CN111514122A

CN111514122A - Application of disulfiram in preparation of drug for treating liposarcoma

Info

Publication number: CN111514122A
Application number: CN202010466547.6A
Authority: CN
Inventors: 王立生; 张�林; 韩冰; 肖凤君; 苗成利
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2020-08-11

Abstract

The invention provides application of disulfiram in preparation of a drug for treating liposarcoma, and relates to the technical field of drug research and development. The invention provides application of disulfiram in preparing a medicament for treating liposarcoma, application in preparing a medicament for inhibiting liposarcoma cell proliferation, application in preparing a medicament for promoting liposarcoma cell apoptosis and application in preparing a medicament for inhibiting liposarcoma cell cloning. In the present invention, the disulfiram inhibits the proliferation and induces apoptosis in the human liposarcoma cell line SW872, the mechanism of which is associated with high expression of a20, but does not die via iron, and DSF has the potential to inhibit SW872 stem cells.

Description

Application of disulfiram in preparation of drug for treating liposarcoma

Technical Field

The invention belongs to the technical field of drug research and development, and particularly relates to application of disulfiram in preparation of a drug for treating liposarcoma.

Background

Retroperidothelial liposarcoma (RPLS) is the most common type of retroperitoneal sarcoma, accounting for approximately 45% of retroperitoneal soft tissue sarcomas. It has no symptoms in the early stage of onset, so that the tumor volume of a patient is often huge when the patient is diagnosed, the patient already invades organs in the abdominal cavity, the complete excision is difficult, the postoperative recurrence is easy, and the prognosis is poor. However, the main treatment means of the disease is surgical resection, and other effective treatment methods are not available, so that a medicine which has an inhibiting effect on the retroperitoneal liposarcoma is urgently needed to be found.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of disulfiram in the preparation of a drug for treating liposarcoma, wherein Disulfiram (DSF) can inhibit the proliferation and induce the apoptosis of human liposarcoma cells, the mechanism of the disulfiram is related to the high expression of a20, but the disulfiram does not die by iron, and DSF has the potential of inhibiting human liposarcoma cell stem cells.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of disulfiram in preparing a medicament for treating liposarcoma.

The invention also provides application of disulfiram in preparing a medicament for inhibiting the cell proliferation of liposarcoma.

The invention also provides application of disulfiram in preparing a medicament for promoting the apoptosis of liposarcoma cells.

The invention also provides application of disulfiram in preparing a drug for inhibiting the cell clone of liposarcoma.

The invention also provides application of disulfiram in preparing a medicament for inducing the expression of A20 to be up-regulated.

The invention also provides a medicament for treating liposarcoma, which takes disulfiram as an effective component.

Preferably, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Preferably, the weight percentage of the disulfiram in the medicine is 0.1-99%.

The invention provides application of disulfiram in preparing a medicament for treating liposarcoma, and in the invention, human liposarcoma cells SW872 is taken as an example for verification, and the DSF is found to have remarkable proliferation inhibition effect (P is less than 0.05) on SW872 cells, wherein the proliferation inhibition effect is 1 mu mol.L^-1Time to IC₅₀And is concentration dependent; DSF causes SW872 cells to shrink and become roundAnd the cell gap increases; after DSF acted on SW872 cells for 24 hours, 1. mu. mol. multidot.L of the control group^-1The apoptosis rate of DSF group cells is obviously increased (P is less than 0.05) and reaches 72.6%, and apoptotic bodies obviously appear; DSF inhibits the formation of SW872 cell clones; a20 is significantly highly expressed at the protein level and mRNA level; fer-1 can not reverse the inhibition effect of DSF and Erastin on SW872 cells; ALDH1 tends to increase and then decrease with increasing DSF concentration. Therefore, DSF can inhibit the proliferation and induce apoptosis of SW872, a human liposarcoma cell line, the mechanism of which is related to high expression of a20 but does not die by iron, and DSF has the potential to inhibit SW872 stem cells.

