Application of Saikosaponin A in the Preparation of Drugs for Treating Oral Squamous Cell Carcinoma and Drug Compositions

Abstract

This invention discloses the application of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma and pharmaceutical compositions, belonging to the field of biomedical technology. The invention discloses the application of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma, determining that saikosaponin A inhibits tumor cell proliferation, migration, and invasion by regulating the phosphorylation level of the PI3K / AKT signaling pathway and downregulating the expression of target genes (MMP12, BMP2, CASP7, IGF1R, and MMP2). The binding stability was verified through molecular dynamics simulations. This provides clear technical parameters for the preparation of highly effective and low-toxicity anti-OSCC drugs, solving the problems of strong drug resistance and unclear mechanisms in existing chemotherapy regimens for oral squamous cell carcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma and pharmaceutical compositions thereof. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, accounting for approximately 90% of all oral malignancies. Its highly aggressive and heterogeneous nature poses a serious threat to patients' lives. Currently, for locally advanced patients, the main treatment regimen is surgical resection combined with radiotherapy and chemotherapy; however, the five-year overall survival rate remains below 50%, and the risk of recurrence and metastasis is high. Chemotherapy resistance is a key factor leading to treatment failure; therefore, developing new and more effective therapeutic drugs and strategies is of significant clinical importance.

[0003] In recent years, traditional Chinese medicine (TCM) has gained increasing attention as an important component of comprehensive cancer treatment, often considered a crucial supplement to traditional therapies. Natural phytochemicals, due to their good biocompatibility and multi-target regulatory properties, have shown great potential in adjuvant cancer therapy. Saikosaponin A, one of the main active ingredients extracted from the TCM herb Bupleurum chinense, has been shown to have inhibitory effects in various cancers, including gastric cancer, pancreatic cancer, cervical cancer, and prostate cancer. However, the quality of the drug is not universally applicable to different cancer sites and subtypes. Currently, the specific role, direct target, and molecular mechanism of saikosaponin A in oral squamous cell carcinoma (OSCC) remain unclear, and there are still research gaps in systematic target validation and pharmacodynamic evidence, which to some extent limits its application in the development of clinical anti-OSCC drugs. Summary of the Invention

[0004] This invention discloses the application of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma, and provides clear technical parameters and pharmacodynamic support for the preparation of highly effective and low-toxicity anti-OSCC drugs, so as to solve the technical bottleneck of existing saikosaponin A in the clinical anti-OSCC drug development.

[0005] The technical solution provided by this invention is as follows: In a first aspect, the present invention discloses the application of saikosaponin A in the preparation of a drug for treating oral squamous cell carcinoma.

[0006] Furthermore, the drug is used to inhibit the proliferation, migration, and invasion of oral squamous cell carcinoma cells.

[0007] Furthermore, the oral squamous cell carcinoma cells are CAL-27 or HSC-3 cells.

[0008] Furthermore, the drug is used to downregulate the expression of one or more genes among MMP12, BMP2, CASP7, IGF1R, and MMP2.

[0009] Furthermore, the drug is used to inhibit the phosphorylation level of the PI3K / AKT signaling pathway.

[0010] On the other hand, the present invention provides a pharmaceutical composition for treating oral squamous cell carcinoma, the pharmaceutical composition comprising an effective dose of saikosaponin A and its pharmaceutically acceptable carrier.

[0011] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention reveals for the first time the new use of saikosaponin A in the treatment of oral squamous cell carcinoma: Through rigorous experiments, this invention has confirmed that saikosaponin A can significantly inhibit the proliferation, migration and invasion of oral squamous cell carcinoma cells, and has no obvious toxicity to normal oral epithelial cells, providing a new potential candidate drug for the treatment of oral squamous cell carcinoma. (2) This invention clarifies for the first time the key target and signaling pathway of saikosaponin A in the fight against oral squamous cell carcinoma: This invention comprehensively utilizes multiple methods such as network pharmacology, bioinformatics, molecular dynamics simulation, and in vitro experiments to systematically reveal for the first time that saikosaponin A exerts its anti-tumor effect by targeting five core genes—MMP12, BMP2, CASP7, IGF1R, and MMP2—and regulating the downstream PI3K / AKT signaling pathway. This elucidation of the mechanism provides a solid theoretical foundation for understanding its efficacy; (3) This invention provides reliable efficacy verification data: This invention not only predicts the binding mode of drugs to targets through computational simulation, but also verifies its regulatory role on the PI3K / AKT pathway and core targets at the protein and mRNA levels through molecular biology experiments such as Western blot and qPCR. The evidence chain is complete and highly credible. Attached Figure Description

