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

Abstract

The invention discloses an application of 1, 3-dicyclohexylbarbituric acid combined with dexamethasone in preparation of drugs for treating leukemia, and experimental detection finds that 1, 3-dicyclohexylbarbituric acid can increase the sensitivity of leukemia to dexamethasone; a glucocorticoid-resistant acute T lymphocytic leukemia cell strain Jurkat is applied in vitro, experimental detection shows that the 1, 3-dicyclohexylbarbituric acid and the dexamethasone have a remarkable synergistic effect on Jurkat cells, and the 1, 3-dicyclohexylbarbituric acid can enhance the sensitivity of the Jurkat cells to the dexamethasone in a concentration and time dependent manner. The combination of 1, 3-dicyclohexylbarbituric acid and dexamethasone can induce the generation of G1 phase retardation of Jurkat cells, and regulates and controls GR / BIM and JAK2 / STAT3 signal transduction to activate a Caspase cascade reaction, thereby inducing the apoptosis of the Jurkat cells. The 1, 3-dicyclohexylbarbituric acid disclosed by the invention has the potential of becoming a dexamethasone sensitizer, and also provides a research basis for the development of drugs for overcoming the drug resistance of glucocorticoid.

Description

Technical Field

[0001] The invention belongs to the technical field of biology and medicine, and specifically 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 malignant tumor of the blood system caused by abnormal proliferation of hematopoietic stem cells, mainly including 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 acute lymphoblastic leukemia cases. The standard treatment regimen for acute T-lymphoblastic leukemia is based on glucocorticoids (GCs) and combined with a variety of chemotherapy drugs, such as vincristine, asparaginase, cytarabine, 6-mercaptopurine and methotrexate. Despite the wide variety of chemotherapy drugs, glucocorticoids remain the first-line treatment. The early response of patients with acute T-lymphoblastic leukemia to glucocorticoids is an important indicator of prognosis, but glucocorticoid resistance is often observed in patients with relapsed or refractory acute T-lymphoblastic leukemia, which is one of the main reasons for relapse in patients with acute T-lymphoblastic leukemia. Overcoming glucocorticoid resistance is extremely important for improving the long-term survival rate of patients with acute T-lymphoblastic leukemia. Therefore, it is urgent to invent a drug that can overcome glucocorticoid resistance for the treatment of acute T-lymphoblastic leukemia. Summary of the invention

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

[0005] The 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 aims at the problem that dexamethasone can cause drug resistance in clinical treatment of acute T lymphocyte leukemia, resulting in poor treatment effect or recurrence after treatment. The drug-resistant strain Jurkat cells of human acute T lymphocyte leukemia cells are selected as an in vitro research model. The experimental results show that 1,3-dicyclohexyl barbituric acid itself has little effect on acute T lymphocyte leukemia cells, and the apoptosis rate is (15.80±1.01)%, but it can significantly induce cell apoptosis after being used in combination with dexamethasone, and the apoptosis rate is (62.62±2.04)%, showing obvious synergistic effect. After the present invention combines 1,3-dicyclohexyl barbituric acid with dexamethasone, the sensitivity of Jurkat cells to dexamethasone can be enhanced, and the dosage of dexamethasone can be reduced. The results of flow cytometry and protein immunoblotting showed that 1,3-dicyclohexylbarbituric acid combined with dexamethasone induced apoptosis of Jurkat cells by inducing G1 arrest of Jurkat cells and regulating GR / BIM and JAK2 / STAT3 signaling to activate the Caspase cascade reaction, indicating that 1,3-dicyclohexylbarbituric acid has the potential to become 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 figure is the verification result of Jurkat cell 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 100μM DEX on Jurkat cell apoptosis and statistical analysis of cell apoptosis; (D, E) are the effect of 1μM DEX on CEM-C7H2 cell apoptosis and statistical analysis of cell apoptosis.

[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.

