Application of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of drugs for treating leukemia

Abstract

The present invention discloses the use of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating leukemia. Experimental testing shows that 1,3-dicyclohexylbarbituric acid can increase the sensitivity of leukemia to dexamethasone. In vitro testing using the glucocorticoid-resistant acute T lymphocytic leukemia cell line Jurkat reveals that 1,3-dicyclohexylbarbituric acid and dexamethasone have a significant synergistic effect on Jurkat cells, and 1,3-dicyclohexylbarbituric acid can enhance the sensitivity of Jurkat cells to dexamethasone in a concentration- and time-dependent manner. The combination of 1,3-dicyclohexylbarbituric acid and dexamethasone can induce G1 arrest in Jurkat cells and regulate GR / BIM and JAK2 / STAT3 signal transduction to activate the caspase cascade reaction, thereby inducing Jurkat cell apoptosis. The 1,3-dicyclohexylbarbituric acid of the present invention has the potential to become a dexamethasone sensitizer, and also provides a research basis for the development of drugs to overcome glucocorticoid resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and medicine, and particularly relates to the application of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating leukemia. Background Art

[0002] Leukemia is a blood system malignancy caused by the abnormal proliferation of hematopoietic stem cells. It mainly includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). Among them, ALL is the most common type of leukemia in children, accounting for 80% of childhood leukemia cases.

[0003] Acute T-lymphoblastic leukemia (T-ALL) is an aggressive disease, accounting for 10% to 15% of childhood ALL cases. Standard treatment for T-ALL is based on glucocorticoids (GCs) combined with a variety of chemotherapeutic agents, such as vincristine, asparaginase, cytarabine, 6-mercaptopurine, and methotrexate. Despite the wide range of chemotherapeutic agents, glucocorticoids remain the first-line treatment. Early response to glucocorticoids is an important prognostic indicator for patients with T-ALL. However, glucocorticoid resistance is frequently observed in patients with relapsed or refractory T-ALL and is one of the main causes of relapse. Overcoming glucocorticoid resistance is crucial for improving the long-term survival of patients with T-ALL. Therefore, the development of a drug that can overcome glucocorticoid resistance for the treatment of T-ALL is urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of 1,3-dicyclohexylbarbituric acid (DL504) in combination with a glucocorticoid drug, dexamethasone (DEX), in the preparation of a drug for treating leukemia.

[0005] The above-mentioned 1,3-dicyclohexylbarbituric acid can enhance the sensitivity of Jurkat cells to dexamethasone. Furthermore, the present invention provides the use of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating acute T lymphocytic leukemia.

[0006] The leukemia treatment drug of the present invention is prepared by combining 1,3-dicyclohexylbarbituric acid and dexamethasone, and adding pharmaceutically acceptable excipients or auxiliary components to form a pharmaceutical preparation; wherein the molar ratio of 1,3-dicyclohexylbarbituric acid to dexamethasone in the pharmaceutical preparation is 10-40:100.

[0007] Furthermore, preferably, the molar ratio of 1,3-dicyclohexylbarbituric acid to dexamethasone in the pharmaceutical preparation is 20-25:100.

[0008] The pharmaceutical preparation of the present invention is any one of oral liquid, injection, tablet, capsule and pill.

[0009] The beneficial effects of the present invention are as follows:

[0010] The present invention addresses the problem of drug resistance to dexamethasone in the clinical treatment of acute T-lymphocytic leukemia, which can lead to poor treatment efficacy or relapse after treatment. Jurkat cells, a drug-resistant strain of human acute T-lymphocytic leukemia cells, were used as an in vitro research model. Experimental results showed that 1,3-dicyclohexylbarbituric acid alone had little effect on acute T-lymphocytic leukemia cells, with an apoptosis rate of (15.80±1.01)%. However, when combined with dexamethasone, 1,3-dicyclohexylbarbituric acid significantly induced cell apoptosis, with an apoptosis rate of (62.62±2.04)%, demonstrating a significant synergistic effect. The present invention combines 1,3-dicyclohexylbarbituric acid with dexamethasone to enhance the sensitivity of Jurkat cells to dexamethasone and reduce the amount of dexamethasone used. Both flow cytometry and protein immunoblotting results showed that 1,3-dicyclohexylbarbituric acid combined with dexamethasone induced apoptosis in Jurkat cells by inducing G1 arrest and regulating GR / BIM and JAK2 / STAT3 signaling to activate the caspase cascade. This indicates that 1,3-dicyclohexylbarbituric acid has the potential to be a dexamethasone sensitizer, providing a research basis for the development of drugs to overcome glucocorticoid resistance, and further providing a theoretical basis for clinical research on overcoming dexamethasone resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The figures are the verification results of Jurkat cells' resistance to DEX; (A) is the effect of different concentrations of DEX on the proliferation of Jurkat and CEM-C7H2 cells; (B, C) are the effect of 100 μM DEX on Jurkat cell apoptosis and statistical analysis of cell apoptosis, respectively; (D, E) are the effect of 1 μM DEX on CEM-C7H2 cell apoptosis and statistical analysis of cell apoptosis, respectively.

