Application of brucea javanica bitter alcohol in preparation of anti-leukemia drugs

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of brucea javanica bitter alcohol in preparation of anti-leukemia medicines. Test results show that the brucea javanica bitter alcohol can inhibit the cell activity of leukemia cells and cause apoptosis and cell cycle arrest of the leukemia cells. The brucea javanica picrol promotes leukemia cell lipid peroxidation and intracellular ferrous ion accumulation to cause ferroptosis by inhibiting protein expression of an antioxidant transcription factor Nrf2 and downstream NQO1 of the antioxidant transcription factor Nrf2, and the brucea javanica picrol and a ferroptosis inducer Erastatin exert the anti-leukemia synergistic effect. The application proves that the brucea javanica picrol has a good anti-leukemia effect on leukemia cells, prompts that a ferroptosis pathway is one of the effective anti-leukemia pathways, is used for preparing the medicine for treating leukemia, and provides a brand-new thought for developing a drug administration scheme for treating leukemia.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of brucea bruceiol in the preparation of anti-leukemia drugs. Background Art

[0002] Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease characterized by the accumulation of undifferentiated and uncontrolled proliferation of hematopoietic progenitor cells. At present, the traditional treatments for AML mainly include conventional chemotherapy, hematopoietic stem cell transplantation, and targeted molecular therapy. Conventional chemotherapy has a certain effect on the treatment of acute myeloid leukemia, but the treatment of relapsed / refractory AML often has a low remission rate, high drug resistance, and poor treatment effect; hematopoietic stem cell transplantation faces a matching dilemma in clinical practice; new targeted drugs represented by venetoclax are prone to drug resistance, relapse, and other problems. Therefore, there is still an urgent need to find a therapeutic drug with anti-leukemia efficacy and a new treatment strategy.

[0003] Brusatol (Bru) is a quassinolide chemical component extracted from Brucea brucea. It is an effective inhibitor of Nrf2 and can sensitize a variety of cancer cells to cisplatin and other chemotherapeutic active molecules. It has been reported that brucea enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. In recent years, the research on the anti-tumor mechanism of brucea has increased. Some literatures have proposed that brucea participates in the ROS-mediated mitochondrial-dependent pathway and inhibits the Nrf2-mediated antioxidant response, causing G0 / G1 cell cycle arrest in human non-small cell lung cancer cells, increasing the level of intracellular reactive oxygen and lipid peroxidation, reducing the level of glutathione (GSH), destroying the mitochondrial membrane potential, and promoting the apoptosis of lung cancer cells. At the same time, brucea can reduce the viability and proliferation of bladder cancer cells T24 and 5637 through the Chac1 / Nrf2 / SLC7A11 pathway, weaken the migration of T24 and 5637 cells, and increase ROS, malondialdehyde and ferrous ions (Fe 2+ ) levels, thereby hindering the progression of bladder cancer. However, little is known about the anti-leukemia effects of bruceiol.

[0004] Conventional chemotherapy drugs kill tumor cells mainly by inducing apoptosis. However, in clinical use, many tumor cells will develop chemotherapy resistance and apoptosis induction defects. Therefore, the use of other forms of cell death provides new insights into overcoming tumor cell resistance and eliminating tumors. Ferroptosis is a new type of programmed cell death that is different from apoptosis, necrosis, autophagy, etc. It is characterized by the accumulation of intracellular ROS, the accumulation of free iron, and iron-dependent membrane structure lipid peroxidation damage. Morphologically, it is mainly manifested as mitochondrial outer membrane rupture, mitochondrial shrinkage, and disappearance or reduction of mitochondrial cristae. Among them, the fatal accumulation of lipid peroxidation is a major feature of ferroptosis. In order to resist oxidative damage and the occurrence of ferroptosis, cells have evolved a variety of antioxidant systems. It is worth noting that cells themselves have many endogenous antioxidant defense systems to control intracellular ROS levels. Nuclear factor erythroid2-related factor 2 (Nrf2) is a key transcription factor for antioxidant regulation of stress, which is encoded by the NFE2L2 (nuclear factor erythroid-derived2-like 2) gene in humans. When intracellular ROS increases, Nrf2 accumulates in the cytoplasm and then translocates to the nucleus. It binds to the antioxidant response element on the promoter of the target gene through heterodimerization with the sMAF protein, inducing the expression of a series of antioxidant genes such as NQO1. Therefore, the Nrf2-mediated antioxidant pathway is considered to be an important defense mechanism against ferroptosis, and the activated Nrf2 pathway in leukemia cells may enhance the resistance of leukemia cells to ferroptosis and lead to chemotherapy resistance. Therefore, further exploration of the specific mechanism of drug-induced ferroptosis in leukemia will help provide more accurate prevention and treatment strategies for leukemia patients. At present, there is no report on the application of brucein to induce ferroptosis in leukemia cells. Summary of the invention

[0005] In view of the above-mentioned technical problems, the object of the present invention is to provide the use of brucein in the preparation of drugs for inhibiting leukemia. Based on this, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides the use of brucea bruceiol in the preparation of a drug for treating leukemia.

