Pharmaceutical composition for inducing leukemia cell copper death and application thereof

Abstract

The invention discloses a pharmaceutical composition for inducing leukemia cell copper death and application thereof, and relates to the technical field of tumor cell apoptosis. The active ingredients of the pharmaceutical composition are oleanolic acid and illlisemom. It is found for the first time that oleanolic acid can significantly up-regulate expression of a copper death key regulatory factor FDX1, when oleanolic acid is used in combination with a copper ion carrier Ilismor, a large synergistic effect can be generated through an FDX1 / DLAT axis, leukemia cells are significantly induced to generate copper death, and therefore in-vitro proliferation and in-vivo tumor forming ability of the leukemia cells are effectively inhibited. The composition provides a new drug development strategy with a clear mechanism for treatment of leukemia, especially drug-resistant leukemia.

Description

Technical Field

[0001] This invention relates to the field of tumor cell apoptosis technology, and more specifically to a pharmaceutical composition that induces copper death in leukemia cells and its application. Background Technology

[0002] Copper death is a novel, copper-dependent programmed cell death mechanism discovered in recent years. Its core mechanism involves the abnormal aggregation of copper ions with lipid-acylated circulating TCA proteins (such as DLAT) during mitochondrial respiration, leading to protein toxicity stress and cell death.

[0003] Frixoprotein 1 (FDX1) is a key upstream regulator in the copper death pathway. It not only reduces copper ions but also regulates the lipid acylation modification of several key proteins, including DLAT. The FDX1 / DLAT functional axis is the core hub for regulating copper death.

[0004] Oleanolic acid induces apoptosis in many cancer cells, including acute myeloid leukemia, liver cancer cells, osteosarcoma cells, non-small cell lung cancer cells, breast cancer cells, gastric cancer cells, pancreatic cancer cells, prostate cancer cells, bladder cancer cells, and colorectal cancer cells. Oleanolic acid and its derivatives induce extrinsic and intrinsic apoptosis through multiple signaling pathways.

[0005] Currently, copper death inducers such as ilismo have anti-tumor effects, but their efficacy is limited when used as monotherapy and they may be accompanied by drug resistance issues. Therefore, developing anticancer drugs or combinations that can effectively enhance copper death, especially by synergistically enhancing FDX1 / DLAT axis function through a clear molecular mechanism, has important clinical significance and broad application prospects for the treatment of malignant tumors. Summary of the Invention

[0006] The purpose of this invention is to provide a pharmaceutical composition that can synergistically induce copper death in leukemia cells.

[0007] Another object of the present invention is to provide the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of leukemia.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention is the first to discover that the natural product oleanolic acid can significantly upregulate the expression of FDX1 in leukemia cells and synergize with the copper ion carrier ilismo, jointly enhancing the copper death effect through the FDX1 / DLAT axis, thereby effectively inhibiting the proliferation and growth of leukemia cells.

[0009] In a first aspect, the present invention provides a pharmaceutical composition comprising an effective dose of oleanolic acid and irismo.

[0010] In a preferred embodiment of the present invention, the molar mass ratio of oleanolic acid to illisoxim is 5:1.

[0011] Furthermore, the working concentration of oleanolic acid is 50 μM, and the working concentration of illismo is 10 nM.

[0012] In a second aspect, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for inducing copper death in tumor cells.

[0013] Furthermore, the pharmaceutical composition induces copper death by upregulating FDX1 expression and / or promoting DLAT esterification modification.

[0014] A third aspect of the invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of malignant tumors.

[0015] In a preferred embodiment of the present invention, the malignant tumor is leukemia.

[0016] Furthermore, the leukemia includes chronic myeloid leukemia and acute myeloid leukemia.

[0017] Furthermore, the drug inhibits the progression of leukemia by suppressing the proliferation of leukemia cells.

[0018] In a preferred embodiment of the present invention, the leukemia cells are any one of the following types: K562 cells, HL-60 cells, K562-Re imatinib-resistant cells, or HL-60-Re imatinib-resistant cells and their resistant cell lines.

[0019] Furthermore, the action of the pharmaceutical composition depends on the FDX1 / DLAT signaling pathway.

[0020] The mechanism of action of this invention is as follows: 1. Oleanolic acid alone can significantly upregulate the expression of FDX1 at the transcriptional and translational levels, thereby promoting lipid acylation of key TCA cycle proteins DLAT and DLST, creating favorable conditions for copper death.

[0021] 2. When oleanolic acid is used in combination with ilismo, the upregulated FDX1 of oleanolic acid significantly enhances the toxic effects induced by copper ions carried by ilismo. FDX1-mediated DLAT esterification modification undergoes abnormal aggregation in the presence of copper ions, leading to protein toxic stress and ultimately synergistically inducing copper death.