[0012] Figure 2Schematic diagram of the inhibition of Jurkat cell viability by different concentrations of DL504 combined with DEX; (A) is the viability of Jurkat cells when treated with DEX (100 μM) alone, DL504 (20 μM) alone, and the combination of the two for 24, 48, 72, and 96 h; (B, C, D, E) are the viability of Jurkat cells when treated with 10, 20, and 40 μM DL504 alone and in combination with DEX (100 μM) for 24, 48, 72, and 96 h, respectively.

[0013] Figure 3 The effect of DL504 combined with DEX on Jurkat cell morphology.

[0014] Figure 4 The effects of DL504 combined with DEX treatment on Jurkat cell cycle and cycle-related proteins for 24 hours; (A) is the flow cytometric graph of Jurkat cell cycle; (B) is the statistical graph of Jurkat cell cycle distribution.

[0015] Figure 5 The figure shows the effects of DL504 combined with DEX on Jurkat cell apoptosis and apoptosis-related proteins after 48 hours of treatment; (A) is the flow cytometry graph of Jurkat cell apoptosis, and (B) is the statistical graph of Jurkat cell apoptosis rate.

[0016] Figure 6 The effects of DL504 combined with DEX on Jurkat cell cycle-related proteins after 24 hours of treatment of Jurkat cells; (A) is the expression of Jurkat cell cycle-related proteins (GAPDH is the internal reference); (B) is the quantitative statistical chart of Jurkat cell cycle regulatory proteins.

[0017] Figure 7 The effects of DL504 combined with DEX on Jurkat cell apoptosis-related proteins after 24 hours of treatment; (A) is the expression of Jurkat cell apoptosis-related proteins (GAPDH is the internal reference); (B) is the quantitative statistical chart of Jurkat cell apoptosis regulatory proteins.

[0018] Figure 8 The effect of DL504 combined with DEX on the GR / BIM pathway proteins in Jurkat cells after 24 hours of treatment; (A) is the expression of GR and p-GR Ser211(B) is the expression level of GR protein (GAPDH is the internal reference); (B) is the expression level of GR protein in the cytoplasm and nuclear proteins (GAPDH is the cytoplasmic internal reference, Lamin B1 is the nuclear internal reference); (C) is the statistical graph of GR in the nucleus and cytoplasm; (D) is the expression level of BIM protein (GAPDH is the internal reference); (E) is the quantitative statistical graph of BIM protein.

[0019] Figure 9 The effects of DL504 combined with DEX on the JAK2 / STAT3 pathway proteins in Jurkat cells after 24 hours of treatment; (A) is the expression of JAK2, p-JAK2, STAT3 and p-STAT3 proteins (GAPDH is the internal reference); (B) is the quantitative statistical graph of JAK2, p-JAK2, STAT3 and p-STAT3. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0021] In a specific embodiment of the present invention, the Jurkat cells are a glucocorticoid-resistant human acute T lymphoblastic leukemia cell line, and the CEM-C7H2 cells are a glucocorticoid-sensitive human acute T lymphoblastic leukemia cell line, both of which are stored in the Guizhou Natural Products Research Center.

[0022] In a specific embodiment of the present invention, the chemical formula of the 1,3-dicyclohexylbarbituric acid is as shown in Formula I:

[0023]

[0024]

[0025] The present invention conducted DEX drug sensitivity experiments on acute T lymphocytic leukemia cells CEM-C7H2 cells (sensitive strain) and Jurkat cells (resistant strain). Figure 1 It can be seen that DEX can significantly inhibit the proliferation of CEM-C7H2 cells, but has no significant effect on the proliferation of Jurkat cells. In addition, 1 μM DEX can significantly induce apoptosis in CEM-C7H2 cells, but 100 μM DEX has no significant effect on apoptosis in Jurkat cells, indicating that the Jurkat cells used in the examples of the present invention are resistant to DEX.

