Application of isoflavone compound in preparation of anti-leukemia medicine

Abstract

The invention discloses application of an isoflavone compound in preparation of an anti-leukemia medicine, and relates to the technical field of medicines. The invention specifically discloses an isoflavone compound with the following structural formula: # imgabs0 #; r1 is selected from H, # imgabs 1 # or # imgabs 2 #; r2 is selected from OH or OCH3; r3 is selected from H or # imgabs3 #; r4 is selected from H or OH. The isoflavone compound or the pharmaceutically acceptable salt thereof can effectively inhibit proliferation of leukemia cells, has small toxic effect on human immortalized epidermal cells, and has good application prospects in preparation of anti-leukemia drugs or inhibition of proliferation of leukemia cells for non-disease treatment.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to the application of an isoflavone compound in the preparation of an anti-leukemia drug. Background Art

[0002] Chronic myeloid leukemia (CML) is a myeloproliferative tumor that is prone to drug resistance during treatment. Tumor multidrug resistance (MDR) is one of the main causes of tumor chemotherapy failure and tumor recurrence. It manifests itself in that when malignant tumor cells are resistant to one chemotherapy drug, they also become resistant to multiple other chemotherapy drugs with different structures and mechanisms of action. One of the main mechanisms leading to multidrug resistance is the overexpression of ABC transporters. Therefore, how to overcome tumor multidrug resistance is a top priority in cancer treatment. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of isoflavone compounds in the preparation of anti-leukemia drugs or in the inhibition of leukemia cell proliferation for non-disease treatment purposes.

[0004] According to the first aspect of the present invention, an isoflavone compound having the following structural formula or a pharmaceutically acceptable salt thereof is used in the preparation of an anti-leukemia drug or in the inhibition of leukemia cell proliferation for non-disease treatment purposes: ; R1 is selected from H, or ; R2 is selected from OH or OCH3; R3 is selected from H or ; R4 is selected from H or OH.

[0005] According to some embodiments of the present invention, the structural formula of the compound is , or .

[0006] According to some embodiments of the present invention, the leukemia is chronic myeloid leukemia.

[0007] According to some embodiments of the present invention, the leukemia cells are chronic myeloid leukemia cells (such as K562 cells).

[0008] According to some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

[0009] According to some embodiments of the present invention, the carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent.

[0010] According to some embodiments of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form selected from tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, dripping pills, dry extracts, injections or infusions.

[0011] According to some embodiments of the present invention, the mass percentage of the isoflavone compound in the drug is 0.1wt% to 99wt%. For example, it can be 0.1wt%, 1wt%, 5wt%, 10wt%, 15wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt%, 95wt%, 99wt%.

[0012] According to the second aspect of the present invention, the use of the compound represented by formula I or a pharmaceutically acceptable salt thereof in any one of items A1) to A13): (Formula I); A1) Preparation of drugs to combat drug-resistant leukemia; A2) Inhibition of proliferation of drug-resistant leukemia cells for non-disease therapeutic purposes; A3) Preparation of leukemia resistance reversal agents; A4) Preparation of leukemia drug sensitizers; A5) Inhibition of the expression of drug-resistant proteins in drug-resistant leukemia cells for non-disease treatment purposes; A6) Preparation of products that promote or induce cell cycle arrest in leukemia cells or drug-resistant leukemia cells; A7) Promoting or inducing cell cycle arrest in leukemia cells or drug-resistant leukemia cells for non-disease treatment purposes; A8) Preparation of products that induce apoptosis in leukemia cells or drug-resistant leukemia cells; A9) Inducing apoptosis of leukemia cells or drug-resistant leukemia cells for non-disease treatment purposes; A10) Preparation of products that inhibit the Keap-1-Nrf2 / ARE signaling pathway; A11) Inhibition of Keap-1-Nrf2 / ARE signaling pathway for non-disease therapeutic purposes; A12) Preparation of products that inhibit MAPK signaling pathways; A13) Inhibition of MAPK signaling pathway in leukemia cells or drug-resistant leukemia cells for non-disease therapeutic purposes.

[0013] According to some embodiments of the present invention, the drug resistance includes resistance to doxorubicin.

[0014] According to some embodiments of the present invention, the leukemia drug comprises doxorubicin.

[0015] According to some embodiments of the invention, the leukemia comprises chronic myeloid leukemia.

[0016] According to some embodiments of the present invention, the leukemia is chronic myeloid leukemia.

[0017] According to some embodiments of the present invention, the leukemia cells are chronic myeloid leukemia cells (such as K562 cells).

[0018] According to some embodiments of the present invention, the drug-resistant leukemia cells include doxorubicin-resistant leukemia cells.

[0019] According to some embodiments of the present invention, the adriamycin-resistant leukemia cells include adriamycin-resistant chronic myeloid leukemia cells (such as K562 / ADR cells).

[0020] According to some embodiments of the present invention, in A5), the drug resistance protein includes at least one of BCRP and P-gp.

