CN111202726A - Application of echinocandin in preparation of anti-esophageal cancer drugs - Google Patents

Abstract

The invention discloses an application of echinocandin in preparation of anti-esophageal cancer drugs, belonging to the technical field of medicines. The invention discovers for the first time that the licochalcone has the inhibiting effect on the proliferation, the metastasis and the invasion of esophageal cancer cells in vitro, and can effectively inhibit the growth of esophageal cancer in vivo. In addition, the invention also discovers that the glycyrrhiza uralensis chalcone and 5-fluorouracil are used together, and the glycyrrhiza uralensis chalcone can increase the sensitivity of esophageal cancer cells to the 5-fluorouracil. Meanwhile, the invention proves that the licochalcone has no obvious side effect while inhibiting the growth of the esophageal cancer through the blood biochemical routine detection of tested animals. The invention provides a new medicine selection for treating esophageal cancer.

Description

Application of echinocandin in preparation of anti-esophageal cancer drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of echinocandin in preparation of an anti-esophageal cancer medicine.

Background

Esophageal cancer is a relatively common tumor of the digestive tract, and has high morbidity and mortality. Currently, the first treatment for esophageal cancer is surgical resection of the lesion and regional lymph nodes. However, since there are no obvious abnormal symptoms in the early stage of the disease, when the patient is diagnosed, the disease is mostly developed to the middle and late stage, and the patient is subjected to surgical radical operation treatment at the time, not only the ideal treatment effect is not achieved, but also the body of the patient is seriously injured. Aiming at the patients, the synchronization therapy is clinically adopted for treating the patients, and common chemotherapeutic drugs are cisplatin and docetaxel. Clinical experience has shown that the desired effect is often not achieved with this treatment, with severe side effects. Therefore, it is important to develop a novel and effective method for treating esophageal cancer.

Natural products are important for the research and development of anticancer drugs due to the advantages of definite anticancer effectiveness, abundance of candidate resources and the like. For example, vincristine, irinotecan, etoposide and paclitaxel are all plant-derived anticancer compounds. Glycyrrhiza extract can exert a variety of biological effects including antioxidant, anti-inflammatory and anti-tumor effects, and is achieved in part by modulating the Nrf2, NO and NF κ B pathways. Licochalcone A (LCA) is a flavonoid isolated from licorice. It has been reported to inhibit cell viability in non-small cell lung cancer and breast cancer; licochalcone a inhibits breast cancer progression by inhibiting the cadherin E and MAPK pathways; licochalcone a inhibits the proliferation of non-small cell lung cancer by inducing G2/M cell cycle arrest and endoplasmic reticulum stress. Echinatin is another natural compound separated from Glycyrrhrizae radix and has molecular formula of C₁₆H₁₄O₄The chemical structural formula is as follows:

disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the echinocandin in preparing the anti-esophageal cancer medicine.

The purpose of the invention is realized by the following technical scheme:

application of echinocandin in preparing anti-esophageal cancer medicine is provided.

The anti-esophageal cancer is used for inhibiting the proliferation, metastasis, invasion or tumor growth of esophageal cancer cells.

The effective concentration of the echinocandin is preferably 10-40 mu M; more preferably 10 to 20 μ M.

The esophageal cancer cell is a human esophageal cancer cell line KYSE30 or KYSE 270.

The anti-esophageal cancer drug preferably further contains 5-fluorouracil.

The preferable proportion of the echinocandin and the 5-fluorouracil is that the molar ratio is 10-40: 0.1 to 2; more preferably, the molar ratio is 10-20: 0.3 to 1.25; most preferably, when the esophageal cancer cells are human esophageal cancer cell line KYSE30, the molar ratio is 20: 1.25; when the esophageal cancer cells are human esophageal cancer cell line KYSE270, the molar ratio is 20: 0.3. the dosage can be properly adjusted according to the sensitivity of the cells to the medicine.

The licochalcone increases the sensitivity of esophageal cancer cells to 5-fluorouracil.

