CN114369637B

CN114369637B - Research method and application of benzbromarone for treating acute myelogenous leukemia

Info

Publication number: CN114369637B
Application number: CN202111556134.8A
Authority: CN
Inventors: 甘滔; 张翔; 王烈峰; 张峰华; 耿戈戈; 丁超; 熊梦婕
Original assignee: Gannan Medical University
Current assignee: Gannan Medical University
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2023-09-12
Anticipated expiration: 2041-12-17
Also published as: CN114369637A

Abstract

The invention discloses a research method and application of benzbromarone for treating acute myelogenous leukemia, wherein the benzbromarone is used as an EYA phosphatase inhibitor, can effectively inhibit the growth of leukemia cell lines, can effectively inhibit the self-renewal capacity of the leukemia cell lines and promote the differentiation of the leukemia cell lines, and can induce apoptosis by disturbing the cell cycle of leukemia cells.

Description

Research method and application of benzbromarone for treating acute myelogenous leukemia

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a research method and application of benzbromarone for treating acute myelogenous leukemia.

Background

Leukemia is a bone marrow-derived cancer characterized by abnormal proliferation of white blood cells and inhibition of functional blood cell production, and is a malignant tumor with a poor prognosis and a high mortality rate. Common leukemias can be divided into four different types: acute Lymphoblastic Leukemia (ALL), acute Myeloid Leukemia (AML), chronic Lymphoblastic Leukemia (CLL), and Chronic Myeloid Leukemia (CML). In China, the incidence rate and the death rate of leukemia in the year are 5.3/10 ten thousand and 3.3/10 ten thousand respectively, and the relative incidence rate is high in the first ten places worldwide. Among the various leukemia subtypes, AML is extremely high in incidence and increases year by year. More than half of patients who die from leukemia annually suffer from AML, a type of leukemia with the worst prognosis and highest mortality. Despite the efficacy of cytarabine and daunorubicin based chemotherapy regimens over the last 40 years, survival rates in AML patients remain paramount, especially in the elderly population. The high degree of genetic heterogeneity makes it very difficult to develop a broad spectrum of drugs against AML. Therefore, through research on AML pathogenesis, common molecules which participate in the regulation of multiple subtype AML simultaneously are found, and the development of effective medicaments which can effectively target the common molecules is significant.

In recent years, a variety of phosphatases including PP2A have been found to play a key role in the development of AML, and can be the subject of targeted therapies, but are difficult to convert to clinic because of the difficulty in obtaining specific inhibitors. The EYA phosphatase family is chemically distinct from other phosphatases, in that cysteine residues are not used in the catalytic process, and belongs to the halohydrin dehalogenase type phosphatases. Therefore, designing inhibitors with their active centers as targets is not prone to the non-specific recognition problems common to phosphatase inhibitors. EYA family members include EYA1-4, all having dual activities of phosphatase and transcription factors, with a very conserved tyrosine phosphatase activity at the C-terminus of the protein structure, and a threonine phosphatase activity domain and a transcriptional activation domain at the N-terminus, which together with partner SIX having DNA binding activity, activate transcription of downstream genes. Functional studies have shown that EYA is involved in a variety of biological processes including organ development, innate immunity, DNA damage repair, angiogenesis, and the like. Currently, high levels of EYA phosphatase expression are found in a variety of human cancers. EYA is capable of driving the development and progression of cancer from many aspects such as cancer cell proliferation, metastasis and angiogenesis, DNA repair, etc., and is therefore considered as a potential drug target in cancer treatment. As early as 2011, thiaman et al found that the EYA1 gene was highly expressed in AML clinical samples, and that overexpression of EYA1 also promoted malignant proliferation of hematopoietic precursor cells. Whereas partner protein SIX of EYA1 was also shown to be involved in regulating the fusion protein MLL-AF 9-initiated AML by maintaining leukemia stem cell homeostasis. Downregulation of EYA1 and SIX1 expression significantly reduced the number of leukemia initiating (stem) cells, thereby inhibiting the onset of MLL-AF9 AML. Another family member EYA2 has also been shown to regulate stem cell self-renewal, and two fusion proteins, PLZF-RARA and E2A-PBX1, directly activate EYA2 expression to promote the development of the corresponding subtype AML. In addition, currently known EYA downstream targets, including NRP1, NRP2, GLI1, CYCLIND1, MYC and the like, are important molecules on which AML depends. It follows that members of the EYA phospholipase family can be involved in the development of a number of different subtypes of AML, and that if specific inhibitors for EYA phosphatases can be found, a broad spectrum treatment of AML is possible.

Disclosure of Invention

The object of the present invention is to find EYA phospholipase specific inhibitors for AML treatment.

