Use of matrine in preparation of medicine for treating oral squamous cell carcinoma and medicine composition

Abstract

The application discloses application of echinacoside in preparation of a medicine for treating oral squamous cell carcinoma and a medicine composition, relates to the technical field of biological medicine, and discloses, for the first time, that echinacoside (ECH) is applied to treatment of oral squamous cell carcinoma (OSCC), and through network pharmacology, in-vitro cell experiments and in-vivo animal experiments, the significant anti-tumor effect is verified, and a new candidate drug for treatment of OSCC is provided.

Description

Technical Field

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

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral and maxillofacial region, accounting for approximately 90% of oral cancers. Its high incidence rate, with 377,713 new cases worldwide in 2020 alone, represents about 2% of all cancers. Its highly aggressive and heterogeneous nature poses a serious threat to patients' lives. Currently, clinical treatment for OSCC primarily employs a comprehensive sequential therapy, with surgical resection as the main approach. Chemotherapy, alone or in combination with other drugs, has become the standard or first-line treatment option for advanced stages. Commonly used chemotherapy drugs have significant side effects, and patients receiving platinum-based therapy are prone to acquired resistance, often leading to treatment failure and tumor recurrence. In recent years, although cancer treatment methods have continuously improved, the improvement in the survival rate of OSCC patients has been limited. The overall 5-year survival rate for patients with advanced OSCC is only about 50%. Therefore, finding a safe and effective new anticancer drug is particularly important.

[0003] In recent years, numerous studies have shown that traditional Chinese medicine (TCM) and its natural products are frequently used as adjunctive therapies for various cancers, such as non-small cell lung cancer, hepatocellular carcinoma, and breast cancer, due to their unique advantages of high efficacy and few side effects. Clinical trials have demonstrated that TCM has lower toxicity and side effects than chemotherapy drugs, thus it is often considered an important supplement to traditional treatment modalities. Echinacoside (ECH) is a natural phenylethyl glycoside derived from traditional Chinese herbs such as Echinacea and Cistanche deserticola. Studies have shown that ECH possesses various pharmacological activities, including anti-inflammatory, antioxidant, neuroprotective, immunomodulatory, and antitumor effects. In anticancer applications, ECH can inhibit the proliferation, invasion, and migration of cancer cells and induce apoptosis, exhibiting significant inhibitory effects on various tumors such as breast cancer, liver cancer, and lung cancer. However, currently, no studies have confirmed the efficacy and mechanism of action of ECH against OSCC. Research gaps remain in systematic target validation and pharmacodynamic evidence for ECH, 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 echinacoside 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 bottlenecks of existing echinacoside 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 use of echinacoside in the preparation of a medicament for treating oral squamous cell carcinoma.

[0006] Furthermore, the drug is used to inhibit the activation of the PI3K / AKT signaling pathway, thereby inhibiting 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 upregulate E-cadherin protein expression and downregulate N-cadherin, Vimentin, MMP-2, and MMP-9 protein expression.

[0009] Furthermore, the drug is used to upregulate the expression of pro-apoptotic proteins Bax and Cleaved-Caspase-9, and downregulate the expression of anti-apoptotic protein Bcl-2.

[0010] Furthermore, the drug is used to induce OSCC cell cycle arrest in the G1 phase.

[0011] 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 echinacoside and a pharmaceutically acceptable carrier thereof.

[0012] 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 application of echinacoside (ECH) in the treatment of oral squamous cell carcinoma (OSCC), and systematically verifies its significant anti-tumor effect through network pharmacology, in vitro cell experiments and in vivo animal experiments, providing a novel candidate drug for the treatment of OSCC; (2) This invention reveals the mechanism of action of ECH against OSCC, namely, by inhibiting the key signaling pathway PI3K / AKT, it affects the biological behavior of tumor cells in multiple dimensions, including inhibiting proliferation, migration and invasion, and inducing apoptosis and cell cycle arrest. The mechanism is clear, providing a solid theoretical basis for clinical application; (3) This invention provides reliable efficacy verification data: In vivo experimental results show that ECH effectively inhibits tumor growth in mice while having no significant toxicity to the major organs (heart, liver, spleen, and lungs) of mice, and no significant abnormalities in blood routine and blood biochemical indicators, demonstrating good safety. This overcomes the shortcomings of existing chemotherapy drugs with large toxic side effects.

