CN113318104A - Application of sulconazole and/or sulconazole salt in preparing medicine for treating esophageal squamous cell carcinoma - Google Patents
Application of sulconazole and/or sulconazole salt in preparing medicine for treating esophageal squamous cell carcinoma Download PDFInfo
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Abstract
The invention relates to the field of biological medicines, in particular to application of sulconazole and/or a sulconazole salt in preparing a medicine for treating esophageal squamous cell carcinoma. The invention provides application of sulconazole and/or sulconazole salt in preparing a medicament for treating esophageal squamous cell carcinoma. The side effects of the sulconazole and/or the sulconazole salt are clearer than those of chemotherapeutic drugs, the safety of the sulconazole and/or the sulconazole salt is proved to be capable of being used for treating esophageal squamous cell carcinoma, remarkably inhibiting the proliferation of esophageal squamous cell carcinoma cells and nude mouse transplantable tumors and inhibiting the metastasis level of esophageal squamous cell carcinoma cells, and predicting the potential of being used for treating patients with esophageal squamous cell carcinoma clinically.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of sulconazole and/or a sulconazole salt in preparing a medicine for treating esophageal squamous cell carcinoma.
Background
Esophageal cancer (esophageal cancer) is a malignant lesion formed by abnormal hyperplasia of esophageal squamous epithelium or glandular epithelium. Esophageal cancer is mainly divided into two main pathological types, Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC).
Esophageal carcinogenesis involves a complex process of multifactorial, multistage, polygenic variation accumulation and interaction with environmental factors, including changes in numerous protooncogenes, oncogenes and proteins at the molecular level, and the effects of long-term adverse living or eating habits (eating foods with a high content of nitrosamines, such as a preference for pickling pickled cabbage or mildewed food, a long-term preference for scalding food, smoking, drinking or other adverse preferences, etc.). Esophageal cancer is hidden, asymptomatic or atypical in early stage, is found to be clinically advanced, and has poor prognosis in general (5-year survival rate is about 13%). At present, the treatment method of esophageal cancer still mainly adopts chemotherapy drugs, and the side effect is large.
Disclosure of Invention
In order to solve the problems, the invention provides application of sulconazole and/or a sulconazole salt in preparing a medicament for treating esophageal squamous cell carcinoma. The side effects of the sulconazole and/or the sulconazole salt are clearer than those of chemotherapeutic drugs, and the drug safety of the sulconazole and/or the sulconazole is proved to be capable of being used for treating esophageal squamous cell carcinoma, and also capable of obviously inhibiting the proliferation of esophageal squamous cell carcinoma cells and nude mouse transplantable tumors and inhibiting the metastasis level of esophageal squamous cell carcinoma cells in cells.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of sulconazole and/or sulconazole salt in preparing a medicament for treating esophageal squamous cell carcinoma.
Preferably, the salt of sulconazole comprises sulconazole nitrate.
The invention provides a medicament for treating esophageal squamous cell carcinoma, which comprises active ingredients of sulconazole and/or salt of sulconazole.
Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials.
Preferably, the dosage form of the medicament comprises an injection.
The invention provides application of sulconazole and/or sulconazole salt in preparing a medicament for promoting esophageal squamous cell carcinoma apoptosis and inhibiting esophageal squamous cell proliferation, metastasis and glucose absorption.
The invention provides a medicament for promoting esophageal squamous cell carcinoma apoptosis and inhibiting esophageal squamous cell proliferation, metastasis and glucose absorption, and the effective components of the medicament comprise sulconazole and/or sulconazole salt.
Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials.
Preferably, the dosage form of the medicament comprises an injection.
Has the advantages that:
the invention provides application of sulconazole and/or sulconazole salt in preparing a medicament for treating esophageal squamous cell carcinoma. The safety of the sulconazole and/or the sulconazole salt is proved, the side effect is clearer than that of chemotherapeutics, and the sulconazole and/or the sulconazole salt can be used for treating esophageal squamous cell carcinoma. The specific embodiment of the invention adopts the esophageal squamous cell carcinoma cell and tumor-bearing nude mouse animal model test to prove that: the sulconazole and/or the salt of the sulconazole can obviously inhibit the proliferation of esophageal squamous cell carcinoma cells and nude mouse transplantable tumors and the metastasis level of esophageal squamous cell carcinoma cells in the cells, and limit the absorption of glucose by the esophageal squamous cell carcinoma cells; the sulconazole and/or the sulconazole salt can remarkably promote the apoptosis of esophageal squamous cell carcinoma cells.
