CN110639002B - Application of carfilzomib in preparation of drug for treating osteosarcoma - Google Patents

Application of carfilzomib in preparation of drug for treating osteosarcoma Download PDF

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CN110639002B
CN110639002B CN201910995348.1A CN201910995348A CN110639002B CN 110639002 B CN110639002 B CN 110639002B CN 201910995348 A CN201910995348 A CN 201910995348A CN 110639002 B CN110639002 B CN 110639002B
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osteosarcoma
carfilzomib
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osteosarcoma cells
osteogenic differentiation
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CN110639002A (en
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胡劲松
张丹
雷莉
陈萍
贾茜
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Xian Jiaotong University
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Abstract

The invention provides a new function of a proteasome inhibitor carfilzomib, which is used for preparing a medicine for treating osteosarcoma: can induce osteogenic differentiation of osteosarcoma, and can be used for treating osteosarcoma. Experiments prove that the carfilzomib with medium and high doses can induce apoptosis of osteosarcoma cells, and the carfilzomib with low concentration can effectively induce osteogenic differentiation of the osteosarcoma cells, and the specific expression is as follows: causing the calcified nodules to increase in osteosarcoma cells F5M2 and U-2 OS; an increase in alkaline phosphatase activity; increased expression of osteogenic differentiation markers (ALP, OPN, Runx 2).

Description

Application of carfilzomib in preparation of drug for treating osteosarcoma
Technical Field
The invention relates to application of carfilzomib in preparation of a drug for treating osteosarcoma
Background
The ubiquitin-proteasome pathway (UPP) is an important proteolytic pathway in the cell of an organism. This hydrolysis process occurs primarily in the 26S protease body, which consists of two 19S regulatory complexes and a 20S catalytic core. The 20S catalytic core body is responsible for decomposing multi-ubiquitination labeled substrate protein into polypeptide fragments, and the polypeptide fragments are of a tubular structure consisting of stacked six-leaf rings containing different alpha subunits and beta subunits, wherein the active subunits are beta 1, beta 2 and beta 5. Research proves that proteasomes are involved in degradation of various proteins (such as NF-kappa B, p53, cyclin and the like) and indirectly regulate biological processes such as cell mitosis, cell apoptosis, signal transduction, protein transmembrane localization and the like, so that the proteasomes are closely related to various diseases such as Alzheimer's disease, cancer and the like. At present, proteasomes have become a new hot spot for research and development of antitumor drugs. The second generation proteasome inhibitor Carfilzomib (CFZ) is an epoxy ketone drug which can irreversibly bind to the N-terminal threonine active site of the proteasome 20S catalytic core body to perform a targeted inhibition effect on the activity of the beta 5 subunit. Many studies have shown that carfilzomib has sustained antitumor activity in the treatment of patients with relapsed, refractory multiple myeloma, has been formally approved by the FDA in the united states in 2012 for the treatment of multiple myeloma, becoming a first-line specific targeted drug for multiple myeloma.
Osteosarcoma (Osteosarcoma) is a common malignant bone tumor which is good for adolescents, the incidence rate is the first of primary bone tumors, and 70% -80% of patients have the age of 10-25 years. Osteosarcoma occurs predominantly in the long metaphysis, rarely in the spine, pelvis and sacrum, and patients with single lesions predominate. Osteosarcoma is not only easy to recur but also has a high early metastasis rate. In the initial osteosarcoma patients, about 10% -20% of patients have distant metastasis and 90% are lung metastasis, and once the osteosarcoma has metastasis and relapse, poor prognosis is often indicated, and the 5-year overall survival rate is 20% -30%. In recent years, with the progress of surgical techniques, the research of effective chemotherapeutic drugs, the application of chemoradiotherapy adjuvant therapy before and after operation and other techniques, the 5-year survival rate of osteosarcoma patients is greatly improved, but the recurrent and metastatic osteosarcoma is still an incurable disease and needs to be overcome urgently.
