CN116850272A - Application of recombinant hirudin in preparation of medicine for treating liver cancer - Google Patents
Application of recombinant hirudin in preparation of medicine for treating liver cancer Download PDFInfo
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- CN116850272A CN116850272A CN202210310179.5A CN202210310179A CN116850272A CN 116850272 A CN116850272 A CN 116850272A CN 202210310179 A CN202210310179 A CN 202210310179A CN 116850272 A CN116850272 A CN 116850272A
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- liver cancer
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- recombinant hirudin
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Classifications
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
- A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Zoology (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The application discloses an application of recombinant hirudin in preparing a medicament for treating liver cancer. The application provides an application of recombinant hirudin in preparing a medicine for treating liver cancer, an application in preparing a medicine for inhibiting liver cancer tumor growth, an application in preparing a medicine for inhibiting liver cancer metastasis, and an application in preparing a medicine for inhibiting liver cancer migration and/or inhibiting liver cancer invasion. The application discovers that: the recombinant hirudin can inhibit proliferation, migration adhesion and invasion of liver cancer cells through targeting thrombin, inhibit angiogenesis and induce apoptosis of tumor cells, and has certain pharmacological activity, safety and effectiveness. The application provides a new strategy for developing therapeutic intervention drugs for hepatocellular carcinoma.
Description
Technical Field
The application belongs to the technical field of biology, and relates to application of recombinant hirudin in preparation of a medicine for treating liver cancer.
Background
Hepatocellular carcinoma (hepatocellular carcinoma, HCC, abbreviated as liver cancer) is the most common primary liver cancer worldwide, and causes of death are secondary to many cancers. Despite significant progress in the diagnosis and treatment of liver cancer, the prognosis is still poor, with all stages taken together, with a total survival rate (OS) of only 12% for 5 years. Therefore, it is particularly necessary to explore new therapeutic approaches to further improve patient prognosis and quality of life.
Hirudin (hirudin) is the main active ingredient of traditional Chinese medicine Hirudo, has effects of removing blood stasis, eliminating mass, and dredging channels, and is a good medicine for treating mass, blood stasis and amenorrhea. Because the hirudin only exists in the salivary glands of the leeches, the content is very small, and Harvey in 1986 utilizes a genetic engineering method to synthesize the recombinant hirudin (Recombinant Hirudin, rH), the problem of difficult source of the natural hirudin is solved. The appearance of recombinant hirudin opens up a new way for researching anticoagulant and antithrombotic medicines.
Disclosure of Invention
The application aims to provide an application of recombinant hirudin in preparing a medicament for treating liver cancer.
The application provides an application of recombinant hirudin in preparing a medicament for treating liver cancer.
The application also provides an application of the recombinant hirudin in preparing a medicament for inhibiting the growth of liver cancer tumors.
The application also provides application of the recombinant hirudin in preparing a medicament for inhibiting liver cancer metastasis.
The application also provides application of the recombinant hirudin in preparing medicines for inhibiting migration and/or invasion of liver cancer.
The application also provides an application of the recombinant hirudin in preparing products; the function of the product is as follows (a) or (b) or (c) or (d):
(a) Inhibiting liver cancer cell blood migration;
(b) Inhibiting lung metastasis and/or brain metastasis of liver cancer;
(c) Inhibiting vascular infiltration of carcinoma in situ of the liver;
(d) Relieving hypercoagulability of blood in liver cancer tumor microenvironment.
The application also provides an application of the recombinant hirudin in preparing products; the product has the functions of inhibiting proliferation of liver cancer cells and/or promoting apoptosis of liver cancer cells and/or inhibiting survival of liver cancer cells and/or inhibiting migration of liver cancer cells and/or inhibiting invasion of liver cancer cells and/or inhibiting adhesion of liver cancer cells.
In any of the above applications, the recombinant hirudin acts through targeted thrombin mediation.
In any of the above applications, the recombinant hirudin acts through the targeting of PAR-1 mediation.
In any of the above applications, the recombinant hirudin acts via the PI3K/Akt pathway.
The application also provides a medicine for treating liver cancer, which contains recombinant hirudin.
The application also provides a medicament comprising recombinant hirudin; the medicine has the functions of inhibiting the growth of liver cancer tumor and/or inhibiting liver cancer metastasis and/or inhibiting liver cancer migration and/or inhibiting liver cancer invasion.
The application also provides a product comprising recombinant hirudin; the function of the product is as follows (a) or (b) or (c) or (d):
(a) Inhibiting liver cancer cell blood migration;
(b) Inhibiting lung metastasis and/or brain metastasis of liver cancer;
(c) Inhibiting vascular infiltration of carcinoma in situ of the liver;
(d) Relieving hypercoagulability of blood in liver cancer tumor microenvironment.
The application also provides a product comprising recombinant hirudin; the product has the functions of inhibiting proliferation of liver cancer cells and/or promoting apoptosis of liver cancer cells and/or inhibiting survival of liver cancer cells and/or inhibiting migration of liver cancer cells and/or inhibiting invasion of liver cancer cells and/or inhibiting adhesion of liver cancer cells.
In any of the above drugs, the recombinant hirudin acts through targeted thrombin mediation.