Drawings

FIG. 1 is a graph showing the effect of DSF on inhibiting the proliferation of SW872 cells;

FIG. 2 is a graph showing the effect of DSF on the morphology of SW872 cells;

FIG. 3 is a graph showing the results of DSF-induced apoptosis of SW872 cells;

FIG. 4 is a diagram of the formation of DSF-induced apoptotic bodies;

FIG. 5 shows that DSF inhibits the formation of SW872 cell clones;

FIG. 6 is a graph showing the change in protein and mRNA levels of A20 after DSF acts on SW 872;

FIG. 7 shows that Fer-1 was unable to reverse the inhibitory effect of DSF on SW872 cells;

FIG. 8 is a graph showing the change in mRNA level of ALDH 1.

Detailed Description

The source of Disulfiram (DSF) is not particularly limited in the present invention and is preferably obtained from apebaio, usa. In the invention, SW872 cells are cultured in vitro, a DMSO group (Con group) and DSF groups with different concentrations are set, and the proliferation inhibition effect on the cells after drug intervention is detected for 24 hours by adopting a CCK8 method; giemsa staining method for observing the effect of DSF on SW872 cell morphology; flow cytometry and DAPI staining to detect apoptosis; a clonogenic experiment detects the influence of DSF on the clonogenic capacity of SW872 cells; detecting the expression of A20 by Westernblotting and RT-PCR; CCK8 method for detecting whether Fer-1 can reverse DSF and ErastinInhibition of SW872 cells; the RT-PCR method detects the expression of DSF on the mRNA level of ALDH1 in SW872 cells. The result shows that the DSF has obvious proliferation inhibiting effect (P is less than 0.05) on SW872 cells, and the concentration is 1 mu mol.L^-1Time to IC₅₀And is concentration dependent; DSF caused SW872 cells to shrink round and the intercellular space to increase; after DSF acted on SW872 cells for 24 hours, 1. mu. mol. multidot.L of the control group^-1The apoptosis rate of DSF group cells is obviously increased (P is less than 0.05) and reaches 72.6%, and apoptotic bodies obviously appear; DSF inhibits the formation of SW872 cell clones; a20 is significantly highly expressed at the protein level and mRNA level; fer-1 can not reverse the inhibition effect of DSF and Erastin on SW872 cells; ALDH1 tends to increase and then decrease with increasing DSF concentration. In conclusion, DSF can inhibit the proliferation of SW872 and induce the apoptosis of human liposarcoma cell line, the mechanism of the DSF is related to the high expression of A20 but does not die through iron, and the DSF has the potential of inhibiting SW872 stem cells, so the DSF can be used for preparing the medicine for treating liposarcoma.

The invention also provides application of disulfiram in preparing a medicament for inhibiting the cell proliferation of liposarcoma. The application of the present invention is preferably the same as described above and will not be described further herein.

The invention also provides application of disulfiram in preparing a medicament for promoting the apoptosis of liposarcoma cells. The application of the present invention is preferably the same as described above and will not be described further herein.

The invention also provides application of disulfiram in preparing a drug for inhibiting the cell clone of liposarcoma. The application of the present invention is preferably the same as described above and will not be described further herein.

The invention also provides application of disulfiram in preparing a medicament for inducing the expression of A20 to be up-regulated. The application of the present invention is preferably the same as described above and will not be described further herein.

The invention also provides a medicament for treating liposarcoma, which takes disulfiram as an effective component. The medicament of the invention preferably also comprises pharmaceutically acceptable auxiliary materials, the type and source of the auxiliary materials are not specially limited, and the conventional auxiliary materials in the field can be utilized aiming at different medicament forms. The weight percentage of disulfiram in the medicine is preferably 0.1-99%.

The use of disulfiram in the preparation of a medicament for the treatment of liposarcoma according to the present invention is described in more detail below with reference to the examples, but these should not be construed as limiting the scope of the invention.

