Application of benzethonium chloride as STAT3 inhibitor in preparation of medicine for treating head and neck tumors

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of benzethonium chloride as an STAT3 inhibitor in preparation of a medicine for treating head and neck tumors. The invention proves that BZN is directly combined with STAT3 protein to reduce tyrosine phosphorylation and nuclear translocation of the STAT3 protein, so that expression of downstream MCL-1 is reduced, and head and neck tumor cells are subjected to apoptosis. According to the application disclosed by the invention, the STAT3 is determined to be a potential target of the BZN, and the BZN is proved to be combined with STAT3 protein in head and neck tumor cells FaDu and CAL-27, so that the CAL-27 and FaDu cells are subjected to apoptosis, and an anti-tumor effect is achieved. BZN is expected to become a new action target of STAT3.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of benzethonamine as a STAT3 inhibitor in the preparation of a drug for treating head and neck tumors. Background Art

[0002] Head and neck tumors mainly occur in the oral cavity, nose and pharynx, of which head and neck squamous cell carcinoma (HNSCC) accounts for more than 90%. Head and neck squamous cell carcinoma is a malignant disease originating from the head and neck mucosa, squamous cells and their secretions, and is one of the common head and neck cancers. The causes of head and neck squamous cell carcinoma are very complex, involving multiple factors such as eating habits, smoking, drinking, and infection with human papillomavirus. There are usually no symptoms in the early stages and the development rate is slow. As the disease progresses, symptoms such as difficulty swallowing and neck lumps often appear. Most HNSCC patients will progress to the local advanced stage, and the recurrence rate is high. About 10% of HNSCC patients will metastasize.

[0003] Currently, the main clinical treatments for HNSCC patients are surgical resection, adjuvant radiotherapy and chemotherapy, and combined systemic therapy. However, for patients with recurrent or metastatic HNSCC (R / M HNSCC), multiple conventional treatment options have failed to improve overall survival (OS). For patients with recurrent and metastatic disease, this has caused great economic and mental burdens and pain to society and families.

[0004] Benzethonium chloride (BZN) is a nonionic surfactant with a broad-spectrum antibacterial effect. It is an antifungal and antibacterial compound that can effectively penetrate into bacterial cells and interfere with their metabolic processes. When used for skin and mucous membrane disinfection, it can quickly kill most Gram-positive and Gram-negative bacteria, including drug-resistant strains such as Staphylococcus aureus and Pseudomonas aeruginosa. Benzethonium chloride can be used to treat a variety of infectious diseases caused by bacteria, such as urethritis, vaginitis, etc. The clinical manifestations of these diseases may include symptoms such as frequent urination, urgency, dysuria, and abnormal leucorrhea.

[0005] STAT3 is a protein composed of 770 amino acids, characterized by the presence of 6 functional conserved domains, among which SH2 is the most conserved STAT domain, which plays a vital role in signal transduction by binding to specific phosphotyrosine motifs. STAT3 is related to cell proliferation, differentiation, epithelial-mesenchymal transition, etc., and is also involved in tumor immunity. STAT3 is constitutively activated in tumor immune cells and participates in tumor immune surveillance. STAT3 mainly exerts its specific functions by activating phosphorylation, dimerization, nuclear translocation and binding to DNA, activating the expression of downstream proliferation and survival genes such as c-myc, bcl-XL and mcl-1.

[0006] BZN is approved for anti-infection and has been reported to play a role in a variety of tumors, but its mechanism of action in HNSCC is still unclear. Therefore, it is urgent to study the relationship between benzethonyl chloride and STAT3 and use benzethonyl chloride in the preparation of STAT3 inhibitors, thereby providing a clinical application basis for the prevention and treatment of HNSCC. Summary of the invention

[0007] In view of the above-mentioned deficiencies, the present invention provides the use of benzethonium chloride as a STAT3 inhibitor in the preparation of a drug for treating head and neck tumors. The present invention provides the use of benzethonium chloride in the preparation of a STAT3 inhibitor. The present invention confirms that BZN directly binds to the STAT3 protein, reduces its tyrosine phosphorylation and nuclear translocation, and then reduces the expression of downstream MCL-1, leading to apoptosis of head and neck tumor cells. The present invention determines that STAT3 is a potential target of BZN, and confirms that BZN binds to the STAT3 protein in the head and neck tumor cells FaDu and CAL-27 cells, causing apoptosis of CAL-27 and FaDu cells, and exerts an anti-tumor effect. BZN is expected to become a new target of STAT3.