The induced differentiation therapy is a new hotspot of tumor therapy and aims to treat and cure tumors by inducing and differentiating tumor cells into normal cells under the action of an induced differentiation agent. In the existing in vitro research, the aim of treating diseases such as clear cell sarcoma, medulloblastoma, granulocyte leukemia and the like can be achieved by inducing differentiation. There have also been recent studies reporting the induction of differentiation in osteosarcoma therapy. Sramek M and other researches find that methotrexate can promote osteogenic differentiation of osteosarcoma cells and expression of genes related to osteogenic differentiation, and that methotrexate and all-trans retinoic acid have curative effect on osteosarcoma (Sramek M.et al cancer Cell int.2016Feb; 16: 14). Liu C and the like found that galangin can obviously inhibit the proliferation of human osteosarcoma cells and promote osteogenic differentiation of the osteosarcoma cells and the expression of osteogenic differentiation markers (Liu C.et al.biomed Pharmacother.2017 May; 89: 1415-1421). Therefore, the search for a high-efficiency medicament for treating osteosarcoma by inducing differentiation has very important theoretical and clinical significance.
Disclosure of Invention
The invention aims to provide application of carfilzomib in preparation of a medicine for treating osteosarcoma, and finds that the carfilzomib with medium and high doses of proteasome inhibitor can effectively induce apoptosis of osteosarcoma cells, and the carfilzomib with low concentration can also induce osteogenic differentiation of the osteosarcoma cells. The invention is expected to provide a new clinical treatment mode for osteosarcoma treatment: the purpose of treating osteosarcoma is achieved by inducing the osteogenic differentiation of osteosarcoma.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
use of carfilzomib in the manufacture of a medicament for the treatment of osteosarcoma.
A further improvement of the present invention resides in the use of carfilzomib for the manufacture of a medicament for inducing osteogenic differentiation in osteosarcoma cells.
A further improvement of the invention resides in the use of carfilzomib in the manufacture of a medicament for inducing apoptosis in osteosarcoma cells.
In a further development of the invention, the osteosarcoma cell is an F5M2 or U-2OS cell line.
The invention further improves the application of carfilzomib in preparing the medicine with killing effect on osteosarcoma cells.
A further improvement of the invention resides in the use of carfilzomib in the manufacture of a medicament for causing the increase of calcified nodules in osteosarcoma cells.
A further improvement of the present invention resides in the use of carfilzomib for the manufacture of a medicament for causing an increase in alkaline phosphatase activity in osteosarcoma cells.
A further improvement of the present invention resides in the use of carfilzomib for the manufacture of a medicament for causing increased expression of osteogenic differentiation markers in osteosarcoma cells.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a new function of proteasome inhibitor carfilzomib: can induce osteogenic differentiation of osteosarcoma, and can be used for treating osteosarcoma. Experiments prove that the carfilzomib with medium and high doses can induce apoptosis of osteosarcoma cells, and the carfilzomib with low concentration can effectively induce osteogenic differentiation of the osteosarcoma cells, and the specific expression is as follows: causing the calcified nodules to increase in osteosarcoma cells F5M2 and U-2 OS; an increase in alkaline phosphatase activity; increased expression of osteogenic differentiation markers (ALP, OPN, Runx 2).
The low-concentration carfilzomib can induce cells to generate osteogenic differentiation, so that the possibility is provided for clinical treatment of osteosarcoma, and a new clinical medicine is expected to be provided for osteosarcoma treatment.
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FIG. 1 shows the results of different concentrations of Carfilzomib (CFZ) induced apoptosis of osteosarcoma cells F5M2 and U-2 OS. In the figure, A is the flow detection result of F5M2 cell apoptosis induced by carfilzomib with different concentrations, B is the flow detection result of U-2OS cell apoptosis induced by carfilzomib with different concentrations, C is the statistical result of three independent experiments in the figure A, and D is the statistical result of three independent experiments in the figure B.