[0021] According to some embodiments of the present invention, in A6) or A7), the inducing cell cycle arrest in leukemia cells or drug-resistant leukemia cells is inducing leukemia cell cycle arrest in the G2 / M phase.

[0022] According to some embodiments of the present invention, in A8) or A9), the inducing apoptosis of leukemia cells or drug-resistant leukemia cells is achieved by inhibiting or downregulating the expression of at least one of the apoptosis-related proteins pro-caspase 3, pro-caspase 8 and pro-caspase9.

[0023] According to some embodiments of the present invention, in A10) or A11), the inhibition of the Keap-1-Nrf2 / ARE signaling pathway includes inhibiting or down-regulating the expression of at least one of Nrf2, HO-1, and Keap-1.

[0024] According to some embodiments of the present invention, in A12) or A13), the inhibition of MAPK signaling pathway includes inhibiting the phosphorylation of at least one of JNK, ERK, and p38.

[0025] According to some embodiments of the present invention, the product is selected from a kit, a drug or a reagent.

[0026] According to some embodiments of the present invention, the product is a drug; the drug also includes a pharmaceutically acceptable carrier.

[0027] According to some embodiments of the present invention, the carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent.

[0028] According to some embodiments of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form. The dosage form is an oral dosage form or a non-oral dosage form. The dosage form is selected from tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, dripping pills, dry extracts, injections or infusions.

[0029] According to some embodiments of the present invention, the effective concentration of the compound represented by Formula I is 5 μM to 400 μM.

[0030] According to some embodiments of the present invention, the target is leukemia cells, and the concentration of the compound represented by Formula I is 5 μM to 150 μM. For example, it can be 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM or 150 μM.

[0031] According to some embodiments of the present invention, the target is drug-resistant leukemia cells, and the concentration of the compound represented by Formula I is 5 μM to 400 μM. For example, it can be 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM or 400 μM.

[0032] According to some embodiments of the present invention, the carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent.

[0033] According to some embodiments of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form selected from tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, dripping pills, dry extracts, injections or infusions.

[0034] According to some embodiments of the present invention, the mass percentage of the compound represented by Formula I in the drug is 0.1wt% to 99wt%. For example, it can be 0.1wt%, 1wt%, 5wt%, 10wt%, 15wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt%, 95wt%, 99wt%.

[0035] The embodiments of the present invention have at least the following beneficial effects: Isoflavones FA-7, FA-17 and TMI isolated from the fruit of Ficus alpina can effectively inhibit the proliferation of leukemia cells and have low toxicity to human immortalized epidermal cells. Among them, isoflavone TMI has a significant inhibitory effect on drug-resistant leukemia cells, reducing cell viability in a concentration- and time-dependent manner; it inhibits cell proliferation by blocking the cell cycle, reduces the expression of pro-caspase 3, pro-caspase 8 and pro-caspase 9, and induces cell apoptosis. In addition, isoflavone TMI can also overcome the resistance of doxorubicin-resistant leukemia cells (such as K562 / ADR cells) by directly targeting and inhibiting Nrf2 and downregulating the expression of drug-resistant proteins such as BCRP and P-gp. The inhibitory effect of isoflavone TMI is stronger than that of doxorubicin.