[0012] Figure 1 This is a heatmap showing the correlation between the expression of the core targets (MMP12, BMP2, CASP7, IGF1R, and MMP2) and immune cell infiltration in Example 1. Figure 2 The figures show the molecular docking results of the core target and saikosaponin A in Example 1, where a is the molecular docking result of BMP2-saikosaponin A; b is the molecular docking result of CASP7-saikosaponin A; c is the molecular docking result of IGF1R-saikosaponin A; d is the molecular docking result of MMP2-saikosaponin A; and e is the molecular docking result of MMP12-saikosaponin A. Figure 3The graph shows the molecular dynamics simulation results of the core target and saikosaponin A in Example 1. In the graph, a is the RMSD curve of saikosaponin A and the core target; b is the SASA curve of saikosaponin A and the core target; c is the Rg curve of saikosaponin A and the core target; d is the hydrogen bond count of saikosaponin A and the core target; and e~i are Gibbs energy landscapes of the five core targets and saikosaponin A.

[0013] Figure 4 The graph shows the cell viability of three cell types (CAL-27, HSC-3, and HOK) after treatment with different concentrations of saikosaponin A, as determined by the CCK-8 assay in Example 2.

[0014] Figure 5 This is a diagram showing the colony-forming ability of CAL-27 and HSC-3 cells treated with different concentrations of saikosaponin A in Example 2.

[0015] Figure 6 This is a graph showing the cell migration ability of CAL-27 and HSC-3 cells after treatment with different concentrations of saikosaponin A in Example 2.

[0016] Figure 7 This is a graph showing the cell invasion ability of CAL-27 and HSC-3 cells after treatment with different concentrations of saikosaponin A in Example 2.

[0017] Figure 8 This image shows the protein bands of PI3K, p-PI3K, AKT, and p-AKT in CAL-27 cells in Example 2, along with relative grayscale analysis and relative protein expression analysis.

[0018] Figure 9 This image shows the protein bands of PI3K, p-PI3K, AKT, and p-AKT in HSC-3 cells in Example 2, along with relative grayscale analysis and relative protein expression analysis.

[0019] Figure 10 This is a graph showing the mRNA expression analysis of MMP12, BMP2, CASP7, IGF1R, and MMP2 in CAL-27 cells in the control group and the 10µmol / L saikosaponin A treatment group in Example 2.

[0020] Figure 11 This is a graph showing the mRNA expression analysis of MMP12, BMP2, CASP7, IGF1R, and MMP2 in the control group and the 10µmol / L saikosaponin A treatment group of HSC-3 cells in Example 2. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and various embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0022] Unless otherwise stated, the terms or phrases used herein should not be considered uncertain or unclear unless specifically defined, and should be understood in their ordinary sense. When trade names appear in this document, they are intended to refer to the corresponding product or its active ingredient.

[0023] Unless otherwise stated, all reagents and raw materials used in this invention are commercially available.

[0024] Example 1 This embodiment analyzes the feasibility of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma based on the molecular level of targets and pathways in network pharmacology and bioinformatics. The analysis process is as follows: (1) Acquisition of target of saikosaponin A: 120 potential targets of saikosaponin A were screened and integrated through Swiss Target Prediction, Pharm Mapper and SEA database.

[0025] (2) Target acquisition for oral squamous cell carcinoma: The GSE30784 gene chip dataset was downloaded from the GEO database. Differentially expressed genes were screened using the R language "limma" package (adj.P<0.05 and |log2FC|>0.5); the yellow module genes most relevant to the disease were identified by weighted gene co-expression network analysis (WGCNA). The intersection of the saikosaponin A target, differentially expressed genes, and WGCNA module genes was used to obtain 50 potential therapeutic targets.