In any of the above products, the recombinant hirudin acts through targeted thrombin mediation.
In any of the above drugs, the recombinant hirudin acts through the mediation of targeting PAR-1.
In any of the above products, the recombinant hirudin acts through the targeting of PAR-1 mediation.
In any of the above drugs, the recombinant hirudin acts through the PI3K/Akt pathway.
In any of the above products, the recombinant hirudin acts through the PI3K/Akt pathway.
Any of the above liver cancers may specifically be hepatocellular carcinoma.
Specifically, the recombinant hirudin is a product of Tianjin harmony civil biotechnology limited company.
Specifically, the recombinant hirudin is shown as a sequence 1 in a sequence table in a patent publication text of CN 101250530B.
Specifically, the recombinant hirudin is expressed by a DNA molecule shown as a sequence 2 of a sequence table in a patent publication text of CN 101250530B.
Specifically, the recombinant hirudin is obtained by expressing Hansenula polymorpha 205-17CGMCC No.2424 in CN101250530B patent publication text.
Specifically, the recombinant hirudin is obtained in the 0084 section in the patent publication text of CN 101250530B.
In practice, the recombinant hirudin may be administered to a patient directly or after admixture with a suitable carrier or excipient for therapeutic purposes. The carrier materials herein include, but are not limited to, water soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl ethyl cellulose, etc.). The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injection and the like. Can be common preparation, slow release preparation, controlled release preparation and various microparticle administration systems. For the purpose of shaping the unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets. For the purpose of formulating the unit dosage form into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. For preparing a unit dosage form into a suppository, various carriers well known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. For preparing unit dosage forms into injectable preparations such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired. The dosage forms can be used for administration by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like.
The dosage of recombinant hirudin to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the particular active ingredient used, the route and number of administrations, etc. The above-mentioned doses may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.
The inventors of the present application conducted in vitro experiments and in vitro experiments, and the results showed that: recombinant hirudin (rH) can inhibit proliferation, migration adhesion and invasion of liver cancer cells through targeted thrombin, inhibit angiogenesis and induce apoptosis of tumor cells, and has certain pharmacological activity, safety and effectiveness. The application provides a new strategy for developing therapeutic intervention drugs for hepatocellular carcinoma.
Drawings
FIG. 1 shows the results of cell proliferation rate and IC in example 1 50 And (5) value results.
FIG. 2 shows the results of the cell proliferation rate in example 2.
FIG. 3 is the result of the scratch test in example 2 to determine tumor cell migration.
FIG. 4 shows the results of the Transwell assay of example 2 for tumor cell invasion.
FIG. 5 shows the result of the adhesion of recombinant hirudin to BEL-7402 cells in example 2.
FIG. 6 shows the results of detection of caspase-3, bax and Bcl-2 expression in example 2.
FIG. 7 shows the results of the TUNEL-positive cell count assay in example 2.
FIG. 8 shows the effect of the PI3K/Akt pathway of example 2 on recombinant hirudin-induced apoptosis.
FIG. 9 shows the results of the cell proliferation rate in example 3.
FIG. 10 is the result of the scratch test in example 3.
FIG. 11 shows the result of the Transwell experiment in example 3.
FIG. 12 is the result of the adhesion test in example 3.
FIG. 13 shows the results of detection of caspase-3 and Bax expression in example 3.
FIG. 14 shows the results of the TUNEL-positive cell count assay in example 3.
FIG. 15 shows the result of the inhibition of thrombin-induced tumor angiogenesis by recombinant hirudin in example 3.
Figure 16 shows the change in animal body weight and tumor volume as a function of time for administration in step one of example 4.
Fig. 17 is a photograph of the sacrificed animals and a photograph of the tumor tissue in step one of example 4.
FIG. 18 is a photograph of H & E staining of tumor tissue in step one of example 4.
FIG. 19 is a graph showing the change in animal body weight with time of administration in step two of example 4.
FIG. 20 is a photograph of H & E staining in step two of example 4.
Figure 21 is a graph showing the change in animal body weight over time in step three of example 4.
FIG. 22 is the results of the liver/body ratio and liver volume of the animals in step three of example 4.
FIG. 23 is a photograph of the liver and H & E staining in step three of example 4.
FIG. 24 is a photograph of the liver and absorbance results of the homogenized supernatant in step three of example 4.
FIG. 25 shows the results of Westren Blot detection in step three of example 4 (caspase-3, bax and Bcl-2).
FIG. 26 shows the effect of recombinant hirudin on TUNEL-positive area in a model mouse for carcinoma in situ in step three of example 4.
FIG. 27 is the result of Westren Blot detection in step three of example 4 (phosphorylated PI3K and phosphorylated Akt).