[0036] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The effect of TMI on the activity of K562 / ADR and K562 cells; A: K562 / ADR cells, B: K562 cells; Figure 2 The expression of drug-resistant protein P-gp in K562 / ADR and K562 cells; A: western blot results, B: quantification results of the relative expression of P-gp in A; ** indicates a significant difference relative to the K562 / ADR group ( p <0.01); Figure 3 The effect of TMI on the expression of drug-resistant proteins BCRP and P-gp in K562 / ADR cells; A: western blot results, B: quantification results of the relative expression of P-gp in A, C: quantification results of the relative expression of BCRP in A; * indicates significant difference relative to the control group ( p<0.05, ** indicates a significant difference relative to the control group ( p <0.01); Figure 4 The effect of TMI on the cell cycle of K562 / ADR cells and K562 cells; A: Western blot results of K562 / ADR cells, B: quantification results of the relative expression of cell cycle proteins in A, C: Western blot results of K562 cells, D: quantification results of the relative expression of cell cycle proteins in C; * indicates significant difference relative to 0h ( p <0.05, ** indicates a significant difference relative to 0 h ( p <0.01); Figure 5 The effect of TMI on the expression of apoptosis-related proteins in K562 / ADR cells and K562 cells; A: western blot results, B: quantification results of the relative expression of apoptosis-related proteins in K562 / ADR cells corresponding to A, C: quantification results of the relative expression of apoptosis-related proteins in K562 cells corresponding to A; * indicates significant difference relative to the control group ( p <0.05, ** indicates a significant difference relative to the control group ( p <0.01); Figure 6 The effect of TMI on the expression of apoptosis-related proteins in K562 / ADR cells and K562 cells before and after treatment with pan-caspase inhibitors; A: western blot results, B: quantification results of the relative expression of apoptosis-related proteins in K562 / ADR cells corresponding to A, C: quantification results of the relative expression of apoptosis-related proteins in K562 cells corresponding to A; ** indicates a very significant difference relative to the control group ( p <0.01); # indicates significant difference ( p <0.05, ## indicates a very significant difference ( p <0.01); Figure 7 The effect of TMI on the expression of drug-resistant proteins BCRP and P-gp in K562 / ADR cells before and after treatment with pan-caspase inhibitors; A: western blot results, B: quantification results of the relative expression of P-gp and BCRP in A; * indicates significant difference relative to the control group ( p <0.05), ns means no significant difference; Figure 8Effects of TMI on MAPK pathway-related proteins in K562 / ADR cells and K562 cells; A: western blot results, B, D, F: quantification results of relative expression levels of p-JNK, p-ERK, and p-p38 in K562 / ADR cells corresponding to A, C, E, G: quantification results of relative expression levels of p-JNK, p-ERK, and p-p38 in K562 cells corresponding to A; * indicates significant difference relative to the control group ( p <0.05, ** indicates a significant difference relative to the control group ( p <0.01); Fig. 9 The effect of TMI on the survival rate of K562 / ADR cells and K562 cells before and after treatment with different agonists or inhibitors; A: TBHQ, B: anisomycin, C: SB203580; ** indicates a significant difference relative to the control group ( p <0.01); ns indicates no significant difference, # indicates a significant difference ( p <0.05, ## indicates a very significant difference ( p <0.01); Fig.10 Effects of TMI on Keap-1-Nrf2 / ARE signaling pathway in K562 / ADR cells and K562 cells; A: western blot results, B: quantification results of relative expression of proteins related to Keap-1-Nrf2 / ARE signaling pathway in K562 / ADR cells corresponding to A, C: quantification results of relative expression of proteins related to Keap-1-Nrf2 / ARE signaling pathway in K562 cells corresponding to A; * indicates significant difference relative to control group ( p <0.05, ** indicates a significant difference relative to the control group ( p <0.01); Fig.11 Effects of TMI on the expression of apoptosis-related proteins in K562 / ADR cells and K562 cells before and after TBHQ treatment; A: western blot results, B: quantification results of the relative expression of apoptosis-related proteins in K562 / ADR cells corresponding to A, C: quantification results of the relative expression of apoptosis-related proteins in K562 cells corresponding to A; * indicates significant difference relative to the control group ( p <0.05, ** indicates a significant difference relative to the control group ( p <0.01); # indicates significant difference ( p<0.05, ## indicates a very significant difference ( p <0.01); Fig.12 The effect of TMI on the expression of drug resistance-related proteins in K562 / ADR cells before and after TBHQ treatment; A: western blot results, B: quantification results of the relative expression of P-gp in A, C: quantification results of the relative expression of BCRP in A; * indicates significant difference relative to the control group ( p <0.05, ** indicates a significant difference relative to the control group ( p <0.01); # indicates significant difference ( p <0.05, ## indicates a very significant difference ( p <0.01); Fig.13 The effect of TMI on the expression of Nrf2 protein in K562 / ADR cells and K562 cells before and after Z-VAD or TBHQ treatment; A: western blot results, B: quantification results of the relative expression of Nrf2 protein affected by Z-VAD in A, C: quantification results of the relative expression of Nrf2 protein affected by TBHQ in A; * indicates significant difference relative to the control group ( p <0.05, ** indicates a significant difference relative to the control group ( p <0.01); ns indicates no significant difference, # indicates a significant difference ( p <0.05); Fig.14 The results of the protein thermal stability (CETSA) experiment verifying the binding of TMI to K562 / ADR cells and Nrf2 protein in K562 cells; A: western blot results, B: quantification results of the relative expression of Nrf2 protein in K562 / ADR cells treated with TMI at different temperatures in A, C: quantification results of the relative expression of Nrf2 protein in K562 cells treated with TMI at different temperatures in A; Fig.15 The results of CETSA experiment verification of the binding of TMI to p-ERK and ERK proteins in K562 / ADR cells; A: western blot results, B: quantification results of the relative expression of ERK protein in K562 / ADR cells treated with TMI at different temperatures in A, C: quantification results of the relative expression of p-ERK in K562 / ADR cells treated with TMI at different temperatures in A. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0040] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0041] Unless otherwise specified, "1640 complete medium" in this article refers to 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

[0042] Unless otherwise specified, K562 cells were cultured and passaged using 1640 complete medium. K562 / ADR cells were cultured and passaged using 1640 complete medium containing 2 μM doxorubicin to maintain the drug resistance of the cells. Ten days before the start of the experiment, the culture medium of K562 / ADR cells was replaced with 1640 complete medium without doxorubicin for drug-free culture.