FIG. 2 shows that alizarin red staining low detection concentration Carfilzomib (CFZ) treated osteosarcoma cells F5M2 and U-2OS cells show that red calcified nodules are increased significantly. Among them, Osteogenic Induction Medium (OIM) was used as a positive control.
FIG. 3 shows that the alkaline phosphatase activity in cells is remarkably enhanced by Gomori calcium cobalt method for staining and detecting osteosarcoma cells F5M2 and U-2OS cells treated by low-concentration Carfilzomib (CFZ) after dosing. Among them, Osteogenic Induction Medium (OIM) was used as a positive control.
FIG. 4 shows that the expression level of osteogenic differentiation markers (ALP, OPN and Runx2) in osteosarcoma cells F5M2 and U-2OS is remarkably increased after low-concentration Carfilzomib (CFZ) treatment by Western blotting detection.
Detailed Description
The invention discloses a new function of a proteasome inhibitor carfilzomib for treating osteosarcoma. The carfilzomib can induce apoptosis of osteosarcoma cells, and the low-concentration carfilzomib can also induce osteogenic differentiation of the osteosarcoma cells, specifically to cause calcified nodule increase in the osteosarcoma cells F5M2 and U-2 OS; an increase in alkaline phosphatase activity; increased expression of osteogenic differentiation markers (ALP, OPN, Runx 2).
The invention adopts Annexin V-FITC/7-AAD double-staining flow cytometry to detect the killing effect of carfilzomib on osteosarcoma cells. An alizarin red staining experiment was used to detect the effect of low concentration carfilzomib on the generation of calcified nodules in osteosarcoma cells. A Gomori calcium cobalt method staining experiment is adopted to detect the influence of low-concentration carfilzomib on alkaline phosphatase activity in osteosarcoma cells. Immunoblotting experiments were used to detect the effect of low concentrations of carfilzomib on the expression levels of osteogenic differentiation markers in osteosarcoma cells. The invention will now be described in further detail with reference to specific examples (human osteosarcoma cell lines F5M2, U-2OS used in the experiments) which are given by way of illustration and not by way of limitation.
1. Annexin V-FITC/7-AAD double-staining flow cytometry is adopted to detect the killing effect of carfilzomib on osteosarcoma cells.
1) Treatment of osteosarcoma cells with varying concentrations of carfilzomib
Collecting osteosarcoma cells F5M2 and U-2OS, observing and counting cells under microscope, and adjusting cell density to 0.2 × 106Perml, inoculated into 6-well plates, incubated at 37 ℃ with 5% CO2Culturing in a constant temperature incubator.
② 24h later, adding carfilzomib into osteosarcoma cells F5M2 and U-2OS which grow in logarithmic phase, so that the final concentration of the medicine is 0, 5, 10, 25, 50 and 100 nmol/L. The cells were cultured for 24 h.
2) Detection of osteosarcoma apoptosis by Annexin V-FITC/7-AAD double-staining flow cytometry
Collecting cell culture medium supernatant of each group to a centrifuge tube.
② digesting the cells by pancreatin, collecting each group of cell suspension to a centrifuge tube, combining with the culture medium supernatant collected in the previous step, placing the mixture in a desk centrifuge for centrifugation at 1200rpm for 5min, and discarding the supernatant.
And thirdly, 2-3mL of precooled PBS buffer solution is added into each tube to resuspend the cells, the cells are fully vortexed, the tubes are placed in a desktop centrifuge for centrifugation at 1200rpm for 5min, supernatant is discarded, serum in the culture medium is fully washed away, and interference on subsequent staining is reduced.
And fourthly, suspending the cells in 50 mu L Binding buffer, adding 2.5 mu L Annexin V-FITC dye solution and 0.5 mu L7-AAD dye solution into each tube, carrying out vortex mixing, and then carrying out dark dyeing on ice for 15 min.
Before detection, 300 mu L of Binding buffer is added in each tube.
Sixthly, detecting on the machine. The same experiment was repeated three times. Flow assay data were plotted using Flowjo v7.6.2 analysis and the proportion of apoptosis in each group was calculated.