[0022] 3. In vitro and in vivo experiments confirmed that knocking down FDX1 or overexpressing DLAT could significantly reverse copper death and antitumor effects induced by oleanolic acid or the oleanolic acid / ilimismo combination, thus proving that the effect of this combination depends on the FDX1 / DLAT signaling axis.

[0023] As can be seen from the above technical solution, compared with the prior art, the present invention has the following advantages and effects: 1. The combination of oleanolic acid and ilimismo showed a significant synergistic anti-leukemia effect in both in vitro cell experiments and in vivo animal models, with effects far superior to single-drug treatment.

[0024] 2. This invention reveals for the first time the molecular mechanism by which oleanolic acid induces copper death by upregulating FDX1 and acting synergistically with ilismo on the FDX1 / DLAT axis, providing a solid theoretical basis for clinical combination therapy.

[0025] 3. This combined strategy also showed good results in drug-resistant leukemia cell lines, providing new ideas and directions for solving the problem of tumor drug resistance.

[0026] 4. Oleanolic acid is a natural product with good safety profile, and its combination with existing chemotherapy drugs may reduce their toxic side effects. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 Proliferation curves of K562 and HL-60 cells treated with oleanolic acid; Figure 1 A shows the proliferation curve of K562 cells, and 1B shows the proliferation curve of HL-60 cells; ES concentration was 10 nM, OA concentration was 50 µM, and treatment time was 24, 48, and 72 h. **P<0.01 vs Control; #P<0.05, ###P<0.001 vs ES treatment group (n=3).

[0029] Figure 2 Colony formation diagrams for K562 and HL-60 cells; ES concentration 10 nM, OA concentration 50 µM, **P<0.01 vs Control; #P<0.05, ###P<0.001 vs ES treatment group (n=3).

[0030] Figure 3EDU staining images of K562 and HL-60 cells; ES concentration was 10 nM, and OA concentration was 50 µM.

[0031] Figure 4 A statistical graph showing the positive rates of K562 and HL-60 cells; Figure 4 A is a statistical graph showing the positive rate of K562 cells. Figure 4 B is a statistical graph of HL-60 cell positivity rate; treatment time: 24 h; ES concentration: 10 nM, OA concentration: 50 µM, **P<0.01 vs Control; #P<0.05, ###P<0.001 vs ES treatment group (n=3).

[0032] Figure 5 Figure showing the effect of oleanolic acid in enhancing the inhibitory effect of ilimasmo on tumor growth in a mouse model of leukemia.

[0033] Figure 6 A bar chart illustrating the accumulation of copper death markers in leukemia cells mediated by oleanolic acid and ilismo. Figure 6 A shows the Cu2+ levels in K562 cells; Figure 6 B is a graph showing the Cu2+ levels in HL-60 cells; Figure 6 C represents the PA level in K562 cells; Figure 6 D represents the PA level map of HL-60 cells; Figure 6 E represents the α-KG level in K562 cells; Figure 6 F represents the α-KG level in HL-60 cells; ES concentration was 10 nM, OA concentration was 50 µM, and treatment time was 24 h; **P<0.01, ***P<0.001 vs Control; ###P<0.001 vs ES treatment group (n=3).

[0034] Figure 7 A bar chart showing the horizontal histogram of copper death markers in tumor cells of a mouse model of leukemia enhanced by oleanolic acid and ilimismo-mediated leukemia. Figure 7 A is a horizontal bar chart of copper. Figure 7 B is a bar chart showing pyruvate levels. Figure 7 C represents the bar chart of α-ketoglutarate levels; **P<0.01; ***P<0.001, ES or OA group vs Control, ES+OA group vs ES treatment group (n=5); **P<0.01 vs Control; ###P<0.001 vs ES treatment group (n=5).

[0035] Figure 8 Figure 1 shows the FDX1 mRNA level of oleanolic acid in K562 and HL-60 cells. Figure 8 A shows the FDX1 mRNA level in K562 cells. Figure 8 B shows the FDX1 mRNA level in HL-60 cells; OA concentration was 50 µM, treatment time was 24 h; *P<0.05, **P<0.01, ***P<0.001 vs Control group (n=3).

[0036] Figure 9 Figure 1 shows the FDX1 protein level of oleanolic acid in K562 and HL-60 cells. Figure 9 A shows the FDX1 protein level in K562 cells. Figure 9 B shows the FDX1 protein level in HL-60 cells; OA concentration was 50 µM, treatment time was 24 h; *P<0.05, **P<0.01, ***P<0.001 vs Control group (n=3).