[0043] Example 1 This example provides an isoflavone compound of Ficus microcarpa (denoted as: TMI), the structural formula of which is shown in Formula I; (Formula Ⅰ).

[0044] The preparation method of isoflavone TMI refers to the method disclosed in the literature "Research on Active Ingredients of Banyan Fruit" (Yao Jiaming. Research on Active Ingredients of Banyan Fruit [D]. Guangdong Pharmaceutical University, 2021. DOI: 10.27690 / d.cnki.ggdyk.2021.000257.).

[0045] Example 2 This example provides an isoflavone compound of Ficus microcarpa (denoted as: FA-7), the structural formula of which is shown in Formula II; (Formula II).

[0046] The preparation method of isoflavone FA-7 refers to the method disclosed in the literature "Research on Active Ingredients of Banyan Fruit" (Yao Jiaming. Research on Active Ingredients of Banyan Fruit [D]. Guangdong Pharmaceutical University, 2021. DOI: 10.27690 / d.cnki.ggdyk.2021.000257.).

[0047] Example 3 This example provides an isoflavone compound of Ficus microcarpa (denoted as: FA-17), the structural formula of which is shown in Formula III; (Formula III).

[0048] The preparation method of isoflavone FA-17 refers to the method disclosed in the literature "Research on Active Ingredients of Banyan Fruit" (Yao Jiaming. Research on Active Ingredients of Banyan Fruit [D]. Guangdong Pharmaceutical University, 2021. DOI: 10.27690 / d.cnki.ggdyk.2021.000257.).

[0049] Experimental Example 1 Cells (K562 / ADR, K562 and Hacat) in the logarithmic growth phase were inoculated into 96-well plates, and 90 μL of cell suspension (cell density was 5 × 10 3 cells / mL), with 3 replicate wells in each group, and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. The next day, 10 μL of different concentrations of isoflavones (TMI, FA-7 or FA-17) were added to each well, and the final concentrations of isoflavones were 0, 12.5, 25, 50 and 100 μM, respectively; a positive control group (doxorubicin) was set up at the same time. After incubation for 48 h, the cell survival rate was detected by MTT method, and the IC 50 .

[0050] The results are shown in Table 1.

[0051] Table 1

[0052] TMI showed a significant inhibitory effect on K562 / ADR cell proliferation, and the effect was comparable to that of the positive drug doxorubicin, far superior to FA-7 and FA-17, and its toxicity to Hacat (human immortalized epidermal cells) was much lower than that of doxorubicin. This indicates that TMI has the effect of inhibiting the proliferation of leukemia cells K562 and drug-resistant leukemia cells K562 / ADR.

[0053] Experimental Example 2 Cells (K562 / ADR, K562) in the logarithmic growth phase were inoculated into a 96-well plate, and 90 μL of cell suspension (cell density was 5×10 3 cells / mL), with 3 replicate wells in each group, and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. The next day, 10 μL of different concentrations of isoflavone TMI (0, 12.5, 25, 50 and 100 μM) was directly added to each well, and then incubated for 24, 48 and 72 h. Then, 10 μL of MTT reagent was added to each well, and cultured at 37°C for 4 h. The supernatant was removed, and 150 μL of DMSO was added to each well and shaken on a shaker for 10 min. The absorbance of each well was measured at 490 nm using an enzyme marker to detect cell activity and calculate the cell survival rate.

[0054] The results are as follows Figure 1 shown.

[0055] TMI reduced the viability of K562 and K562 / ADR cells in a concentration- and time-dependent manner, indicating that TMI can effectively kill drug-resistant leukemia cells and general leukemia cells, and has a certain anti-leukemia drug resistance effect.

[0056] Experimental Example 3 1. Take K562 / ADR and K562 cells in the logarithmic growth phase and inoculate them separately (inoculation concentration is 5×10 5 cells / well) in a 6-well plate and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. The cells in the wells were collected the next day for subsequent experiments. The expression of P-gp protein in the cells was detected by Western blotting (GAPDH was used as an internal reference, the primary antibodies P-gp and GAPDH were purchased from Wuhan Tri-Tek (Wuhan, China), the primary antibody GAPDH was diluted at a ratio of 1:5000, and the primary antibody P-gp was diluted at a ratio of 1:2000 before use; the secondary antibodies used were rabbit and mouse antibodies, purchased from Cell Signaling Technology (Danvers, USA), and the antibodies were diluted at a ratio of 1:2000 before use).

[0057] The results are as follows Figure 2 shown.

[0058] The expression of P-gp resistance protein in K562 / ADR cells was significantly higher than that in K562 cells, which indicated that the drug resistance of K562 / ADR cells was related to the high expression of resistance protein.