Drawings
FIG. 1 is a typical graph of cell viability at different drug concentrations;
FIG. 2 is a schematic diagram of cell clone number, wherein A is a statistical histogram of clone number of cultured KYSE30 cells treated with 0. mu.M and 20. mu.M of sulconazole nitrate, B is a statistical histogram of clone number of cultured KYSE150 cells treated with 0. mu.M and 20. mu.M of sulconazole nitrate, C is a typical picture of actual clone condition of cells under the condition of data source of A picture, and D is a typical picture of actual clone condition of cells under the condition of data source of B picture;
FIG. 3 is a schematic representation of the healing area of a scratch, wherein A is a statistical histogram of the healing change of 0 μ M and 20 μ M sulconazole nitrate in KYSE30 cells at 0 and 24 hours respectively, B is a statistical histogram of the healing change of 0 μ M and 20 μ M sulconazole nitrate in KYSE150 cells at 0 and 24 hours respectively, C is a real cell picture of the healing change of 0 μ M and 20 μ M sulconazole nitrate in KYSE30 cells at 0 and 24 hours respectively, and D is a real cell picture of the healing change of 0 μ M and 20 μ M sulconazole nitrate in KYSE150 cells at 0 and 24 hours respectively;
FIG. 4 is a graph of a cell transfer experiment, wherein A is a histogram of transferred cells 48 hours after KYSE30 cells were treated with DMSO nitrate and 30 μ M sulconazole nitrate; b is a transfer cell histogram after KYSE150 cells are treated by dimethyl sulfoxide and 30 mu M of sulconazole nitrate for 48 hours; c is a real cell picture of transferred cells after KYSE30 cells are treated by dimethyl sulfoxide and 30 mu M of sulconazole nitrate for 48 hours; d is a real cell picture of transferred cells after KYSE150 cells are treated by dimethyl sulfoxide and 30 mu M of sulconazole nitrate for 48 hours;
FIG. 5 is a diagram of a flow cytometry experiment, wherein A is a diagram of apoptosis detection results and B is a diagram of statistical apoptosis ratio quantification;
FIG. 6 is a graph of a glucose uptake assay, wherein A is a histogram of glucose uptake change of esophageal cancer cell KYSE30 and B is a histogram of glucose uptake change of esophageal cancer cell KYSE 150;
FIG. 7 is a graph showing the change of the tumor volume and weight after 30 days by intraperitoneal injection of drug-dissolving agents dimethyl sulfoxide and sulconazole nitrate (10mg/kg) every 3 days after nude mice are inoculated with KYSE30 and KYSE150, wherein A is an actual graph of the volume and weight of the tumor formed by KYSE30, B is a graph of the volume change of the tumor formed by KYSE30, C is a graph of the weight change of the tumor formed by KYSE30, D is an actual graph of the volume and weight of the tumor formed by KYSE150, E is a graph of the volume change of the tumor formed by KYSE150, and F is a graph of the weight change of the tumor formed by KYSE 150.
Detailed Description
The invention provides application of Sulconazole (Sulconazole) and/or a salt of Sulconazole in preparing a medicament for treating esophageal squamous cell carcinoma, wherein the salt of Sulconazole preferably comprises Sulconazole nitrate (Sulconazole nitrate). Sulconazole has proven its drug safety, its side effects are more clear than those of chemotherapeutic drugs, and it can be used to treat esophageal squamous cell carcinoma cells; the source of the sulconazole and/or the salt of the sulconazole is not particularly limited in the invention, and the sulconazole and/or the salt of the sulconazole can be purchased by the technicians in the field in a conventional way; in a specific embodiment of the invention, the sulconazole nitrate is preferably purchased from MedChemexpress under the trade designation HY-B1460A; the chemical formula of the sulconazole nitrate is preferably shown as a formula I:
the specific embodiment of the invention adopts the esophageal squamous cell carcinoma cell and tumor-bearing nude mouse animal model test to prove that: the sulconazole nitrate can obviously inhibit the proliferation and metastasis levels of esophageal squamous cell carcinoma cells and nude mouse transplantable tumors, so that the sulconazole nitrate can be used for preparing a medicament for treating esophageal squamous cell carcinoma.
