Application of vilazodone in preparation of sensitizing drug for resisting drug resistance of oral cancer chemotherapeutic drug

Abstract

The invention provides an application of vilazodone in preparation of a sensitizing drug for resisting drug resistance of an oral cancer chemotherapeutic drug. The vilazodone can increase the sensitivity of cisplatin-resistant oral cancer cells to cisplatin, and when the vilazodone is used for treating the cisplatin-resistant oral cancer, compared with the single use of cisplatin, the combination of the vilazodone and cisplatin can significantly inhibit the proliferation and clone formation of the cisplatin-resistant oral cancer, so that the curative effect of the cisplatin-resistant oral cancer is improved. The compound has a good application prospect in the aspect of developing drugs for enhancing the drug resistance of anti-oral cancer chemotherapy drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the application of vilazodone in the preparation of a sensitizing drug for resisting oral cancer chemotherapy drug resistance. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) is the most common type of cancer in the head and neck, and the sixth largest malignant tumor in the world. The annual incidence and mortality rates are increasing. The main sites of occurrence are the oral cavity, nasal cavity, pharynx, and larynx. Among them, oral squamous cell carcinoma (OSCC) is the main type of HNSCC, accounting for about 40%. Risk factors for oral cancer include smoking, alcoholism, long-term bad living habits, viral infection, age, and gender. Among them, smoking and alcoholism are the most common carcinogenic factors and the main causes of death in cancer patients. The occurrence of oral cancer is closely related to infection with human papillomavirus (HPV). Oral cancer infected with HPV mainly occurs in the palatine tonsils and lingual tonsils in the oropharynx. In general, the incidence of oral cancer in men is higher than that in women. The incidence of oral cancer increases with age and is more common in adults aged 50 to 70 years old. However, due to HPV infection, the incidence of young patients increases, and the number of young patients under 40 years old is increasing. Oral cancer tumors are highly malignant, with a five-year survival rate of less than 40%, and the overall prognosis of patients is poor. If diagnosed in the early stages (stages I and II), the survival rate can exceed 80%. However, there is currently no effective screening strategy because most patients have no symptoms in the early stages and do not pay attention until obvious symptoms such as pain or bleeding appear. As a result, up to 50% of oral cancers are diagnosed in the late stages (stages III and IV), resulting in an increase in the mortality rate of oral cancer [1].

[0003] Currently, advanced oral cancer is mainly treated by surgical resection, radiotherapy, chemotherapy or a more comprehensive approach. However, despite the various treatments adopted over the past few decades, the overall survival rate of oral cancer remains low. Chemotherapy is currently the most common treatment option for oral cancer. Chemotherapy drugs include 5-fluorouracil (5-FU), platinum drugs, paclitaxel (PTX) and doxorubicin (Dox). However, most patients will develop drug resistance, which is the main obstacle to the failure of cancer chemotherapy. Therefore, it is extremely important and urgent to explore drugs with significant effects and few toxic side effects to increase the sensitivity of tumor cells to chemotherapeutic drugs and solve the problem of drug resistance.

[0004] Vilazodone is a drug used to treat depression and belongs to the class of serotonin modulators. It has been clinically approved for use in the United States to treat depression and has the advantages of no serious adverse reactions and good clinical tolerance. Currently, there are no reports on whether vilazodone can increase the sensitivity of tumor cells to chemotherapy drugs and solve the problem of oral cancer resistance to cisplatin. Summary of the invention

[0005] In view of this, in order to solve the above technical problems, the present invention provides the use of vilazodone in the preparation of a sensitizing drug for resisting oral cancer chemotherapy drugs. The present invention shows that vilazodone can increase the sensitivity of cisplatin-resistant oral cancer cells to cisplatin. When used to treat cisplatin-resistant oral cancer, compared with cisplatin alone, the addition of vilazodone can significantly inhibit the proliferation and clone formation of resistant cells, and has good application prospects in the development of sensitizing drugs for resisting oral cancer chemotherapy drugs.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] The present invention provides, on one hand, the use of vilazodone in the preparation of a sensitizing drug for resisting oral cancer chemotherapy drug resistance.

[0008] Furthermore, the anti-oral cancer chemotherapy drug resistance is anti-oral cancer cisplatin chemotherapy drug resistance.

[0009] Furthermore, vilazodone reversed the chemotherapy resistance of cisplatin-resistant oral cancer cells by inhibiting the expression of the transporter protein MDR1 in resistant cells and upregulating the expression of the DNA damage marker protein H2AX.

[0010] Furthermore, vilazodone increased the sensitivity of oral cancer cisplatin-resistant cells to cisplatin.

[0011] Furthermore, vilazodone inhibits the proliferation and clone formation of cisplatin-resistant oral cancer cells.

[0012] Furthermore, the combination of vilazodone and cisplatin inhibited the proliferation and clone formation of cisplatin-resistant oral cancer cells.

[0013] Furthermore, the sensitization drug comprises vilazodone as an active ingredient and a pharmaceutically acceptable carrier.