Example 1

1.1 drugs, cells, Primary reagents and instruments

Liposarcoma cell line SW872 was purchased from American Type Culture Collection (ATCC); DMEM (Gibco, usa); fetal bovine serum (Chinese ilex); pancreatin (Hyclone, usa); double antibody (Hyclone, usa); disulfiram (DSF) and the iron death inducer Erastin were purchased from apebaio, usa; the iron death inhibitor, Fer-1, was purchased from Sigma; CCK-8 kit (Bimake, USA); giemsa (soleagbao, china); DAPI dye liquor (kaikyl, china); double-staining apoptosis kit (GenStar, china); reverse transcription kit (chinese all-gold); SBYR fluorescent quantitative PCR kit (chinese Genstar); ALDH1, A20 and ACTB primers were synthesized by the Beijing Biotech primer Synthesis part; a20, GAPDH antibody (us CST); horseradish peroxidase (HRP) -labeled goat anti-rabbit antibody (china sequoia golden bridge); RIPA lysate and protease inhibitor (chinese bi yun day). Secondary biosafety cabinets (ESCO, usa); centrifuge, CO₂Cell culture boxes, multifunctional full-wavelength plate readers (Thermo corporation, usa); an optical inverted microscope (Olympus, japan); FACSCalibur flow cytometer (BD corporation, usa); a protein vertical electrophoresis apparatus, a protein vertical electrotransfer apparatus (Bio-Rad, USA); 7500Fast real time quantitative PCR instrument (Applied Biosystems, USA); gel imaging system (Tanon, china).

The following experiments were repeated three times, with data in the form of mean ± standard deviation. Statistical analysis is carried out by adopting SPSS24.0 software, and T test is adopted for pairwise comparison among groups, and the difference with P <0.05 has statistical significance.

1.2 culture of SW872 cells

The cells were removed from liquid nitrogen, rapidly lysed in a 37 ℃ water bath, transferred to DMEM containing 10% FBS, and placed in CO₂And (5) in a cell culture box, changing the liquid after the wall is attached. Passage every other day, cell adherenceGrowing in spindle shape.

1.3 CCK8 method for detecting inhibition effect of DSF on SW872 at different concentrations

SW872 cells in logarithmic growth phase were routinely digested, counted and cell density adjusted to 1 × 10⁵One per mL, and adding the mixture to obtain final concentrations of 0, 1, 2.5, 5 and 10 mu mol & L^-1The DSF (total serum glucose) of (1) was seeded into a 96-well plate at 100. mu.L/well, 3 wells per group, and placed at 37 ℃ in 5% CO₂And (5) culturing for 24 hours in a saturated humidity incubator. After 24h, adding 10uL of CCK8 into each well, incubating for 3 hours at 37 ℃, detecting by using an enzyme labeling instrument, wherein the wavelength is 450nm, and the result is expressed by the inhibition rate of cell proliferation: [1- (OD value of experimental group-OD value of blank control group)/(OD value of control group-OD value of blank control group)]×100％。

As shown in FIG. 1, the growth inhibition rate of SW872 cells by DSF was higher at 1. mu. mol. multidot.L as the concentration of DSF increased 24 hours after the DSF acted on SW872 cells^-1Time to IC₅₀。

1.4 microscopic examination of the Effect of different concentrations of DSF on the form of SW872

SW872 cells in logarithmic growth phase were routinely digested, counted and cell density adjusted to 1 × 10⁵one/mL, and adding the mixture to obtain final concentrations of 0, 1 and 2.5 mu mol & L ^-12 mL/well into 6-well plates, and giemsa staining after 24 hours: the supernatant was aspirated, the cells were washed once with PBS, fixed with methanol for 20-30 minutes, methanol was discarded, PBS was washed 3 times, then stained with gimsa for 30 minutes, after 30 minutes the plates were rinsed with small tap water, air dried naturally, and photographed under microscope.

As shown in FIG. 2, after SW87224 hours, the DSF was stained with Giemsa and observed under the microscope (20X), and SW872 was shrunk and rounded under the action of DSF, and the cell gap was increased, as compared with the control (DMSO group).