Fig. 28 is the result of the time to bleed from the tip of the mouse tail in step three of example 4.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. In an embodiment, TUNEL apoptosis assay kit is used to detect apoptosis. 5-fluorouracil (CAS No. 51-21-8): beijing Soy Bao technology Co., ltd. The recombinant hirudin used in the examples was obtained as a product of Tianjin and well-established biotechnology Co., ltd (see patent publication No. CN101250530B, namely, recombinant hirudin obtained in paragraph 0084 therein, which patent publication No. 200810103154.8). BEL-7402 cells (human liver cancer cells), huh-7 cells (human liver cancer cells), hepG2 cells (human liver cancer cells), L-O2 cells (human normal liver cells), shanghai cell biology institute of Chinese academy of sciences. Cell culture conditions: 37 ℃ and 5% CO 2 In an incubator with 100% humidity. Complete culture solution: comprises 1% penicillin-streptomycin and 10% fetal bovine serum, and the balance of RPMI-1640 culture medium. Serum-free medium: contains 1% penicillin-streptomycin and the balance of RPMI-1640 culture medium. Experimental data in the examples are expressed as Mean ± standard error (Mean ± SD), analyzed using SPSS 21.0 statistical software; the average value between the two groups adopts t detection and p<0.05 toolHas statistical significance.
EXAMPLE 1 recombinant hirudin IC for three liver cancer cells 50 Value of
Test cells: BEL-7402 cells, huh-7 cells, hepG2 cells.
Test drug: recombinant hirudin.
1. The test cells were seeded in 96-well cell culture plates (4000-5000 cells per well) and cultured with complete culture medium until the cells had adhered to the wall and a growth density of about 50%.
2. After completion of step 1, the culture supernatant was discarded, and 100. Mu.L of serum-free medium containing the test drug was added to each well and cultured for 24 hours. Different concentrations of the test drug were set, and a control was set without the test drug added. 6 duplicate wells were set per concentration treatment.
3. After completion of step 2, the culture supernatant was discarded, washed with PBS buffer, and then 100. Mu.L of CCK-8 solution was added to each well, followed by incubation for 1-2 hours.
4. After the completion of step 3, the absorbance at 450nm (OD value) was measured by a microplate reader, and the cell proliferation rate was calculated.
Cell proliferation rate= (OD Control wells –OD Medicine hole )÷OD Control wells ×100％。
The drug concentration at 50% cell proliferation rate is IC 50 Values.
Cell proliferation rate results and IC 50 The results are shown in FIG. 1. IC of recombinant hirudin on BEL-7402 cells 50 The value was 192.8. Mu. Mol/L. IC of recombinant hirudin on HepG2 cells 50 The value was 241.9. Mu. Mol/L. IC of recombinant hirudin on Huh-7 cells 50 The value was 387. Mu. Mol/L.
Example 2 Effect of recombinant hirudin on liver cancer cells (cell test)
1. Effect of recombinant hirudin on cell proliferation
Test cells: BEL-7402 cells, L-O2 cells.
Test drug: recombinant hirudin.
The test method was the same as in example 1.
The results of the cell proliferation rate are shown in FIG. 2 (vsIn comparison with the control group, * p<0.05). Recombinant hirudin had no significant effect on the proliferation and growth of L-O2 cells (left panel), only at higher doses (160. Mu.M and 320. Mu.M) had a certain inhibitory effect. The recombinant hirudin produced an inhibitory effect on BEL7402 cells at an administration concentration of 20. Mu.M (right panel), with the inhibition rate increasing with increasing concentration, with a dose-dependent effect.
2. Effect of recombinant hirudin on BEL-7402 cell migration and invasion
Test drug: recombinant hirudin or 5-fluorouracil (positive control drug).
1. Scratch assay for determination of tumor cell migration
(1) Three horizontal lines were drawn on average after 6-well plates with markers, and BEL-7402 cells in the logarithmic phase were then seeded into 6-well plates (3X 10 per well) 5 -5×10 5 Cells) were cultured using complete culture broth to a cell density of greater than 70%.
(2) After the step (1) is completed, the culture supernatant is discarded, scratches are performed on the cell surface perpendicular to the well line by using a sterile gun head, and then the scratched cells and fragments are removed by washing with PBS buffer for 2 to 3 times.
(3) After the step (2) is completed, adding serum-free culture solution containing the tested medicine, marking the intersection point of the transverse line and the scratch by using a marker pen, and photographing and recording for 0h under an inverted microscope. Different concentrations of the test drugs were set, and a Control (Control) was set without adding the test drugs. 3 duplicate wells were set per concentration treatment.
(4) After the step (3) is completed, culturing is continued for 6 hours, 12 hours or 24 hours, photographing and observing are carried out under an inverted microscope, and the migration condition of the cells is recorded.
The results are shown in figure 3 (compared to the control group, * p<0.05， ** p<0.01)。
2. determination of tumor cell invasion by Transwell experiments
(1) A transwell cell having a membrane pore size of 8 μm was selected, matrigel was diluted to 8-fold volume with serum-free culture medium, and then the upper cell surface of the bottom membrane was coated at 40. Mu.l/cell, and left standing at 37℃for 3 hours to polymerize Matrigel into a gel.
(2) After completion of step (1), 600. Mu.l of RPMI-1640 medium containing 10% FBS was added to the lower chamber, the forceps were sterilized, the chamber was gently placed in an orifice plate, BEL-7402 cells in the logarithmic phase (1.5X10 cells per orifice) were added to the upper chamber 5 Individual cells), and the test drug-containing serum-free medium was used for 24 hours. Different concentrations of the test drugs were set, and a Control (Control) was set without adding the test drugs. 3 duplicate wells were set per concentration treatment.