[0014] Furthermore, the dosage form of the sensitizing drug is one of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, wine preparations, tinctures, dew preparations, and membrane preparations.

[0015] Furthermore, the sensitization drug is administered orally or by injection.

[0016] Another aspect of the present invention provides the use of vilazodone and cisplatin in combination in the preparation of a drug for treating oral cancer, wherein the vilazodone and cisplatin in combination reduces the drug resistance of oral cancer cells to cisplatin.

[0017] Compared with the prior art, the use of vilazodone in the present invention in the preparation of a sensitizing drug for resisting oral cancer chemotherapy drug resistance has the following advantages:

[0018] The present invention provides a new use of vilazodone, namely, the use of vilazodone in the preparation of a sensitizing drug for resisting oral cancer chemotherapy drug resistance. In the present invention, vilazodone can increase the sensitivity of cisplatin-resistant oral cancer cells to cisplatin. When used to treat cisplatin-resistant oral cancer, compared with cisplatin alone, the combination of vilazodone and cisplatin can significantly inhibit the proliferation and clone formation of resistant cells, providing a new research direction for the development of sensitizing drugs for resisting oral cancer chemotherapy drug resistance, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 is the IC of cisplatin against parental oral cancer CAL27 cells 50 ;

[0021] Figure 2 IC is the inhibitory activity of cisplatin on cisplatin-resistant CAL27 / DDP cells 50 ;

[0022] Figure 3 Western blot was used to detect the effect of vilazodone on the expression of MDR1 and H2AX proteins in oral cancer cell line CAL-27 and cisplatin-resistant oral cancer cell line CAL27 / DDP;

[0023] Figure 4 Cell proliferation after treatment of cisplatin-resistant oral cancer cell CAL27 / DDP with control group, cisplatin (2 μg / mL), vilazodone (10 μM), and cisplatin combined with vilazodone;

[0024] Figure 5 This is the cell clone formation after the cisplatin-resistant oral cancer cell CAL27 / DDP was treated with the control group, cisplatin (2μg / mL) group, vilazodone (10μM) group, and cisplatin combined with vilazodone group. DETAILED DESCRIPTION

[0025] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0026] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings.

[0027] Example 1

[0028] Establishment of oral cancer CAL27 cisplatin-resistant cells.

[0029] The cisplatin-resistant strain of CAL27 cells was induced by the concentration gradient increasing method, and the specific implementation method was as follows:

[0030] Take the CAL27 cell line in the logarithmic growth phase, and wait until it grows to about 70% and then perform the following operations: add culture medium containing cisplatin (concentration of 0.1 mg / L) for 48 hours; discard the culture medium containing the drug, add fresh culture medium, and continue to culture; wait for it to resume normal growth, digest and subculture; then treat with culture medium containing cisplatin (concentration of 0.2 mg / L) for 48 hours, repeat and gradually increase the concentration of cisplatin until the cells can no longer tolerate it. After the induction of cisplatin-resistant cells is completed, take the parent CAL27 cells and cisplatin-resistant cells and evenly inoculate them in a 96-well plate at a density of 5000 cells / well, add different concentrations of cisplatin and treat for 48 hours, with three replicates in each group. After 48 hours, add 20μL MTT (5mg / mL) and treat for 4 hours, then use an enzyme reader to test the 570nm OD value of each well, and calculate the IC 50 .

[0031] The experimental results are as follows Figure 1 and Figure 2 As shown, the IC of cisplatin against parental oral cancer CAL27 cells 50 The value was 1.637 μg / mL ( Figure 1 ), IC of cisplatin against drug-resistant oral cancer CAL27 cells 50 The value was 5.077 μg / mL ( Figure 2 ), the established CAL27 cisplatin-resistant cell resistance index was 3.1.

[0032] Example 2

[0033] Vilazodone increases the uptake of cisplatin by cisplatin-resistant oral cancer CAL-27 cells in vitro and promotes DNA damage in resistant cells.

[0034] Western blot was used to detect the effect of vilazodone on the expression of MDR1 and H2AX proteins in oral cancer cell line CAL-27 and cisplatin-resistant oral cancer cell CAL27 / DDP. The specific implementation method is as follows:

[0035] After the cells were inoculated in 6-well plates and cultured as monolayer cells, the solution was discarded. CAL27 cells and CAL27 / DDP cells were set up as control group and vilazodone group (10 μM) respectively. After drug treatment, they were cultured in a 37°C, CO2 (5%) incubator for 48 h. The proteins in the cells of different treatment groups were extracted and analyzed by western blot. The whole cell lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membrane, and then sealed with 5% fat-free milk. The membrane was incubated with monoclonal antibodies against GAPDH, MDR1 and H2AX at 4°C overnight. The membrane was further incubated with secondary antibodies (affinity, 1:10000). Finally, the membrane was placed in an electrophoresis gel imaging system (ChemiScope 6000, CLIX, Shanghai, China).