[0037] Figure 10 The transfection efficiency of the FDX1 overexpression vector in K562 and HL-60 cells is shown in the figure. Figure 10 A shows the transfection efficiency in K562 cells. Figure 10 B shows the transfection efficiency in HL-60 cells.

[0038] Figure 11 The interference efficiency of siFDX1 in K562 and HL-60 cells is shown. Figure 11 A represents the interference efficiency in K562 cells. Figure 11 B shows the interference efficiency in HL-60 cells.

[0039] Figure 12 Plots showing the levels of DLAT and DLST esterification modification in K562 and HL-60 cells; Figure 12 A shows a comparison of Western blot images of DLAT and DLST esterified proteins in K562 and HL-60 cells; Figure 12 B shows the level of DLAT lipophilization modification in K562 cells; Figure 12 C represents the level of DLST esterification modification in K562 cells; Figure 12 D represents the level of DLAT lipoylation modification in HL-60 cells; Figure 12 E represents the level of DLST esterification modification in HL-60 cells; *P<0.05, **P<0.01 vs NC orsiNC group.

[0040] Figure 13 This is a graph showing the levels of DLAT and DLST esterification modification in K562 cells after treatment with oleanolic acid and siFDX1. Figure 13 A shows a comparison of Western blot images of DLAT and DLST esterified proteins in K562 cells after treatment. Figure 13 B shows the level of DLAT esterification modification in K562 cells after treatment. Figure 13 C represents the level of DLST esterification modification in K562 cells after treatment; Figure 13 D represents the expression level of FDX1 protein in K562 cells after treatment; OA concentration was 50 µM, treatment time was 24 h; ***P<0.001 vs Control; ###P<0.001 vs OA treatment group (n=3).

[0041] Figure 14 This is a graph showing the levels of DLAT and DLST esterification modification in HL-60 cells after treatment with oleanolic acid and siFDX1. Figure 14 A shows a comparison of Western blot images of DLAT and DLST esterified proteins in HL-60 cells after treatment. Figure 14 B shows the level of DLAT esterification modification in HL-60 cells after treatment; Figure 14 C represents the level of DLST esterification modification in HL-60 cells after treatment; Figure 14 D represents the expression level of FDX1 protein in K562 cells after treatment; OA concentration was 50 µM, treatment time was 24 h; ***P<0.001 vs Control; ###P<0.001 vs OA treatment group (n=3).

[0042] Figure 15 The proliferation curves of K562 and HL-60 cells after treatment with oleanolic acid for 24, 48, and 72 h are shown. Figure 15 A represents the proliferation curve of K562 cells after treatment. Figure 15 B represents the proliferation curve of HL-60 cells after treatment.

[0043] Figure 16 Colony formation diagrams for K562 and HL-60 cells; ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs OA treatment group (n=3).

[0044] Figure 17 EDU staining images of K562 and HL-60 cells.

[0045] Figure 18 A statistical chart showing the positive rate; Figure 18 A is a statistical graph showing the positive rate of K562 cells. Figure 18 B is a statistical graph of HL-60 cell positivity rate; OA concentration was 50 µM, treatment time was 24 h; ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs OA treatment group (n=3).

[0046] Figure 19 This diagram illustrates how oleanolic acid inhibits the in vivo growth of leukemia cells via the FDX1 / DLAT axis.

[0047] Figure 20 Bar graph showing oleanolic acid promoting copper death in leukemia cells via the FDX1 / DLAT axis; Figure 20 A represents K562 cell Cu2 + Horizontal map; Figure 20 B represents HL-60 cells with Cu2. + Horizontal map; Figure 20 C represents the PA level in K562 cells; Figure 20 D represents the PA level map of HL-60 cells; Figure 20 E represents the α-KG level in K562 cells; Figure 20 F represents the α-KG level in HL-60 cells; OA concentration was 50 µM, and treatment time was 24 h; **P<0.01, ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs OA treatment group (n=3).