[0026] Example 1

[0027] Effect of 1,3-dicyclohexylbarbituric acid on overcoming dexamethasone resistance in Jurkat cells

[0028] 1. Experimental Materials

[0029] Cell lines: Jurkat cells (drug-resistant strain) and CEM-C7H2 cells (sensitive strain) were stored at the Guizhou Natural Products Research Center. Jurkat and CEM-C7H2 cells were cultured in RPMI 1640 medium supplemented with 5% FBS in a 37°C incubator with 4.5% CO2.

[0030] Experimental reagents: Fetal bovine serum (FBS) was purchased from BI; RPMI 1640 medium was purchased from Gibco; Annexin V-FITC apoptosis detection kit was purchased from BD; 1,3-dicyclohexylbarbituric acid (DL504) was provided by Guizhou Natural Products Research Center; dexamethasone (DEX) standard, propidium iodide (PI), thiazolyl blue (MTT), dimethylsulfoxide (DMSO), Trition X-100, PMSF, BCA protein concentration detection kit and bovine serum albumin (BSA) were all purchased from Beijing Solebao Company; RNase A was purchased from Dalian Bao Biotechnology Company; IP cell lysate, SDS-PAGE gel rapid preparation kit and SDS-PAGE protein loading buffer (5X) were all purchased from Shanghai Biyuntian Biotechnology Company; skim milk powder was purchased from Yili Co., Ltd.; primary antibody GAPDH was purchased from Affinity Company; c-Myc, CDK2 and Cyclin E1 was purchased from Abcam; Bcl-XL, Bcl-2, Caspase 3, Caspase 9 and PARP were purchased from CST.

[0031] Preparation of 5 μg / mL MTT solution: Weigh 0.2 g MTT, dissolve in 40 mL 1× PBS, filter sterilize with a 0.22 μm filter, and store at 4°C in the dark.

[0032] The drugs in the DEX group, DL504 group and combination group (DL504+DEX) were prepared into solutions of the required concentrations using DMSO as the solvent.

[0033] 2. Experimental methods

[0034] (1) Effects of DL504 combined with DEX on the growth of Jurkat cells

[0035] Jurkat cells growing exponentially were taken out and plated at 1×10 5The cells were seeded at a density of 100 μg / mL in a 96-well plate, and 90 μL of cell suspension was added to each well. They were set up as DMSO group (blank control), DEX group, DL504 group and combination group (DL504+DEX), with 4 replicate wells in each group. The DEX concentration in the DEX group was 100 μM, and the DL504 concentration in the DL504 group was divided into 10, 20 and 40 μM. The combination group was composed of different concentrations of DL504 combined with 100 μM DEX. Jurkat cells were treated for 24, 48, 72 and 96 hours, respectively. 10 μL of 5 μg / mL MTT solution was added for routine incubation for 4 hours, and 100 μL / well of the triple solution was added and incubated overnight. The OD value of each well was measured at a wavelength of 570 nm using an enzyme reader. The cell survival rate was calculated according to formula (1) and obtained Figure 2 CompuSyn software was used to calculate the combination index (CI) of the interaction between DL504 and DEX. CI = 1 indicates an additive effect; CI > 1 indicates an antagonistic effect; CI < 1 indicates a synergistic effect, as shown in Table 1.

[0036]

[0037] Table 1 CI values ​​of Jurkat cells treated with different concentrations of DL504 combined with DEX for 48 and 72 h

[0038]

[0039] Depend on Figure 2 Compared with DMSO, DL504, or DEX alone, DL504 combined with DEX significantly inhibited Jurkat cell proliferation, with cell viability rates at 24, 48, 72, and 96 hours reaching (94.12±3.80)%, (61.03±9.34)%, (24.64±3.45)%, and (17.55±2.15)%, respectively. Furthermore, different concentrations of DL504 (10, 20, and 40 μM) alone or in combination with DEX (100 μM) were tested for different treatment times. The results showed that different concentrations of DL504 combined with DEX significantly inhibited Jurkat cell proliferation at 48, 72, and 96 hours. This suggests that DL504 enhances Jurkat cell sensitivity to DEX in a concentration- and time-dependent manner. The results in Table 1 show that the CI values ​​of Jurkat cells treated with different concentrations of DL504 (10, 20 and 40 μM) combined with DEX (100 μM) for 48 and 72 h were all less than 1, indicating a synergistic effect.