[0008] The technical solution of the present invention includes:

[0009] In a first aspect, the present invention provides the use of benzethonium chloride in the preparation of a STAT3 inhibitor.

[0010] Specifically, the benzethonamine binds to STATS, reduces STATS tyrosine phosphorylation and nuclear translocation, and inhibits downstream protein expression.

[0011] In a second aspect, the present invention provides a STAT3 inhibitor, wherein the STAT3 inhibitor contains benzethonium chloride.

[0012] In a third aspect, the present invention provides benzethonium chloride as a STAT3 inhibitor and / or the use of the above-mentioned STAT3 inhibitor in the preparation of anti-tumor drugs.

[0013] Specifically, the anti-tumor drugs include drugs for preventing, treating or assisting in the treatment of tumors.

[0014] Preferably, the tumor is a head and neck tumor.

[0015] Preferably, the head and neck tumors include but are not limited to: one or more of FaDu, CAL-27, and HK-1.

[0016] Specifically, the anti-tumor drug exerts an anti-tumor effect through at least one of the following:

[0017] (1) Promote apoptosis of tumor cells; promote the reduction of tumor cell proliferation;

[0018] (2) Reduce the expression of anti-apoptotic proteins in tumor cells; promote the expression of pro-apoptotic proteins in tumor cells;

[0019] (3) directly binds to STAT3 protein;

[0020] (4) Inhibit STAT3 phosphorylation in tumor cells.

[0021] Specifically, the benzethonamine or STAT3 inhibitor is used as the sole or main active ingredient of the anti-tumor drug.

[0022] Specifically, the anti-tumor drug also includes pharmaceutically acceptable excipients.

[0023] Preferably, the pharmaceutically acceptable excipients include one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

[0024] Preferably, the pharmaceutically acceptable excipient is at least one of lactose, mannose, mannitol, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, vitamin C, vitamin E, citric acid, phosphoric acid, sodium benzoate, potassium sorbate, Tween-80, poloxamer, glutamic acid and glycine.

[0025] Specifically, the dosage forms of the anti-tumor drug include injections, tablets, capsules, granules, powders, creams, ointments, gels or sprays.

[0026] Specifically, the administration of the anti-tumor drug includes oral administration, intravenous injection, local injection, intradermal injection or subcutaneous injection.

[0027] Specifically, the anti-tumor drug can also be used in combination with other anti-tumor drugs.

[0028] Preferably, the other anti-tumor drugs include, but are not limited to, one or more of cisplatin, carboplatin, paclitaxel, docetaxel, 5-fluorouracil, cetuximab, nimotuzumab, pembrolizumab, nivolumab, methotrexate, cyclophosphamide, vincristine, and afatinib.

[0029] Preferably, the combined use includes simultaneous use or sequential use.

[0030] In a fourth aspect, the present invention provides an anti-tumor drug comprising: benzethonamine chloride and / or the STAT3 inhibitor according to claim 3.

[0031] Specifically, the anti-tumor drug also includes pharmaceutically acceptable excipients.

[0032] Preferably, the pharmaceutically acceptable excipients include one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

[0033] Preferably, the pharmaceutically acceptable excipient is at least one of lactose, mannose, mannitol, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, vitamin C, vitamin E, citric acid, phosphoric acid, sodium benzoate, potassium sorbate, Tween-80, poloxamer, glutamic acid and glycine.