[0048] Figure 21 Oleanolic acid promotes copper death in leukemia cells via the FDX1 / DLAT axis; Figure 21 A shows the Cu2+ level in tumor tissue; Figure 21 B shows the pyruvate level in tumor tissue; Figure 21 C represents the α-ketoglutarate level in tumor tissue; **P<0.01 vs Control; ##P<0.01 vs OA treatment group (n=5). Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Source of experimental materials: The cell lines used in the following examples are human chronic myeloid leukemia cell line K562, human acute promyelocytic leukemia cell line HL-60, and their corresponding drug-resistant cell lines K562-Re and HL-60-Re, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The compounds used were oleanolic acid (OA, MCE, purity ≥98%) and irismo (ES, MCE, purity ≥99%). OA was prepared as a 50 mM stock solution with DMSO, and ES was prepared as a 10 mM stock solution with DMSO. After aliquoting, the solutions were stored at -20°C. The experimental animals were 4-week-old male BALB / c nude mice, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0051] Experimental data analysis: The data in the following examples are the average of at least three independent experiments, analyzed using GraphPad Prism 7.0 and SPSS 19.0 software, and statistical significance is defined by standard deviation. Statistical differences between data were tested using the Student's test or one-way ANOVA. *P<0.05 or #P<0.05 was considered statistically significant.

[0052] Example 1: Oleanolic acid enhances the inhibitory effect of ilismo on the proliferation of leukemia cells.

[0053] Cloning experiments: Collect cells in the logarithmic growth phase and resuspend them. After appropriate dilution, count the cells and adjust the density to 1×10⁻⁶. 4 Cells / mL; blank control group (cells, culture medium), ES single-drug group (10 nM), OA single-drug group (50 μM), and OA (50 μM) + ES (10 nM) combined group were set up. Cell suspension was pre-warmed at 37 °C for later use; 5% agar was prepared, sterilized and heated until completely melted, cooled, and 1 mL of uncured agar was mixed with 9 mL of pre-warmed complete culture medium. Immediately, 2 mL was added to each well of a 12-well plate and allowed to solidify at room temperature as the bottom agar. 9.4 mL of pre-warmed cell suspension was quickly mixed with 0.6 mL of slightly cooled 5% agar solution, and 1 mL was added to each well. The plate was allowed to solidify at room temperature as the top agar. The culture plates were placed in a 37 °C incubator for 2 weeks; cell clones were observed and counted under a microscope, with more than 50 cells counted as one clone; crystal violet staining was used, and finally, photographs were taken.

[0054] EDU proliferation experiment: Drug administration: After centrifugation and resuspending, cells were seeded into 6-well plates. The following groups were established: a blank control group (cells, culture medium), an ES single-drug group (10 nM), an OA single-drug group (50 μM), and a combined OA (50 μM) + ES (10 nM) group. After the desired drug treatment, EDU working solution was added to a final concentration of 10 μM, and incubation continued for 2 h. Cells were centrifuged to remove culture medium and staining solution, resuspended in fixative, and fixed at room temperature for 15 min. The fixative was then removed by centrifugation, and the cells were washed three times with washing buffer for 3 min each time. After removing the washing buffer by centrifugation, 1 mL of permeabilization buffer was added to each well, and the cells were incubated at room temperature for 10 min. The permeabilization buffer was then removed by centrifugation, and the cells were washed twice with washing buffer for 3 min each time. Click reaction buffer, CuSO4, Azide 594, and Clickadditive solution were mixed according to the manufacturer's instructions. Cells were centrifuged to remove the washing buffer, and 500 μL of the above reaction solution was added to each well. The cells were incubated at room temperature in the dark for 30 min. The reaction solution was removed, and the cells were washed three times with washing buffer for 3 min each time. 1 mL of the above reaction solution was added to each well. Hoechst 33342 was incubated at room temperature in the dark for 10 min; washed three times with washing solution for 3 min each time; and finally observed and photographed under a fluorescence microscope.

[0055] Tumor formation experiment in nude mice: Four-week-old male BALB / c nude mice (approximately 18±2 g) were purchased and acclimatized to a clean environment for one week before experiments were conducted. Cells were collected and counted after specific transfection, and resuspended in serum-free medium to a concentration of 1×10⁶ cells / mL. 7 / 100 μL, keep on ice for later use. Use a sterile syringe to draw 100 μL of cell suspension and inject it into the back of the mouse. Starting on day 5 after cell inoculation, tumor size was measured and volume calculated every 5 days: Volume (mm3) = (width2 × length) / 2. Starting on day 5, mice were treated with the following medications: Ilisimor (ES) was administered subcutaneously at a concentration of 10 mg / kg body weight (dissolved in physiological saline) every 3 days; Oleanolic acid (OA) was administered by gavage at a concentration of 20 mg / kg body weight (dissolved in physiological saline) every 2 days. In the combined treatment group, Ilisimor was administered subcutaneously at a concentration of 10 mg / kg body weight (dissolved in physiological saline) every 3 days, and Oleanolic acid was administered by gavage at a concentration of 20 mg / kg body weight (dissolved in physiological saline) every 2 days. Control group mice were injected with an equal volume of physiological saline. Thirty days after cell inoculation, mice were euthanized by mortise and tenoning, and tumors were collected, photographed, and weighed. The tumors were then divided into two parts: one part was fixed in 4% paraformaldehyde, and the other part was stored at -80 °C for protein and RNA expression analysis.