[0040] (2) Effects of DL504 combined with DEX on Jurkat cell morphology

[0041] Take out the logarithmically growing Jurkat cells and press 5×10 5The cells were seeded in 6-well plates. DMSO control group (blank control), DEX (100 μM) single-use group, DL504 (20 μM) single-use group and combined-use group (100 μM DEX + 20 μM DL504) were set up respectively. Jurkat cells were treated for 24 and 48 hours. Cell morphology was observed and photographed using an inverted microscope. Figure 3 .

[0042] Depend on Figure 3 It can be seen that DEX (100μM), DL504 (20μM) single-use group or combined group (100μM DEX+20μM DL504) treatment for 24h had no significant effect on the morphology of Jurkat cells; after treating Jurkat cells for 48h, compared with the DMSO group, DEX or DL504 single-use group had no significant effect on the morphology of Jurkat cells, but compared with DMSO, DEX or DL504 alone, the Jurkat cell morphology induced by DL504 and DEX was wrinkled and fragmented.

[0043] (3) Effects of DL504 combined with DEX on the Jurkat cell cycle

[0044] Take out the logarithmically growing Jurkat cells and culture them at a rate of 5×10 5 Each well was seeded in a 6-well plate. DMSO control group (blank control), DEX (100 μM) single-use group, DL504 (20 μM) single-use group and combined group (100 μM DEX + 20 μM DL504) were set up respectively. Jurkat cells were treated for 24 hours, the cells were collected, washed twice with pre-cooled 1× PBS, resuspended with a volume concentration of 70% ethanol aqueous solution and fixed at -20°C overnight, then centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, washed twice with pre-cooled 1× PBS, and 500 μL periodic staining solution (0.1 mg / mL RNase A, 50 μg / mL PI and 0.05% Triton X-100) was added, stained at room temperature in the dark for 15 minutes, centrifuged at 1500 rpm for 3 minutes to discard the dye, resuspended with pre-cooled 1× PBS and filtered with a 200-mesh filter. Finally, flow cytometry was used for analysis. The results are shown in the figure. Figure 4 .

[0045] Depend on Figure 4 As shown, compared with the DMSO group, DEX or DL504 alone did not significantly induce G1 arrest in Jurkat cells. However, the combination of DEX and DL504 significantly induced G1 arrest in Jurkat cells. This suggests that DL504 enhances the inhibitory effect of DEX on Jurkat cell proliferation by affecting the cell cycle.

[0046] (4) Effects of DL504 combined with DEX on apoptosis of Jurkat cells

[0047] Take out the logarithmically growing Jurkat cells and press 5×10 5 100μM DEX (100μM) alone, DL504 (20μM) alone, and a combination group (100μM DEX + 20μM DL504) were set up respectively. Jurkat cells were treated for 48 hours, collected, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, washed once with pre-chilled 1× PBS, resuspended in 1mL pre-chilled 1× PBS and transferred to a 1.5mL EP tube, centrifuged at 1000rpm for 5 minutes, removed the supernatant, resuspended in 1× Binding Buffer, and 1.5μL each of FITC-Annexin V and PI were added and mixed. The cells were stained at room temperature in the dark for 15 minutes, centrifuged at 1000rpm for 5 minutes to remove the dye, and resuspended in × Binding Buffer. Finally, the cells were analyzed by flow cytometry.

[0048] Depend on Figure 5 Results: Jurkat cells treated with DMSO, DEX (100 μM), DL504 (20 μM) alone, or in combination (100 μM DEX + 20 μM DL504) for 48 hours showed apoptosis rates of (5.57 ± 0.89)%, (6.70 ± 2.35)%, (15.80 ± 1.01)%, and (62.62 ± 2.04)%, respectively. Compared with DMSO, DEX, or DL504 alone, the combination of DL504 and DEX significantly induced apoptosis in Jurkat cells. This suggests that DL504 can enhance the DEX-induced apoptosis effect in Jurkat cells and has the potential to serve as a DEX sensitizer.