1.5 AnnexinV-FITC/PI double staining method for detecting influence of DSF on SW872 cell apoptosis

SW872 cells were taken in logarithmic growth phase at 3 × 10⁵Perwell in 6-well plates, 2mL system. Let 0. mu. mol. L^-1Group (DMSO), 1. mu. mol. L^-1Group of DSFs, 24After h, the cells were harvested, washed twice with ice PBS and resuspended with 100. mu.L of 1 × binding buffer, 5. mu.L of LannexinV-FITC was added, incubated for 15 minutes at room temperature in the dark, 2. mu.L of LPI was added, incubated for 5 minutes at room temperature, resuspended with 400. mu.L of LPBS, immediately tested on the machine and the FITC positive cell fraction was calculated.

As a result, as shown in FIG. 3, it was found that the apoptotic effect of DSF on SW872 was significantly increased by DSF at 1. mu. mol. L when the apoptotic effect of DSF on SW87224 hours was measured by flow cytometry^-1Under the action of DSF, the apoptosis rate of SW872 reaches 72.6%.

1.6 DAPI staining

Pre-treating the slide: the climbing piece with the diameter of 1cm is soaked in alcohol for 24h, then soaked in pure water for 1h, and placed in a 6-hole plate after being dried. Plate paving: the plate paving method is the same as 1.5, and the group is as follows: 0. mu. mol. L^-1Group (DMSO), 1. mu. mol. L^-1Group, 2.5. mu. mol. L^-1And (4) grouping. After 24 hours, wash with PBS 1 time, after fixation with 4% fixative for 30 minutes wash with PBS. Then clamping the climbing film out by a small forceps, placing the cell surface downwards on a cut sealing film, and injecting 30 mu L of DAPI working solution from the edge to keep out of the sun for 5 minutes; then, the cells were punched out with 200. mu.L of PBS, aspirated off and washed three times. Finally, sealing the film and observing under a fluorescence microscope.

The results are shown in fig. 4, DSF induced the appearance of apoptotic bodies by DAPI staining after SW87224 hours after DSF, and this was more evident with increasing concentration: cell contraction, cell nucleus fixation.

1.7 clone formation assay

Cells in logarithmic growth phase were digested, centrifuged, counted, plated and plated in 24-well plates, 500 cells per well, 500 μ L system (DMEM with 10% FBS), 3 replicate wells. After the cells adhere to the wall, the final concentration of the mixture is 0(DMSO group), 0.1 and 0.25 mu mol. L^-1After 6 days of DSF addition, the DMSO groups showed macroscopic colonies, so giemsa staining, photographing (20 ×), and counting clones with cell number of 60 or more under a microscope were performed.

The results are shown in FIG. 5, where the concentration of the catalyst in the DSF is 0. mu. mol. L^-1Form when it is about129 clones in DSF 0.1. mu. mol. L^-1About 17 clones were formed at DSF 0.25. mu. mol. L^-1Almost no SW872 clone was formed.

1.8 RT-PCR method for detecting expression of A20 and ALDH1 in cells

Digesting, centrifuging, counting, adding medicine and adjusting the concentration of the medicine to 0(DMSO), 1, 5, 10 mu mol. L^-1Spreading in 6-well plates, each well having 5 × 10⁵Cells, 2mL system (DMEM with 10% FBS). After 12 hours, the cells were digested and centrifuged, RNA was extracted by Trizol method, and 1. mu.g of RNA was reverse-transcribed into cDNA and subjected to RT-PCR.

The primer sequences are as follows:

a20 upstream primer (SEQ ID NO. 1): 5'-TGCTGCCCTAGAAGTACAATAGGAA-3' the flow of the air in the air conditioner,

downstream primer (SEQ ID NO. 2): 3 '-GCAGCTGGTTGAGTTTATGCAAG-5';

ALDH1A1 upstream primer (SEQ ID NO. 3): 5'-TTGGAATTTCCCGTTGGTTA-3' the flow of the air in the air conditioner,

downstream primer (SEQ ID NO. 4): 3 '-CTGTAGGCCCATAACCAGGA-5';

ALDH1A3 upstream primer (SEQ ID NO. 5): 5'-GCCCTTTATCTCGGCTCTCT-3' the flow of the air in the air conditioner,

downstream primer (SEQ ID NO. 6): 3 '-CGGTGAAGGCGATCTTGT-5';

ACTB upstream primer (SEQ ID NO. 7): 5'-CATCCTCACCCTGAAGTACCC-3' the flow of the air in the air conditioner,

downstream primer (SEQ ID NO. 8): 3 '-AGCCTGGATAGCAACGTACATG-5'.

