Application of SR-B1 inhibitor in treating acute myelogenous leukemia

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of an SR-B1 inhibitor BLT-1 in preparation of medicines for treating acute myelogenous leukemia. The invention aims to provide a novel treatment method for treating acute myelogenous leukemia. The invention firstly discovers that the SR-B1 inhibitor BLT-1 has the effects of inhibiting the proliferation of acute myelogenous leukemia cells, promoting the apoptosis of the acute myelogenous leukemia cells, improving the individual level and the like, and can provide a medicine for treating acute myelogenous leukemia. A new thought is provided for further researching an acute myelogenous leukemia mechanism and developing an acute myelogenous leukemia medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and discloses an application of an SR-B1 inhibitor in treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a type of malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in the bone marrow and other hematopoietic tissues due to uncontrolled proliferation, differentiation disorders, and blocked apoptosis, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoietic function. AML is the most common acute leukemia in adults and is also a new malignant tumor. The treatment of AML includes intensive induction therapy, non-intensive therapy, hematopoietic cell transplantation, and immunotherapy, which have improved the complete remission (CR) and 5-year disease-free survival (DFS) of AML patients. However, AML drug resistance and high relapse rate are still serious problems faced by patients. In recent years, although new immunotherapies have achieved certain results, they still face challenges in the treatment of AML, such as clarifying therapeutic targets and overcoming the lack of specific markers for AML mutations. These are still difficulties that the scientific community needs to work hard to overcome in the future.

[0003] The scavenger receptor (SR) family is a class of glycoproteins mainly located on the surface of macrophages and is an important pattern recognition receptor in innate immunity. Among them, SR-B1, as a high-density lipoprotein receptor, mediates the selective uptake or influx of high-density lipoprotein cholesterol (HDL-C) into cells and tissues, promotes the efflux of cholesterol from peripheral tissues (including macrophages) back to the liver; it also promotes the uptake of fat-soluble vitamins and the entry of viruses into host cells. The role of SR-B1 in a variety of diseases has been studied, such as colon cancer, breast cancer, prostate cancer, ovarian cancer, leukemia, etc. Studies have shown that SR-B1 is highly expressed in hematological malignant tumor cells, can recruit cholesterol required for cell division, promote tumor cell proliferation and cancer development. So far, there has been no report on the role of the SR-B1 gene in acute myeloid leukemia. Summary of the invention

[0004] The purpose of the present invention is to provide an application of SR-B1 gene in the treatment of acute myeloid leukemia, and to provide a therapeutic drug for acute myeloid leukemia to solve the technical problem that the drugs for treating acute myeloid leukemia in the prior art need to be further developed.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] Application of SR-B1 inhibitor BLT-1 in the preparation of medicine for treating acute myeloid leukemia.

[0007] The principles and advantages of this technical solution are:

[0008] The inventors conducted a large number of experiments to verify and explore the role of SR-B1 inhibitor BLT-1 in the treatment of acute myeloid leukemia, as follows:

[0009] (1) Effect of SR-B1 inhibitor BLT-1 on proliferation of acute myeloid leukemia: Acute myeloid leukemia cells were incubated with different concentrations of SR-B1 inhibitor BLT-1 for 48 h, and the cell proliferation ability was detected using a cell counting kit (CCK-8).

[0010] (2) Effect of SR-B1 inhibitor BLT-1 on apoptosis of acute myeloid leukemia cells: Flow cytometry (FCM) was used to detect apoptosis of acute myeloid leukemia cells after co-incubation with SR-B1 inhibitor BLT-1.

[0011] (3) Effects of SR-B1 inhibitor BLT-1 on acute myeloid leukemia model mice: First, a blood routine test was used to detect changes in various cells in the blood of mice. Second, blood smears and bone marrow smears were used to observe the number of leukemia cells and the proportion of primitive cells in the blood and bone marrow of mice; the weight changes and survival of mice were counted. Finally, the organ-to-body ratio of mice was calculated and H&E staining was used to observe the differences in the organs of mice.

[0012] Compared with the prior art, this technical solution has the following beneficial effects.

[0013] The present invention reports for the first time that the SR-B1 inhibitor BLT-1 can inhibit the in vitro proliferation of acute myeloid leukemia cells by promoting apoptosis of acute myeloid leukemia cells. In addition, the present invention reports for the first time that the SR-B1 inhibitor BLT-1 can reduce the proportion of primitive cells in leukemia mice, prolong the survival of leukemia mice, and effectively improve the organ lesions of leukemia mice. The above findings can provide a therapeutic drug for acute myeloid leukemia, providing new ideas for further research on the onset of acute myeloid leukemia; at the same time, the SR-B1 inhibitor BLT-1 can be used in the screening and testing of new anti-acute myeloid leukemia drugs, providing guidance for the development of new drugs for the treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the chemical structural formula of SR-B1 inhibitor BLT-1; Figure 2 This is a graph showing the anti-cell proliferation results of different acute myeloid leukemia cells in Example 1 detected by CCK-8 test after incubation with SR-B1 inhibitor BLT-1 for 48 hours; Figure 3 This is a result diagram of the detection of cell apoptosis by flow cytometry after the acute myeloid leukemia cells C1498 in Example 2 were incubated with different concentrations of the SR-B1 inhibitor BLT-1 for 48 hours; Figure 4 The statistical graphs of the blood routine test results of the blank group, model group and BLT-1 group mice in Example 3; Figure 5 The blood smear and bone marrow smear results and statistical graphs of the blank group, model group and BLT-1 group of mice in Example 3; Figure 6 The figure is a statistical graph of the weight change curve and survival time curve of the blank group, model group and BLT-1 group mice in Example 3; Figure 7 These are pictures of the organs, statistical results of organ-to-body ratios, and H&E staining results of the organs of the blank group, model group, and BLT-1 group of mice in Example 3. DETAILED DESCRIPTION