[0013] (4) Echinacoside is derived from traditional Chinese medicine such as Cistanche deserticola. It is widely available and easy to obtain, and has good prospects for drug development. Attached Figure Description

[0014] Figure 1 This is a diagram of the ECH-OSCC-target-pathway network constructed in Example 1; Figure 2 This is a bar chart of GO enrichment analysis of 10 target sites in Example 1; Figure 3 This is a bubble chart of KEGG pathway enrichment analysis performed on 20 pathways in Example 1; Figure 4 The figures show the molecular docking results of the 10 core targets in Example 1 with echinacoside. Specifically, A represents the molecular docking results of MMP9-echinacoside; B represents the molecular docking results of APP-echinacoside; C represents the molecular docking results of TNF-echinacoside; D represents the molecular docking results of IL2-echinacoside; E represents the molecular docking results of MMP2-echinacoside; F represents the molecular docking results of SIRT1-echinacoside; G represents the molecular docking results of BDNF-echinacoside; H represents the molecular docking results of MAPK3-echinacoside; I represents the molecular docking results of CASP3-echinacoside; and J represents the molecular docking results of HMOX1-echinacoside. Figure 5 The graph shows the cell viability of three cell types (CAL-27, HSC-3, and HOK) after treatment with different concentrations of echinacoside, as determined by the CCK-8 assay in Example 2. Figure 6 The cell proliferation of CAL-27 and HSC-3 cells in Example 2 at different drug concentrations for 0, 24, 48, and 72 hours, and statistical line graphs are shown. Figure 7 This is a diagram showing the cloning results of CAL-27 and HSC-3 cells treated with different drug concentrations for 24 hours in Example 2; Figure 8 The images shown are gel images and bar charts of PCNA protein expression detected by Western blot in CAL-27 and HSC-3 cells after treatment with different concentrations of drugs for 24 hours in Example 2. Figure 9 The figures show the scratch test results of CAL-27 and HSC-3 cells after 24 hours of treatment with different concentrations of drugs in Example 2, as well as the statistical bar chart of cell migration rate in each group. Figure 10 The figures show the Transwell experiment results of CAL-27 and HSC-3 cells after 24 hours of treatment with different concentrations of drugs in Example 2, as well as the bar charts showing the statistical significance of cell invasion ability in each group. Figure 11The images shown are gel images and bar charts obtained by Western blot analysis of the expression of relevant proteins in CAL-27 and HSC-3 cells after treatment with different concentrations of drugs for 24 hours in Example 2. Figure 12 The graph and bar chart show the apoptotic cell count of CAL-27 and HSC-3 cells after 24 hours of treatment with different drug concentrations in Example 2, as detected by flow cytometry. Figure 13 The images shown are gel images and bar charts of apoptotic proteins in CAL-27 and HSC-3 cells after treatment with different concentrations of drugs for 24 hours, as detected by Western blotting. Figure 14 The graph shows the cell cycle status of CAL-27 and HSC-3 cells after 24 hours of treatment with different drug concentrations, as well as the statistical bar chart. Figure 15 The images shown are gel images and statistical bar charts of P-PI3K / PI3K and P-AKT / AKT protein gels and graphs of CAL-27 and HSC-3 cells after 24 hours of treatment with different concentrations of drugs, as detected by Western blotting. Figure 16 The images and bar charts of PI3K, P-PI3K, AKT and P-AKT proteins in CAL-27 and HSC-3 cells after co-treatment with 740-YP and 100μM ECH for 24h are obtained by Western blotting. Figure 17 The following is a comparison of tumor control data in mice under different treatment groups in Example 3: A shows tumor photographs of different treatment groups; B shows the tumor volume change curves of different treatment groups over 14 days; and C shows the tumor weight statistics of different treatment groups after 14 days. Figure 18 This is a graph showing the expression of PCNA, Vimintin, and Bcl-2 in different treatment groups after the completion of the in vivo mouse experiment in Example 3; Figure 19 HE staining images of heart, liver, spleen and lung tissues from different treatment groups after the completion of the in vivo experiments in mice in Example 3. Detailed Implementation

[0015] 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.

[0016] 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.

[0017] Unless otherwise stated, all reagents and raw materials used in this invention are commercially available.

[0018] Example 1 This embodiment analyzes the feasibility of echinacoside 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) Echinacoside target acquisition: The SMILE numbers of ECH were searched in the Swiss Target Prediction and CTD databases. After removing duplicates, 113 targets were selected.

[0019] (2) Obtaining target for oral squamous cell carcinoma: The Gene Card, DISGENET and OMIM databases were searched for "oral squamous cell carcinoma". After removing duplicates, 6796 OSCC-related targets were obtained. The selected ECH targets and disease targets were imported into the MicroBioinformatics online tool, and a total of 91 common targets were obtained.