[0034] The beneficial effects of the present invention are:

[0035] The present invention confirms that BZN directly binds to the STAT3 protein, and the binding protein is mainly enriched in the JAK / STAT3 pathway; it reduces its 705 site tyrosine phosphorylation and nuclear translocation, thereby reducing the expression of downstream MCL-1 and BCL-2, and inhibiting the proliferation of FaDu and CAL-27 cells. The BZN of the present invention can significantly inhibit the expression of STAT3 protein levels, and STAT3 is determined to be a potential target of BZN. The BZN of the present invention inhibits STAT3 dimerization, nuclear entry, and phosphorylation, causing FaDu and CAL-27 cells to undergo apoptosis, thereby exerting an anti-cancer effect. In summary, the BZN of the present invention can cause apoptosis of head and neck tumor cells by inhibiting STAT3, thereby exerting an anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Benzethonium chloride inhibits cancer cell proliferation in a concentration-dependent manner. In the figure, A is CAL-27 cells; B is FaDU cells; C is HK-1 cells; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0037] Figure 2 Benzethonamine inhibits cancer cell proliferation in a time-dependent manner; in the figure, A is CAL-27 cells; B is FaDU cells; C is HK-1 cells; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0038] Figure 3 The figure shows the effect of different concentrations of benzethonium chloride on the cell wound healing ability; A is a photo of cell wound healing observed under a microscope; B is the wound healing area of ​​CAL-27 cells; C is the wound healing area of ​​FaDU cells; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0039] Figure 4 The figure shows the effect of different concentrations of benzethonium chloride on cell migration ability; A is a photo of cell migration ability observed under a microscope; B is the number of CAL-27 cell migration; C is the number of FaDU cell migration; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0040] Figure 5 The figure shows the effect of different concentrations of benzethonium chloride on the cell clone formation ability; A is a photo of the cell clone formation ability observed under a microscope; B is the relative clone number of CAL-27 cells; C is the relative clone number of FaDU cells; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0041] Figure 6 The results of Annexin V-FITC / PI double staining method; A in the figure shows the effect of BZN on apoptosis of CAL-27 cells; B shows the effect of BZN on apoptosis of FaDU cells; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0042] Figure 7The results of Annexin V-FITC / PI double staining method; A in the figure shows the effect of BZN and Z-VAD on apoptosis of CAL-27 cells; B shows the effect of BZN and Z-VAD on apoptosis of FaDU cells; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0043] Figure 8 Figure 2 is a cell cycle result diagram; A is the CAL-27 cell cycle result; B is the FaDU cell cycle result; ns represents no significant difference; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001.

[0044] Fig. 9 This is the result of detecting the effects of different inhibitors on BZN through CCK8; ns in the figure means no significant difference; * means P<0.05; ** means P<0.01; *** means P<0.001; **** means P<0.0001.

[0045] Fig.10 These are the WB measurement results of apoptosis-related proteins Caspase3, BAK, BCL-2, MCL-1, and XIAP.

[0046] Fig.11 The results of BZN and STAT3 related proteins are shown in Figure 2 .

[0047] Fig.12 This is a diagram showing the results of IP experiments verifying that BZN affects STAT3 dimerization.

[0048] Fig.13 This is a validation diagram of the binding of BZN and STAT3.

[0049] Fig.14 BZN inhibited tumor growth. *** in the figure represents P < 0.001.

[0050] Fig.15 Neferine inhibits cancer cell proliferation in a concentration-dependent manner.

[0051] Fig.16 This is a diagram showing the results of IP experiments to verify the effect of Neferine on STAT3 dimerization. DETAILED DESCRIPTION

[0052] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0053] The cell sources used in the present invention are shown in Table 1:

[0054] Table 1 Cell sources

[0055] name factory CAL-27 cell line Wuhan Prosai Life Science Co., Ltd. FaDu cell line Wuhan Prosai Life Science Co., Ltd. HK-1 cell line Wuhan Prosai Life Science Co., Ltd.

[0056] The sources of antibodies used in the present invention are shown in Table 2:

[0057] Table 2 Antibody sources

[0058]

[0059] The sources of the reagents used in the present invention are shown in Table 3:

[0060] Table 3 Sources of reagents

[0061]

[0062]

[0063] Example 1 Benzethonium chloride inhibits the proliferation of cancer cells

[0064] 1.1 Benzethonium chloride inhibits cancer cell proliferation in a concentration-dependent manner

[0065] CCK8 kit was used to detect the effect of BZN on the proliferation of CAL-27, FaDu, and HK-1 cells. 1×10 4 Cells were added in triplicate. CAL-27, FaDu, and HK-1 cells were given 100 μL of corresponding concentrations of benzethonium chloride (15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, and 45 μM), and then 100 μL of working solution (CCK-8 reagent: DMEM medium volume ratio of 1:9) was added. DMSO was used as a control, and the cells were incubated at 37°C with 5% CO 2 After incubation for 48 h, the OD value was measured at 450 nm.