The invention provides a medicament for treating esophageal squamous cell carcinoma, which comprises active ingredients of sulconazole and/or a salt of sulconazole, wherein the salt of sulconazole preferably comprises sulconazole nitrate. In the present invention, the medicament preferably further comprises pharmaceutically acceptable excipients; the dosage form of the medicament preferably comprises an injection. When the sulconazole nitrate is used for cell viability tests, the concentration is 20 mu M, and the dosage is 10mg/kg when the nude mouse xenograft tests are carried out, so that the effect of inhibiting the growth of cancer cells can be achieved.
The invention provides application of sulconazole and/or a salt of sulconazole in preparing a medicament for promoting esophageal squamous cell carcinoma apoptosis and inhibiting esophageal squamous cell proliferation, metastasis and glucose absorption, wherein the salt of sulconazole preferably comprises sulconazole nitrate.
The invention provides a medicament for promoting esophageal squamous cell carcinoma apoptosis and inhibiting esophageal squamous cell proliferation, metastasis and glucose absorption, wherein the effective component of the medicament comprises sulconazole and/or a salt of sulconazole, and the salt of sulconazole preferably comprises sulconazole nitrate. In the present invention, the medicament preferably further comprises pharmaceutically acceptable excipients; the dosage form of the medicament preferably comprises an injection. When the sulconazole nitrate is used for cell viability test, the concentration is 20 mu M, and the dosage is 10mg/kg when a nude mouse xenograft test is carried out, so that the effect of inhibiting the growth of cancer cells can be achieved; when the concentration of the cell scratch healing experiment is 20 mu M, the effect of inhibiting the transfer of esophageal squamous cell carcinoma cells in a cell culture dish can be achieved; the concentration of 50 mu M in the flow cytometry experiment can achieve the aim of promoting the death rate of cancer cells; when a glucose uptake assay experiment was performed, 30 μ M and 50 μ M of sulconazole nitrate significantly inhibited glucose uptake by both KYSE30 and KYSE150 cells.
When the cell viability test is carried out, the adopted cell line preferably comprises KYSE30, KYSE150, KYSE450, TE1, TE3 and TE 5; the invention has no special requirements on the sources of KYSE30, KYSE150, KYSE450, TE1, TE3 and TE5, and is obtained by conventional purchase by technicians in the field, wherein KYSE30, KYSE150 and KYSE450 are from Japanese Cell Bank of Japanese research biosources Cell Bank; TE1, TE3 and TE5 were from Japanese Cell Resource Center for biological Research.
For further illustration of the present invention, the application of sulconazole and/or sulconazole salt provided by the present invention in preparing a drug for treating esophageal squamous cell carcinoma will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Cell viability assay (MTS assay)
To measure the sensitivity of different Cell lines (KYSE30, KYSE150, KYSE450 from Japanese Cell Bank Japan Collection Bioresources Cell Bank; TE1, TE3 and TE5 from Japanese Cell Resource Center for biological Research) to the drug tioconazole nitrate, the cells were digested and cultured in RPMI Medium1640 Medium (Purchase: Yinzi Jie (Shanghai) Co., Ltd., Cat: 11875119, Brand: GIBCO) containing 10% serum to form a single Cell suspension, the cells were counted and seeded in 96-well plates at 10000 cells per well. The volume of the medium per well was 100. mu.l. The cells were cultured for 12 hours to adhere to the wall, and then replaced with RPMI Medium1640 Medium containing 0. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M and 100. mu.M of sulconazole nitrate, respectively (Purchase Co., Ltd.: Yiwei Ji (Shanghai) trade Co., Ltd., Cat: 11875119, brand: GIBCO). After 24 hours of continuous culture, 20. mu.l of MTS (cat # G3581, company: Promega) was added to each well and incubated for 2 hours. The absorbance of each well was measured at a wavelength of 492nm using an MK3 microplate reader from Thermo Scientific. The specific procedures refer to SLC52A3 expression is activated by NF-kappaB p65/Rel-B and serves as amylostic biomarker in the expression vector cancer (Long, L.et al. cell Mol Life Sci 75, 2643-.
TABLE 1 cell viability ratio at different drug concentrations
As is clear from table 1 and fig. 1, with increasing drug concentration, sulconazole nitrate gradually and significantly inhibited the survival of cancer cells of esophageal squamous cell lines KYSE30, KYSE150, KYSE450, TE1, TE3 and TE 5.