[0056] Test results are as per the instruction manual. Figure 1-4As shown in the figure, compared with the control group, ilisimo treatment significantly reduced the number of EDU-positive K562 and HL-60 leukemia cells; the combined treatment group showed significantly stronger inhibition of cell viability, blockade of colony formation, and inhibition of cell proliferation than the individual single-drug groups, indicating that ES can inhibit the proliferation of leukemia cells in vitro; oleanolic acid enhanced the inhibitory effect of the copper death inducer ilisimo on the proliferation of leukemia cells.

[0057] As attached Figure 5 As shown, the combined treatment of OA and ES significantly inhibited tumor growth in the K562 nude mouse model compared to each single-drug group, indicating that the two have a synergistic effect.

[0058] Example 2: Oleanolic acid enhances the accumulation of copper death markers in leukemia cells mediated by ilismo.

[0059] Cu level detection: Homogenize fresh tissue with double-distilled water, centrifuge at 5000 rpm for 5 min, and transfer the supernatant to EP tubes for analysis; Cell sample preparation: collect approximately 2 × 10⁶ cells / mL. 6 Cells were homogenized with 0.15 mL of double-distilled water, centrifuged at 5000 rpm for 5 min, and the supernatant was transferred to EP tubes for testing. Standard solutions were 0 µM, 0.5 µM, 1 µM, 2 µM, 2.5 µM, 3 µM, 4 µM, and 5 µM. 100 μL of standard solution or test sample was added to the corresponding wells of a 96-well plate, with three replicates per group. 50 μL of chromogenic reagent was added to each well, covered with a protective membrane, and incubated at 37°C for 5 min. The OD value of each well was measured at 580 nm using a microplate reader. A standard curve was plotted based on the standard measurements, and the Cu concentration in each sample was calculated. The protein content (gprot / L) of each group of samples was determined using a BCA kit. The copper ion content in the cells was calculated.

[0060] Detection of pyruvic acid (PA) content: Collect approximately 5 × 10 6Cells were subjected to sonication for 5 min with 1 mL of Extraction Buffer, followed by 30 min on ice and centrifugation at 4000 g for 10 min. The supernatant was collected and placed in an EP tube and kept on ice. Approximately 100 mg of fresh tissue was weighed and placed in a grinding rod with 1 mL of Extraction Buffer. The mixture was ground and centrifuged at 4000 g for 10 min on ice. The supernatant was collected and placed in an EP tube and kept on ice. 75 μL of standard or sample was added to each well of a 96-well plate, and an equal volume of Extraction Buffer was added to each blank well. Then, 25 μL of reaction solution A was added to each well, mixed, and allowed to stand at room temperature for 2 min. Reaction solution B was added to each well, mixed, and the absorbance was measured at 520 nm. A standard curve was plotted and the PA content in each sample was calculated.

[0061] Detection of α-ketoglutarate (α-KG): α-KG standards of 1 nmol / μL and 0.1 nmol / μL were prepared and added to each well of a 96-well plate to obtain the following concentration gradient: 0 nmol / well, 0.2 nmol / well, 0.4 nmol / well, 0.6 nmol / well, 0.8 nmol / well, and 1 nmol / well, with a final solution volume of 50 μL in each well. Cells (approximately 2 × 10⁻⁶ cells) were collected. 6(Each group) Wash once with pre-chilled PBS, resuspend cells in 500 μL of pre-chilled α-KG assay buffer, and rapidly and repeatedly pipette to lyse the cells; centrifuge the sample at 13000 rpm for 3 min at 4 °C to remove all insoluble matter; collect the supernatant in a clean EP tube and store on ice for later use. Weigh approximately 20 mg of fresh tissue, wash once with pre-chilled PBS, place in 500 μL of pre-chilled α-KG assay buffer, and homogenize using a homogenizer; collect the homogenate and centrifuge at 13000 rpm for 3 min at 4 °C, collect the supernatant in a new EP tube, and store on ice for later use. Add pre-cooled PCA to the prepared sample and vortex to mix. Incubate on ice for 5 min. Centrifuge at 13000 rpm for 2 min and transfer the supernatant to a new EP tube. Add pre-cooled 2 mol / L KOH to precipitate excess PCA. Centrifuge at 13000 rpm for 15 min and collect the supernatant to a new tube and place on ice. Add the sample to the detection wells, with a total volume of 50 μL. Prepare the reaction mixture by calculating and preparing sufficient reaction solution according to the number of standards and samples. Add the reaction solution to the standard and sample wells, with a volume of 50 μL per well. Add 50 μL of background reaction solution to the sample background wells. React at 37 °C in the dark for 30 min. Detect the fluorescence value using a microplate reader (excitation / emission = 535 / 587 nm). Plot a standard curve and calculate the α-KG content in each group of samples.