[0059] 2. Take K562 / ADR cells in the logarithmic growth phase and inoculate them (the inoculation concentration is 5×10 5cells / well) in a 6-well plate and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. The culture medium was discarded every other day, and 2 mL of 1640 culture medium containing different concentrations (25, 50 and 100 μM) of isoflavone TMI was added to each well. A positive control group (ADR group, 50 μM doxorubicin) and a blank control group (control group, 1640 culture medium without TMI and doxorubicin) were set up. After incubation for 24 h, the expression of BCRP and P-gp proteins in the cells was detected by western blot (GAPDH was used as an internal reference. The primary antibodies P-gp, BCRP and GAPDH were purchased from Wuhan Tri-Tac (Wuhan, China), and the primary antibody GAPDH was diluted at a ratio of 1:5000 before use. The primary antibodies P-gp and BCRP were diluted at a ratio of 1:2000; the secondary antibodies were rabbit and mouse antibodies, purchased from Cell Signaling Technology (Danvers, USA), and the antibodies were diluted at a ratio of 1:2000 before use).

[0060] The results are as follows Figure 3 shown.

[0061] TMI can effectively inhibit the expression of drug-resistant proteins such as BCRP and P-gp in K562 / ADR. Under 50 μM TMI treatment, the inhibitory effect on P-gp protein is even stronger than that of the positive control doxorubicin. This shows that TMI overcomes the drug resistance of K562 / ADR cells by inhibiting the expression of drug-resistant proteins such as BCRP and P-gp.

[0062] Experimental Example 4 K562 / ADR and K562 cells in the logarithmic growth phase were inoculated (inoculation concentration was 5×10 5cells / well) in a 6-well plate and cultured overnight in an incubator. The culture medium was discarded the next day, and 2 mL of 1640 medium containing the specified concentration of isoflavone TMI (K562 / ADR: 60 μM, K562: 30 μM) was added to each well. After incubation for 0, 12, 18, 24, and 36 h, the expression of G2 / M phase-related cycle proteins (Cyclin B1, p-cdc2, Cyclin A2, and p-HistineH3) was detected by western blot (GAPDH was used as an internal reference, and the primary antibody GAPDH was purchased from Wuhan Tri-Tek (Wuhan, China). The other primary antibodies were from the Cell Cycle Regulation Antibody Sampling Kit II, and the secondary antibodies were rabbit and mouse antibodies, all purchased from Cell Signaling Technology (Danvers, USA). The primary antibody GAPDH was diluted at a ratio of 1:5000 before use, the other primary antibodies were diluted at a ratio of 1:1000, and the secondary antibodies were diluted at a ratio of 1:2000).

[0063] The results are as follows Figure 4 shown.

[0064] Compared with cells before TMI treatment, the protein expressions of Cyclin B1, p-cdc2, Cyclin A2 and p-Histine H3 in K562 / ADR were decreased after TMI treatment for 12, 18, 24 and 36 h. In K562 cells, the expression of Cyclin B1 and p-cdc2 proteins increased significantly at 24 h, while decreased at 36 h; meanwhile, TMI upregulated the protein expression of Cyclin A2, and the p-Histine H3 protein decreased significantly at 18 h, and then the protein expression level recovered. This indicates that TMI inhibits cell proliferation by inducing cell cycle arrest in G2 / M phase in K562 / ADR and K562 cells.

[0065] Experimental Example 5 1. Take K562 / ADR and K562 cells in the logarithmic growth phase (seeding concentration is 5×10 5cells / well) in a 6-well plate and cultured in an incubator overnight. The culture medium was discarded every other day, and 2 mL of 1640 medium containing TMI at the specified concentrations (0, 25, 50, and 100 μM for K562 / ADR cells and 0, 15, 30, and 60 μM for K562 cells) was added to each well. A positive control group (50 μM doxorubicin for K562 / ADR and 2 μM doxorubicin for K562) was set up. After 24 h of incubation, the expression of apoptosis-related proteins (pro-caspase 3, pro-caspase 8, and pro-caspase 9) was detected by western blot (GAPDH was used as an internal reference, and the primary antibodies pro-caspase 8, pro-caspase 9, and GAPDH were purchased from Wuhan Tri-Tek (Wuhan, China), and the primary antibody pro-caspase 3, secondary antibody rabbit antibody, and secondary antibody mouse antibody were purchased from Cell Signaling Technology (Danvers, USA), the primary antibody GAPDH was diluted at 1:5000, the secondary antibodies rabbit and mouse were diluted at 1:2000, and the rest of the antibodies were diluted at 1:1000).

[0066] The results are as follows Figure 5 shown.

[0067] TMI significantly reduced the expression of pro-caspase 3, pro-caspase 8, and pro-caspase 9 proteins in K562 / ADR cells and K562 cells. In contrast, 50 μM doxorubicin did not induce apoptosis in K562 / ADR cells. This indicates that TMI can induce apoptosis in leukemia cells.