Example 2
Cell clone formation assay
In a 12-well plate, 500-1,000 KYSE30 or KYSE150 cells are inoculated in each well. After the cells were attached to the surface, a sulconazole nitrate drug was added to the RPMI Medium1640 cell culture solution (Purchase manufacturer: Yiwei Jie Co., Ltd., Cat: 11875119, brand: GIBCO) at a concentration of 20. mu.M, and the mixture was cultured for 12 hours. The RPMI Medium1640 culture Medium containing the drug was replaced with RPMI Medium1640 Medium (Purchase, Uighur) trade Co., Ltd., Cat No. 11875119, brand: GIBCO) and the cells were cultured for 2 weeks, with Dimethyl sulfoxide (DMSO, full name, Dimethyl sulfoxide, Purchase, Cat No. D4540-1L, Brand: sigma) as a negative control. After washing with 4 ℃ precooled PBS, the Cells were fixed with 4 ℃ precooled methanol and glacial acetic acid at a ratio of 3:1 for 20 minutes and stained with violet crystals for 15 minutes, in particular in Riboflavin deletion proteins mutagenesis in HEK293 tag NIH3T3 Cells by staining and Regulating Cell Cycle-Related genetic transformation (Long, L., et al JN UTr,2018.148(6): p.834-843). Colonies were photographed by ChemiDoc Touch (Bio-Rad), and the number of clones was counted by Image J software (national institute of health, Bethesda, Md., USA) and repeated 3 times for each experiment, and the results are shown in Table 2, Table 3 and FIG. 2.
TABLE 2 KYSE30 cell clone number schematic table
TABLE 3 KYSE150 cell clone number schematic table
As is clear from tables 2, 3 and 2, the number of cell clones formed by both the KYSE30 and KYSE150 cells was significantly inhibited by 20 μ M of sulconazole nitrate.
Example 3
Scratch healing test
KYSE30 or KYSE150 cells (from Japanese Cell Bank Japanese Collection of Research Bioresources Cell Bank) were seeded in 6-well plates and cultured in serum-free RPMI Medium1640 Medium for 12 hours. A straight line was drawn with a sterile pipette tip. Cells were washed with PBS and then cultured in RPMI Medium1640 Medium of 2% fetal bovine serum. For specific steps, the RPMI Medium1640 culture Medium containing 0. mu.M and 20. mu.M of sulconazole nitrate drug is added to the cell dish for culturing KYSE30 or KYSE150 cells for 0 hour and 24 hours, and then microscopic observation is carried out on the designated area by using a microscope, and dimethyl sulfoxide is used as a negative control, and the method refers to "RAC 1 inhibition reactions in microbial cells and antibodies in biochemical enzymes" (Zeng, R.J., et al. mol Oncol,2019.13(9): p.2010-2030 "). The scratch healing area was calculated using ImageJ software (national institute of health, Bethesda, MD, USA) and the results are shown in table 4, table 5 and fig. 3.
TABLE 4 scratch healing area of KYSE30
TABLE 5 scratch healing area of KYSE150
As is clear from tables 4, 5 and 3, the 20 μ M sulconazole nitrate significantly suppressed the migration rate of both KYSE30 and KYSE150 cells in the cell culture plate.
Example 4
Cell transfer assay
To measure the effect of the drug tioconazole nitrate on the motility of cell lines, esophageal cancer cells KYSE30 and KYSE150 were starved for 12 hours in serum-free RPMI Medium1640 Medium, lysed into Transwell cells at 5 million cells per well, fixed, stained and imaged after 48 hours treatment with dimethylsulfoxide (control) and 30 μ M tioconazole nitrate, respectively, and ten field cells were counted randomly per cell, as shown in table 6 and fig. 4.
TABLE 6 KYSE30 and KYSE150 cell transfer number statistical table
As is clear from table 6 and fig. 4, 30 μ M of sulconazole nitrate significantly inhibited the degree of transfer of both KYSE30 and KYSE150 cells in the Transwell chamber.
Example 5
Flow cytometry experiments
The control group is treated by dimethyl sulfoxide, esophageal cancer cells KYSE30 and KYSE150 are respectively treated by 50 mu M of sulconazole nitrate for 24 hours (three samples in each group), the cells are treated according to the instruction of an apoptosis kit (company: Biyun, Cat. No. C1062S), and the apoptosis level is detected by a flow cytometer. The results are shown in Table 7 and FIG. 5, wherein A in FIG. 5 is a graph of the results of apoptosis assays. In FIG. 5, B is a quantitative graph of the ratio of statistical apoptosis.