(3) After the step (2) is completed, the residual culture medium liquid in the upper chamber is sucked, the cells in the upper chamber are gently wiped off by a cotton swab, 1ml of methanol is added for fixing for 10min to 20min, the fixing liquid is discarded, the PBS buffer solution is used for cleaning for 2 to 3 times, the crystal violet dye is used for dyeing for 30min, the PBS buffer solution is used for cleaning for 3 times, the crystal violet and nonspecific binding dye which are not bound with the cells are washed off, and then the cells are placed on a glass slide for observation under an inverted microscope, and random 5 interfaces are photographed and counted.
The results are shown in figure 4 (compared to the control group, * p<0.05， ** p<0.01)。
the result of the second step shows that: compared with the control group, the recombinant hirudin with the concentration of 40, 80 and 160 mu M remarkably reduces BEL-7402 cell wound healing, reduces the number of cell penetrating the membrane and has better effect than 5-fluorouracil. It is shown that recombinant hirudin inhibits the migration and invasive capacity of cancer cells and is dose-dependent.
3. Effect of recombinant hirudin on BEL-7402 cell adhesion
Test drug: recombinant hirudin or 5-fluorouracil (positive control drug).
1. The FN fibronectin (Beijing Soy Bao technology Co., ltd.) was diluted to 10. Mu.g/ml with serum-free medium to obtain FN diluent.
2. A96-well plate was prepared, 50. Mu.l of FN dilution was added to each well, and the mixture was allowed to stand at 4℃overnight.
3. After completion of step 1, the liquid in the wells was discarded, washed with 1% BSA solution, and then 50. Mu.l of 1% BSA solution was added to each well and incubated at 37℃for 1 hour.
4. After step 3 is completed, the liquid in the wells is discarded, and 4×10 liquid is added to each well 4 BEL-7402 cells containing the test agentThe serum-free culture solution is cultured for 2 hours. Different concentrations of the test drugs were set, and a Control (Control) was set without adding the test drugs. 3 duplicate wells were set per concentration treatment.
5. After step 4 is completed, the liquid in the wells is discarded, the wells are washed 3 times by using PBS buffer solution to wash off the cells which are not adhered, the cells are fixed by using cell fixing solution for 15-30min, then the fixed solution is washed off, the cells are dyed by using 0.5% crystal violet solution for 20min, then the cells are washed 2-3 times by using PBS buffer solution, 4 fields of view are randomly selected in a 96-well plate, and the cells are photographed and counted.
6. After completion of step 5, the cells stained with crystal violet were soaked in 30% acetic acid solution, the dye was extracted, and absorbance was measured at 600nm with an enzyme-labeled instrument.
The results are shown in figure 5 (compared to the control group, * p<0.05， ** p<0.01). The result shows that BEL-7402 cells have obvious central aggregation and adhesion phenomenon under the induction of adhesion factors, and the recombinant hirudin treatment can obviously relieve the adhesion phenomenon of cancer cells and matrixes and is dose-dependent.
4. Recombinant hirudin-induced apoptosis of BEL-7402 cells
To further investigate the mechanism by which recombinant hirudin induces BEL-7402 cell death and exerts protective effects, the inventors examined the expression of anti-apoptotic and pro-apoptotic proteins. Recombinant hirudin was found to significantly increase the expression of cleaved caspase-3 and Bax, decreasing the expression of Bcl-2, with concomitant increase in TUNEL positive cell numbers. The results are shown in figure 6 (compared to the control group, * p<0.05， ** p<0.01 Fig. 7 (compared to the control group, ** p<0.01)。
the PI3K/Akt signaling pathway plays an important role in regulating cell cycle, proliferation and apoptosis. To investigate the role of the PI3K/Akt pathway in recombinant hirudin-induced apoptosis, PI3K and Akt activation was detected by western blotting using phosphorylated antibodies. The results are shown in figure 8 (compared to the control group, * p<0.05， ** p<0.01). Treatment with recombinant hirudin reduced the levels of phosphorylated PI3K and phosphorylated Akt in BEL-7402 cells.
The results of example 2 show that: recombinant hirudin (rH) can inhibit proliferation of liver cancer cell BEL-7402, but does not affect growth and proliferation of normal liver cell; meanwhile, rH administration can obviously inhibit migration, invasion and adhesion of tumor cells and induce apoptosis of the tumor cells, and is dose-dependent.
Example 3, inhibition of cells is mediated by targeting Thrombin (Thrombin): beijing Soy Bao technology Co., ltd.
1. Recombinant hirudin can inhibit abnormal growth of tumor cells induced by thrombin
To determine the specific mechanism of the protective action of recombinant hirudin, the inventors further induced BEL-7402 cells in vitro with thrombin (1U/mL) and conducted experiments related to cell proliferation and migration.
Test methods see example 2.