[0066] The results of the test are as follows Figure 1As shown, the results showed that benzethonium chloride could inhibit the proliferation of CAL-27, FaDu and HK-1 cells in a concentration-dependent manner. After incubation for 48 hours, the IC50 of benzethonium chloride for CAL-27 cells was 13.75, the IC50 for FaDu cells was 14.37, and the IC50 for HK-1 cells was 12.52.

[0067] 1.2 Benzethonyl chloride inhibits cancer cell proliferation in a time-dependent manner

[0068] CCK8 kit was used to detect the effect of BZN on the proliferation of CAL-27, FaDu, and HK-1 cells. 1×10 4 CAL-27, FaDu, and HK-1 cells were given 100 μL of 20 μM benzethonium chloride, and then 100 μL of working solution (CCK-8 reagent: DMEM culture medium volume ratio of 1:9) was added, and DMSO containing the same volume concentration was used as a control. 37°C, 5% CO 2 The cells were incubated and the OD value was measured at 450 nm at 24, 48, and 72 hours of incubation.

[0069] The results of the test are as follows Figure 2 As shown, the results showed that benzethonamine incubation for 24-72h could inhibit the proliferation of CAL-27, FaDu, and HK-1 cells in a time-dependent manner.

[0070] Example 2 Benzethonyl chloride inhibits cancer cell healing

[0071] Press 5×10 5 CAL-27 or FaDu cells were seeded into 6-well plates and incubated at 37°C with 5% CO. 2 Culture in an incubator for 24-48 hours until the cells reach 100% confluence. Scrape the cell monolayer with a 200μL pipette tip to form a gap, and then wash the cells with PBS. Replace the culture medium with serum-free DMEM medium to inhibit cell proliferation. The drug group was prepared with serum-free DMEM medium to a concentration of 20μM BZN, and the control group was DMSO of the same concentration volume. Observe and photograph the gap at 0h, 12h, and 18h ​​after scratching, and calculate the scratch area using ImageJ software.

[0072] The results are as follows Figure 3 As shown, benzethonamine can significantly inhibit the wound healing of CAL-27 cells and FaDu cells.

[0073] Example 3 Effect of benzethonium chloride on the migration ability of cancer cells

[0074] CAL-27 and FaDu cells were digested and centrifuged and then resuspended in serum-free DMEM medium. 2×10 4 CAL-27 or FaDu cells were aspirated into the upper chamber of the transwell without Matrigel to evaluate cell migration ability. 600 μL DMEM medium containing 10% FBS was added to the lower chamber to stimulate cell migration. The drug group used DMEM medium to prepare 20 μM BZN, and the control group used DMEM medium to prepare the same concentration of DMSO. The cells were incubated at 37°C and 5% CO 2 After 48 hours of culture in the incubator, the transwell chamber was fixed with 4% paraformaldehyde for 30 minutes and then stained with 1% crystal violet for 15 minutes. The cells placed on the upper surface of the transwell membrane were wiped with a cotton swab, and the cells moved to the lower surface of the membrane were photographed under a microscope.

[0075] The results are as follows Figure 4 As shown, benzethonamine can significantly inhibit the migration of CAL-27 cells and FaDu cells.

[0076] Example 4 Effect of benzethonium chloride on the colony-forming ability of cancer cells

[0077] CAL-27 cells or FaDu cells were seeded in 6-well plates, with 1×10 cells per well. 3 The cells were treated with corresponding concentrations of BZN (0, 2, 3 μM) at 37°C and 5% CO 2 The cells were cultured for 14 days under the same conditions, washed with PBS and fixed with 4% paraformaldehyde for 15 min, stained with 1% crystal violet for 15 min, and photographed under a microscope.

[0078] The results are as follows Figure 5 As shown, benzethonium chloride inhibited the colony-forming ability of CAL-27 cells and FaDu cells.