TABLE 7 KYSE30 and KYSE150 cell death rate statistics table
As is clear from table 7 and fig. 5, 50 μ M of sulconazole nitrate significantly promoted the death rate of both KYSE30 and KYSE150 cells.
Example 6
Glucose uptake assay
Esophageal cancer cells KYSE30 and KYSE150 were treated with 0. mu.M, 30. mu.M, and 50. mu.M of sulconazole nitrate for 24 hours (three samples per group), and the change in Glucose Uptake of the cells was detected using a kit (Glucose-uplink Glucose assay, cat # J1342, Promega). The results are shown in Table 8 and FIG. 6.
TABLE 8 statistical Table of glucose uptake in KYSE30 and KYSE150 cells
As is clear from table 8 and fig. 6, 30 μ M and 50 μ M of sulconazole nitrate significantly inhibited the uptake of glucose in both KYSE30 and KYSE150 cells, and further, the effect of inhibiting esophageal cancer cells was achieved.
Example 7
Nude mouse xenograft experiment
Animal experiments were carried out according to protocols approved by the ethical committee for laboratory animals in the medical laboratory of Shantou university school of medicine, with specific procedures being referenced to RAC1 inhibition transformations in tissue resistance in esophageal cells, cartinosa and indexes down regulation of hydrolytic enzymes (Zeng, R.J., et al. mol Oncol,2019.13(9): p.2010-2030). Nude mice (Experimental animal technology Co., Ltd. Beijing vitamin River, China) of 3-5 weeks are randomly divided into 4 groups (1 group is KYSE30 dimethyl sulfoxide group, 2 group is KYSE30 sulfur nitrate conazole group, 3 group is KYSE150 dimethyl sulfoxide group and 4 group is KYSE150 sulfur nitrate conazole group), 5 mice in each group are respectively treated with KYSE30(2 x 106One) and KYSE150 (1X 10)6One) cells were injected into mouse axilla. When the average tumor volume reaches 50mm3Drug injections were started, with 2 and 4 groups intraperitoneally injected with sulconazole nitrate (10mg/kg) every 3 days, and 1 and 3 groups injected with dimethyl sulfoxide (150 μ L) every 3 days. Tumor volume was measured every 3 days according to the following formula (Width)2X length)/2. 30 days after inoculation, the mice were euthanized with carbon dioxide, tumors were excised and weighed, and the results are shown in tables 9 to 12 and fig. 7.
TABLE 9 statistical table of nude mouse transplanted tumor volume of KYSE30 cells (mm)3)
TABLE 10 statistical table of nude mouse transplanted tumor volume of KYSE150 cells (mm)3)
TABLE 11 KYSE30 weight statistics Table for nude mouse transplanted tumor (g)
TABLE 12 KYSE150 cell nude mouse transplanted tumor weight statistical Table (g)
As is apparent from tables 9 to 12 and fig. 7, in the nude mouse xenograft experiment, sulconazole nitrate (10mg/kg) significantly suppressed the volume and weight of the subcutaneous transplantable tumor formation of KYSE30 and KYSE150 cells, respectively, in nude mice.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. Use of sulconazole and/or a salt of sulconazole in the manufacture of a medicament for the treatment of esophageal squamous cell carcinoma.
2. The use of claim 1, wherein the salt of sulconazole comprises sulconazole nitrate.
3. A medicament for treating esophageal squamous cell carcinoma, which is characterized in that the effective component of the medicament comprises sulconazole and/or a salt of sulconazole.
4. The medicament of claim 3, further comprising a pharmaceutically acceptable excipient.
5. The medicament of claim 3 or 4, wherein the dosage form of the medicament comprises an injection.
6. Application of sulconazole and/or sulconazole salt in preparing medicine for promoting esophageal squamous cell carcinoma apoptosis and inhibiting esophageal squamous cell proliferation, metastasis and glucose absorption.
7. A medicine for promoting esophageal squamous cell carcinoma apoptosis and inhibiting esophageal squamous cell proliferation, metastasis and glucose absorption is characterized in that the effective component of the medicine comprises sulconazole and/or sulconazole salt.
8. The medicament of claim 7, further comprising a pharmaceutically acceptable excipient.
9. The medicament of claim 7 or 8, wherein the dosage form of the medicament comprises an injection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114948947A (en) * | 2021-12-31 | 2022-08-30 | 广州医科大学附属第五医院 | Application of fenticonazole nitrate in preparation of antitumor drugs |
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