、MET 660 Multiple contacts with PSE2 ( Figure 3 B). Figure 3 C shows the interactions that occurred for more than 10.0% of the simulation time (i.e., the interaction time exceeded 10 ns in 100 ns) in the selected trajectory. The results show that PSE2 interacts with the amino acid residue TYR of the STAT3 protein A chain. 657 (35%), MET 660 (26% and 28%), SER 636 (14%), GLN 635 (20%), LYS 626 (24% and 20%) directly form hydrogen bonds, form one hydrogen bond within the molecule (94%), and also form a hydrogen bond with LYS 658 、TRP 623 , GLU 625 ASP 627 The water bridge is formed (the simulation time can be used to quantitatively evaluate the stability or strength of the interaction). CETSA experiments showed that 0.75 μM PSE2 prevented the thermal denaturation of STAT3 protein, revealing that PSE2 can target and bind to STAT3 protein at the cellular level ( Figure 3 D). Transwell results showed that 0.75μM PSE2 significantly inhibited the invasion and migration of TNBC cells (P<0.05); after knocking down STAT3, the inhibitory effect of PSE2 was weakened (P<0.05) ( Figure 4 A-4B). Therefore, STAT3 is likely to be the core target of PSE2 in exerting its anti-TNBC effect.

[0066] from Figure 2 It can be seen that STAT3 is the direct core target of PSE2. Figure 2A: Venn diagram showing the shared genes between TNBC metastasis-associated genes and PSE2 target genes predicted by network pharmacology analysis. B: 11 core target genes were identified from the 41 shared genes using a threshold-based approach using the Centiscape 2.2 plugin in Cytoscape 3.10.1 software. C: Molecular docking results of PSE2 with the active sites of these 11 core targets. D: 2D and 3D images of PSE2 docking with STAT3 protein.

[0067] from Figure 3 It can be seen that PSE2 can stably bind to STAT3 protein. Figure 3 A: Comprehensive molecular dynamics simulation results of the interaction between PSE2 and STAT3. B: The entire trajectory of the interaction between PSE2 and specific amino acids in STAT3. C: Schematic diagram of the interaction between PSE2 and STAT3 residues. D: CETSA analysis of the thermal stability of STAT3 in MDA-MB-231 cells before and after treatment with 0.75 μM PSE2 at different temperatures.

[0068] from Figure 4 It can be seen that STAT3 is the key target of PSE2 in inhibiting TNBC cell invasion and migration. Figure 4 A: Transwell assay to detect the invasion and migration levels of MDA-MB-231 cells in the control and STAT3 knockdown groups treated with 0.75 μM PSE2. B: Figure 4 Results in A were quantified: **P < 0.01, compared with the control group. NS indicates no significant difference.

[0069] Example 3

[0070] PSE2 significantly inhibited p-STAT3 (Tyr 705 ) and STAT3 nuclear transcription.

[0071] The experimental method is as follows:

[0072] First, Western blot was used to detect p-STAT3 (Tyr 705 )、p-STAT3(Ser 727 ) and STAT3 protein levels. Because STAT3, as a transcription factor, binds to specific DNA response elements to regulate target gene expression, we transiently transfected HEK-293T cells expressing low STAT3 with pGL3-STAT3, STAT3C, and Renilla luciferase plasmids and performed luciferase reporter assays to further investigate whether PSE2 inhibits STAT3-dependent transcriptional activation.

[0073] 1. Western blot detection of p-STAT3 (Tyr 705 )、p-STAT3(Ser 727 ) and STAT3 protein levels:

[0074] After incubation with a certain concentration of PSE2 for 48 h, TNBC cells were lysed and quantified. Proteins were separated by 12.5% ​​SDS-polyacrylamide gel electrophoresis, and the membranes were transferred to PVDF membranes activated with methanol at 100 V for 2 h. The membranes were blocked with 5% skim milk at room temperature for 1 h. p-STAT3 (Tyr 705 )、p-STAT3(Ser 727 ), STAT3 primary antibody and horseradish peroxidase-labeled secondary antibody incubation, and protein bands were visualized and analyzed using a protein developer.

[0075] 2. Dual luciferase reporter gene assay:

[0076] HEK-293T cells were co-transfected with 50 ng of pGL3-STAT3 reporter gene plasmids, 50 ng of STAT3C, and 40 ng of Renilla luciferase as an internal control. After the cells stably expressed the STAT3 reporter gene, they were treated with various concentrations of PSE2 for 24 hours. The cells were lysed, and luciferase activity was measured within 24 hours using a dual-luciferase reporter gene assay kit.