The chemical structural formula of the echinocandin is as follows:

the anti-esophageal cancer drug contains at least one or two of the licochalcone or the drug salt thereof.

The weight ratio of the echinocandin to the individual body weight of the mice taking the echinocandin is 20-50 mg/kg.

The anti-esophageal cancer drug also contains a pharmaceutically acceptable carrier and an excipient.

The carrier is preferably sodium carboxymethyl cellulose.

The sodium carboxymethylcellulose is 0.5% sodium carboxymethylcellulose aqueous solution.

The anti-esophageal cancer drug is a tablet, a capsule, a dropping pill, a granule or an oral liquid.

Compared with the prior art, the invention has the following advantages and effects:

the echinocandin is a natural compound separated from the Chinese herbal medicine liquorice, and the molecular mechanism of the echinocandin for resisting cancer is still unclear. The invention finds that the licochalcone has the inhibition effect on the proliferation, the metastasis and the invasion of esophageal cancer cells in vitro, and can effectively inhibit the growth of esophageal cancer in vivo. The echinocandin obviously inhibits the cell tumorigenicity of the esophageal cancer, has no obvious toxic or side effect, and can provide a new medicine selection for the clinical treatment of the esophageal cancer.

The invention also discovers that the licochalcone can increase the sensitivity of esophageal cancer cells to 5-fluorouracil, and can be used for clinical treatment of esophageal cancer by using the licochalcone and 5-fluorouracil in a combined mode.

Drawings

FIG. 1 is a graph showing the results of experiments on esophageal cancer cells KYSE30 and KYSE270 CCK-8 at different concentrations of licochalcone.

FIG. 2 is a graph showing the results of experiments on the formation of single clones of esophageal cancer cells KYSE30 and KYSE270 at different concentrations of licochalcone.

FIG. 3 is a graph showing the results of experiments on the metastasis of esophageal cancer cells KYSE30 and KYSE270 at different concentrations of licochalcone.

FIG. 4 is a graph showing the results of experiments on the invasion of esophageal cancer cells KYSE30 and KYSE270 at different concentrations of licochalcone.

FIG. 5 is a graph showing the results of experiments for detecting the expression levels of Vimentin, β -catenin and E-cadherin in esophageal cancer cells KYSE30 and KYSE270 under different concentrations of licochalcone.

FIG. 6 is a graph showing the results of experiments on esophageal cancer cells KYSE30 and KYSE270 CCK-8 under different reagent treatments.

FIG. 7 is a graph showing the experimental results of the monoclonal formation of esophageal cancer cells KYSE30 and KYSE270 under different reagent treatments.

FIG. 8 is a graph showing the results of experiments for detecting the expression levels of clean caspase-3, caspase-3 and clean PARP proteins in esophageal cancer cells KYSE30 and KYSE270 under different reagent treatments.

FIG. 9 is a graph showing the results of changes in tumor volume at different concentrations of licochalcone.

FIG. 10 is a graph showing the results of body weight changes in nude mice at different concentrations of licochalcone.

FIG. 11 is a graph showing the results of changes in the morphology of important tissues (kidney, lung, liver) at different concentrations of licochalcone.

FIG. 12 is a graph showing the results of experiments for detecting the expression levels of P-AKT, P-mTOR, mTOR and LC3 proteins in esophageal cancer cells KYSE30 and KYSE270 at different concentrations of licochalcone.