[0026] (3) PPI Network and Enrichment Analysis: Fifty potential targets were imported into the STRING database to construct a protein-protein interaction (PPI) network, which was then visualized using Cytoscape software. Enrichment analysis of the GO and KEGG pathways was performed using the DAVID database. The results showed that these targets were mainly enriched in cancer-related signaling pathways such as PI3K / AKT.

[0027] (4) Machine learning screening of key targets: LASSO regression, SVM-RFE and random forest were used to screen 50 potential targets. By taking the intersection of the screening results of the three algorithms, MMP12, BMP2, CASP7, IGF1R and MMP2 were finally identified as core targets.

[0028] (5) Immune infiltration analysis: The CIBERSORT algorithm was used to analyze the infiltration of 22 immune cell types in the GSE30784 dataset, and the correlation between the expression of 5 core targets and immune cell infiltration was analyzed. The results are as follows: Figure 1 As shown, the relative abundance of memory B cells, naive B cells, resting dendritic cells, M2 macrophages, resting mast cells, CD4 naive T cells, and CD8 T cells was significantly negatively correlated with the expression levels of all hub genes, while the relative abundance of activated dendritic cells, M0 macrophages, M1 macrophages, and activated mast cells was significantly positively correlated with the expression levels of all hub genes.

[0029] (6) Molecular docking technology was used to verify the binding characteristics of saikosaponin A to the core targets. First, the three-dimensional crystal structures of five key targets were obtained from the PDB protein structure database: MMP12 (1y93), BMP2 (4n1d), CASP7 (4jr2), IGF1R (1imx), and MMP2 (1gen). Based on topological parameters, saikosaponin A was selected as a candidate ligand molecule. Semi-flexible docking calculations were performed using the AutoDock tool, such as... Figure 2 The results show that the binding free energy of all ligand-receptor complexes is below -5 kcal / mol, demonstrating the molecular interaction modes of representative complexes, including key forces such as hydrogen bond networks, π-π stacking, and hydrophobic cavity binding.

[0030] (7) Molecular dynamics simulations were conducted to analyze the optimal binding conformations of saikosaponin A with MMP12, BMP2, CASP7, IGF1R, and MMP2 to reveal the dynamic binding characteristics of their complexes. A simulation system with a duration of 100 ns was constructed, and the root mean square deviation (RMSD) was used to assess the stability of the system. It was found that the RMSD values ​​of the five complexes stabilized after 20 ns (…). Figure 3 a) indicates that no significant conformational drift occurred in the protein backbone. SASA refers to the surface area of ​​a molecule that can be contacted by solvent molecules, and it is an important parameter describing the solvation properties of a molecule. The SASA parameters of these five complexes fluctuated little within 100 ns, indicating that the complexes were relatively stable. The radius of gyration (Rg) reflects the binding tightness and constraint of the system. A higher Rg value is associated with a better chance of generating flexible ligands. Therefore, the smaller the Rg value, the more compact the molecule. The lowest Rg value of IGF1R-SaikosaponinA is 11 Å, indicating relatively good binding; while the Rg value of CASP7-SaikosaponinA is relatively high, indicating relatively poor surface binding; the Rg values ​​of the above five complexes tend to be stable within 100 ns. Figure 3c). Hydrogen bonds are among the strongest non-covalent interactions; the more hydrogen bonds present, the stronger the binding force. Hydrogen bond dynamics analysis shows that 7 hydrogen bonds can form between saikosaponin A and MMP12, 5 hydrogen bonds between saikosaponin A and BMP2, 3 hydrogen bonds between saikosaponin A and CASP7, 6 hydrogen bonds between saikosaponin A and MMP2, and 2 hydrogen bonds between saikosaponin A and IGF1R. Figure 3 d). Free energy landscape analysis further confirmed that all five complexes exhibit a single energy funnel characteristic in the RMSD-Rg two-dimensional phase space. Figure 3 e~i).