[0007] In the technical solution of the above application, brucea javanica inhibits the activity of leukemia cells.

[0008] In the technical solution of the above application, brucea javanica promotes apoptosis of leukemia cells.

[0009] In the above-mentioned technical solution, brucea javanica causes cell cycle G0 / G1 phase arrest of leukemia cells.

[0010] The second aspect of the present invention provides the use of brucea ol in the preparation of anti-leukemia drugs by inducing ferroptosis of leukemia cells by inhibiting the Nrf2 / NQO1 pathway.

[0011] In the technical solution of the above application, brucea javanica inhibits the protein expression of ferroptosis-related regulatory molecules Nrf2 and NQO1.

[0012] In the technical solution of the above application, brucea javanica promotes the upregulation of lipidROS levels in leukemia cells.

[0013] In the technical solution of the above application, brucea javanica promotes the accumulation of ferrous ions in leukemia cells.

[0014] In the technical scheme of the above application, brucein and the ferroptosis inducer Erastin synergistically inhibit the proliferation of leukemia cells and promote the upregulation of lipid ROS levels in leukemia cells.

[0015] In any of the above-mentioned technical solutions for application, the leukemia is acute myeloid leukemia.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides new insights into the role of brucein in regulating the occurrence and development of leukemia cells, and provides new targets and strategies for improving the chemotherapy efficacy of leukemia patients. The present invention shows that brucein can inhibit the proliferation ability of leukemia cells and promote the apoptosis and cell cycle arrest of leukemia cells. Brucein inhibits the Nrf2 / NQO1 pathway, promotes the increase of lipid ROS levels, promotes the accumulation of ferrous ions, causes ferroptosis of leukemia, and can synergistically inhibit the activity of leukemia cells and enhance the ferroptosis of leukemia cells with the ferroptosis inducer Erastin. Brucein can be used as a drug for preparing a drug for treating leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is the chemical formula of brucea javanica.

[0018] Figure 1 B. Figure 1 C and Figure 1 D is a graph showing the cell viability results of acute myeloid leukemia cell lines HL-60 and NB4 cells and bone marrow stromal cells HS-5 cells treated with different concentrations of brucea brucei for 24 h, 48 h and 72 h in the embodiments of the present invention.

[0019] Figure 1 E is the statistical result table of the half-inhibitory concentration (IC50) of brucea alcohol in these three cell types.

[0020] Figure 2 A is a graph showing the results of brucea ol-induced apoptosis of HL-60 and NB4 cells in an example of the present invention.

[0021] Figure 2 B is a graph showing the cell cycle results of HL-60 and NB4 cells after 24 hours of treatment with brucea brucei in an example of the present invention.

[0022] Figure 3 A and Figure 3 B respectively shows the expression of Nrf2 and NQO1 proteins in HL-60 and NB4 cells after 24 hours of treatment with brucea brucei in the present invention.

[0023] Figure 4 A is a graph showing the results of intracellular lipidROS detection in HL-60 and NB4 cells after 24 hours of treatment with brucea brucei in an example of the present invention.

[0024] Figure 4 B is a graph showing the results of detecting the intracellular ferrous ion content in HL-60 and NB4 cells after 24 hours of treatment with brucea brucei in an example of the present invention.

[0025] Figure 5 A is a graph showing changes in cell activity in HL-60 cells and NB4 cells after stimulation with brucea alone, erastin alone, or brucea combined with erastin for 24 hours in the examples of the present invention.

[0026] Figure 5 B is the combination index (CI) results of bruceiol and erastin in HL-60 cells and NB4 cells respectively in the examples of the present invention.

[0027] Figure 5 C is a graph showing the results of lipid ROS detection in HL-60 cells and NB4 cells after 24 hours of stimulation and culture with brucea brucea alone, Erastin alone, or brucea brucea combined with Erastin in the examples of the present invention.