[0049] (5) Effects of DL504 combined with DEX on Jurkat cell cycle, apoptosis and related pathways

[0050] ① Extraction of total cell protein

[0051] Jurkat cells growing exponentially were taken out and 1.5×10 6Jurkat cells were seeded into 60mm cell culture dishes. A DMSO control group (blank control), a DEX (100μM) alone group, a DL504 (20μM) alone group, and a combination group (100μM DEX + 20μM DL504) were set up. Jurkat cells were treated for 24 hours, harvested, centrifuged at 1500rpm for 5 minutes, and the supernatant removed. The cells were washed once with pre-chilled 1× PBS, centrifuged at 1500rpm for 5 minutes, and the supernatant removed. The cells were resuspended in 1mL of pre-chilled 1× PBS and transferred to a 1.5mL EP tube. The cells were centrifuged at 1500rpm for 5 minutes and the supernatant removed. IP lysis buffer containing PMSF (10μL PMSF per 1mL IP lysis buffer) was added according to the cell number. The cells were lysed on ice for at least 30 minutes, centrifuged at 12000rpm for 15 minutes, and the supernatant was collected to obtain the protein sample.

[0052] ②Isolation and extraction of Jurkat cell nucleocytoplasmic proteins

[0053] Jurkat cells growing exponentially were taken out and 1.5×10 6 Jurkat cells were seeded into 60 mm cell culture dishes. A DMSO control group (blank control), a DEX (100 μM) alone group, a DL504 (20 μM) alone group, and a combination group (100 μM DEX + 20 μM DL504) were set up. Jurkat cells were treated for 24 hours and then harvested. 120 μL of Cytoplasmic Protein Extraction Reagent A containing PMSF was added to the cell pellet. The cells were vigorously vortexed to resuspend and lysed on ice for 12 minutes. Six μL of Cytoplasmic Protein Extraction Reagent B was added, and the cells were vigorously vortexed for 10 seconds, ice-cooled for 2 minutes, and centrifuged at 13,400 rpm for 5 minutes. The supernatant (cytoplasmic protein) was collected. The supernatant was removed as much as possible by aspiration, and 40 μL of Nuclear Protein Extraction Reagent containing PMSF was added to the pellet. The pellet was vigorously vortexed for 20 seconds, then ice-cooled for 30 minutes (vortexing vigorously for 20 seconds every 2 minutes). The pellet was centrifuged at 13,400 rpm for 5 minutes, and the supernatant (nuclear protein) was collected.

[0054] ③ Protein concentration determination

[0055] The concentration of protein samples was quantified strictly according to the instructions of the BCA protein concentration assay kit.

[0056] ④ Protein denaturation treatment

[0057] The protein sample was measured and added with 5× Loading Buffer at a volume ratio of 5× Loading Buffer: protein sample = 1:4, mixed thoroughly, and boiled on a metal hot plate at 95°C for 5 min to denature the sample. The treated sample was then stored at -20°C until use.

[0058] ⑤SDS-PAGE electrophoresis

[0059] Prepare a separating gel with a polyacrylamide gel concentration of 8% to 12% and a stacking gel with a polyacrylamide gel concentration of 5% based on the protein's molecular weight. Once the gels are prepared, secure the laminated glass plates to the corresponding positions in the electrophoresis tank. Add electrophoresis buffer to the inner and outer reservoirs. Remove the sample comb and use a micropipette to draw 50 μg of the treated sample. Connect the power supply and run electrophoresis at a constant voltage of 100 V until the bromophenol blue indicator reaches 1 cm below the bottom, then stop the electrophoresis.

[0060] ⑥ Transfer

[0061] Cut a PVDF membrane (8 cm x 5 cm) the same size as the electrophoresis gel and activate it with methanol for 30 seconds. Place the gel, pre-soaked PVDF membrane, filter paper, and sponge pad in the order of sponge pad, filter paper, polyacrylamide gel, PVDF membrane, filter paper, and sponge pad, forming a sandwich structure. Use a glass rod to remove air bubbles between the PVDF membrane or filter paper and the gel. Hold the sandwich structure between a plexiglass support plate and place it in the electrophoresis tank, with the PVDF membrane facing the anode. Add 1× Western Blot Rapid Transfer Buffer and transfer at a constant current of 300 mA for 1 hour.

[0062] ⑦ Closed

[0063] After the transfer, the PVDF membrane was removed and placed in an antibody incubation box, and blocked with 3% BSA on a shaker at room temperature for 60 min.

[0064] ⑧Incubate with primary antibody

[0065] Remove the blocked PVDF membrane, wash it with 1×TBS for 5 minutes, discard the 1×TBS, add the primary antibody and incubate it at 4°C overnight.

[0066] ⑨Incubation with secondary antibody

[0067] The primary antibody was recovered and washed three times with 1× TBS for 5 min each time. The diluted secondary antibody was added and incubated on a shaker at room temperature in the dark for 1.5 h. The membrane was then washed three times with 1× TBS for 5 min each time.