[0031] The results above demonstrate that this study successfully predicted 50 potential targets of saikosaponin A for OSCC, identified PI3K / AKT as the key signaling pathway, and screened out five core pivot genes: MMP12, BMP2, CASP7, IGF1R, and MMP2. Molecular docking results showed that the binding free energy of saikosaponin A to the five targets was below -5 kcal / mol, indicating stable binding. Molecular dynamics simulations showed that the RMSD of each complex tended to stabilize after 20 ns, and the SASA and Rg parameters fluctuated little, indicating that the complex system was stable. Saikosaponin A has the potential to be used to prepare drugs for the treatment of oral squamous cell carcinoma at the molecular level.

[0032] Example 2 This embodiment analyzes the feasibility of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma at the cellular level. The analysis process is as follows: (1) To investigate the effects of saikosaponin A on the activity of OSCC cell lines CAL-27 and HSC-3, as well as human normal keratinocytes (NOK), the cells were treated with different concentrations of saikosaponin A (0, 5, 10, 15, 20, 25 μmol / L) for 24 hours. Subsequently, the activity was measured using the CCK-8 assay. Figure 4 The results showed that the viability of CAL-27 and HSC-3 cells gradually decreased with increasing concentration of saikosaponin A. However, when human NOK cells were treated with concentrations below 20 μmol / L, saikosaponin A did not significantly inhibit HOK cell viability. This indicates that low concentrations of saikosaponin A have no significant toxicity to normal oral epithelial cells. To further elucidate the effect of saikosaponin A on the proliferation ability of OSCC cells, a colony formation assay was used to detect the colony formation ability of CAL-27 and HSC-3 cells under different concentrations of saikosaponin A treatment. Figure 5The results showed that saikosaponin A had a concentration-dependent inhibitory effect on the proliferation of OSCC cells. Notably, treatment with 15 μmol / L saikosaponin A almost completely inhibited the proliferation of cancer cells. These results demonstrate the concentration-dependent inhibitory effect of saikosaponin A on OSCC cell proliferation.

[0033] To investigate the effects of saikosaponin A on the migration and invasion abilities of OSCC cells, a scratch assay was performed to observe the migration ability of OSCC cells after treatment with different concentrations of saikosaponin A (0, 5, 10 μmol / L). Figure 6 The results showed that saikosaponin A significantly inhibited the migration of CAL-27 and HSC-3 cells in a concentration-dependent manner. Furthermore, Transwell migration and invasion assays were performed, and the results showed that the migration and invasion abilities of both CAL-27 and HSC-3 cells decreased after treatment with saikosaponin A. Figure 7 ).

[0034] Western blot analysis showed that treatment with different concentrations of saikosaponin A (0, 5, 10, 15 μmol / L) reduced the protein expression levels of p-PI3K and p-AKT in CAL-27 and HSC-3 cells (P < 0.05), and the reduction was concentration-dependent. Figure 8 , 9 The primer sequences used in this study are listed in Supplementary Table S2. Saikosaponin A significantly downregulated the mRNA expression of MMP12, BMP2, CASP7, IGF1R, and MMP2 in CAL-27 and HSC-3 cells (p<0.05). Figure 10 , 11 ).

[0035] It is evident that at the cellular level, saikosaponin A effectively inhibits the proliferation, migration, and invasion of oral squamous cell carcinoma by targeting core genes such as MMP12, BMP2, CASP7, IGF1R, and MMP2, thereby suppressing the PI3K / AKT signaling pathway. Therefore, saikosaponin A or its pharmaceutical compositions can be used to prepare drugs for the treatment of oral squamous cell carcinoma.

[0036] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. Application of saikosaponin A in the preparation of drugs for treating oral squamous cell carcinoma.

2. The application as described in claim 1, characterized in that, The drug is used to inhibit the proliferation, migration, and invasion of oral squamous cell carcinoma cells.

3. The application as described in claim 2, characterized in that, The oral squamous cell carcinoma cells are CAL-27 or HSC-3 cells.

4. The application as described in claim 1, characterized in that, The drug is used to downregulate the expression of one or more genes among MMP12, BMP2, CASP7, IGF1R, and MMP2.

5. The application as described in claim 1, characterized in that, The drug is used to inhibit the phosphorylation level of the PI3K / AKT signaling pathway.

6. A pharmaceutical composition for treating oral squamous cell carcinoma, characterized in that, The pharmaceutical composition comprises an effective dose of saikosaponin A and its pharmaceutically acceptable carrier.

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