Oral squamous cell carcinoma inhibitor and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly discloses an oral squamous cell carcinoma inhibitor and application thereof. The inhibitor takes rutin as an active component. In-vitro experiments prove that rutin can significantly inhibit proliferation, clone formation, migration and invasion ability of OSCC cells in a dose-dependent manner, and effectively induce cell apoptosis. The action mechanism of the rutin is as follows: the rutin can directly target STAT3 protein and inhibit phosphorylation of the STAT3 protein so as to regulate and control a downstream BCL2 / CASP3 apoptosis pathway, thereby playing a role in treating OSCC. Molecular docking results further show that rutin and STAT3 have strong binding capacity. The invention discloses a new application of rutin as an STAT3 inhibitor in treating oral squamous cell carcinoma, and provides a new strategy and a candidate compound for developing high-efficiency and low-toxicity OSCC treatment medicines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an inhibitor of oral squamous cell carcinoma and its application. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is the most common highly malignant tumor of the head and neck, characterized by its aggressiveness and tendency to metastasize, resulting in a generally poor prognosis for patients. Currently, surgery, radiotherapy, and chemotherapy are the main treatment methods, but these methods still face challenges such as high postoperative recurrence rates, strong chemotherapy resistance, and significant systemic toxicity, making it difficult to effectively improve the five-year survival rate. Therefore, developing novel, highly effective, and low-toxicity treatment strategies is an urgent clinical need.

[0003] Signal transducer and activator of transcription 3 (STAT3), as a key molecule regulating cell proliferation, survival and apoptosis, is continuously activated in various cancers, including OSCC. It can drive the progression of malignant tumors by promoting tumor proliferation, inhibiting apoptosis and enhancing invasiveness. This indicates that STAT3 is a highly promising specific target for the treatment of OSCC, and the development of inhibitors targeting STAT3 is expected to become a new direction for the treatment of OSCC.

[0004] Rutin is a natural flavonoid compound widely found in plants, possessing various pharmacological activities such as anti-inflammatory, antioxidant, neuroprotective, and antitumor effects. In particular, rutin has shown good anticancer effects in various malignant tumors such as glioblastoma, prostate cancer, gastric cancer, hepatocellular carcinoma, and colon cancer. Furthermore, due to its natural origin, it has the advantages of low toxicity and high safety, providing a good foundation for its development as an antitumor drug.

[0005] However, to date, no studies have disclosed that rutin can exert a therapeutic effect on OSCC by inhibiting STAT3 activity, nor have there been any reports of combining rutin with the STAT3 target for OSCC treatment. There is still a significant technological gap in this field. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide an oral squamous cell carcinoma inhibitor and its application, and to propose a new use of rutin as a STAT3 inhibitor for the treatment of OSCC, aiming to fill the gap in the existing technology and provide a new, highly effective and low-toxicity treatment strategy for OSCC.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides an inhibitor for oral squamous cell carcinoma and its application.

[0009] Furthermore, the inhibitor is rutin.

[0010] Furthermore, the rutin is characterized in that it is used to treat OSCC by inhibiting the proliferation and migration activity of OSCC cells and promoting their apoptosis.

[0011] Furthermore, the rutin is characterized in that it is used to treat OSCC by targeting STAT3 and inhibiting its phosphorylation.

[0012] Furthermore, the dosage form of the drug is tablets, capsules, granules, injections, or sprays.

[0013] Furthermore, the method of administration of the drug is oral or non-gastrointestinal.

[0014] The present invention discloses the following technical effects:

[0015] This invention, through in vitro cell experiments, confirms that rutin significantly inhibits the proliferation activity, single-cell colony formation, migration, and transmembrane invasion of OSCC cells in a dose-dependent manner. Furthermore, rutin also promotes OSCC cell apoptosis and targets STAT3 while inhibiting its phosphorylation. These results indicate that rutin can be used as a STAT3 inhibitor for the treatment of OSCC, thus providing a novel, highly effective, and low-toxicity treatment strategy for OSCC. Attached Figure Description

[0016] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0017] Figure 1 This invention uses CCK-8 reagent to detect the effect of rutin on the proliferation of SCC-15 cells (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no label indicates no statistical difference);

[0018] Figure 2 This invention uses a plate cloning method to detect the effect of rutin on the single-cell proliferation of SCC-15 cells (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no label indicates no statistical difference);

[0019] Figure 3 This invention uses a wound healing assay to detect the effect of rutin on the migration of SCC-15 cells (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no label indicates no statistical difference);

[0020] Figure 4This invention verifies the effect of rutin on the migration of SCC-15 cells using Transwell assays (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no label indicates no statistical difference);

[0021] Figure 5 This invention uses flow cytometry to detect the effect of rutin treatment on apoptosis in SCC-15 cells (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no marker indicates no statistical difference);

[0022] Figure 6 This invention uses RT-qPCR to analyze the effect of rutin treatment on the relative mRNA expression levels of apoptosis-related genes BCL2 and CASP3 in SCC-15 cells (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no label indicates no statistical difference);

[0023] Figure 7 This invention uses Western blot to detect the relative expression levels of apoptosis-related genes and the phosphorylated protein of the target gene STAT3 in SCC-15 cells after rutin treatment (compared with the control group, * indicates p<0.05, ** indicates p<0.01, no label indicates no statistical difference).