The results of the cell proliferation rate are shown in figure 9 (compared to thrombin-induced group, ** p<0.01; in comparison with the control group, ## p<0.01). The results of the scoring experiments are shown in figure 10 (compared to thrombin-induced group, ** p<0.01; in comparison with the control group, # p<0.05). The results of the Transwell experiments are shown in figure 11 (compared to thrombin-induced group, ** p<0.01; in comparison with the control group, ## p<0.01). The adhesion test results are shown in figure 12 (compared to thrombin-induced group, ** p<0.01; in comparison with the control group, ## p<0.01)。
the results show that: thrombin induction can obviously promote proliferation and wound healing of tumor cells, and improve invasion capacity of the tumor cells penetrating through matrixes and adhesion effect with the matrixes; the protective effect of the recombinant hirudin can be partially antagonized along with the induction of thrombin, so that the protective effect generated at a lower dosage (20 mu M) is eliminated, but along with the increase of the administration concentration of the recombinant hirudin, the abnormal growth of tumor cells caused by the induction of thrombin can be effectively reversed, and the recombinant hirudin has certain dose dependency.
2. Recombinant hirudin can reverse reduction of thrombin-induced tumor cell apoptosis
The apoptosis promoting protein Bax and the cleaved caspase-3 protein are obviously reduced under the action of thrombin, which indicates that the induction of thrombin can reduce the apoptosis phenomenon in tumor cells and inhibit the apoptosis promoting effect of recombinant hirudin on the tumor cells; however, with the administration of recombinant hirudin drug, the above phenomenon was effectively improved, accompanied by an increase in the number of TUNEL positive cells, demonstrating that recombinant hirudin can effectively reverse the decrease in thrombin-induced tumor cell apoptosis.
The results are shown in figure 13 (compared to thrombin-induced group, * p<0.05， ** p<0.01; in comparison with the control group, ## p<0.01 Fig. 14).
3. Recombinant hirudin can inhibit thrombin-induced tumor angiogenesis
The abnormal angiogenesis is an important influencing factor in the tumor microenvironment, and because of the characteristic, a great amount of growth factors, cell chemotactic factors and immunoinflammatory reactions generated by various proteolytic enzymes exist in the tumor microenvironment, and the characteristics are very favorable for proliferation, invasion, adhesion, angiogenesis and radioresistance chemotherapy of tumors, and promote the generation and metastasis of malignant tumors, wherein Ang-I, ang-II and VEGFA are proteins with very close relationship with angiogenesis and vascular maturation. In order to determine the effect of recombinant hirudin on reverse thrombin-induced angiogenesis, the inventors further examined the change in the above index.
The results are shown in figure 15 (compared to thrombin-induced group, * p<0.05， ** p<0.01; in comparison with the control group, ## p<0.01). The significant increase in thrombin receptor PAR-1 in response to thrombin induction, with concomitant significant increases in Ang-I, ang-II and VEGFA expression, compared to the uninduced group, suggests that thrombin may induce the above protein expression, further promoting tumor neovascularization. The administration of the recombinant hirudin can obviously inhibit the expression of PAR-1 protein and relieve the phenomenon, so that the angiogenesis-related protein is reduced to the original level or even below the original level, which proves that the recombinant hirudin can effectively inhibit the angiogenesis induced by thrombin.
The results of example 3 show that: recombinant hirudin (rH) can inhibit proliferation, migration, invasion and adhesion of BEL-7402 cells, induce apoptosis of tumor cells, and effectively inhibit expression of angiogenesis-related proteins in tumor cells in a dose-dependent manner by treating the recombinant hirudin (rH); furthermore, the antitumor effect of recombinant hirudin is exerted by targeted inhibition of thrombin.
Example 4 recombinant hirudin inhibits tumor growth and angiogenesis in vivo
The test animals are 4-6 weeks male BALB/c-nude mice; university of south Beijing—south Beijing biomedical research institute, animal use license number: SCXK (Su) 2015-0001.SPF environment feeding.
1. Recombinant hirudin for inhibiting growth of nude mice axillary transplanted tumor
1. Establishment of nude mouse xenograft tumor model
(1) BEL-7402 cells in logarithmic growth phase were taken, digested, collected by centrifugation, washed with PBS buffer, and resuspended in Matrigel dilution (Matrigel was diluted 8-fold volume with serum-free medium) to give 5X 10 cells 8 cell suspension of cells/mL.
(2) The right axilla of the test animal was inoculated subcutaneously with 100. Mu.L/min of the cell suspension prepared in step (1). Normal feeding, about one week, the induration with the size of rice grains can be observed under the skin of the inoculated part, and the nude mice xenograft tumor model is successfully established.
2. Drug administration and drug efficacy evaluation
Taking a nude mice xenograft tumor model, and feeding normally until tumor volume reaches 100mm 3 . Animals were then divided into six groups for group administration. Tumor volume v=ab 2 2; a: maximum diameter of transplanted tumor, b: maximum transverse diameter of the transplanted tumor.
Model set (Model): subcutaneous administration was twice daily (9:00/17:30), with 0.4ml of physiological saline administered each time;
recombinant hirudin treatment group 1 (0.2 mg/kg): the single administration dosage of the recombinant hirudin is 0.2mg/kg;
recombinant hirudin treatment group 2 (0.4 mg/kg): the single administration dosage of the recombinant hirudin is 0.4mg/kg;
recombinant hirudin treatment group 3 (0.8 mg/kg): the single administration dosage of the recombinant hirudin is 0.8mg/kg;
recombinant hirudin treatment group 4 (1.6 mg/kg): the single administration dosage of the recombinant hirudin is 1.6mg/kg;
5-Fu positive control group (20 mg/kg): the single dose of 5-fluorouracil was 20mg/kg.