[0028] Figure 5 D is a graph showing changes in cell activity in NB4 cells after co-culture of brucein, erastin, and ferroptosis inhibitor ferrostatin-1 (Fer-1) in an example of the present invention. DETAILED DESCRIPTION The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention. The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0029] Example 1. Application of brucein in inhibiting leukemia cell viability, promoting leukemia cell apoptosis, and cell cycle arrest 1.1 Experimental methods 1.1.1 Drugs The drug Brusatol (Bru, product number HY-19543, purity 99.89%, batch HY-19543-33) was purchased from MedChemExpress (MCE) and the drug chemical formula is as follows Figure 1 As shown in A. 1.1.2 Cell culture Human acute myeloid leukemia cells HL-60 and NB4 cells and human bone marrow stromal cells HS-5 cells were purchased from the American Type Culture Collection (ATCC). HL-60 and NB4 cells were cultured in RPMI-1640 medium containing 10% South American fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL), and HS-5 cells were cultured in DMEM medium containing 10% South American fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator with 5% CO. 2 The cells were subcultured every 1 day on average to maintain logarithmic growth. 1.1.3 CCK-8 assay to detect cell proliferation HL-60 and NB4 cells were cultured at 1.0 × 10 4 Cells were seeded in 96-well plates at 3.0 × 10 cells per well or HS-5 cells at 3.0 × 10 3 The cells were inoculated in a 96-well plate, with 5 replicate wells in each group. The cell lines were treated with brucea ol in concentration gradient and time gradient. When the culture reached the corresponding time, 10 μL of CCK-8 reagent was added to each well, and the cells were mixed and cultured for 3 hours. The absorbance (OD) value was detected at a wavelength of 450 nm. 1.1.4 Statistical results Calculation of IC50 of brucein in these three cell types IC50 was calculated based on the cell proliferation curve, as shown in the attached Figure 1 As shown in E. 1.1.5 FCM assay to detect cell apoptosis Collect the cells of each group treated with brucein in a centrifuge tube, centrifuge at 500 rpm for 5 min, remove the supernatant, resuspend and wash the cell pellet with pre-cooled PBS, centrifuge at 3000 rpm for 5 min, remove the supernatant, repeat three times; take 5×10 5 Resuspend the cells in PBS at 300g for 5 min, discard the supernatant and add 500μL diluted 1×Annexin V Binding Buffer to resuspend the cells; then add 5μL Annexin V-APC and 5μL DAPI-PB450 staining solution to the cell suspension, gently vortex to mix, incubate at room temperature in the dark for 15 min, and detect immediately after the reaction is completed. 1.1.6 FCM assay to detect cell cycle Collect the cells of each group treated with bruceiol, wash twice with pre-cooled PBS, 500rpm×5min; retain the cell pellet, add 500μL 75% ethanol to fix the cells, and incubate at 4℃ overnight; discard the supernatant, add 100μL RNase A, incubate at 37℃ in the dark for 30min; discard the supernatant, add 400μL PI, incubate at 4℃ in the dark for 30min, and detect immediately after the reaction is completed. 1.2 Experimental Results 1.2.1 Effect of brucein on proliferation of acute myeloid leukemia cells To observe the effect of brucein on the proliferation of leukemia cells in vitro, we used CCK-8 assay to detect the effect of brucein on the cell viability of leukemia cells. Figure 1 B and Figure 1 As shown in C, after treatment with brucein, the cell viability of HL-60 and NB4 cells (human leukemia cells) decreased with both concentration gradient and time gradient. Figure 1 As shown in D, the cell viability of human bone marrow stromal cells HS-5 cells did not change much. The IC50 values ​​of these three cell lines were calculated by cell proliferation curves. It was found that after 24 hours of treatment with brucein, the IC50 values ​​of HL-60, NB4 and HS-5 cells were 68.19nM, 74.29nM and 368.3nM, respectively; after 48 hours of treatment, the IC50 values ​​were 52.96nM, 34.96nM and 280.5nM, respectively; after 72 hours of treatment, the IC50 values ​​were 18.86nM, 30.51nM and 142.2nM, respectively. These findings indicate that brucein has the ability to inhibit the growth of leukemia cells in vitro. 1.2.2 Effect of brucein on apoptosis of acute myeloid leukemia cells To observe the effect of brucein on apoptosis of leukemia cells, we used FCM experiments to detect the changes in apoptosis of HL-60 and NB4 cells after treatment with different concentrations of brucein (0, 50, 100 nM) for 24 h. Figure 2 It can be seen from the results of A that when the concentration of bruceiol was 50nM and 100nM, the apoptosis rates of HL-60 cells were 24.50% and 46.77%, respectively, which were higher than that of the group without drug addition (7.77%); the apoptosis rates of NB4 cells were 9.63% and 27.62%, respectively, which were higher than that of the group without drug addition (4.10%). 1.2.3 Effect of brucein on the cell cycle of acute myeloid leukemia cells To observe the effect of brucein on the cell cycle of leukemia cells, we used FCM experiments to detect the changes in the cell cycle of HL-60 and NB4 cells after treatment with different concentrations of brucein (0, 50, 100 nM) for 24 hours. Figure 2 It can be seen from the results of B that when the concentration of bruceiol was 50nM and 100nM, the number of G0 / G1 phase cells in HL-60 cells increased (62.48% and 80.46%, respectively) compared with the non-drug group (44.73%); compared with the non-drug group (57.70%), the number of G0 / G1 phase cells in NB4 cells increased (65.16% and 71.43%, respectively).