[0079] Example 5 Benzethonium chloride induces apoptosis in cancer cells

[0080] 5.1 Effect of benzethonium chloride on cancer cell apoptosis

[0081] Cell apoptosis was measured using the Annexin-V apoptosis detection kit. 2 × 10 cells were plated per well in a 12-well plate. 5 CAL-27 or FaDu cells were treated with benzethonium chloride (30, 45 μM), and all cells were harvested 48 h later and then incubated with annexin V and propidium iodide (PI) for 15 min in the dark. Annexin V or PI staining alone was used as a control. Cell apoptosis was assessed by flow cytometry. The experiment was performed in triplicate.

[0082] The results of the test are as follows Figure 6 As shown, Figure 6 The effect of different concentrations of benzethonium chloride on apoptosis of CAL-27 or FaDu cells is shown. It can be seen that as the concentration of benzethonium chloride increases, the proportion of early apoptosis and late apoptosis increases.

[0083] 5.2 Effects of benzethonamine plus Z-VAD on cancer cell apoptosis

[0084] Cell apoptosis was measured using the Annexin-V apoptosis detection kit. 2 × 10 cells were plated per well in a 12-well plate. 5 CAL-27 or FaDu cells were treated with DMSO as a control. One experimental group was treated with 25 μM benzethonium chloride, and the other group was treated with 20 μM Z-VAD on the basis of 25 μM BZN. All cells were harvested after 48 h of incubation in the incubator and then incubated with annexin V and propidium iodide (PI) for 15 min in the dark. Annexin V or PI staining alone was used as a control. Cell apoptosis was evaluated by flow cytometry. The experiment was performed in triplicate.

[0085] The results of the test are as follows Figure 7 As shown, the results showed that adding 20 μM Z-VAD on the basis of 25 μM benzethonamine could reduce the effect of BZN drug. It shows that Z-VAD apoptosis inhibitor reduces the cell apoptosis caused by BZN.

[0086] Example 6 Benzethonamine causes G1 and S phase arrest in CAL-27 and FaDu cells

[0087] CAL-27 or FaDu cells were treated with 5 μM or 10 μM BZN for 48 h, respectively, and the control group was given the same concentration of DMSO. The cells were fixed and stained with propidium iodide (PI) staining buffer at room temperature in the dark for 10 min. Cell cycle distribution was measured using flow cytometry. The experiment was performed in triplicate.

[0088] Cell cycle results are shown in Figure Figure 8 As shown, the results showed that different drug concentrations of BZN caused CAL-27 and FaDu cells to be arrested in the G1 phase and S phase, specifically manifested in an increase in the G1 phase and S phase.

[0089] Example 7 Effect of benzethonium chloride on different inhibitors

[0090] CCK8 kit was used to detect the effects of different death inhibitors on the proliferation of CAL-27 and FaDu after BZN treatment. 1×10 4CAL-27 and FaDu cells were treated with 20 μM BZN, 20 μM Z-AVD, 20 μM ferr-1, 5 mM 3-MA or 20 μM Nerc-1, or 20 μM BZN and 20 μM Z-AVD, 20 μM ferr-1, 5 mM 3-MA or 20 μM Nerc-1, respectively. DMSO was used as a control. The cells were cultured at 37°C with 5% CO. 2 After incubation in the incubator for 48 hours, the culture medium was removed and 100 μL of working solution (CCK-8 reagent: DMEM culture medium volume ratio was 1:9) was added. After incubation for 2 hours, the OD value was measured at 450 nm.

[0091] The results of the test are as follows Fig. 9 As shown, after using the apoptosis inhibitor Z-VAD, ferroptosis inhibitor Ferr-1, autophagy inhibitor 3-MA, and necroptosis inhibitor Nerc-1, the results showed that the apoptosis inhibitor Z-VAD could reduce the cell death caused by BZN.

[0092] Example 8 WB determination of related proteins

[0093] CAL-27 or FaDu cells were plated at 5 × 10 5 / well, and 0μM, 10μM, and 20μM BZN were given on the next day. After incubation for 48 hours, the cells were centrifuged and washed with PBS, lysed on ice for half an hour using RIPA, centrifuged at 12000rpm for 15min at 4℃, and the protein concentration was determined using a BCA kit. The cells were placed in a 95℃ metal bath for 5min and stored at -20℃.

[0094] The samples were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred onto PVDF membranes. After blocking with 5% skim milk for 2 hours, the membranes were incubated with primary antibodies at 4°C overnight and washed 3 times with 1x Tris-buffered saline and Tween for 5 minutes each. The membranes were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. Chemiluminescent development kits were used for visualization.