The recombinant hirudin-treated group was subcutaneously administered twice daily (9:00/17:30) in a volume of 0.4ml each time (in physiological saline).
5-Fu positive control group was administered once daily.
The group administration was continued for 28 days. Animals were weighed every 4 days and the volume of the transplanted tumor was measured. After 28 days of group dosing, animals were sacrificed and photographed, tumor tissue was peeled off and photographed, and tumor tissue was H & E stained.
The change in animal body weight and tumor volume of the grafts with time of administration is shown in figure 16 (n=2).
Photographs of sacrificed animals and photographs of tumor tissues are shown in fig. 17.
A photograph of H & E staining of tumor tissue is shown in FIG. 18.
The results show that: the weight of mice in the model group shows a descending trend, the weight change of each administration group is basically kept stable during the administration period, and the tumor volume increasing trend is obviously weakened compared with that of the model group, so that the administration of the recombinant hirudin can protect the weight reduction of nude mice caused by tumor transplantation, and simultaneously can slow down the tumor increasing trend, and has good anti-tumor effect. The H & E staining results of tumor tissue showed: the cells in the tumor tissue of the model group have obvious phenomena of chromatin edge collection, nuclear shrinkage and the like, the cancer cells have obvious atypical property, and the recombinant hirudin treatment can obviously relieve the occurrence of the phenomena.
2. Recombinant hirudin for inhibiting blood metastasis of nude mice BEL-7402 liver cancer cells
1. BEL-7402 cells in logarithmic growth phase are taken, digested, and then centrifuged to collect the cells, which are resuspended with physiological saline to obtain a cell suspension.
2. The tail vein injection of the test animal injects the cell suspension prepared in the step 1, 5×10 5 Individual cells/individual.
3. After 12 hours from the completion of step 2, the drugs were divided into five groups and administered in groups.
Model set (Model): subcutaneous administration was twice daily (9:00/17:30), with 0.4ml of physiological saline administered each time;
recombinant hirudin treatment group 1 (0.4 mg/kg): the single administration dosage of the recombinant hirudin is 0.4mg/kg;
recombinant hirudin treatment group 2 (0.8 mg/kg): the single administration dosage of the recombinant hirudin is 0.8mg/kg;
recombinant hirudin treatment group 3 (1.6 mg/kg): the single administration dosage of the recombinant hirudin is 1.6mg/kg;
5-Fu positive control group (20 mg/kg): the single dose of 5-fluorouracil was 20mg/kg.
The recombinant hirudin-treated group was subcutaneously administered twice daily (9:00/17:30) in a volume of 0.4ml each time (in physiological saline).
5-Fu positive control group was administered once daily.
The group administration was continued for 28 days. Animals were weighed every 4 days. After 28 days of group administration, animals were sacrificed by cervical removal, liver, lung and brain tissues were removed, part of the tissues were fixed with 4% formaldehyde, and H & E staining was performed after paraffin embedding.
The change in animal body weight with time of administration is shown in fig. 19 (n=6). Within 14 days, the body weight of each group of mice is kept unchanged or stably increased; after 14 days, the weight of the mice in the model group is obviously reduced, the weight of the recombinant hirudin low-dose group and the weight of the recombinant hirudin medium-dose group are reduced but the tendency is lower than that of the mice in the model group, and the weight of the mice in the high-dose group and the weight of the mice in the positive control group are kept stable. After 14 days, the mice showed obvious cachexia due to metastasis of tumor cells, and the administration of recombinant hirudin could attenuate or even reverse the situation.
The photograph of the H & E staining is shown in FIG. 20. The organs of the mice in the model group are transferred to different degrees, the tumor cells are arranged in a nest shape, a sheet shape and a strip shape, the multiple nuclei are split, and the symptoms are obviously improved along with the increase of the administration concentration of the recombinant hirudin.
3. Recombinant hirudin effect for inhibiting in-situ cancer of BEL-7402 cell liver of nude mice
1. BEL-7402 cells in logarithmic growth phase are taken, digested, and then centrifuged to collect the cells, which are resuspended with physiological saline to obtain a cell suspension.
2. After the test animals (about 20g of body weight) were anesthetized, the animals were fixed on an operating table in a supine position, skin in the upper abdominal operation area was sterilized with iodophor and 70% ethanol solution, then an oblique incision of about lcm was made at the left liver portion of the upper abdomen, the abdominal cavity was exposed layer by layer, and left lobe of the liver was pulled out of the abdominal cavity with a sterile cotton swab. The cell suspension prepared in step 1 was then injected into the liver (4X 10) 6 Individual cells/individual), pressed with a cotton swab to stop bleeding, sutured with 5-0 sutures, and the wound swabbed with gentamicin to prevent infection.
3. Group administration
Blank (Control) test animals were treated according to step 2, but without injection of cell suspension.