[0024] Figure 8 This invention uses molecular docking technology to predict the docking site between STAT3 and rutin. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the accompanying drawings. This embodiment is implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. The chemical reagents used in the embodiments are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art. The specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0026] Please refer to the following: Figures 1-8 This example illustrates an oral squamous cell carcinoma inhibitor and its application, including the following steps:

[0027] Cell culture

[0028] This invention uses the human oral squamous cell carcinoma cell line SCC-15 as an in vitro model. SCC-15 cells are cultured in 10% FBS + DMEM / F12 medium.

[0029] CCK-8 cell proliferation activity assay

[0030] SCC-15 cells were seeded in 96-well plates and cultured for 6 hours. Then, different concentrations (0, 200, 400, 600, 800 μM) of rutin were added for 0, 24, and 48 hours, respectively, followed by incubation with CCK8 reagent for 1 hour. Absorbance at 450 nm was recorded using a microplate reader to analyze changes in cell viability and calculate the IC50 of rutin. 50 Values. The results showed that rutin inhibited the proliferation of SCC-15 cells in a dose-dependent manner (see [reference needed]). Figure 1 ). Calculations showed that rutin has an IC50 value for SCC-15 cells. 50 The value is 400 μM.

[0031] Plate cloning experiment

[0032] SCC-15 cells were seeded in 6-well plates and treated with different concentrations (0, 200, 400, 600, 800 μM) of rutin for 10 days, with the medium changed every 3 days. Afterwards, the cells were fixed, stained, and observed, and the number of colonies on each plate was recorded. The results showed that rutin inhibited the single-cell colony formation ability of SCC-15 cells in a dose-dependent manner (see...). Figure 2 ).

[0033] Wound healing experiment

[0034] SCC-15 cells were seeded in 6-well plates and cultured until 80% confluence was achieved. Scratches were then created using a sterile pipette tip, and the complete culture medium was replaced with serum-free medium. Different concentrations (0, 200, 400, 600, 800 μM) of rutin were added for treatment. Wound healing was recorded at 0 and 12 hours. The results showed that rutin inhibited SCC-15 cell migration in a dose-dependent manner (see...). Figure 3 ).

[0035] Transwell experiment

[0036] Pre-infiltrate 24-well plates and Transwell chambers. Resuspend SCC-15 cells in serum-free medium and seed them in the upper chamber of the Transwell. Add high-serum-concentration medium containing different concentrations (0, 200, 400, 600, 800 μM) of rutin to the lower layer, and incubate for 24 hours. After incubation, fix, stain, and remove unmigrated cells from the upper chamber. Observe the cell migration results under a microscope. The results show that rutin inhibits the cell migration and cell-penetrating ability of SCC-15 cells in a dose-dependent manner (see...). Figure 4 ).

[0037] Flow cytometry analysis of apoptosis experiments

[0038] SCC-15 cells were seeded in 6-well plates and cultured with different concentrations (0, 400 μM) of rutin until 80% confluence. The cells were then collected and resuspended in a small amount of binding buffer. Annexin V-FITC and PI (pigmentation inhibitors) were added, and the cells were incubated at room temperature in the dark for 10 minutes. After adding an appropriate amount of binding buffer and mixing thoroughly, flow cytometry was performed. FITC green fluorescence was detected using the FL1 channel, and PI red fluorescence was detected using the FL3 channel. The excitation wavelength was 488 nm. Data analysis was then performed using Flowjo software. The results showed that rutin significantly promoted SCC-15 cell apoptosis (see...). Figure 5 ).

[0039] Real-time quantitative PCR experiment

[0040] SCC-15 cells were seeded in 6-well plates and treated with different concentrations (0, 400 μM) of rutin for 48 hours. Total RNA was then extracted from the cells manually. The extracted total RNA was reverse transcribed into cDNA according to the instructions of the BioSharp Reverse Transcription Kit (with dsDNase). Primers for BCL2 and CASP3 were pre-designed, and the reaction solution was prepared according to the instructions of the BioSharp TranStart Top Green qPCR SuperMix. After precise spotting on ice and sealing, the cells were placed in an RT-qPCR machine. Data were analyzed using Piko Real Software. The results showed that rutin significantly inhibited the mRNA expression level of BCL2 and increased the mRNA expression level of CASP3 (see [link to Piko Real Software]). Figure 6 This result indicates that rutin effectively activates the intrinsic apoptosis pathway in SCC-15 cells by downregulating the anti-apoptotic gene BCL2 and upregulating the mRNA expression of the pro-apoptotic gene CASP3.