[0062] Experiments show that, as shown in the attached Figure 6 As shown, in K562 and HL-60 cells, the combined use of oleanolic acid and ilipin not only synergistically promoted intracellular copper accumulation but also more effectively downregulated pyruvate and α-ketoglutarate levels. This collectively confirms that oleanolic acid is an effective sensitizer for ilipin-induced copper death. In the K-562 cell tumorigenesis experiment in nude mice, as shown in the attached figure… Figure 7 As shown, compared with the control group, treatment with oleanolic acid or ilipin alone increased copper accumulation in tumor tissue; compared with treatment alone, combined treatment with oleanolic acid and ilipin enhanced the promoting effect of ilipin on copper accumulation (see attached diagram). Figure 7 A). In tumor tissues, compared with the control group, treatment with oleanolic acid or ilisimo alone significantly downregulated the levels of pyruvate and α-ketoglutarate; compared with treatment alone, combined treatment with oleanolic acid and ilisimo enhanced the inhibitory effect of ilisimo on the levels of pyruvate and α-ketoglutarate (see appendix). Figure 7 (B and C). These results indicate that oleanolic acid enhances the levels of copper death markers in tumors in leukemia model mice mediated by ilisimo.

[0063] Example 3: Oleanolic acid promotes FDX1 expression in leukemia cells. RNA extraction and RT-qPCR: K562 and HL-60 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated at 37°C in a 5% CO2 incubator. Experimental group: cells were treated with 50 μM OA for 24 hours; control group: an equal volume of DMSO solvent was added. After cell collection, the medium was discarded, and the cells were washed twice with PBS. 1 mL of Trizol was added to lyse the cells, and the cells were incubated on ice for 10 minutes. 200 μL of chloroform was added, and the cells were centrifuged (12,000 rpm, 10 minutes). The supernatant was collected, and an equal volume of isopropanol was added to precipitate RNA. After centrifugation, the RNA was washed and dissolved in DEPC water to complete RNA extraction. 1 μg of RNA was used for reverse transcription according to the reverse transcription kit instructions: OligodT and dNTP mixture was added, and the reaction was carried out at 70°C for 5 minutes; RT buffer and RNasin were added, and the reaction was carried out at 42°C for 30 minutes, followed by inactivation at 85°C for 2 minutes to complete cDNA synthesis.

[0064] The qPCR reaction system used cDNA template, with a volume of 1 μg. The upstream primer for FDX1 is (5'-TTCAACCTGTCACCTCATCTTTG-3', as shown in SEQ ID No. 1), and the amount used is 0.5 μL; The downstream primer for FDX1 is (5'-TGCCAGATCGAGCATGTCATT-3', as shown in SEQ ID No. 2), and the amount used is 0.5 μL; The volume of SYBR Green premixed solution was 10 μL, and ddH2O was added to bring the volume to 20 μL. The above system was placed in a PCR instrument, and Ct values ​​were obtained using 2-... The Ct formula is used for calculation.

[0065] Protein extraction and Western blotting: Cells were washed with PBS, then lysed with RIPA lysis buffer (containing protease inhibitors) on ice for 20 minutes; centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant was collected; protein concentration was determined by BCA method, and 5× loading buffer was added, followed by denaturation at 100°C for 5 minutes. A 10% separating gel and a 5% stacking gel were prepared; 35 μg of protein was loaded into each well, and electrophoresis was performed at a constant current of 60 mA until bromophenol blue reached the bottom of the gel; the gel was transferred to a PVDF membrane (200 mA, 1 hour) using transfer buffer containing 20% ​​methanol. The membrane was blocked with rapid blocking buffer for 20 minutes; FDX1 primary antibody (1:1000 dilution) was added, and the membrane was incubated overnight at 4°C; the cells were washed three times with PBST, and HRP-labeled secondary antibody (1:5000 dilution) was added, and the membrane was incubated at room temperature for 1 hour; after incubation with ECL chemiluminescence buffer, band images were acquired using a gel imaging system; the internal control protein was β-actin.

[0066] The results of RT-qPCR and Western blot experiments showed, as shown in the attached... Figure 8 With appendix Figure 9 As shown, in K562 and HL-60 leukemia cells, oleanolic acid treatment significantly increased FDX1 expression at both mRNA and protein levels compared to the control group. These results indicate that oleanolic acid promotes FDX1 expression in leukemia cells.