The technical scheme adopted by the invention is as follows:

the research method of the tribromone for treating the acute myelogenous leukemia comprises the following steps of:

step 1: two human AML cell lines were selected, wherein HL-60 represents a karyotype normal subtype and THP-1 represents a karyotype abnormal subtype, and both AML cells were cultured in RPMI 1640 medium containing 10% FBS, 100U/mL penicillin and 100 μg/mL streptomycin at 5% CO2 at 37deg.C;

step 2: dissolving tribromone powder with cell culture grade DMSO to prepare 100mM working solution, and diluting with complete culture medium as required in subsequent experiments;

step 3: taking THP-1 cells in logarithmic growth phase, inoculating 5000 cells in each well of a 96-well plate, inoculating 18 wells in each plate, and inoculating 3 plates in total; co-culture was performed by adding 0. Mu.M, 50. Mu.M, 75. Mu.M, 100. Mu.M, 150. Mu.M and 200. Mu.M benzbromarone for 24, 48 and 72 hours, respectively, with one 96-well plate for each time point and 3 multiplex wells for each concentration; after co-culture, determining the total number of living cells by using a CCK8 kit, and calculating the relative activity of the cells after the phenylbromarone treatment;

step 4: taking HL-60 cells in logarithmic growth phase, inoculating 5000 cells in each well of a 96-well plate, inoculating 18 wells in each plate, inoculating 3 plates altogether, adding 0 mu M, 20 mu M, 40 mu M, 60 mu M, 80 mu M and 100 mu M benzbromarone respectively, co-culturing for 24, 48 and 72 hours, wherein each time point corresponds to one 96-well plate, and 3 compound wells are arranged in each concentration; after co-culture, determining the total number of living cells by using a CCK8 kit, and calculating the relative activity of the cells after the phenylbromarone treatment;

step 5: the HL-60 cell strain is used for continuing the subsequent experiments, and the following experiments are selectedEstablishing a high-low concentration gradient between 80 mu M and 40 mu M, taking 9 parts, mixing 1000 HL-60 cells each with 1mL of methylcellulose semisolid culture medium, and simultaneously adding 0 mu M, 40 mu M and 80 mu M benzbromarone respectively, wherein three parallel replicates are arranged for each concentration; adding the cells, benzbromarone and semi-solid medium mixture into a culture dish, and adding 5% CO at 37deg.C ₂ Culturing for 14 days under the condition, and observing and calculating the number of generated cell colonies and the distribution condition of different forms of colonies under a microscope;

step 6: taking HL-60 cells in logarithmic growth phase, inoculating 2×10 into each 6-well plate ⁶ The method comprises the steps of culturing cells in a complete culture medium, adding 0 mu M, 40 mu M and 80 mu M benzbromarone respectively, co-culturing for 72 hours, and setting 3 compound holes for each action concentration; after 72 hours, determining the DNA content in the cells by using a PI cell cycle and apoptosis detection kit so as to judge the distribution condition of the cell cycle;

step 7: taking HL-60 cells in logarithmic growth phase, inoculating 2×10 into each 6-well plate ⁶ The method comprises the steps of culturing cells in a complete culture medium, adding 0 mu M, 40 mu M and 80 mu M benzbromarone respectively, co-culturing for 72 hours, and setting 3 compound holes for each action concentration; cells were collected 72 hours later and were subjected to PI and Annexin V double-staining flow assay to assess apoptosis levels;

step 8: taking HL-60 cells in logarithmic growth phase, inoculating 2×10 into each 6-well plate ⁶ The method comprises the steps of culturing cells in a complete culture medium, adding 0 mu M, 40 mu M and 80 mu M benzbromarone respectively, co-culturing for 72 hours, and setting 3 compound holes for each action concentration; after 72 hours, cells were collected, proteins were extracted, and the change in expression level of known EYA downstream targets was detected using Western immunoblotting.

Another object of the invention is to provide the use of benzbromarone in the treatment of acute myelogenous leukemia.

Preferably, benzbromarone acts as an inhibitor of EYA phosphatase.

Preferably, the medicament for treating acute myelogenous leukemia comprises the benzbromarone as the only active ingredient.

The invention has the beneficial effects that: the research method and application of the benzbromarone provided by the invention for treating acute myelogenous leukemia have the following obvious effects:

the benzbromarone serving as an EYA phosphatase inhibitor can effectively inhibit the growth of a leucocyte strain, can effectively inhibit the self-renewal capacity of the leucocyte strain and promote the differentiation of the leucocyte strain, and induces apoptosis by disturbing the cell cycle of the leucocyte, and simultaneously causes the expression disorder of a known EYA downstream target molecule.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart showing the method for researching the use of the benzbromarone in acute myelogenous leukemia.