[0036] The experimental results are as follows Figure 3 As shown, vilazodone had no effect on the expression of MDR1 and H2AX proteins in parental CAL27 cells. Compared with parental CAL27 cells, MDR1 expression was upregulated and H2AX expression was downregulated in cisplatin-resistant CAL27 / DDP cells, indicating that the drug efflux capacity of resistant cells was improved and DNA damage was inhibited. After resistant cells were treated with vilazodone, vilazodone reduced the expression of MDR1 in resistant cells and upregulated the expression of H2AX, indicating that vilazodone can inhibit the efflux of cisplatin in resistant cells and promote DNA damage. The above results show that vilazodone can reverse the chemotherapy resistance of cisplatin-resistant oral cancer cells.

[0037] Example 3

[0038] Vilazodone significantly inhibited the proliferation of cisplatin-resistant oral cancer cells at the in vitro cell level.

[0039] The MTT proliferation curve method was used to evaluate the effect of vilazodone on the proliferation ability of cisplatin-resistant oral cancer cell line CAL-27 / DDP cells. The specific implementation method is as follows:

[0040] The cells were seeded in a 96-well plate and cultured. After the monolayer cells adhered to the wall, the liquid was discarded. A control group, a cisplatin (2 μg / mL) group, a vilazodone (10 μM) group, and a cisplatin combined with vilazodone group were set up respectively. After drug treatment, the cells were cultured in a 37°C, CO2 (5%) incubator for 0, 24, 48, and 72 h, the culture medium was discarded, 20 μL of a 5 mg / ml MTT solution was added, and the absorbance of each well was detected at a wavelength of 570 nm using an enzyme reader.

[0041] The cell survival rate represented by the absorbance of the control group was 100%, and the absorbance of the remaining drug-added wells / the absorbance of the control group represented the survival rate of the cells after being affected by the drugs. The cell proliferation curve was drawn using GraphpadPrism 5 software, and each index was expressed as mean ± standard deviation.

[0042] The experimental results are as follows Figure 4 As shown, there was no significant change in the proliferation ability of resistant cells after cisplatin treatment. Vilazodone alone could inhibit the proliferation of resistant cells, and compared with the cisplatin alone group, the combination of vilazodone and cisplatin could significantly inhibit the proliferation ability of resistant cells.

[0043] Example 5

[0044] Vilazodone significantly inhibited the clone-forming ability of cisplatin-resistant oral cancer cells at the in vitro cell level.

[0045] The clone formation method was used to evaluate the effect of vilazodone on the clone formation ability of cisplatin-resistant oral cancer cell line CAL-27 / DDP cells. The specific implementation method is as follows:

[0046] 500 cells were inoculated in 6-well plates for culture. After the cells adhered, the liquid was discarded. A control group, a cisplatin (2 μg / mL) group, a vilazodone (10 μM) group, and a cisplatin combined with vilazodone group were set up respectively. After drug treatment, the cells were placed in a 37°C, CO2 (5%) incubator for 14 days, the culture medium was discarded, and after washing with PBS, cold methanol was added to fix the cells for 10 minutes, followed by staining with crystal violet for 15 minutes. After washing away the excess dye with PBS, the cells were photographed and the number of clones was counted.

[0047] The experimental results are as follows Figure 5 As shown, there was no significant change in the clone-forming ability of resistant cells after treatment with cisplatin. Vilazodone alone could inhibit the clone-forming ability of resistant cells, and compared with the cisplatin alone group, the combination of vilazodone and cisplatin could significantly inhibit the clone-forming ability of resistant cells.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of vilazodone in the preparation of sensitizing drugs against oral cancer chemotherapy drug resistance.

2. The use according to claim 1, characterized in that: The anti-oral cancer chemotherapy drug resistance is anti-oral cancer cisplatin chemotherapy drug resistance.

3. The use according to claim 1, characterized in that: Vilazodone reverses the chemotherapy resistance of cisplatin-resistant oral cancer cells by inhibiting the expression of the transporter protein MDR1 in resistant cells and upregulating the expression of the DNA damage marker protein H2AX.

4. The use according to claim 1, characterized in that: Vilazodone increases the sensitivity of cisplatin-resistant oral cancer cells to cisplatin.

5. The use according to claim 1, characterized in that: Vilazodone inhibits the proliferation and clone formation of cisplatin-resistant oral cancer cells.

6. The use according to claim 1, characterized in that: The combination of vilazodone and cisplatin inhibits the proliferation and clone formation of cisplatin-resistant oral cancer cells.

7. The use according to any one of claims 1 to 6, characterized in that: The sensitization drug comprises vilazodone as an active ingredient and a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that: The dosage form of the sensitization drug is one of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, wine preparations, tinctures, dew preparations, and film preparations.

9. The use according to claim 7, characterized in that: The administration route of the sensitization drug is oral administration or injection.

10. Use of vilazodone and cisplatin in combination in the preparation of a drug for treating oral cancer, wherein the vilazodone and cisplatin in combination reduces the drug resistance of oral cancer cells to cisplatin.

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