[0067] Example 4: FDX1 mediates lipophilization of DLAT and DLST, key proteins in the TCA cycle, in leukemia cells. Cell transfection: Plasmid transformation and extraction: The FDX1 overexpression vector was transformed into E. coli Trans5α competent cells and plated on LB solid medium containing ampicillin, and cultured at 37°C for 12-16 hours. Single colonies were picked and inoculated into LB liquid medium, and cultured at 37°C with shaking for 12 hours. Plasmids were extracted using a plasmid miniprep kit, and after concentration determination, stored at -20°C. K562 and HL-60 cells in logarithmic growth phase were used to extract plasmids at a density of 10⁻⁶ cells per well. 6 Cells were seeded in 6-well plates; transfection complexes were prepared as follows: Solution A: 4 μg plasmid (pcDNA-FDX1 or empty vector) or 200 pmol siRNA (siFDX1-2 or siNC) was mixed with 50 μL Opti-MEM; Solution B: 8 μL Lipofectamine 3000 was mixed with 50 μL Opti-MEM and incubated at room temperature for 5 minutes; Solution A and Solution B were mixed and incubated at room temperature for 20 minutes, then cell culture medium was added; 6 hours after transfection, the medium was replaced with complete medium and cultured for another 48 hours for subsequent experiments.

[0068] Protein sample preparation and Western blotting: Collect transfected cells, wash with PBS, add RIPA lysis buffer (containing protease inhibitor), and lyse on ice for 20 minutes; centrifuge at 4°C (12,000 rpm, 15 minutes), collect the supernatant; determine protein concentration using the BCA method, add 5× loading buffer, and heat at 100°C for 5 minutes for denaturation. Prepare 10% separating gel and 5% stacking gel; load 30 μg of protein into each well, electrophoresis at a constant current of 60 mA until bromophenol blue reaches the bottom of the gel; transfer to a PVDF membrane (200 mA, 1 hour), using transfer buffer containing 20% ​​methanol. The membrane was blocked with rapid blocking buffer for 20 minutes; primary antibodies FDX1 (1:1000), DLAT (1:1000), DLST (1:1000), Lip-DLAT (1:800), and Lip-DLST (1:800) were added, and the membrane was incubated overnight at 4°C; the membrane was washed three times with PBST, and HRP-labeled secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 hour; after incubation with ECL chemiluminescence buffer, band images were acquired using a gel imaging system; internal control protein: β-actin (1:5000). The groups were: empty vector control group (NC): transfected with pcDNA 3.1+ empty vector; FDX1 overexpression group (OE-FDX1): transfected with pcDNA-FDX1; negative interference control group (siNC): transfected with siNC; FDX1 interference group (siFDX1-2): transfected with siFDX1-2.

[0069] FDX1 Overexpression and Interference Efficiency Verification: Western blot results showed that, compared with the NC group, FDX1 protein expression was significantly upregulated in the OE-FDX1 group (**P<0.01); compared with the siNC group, FDX1 protein expression was significantly inhibited in the siFDX1-2 group (*P<0.001), as shown in the attached figure. Figure 10 and attached Figure 11 As attached Figure 12 In K562 and HL-60 cells, the total protein expression of DLAT and DLST in the OE-FDX1 group was not significantly different, but the band intensity of Lip-DLAT and Lip-DLST was significantly increased; in the siFDX1-2 group, the band intensity of Lip-DLAT and Lip-DLST was significantly weakened; quantitative analysis showed that the level of lipid acylation modification was positively correlated with FDX1 expression (P<0.05, *P<0.01).

[0070] This embodiment, through genetic methods combined with the detection of lipid acylation modification-specific antibodies, confirms that FDX1 directly mediates the lipid acylation modification of the key TCA cycle proteins DLAT and DLST in leukemia cells without affecting their total protein expression levels. This result provides mechanistic evidence for FDX1's role as a key regulator of copper death.

[0071] Example 5: Oleanolic acid promotes the lipid acylation modification of DLAT and DLST in leukemia cells via FDX1.

[0072] For usage instructions, please refer to Example 4.

[0073] The Western blot results are shown in the attached figure. Figure 13 and attached Figure 14 As shown, in K562 and HL-60 leukemia cells, compared with the control group, oleanolic acid upregulated FDX1 expression, had no effect on DLAT and DLST protein expression, but significantly increased their lipid acylation modification levels (Lip-DLAT and Lip-DLST). Knockdown of FDX1 blocked the promoting effect of oleanolic acid on FDX1 expression and the lipid acylation modification levels of DLAT and DLST.