As shown in FIG. 1, the apoptosis of osteosarcoma cells F5M2 and U-2OS can be induced by high-concentration carfilzomib, while apoptosis of osteosarcoma cells F5M2 and U-2OS can not be induced by low-concentration carfilzomib.
2. The influence of low-concentration carfilzomib on the generation of calcified nodules in osteosarcoma cells was detected by alizarin red staining experiment.
Collecting osteosarcoma cells F5M2 and U-2OS, observing and counting cells under microscope, and adjusting cell density to 0.1 × 106Perml, inoculated into 6-well plates, incubated at 37 ℃ with 5% CO2Culturing in a constant temperature incubator.
② 24h later, adding carfilzomib into osteosarcoma cells F5M2 and U-2OS which grow in logarithmic phase, so that the final concentration of the medicine is 0, 5, 10, 25, 50 and 100 nmol/L.
And thirdly, continuously culturing the cells for 6 days, and replacing the corresponding culture medium every 3 days.
Fourthly, the culture medium in the culture dish is slightly sucked up, and the cells are washed for 2 times by PBS buffer solution. Then, 1mL of 4% paraformaldehyde solution was added, and the cells were fixed at room temperature for 15 min. The paraformaldehyde solution was discarded and the cells were washed 2 times with PBS buffer.
Fifthly, adding l mL alizarin red staining solution into each culture dish to enable the bottom of the dish to be completely covered (the alizarin red staining solution is in full contact with cells by light shaking), and staining for 30min at room temperature.
Sixthly, the staining solution is removed, and the cells are washed 3 times with PBS. After the cover of the culture dish is opened, the culture dish is placed under a cell inverted microscope, the staining effect of the calcified nodules is observed through a low power microscope (multiplied by 10 and multiplied by 20), and the staining effect is photographed and recorded. Calcified nodules appeared bright red.
The results of the experiment are shown in FIG. 2, and the low concentration of carfilzomib caused the increase of calcified nodules in osteosarcoma cells F5M2, U-2OS with Osteogenesis Inducing Medium (OIM) as a positive control.
3. A Gomori calcium cobalt method staining experiment is adopted to detect the influence of low-concentration carfilzomib on the activity of alkaline phosphatase in osteosarcoma cells.
Culturing osteosarcoma cells F5M2 and U-2OS to logarithmic growth phase, observing and counting cells under microscope, and regulating cell density to 0.1 × 106/mL, inoculated into a 35mm dish, placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator.
② adding carfilzomib into osteosarcoma cells F5M2 and U-2OS which grow in logarithmic phase, so that the final concentration of the medicine is 0, 2.5, 5 and 10 nmol/L. Osteogenic Induction Medium (OIM) was used as a positive control.
③ continuously treating the cells with carfilzomib with different concentrations for 6 days, and replacing the corresponding culture medium every 3 days.
Fourthly, the culture medium in the culture dish is slightly sucked up, and the cells are washed for 2 times by PBS buffer solution. 1mL of 70% ethanol solution was added and the cells were fixed at room temperature for 10 min. Cells were washed 1 time with distilled water. A further l mL of sodium β -glycerophosphate solution was added to each dish and incubated for 6h at 37 ℃. Washing with distilled water several times.
Fifthly, adding 1mL of cobalt nitrate solution into each culture dish, and standing for 3-5min at room temperature. Washing with distilled water several times.
Sixthly, adding 1mL of ammonium sulfide solution into each culture dish, and standing for 2min at room temperature.
Seventhly, after washing the cells with distilled water, observing the cells under an inverted microscope, and photographing and recording the cells.
The results of the experiment are shown in FIG. 3, and the low concentration of carfilzomib caused an increase in alkaline phosphatase activity in osteosarcoma cells F5M2, U-2OS, using Osteogenic Induction Medium (OIM) as a positive control.
4. The influence of low-concentration carfilzomib on the expression level of osteogenic differentiation markers in osteosarcoma cells is detected by adopting an immunoblotting experiment.