Model set (Model): the experimental animals of step 2 were completed and were subcutaneously administered twice daily (9:00/17:30) with 0.4ml of physiological saline each time;
recombinant hirudin treatment group 1 (0.4 mg/kg): finishing the experimental animal in the step 2, wherein the single administration dosage of the recombinant hirudin is 0.4mg/kg;
recombinant hirudin treatment group 2 (0.8 mg/kg): finishing the experimental animal in the step 2, wherein the single administration dosage of the recombinant hirudin is 0.8mg/kg;
recombinant hirudin treatment group 3 (1.6 mg/kg): finishing the experimental animal in the step 2, wherein the single administration dosage of the recombinant hirudin is 1.6mg/kg;
5-Fu positive control group (20 mg/kg): the experimental animal completed step 2, the single administration dose of 5-fluorouracil was 20mg/kg.
The recombinant hirudin-treated group was subcutaneously administered twice daily (9:00/17:30) in a volume of 0.4ml each time (in physiological saline).
5-Fu positive control group was administered once daily.
The group administration was continued for 28 days.
4. During the group dosing, animals were weighed every 4 days. The change in body weight with time is shown in fig. 21 (n=8). Compared with a blank group, the weight of the nude mice in the model group shows a remarkable decrease trend and death caused by cachexia, and the administration of the recombinant hirudin can remarkably improve the situation, so that the weight decrease trend of the nude mice at medium and high doses is improved.
5. After 28 days of group administration, the animals were sacrificed by cervical removal, liver tissues were taken, the liver/body ratio was calculated, and the liver volume was calculated by a drainage method. The results are shown in figure 22 (compared to the model tumor-bearing group, * p<0.05， ** p<0.01; in contrast to the blank control, ## p<0.01 (n=8). Compared with the blank, the model group has obviously raised liver/body ratio and liver volume, the above situation is improved and dose dependency is presented along with the administration of the recombinant hirudin, and the high-dose recombinant hirudin can adjust the liver body ratio and liver volume of tumor-bearing mice to approach the normal level.
6. After 28 days of group administration, animals were sacrificed by cervical removal, livers were taken and photographed, liver, lung and brain tissues were taken, fixed with 4% formaldehyde, and H & E stained after paraffin embedding. Photographs of the liver and H & E staining are shown in FIG. 23. As shown, the model group showed extensive tumor growth infiltration and multiple tumor growth foci in the liver compared to the blank group, while the lung and brain tissues showed significant metastasis and inflammatory lesions. With the increase of the administration concentration of the recombinant hirudin, the symptoms are obviously improved, the areas of liver tumors in medium-dose and high-dose groups are obviously reduced, the number of growing foci is obviously reduced, and the metastasis of lung and brain tissue tumors is obviously reduced, which indicates that the recombinant hirudin can improve the symptoms of BEL-7402 cell liver in-situ cancer and increase the survival rate.
7. After 28 days of group administration, evans Blue dye solution was injected into the tail vein (100. Mu.l of Evans Blue dye solution was injected into each mouse, evans Blue was diluted to 20. Mu.g/. Mu.l with physiological saline; eyes, ears and limbs of the mice were observed to turn Blue immediately after injection), after 4 hours of injection, the experimental animals were anesthetized, and then opened, and the right ventricle was injected with physiological saline (10 mL each) for liver perfusion until the liquid flowing out of the right ventricle was free of blood. Weighing liver, draining, soaking in 150mg/2mL of N, N-dimethylformamide solution, homogenizing, extracting in water bath at 60deg.C for 24 hr, centrifuging at 12000g for 20minThe supernatant was measured for absorbance at 600nm using an ultraviolet spectrophotometer. The liver photographs and absorbance results of the homogenate supernatants are shown in figure 24 (compared to model tumor-bearing groups, ** p<0.01; in contrast to the blank group, ## p<0.01 (n=3). The results show that the model group nude mice have obvious vascular leakage condition due to the influence of tumors. The administration of the recombinant hirudin obviously changes the tumor size and simultaneously obviously reduces the extravasation of Evans Blue dye, which proves that the recombinant hirudin can inhibit vascular infiltration of BEL-7402 cell hepatocarcinoma in situ and has certain dose dependency.
8. After 28 days of grouped administration, the animals were sacrificed by cervical removal, tumor tissues on the liver were taken, total proteins were extracted, and Westren Blot detection was performed. The results of the Westren Blot detection are shown in figure 25 (compared to the model tumor bearing group, * p<0.05， ** p<0.01 (n=3) and figure 27 (compared to the model tumor-bearing group, * p<0.05， ** p<0.01 (n=3). The effect of recombinant hirudin on the TUNEL-positive area of the in situ carcinoma model mouse is shown in FIG. 26. The results show that the pro-apoptotic protein Bax and cleaved caspase-3 are low expressed in tumor tissues, but with recombinant hirudin treatment, protein levels are significantly elevated compared to the non-dosed model group, with the anti-apoptotic protein Bcl-2 exhibiting the opposite trend; meanwhile, the recombinant hirudin can reduce the levels of phosphorylated PI3K and phosphorylated Akt in vivo, and is accompanied by the increase of TUNEL positive cells of tumor tissues, and the results show the same trend as in vitro results.