[0074] Example 6: Oleanolic acid inhibits the in vitro proliferation of leukemia cells via the FDX1 / DLAT axis. The experimental method is the same as in Example 1.

[0075] The results of CCK-8 are shown in the attached figure. Figure 15 As shown, compared with the control group, oleanolic acid treatment significantly inhibited the activity of K562 and HL-60 leukemia cells, while overexpression of DLAT or interference with FDX1 blocked the inhibitory effect of oleanolic acid on leukemia cell activity. Colony formation assays showed, as shown in the attached... Figure 16 As shown, compared with the control group, oleanolic acid treatment significantly inhibited the clonogenic ability of K562 and HL-60 leukemia cells, while overexpression of DLAT or interference with FDX1 weakened this inhibitory effect. (See attached image) Figure 17 and attached Figure 18 As shown, compared with the control group, oleanolic acid treatment significantly reduced the number of EDU-positive K562 and HL-60 leukemia cells, while overexpression of DLAT or interference with FDX1 significantly blocked the effect of oleanolic acid. These results indicate that oleanolic acid inhibits leukemia cell proliferation through the FDX1 / DLAT cascade.

[0076] Example 7: Oleanolic acid inhibits the in vivo growth of leukemia cells via the FDX1 / DLAT axis. Tumorigenesis experiments were conducted in nude mice using K562 cells, DLAT-overexpressing cells, and FDX1-knockdown cells. Mice were divided into four groups: K562 control tumor (Control), K562 tumor OA treatment group, DLAT-overexpressing (DLAT) tumor + oleanolic acid treatment group, and FDX1-interference (siFDX1) tumor + OA treatment group.

[0077] The results are attached. Figure 19 The results showed that oleanolic acid treatment significantly inhibited tumor size, volume, and weight compared to the control group. These results indicate that oleanolic acid inhibits the in vivo growth of leukemia cells through the FDX1 / DLAT axis.

[0078] Example 8: Oleanolic acid promotes copper death in leukemia cells via the FDX1 / DLAT axis.

[0079] The experimental method is the same as in Example 2.

[0080] The results are attached. Figure 20 As shown, in K562 and HL-60 leukemia cells, oleanolic acid treatment significantly upregulated intracellular copper levels compared to the control group (see attached figure). Figure 20 (A and B). Meanwhile, in K562 and HL-60 leukemia cells, the levels of the copper death-related markers pyruvate and α-ketoglutarate were inhibited by oleanolic acid (see appendix). Figure 20 CF). Overexpression of DLAT or interference with FDX1 significantly blocked the regulatory effects of oleanolic acid on copper, pyruvate, and α-ketoglutarate levels (see appendix). Figure 20 These results indicate that oleanolic acid promotes copper death in leukemia cells via the FDX1 / DLAT axis.

[0081] In the K562 cell tumorigenesis experiment in nude mice, mice were divided into K562 control tumor (Control), K562 tumor OA treatment group, DLAT overexpression (DLAT) tumor + OA treatment group, and FDX1 interference (siFDX1) tumor + OA treatment group. siRNA was administered via tail vein injection at a concentration of 5 µmol / kg body weight (dissolved in physiological saline) every 3 days.

[0082] The results are attached. Figure 21 As shown, in tumor tissues, compared with the control group, oleanolic acid treatment significantly upregulated intracellular copper levels and inhibited the levels of pyruvate and α-ketoglutarate (see attached diagram). Figure 21 AC). Overexpression of DLAT or interference with FDX1 can significantly block the effects of oleanolic acid in animals (see appendix). Figure 21 These results indicate that oleanolic acid promotes copper death in leukemia cells via the FDX1 / DLAT axis.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A pharmaceutical composition for inducing copper death in leukemia cells, characterized in that, The pharmaceutical composition includes oleanolic acid and ilimismo.

2. The pharmaceutical composition according to claim 1, characterized in that, The molar mass ratio of oleanolic acid to illisoxim is 5:

1.

3. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition induces copper death by upregulating FDX1 expression and / or promoting DLAT esterification modification.

4. The use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating leukemia, characterized in that, The leukemias mentioned include chronic myeloid leukemia and acute myeloid leukemia.

5. The application according to claim 4, characterized in that, The drug inhibits the progression of leukemia by suppressing the proliferation of leukemia cells.

6. The application according to claim 4, characterized in that, The leukemia cells are any one of K562 cells, HL-60 cells, K562-Re imatinib-resistant cells, or HL-60-Re imatinib-resistant cells.

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