[0020] (3) PPI Network and Enrichment Analysis: 91 common targets were imported into the STRING database to construct a protein-protein interaction (PPI) network, which was then visualized using Cytoscape software. Using a built-in Cytoscape plugin, the top 10 targets were selected as core targets based on their degree values: TNF, CASP3, MAPK3, MMP9, HMOX1, APP, MMP2, BDNF, SIRT1, and IL2. These 91 common targets were then imported into Cytoscape 3.7.2 software to obtain the ECH-OSCC-target-pathway network diagram (Figure 1). The diagram shows that ECH can act on OSCC through multiple targets.

[0021] (4) KEGG and GO enrichment analysis: To better understand the biological processes and potential mechanisms of ECH anti-OSCC, enrichment analysis was performed on 91 targets using the DAVID database. GO enrichment analysis showed 260 entries related to biological processes (BP), 82 entries related to cellular components (CC), and 58 entries related to molecular functions (MF). The top 10 GO entries were used to create a bar chart. Figure 2KEGG pathway enrichment analysis yielded 130 signaling pathways. Based on P < 0.01, the top 20 pathways were selected and plotted in a bubble diagram (Figure 3). The size of the bubble reflects the number of genes enriched in that pathway, and the intensity of the bubble color reflects the significance of the enrichment. Larger bubbles indicate a greater number of enriched genes; darker bubbles indicate a higher degree of enrichment significance. These pathways primarily involve cancer signaling pathways and the PI3K / AKT signaling pathway.

[0022] (5) Molecular docking technology was used to verify the binding characteristics of echinacoside to the core targets. ECH was molecularly docked with the top 10 core targets in the PPI network using AutoDock software. The results are shown in Table 1. The lower the binding energy between the compound and the core target, the more stable the binding between the compound and the target protein. The molecular docking results show that ECH has good binding activity with these key targets, with binding energies for MMP9, APP, TNF, IL2, MMP2, SIRT1, BDNF, MAPK3, and CASP3 all below -5.0 kcal·mol⁻¹. -1 The stronger affinity with ECH suggests that ECH may exert its anti-OSCC effect by acting on the above key targets. Finally, the docking results were visualized using PyMOL software. Figure 4 ).

[0023] Table 1 Binding energies of ECH with 10 core targets

[0024] The results above show that this study successfully predicted 91 potential targets of echinacoside for oral squamous cell carcinoma (OSCC), identified PI3K / AKT as the key signaling pathway, and screened out 10 core hub genes including TNF, CASP3, MAPK3, MMP9, HMOX1, APP, MMP2, BDNF, SIRT1, and IL2. Molecular docking results showed that the binding free energy of echinacoside to the nine targets was all below -5 kcal / mol, indicating stable binding. Echinacoside has the potential to be used to prepare drugs for the treatment of oral squamous cell carcinoma at the molecular level.

[0025] Example 2 This embodiment analyzes the feasibility of echinacoside 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 echinacoside 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 echinacoside (0, 25, 50, 100, 400, 800, 1600 μM) for 24 hours. Cell viability was then determined using the CCK-8 assay. Figure 5 The results showed that, compared with the control group, with the increase of ECH drug concentration, the viability of CAL27 and HSC3 cells was significantly reduced, and the IC50 value was lower. 50 The concentrations were 213.54 μM and 187.99 μM, respectively. Lower concentrations of ECH showed relatively low cytotoxicity to normal oral keratinocytes (HOK). However, when the drug concentration exceeded 400 μM, the cytotoxicity to HOK cells increased significantly. Therefore, concentrations of 50, 100, and 200 μM were chosen for subsequent experiments. Cell proliferation was detected at 0, 24, 48, and 72 hours after drug administration using an Incucyte live cell analyzer. Figure 6 The results showed that as the concentration of ECH drug increased, the cell growth and proliferation rate gradually decreased. Figure 7 Cloning experiments showed that after 24 hours of ECH treatment, the number and size of OSCC cell colonies gradually decreased. Western blot analysis of proliferating cell nuclear antigen (PCNA) protein expression revealed... Figure 8 The results showed that PCNA protein expression decreased with increasing ECH concentration.

[0026] (2) To investigate the effects of echinacoside on the migration and invasion abilities of OSCC cells, scratch assays and Transwell assays were performed, such as... Figure 9 The results showed that, compared with the control group, the cell migration rate in all groups was significantly decreased, and this decrease was concentration-dependent. Figure 10 The results showed that, compared with the control group, the number of cells that invaded the cell membrane was significantly reduced in all groups, and this decrease was concentration-dependent. Since E-cadherin, N-cadherin, Vimentin, MMP-2, and MMP-9 are EMT-related proteins, we further examined the expression of these proteins in cells, and the results are as follows: Figure 11 As shown, compared with the control group, the expression of invasion-promoting factors N-cadherin, Vimentin, MMP-2, and MMP-9 was significantly reduced in the ECH group, while the expression of invasion-inhibiting factor E-cadherin was significantly increased, indicating that ECH inhibits OSCC cell migration and invasion.