[0041] Western blot analysis

[0042] SCC-15 cells were seeded in 6-well plates and treated with different concentrations (0 and 400 μM) of rutin for 48 hours. Total protein was then extracted, and BCA protein concentration was determined. The total protein at appropriate concentrations was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After incubation with specific primary and secondary antibodies, the membrane was exposed to light. Results showed that rutin significantly inhibited STAT3 phosphorylation, suppressed BCL2 protein expression, and increased CASP3 protein expression (see [link to relevant documentation]). Figure 7This result indicates that rutin is an effective inhibitor of the STAT3 signaling pathway. It specifically activates the intrinsic apoptotic pathway in SCC-15 cells by inhibiting STAT3 phosphorylation, thereby downregulating the expression of its downstream anti-apoptotic target protein BCL2 and upregulating the expression of the apoptosis executive protein CASP3. This elucidates the core molecular mechanism by which rutin exerts its anti-OSCC effect.

[0043] Molecular docking prediction

[0044] The 3D structure of rutin was retrieved from the TCMSP database (https: / / www.tcmsp-e.com / ) and the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The 3D structure of STAT3 was downloaded from the RCSB PDB database (https: / / www.rcsb.org / ) and pretreated with Pymol software, including dehydration and hydrogenation. The binding pocket of STAT3 was predicted from the Prankwed database (https: / / prankweb.cz / ). The pretreated STAT3 3D file was imported into AutoDock software for docking analysis with rutin, and the binding energy was recorded. The docking data was then imported into Pymol software for visualization. The results showed that the binding energy of rutin to STAT3 was -7.8 kcal / mol, indicating a strong binding interaction. The docking site of rutin to STAT3 is also shown in the attached figure. Figure 8 ).

[0045] This invention demonstrates, through in vitro cell models, that rutin significantly inhibits the proliferation, single-cell colony formation, migration, and transmembrane invasion of oral squamous cell carcinoma (OSCC) cells in a dose-dependent manner. It also reveals that rutin exerts its anti-tumor effect by targeting STAT3 protein, inhibiting its phosphorylation, and thereby regulating the BCL2 / CASP3 apoptosis pathway to induce apoptosis. In summary, this invention provides an inhibitor of oral squamous cell carcinoma with rutin as the active ingredient. This inhibitor inhibits the proliferation and migration of OSCC by targeting STAT3 protein, inhibiting its phosphorylation and activation, and thereby regulating the BCL2 / CASP3 signaling axis to activate the intrinsic apoptosis pathway of cancer cells. This invention also provides the application of the inhibitor in the preparation of drugs for the prevention and / or treatment of oral squamous cell carcinoma, particularly oral squamous cell carcinoma with abnormal activation of the STAT3 signaling pathway.

[0046] The various embodiments of the present invention have been described above. These embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art can make equivalent substitutions or modifications based on the technical solutions and inventive concepts of the present invention, and all such modifications or substitutions should fall within the protection scope of the appended claims.

Claims

1. An inhibitor for oral squamous cell carcinoma (OSCC), characterized in that, The inhibitor uses rutin as its active ingredient. Rutin binds to STAT3 protein with an energy lower than -7.8 kcal / mol. By targeting and inhibiting STAT3 protein phosphorylation, it achieves dose-dependent inhibition of OSCC, and its IC50 value is [not specified]. 50 The value is 200 μM.

2. The inhibitor as described in claim 1, characterized in that, The inhibitor promotes OSCC apoptosis by inhibiting the proliferation and migration of OSCC cells and by activating the apoptosis pathway by inhibiting STAT3 phosphorylation.

3. The inhibitor as described in claim 1, characterized in that, The OSCC mentioned is an OSCC with abnormal activation of the STAT3 signaling pathway.

4. A pharmaceutical composition, characterized in that, The invention comprises a therapeutically effective amount of an OSCC inhibitor as described in any one of claims 1-3, and one or more pharmaceutically acceptable carriers or excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, granules, injections, or sprays, and the administration route is oral or non-gastrointestinal.

6. The use of an oral squamous cell carcinoma (OSCC) inhibitor as described in claim 1.

7. The application as described in claim 6, characterized in that, The drug is used to inhibit the proliferation and migration of OSCC cells.

8. The application as described in claim 6, characterized in that, The drug is used to promote apoptosis in OSCC cells.

9. The application as described in claim 6, characterized in that, The drug is used to inhibit the phosphorylation of STAT3.

10. The application as described in claim 6, characterized in that, The OSCC mentioned is an OSCC with abnormal activation of the STAT3 signaling pathway.

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