CN112057461A - Application of doramectin in treating esophageal cancer - Google Patents
Application of doramectin in treating esophageal cancer Download PDFInfo
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- CN112057461A CN112057461A CN202010957785.7A CN202010957785A CN112057461A CN 112057461 A CN112057461 A CN 112057461A CN 202010957785 A CN202010957785 A CN 202010957785A CN 112057461 A CN112057461 A CN 112057461A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention relates to the technical field of new application of medicaments, in particular to new application of doramectin in treating esophageal cancer. The invention examines the effect of doramectin on the esophageal cancer resistance through in vivo and in vitro experiments. Doramectin can inhibit the proliferation and migration of esophageal cancer, and promote apoptosis and autophagy of esophageal cancer cells. Therefore, doramectin can be used for preparing a medicament for treating the esophageal cancer, and has good effect on treatment and prognosis of the esophageal cancer.
Description
Technical Field
The invention relates to the technical field of new application of medicaments, in particular to application of doramectin in treating esophageal cancer.
Background
Esophageal cancer is one of the most common and fatal malignant tumors in the world, and has a very low 5-year survival rate due to its high incidence rate and difficulty in making a diagnosis at an early stage. At present, the means for treating esophageal cancer mainly comprises surgical operation, and although the surgical resection rate is higher than before, the overall treatment effect is still unsatisfactory.
Doramectin: (Doramectin, DRM) is a third-generation derivative of avermectin, belongs to macrolide antiparasitic drugs, has a chemical name of 25-cyclohexane-5-O-demethyl-25-abamectin B1, and has a molecular formula of C50H74O14The broad inhibition of parasite activity in and out of animals is used in large quantities in the livestock industry.
To date, no effect of doramectin in the treatment of esophageal cancer has been reported.
Disclosure of Invention
The present invention aims to provide the application of doramectin in treating esophageal cancer so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
the application of doramectin in treating esophageal cancer and the application of doramectin as a medicament for treating esophageal cancer.
Preferably, the doramectin has a structural formula:
preferably, the medicament for treating esophageal cancer comprises doramectin and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutically acceptable carrier comprises one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants or flavoring agents; the excipient is one or a combination of mannitol, lactose, starch, dextran or microcrystalline cellulose; the disintegrating agent is one or more of polyvinylpyrrolidone, carboxymethyl cellulose, sodium carboxymethyl cellulose or hydroxypropyl methyl cellulose; the lubricant is talcum powder or magnesium stearate.
Preferably, the medicament for treating esophageal cancer is in the form of any one of injection, tablet, granule, pill, capsule, suspension, emulsion, ointment, cream and transdermal patch.
Preferably, the medicament for treating the esophageal cancer is an esophageal cancer cell proliferation inhibitor.
Preferably, the medicament for treating esophageal cancer is an esophageal cancer cell migration inhibitor.
Preferably, the medicament for treating the esophageal cancer is an esophageal cancer apoptosis promoter.
Preferably, the medicament for treating the esophageal cancer is an autophagy promoter for the neuroesophageal cancer cells.
Compared with the prior art, the invention has the beneficial effects that:
the doramectin used in the invention can inhibit the proliferation and migration of esophageal cancer and promote the apoptosis and autophagy of esophageal cancer cells. Therefore, doramectin can be used for preparing a medicament for treating the esophageal cancer, and has good effect on treatment and prognosis of the esophageal cancer.
Drawings
FIG. 1 shows the results of MTT assay for the in vitro proliferation of doramectin on Eca109, EC9706 and HECC cells, which inhibits the viability of Eca109 and EC9706 cells in a time and dose dependent manner. However, it had a minor effect on the proliferation of normal astrocyte HECCs, where panels a, b, c are the results of Eca109, EC9706 and HECC cells treated with doramectin for 24h, 48h and 72h, respectively.
Fig. 2 is a result of observing migration inhibition of Eca109 and EC9706 cells by doramectin in a scratch experiment, wherein fig. a: results of Eca109 cells after about 24h treatment with doramectin; and (b) figure: results of EC9706 cells after 24h treatment with doramectin; and (c) figure: statistical results of cell mobility of Eca109 cells after 24h treatment with doramectin; FIG. d: statistics of cell mobility of EC9706 cells after doramectin treatment for 24 h.
Fig. 3 is the results of transmission electron microscopy of apoptotic body autophagosomes in Eca109 and EC9706 cells, wherein panel a: eca109 cell control group; and (b) figure: EC9706 cell control group; and (c) figure: after the Eca109(40 mu M) cells are treated by doramectin for 48 hours, the cell morphology is observed by a transmission electron microscope; FIG. d: after EC9706 (20. mu.M) cells were treated with doramectin for 48h, the morphology of the cells was observed by transmission electron microscopy.