9. Recombinant hirudin for changing in-situ cancer tumor-bearing rat tail bleeding time
Nude mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) 2h after the last administration. The tail diameter of each animal was measured 1cm from the tail tip with a vernier caliper and recorded, and mice with tail diameters of 1.108-1.110mm were screened for the next experiment. The animals are rapidly sheared by using surgical scissors at a position 1.5cm away from the tail tip, when blood begins to appear, the tail is dipped once every 30 seconds by using the prepared filter paper until the tail stops bleeding. The time to tail tip bleeding (i.e., tail stopped bleeding time-starting bleeding time) was recorded for each group of mice.
The results are shown in figure 28 (compared to the model tumor-bearing group, * p<0.05， ** p<0.01; in contrast to the blank group, ## p<0.01 (n=8). The model group tumor-bearing mice have the advantages that due to the influence of tumor microenvironment, in-vivo blood shows a hypercoagulable state, compared with a blank group dipped with a filter paper sheet for bleeding from the tail tip of a mouse, the blood trace showing time on the filter paper sheet of the model group is obviously reduced, after the recombinant hirudin is given, compared with the model group, the bleeding time of the tail of the naked mouse is obviously prolonged, but the conditions of subcutaneous bleeding points, uncontrollable bleeding and the like do not appear, so that the recombinant hirudin can relieve the hypercoagulable state of the tumor microenvironment, and has certain safety.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. Application of recombinant hirudin in preparing medicine for treating liver cancer is provided.
2. Application of recombinant hirudin in preparing medicine for inhibiting liver cancer tumor growth is provided.
3. Application of recombinant hirudin in preparing medicine for inhibiting liver cancer metastasis is provided.
4. The application of recombinant hirudin in preparing medicine for inhibiting liver cancer migration and/or inhibiting liver cancer invasion is provided.
5. The application of recombinant hirudin in preparing products; the function of the product is as follows (a) or (b) or (c) or (d):
(a) Inhibiting liver cancer cell blood migration;
(b) Inhibiting lung metastasis and/or brain metastasis of liver cancer;
(c) Inhibiting vascular infiltration of carcinoma in situ of the liver;
(d) Relieving hypercoagulability of blood in liver cancer tumor microenvironment.
6. The application of recombinant hirudin in preparing products; the product has the functions of inhibiting proliferation of liver cancer cells and/or promoting apoptosis of liver cancer cells and/or inhibiting survival of liver cancer cells and/or inhibiting migration of liver cancer cells and/or inhibiting invasion of liver cancer cells and/or inhibiting adhesion of liver cancer cells.
7. A medicament for treating liver cancer, which contains recombinant hirudin.
8. A medicament comprising recombinant hirudin; the medicine has the functions of inhibiting the growth of liver cancer tumor and/or inhibiting liver cancer metastasis and/or inhibiting liver cancer migration and/or inhibiting liver cancer invasion.
9. A product comprising recombinant hirudin; the function of the product is as follows (a) or (b) or (c) or (d):
(a) Inhibiting liver cancer cell blood migration;
(b) Inhibiting lung metastasis and/or brain metastasis of liver cancer;
(c) Inhibiting vascular infiltration of carcinoma in situ of the liver;
(d) Relieving hypercoagulability of blood in liver cancer tumor microenvironment.
10. A product comprising recombinant hirudin; the product has the functions of inhibiting proliferation of liver cancer cells and/or promoting apoptosis of liver cancer cells and/or inhibiting survival of liver cancer cells and/or inhibiting migration of liver cancer cells and/or inhibiting invasion of liver cancer cells and/or inhibiting adhesion of liver cancer cells.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210310179.5A CN116850272A (en) | 2022-03-28 | 2022-03-28 | Application of recombinant hirudin in preparation of medicine for treating liver cancer |
| PCT/CN2023/083725 WO2023185681A1 (en) | 2022-03-28 | 2023-03-24 | Use of recombinant hirudin in preparation of drug for treating liver cancer |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210310179.5A CN116850272A (en) | 2022-03-28 | 2022-03-28 | Application of recombinant hirudin in preparation of medicine for treating liver cancer |
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| Publication Number | Publication Date |
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| CN116850272A true CN116850272A (en) | 2023-10-10 |
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| CN202210310179.5A Pending CN116850272A (en) | 2022-03-28 | 2022-03-28 | Application of recombinant hirudin in preparation of medicine for treating liver cancer |
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| CN (1) | CN116850272A (en) |
| WO (1) | WO2023185681A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU659432B2 (en) * | 1991-03-08 | 1995-05-18 | Novartis Ag | A method for the inhibition or prevention of tumor cell metastasis with hirudin |
| CN105055665A (en) * | 2015-08-28 | 2015-11-18 | 潍坊医学院附属医院 | Anti-hepatoma traditional Chinese medicine preparation |
| CN108421006A (en) * | 2018-06-13 | 2018-08-21 | 张玉梓 | A kind of drug accelerating apoptosis of tumor cells |
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| WO2023185681A1 (en) | 2023-10-05 |
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