[0068] ⑩ Target protein visualization

[0069] The secondary antibody was recovered and washed three times with 1× TBS in the dark for 5 minutes each time. Finally, the target protein was imaged using the Odyssey & CLX dual-color infrared imaging system.

[0070] Depend on Figure 6As shown in the figure, Jurkat cells were treated with DMSO, DEX (100μM), DL504 (20μM) alone or in combination (100μM DEX + 20μM DL504) for 24 hours. The combination of DL504 and DEX significantly downregulated the expression levels of c-Myc, CDK2, and Cyclin E1, consistent with the downregulation of cell cycle regulatory genes known to be involved in the G1 / S transition. This suggests that the combination of DL504 and DEX induces G1 arrest in Jurkat cells by regulating cell cycle-related proteins.

[0071] As shown in Figure 7, Jurkat cells were treated with DMSO, DEX (100 μM), DL504 (20 μM), or a combination group (100 μM DEX + 20 μM DL504) for 24 hours. Compared with DMSO, DEX, or DL504 alone, the combination of DEX and DL504 significantly downregulated the expression levels of anti-apoptotic proteins Bcl-XL and Bcl-2, and significantly upregulated the expression levels of apoptotic proteins Cleaved Caspase 9, Cleaved Caspase 3, and Cleaved PARP. This suggests that the combination of DL504 and DEX induces apoptosis in Jurkat cells by activating the mitochondrial apoptosis pathway.

[0072] As shown in Figure 8, after DMSO, DEX (100 μM), DL504 (20 μM) or the combination group (100 μM DEX + 20 μM DL504) were used to treat Jurkat cells for 24 hours, DEX, DL504 alone or in combination had no significant effect on GR expression. Ser211 Activation of phosphorylation sites is a hallmark of GR activation. DEX alone and in combination increased p-GR expression, indicating that GR was activated by both groups. GCs enter cells and bind to cytoplasmic GR. Once activated, GR translocates to the nucleus and binds to the GC response element (GRE) to activate target genes. Western blot analysis of nucleocytoplasmic proteins revealed that DEX significantly induced GR nuclear translocation, and DL504 enhanced DEX-induced GR nuclear translocation. GR transcription is active in DEX-resistant ALL xenografts, and resistance is associated with an inability to induce expression of the pro-apoptotic protein BIM. Western blot results showed that the combination of DL504 and DEX significantly upregulated BIM expression. These results suggest that DL504 enhances DEX-induced GR nuclear translocation and BIM expression, thereby promoting apoptosis and inhibiting cell proliferation in Jurkat cells.

[0073] Figure 9 shows that abnormal JAK2 / STAT3 stimulation is closely associated with cancer cell survival and apoptosis. Western blot results show that compared with DMSO, DEX, and DL504 alone, the combination of DEX and DL504 significantly downregulated p-JAK2 and p-STAT3, but had no significant effect on JAK2 and STAT3. These results suggest that the DL504 combined with DEX-induced apoptosis in Jurkat cells is mediated by affecting the JAK2 / STAT3 pathway.

[0074] The above examples demonstrate that the combination of DL504 and DEX exhibits a significant synergistic effect on Jurkat cells. DL504 enhances Jurkat cell sensitivity to DEX by inducing G1 arrest and modulating GR / BIM and JAK2 / STAT3 signaling to activate the caspase cascade, thereby inducing apoptosis. Therefore, DL504 has the potential to be a DEX sensitizer and provides a research foundation for the development of drugs to overcome glucocorticoid resistance.

Claims

1. Use of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating leukemia, wherein the leukemia is acute T-lymphocytic leukemia.

2. The use of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating leukemia according to claim 1, characterized in that: The drug is prepared by combining 1,3-dicyclohexylbarbituric acid and dexamethasone, and adding pharmaceutically acceptable excipients or auxiliary components to form a pharmaceutical preparation; wherein the molar ratio of 1,3-dicyclohexylbarbituric acid to dexamethasone in the pharmaceutical preparation is 10-40:

100.

3. The use of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating leukemia according to claim 2, characterized in that: The molar ratio of 1,3-dicyclohexylbarbituric acid to dexamethasone in the pharmaceutical preparation is 20-25:

100.

4. The use of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of a drug for treating leukemia according to claim 2, characterized in that: The pharmaceutical preparation is any one of oral solution, injection, tablet, capsule and pill.

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