[0030] Example 2. Effect of brucein on the Nrf2 / NQO1 antioxidant pathway in leukemia cells 2.1 Experimental methods 2.1.1 Western blotting to detect the expression levels of ferroptosis-related molecules Nrf2 and NQO1 To investigate the mechanism of BRU-induced ferroptosis in HCC cells, the protein levels of Nrf2 and NQO1 were detected by Western blotting. Leukemia cells treated with brucein (0, 50, 100 nM) for 24 h were collected in centrifuge tubes, centrifuged at 1,000 rpm × 5 min to remove the supernatant, resuspended in 1 mL of pre-cooled PBS, centrifuged at 4,000 rpm × 5 min to remove the supernatant, and washed three times. The cell pellet was retained, and protein lysis buffer (prepared with RIPA lysis buffer and protease inhibitor PMSF at 100:1) of about 2-3 times the volume of the pellet was added, and the mixture was thoroughly pipetted and mixed; the cells were lysed on ice for 30 min, vortexed every 5 min, and repeated 6 times, and centrifuged at 4°C and 12,000 rpm for 30 min; the supernatant was aspirated into a pre-cooled new EP tube, and the protein concentration was detected by BCA method. The remaining protein sample was added with 1 / 4 volume of 5× loading buffer of the supernatant, boiled and denatured, and stored at -40°C. Equal amounts of protein (50 μg in total in each well) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated with a specific primary antibody at 4°C overnight and then incubated with the corresponding secondary antibody at room temperature for 1 hour. The membrane was rinsed three times with PBST buffer and developed with supersignal chemiluminescent substrate to detect the protein expression levels of Nrf2 protein, an antioxidant factor against ferroptosis, and its downstream NQO1 (β-Actin was an internal reference protein). 2.2 Experimental Results 2.2.1 Bruceol reduces the expression levels of Nrf2 and NQO1 proteins in leukemia cells like Figure 3 A and Figure 3 As shown in B, after 24h treatment with different concentrations of bruceiol (0, 50, 100 nM), the expression levels of Nrf2 and NQO1 proteins in HL-60 and NB4 cells decreased significantly in a concentration gradient, and the difference was statistically significant (P<0.05), suggesting that bruceiol inhibits the Nrf2 / NQO1 pathway and may induce ferroptosis in leukemia cells.