[0077] The experimental results are as follows:

[0078] We investigated the effect of PSE2 on STAT3 phosphorylation in HS-578T and MDA-MB-231 cells. 705 Phosphorylation is closely related to the nuclear function of STAT3, and Ser 727 Phosphorylation regulates the activity of the mitochondrial electron transport chain. Unlike many STAT3 inhibitors, PSE2 also significantly inhibits STAT3 Tyr705 and STAT3 Ser727 Phosphorylation (P<0.05) ( Figure 5 A). STAT3, as a transcription factor, binds to specific DNA response elements and regulates the expression of target genes. We further investigated whether PSE2 inhibits STAT3-dependent transcriptional activation by using luciferase gene reporter experiments. HEK-293T cells with low STAT3 expression were transiently co-transfected with pGL3-STAT3, STAT3C, and Renilla luciferase plasmids. Compared with the 0 μM PSE2-treated group, cells treated with different concentrations of PSE2 had lower luciferase activity, indicating that PSE2 significantly inhibited STAT3 transcriptional activity (P<0.05) ( Figure 5 B).

[0079] from Figure 5 It can be seen that PSE2 significantly inhibits p-STAT3 (Tyr 705 ) and STAT3 nuclear transcription. Figure 5 Middle, A: After PSE2 was applied to HS-578T and MDA-MB-231 cells for 24 h, the expression of p-STAT3 (Tyr 705 )、p-STAT3(Ser 727 ) and STAT3 protein levels. B: STAT3C, pGL3-STAT3 promoter, and Renilla luciferase (as a reference) plasmids were transiently co-transfected into HEK-293T cells. Luciferase activity was measured 24 hours after PSE2 treatment as described above. *P < 0.05, **P < 0.01, statistically significant differences compared to the 0 μM PSE2-treated group. NS indicates no significant difference.

[0080] Example 4

[0081] PSE2 inhibits p-STAT3 (Ser 727 ) and mitochondrial oxidative phosphorylation

[0082] The experimental method is as follows:

[0083] TNBC cells were seeded on cell culture plates and treated with PSE2 for a period of time. Oligomycin (a phosphorylation inhibitor), FCCP (a mitochondrial oxidative phosphorylation uncoupler), and antimycin A + rotenone (a mitochondrial respiratory chain inhibitor), or glucose, oligomycin, and 2-deoxy-D-glucose (a glycolysis inhibitor) were injected sequentially. The effect of PSE2 on mitochondrial STAT3 function was assessed by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, a glycolysis indicator) using a Seahorse XFe96 analyzer.

[0084] Mitochondrial bioenergetics analysis:

[0085] Mitochondrial bioenergetics were assessed using OCR and ECAR using a Seahorse XFe96 analyzer. OCR reflects mitochondrial oxidative phosphorylation. In this experiment, 2×10 4MDA-MB-231 cells were seeded onto cell culture plates. After 1 hour of PSE2 treatment, the medium in the plates was replaced with fresh assay medium using the instrument's fluid exchange procedure. Following basal respiration, oligomycin, FCCP, and antimycin A + rotenone were sequentially injected according to the manufacturer's protocol to measure ATP-associated respiration and backup respiration. ECAR reflects glycolysis. In this experiment, the cell density per well and the duration of PSE2 treatment were consistent with those used to measure OCR. Glucose, oligomycin, and 2-deoxy-D-glucose were sequentially injected to assess cellular glycolysis and the ability of cells to resume glycolysis.

[0086] The experimental results are as follows:

[0087] First, we studied the effect of PSE2 on two major energy production pathways in TNBC cells: mitochondrial oxidative phosphorylation and glycolysis. Oxygen consumption rate (OCR) and ECAR are indicators of oxidative phosphorylation and glycolysis, respectively. Figure 6 As shown in Figure A, PSE2 inhibited oxidative phosphorylation in TNBC cells in a concentration-dependent manner. To verify whether the inhibitory effect was related to STAT3, we constructed MDA-MB-231 cells with stable STAT3 knockdown. The oxidative phosphorylation level in cells with stable STAT3 knockdown was significantly inhibited ( Figure 6 B) PSE2-treated STAT3 stably knocked-down MDA-MB-231 cells did not show significant inhibition of oxidative phosphorylation ( Figure 6 C). However, PSE2 had no significant effect on glycolysis in MDA-MB-231 cells ( Figure 6 D).