[0068] 2. Take K562 / ADR and K562 cells in the logarithmic growth phase (seeding concentration is 5×10 5cells / well) in a 6-well plate and cultured overnight in an incubator. The next day, the culture medium was discarded, and 2 mL of 1640 medium containing pan-caspase inhibitor Z-VAD-FMK (abbreviated as: Z-VAD, purchased from Selleck Chemicals (Houston, TX, USA) was added to each well for pretreatment for 4 h. Then, the isoflavone TMI of the specified concentration was directly added to each well (the final concentration for K562 / ADR cells was 60 μM, and the final concentration for K562 cells was 30 μM). At the same time, a blank control group (control group, K562 / ADR cells or K562 cells without any treatment), a negative control group (Z-VAD-FMK group, only pretreated with Z-VAD-FMK) and a TMI group (no Z-VAD-FMK pretreatment, only the same TMI treatment) were set up. After incubation for 24 h, the expression of apoptosis-related proteins (PARP-1, pro-caspase 3, pro-caspase 8 and pro-caspase 9) and the expression of BCRP and P-gp proteins were detected by western blot (GAPDH was used as an internal reference, the primary antibodies PARP-1, pro-caspase 8, pro-caspase 9 and GAPDH were purchased from Wuhan Tri-Cell (Wuhan, China), the primary antibody pro-caspase3, secondary rabbit antibody and secondary mouse antibody were purchased from Cell Signaling Technology (Danvers, USA), the primary antibody GAPDH was diluted at 1:5000, the secondary rabbit antibody, secondary mouse antibody and primary antibody PARP-1 were diluted at 1:2000, and the other antibodies were diluted at 1:1000 before use) and the expression of BCRP and P-gp proteins.

[0069] The results are as follows Figure 6 and Figure 7 shown.

[0070] Compared with the TMI group, Z-VAD-FMK pretreatment significantly restored the expression of pro-caspase3, pro-caspase 8, and pro-caspase 9 proteins in K562 / ADR and K562 cells, and reduced the expression of cleaved-PARP-1 protein, indicating that TMI induces caspase-dependent apoptosis in K562 / ADR and K562 cells, leading to apoptosis.

[0071] Z-VAD-FMK pretreatment had no significant effect on the expression of BCRP and P-gp proteins in K562 / ADR cells, indicating that caspase-dependent apoptosis may not be the main reason for TMI to overcome the multidrug resistance of K562 / ADR cells.

[0072] Experimental Example 6 1. Take K562 / ADR and K562 cells in the logarithmic growth phase (seeding concentration is 5×10 5 cells / well) in a 6-well plate and cultured overnight in an incubator. The culture medium was discarded the next day, and 2 mL of 1640 medium containing the specified concentration of isoflavone TMI (0, 25, 50, and 100 μM for K562 / ADR cells and 0, 15, 30, and 60 μM for K562 cells) was added to each well. A positive control group (K562 / ADR: 50 μM doxorubicin, K562: 2 μM doxorubicin) was set up at the same time. After 24 h of co-incubation, the phosphorylation levels of ERK, p38, and JNK proteins in the MAPK pathway were detected by Western blotting (GAPDH was used as an internal reference, the primary antibodies JNK, ERK, and GAPDH were purchased from Wuhan Tri-Tek (Wuhan, China), the primary antibodies p-JNK, p-ERK, p38, p-p38, and the secondary antibodies rabbit and mouse were purchased from Cell Signaling Technology (Danvers, USA). The primary antibodies GAPDH and ERK were diluted at 1:5000, the primary antibody JNK was diluted at 1:8000, the secondary antibody was diluted at 1:2000, and the remaining antibodies were diluted at 1:1000 before use).

[0073] The results are as follows Figure 8 shown.

[0074] TMI had a significant effect on the phosphorylation of ERK, p38 and JNK proteins in the MAPK pathway of K562 / ADR and K562 cells.

[0075] 2. Take K562 / ADR and K562 cells in the logarithmic growth phase (seeding concentration is 5×10 5cells / well) in a 6-well plate and cultured overnight in an incubator. The next day, the culture medium was discarded, and 2 mL of 1640 medium containing JNK agonist anisomycin (purchased from MedChemExpress (China), 30 nM), ERK agonist TBHQ (purchased from MedChemExpress (China), K562 / ADR cells: 2.5 μM, K562 cells: 5 μM) or p38 inhibitor SB203580 (purchased from MedChemExpress (China), K562 / ADR cells: 5 μM, K562 cells: 3 μM) was added to each well for pretreatment for 4 hours. Then, a certain concentration of TMI (the final concentration for K562 / ADR cells was 60 μM, and the final concentration for K562 cells was 30 μM) was directly added to each well. After incubation for 48 hours, the survival rate of leukemia cells was detected by MTT method. At the same time, a blank control group (control group, K562 / ADR cells or K562 cells without any treatment), a negative control group (TBHQ group, only TBHQ pretreatment; anisomycin group, only anisomycin pretreatment; SB203580 group, only SB203580 pretreatment) and a TMI group (no TBHQ, anisomycin or SB203580 pretreatment, only the same TMI treatment) were set up.

[0076] The results are as follows Fig. 9 shown.