[0031] Example 3. Application of brucein in inducing ferroptosis in leukemia cells 3.1 Experimental methods 3.1.1 FCM detection of cellular lipid ROS content C11-BODIPY 581 / 591 was dissolved in DMSO to 10mM and stored in a -20℃ refrigerator away from light. HL-60 or NB4 cells of appropriate density were cultured in 6-well plates. After treatment with brucein (0, 50, 100nM) for 24h, dissolved C11-BODIPY 581 / 591 was added to make the final concentration reach 5μM and incubated at 37℃ for 1h. Each group was collected and washed 3 times with pre-cooled PBS to remove excess dye. The cells were resuspended in 500μL PBS at 1,000rpm×5min and immediately tested on the machine. 3.1.2 Colorimetric method for detecting cellular ferrous ion content Collect the pre-treated cells from each group, wash with PBS 3 times, 1,000rpm×5min. Discard the supernatant, add 0.2mL of reagent 1 to the cell pellet, mix thoroughly, place the sample on ice for 10min, then centrifuge at 1,5000g for 10min, and draw the supernatant into a new EP tube for later use. Set up standard wells, control wells, and assay wells, and add the reagents to the corresponding wells of the ELISA plate according to the order in Table 3-1: Table 3-1 Determination of ferrous ion content Incubate at 37°C for 10 min, and use an ELISA reader to measure the OD value of each well at 593 nm. Calculate the intracellular ferrous ion content (nmol / 10 6 )=[(△Ab)÷a]÷(N÷V)×f. 3.1.3 CCK-8 assay to detect cell proliferation HL-60 and NB4 cells were cultured at 1.0 × 10 4 The number of cells was inoculated in a 96-well plate, with 5 replicate wells in each group. The cell line was treated with brucein combined with the ferroptosis inducer Erastin in a concentration gradient for 24 hours. When the culture reached the corresponding time, 10 μL of CCK-8 reagent was added to each well, mixed and cultured for another 3 hours; the absorbance (OD) value was detected at a wavelength of 450 nm. 3.1.4 SynergyFinder calculates the combination index value (CI value) The combined drug index was calculated based on the cell proliferation curve using the SynergyFinder web application to evaluate the combined effect of brucein and erastin on leukemia cells. The two drugs were judged to have a synergistic effect when the CI value < 1. 3.1.5 FCM detection of cellular lipid ROS content C11-BODIPY 581 / 591 was dissolved in DMSO to 10mM and stored in a -20℃ refrigerator away from light. HL-60 or NB4 cells of appropriate density were cultured in 6-well plates. After 24h of treatment with brucein and erastin alone or in combination, dissolved C11-BODIPY 581 / 591 was added to make the final concentration reach 5μM and incubated at 37℃ for 1h. Each group was collected and washed 3 times with pre-cooled PBS to remove excess dye, and the cells were resuspended in 500μL PBS at 1,000rpm×5min and immediately tested on the machine. 3.1.6 CCK-8 assay to detect cell proliferation NB4 cells were seeded in a 96-well plate at a cell number of 1.0×104 per well, with 5 replicate wells in each group. The cell line was treated with brucein (20nM) combined with ferroptosis inducer Erastin (10μM) and ferroptosis inhibitor Fer-1 (10μM) in a concentration gradient for 24h. When the culture reached the corresponding time, 10μL of CCK-8 reagent was added to each well, mixed and cultured for another 3h; the absorbance (OD) value was detected at a wavelength of 450nm. 3.2 Experimental Results 3.2.1 Bruceol increases lipid ROS levels in leukemia cells Ferroptosis depends on the accumulation of iron and lipid peroxidation in cells. We used C11-BODIPY 581 / 591 fluorescent probe to detect the level of lipid ROS in leukemia cells after bruceiol treatment. Figure 4 As shown in A, after being treated with different concentrations of bruceiol (0, 50, 100 nM) for 24 h, the levels of lipid ROS in HL-60 and NB4 cells increased significantly along the drug concentration gradient (P<0.05). 3.2.2 Brucea brucei increases ferrous ion content in leukemia cells Ferrous ions (Fe 2+ ) participates in the ferroptosis process and is an important indicator of ferroptosis. We used a colorimetric method to detect Fe in leukemia cells after brucein treatment. 2+ Level. Figure 4 As shown in B, after being treated with different concentrations of brucein (0, 50, 100 nM) for 24 h, the expression of Fe 2+ The level increased significantly with the drug concentration gradient (P<0.05). The above results suggest that brucein induces ferroptosis of leukemia cells. 3.2.3 Bruceol and Erastin, an inducer of ferroptosis, synergistically kill leukemia cells and enhance ferroptosis of leukemia cells Erastin is a classic ferroptosis inducer. We used brucein and erastin to treat leukemia cells. Figure 5 A and Figure 5 As shown in Figure B, it was found that brucein could synergistically kill leukemia cells with Erastin. Moreover, compared with single drug use, the combined drug treatment caused a significant increase in the level of lipid ROS in leukemia cells, and the killing effect of the combined drug treatment on leukemia cells could be reversed by the ferroptosis inhibitor Fer-1. The above results suggest that brucein enhances the sensitivity of leukemia cells to ferroptosis. The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. Application of bruceiol in the preparation of anti-leukemia drugs.

2. The use according to claim 1, characterized in that: The invention discloses an application of brucein in preparing a drug for inhibiting the growth of leukemia cells, wherein the leukemia cells are acute myeloid leukemia cells including HL-60 and NB4.

3. The use according to claim 2, characterized in that: Application of brucein in the preparation of drugs for inhibiting the activity of leukemia cells.

4. The use according to claim 2, characterized in that: Application of brucein in the preparation of drugs for promoting apoptosis of leukemia cells.

5. The use according to claim 2, characterized in that: Application of brucea bruceiol in the preparation of drugs for promoting cell cycle arrest of leukemia cells.

6. The use according to claim 2, characterized in that: The targets of bruceiol in leukemia cells are ferroptosis-related molecules Nrf2 and NQO1.

7. The use according to claim 2, characterized in that: Application of brucein in the preparation of drugs that induce ferroptosis of leukemia cells.

8. The use according to claim 7, characterized in that: Role of bruceiol in the preparation of drugs that enhance the sensitivity of leukemia cells to ferroptosis.

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