Data obtained use 2^-ΔCтIn this case, the target gene C t value is not expressed in the reference gene C t value.

As shown in FIGS. 6 and 8, after SW87224 hours, the A20 in the DSF group was significantly up-regulated in mRNA level; after the DSF acted on SW87212 hours, the levels of ALDH1 in SW872 cells acted on by DSF at different concentrations were compared, and it was found that the levels of ALDH1 tended to increase and then decrease with increasing concentration.

1.9 Westernblotting method for detecting expression of A20 in cells

The cells in logarithmic growth phase were digested, centrifuged, counted, DSF added and drug concentrations adjusted to 0(DMSO), 1, 5, 10 μmol · L^-1Spreading in 6-well plates, each well having 5 × 10⁵Individual cells, 3-well, 2mL system (DMEM with 10% FBS). After 24 hours, the supernatant was discarded, cells were lysed using RIPA, protein amount was determined by BCA method, and pre-experiment, loading with the same protein amount, to ensure internal reference consistency. After the protein sample is boiled and denatured at high temperature, the protein sample is incubated overnight at 4 ℃ by SDS-PAGE electrophoresis, electrotransfer, membrane transfer and primary antibody (A20 and internal reference GAPDH, diluted by 1: 1000), the protein sample is incubated for 2 hours by a shaking table with secondary antibody (goat anti-rabbit antibody marked by HRP, diluted by 1: 5000), a protein band is developed by a gel imager after ECL (electrochrome), and the size of the band of the target protein is observed under the condition that the internal reference is consistent.

The results are shown in fig. 6, and a20 protein level in DSF group was significantly up-regulated after DSF treatment for SW87224 hours.

1.10 CCK8 method for detecting whether Fer-1 can reverse the effect of DSF and Erastin on SW872

SW872 cells in logarithmic growth phase were routinely digested, counted and cell density adjusted to 1 × 10⁵one/mL/Ep tube, and adding final concentrations of 0, 1, 2.5, and 5. mu. mol. L^-1The DSF or Erastin is planted into a 96-well plate at 100 mu L/well, and each group has 3 multiple wells; adding the rest culture solution containing corresponding DSF or Erastin to the culture solution with the final concentration of 1 mu mol & L^-1The Fer-1 is fully and evenly mixed and then planted in the same 96-well plate, 3-multiple-well. After 24 hours, the survival of the cells was examined by the CCK8 method (same procedure as in 1.3).

The results are shown in FIG. 7, 1. mu. mol. L^-1The iron death inhibitor Fer-1 has no reversal effect on the SW872 cell inhibition caused by DSF, and the SW872 cell does not show obvious death phenomenon under the action of the iron death inducer Erastin and can not be reversed by the Fer-1.

In conclusion, DSF has the effects of inhibiting proliferation, promoting apoptosis and inhibiting clonogenic activity in the human liposarcoma cell line SW872, and the specific mechanism of DSF may be related to the expression up-regulation of a20 in SW872 induced by DSF, but not through iron death, which suggests that DSF can be used for the treatment of liposarcoma.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Application of disulfiram in preparation of drug for treating liposarcoma

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Claims

1. The application of disulfiram in preparing medicine for treating liposarcoma.

2. The application of disulfiram in preparing medicine for inhibiting liposarcoma cell proliferation is provided.

3. Application of disulfiram in preparing medicine for promoting liposarcoma apoptosis is provided.

4. The application of disulfiram in preparing medicine for inhibiting the cell cloning of liposarcoma.

5. Use of disulfiram in the manufacture of a medicament for inducing upregulation of A20 expression.

6. The medicine for treating liposarcoma is characterized by taking disulfiram as an active ingredient.

7. The medicament of claim 6, further comprising pharmaceutically acceptable excipients.

8. The medicine according to claim 6, wherein the content of disulfiram in the medicine is 0.1-99% by mass.