FIG. 4 is a Westernblotting test of autophagy protein and autophagy-related protein expression level, and the effect of doramectin on autophagy protein expression of Eca109 and EC9706 cells, wherein, the graph a: after the cells of Eca109 (0. mu.M, 10. mu.M, 20. mu.M and 40. mu.M) are respectively treated by doramectin with different concentrations for 48 hours, the distribution of autophagy protein expression result graphs is obtained; and (b) figure: analysis of the statistics of the expression levels of autophagy and autophagy-related proteins of EC9706 (0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M) cells; and (c) figure: the expression level of autophagy protein and autophagy-related protein of Eca109 cells treated by doramectin; FIG. d: autophagy protein and autophagy-related protein expression levels (P <0.01) of EC706 cells treated with doramectin.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: and (3) verifying that doramectin inhibits the growth of esophageal cancer cells U87 and C6 in vitro.
1. Experimental methods
When the cell density of Eca109, EC9706 and HEEC cells in the logarithmic phase of growth reaches 80-90%, digesting the cells, centrifugally collecting, and blowing into single cell suspension;
the cell density was adjusted to 1.0X 10 cells per 100. mu.L of the culture medium4And uniformly mixing the suspension, sequentially adding the suspension into the holes (100 mu L/hole), culturing overnight, and allowing the cells to be completely attached to the wall, wherein the concentrations of the doramectin drugs of the Eca109 cells are respectively as follows: 0 μ M, 5 μ M, 10 μ M, 20 μ M, 40 μ M, 60 μ M, doramectin drug concentrations of EC9706 and HEEC cells were: 0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 60. mu.M;
the stock culture in the 96-well plate was discarded, and a new culture containing different doses of drugs was added thereto, 100. mu.L per well, 3 duplicate wells per drug concentration were set, and 0. mu.M was used as a control group for the experiment;
the drug-treated cells were placed in an incubator (37 ℃ C., 5% CO)2) Culturing for 24h, 48h and 72 h;
finishing the culture, adding MTT solution with the concentration of 5mg/mL, 20 mu L of MTT solution in each hole, and continuing to culture for 4 hours; carefully remove the liquid in the wells, add 150 μ L DMSO, place on a shaker, shake at room temperature away from light for 12 min;
the microplate reader detects the value of A490, the survival rate and half inhibition rate of the cells of the control group and each drug-added group are calculated, and the experiment is repeated for 3 times.
2. Results of the experiment
The results show that doramectin inhibits Eca109 and EC9706 cell viability in a time and dose dependent manner. However, doramectin has a minor effect on the proliferation of HEEC in normal esophageal cells. The results of MTT analysis gave IC50 values and inhibition of cell proliferation for different time and different cells. Doramectin inhibits the growth of Eca109 and EC9706 cells at lower drug concentrations. From the results, doramectin can obviously inhibit the cell activities of Eca109 and EC 9706.
3. Conclusion
Taken together, these results indicate that doramectin can effectively inhibit the growth of esophageal cancer cells Eca109 and EC 9706.
Example 2: validation that doramectin inhibited the migration of Eca109 and EC9706 cells.
1. Experimental methods
The Eca109 and EC9706 cells in the logarithmic growth phase are digested from the culture bottle, and the cells are uniformly blown by the gun head to be independently and uniformly dispersed in the liquid;
eca109 and EC9706 cells were seeded in six-well plates (1.0X 10)5One/well), culturing overnight until the cells are completely attached to the wall;
old culture medium was aspirated from the wells and Eca109 cells were treated with doramectin (10. mu.M, 20. mu.M, 40. mu.M);
doramectin (5 μ M, 10 μ M, 20 μ M) treated EC9706 cells;
selecting a 1mL gun head, vertically scribing at the bottom of a hole, discarding the culture solution after scribing, washing for 3 times by using sterile PBS, adding the culture solution without serum, observing under an inverted microscope, and recording the scratch width of 0 h;
after 24h incubation, scratch width was recorded by observation under an inverted microscope and photographed, and the experiment was repeated 3 times.
2. Results of the experiment
The result shows that after the cells of the control group without doramectin are cultured for 24 hours, the migration speed is higher, and the finally presented width of the scratch is the minimum; with the increase of the drug dose, it can be found that the width change of the scratched area is minimal when the concentration of doramectin is 40 μ M and 20 μ M, and thus it can be seen that doramectin inhibits the migration and proliferation capacity of Eca109 and EC9706 cells. Compared with the cells of the control group, the migration rates of the Eca109 cells treated by doramectin with 10 μ M, 20 μ M and 40 μ M are 46.10 +/-3.49%, 34.91 +/-3.59%, 26.06 +/-1.76% and 16.50 +/-1.41% respectively; after the EC9706 cells are treated by doramectin with the concentrations of 5. mu.M, 10. mu.M and 20. mu.M, the cell migration rates are 43.06 +/-3.26%, 33.60 +/-2.31%, 15.07 +/-2.49% and 15.07 +/-2.49%, respectively.
3. Conclusion
Taken together, these results indicate that doramectin is effective in inhibiting esophageal cancer cell migration.
Example 3: verification that doramectin induced Eca109 and EC9706 cells to produce apoptosis and autophagy.