[0014] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0015] Example 1: Effect of SR-B1 inhibitor on proliferation of acute myeloid leukemia cells

[0016] 1 Experimental methods

[0017] 1.1 Cell culture

[0018] Acute myeloid leukemia cells C1498 were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in a 37°C incubator containing 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL). Acute myeloid leukemia cells HL-60 were donated by the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, and cultured in a 37°C incubator containing 5% CO2 using 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL). Acute myeloid leukemia cells KG-1a were purchased from Shanghai Fuheng Biotechnology Co., Ltd. and cultured in a 37°C incubator containing 5% CO2 using IMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL).

[0019] 1.2 CCK-8 assay to measure cell proliferation

[0020] The CCK-8 method was used to analyze the effect of SR-B1 inhibitor BLT-1 on the proliferation of acute myeloid leukemia cells C1498, HL-60 and KG-1a. The cells were seeded in 96-well plates, and 200 μL of drug-containing culture medium of different concentrations was added to each well for co-incubation with the cells. The 96-well plates were incubated in a cell culture incubator at 37°C and 5% CO2 for 48 hours. 20 μL of CCK-8 solution was added to each well and incubated in a 37°C, 5% CO2 incubator for 4 hours. The absorbance at 450 nm was measured by a microplate reader and the IC 50 .

[0021] 1.3 Statistical analysis

[0022] All experiments were performed independently for 3 times, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism software was used for statistical analysis, and independent sample t test was used for comparison between groups. P < 0.05 was considered statistically significant.

[0023] 2 Experimental results

[0024] As the instruction manual Figure 2 As shown in (A), the SR-B1 inhibitor BLT-1 can effectively inhibit the proliferation of acute myeloid leukemia cells. Figure 2 (B) shows that the inhibitory effect on acute myeloid leukemia cells increases with the increase of the concentration of SR-B1 inhibitor BLT-1. Figure 2 (C) It can be seen that the IC of acute myeloid leukemia cells C1498, HL-60 and KG-1a after incubation with the SR-B1 inhibitor BLT-1 for 48 h 50 They were 2.92±0.33, 5.38±0.26 and 4.34±0.43 μM respectively.

[0025] Example 2: Effect of SR-B1 inhibitor on apoptosis of acute myeloid leukemia cells

[0026] 1 Experimental methods

[0027] 1.1 Cell culture

[0028] Acute myeloid leukemia cells C1498 were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in a 37°C incubator containing 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1% penicillin□streptomycin solution (100 U / mL).

[0029] 1.2 Cell apoptosis experiment

[0030] (1) Experimental process:

[0031] Acute myeloid leukemia cells C1498 were co-incubated with different concentrations (0, 2, 4, 8 μM) of SR-B1 inhibitor BLT-1 on a 6-well plate for 24 h, and then all the cells and culture medium were transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was removed. The tube was washed with PBS three times, and the supernatant was removed. 500 μL 1×binding buffer was added to resuspend the cells, and the tube was centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was removed. This step was repeated. 500 μL 1×binding buffer was added to resuspend the cells, and the concentration was adjusted to 1-5×10 6 / mL, take 100μL of cell suspension into a 5mL flow cytometry tube, add 5μL Annexin V / FITC, mix well, and incubate at room temperature in the dark for 5min; add 5μL propidium iodide solution (PI) and 400μL PBS, and immediately perform flow cytometry or fluorescence microscopy detection.

[0032] (2) Experimental design:

[0033] ①Blank tube: Negative control group cells, without Annexin V / FITC and PI. Used to adjust voltage. ②Single-stained tube: Positive control group cells, only Annexin V / FITC or only PI. Used to adjust compensation. ③Detection tube: Treated cells, with Annexin V / FITC and PI. After adjusting voltage compensation with blank tube and single-stained tube, obtain the required flow data.

[0034] 1.3 Statistical analysis

[0035] All experiments were performed independently for 3 times, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism software was used for statistical analysis, and independent sample t test was used for comparison between groups. P < 0.05 was considered statistically significant.