[0095] Fig.10 The results of WB determination of apoptosis-related proteins Caspase3, BAK, BCL-2, MCL-1, and XIAP. The results showed that BZN can significantly increase the expression of pro-apoptotic proteins such as Caspase3 and BAX in CAL-27 and FaDu cells, and inhibit the expression of anti-apoptotic proteins such as BCL-2, MCL-1, and XIAP.

[0096] Fig.11The results of BZN and STAT3-related proteins are shown in Figure 2. The results showed that compared with the control group, with the increase of BZN concentration, the protein expression levels of P-Tyr705 STAT3, P-P38, P-JNK, and P-ERK in CAL-27 and FaDu decreased significantly and gradually, showing a concentration-dependent manner. The protein expression levels of STAT3 inhibitors SHP1 and SOCS3 decreased. This shows that BZN can inhibit the STAT3 and MAPK pathways of CAL-27 and FaDu cells, and the expression levels of STAT3 downstream proteins MCL-1 and BCL-2 are also inhibited.

[0097] Example 9 IP experiment verifies that BZN affects STAT3 dimerization

[0098] STAT3-Flag plasmid and STAT3-HA plasmid were co-transfected into CAL-27 and FaDu cells by lentivirus, and 5×10 CAL-27-STAT3-Flag-HA or FaDu-STAT3-Flag-HA cells were plated. 5 / well, and 0μM and 20μM BZN were given on the second day. After incubation for 48h, the cells were centrifuged and washed with PBS, lysed on ice for half an hour using RIPA, centrifuged at 12000rpm for 15min at 4℃, and the protein concentration was determined using the BCA kit. The supernatant of the same concentration was divided into two parts, one of which was added with 50μL Flag beads, shaken overnight at 4℃, then loaded with 5X loading, and placed in a 95℃ metal bath for 5min, and the other part was not overnight, loaded with 5X loading, placed in a 95℃ metal bath for 5min, and stored at -20℃. WB verification.

[0099] The results of the test are as follows Fig.12 As shown, after CAL-27 and FaDu cells transfected with STAT3-Flag plasmid and STAT3-HA plasmid were treated with 20 μM BZN, the expression of HA protein was reduced, indicating that STAT3 autodimerization was reduced after BZN treatment.

[0100] Example 10 Molecular docking diagram of BZN and STAT3

[0101] In order to study whether benzethonium chloride can bind to the STAT3 SH2 domain, the present invention performs a molecular docking experiment, obtains the crystal structure of STAT3 from a protein database, and performs computational modeling with the structure of benzethonium chloride.

[0102] The results of the test are as follows Fig.13As shown, benzethonamine interacts with amino acids on STAT3, such as SER-614, SER-613, SER-611, PRO-639, GLU-638, THR-620, ARG-609, VAL-637, SER-636, LYS-591, etc.

[0103] Example 11 BZN inhibits tumor proliferation

[0104] The present invention used male Reag-1 mice aged 6-8 weeks for the experiments, maintained under standard conditions and cared for according to the institutional guidelines for animal care. All animal experiments were approved by the Ethics Committee of the Animal Experiment Platform of the Hefei Comprehensive National Science Center Institute of Big Health.

[0105] 5×10 6 FaDu cells were resuspended in 100 μL PBS and injected subcutaneously into the flank of mice to establish tumor xenografts. When the tumor xenografts reached 5 mm in diameter, the mice were randomly divided into a treatment group and a model control group. The treatment group was injected intraperitoneally with BZN (5 mg / kg) every two days, while the model control group received only an equal dose of PBS. Body weight and tumor size were measured every two days, and the tumor volume was calculated using the following formula: V (mm 3 )=(length×width 2 ) / 2.

[0106] The results of the test are as follows Fig.14 As shown, BZN can significantly inhibit the growth of head and neck tumors.

[0107] Comparative Example 1 Neferine inhibits cancer cell proliferation

[0108] Neferine is a natural component of Nelumbo nucifera purchased from Source Leaf Biotechnology with product number B20598.