FIG. 2 is a graph showing the results of different concentrations of benzbromarone in inhibiting the growth of THP-1AML cells.

FIG. 3 is a graph showing the results of the inhibition of HL-60AML cell growth by benzbromarone at various concentrations.

FIG. 4 is a graph showing the results of the ability of various concentrations of benzbromarone to reduce HL-60 cell colony formation;

FIG. 5 is a graph showing the results of different concentrations of benzbromarone in inducing HL-60 cell cycle disturbance;

FIG. 6 is a graph showing the results of induction of HL-60 apoptosis by tribromone at various concentrations.

FIG. 7 is a graph showing the results of different concentrations of benzbromarone in eliciting expression disorders of known EYA downstream target molecules.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The EYA phosphatase inhibitor benzbromarone can act on different subtype acute myelogenous leukemia, and can obviously inhibit the growth of leukemia cells. EYA phosphatase family has been shown to be involved in the development of various subtypes of acute myelogenous leukemia, and its chemical structure is distinct from other phosphatases, which can circumvent the non-specific recognition problems common to phosphatase inhibitors. The tribromone is screened to prove that the activity of EYA phosphatase can be effectively inhibited, and the inhibition of EYA does not depend on chelation of metal ions and does not cause nonspecific action of other metalloenzymes. Co-culturing the benzbromarone with different concentrations and AML cells of two subtypes shows that the benzbromarone can effectively inhibit the growth of acute myelogenous leukemia cells by inhibiting self-renewal, promoting differentiation, triggering cell cycle retardation, inducing apoptosis, disturbing key molecule expression and other mechanisms.

Based on the above research results, the invention specifically provides a method for researching the use of benzbromarone in acute myelogenous leukemia, as shown in fig. 1, comprising the following steps:

step 1: two human AML cell lines were selected, of which HL-60 represents the karyotype normal subtype and THP-1 (carrying MLL-AF9 translocation) represents the karyotype abnormal subtype. Both AML cells were cultured in RPMI 1640 medium containing 10% FBS, 100U/mL penicillin, 100. Mu.g/mL streptomycin at 5% CO2,37 ℃.

Step 2 1g BBR powder (Sigma) was dissolved in 23.85ml cell culture grade DMSO to make 100mM working solution, and the subsequent experiments were diluted as needed using complete medium.

Step 3: THP-1 cells in the logarithmic growth phase were seeded in 96-well plates with 5000 cells per well, 18 wells per plate, and 3 plates in total. 0. Mu.M, 50. Mu.M, 75. Mu.M, 100. Mu.M, 150. Mu.M and 200. Mu.M benzbromarone were added for 24, 48 and 72 hours, respectively, one 96-well plate was used at each time point, and 3 replicate wells were set for each concentration of action. After the co-culture is finished, the total number of living cells is measured by a CCK8 kit (Soxhobao), and the relative activity is obtained by dividing the number of the cells treated at different concentrations by the number of the cells of 0 mu M and carrying out normalization treatment (relative viability); the results are shown in fig. 2, where P <0.05 represents P <0.01, and P <0.001. The results show that the inhibition effect of the benzbromarone on THP-1 cells is more remarkable along with the extension of the action time and the increase of the action concentration.

Step 4: HL-60 cells in logarithmic growth phase were taken, 5000 cells were inoculated per well in 96-well plates, 18 wells were inoculated per plate, and 3 plates were inoculated in total. 0. Mu.M, 20. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M and 100. Mu.M benzbromarone were added for 24, 48 and 72 hours, respectively, one 96-well plate was used at each time point, and 3 replicate wells were set for each concentration of action. After the end of co-culture, the total number of living cells was determined with CCK8 kit (Soxhobao) and the relative activity of the benzbromarone treated cells was calculated. The results are shown in fig. 3, where P <0.05 represents P <0.01, and P <0.001. The results show that the inhibition effect of the benzbromarone on HL-60 cells is more remarkable along with the extension of the action time and the increase of the action concentration. HL-60 cells were more sensitive to benzbromarone treatment than THP-1 cells. After 72 hours of treatment, the IC50 of THP-1 was approximately 75. Mu.M, while the IC50 of HL-60 was around 40. Mu.M.