[0088] Figure 6 PSE2 inhibited p-STAT3 (Ser 727 ) and mitochondrial STAT3-dependent oxidative phosphorylation. Figure 6 A: Oxygen consumption rate was measured using a Seahorse XF96 extracellular flux analyzer, revealing that OCR in MDA-MB-231 cells treated with 0.75-1.5 μM PSE2 was suppressed. B: STAT3 knockdown inhibited oxygen consumption rate in MDA-MB-231 cells. C: Oxygen consumption rate in STAT3 knockdown MDA-MB-231 cells before and after PSE2 treatment. D: Extracellular acidification rate in PSE2-treated MDA-MB-231 cells.

[0089] Example 5

[0090] In this example, an experiment was conducted to investigate the effect of PSE2 on the lung and liver metastasis of TNBC in mice.

[0091] The experimental method is as follows:

[0092] Thirteen female immunodeficient (nu / nu) nude mice (18-22 g) were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. and housed in the specific pathogen-free facility of the Experimental Animal Center of Southwest Medical University. 6 Nude mice were randomly divided into two groups: a model group (n=7) and a PSE2 group (n=6). The PSE2 (5 mg / kg) group received intraperitoneal injections of 5 mg / kg of PSE2 every other day until the end of the experiment. The model group received an equal volume of saline every other day until the end of the experiment. Mice were fed a healthy diet and water daily, and their general physical signs were observed for 31 days. After the experiment, mice were sacrificed, and TNBC lung and liver metastases were isolated. Organ tissues, including heart and kidney, were also obtained and fixed with 4% paraformaldehyde. Pathological changes in tumor metastases were examined using hematoxylin-eosin (HE) staining. Pathological changes in multiple normal organ tissues in each group were investigated to assess the toxic effects of PSE2. The number of lung and liver metastases was also determined. This study was reviewed and approved by the Animal Research Ethics Committee of Southwest Medical University (approval number: 20240305-005).

[0093] The experimental results are as follows:

[0094] We established a nude mouse model of MDA-MB-231 cell lung and liver metastasis to explore the therapeutic effect of PSE2 on TNBC lung and liver metastasis mice. The results showed that PSE2 treatment did not observe significant changes in body weight or other toxic signs ( Figure 7 A). After 31 days of the experiment, the mice were killed and it was found that PSE2 significantly inhibited the lung and liver metastasis of TNBC cells and reduced the number of lung and liver metastatic foci ( Figure 7 In addition, intraperitoneal injection of 5 mg / kg PSE2 every other day until the end of the experiment did not cause obvious organic damage to the mouse heart and kidney tissues ( Figure 7 F), suggesting that PSE2 has less toxic side effects.

[0095] Figure 7 PSE2 was shown to effectively inhibit lung and liver metastasis of TNBC in mice. Figure 7Figure 2: A: Body weight changes of mice in the model group (n=7) and the PSE2-treated group (n=6). B: HE staining of the lungs of mice in the model group and the PSE2-treated group. C: Number and quantification of lung metastases in mice in the model group (n=7) and the PSE2-treated group (n=6). D: Number and quantification of liver metastases in mice in the model group (n=7) and the PSE2-treated group (n=6). E: HE staining of the liver in mice in the model group and the PSE2-treated group. F: HE staining of heart, kidney, and other organ tissues in mice in the model group and the PSE2-treated group: *P<0.05, **P<0.01, showing statistically significant differences compared with the model group.

[0096] Data were analyzed using SPSS 13.0 statistical software. Data related to cell lines were repeated at least three times and presented as mean ± SD. Differences between groups were assessed using one-way analysis of variance and LSD post hoc tests (if variances were homogeneous); otherwise, Dunnett's T3 test was used for multiple group comparisons. For animal studies, Student's T test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0097] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0098] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0099] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of a STAT3 dual phosphorylation inhibitor in the preparation of a drug for preventing or treating triple-negative breast cancer, characterized in that: The STAT3 dual phosphorylation inhibitor is pulsatilla saponin E2 and / or a pharmaceutically acceptable salt thereof. The structural formula of pulsatilla saponin E2 is as follows:

2. Use of pulsatilla saponin E2 and / or its pharmaceutically acceptable salt as a STAT3 dual phosphorylation inhibitor in the preparation of a drug for inhibiting the growth or metastasis of triple-negative breast cancer cells.

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