[0109] CCK8 kit was used to detect the effect of Neferine on the proliferation of CAL-27 cells. 1×10 4 CAL-27 cells were treated with 100 μL of neferine (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) at the corresponding concentrations, and DMSO was used as a control at 37°C and 5% CO 2 After incubation for 48 hours, the culture medium was removed and 100 μL of working solution (CCK-8 reagent: DMEM culture medium volume ratio was 1:9) was added. After incubation for 2 hours, the OD value was measured at 450 nm.

[0110] The results of the test are as follows Fig.15As shown, the results showed that with the increase of Neferine drug concentration, the proliferation ability of CAL-27 cells decreased, indicating that Neferine can inhibit the proliferation of cancer cells and CAL-27 cells are concentration-dependent on Neferine.

[0111] Comparative Example 2 IP experiment verifies whether Neferine affects STAT3 dimerization

[0112] STAT3-Flag plasmid and STAT3-HA plasmid were co-transfected into CAL-27 and FaDu cells by lentivirus, and 5×10 CAL-27-STAT3-Flag-HA or FaDu-STAT3-Flag-HA cells were plated. 5 / well, and 0μM and 20μM Neferine were given on the second day. After incubation for 48h, the cells were centrifuged and washed with PBS, lysed on ice for half an hour using RIPA, centrifuged at 4℃, 12000rpm for 15min, and the protein concentration was determined using the BCA kit. The supernatant of the same concentration was divided into two parts, one of which was added with 50μL Flag beads, shaken overnight at 4℃, then loaded with 5X loading, and placed in a 95℃ metal bath for 5min. The other part was not overnight, loaded with 5X loading, placed in a 95℃ metal bath for 5min, and stored at -20℃. WB verification.

[0113] The results of the test are as follows Fig.16 As shown, after CAL-27 and FaDu cells transfected with STAT3-Flag plasmid and STAT3-HA plasmid were treated with 20 μM Neferine, the expression of HA protein in the IP group did not change, indicating that STAT3 autodimerization was not affected after Neferine treatment, indicating that Neferine did not affect STAT3 dimerization in FaDu cells.

[0114] The above results indicate that although Neferine can inhibit the proliferation of CAL-27 cells, it does not affect the STAT3 dimerization of CAL-27 cells and FaDu cells.

[0115] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. Application of benzethonium chloride in the preparation of STAT3 inhibitors.

2. The use according to claim 1, characterized in that: The benzethonamine binds to STATS, reduces STATS tyrosine phosphorylation and nuclear translocation, and inhibits downstream protein expression.

3. A STAT3 inhibitor, characterized in that The STAT3 inhibitor contains benzethonium chloride.

4. Use of benzethonium chloride as a STAT3 inhibitor and / or the STAT3 inhibitor according to claim 3 in the preparation of an anti-head and neck tumor drug.

5. The use according to claim 4, characterized in that: The anti-head and neck tumor drugs include drugs for preventing, treating or assisting in treating head and neck tumors.

6. The use according to claim 4, characterized in that: The anti-head and neck tumor drug has an anti-head and neck tumor effect through at least one of the following: (1) Promote apoptosis of head and neck tumor cells; promote the reduction of head and neck tumor cell proliferation; (2) Reduce the expression of anti-apoptotic proteins in head and neck tumor cells; promote the expression of pro-apoptotic proteins in head and neck tumor cells; (3) directly binds to STAT3 protein; (4) Inhibit STAT3 phosphorylation in head and neck tumor cells.

7. The use according to claim 4, characterized in that: The benzethonamine or STAT3 inhibitor is used as the sole or main active ingredient of the anti-head and neck tumor drug.

8. The use according to claim 4, characterized in that: The anti-head and neck tumor drug also includes pharmaceutically acceptable excipients.

9. The use according to claim 4, characterized in that: The anti-head and neck tumor drug can also be used in combination with other anti-tumor drugs, and the combination includes simultaneous use or sequential use; The other anti-tumor drugs include: one or more of cisplatin, carboplatin, paclitaxel, docetaxel, 5-fluorouracil, cetuximab, nimotuzumab, pembrolizumab, nivolumab, methotrexate, cyclophosphamide, vincristine, and afatinib.

10. An anti-head and neck tumor drug, characterized in that: The anti-head and neck tumor drug comprises benzethonamine chloride and / or the STAT3 inhibitor according to claim 3.

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