1. Experimental methods
Eca109 and EC9706 cells were pre-cultured with doramectin at 40. mu.M and 20. mu.M, respectively, for 48 h;
collecting the culture solution in a culture bottle in a 15mL centrifuge tube, washing cells for 2 times by PBS, adding pancreatin into the culture bottle for digestion, blowing the uniform cells to form single cell suspension, then transferring the single cell suspension into the corresponding centrifuge tube, centrifuging at 1400rpm for 6min, and removing the upper layer liquid;
immediately and slowly dripping 2.5 percent glutaraldehyde at 4 ℃ into the tube, then placing the tube into a refrigerator at 4 ℃ for fixing cells for more than 10 hours in a dark place;
add precooled 0.1M PBS (pH7.2) to the EP tube, mix the cells to achieve the purpose of rinsing, repeat centrifugation twice (1400rpm, 6 min);
carefully remove PBS, add 1% osmic acid to completely submerge the cells, pre-fix the cells for 10 min;
adding the 0.1M PBS, mixing and cleaning the cells, and centrifuging twice under the condition of 1400rpm for 6 min;
dehydrating with 50%, 70%, 90%, 100% ethanol at 4 deg.C for 10min at each concentration;
dehydrating the cells with 100% acetone at room temperature for 5 min;
pure acetone and embedding liquid are used for embedding cells for 1h at room temperature according to the volume ratio of 1: 1;
embedding cells for 1.5h at room temperature according to the volume ratio of 1: 2;
embedding cells at room temperature for 20min according to the volume ratio of 1: 3;
then putting the mixture into an oven to heat for overnight;
slicing, and dyeing by using 3% uranium acetate-lead citrate;
the ultrastructure of the cells was observed by a transmission electron microscope, and the experiment was repeated three times.
2. Results of the experiment
The results show that after doramectin treatment of cells, the cells have obvious characteristics of apoptosis and autophagy, the volume is reduced, the cells have partial damage, nucleolus is solidified and contracted, chromatin is gathered and apoptotic bodies and autophagosome accumulation are generated.
3. Conclusion
As described above, doramectin induces apoptosis and autophagy in esophageal cancer cells.
Example 4: doramectin promotes the influence of the expression level of autophagy proteins and autophagy-related proteins of Eca109 and EC9706 cells.
1. Experimental methods
Culturing Eca109 (0. mu.M, 10. mu.M, 20. mu.M, 40. mu.M) and EC9706 (0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M) cells in log phase with doramectin for 48h, collecting cells, adding cell lysate, and lysing the cells;
and (3) detecting the protein concentration by using a BCA protein detection kit, adding a protein buffer solution, boiling with boiling water for 5min, separating the protein by using polyacrylamide gel electrophoresis, and transferring to a PVDF membrane. Then sealing with 5% skimmed milk powder;
finally, the cells were incubated overnight at 4 ℃ in the primary antibody, and protein bands were detected with the secondary antibody and visualized by a gel imaging analysis system.
2. Results of the experiment
The result shows that the autophagy protein and autophagy-related protein expression amount of EC9706 and Eca109 cells treated by doramectin are dependently changed with the dosage of the medicament, and doramectin can induce the autophagy of esophageal cancer EC9706 and Eca109 cells and influence the expression of autophagy-related protein.
3. Conclusion
In conclusion, doramectin can induce apoptosis and autophagy of EC9706 and Eca109 cells.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. The application of doramectin in treating esophageal cancer is characterized in that: application of doramectin as a medicament for treating esophageal cancer.
2. The doramectin for use in treating esophageal cancer according to claim 1, wherein the doramectin has a structural formula:
3. the doramectin for use in treating esophageal cancer according to claim 2, wherein the medicament for treating esophageal cancer comprises doramectin and a pharmaceutically acceptable carrier.
4. Doramectin for use in treating esophageal cancer according to claim 3, wherein the pharmaceutically acceptable carrier comprises one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants or flavoring agents; the excipient is one or a combination of mannitol, lactose, starch, dextran or microcrystalline cellulose; the disintegrating agent is one or more of polyvinylpyrrolidone, carboxymethyl cellulose, sodium carboxymethyl cellulose or hydroxypropyl methyl cellulose; the lubricant is talcum powder or magnesium stearate.
5. The doramectin for treating esophageal cancer according to claim 2, wherein the dosage form of the drug for treating esophageal cancer is any one of injection, tablet, granule, pill, capsule, suspension, emulsion, ointment, cream and transdermal patch.
6. The doramectin for use in treating esophageal cancer according to claim 2, wherein the drug for treating esophageal cancer is an esophageal cancer cell proliferation inhibitor.
7. The doramectin for use in treating esophageal cancer according to claim 2, wherein the drug for treating esophageal cancer is an esophageal cancer cell migration inhibitor.
8. The doramectin for use in treating esophageal cancer according to claim 2, wherein the drug for treating esophageal cancer is an esophageal cancer apoptosis promoter.
9. The doramectin for use in treating esophageal cancer according to claim 2, wherein the drug for treating esophageal cancer is an autophagy promoter for neuroesophageal cancer cells.
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Application publication date: 20201211 |