FIG. 13 is a graph showing ALT and AST results of blood biochemical routine tests of nude mice at different concentrations of licochalcone.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Experimental materials involved in the examples of the present invention:

human esophageal cancer cell lines KYSE30 and KYSE270 cells were purchased from ATCC;

the echinocandin is purchased from Shanghai ceramic Biotechnology limited;

CCK-8 was purchased from the institute of Homon chemistry, Japan;

female nude mice (balb/c-nu/nu) were purchased from the university of Nanjing model animal institute;

e-cadherin monoclonal antibodies are available from Proteintech Group, β -Catenin, vimentin, caspase-3, cleared caspase-3, PARP, cleared PARP, phophor-AKT, AKT, phophor-mTOR, mTOR, LC3 and β -actin in the United states as Cell Signaling Technology;

DMEM medium, fetal bovine serum, penicillin-streptomycin were purchased from Gibco, usa;

dimethyl sulfoxide (DMSO) was purchased from sigma, usa;

the echinocandin was dissolved in DMSO at a concentration of 50mM and stored in a freezer at-80 ℃.

Example 1 Single drug experiment

CCK-8 experiment: KYSE30 and KYSE270 cells in good growth state were collected, digested, counted, adjusted for cell density, and seeded in a 96-well plate at a cell count of 50. mu.L/1000 cells. After overnight cell attachment, 50mM stock solution of licochalcone was diluted to a concentration gradient in DMEM medium, and 50. mu.L of licochalcone was added to each well. Four cell treatment groups were set with concentrations of 0. mu.M, 10. mu.M, 20. mu.M and 40. mu.M each, 3 duplicate wells per group, and blank wells were set. Adding 10 μ L of CCK-8 to each well at 0, 24, 48, 72, 96 and 120h (keeping away from light during the addition process, avoiding the difference between wells and bubbles) according to CCK-8 kit instruction, and keeping temperature at 37 deg.C and 5% CO₂After incubation for 2h in the incubator, the absorbance of each well was read at a wavelength of 450nm using a multifunctional microplate reader, and the results are shown in FIG. 1.

Monoclonal formation experiments: KYSE30 and KYSE270 cells in good growth state were collected, digested, counted, adjusted for cell density, and seeded in 6-well plates at a cell count of 2mL/1000 cells. After overnight cell adherence, 50mmol/L stock solution of licochalcone is diluted to a certain concentration gradient by DMEM medium, and 2mL of licochalcone is added into each well. Three cell treatment groups were set up with licochalcone concentrations of 0. mu.M, 10. mu.M and 20. mu.M, respectively, 3 duplicate wells in each group at 37 ℃ with 5% CO₂And (5) incubation in an incubator. After 14 days, the 6-well plate was removed, washed 3 times with PBS, fixed with methanol for 15min, stained with 0.4% crystal violet for 10min, rinsed gently under running water to remove excess crystal violet, placed in an oven at 37 ℃ and dried, and the number of monoclonals was counted by naked eyes. The results are shown in FIG. 2.

The CCK-8 experiment result in figure 1 and the monoclonal formation experiment result in figure 2 show that the concentration and time dependence of the echinocandin obviously inhibits the proliferation of esophageal cancer cells.

Transwell experiment: the chamber was placed in a 24-well plate and pre-treated 2h before the experiment. Serum-free blank medium was added to all the migrated cells, 200. mu.L in the upper chamber and 500. mu.L in the lower chamber. The invaded chamber was filled with 200. mu.L of matrigel buffer (matrigel: medium: 1:25) at an appropriate concentration in a serum-free blank mediumThe upper and lower chambers were filled with 500. mu.L of blank medium. All are put at 37 ℃ and 5% CO₂And (5) incubation in an incubator. After 2h, well-grown cells were harvested, digested and collected in a 15mL centrifuge tube, centrifuged at 400g for 5min at room temperature, then washed 2 times with PBS, and the centrifugation was repeated to remove the serum remaining in the medium. The cell density was adjusted by counting with blank medium, the upper chamber was seeded with 200. mu.L/100000 cells (the previous blank medium was discarded), and the lower chamber was changed to complete medium at 20% serum concentration. Four cell treatment groups were set up with licochalcone concentrations of 0. mu.M, 10. mu.M, 20. mu.M and 40. mu.M, 3 duplicate wells in each group, at 37 ℃ with 5% CO₂And (5) incubation in an incubator. After 24h, the upper and lower chambers were discarded, washed 1 time with PBS, and 500. mu.L of methanol was added to the upper and lower chambers, respectively, to immobilize the cells for 15 min. The methanol was discarded and washed 1 time with PBS. Add 500. mu.L of 0.4% crystal violet to the upper and lower chambers, respectively, and stain for 5 min. And finally, soaking with ultrapure water to remove residual crystal violet, wiping off cells attached to the upper chamber with a cotton swab, retaining the passed cells, drying the small chamber in a 37 ℃ oven, taking a picture with a microscope, counting, and determining the inhibition effect of the drug on the cell transfer and invasion capacity according to the number of the passed cells. The results are shown in FIGS. 3 and 4.