[0077] Anisomycin and SB203580 had no effect on the survival rate of leukemia cells, while TBHQ could increase the survival rate of leukemia cells. Since TBHQ is also an Nrf2 agonist, this suggests that the Nrf2-mediated signaling pathway or the MAPK / ERK signaling pathway may be the key factor for TMI to overcome the drug resistance of K562 / ADR cells.

[0078] Experimental Example 7 K562 / ADR and K562 cells in the logarithmic growth phase (seeding concentration of 5×10 5cells / well) in a 6-well plate and cultured overnight in an incubator. The culture medium was discarded the next day, and 2 mL of 1640 medium containing different concentrations of TMI was added to each well (the final concentrations for K562 / ADR cells were 0, 25, 50 and 100 μM, and the final concentrations for K562 cells were 0, 15, 30 and 60 μM). A positive control group (K562 / ADR: 50 μM doxorubicin, K562: 2 μM doxorubicin) was set up at the same time. After 24 hours of co-incubation, the expression of Nrf2, Keap-1 and HO-1 in the cells was detected by Western blotting (GAPDH was used as an internal reference, among which the primary antibodies Nrf2, Keap-1, HO-1 and GAPDH were purchased from Wuhan Tri-Tek (Wuhan, China), and the secondary rabbit antibody and secondary mouse antibody were purchased from Cell Signaling Technology (Danvers, USA), the primary antibodies GAPDH and Nrf2 were diluted 1:5000, the primary antibodies Keap-1 and HO-1 were diluted 1:4000, and the secondary antibodies were diluted 1:2000).

[0079] The results are as follows Fig.10 shown.

[0080] TMI can reduce the protein expression of Nrf2 and HO-1, and the expression of Keap-1 is also reduced. This indicates that TMI induces the degradation of Nrf2 protein in leukemia cells and weakens the inhibitory effect of Keap-1 on Nrf2.

[0081] Experimental Example 8 K562 / ADR and K562 cells in the logarithmic growth phase were inoculated in 6-well plates (inoculation concentration was 5×10 5 cells / well) and cultured in an incubator overnight. The culture medium was discarded the next day, and 2 mL of 1640 medium containing TBHQ (K562 / ADR cells: 2.5 μM, K562 cells: 5 μM) was added to each well for pretreatment for 4 h. Then, a certain concentration of TMI (the final concentration for K562 / ADR cells was 60 μM, and the final concentration for K562 cells was 30 μM) was directly added to each well. After incubation for 24 h, the expression of apoptosis-related proteins (pro-caspase 3, pro-caspase 8, and pro-caspase 9) and drug resistance-related proteins (BCRP and P-gp) were detected by western blot (GAPDH was used as an internal reference).

[0082] The results are as follows Fig.11 and Fig.12 shown.

[0083] Pretreatment with TBHQ significantly restored the expression of pro-caspase 3, pro-caspase 8, and pro-caspase 9 proteins in K562 / ADR and K562 cells.

[0084] TBHQ can restore the expression of drug resistance-related proteins (BCRP, P-gp) in K562 / ADR cells, indicating that TBHQ reduces the effect of TMI in overcoming drug resistance in K562 / ADR cells.

[0085] Experimental Example 9 Nrf2 is a transcription factor that regulates the expression of various detoxification and antioxidant genes and plays a key role in cell defense and oxidative stress. For normal cells, the activation of Nrf2 can maintain the redox homeostasis of cells. However, in tumor cells, Nrf2 can promote the expression of drug efflux transporters, detoxification enzymes and antioxidant defense mechanism-related genes, allowing tumor cells to escape the effects of drugs, leading to the emergence of treatment resistance.

[0086] 1. Take K562 / ADR and K562 cells in the logarithmic growth phase (seeding concentration is 5×10 5 cells / well) in a 6-well plate and cultured overnight in an incubator. The culture medium was discarded the next day, and 2 mL of 1640 medium containing pan-caspase inhibitor Z-VAD-FMK (K562 / ADR cells: 4 μM, K562 cells: 3 μM) or TBHQ (K562 / ADR cells: 2.5 μM, K562 cells: 5 μM) was added to each well for pretreatment for 4 h, and then a certain concentration of isoflavone TMI was directly added to each well (the final concentration for K562 / ADR cells was 60 μM, and the final concentration for K562 cells was 30 μM). At the same time, a blank control group (control group, K562 / ADR cells or K562 cells without any treatment), a negative control group (Z-VAD-FMK group only pretreated with Z-VAD-FMK or TBHQ group only pretreated with TBHQ) and a TMI group (no Z-VAD-FMK pretreatment or TBHQ pretreatment, only the same TMI treatment) were set up. After incubation for 24 h, the expression of Nrf2 in cells was detected by western blot (GAPDH was used as an internal reference).

[0087] The results are as follows Fig.13 shown.