Step 5: subsequent experiments were continued using a HL-60 cell line more sensitive to benzbromarone inhibition, and a high-low concentration gradient was established at two concentrations of 80 μm and 40 μm. 9 parts of HL-60 cells (1000 cells each) were mixed with 1mL of methylcellulose semi-solid medium (HSC 003, R & D) and 0. Mu.M, 40. Mu.M and 80. Mu.M phenylbromarone were added, respectively, and three replicates were set per concentration. The cells, benzbromarone and semi-solid medium mixture were added to a 3.5cm dish and after 14 days of incubation at 37℃and 5% CO2, the number of Colonies (CFU) generated and the distribution of colonies of different forms were counted by microscopic observation. The round transparent arrangement of cells in the colony is compact into a progenitor-like (blast-like) cell colony, and if the cell shape in the colony is irregular, the adherent dispersion distribution is a marrow-like (myeoid-like) cell colony. The number of colonies is shown in fig. 4 (left), P <0.05, P <0.01, and P <0.001. The results show that tribromone inhibition of EYA1 phosphatase activity significantly reduced the colony forming ability of HL-60 cells, 40. Mu.M of tribromone treatment reduced the total number of colonies by 40% and 80. Mu.M of tribromone treatment reduced the total number of colonies by 70%. The colony morphology is shown in fig. 4 (right), and the result shows that the inhibition of EYA phosphatase activity by benzbromarone can promote the differentiation of leukemia cells. Either the reduction in colony count or the appearance of differentiated colonies suggests that benzbromarone may regulate the ability of leukemia to self-renew.

Step 6: culturing under unchanged conditions, collecting HL-60 cells in logarithmic growth phase, inoculating 2×10 cells into each 6-well plate ⁶ Cells were cultured in 4mL complete medium, 0. Mu.M, 40. Mu.M and 80 μm phenylbromarone were added for co-culture for 72 hours, respectively, and 3 duplicate wells were set for each concentration of action. After 72 hours, determining the DNA content in the cells by using a PI cell cycle and apoptosis detection kit (Biyun) so as to judge the distribution condition of the cell cycle; as shown in fig. 5, the difference between the same cell cycle ratio of the 40 μm group or the 80 μm group and the 0 μm group was statistically analyzed, and represented by P<0.01 represents P<0.001. The results show that the tribromone treatment significantly increases the sub-G1 phase ratio, and simultaneously reduces the proportion of cells in the G1 phase, so that the cell cycle of HL-60 cells is disordered.

Step 7: culturing under unchanged conditions, collecting HL-60 cells in logarithmic growth phase, inoculating 2×10 cells into each 6-well plate ⁶ Cells were cultured in 4mL complete medium, 0. Mu.M, 40. Mu.M and 80. Mu.M benzbromarone were added for co-culture for 72 hours, and 3 duplicate wells were set for each concentration of effect. Cells were collected after 72 hours and subjected to PI and Annexin V double-stained flow analysis (Annexin-V-FITC Apoptosis Detection Kit, eBioscience Co.). As shown in FIG. 6, PI-Annexin V+ isDefined as early apoptotic (early apoptotic) cells, pi+annexin v+ defined as late apoptotic (late apoptotic) cells, representing P<0.05 represents P<0.01 represents P<0.001. The results showed that after 72 hours of benzbromarone treatment, cells were all elevated both early and late in apoptosis, but the accumulation of cells at late apoptosis was predominant.

Step 8: culturing under unchanged conditions, collecting HL-60 cells in logarithmic growth phase, inoculating 2×10 cells into each 6-well plate ⁶ Cells were cultured in 4mL complete medium, 0. Mu.M, 40. Mu.M and 80 μm phenylbromarone were added for co-culture for 72 hours, respectively, and 3 duplicate wells were set for each concentration of action. After 72 hours, cells were collected, proteins were extracted, and the change in expression level of known EYA downstream targets was detected using Western immunoblotting. The results are shown in FIG. 7, where the benzbromarone treatment significantly down-regulates the protein levels of the known EYA phosphatase downstream target molecules C-MYC, CYCLIN D1, GLI1, NRP 1.

The invention relates to the technical field of pharmacy, in particular to application of benzbromarone in treating acute myelogenous leukemia, and the benzbromarone is used as an EYA phosphatase inhibitor, so that a broad-spectrum effective medicament is provided for treating the leukemia.

The foregoing is merely illustrative of the present invention and not restrictive, and other modifications and equivalents thereof may occur to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. Application of tribromone in preparing medicine for treating acute myelogenous leukemia is provided.

2. Use of tribromone in the manufacture of a medicament for the treatment of acute myelogenous leukemia according to claim 1, characterized in that tribromone acts as an inhibitor of EYA phosphatase.

3. Use of benzbromarone according to claim 1 or 2, in the preparation of a medicament for the treatment of acute myelogenous leukemia, characterized in that said medicament for the treatment of acute myelogenous leukemia comprises said benzbromarone as sole active ingredient.