Western blot experiment: collecting KYSE30 and KYSE270 cells with good growth state, digesting, counting, adjusting cell density to 2mL/2 × 10⁵The number of individual cells was seeded in 6-well plates. After overnight cell adherence, 50mmol/L stock solution of licochalcone is diluted to a certain concentration gradient by DMEM medium, and 2mL of licochalcone is added into each well. Setting four cell treatment groups, the concentrations of licochalcone are 0 μ M, 10 μ M, 20 μ M and 40 μ M respectively, placing at 37 deg.C and 5% CO₂And (5) incubation in an incubator. After 48h, collecting a proper amount of cells in a 1.5mL centrifuge tube, adding a proper amount of protein lysate (adding a protease inhibitor PMSF according to a ratio of 1:100 in advance), intermittently shaking the lysed cells on a vortex oscillator for 40min until the cells are completely lysed, centrifuging at 12000rpm at 4 ℃ for 1h, and transferring the supernatant into a new centrifuge tube. The BCA method is used for detecting the protein concentration in the cracking sample, ultrapure water and Loading buffer (4X) are used for adjusting the sample to the same concentration, and 10% SDS-PAGE is used after boilingGel electrophoresis (total loading protein about 30 μ g), transferring the Thermo protein semi-dry transfer membrane system onto PVDF membrane, sealing with 5% skimmed milk at room temperature for 1h, incubating overnight at 4 deg.C for the first antibody (Vimentin 1:1000, β -Catenin 1:1000, E-cadherin 1:5000, β -Catenin 1:1000), recovering the first antibody, washing the membrane with 1 ‰ TBST for 5 times each for 5min, adding HRP-labeled corresponding secondary antibody (antibody diluted in 5% skimmed milk powder at a dilution ratio of 1: 2000-3000), incubating with a shaker at room temperature for l h, washing with 1 ‰ TBST for 5 times each for 5min, developing ECL solution in a gel imaging system, analyzing the target protein band with Image Lab software, and obtaining the result shown in FIG. 5.

FIG. 3 shows migration and invasion results and FIG. 4 shows that the concentration-dependent inhibition of esophageal cancer cell migration and invasion by the licochalcone is shown, and the increase of E-cadherin expression and the decrease of β -catenin and Vimentin expression in FIG. 5 show that the licochalcone can inhibit the esophageal cancer cell migration and invasion by reversing EMT process of esophageal cancer.

EXAMPLE 2 Combined drug administration experiment

CCK-8 experiment: KYSE30 and KYSE270 cells in good growth state were collected, digested, counted, adjusted for cell density, and seeded in a 96-well plate at a cell count of 50. mu.L/1000 cells. After overnight cell adherence, 50mM glycyrrhiza uralensis chalcone stock solution is diluted to a certain concentration gradient by a DMEM medium, simultaneously 100mM 5-fluorouracil stock solution is diluted to a certain concentration gradient by a DMEM medium, and 50 mu L of working solution is added into each hole. Four cell treatment groups were set, a control group (DMSO), a 5-fluorouracil group (concentration of 1.25 μmol/L in KYSE30, and concentration of 0.3 μmol/L in KYSE 270), a licochalcone group (Echinatin ═ 20 μmol/L), a combination group (Echinatin +5-FU ═ 20 μmol/L +1.25 μmol/L in KYSE30, and Echinatin +5-FU ═ 20 μmol/L +0.3 μmol/L in KYSE 270), and 3 duplicate wells per group. Adding 10 μ L of CCK-8 to each well at 0, 24, 48, 72, 96 and 120h (keeping away from light during the addition process, avoiding the difference between wells and bubbles) according to CCK-8 kit instruction, and keeping temperature at 37 deg.C and 5% CO₂After incubation in the incubator for 2h, the absorbance of each well was read at a wavelength of 450nm using a multifunctional microplate reader. The results are shown in FIG. 6.