1) Low concentration of carfilzomib for treating osteosarcoma cells
Collecting osteosarcoma cells F5M2 and U-2OS, observing and counting cells under microscope, and adjusting cell density to 0.2 × 106Perml, inoculated into 6-well plates, incubated at 37 ℃ with 5% CO2Culturing in a constant temperature incubator.
② 24h later, adding carfilzomib into osteosarcoma cells F5M2 and U-2OS which grow in logarithmic phase, so that the final concentration of the medicine is 0, 5, 10, 25, 50 and 100 nmol/L. The cells were cultured for 24 h.
2) Western blotting detection of osteosarcoma cell osteogenic differentiation marker expression
Extracting protein: the medium in each group of dishes was aspirated, 1mL of PBS buffer was added, and the mixture was washed by gentle shaking and discarded. Add 100. mu.L commercial RIPA lysate containing protease inhibitors and phosphatase inhibitors, scrape the cells with a cell scraper, transfer the solution to a 1.5mL EP tube labeled with the sample name, and lyse on ice for 20 min. The samples were centrifuged for 15min at 12000rpm in a 4 ℃ pre-chilled cryocentrifuge. At the end, the protein supernatant was aspirated and transferred to a new 1.5mL EP tube.
② protein quantification: and (3) carrying out protein quantification by adopting a BCA protein quantification kit, calculating the concentration of each sample protein according to a standard curve, supplementing a corresponding volume of RIPA lysate into a high-concentration sample, and adjusting to the same concentration. Based on the final volume of each protein sample, it was mixed with 5 × Loading Buffer according to 4: 1, and boiling in 100 ℃ boiling water for 10min for protein denaturation.
③ SDS-PAGE gel electrophoresis: and sequentially loading the protein marker and each histone sample, wherein the loading amount is 30-50 mu g/hole, switching on a power supply, regulating the voltage to 80V, and starting electrophoresis. When the sample runs out of the concentrated gel, the voltage can be increased to 120V.
Fourthly, transferring the film: after electrophoresis is finished, protein is transferred to the PVDF membrane by using a membrane transfer device, the current is adjusted to be 200mA, and the membrane transfer time is adjusted according to different molecular weights.
Sealing: 5% skimmed milk was sealed at room temperature for 1 h.
Sixthly, antibody incubation: milk was discarded and PBST washed 3 times for 5min each. Corresponding commercial antibodies ALP, OPN, Runx2 and β -actin were added and incubated overnight in a shaker at 4 ℃. The next day, primary antibody was recovered and PBST washed 3 times for 5min each. Adding corresponding secondary mouse or rabbit antibodies, and incubating for 1h at normal temperature in a shaking table.
And (c) chemiluminescence.
As shown in FIG. 4, the low concentration of carfilzomib resulted in increased expression of osteogenic differentiation markers (ALP, OPN, Runx2) in osteosarcoma cells F5M2, U-2 OS.
5. Statistical analysis
The experimental results were statistically analyzed using GraphPad Prism 5, and the Two-tailed unpaired t-test was used for group comparisons, with P < 0.05 considered statistically significant.
The experimental results fully prove that the intermediate and high dose of the proteasome inhibitor carfilzomib can induce the apoptosis of osteosarcoma cells, and the low concentration carfilzomib also has the effect of inducing the osteogenic differentiation of the osteosarcoma cells.
Carfilzomib in the present invention can be various forms of carfilzomib and derivatives thereof.

Claims (4)

1. Application of carfilzomib in preparation of a reagent for promoting osteogenic differentiation of osteosarcoma.
2. Use according to claim 1, wherein carfilzomib is used in the manufacture of a medicament for causing an increase in calcified nodules in osteosarcoma cells.
3. Use according to claim 1, wherein carfilzomib is used in the manufacture of a medicament for causing an increase in alkaline phosphatase activity in osteosarcoma cells.
4. Use according to claim 1, wherein carfilzomib is used in the manufacture of a medicament for causing increased expression of osteogenic differentiation markers in osteosarcoma cells.
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