[0027] (3) We used flow cytometry to confirm that the anticancer properties of ECH are related to the induction of apoptosis and the regulation of the cell cycle. For example... Figure 12As shown, compared with the control group, the number of apoptotic cells in the ECH treatment group was significantly increased, and these changes were dose-dependent. We further investigated the molecular mechanism by which ECH promotes OSCC cell apoptosis. Members of the Bcl-2 family play an important role in regulating apoptosis. Therefore, we investigated the expression levels of Bcl-2, Bax, and Cleaved-Caspase-9 in OSCC cells before and after ECH treatment. Figure 13 The Western blot results shown indicate that ECH increases the expression of pro-apoptotic proteins Bax and Cleaved-Caspase-9 in CAL27 and HSC3 cells, while decreasing the expression of the anti-apoptotic protein Bcl-2. Figure 14 The cell cycle distribution results shown indicate that ECH induces G1 phase arrest in OSCC cells, accompanied by a decrease in G2 phase cells.

[0028] (4) To confirm that ECH inhibits OSCC cell proliferation by suppressing the PI3K / AKT pathway, this study investigated the effect of ECH on PI3K / AKT expression. Figure 15 The results showed that ECH treatment significantly reduced the expression of p-PI3K and p-AKT, while total PI3K and total AKT showed no significant changes. To further confirm whether the anticancer efficacy of ECH is related to the PI3K / AKT pathway, we treated cells with ECH and ECH combined with the PI3K inhibitor 740Y-P, respectively. Figure 16 The Western blot results shown indicate that 740Y-P pretreatment reversed the inhibitory effect of ECH on p-PI3K and p-AKT expression.

[0029] Example 3 This embodiment studies the feasibility of using echinacoside in the preparation of drugs for treating oral squamous cell carcinoma based on in vivo experiments in mice: A mouse HSC3 xenograft model was established to further investigate the antitumor activity of ECH in vivo. When the tumor volume reached 100 mm... 3 At approximately 2:00 AM, the experimental group received an intraperitoneal injection of ECH (20 mg / kg / day), while the control group received an injection of normal saline (20 mg / kg / day). Tumor volume was measured every two days in both groups. After two weeks of continuous administration, vital organs and tumor tissue were collected. Results are as follows: Figure 17 As shown, the volume and mass of the tumors in the experimental group were significantly smaller than those in the control group.

[0030] The effects of ECH on the expression of proteins related to tumor proliferation, invasion, and apoptosis were further investigated using immunohistochemistry. The results are as follows: Figure 18 As shown in Table 2, the positive expression levels of PCNA, Vimintin, and Bcl-2 were reduced in tumors treated with ECH, which is consistent with the results of in vitro studies.

[0031] Table 2. Expression levels of PCNA, Vimintin, and Bcl-2 in experimental mice.

[0032] To investigate whether ECH has toxic effects on mice, we analyzed the vital organs of the experimental group mice, such as... Figure 19 HE staining results showed no significant histological differences in the heart, liver, spleen, and lung tissues between the two groups, with only mild edema observed in the renal tubular epithelial cells of the experimental group. Simultaneously, we verified whether ECH affected blood biochemical and routine blood parameters in mice, as shown in Tables 3 and 4. No significant differences were found in routine blood parameters and blood biochemical parameters between the experimental and control groups, indicating that ECH does not cause damage to vital organs or alter blood parameters.

[0033] Table 3. Levels of relevant indicators in the blood routine tests of laboratory mice

[0034] Table 4. Expression levels of blood biochemical markers ALP, ALT, CR and TBIL in experimental mice

[0035] Note: ALP: alkaline phosphatase, ALT: alanine aminotransferase, CR: creatinine, TBIL: total bilirubin. Results are expressed as mean ± standard deviation (n=3).

[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 echinacoside 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 activation of the PI3K / AKT signaling pathway, thereby inhibiting 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 upregulate E-cadherin protein expression and downregulate N-cadherin, Vimentin, MMP-2, and MMP-9 protein expression.

5. The application as described in claim 1, characterized in that, The drug is used to upregulate the expression of pro-apoptotic proteins Bax and Cleaved-Caspase-9, and downregulate the expression of anti-apoptotic protein Bcl-2.

6. The application as described in claim 1, characterized in that, The drug is used to induce OSCC cell cycle arrest in the G1 phase.

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

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