Monoclonal formation experiments: KYSE30 and KYSE270 cells in good growth state were collected, digested, counted, adjusted for cell density, and seeded in 6-well plates at a cell count of 2mL/1000 cells. After overnight cell adherence, working solution was prepared as above, and 2mL of working solution was added to each well. Four cell treatment groups were set, a control group (DMSO), a 5-fluorouracil group (concentration of 1.25 μmol/L in KYSE30 and 0.3 μmol/L in KYSE 270), a licochalcone group (Echinatin 20 μmol/L), a combination group (Echinatin +5-FU 20 μmol/L +1.25 μmol/L in KYSE30 and Echinatin +5-FU 20 μmol/L +0.3 μmol/L in KYSE 270). At 37 ℃ with 5% CO₂And (5) incubation in an incubator. After 14 days, the 6-well plate was removed, washed 3 times with PBS, fixed with methanol for 15min, stained with 0.4% crystal violet for 10min, rinsed gently under running water to remove excess crystal violet, placed in an oven at 37 ℃ and dried, and the number of monoclonals was counted by naked eyes. The results are shown in FIG. 7.

Western blot experiment: collecting KYSE30 and KYSE270 cells with good growth state, digesting, counting, adjusting cell density to 2mL/2 × 10⁵The number of individual cells was seeded in 6-well plates. After overnight cell adherence, mixed working solution was prepared as above, and 2mL of the solution was added to each well. The following procedure was performed in the same manner as in Western blot (concerning primary antibody concentrations caspase-31: 1000, clear caspase-31: 1000, PARP 1:1000) in example 1, and the results are shown in FIG. 8.

FIG. 6CCK-8 and FIG. 7 shows that the combination of licochalcone and 5-fluorouracil can enhance the killing effect of 5-fluorouracil on esophageal cancer cells. In FIG. 8, the expression level of both clear PARP and clear caspase3 in the combination group was increased, which indicates that the sensitivity of esophageal cancer cells to 5-fluorouracil can be increased by inducing apoptosis in the cells.

Example 3 in vivo experiments

Construction of subcutaneous model and treatment of nude mice with different drug concentrations

18 nude mice (balb/c-nu/nu) with age of 6-8 weeks and female animals are selected, 6 control groups and 12 experimental groups are set, and a subcutaneous tumor model of the tumor is constructed.

(1) Injecting 5X 10 subcutaneously into each nude mouse⁶The number of esophageal cancer cells KYSE270 is smallThe cells were resuspended in 100. mu.L of Matrigel mixture (PBS: Matrigel: 1 by volume, pH of PBS (1X) 7.2. + -. 0.1)

(2) Before the experiment, the nude mouse is anesthetized, the anesthesia degree is evaluated through painless and painful stimulation, and the nude mouse is determined to be in an anesthetic state;

(3) 18 nude mice were injected subcutaneously with resuspended cells using a 25G needle microinjector.

Treatment of echinocandin: injecting KYSE270 cells subcutaneously into nude mice for a week, and randomly grouping and administrating when the tumor body diameter reaches about 5 mm. The medicine formula is that the licochalcone powder is dissolved in 0.5% sodium carboxymethylcellulose aqueous solution (CMC-Na) to prepare stock solution, and the concentration of the licochalcone is set to be 0mg/kg, 20mg/kg and 50 mg/kg; the administration was by intragastric administration, 100. mu.L/mouse, and the body weight and tumor volume of nude mice were measured every 2 days. The change in tumor volume is shown in FIG. 9. The change in body weight of nude mice is shown in FIG. 10. Changes in the morphology of the vital tissues (kidney, lung, liver) are shown in figure 11.