[0088] Z-VAD-FMK pretreatment did not significantly affect the expression of Nrf2 protein in K562 / ADR cells treated with TMI, while TBHQ activated the expression of Nrf2 protein in K562 / ADR cells treated with TMI. This indicates that TMI's overcoming of K562 / ADR cell resistance is related to Nrf2 degradation.

[0089] 2. Collect K562 / ADR and K562 cells separately, wash with PBS, and then add TMI-containing PBS cell suspension (about 1x10 7 cells, K562 / ADR: 330 μM, K562: 130 μM) were treated at 37°C for 4 h; and DMSO treatment was set as a blank control group (Control group). Then the cells were heated at different temperatures (37°C, 40°C, 45°C, 50°C, 55°C, 60°C and 65°C) for 3 min, PMSF was added, and then briefly vortexed in ice bricks for 3 min. The cells were repeatedly frozen and thawed three times between liquid nitrogen and a 25°C thermal cycler. Then the samples containing cell lysate were centrifuged at 4°C and 13000 rpm for 20 min, the supernatant was collected, an appropriate amount of SDS-PAGE Protein Sampling Buffer (5X) was added, and it was heated at 100°C for 5 min. The protein expression of Nrf2 in K562 / ADR cells and K562 cells, as well as the protein expression of p-ERK and ERK in K562 / ADR cells were detected by western blot.

[0090] The results are as follows Fig.14 and Fig.15 shown.

[0091] Compared with the Control group, the degradation degree of Nrf2 protein in the TMI group decreased with the increase of temperature, and the thermal melting curve shifted to the right; while there was no significant difference in the protein expression levels of ERK and p-ERK between the two groups. This indicates that TMI directly binds to Nrf2 protein, making Nrf2 protein more stable.

[0092] In summary, TMI overcomes K562 / ADR resistance by directly targeting and inhibiting Nrf2 protein, ultimately promoting cell death.

[0093] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of an isoflavone compound having the following structural formula or a pharmaceutically acceptable salt thereof in the preparation of an anti-leukemia drug or in the inhibition of leukemia cell proliferation for non-disease treatment purposes: ； R1 is selected from H, or ; R2 is selected from OH or OCH3; R3 is selected from H or ; R4 is selected from H or OH.

2. The use according to claim 1, characterized in that: The structural formula of the compound is , or ; Preferably, the leukemia is chronic myeloid leukemia; Preferably, the leukemia cells are chronic myeloid leukemia cells.

3. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in any one of items A1) to A13): (Formula I); A1) Preparation of drugs to combat drug-resistant leukemia; A2) Inhibition of proliferation of drug-resistant leukemia cells for non-disease therapeutic purposes; A3) Preparation of leukemia resistance reversal agents; A4) Preparation of leukemia drug sensitizers; A5) Inhibition of the expression of drug-resistant proteins in drug-resistant leukemia cells for non-disease treatment purposes; A6) Preparation of products that promote or induce cell cycle arrest in leukemia cells or drug-resistant leukemia cells; A7) Promoting or inducing cell cycle arrest in leukemia cells or drug-resistant leukemia cells for non-disease treatment purposes; A8) Preparation of products that induce apoptosis in leukemia cells or drug-resistant leukemia cells; A9) Inducing apoptosis of leukemia cells or drug-resistant leukemia cells for non-disease treatment purposes; A10) Preparation of products that inhibit the Keap-1-Nrf2 / ARE signaling pathway; A11) Inhibition of Keap-1-Nrf2 / ARE signaling pathway for non-disease therapeutic purposes; A12) Preparation of products that inhibit MAPK signaling pathways; A13) Inhibition of MAPK signaling pathway in leukemia cells or drug-resistant leukemia cells for non-disease therapeutic purposes.

4. The use according to claim 3, characterized in that: The drug resistance includes resistance to doxorubicin; And / or, the leukemia drug includes doxorubicin.

5. The use according to claim 3, characterized in that: A5), wherein the drug resistance protein comprises at least one of BCRP and P-gp.

6. The use according to claim 3, characterized in that: In A6) or A7), the inducing cell cycle arrest in leukemia cells or drug-resistant leukemia cells is inducing leukemia cell cycle arrest in the G2 / M phase.

7. The use according to claim 3, characterized in that: A8) or A9), the inducing apoptosis of leukemia cells or drug-resistant leukemia cells is achieved by inhibiting or downregulating the expression of at least one of the apoptosis-related proteins pro-caspase 3, pro-caspase 8 and pro-caspase 9.

8. The use according to claim 3, characterized in that: A10) or A11), wherein the inhibition of the Keap-1-Nrf2 / ARE signaling pathway comprises inhibiting or down-regulating the expression of at least one of Nrf2, HO-1, and Keap-1.

9. The use according to claim 3, characterized in that: A12) or A13), wherein the inhibition of MAPK signaling pathway comprises inhibiting the phosphorylation of at least one of JNK, ERK, and p38.

10. The use according to claim 3, characterized in that: The product is selected from a kit, a drug or a reagent.

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