[0036] 2 Experimental results

[0037] The results of the cell apoptosis experiment are as shown in the instructions. Figure 3 As shown in (A)(B)(C)(D), the SR-B1 inhibitor BLT-1 can promote the apoptosis of acute myeloid leukemia cells. As the concentration of the SR-B1 inhibitor BLT-1 increases, the apoptosis rate of acute myeloid leukemia cells C1498 increases. Figure 3 (E) It can be seen that the apoptosis rates of acute myeloid leukemia cells C1498 at 0, 2, 4, and 8 μM SR-B1 inhibitor BLT-1 concentrations were 6.80±0.99%, 8.60±1.07%, 12.5±1.27%, and 23.35±1.95%, respectively.

[0038] Example 3: Effects of SR-B1 inhibitor on acute myeloid leukemia model mice

[0039] 1 Experimental methods

[0040] 1.1 Establishment of mouse leukemia model

[0041] Acute myeloid leukemia C1498 cells with good growth status were collected at a number of 2×10 6 The mice were randomly divided into a blank group (normal saline injected into the tail vein), a model group, and a BLT-1 group (25 mg / kg / d). After 24 hours, the mice were treated with equal volumes of solvent and BLT-1 (25 mg / kg) every day for 14 consecutive days. Peripheral blood smears and blood routine tests were performed after blood was collected from the eye sockets at 0, 10, 20, and 40 days, respectively, to observe the changes in white blood cells and immature cells in the blood; in addition, the mice were weighed every 3 days, and the survival and death of the mice were recorded and statistical analysis of the survival period was performed. When the mice were on the verge of death, their spleens and livers were taken for photographing and weighing, and the spleens and livers of the mice were stained with eosin to observe the infiltration of leukemia cells under a microscope. At the same time, the bone marrow of the mice was taken for bone marrow cell smears, and the proportion of immature cells in the bone marrow cells of the mice was examined and counted under a microscope.

[0042] 1.2 Statistical analysis

[0043] All experiments were performed independently for 3 times, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism software was used for statistical analysis, and independent sample t test was used for comparison between groups. P < 0.05 was considered statistically significant.

[0044] 2 Experimental results

[0045] Attached to the instruction manual Figure 4 It can be seen that compared with the blank group, the number of monocytes in the model group increased significantly (p < 0.01), and there was no significant change in the number of monocytes in the BLT-1 group (p > 0.05); in addition, compared with the model group, the number of monocytes in the BLT-1 group decreased significantly (p < 0.01). Figure 5 (C) The results showed that compared with the control group, the number of leukocytes in the model group increased significantly (p < 0.05), while there was no significant difference in the BLT-1 group (p > 0.05); compared with the model group, the number of leukocytes in the BLT-1 group decreased significantly (p < 0.05). Figure 5(D) The results showed that compared with the control group, the number of immature cells in the model group and the BLT-1 group increased significantly (p < 0.001), but the increase in the BLT-1 group was less than that in the model group; compared with the model group, the number of immature cells in the BLT-1 group decreased significantly (p < 0.05). Figure 6 The survival curve results show that compared with the model group, the BLT-1 group can effectively prolong the survival time of mice (p < 0.001). Figure 7 The results of (A) and (B) showed that compared with the blank group, the model group mice had enlarged liver and spleen and a large number of white nodules in the liver, while the BLT-1 group had no obvious enlargement and organ lesions. Figure 7 (C) The H&E staining results showed that compared with the blank group, a large number of leukocytes infiltrated the lungs, livers, spleens, and bone marrows of the mice in the model group, and the lung structure and liver lobule structure were destroyed, while no obvious leukocyte infiltration and organ structure destruction were observed in the BLT-1 group.

Claims

1. Application of SR-B1 inhibitors in the preparation and treatment of acute myeloid leukemia.

2. Use of the SR-B1 inhibitor according to claim 1 in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The SR-B1 inhibitor is BLT-1, which is a compound with the following structure:

3. Use of the SR-B1 inhibitor according to claim 2 in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The substance has an inhibitory effect on the proliferation of acute myeloid leukemia cells.

4. Use of the SR-B1 inhibitor according to claim 3 in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The substance can promote apoptosis of acute myeloid leukemia cells.

5. Use of the SR-B1 inhibitor according to claim 3 in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The cells include at least one of HL-60, KG-1a, C1498 and other acute myeloid leukemia cells.

6. A medicament comprising one or more SR-B1 inhibitors or pharmaceutically acceptable salts thereof as claimed in claim 2 and a pharmaceutically acceptable carrier.

7. The use according to claims 2 and 6, wherein the drug is administered orally: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, effervescent tablets, sublingual tablets, capsules, hard capsules, soft capsules, microcapsules, microcapsules, microspheres, granules, pills, pellets, powders, oral liquids, suspensions, solutions, and sustained-release or controlled-release preparations.

8. The use according to claims 2 and 6, wherein the drug is administered by inhalation: aerosol and sustained-release or controlled-release preparations.

9. The use according to claims 2 and 6, wherein the drug is administered intravenously: injection, injection emulsion, lyophilized powder injection, and sustained-release or controlled-release preparation.

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