(4) Western blot detection of nude mouse tumor tissues

Shearing a proper amount of tissue sample by using scissors, placing the tissue sample into a 1.5mL centrifuge tube, adding a corresponding amount of protein lysate (adding a protease inhibitor PMSF according to a ratio of 1:100 in advance) according to the amount of 100 mu L/mg of tissue, grinding the sample on ice by using a grinding rod until no obvious blocky tissue exists, standing on ice for 10min to further crack the tissue, and centrifuging at 4 ℃ and 12000rpm for 1 h. The supernatant was transferred to a new 1.5mL centrifuge tube. The BCA kit detects protein concentration, samples are prepared, electrophoresis is carried out, membrane transfer is carried out, 5% skimmed milk powder is sealed, primary antibodies ((p-AKT 1:2000, AKT 1:1000, p-mTOR 1:1000, mTOR 1:1000, LC 31: 1000) are incubated overnight, TBST is washed for 5 times, secondary antibodies are incubated for 1h, ECL luminescent solution is exposed and developed (detailed steps are shown in the Western blot operation), and the result is shown in figure 12.

(5) Biochemical routine detection of blood of nude mouse

After 14 days of administration, blood was collected from nude mice, serum was separated by centrifugation at 10000g for 1min, and then the samples were sent to Wuhan Google Biotech Co., Ltd for biochemical analysis of blood, and blood from nude mice in 0mg/kg group was used as a control. The results are shown in FIG. 13.

FIG. 9 shows that the experiment results of the change of subcutaneous tumor volume in nude mice treated with different concentrations of licochalcone show that licochalcone can significantly inhibit the esophageal cancer cell tumorigenic ability. FIG. 12 shows changes in P-AKT and P-mTOR, indicating that licochalcone mediates inhibition of esophageal cancer growth through the AKT/mTOR pathway. The change of the weight of the nude mice in fig. 10, the change of the shape of the important tissues (kidney, lung and liver) in fig. 11 and the change of ALT and AST in the conventional blood detection of the nude mice in fig. 13 have no obvious difference, which indicates that the licochalcone has no obvious toxic or side effect on the nude mice.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. Application of echinocandin in preparing anti-esophageal cancer medicine is provided.

2. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that: the anti-esophageal cancer is used for inhibiting the proliferation, metastasis, invasion or tumor growth of esophageal cancer cells.

3. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that: the effective concentration of the echinocandin is 10-40 mu M; further 10 to 20 μ M.

4. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that: the anti-esophageal cancer drug also contains 5-fluorouracil.

5. The use of the licochalcone according to claim 4 for the preparation of an anti-esophageal cancer medicament, wherein: the licochalcone increases the sensitivity of esophageal cancer cells to 5-fluorouracil.

6. The use of the licochalcone according to claim 5 for the preparation of an anti-esophageal cancer medicament, wherein: the proportion of the echinocandin and the 5-fluorouracil is that the molar ratio is 10-40: 0.1 to 2; further comprising the following components in a molar ratio of 10-20: 0.3 to 1.25.

7. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that: the anti-esophageal cancer drug contains at least one or two of the licochalcone or the drug salt thereof.

8. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that: the weight ratio of the echinocandin to the individual body weight of the mice taking the echinocandin is 20-50 mg/kg.

9. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that:

the anti-esophageal cancer drug also contains a pharmaceutically acceptable carrier and an excipient.

10. The use of the licochalcone according to claim 1 for the preparation of an anti-esophageal cancer medicament, characterized in that:

the anti-esophageal cancer drug is a tablet, a capsule, a dropping pill, a granule or an oral liquid.

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