[0013] Figure 3 It is the effect of DL504 combined with DEX on the morphology of Jurkat cells.

[0014] Figure 4 The figure shows the effect 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 effect of DL504 combined with DEX on Jurkat cell apoptosis and apoptosis-related proteins after 48 hours of treatment of Jurkat cells; (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 figure shows the effect 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 figure shows the effect of DL504 combined with DEX on apoptosis-related proteins in Jurkat cells after 24 hours of treatment; (A) is the expression of apoptosis-related proteins in Jurkat cells (GAPDH is the internal reference); (B) is the quantitative statistical chart of apoptosis regulatory proteins in Jurkat cells.

[0018] Figure 8 The effect of DL504 combined with DEX on the GR / BIM pathway proteins of Jurkat cells after 24 hours of treatment; (A) is GR and p-GR Ser211(A) 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] Fig. 9 The effect 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 chart of JAK2, p-JAK2, STAT3 and p-STAT3. DETAILED DESCRIPTION

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

[0021] In a specific embodiment of the present invention, the Jurkat cell is a human acute T lymphoblastic leukemia cell line resistant to glucocorticoids, and the CEM-C7H2 cell is a human acute T lymphoblastic leukemia cell line sensitive to glucocorticoids, 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 a DEX drug sensitivity experiment 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 of CEM-C7H2 cells, but 100 μM DEX has no significant effect on the apoptosis of 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 the resistance of Jurkat cells to dexamethasone

[0028] 1. Experimental Materials

[0029] Cell lines: Jurkat cells (resistant strain) and CEM-C7H2 cells (sensitive strain) of acute T lymphoblastic leukemia cells were stored in Guizhou Natural Products Research Center. Jurkat cells and CEM-C7H2 cells were cultured in RPMI 1640 medium containing 5% FBS in a cell culture incubator at 37°C with 4.5% CO2.

[0030] Experimental reagents: Fetal bovine serum (FBS) was purchased from BI; RPMI 1640 culture medium was purchased from Gibco; AnnexinⅤ-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), dimethyl sulfoxide (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 and sterilize with a 0.22 μm filter, and store at 4°C away from light.

[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 solvent.

[0033] 2. Experimental methods

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

[0035] Jurkat cells growing in logarithmic growth were taken out and plated at 1×10 5The cells were inoculated at a density of 100 μM / mL in a 96-well plate, and 90 μL of cell suspension was added to each well. The plates were divided into 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, the DL504 concentration in the DL504 group was 10, 20 and 40 μM, and 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 h, respectively, and 10 μL of 5 μg / mL MTT solution was added for routine incubation for 4 h. 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 the result was: Figure 2 CompuSyn software was used to calculate the combination index (CI) of the interaction between DL504 and DEX. CI = 1 is additive; CI > 1 is antagonistic; CI < 1 is synergistic, 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 It can be seen that compared with the DMSO, DL504 or DEX groups alone, DL504 combined with DEX can significantly inhibit the proliferation of Jurkat cells, and the cell survival rates at 24, 48, 72 and 96 hours are (94.12±3.80)%, (61.03±9.34)%, (24.64±3.45)% and (17.55±2.15)%, respectively. In addition, different concentrations of DL504 (10, 20 and 40 μM) alone or combined with DEX (100 μM) were tested for different time periods to treat Jurkat cells. The results showed that different concentrations of DL504 combined with DEX can significantly inhibit the proliferation of Jurkat cells at 48, 72 and 96 hours. This suggests that DL504 enhances the sensitivity of Jurkat cells 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 inoculated into 6-well plates. The 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 h, and 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) alone 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 alone 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 combined with DEX was wrinkled and fragmented.

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

[0044] Take out the logarithmically growing Jurkat cells and culture them at the rate of 5×10 5 100 μM 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, and Jurkat cells were treated for 24 hours, and the cells were collected and washed twice with pre-cooled 1×PBS, resuspended with 70% ethanol aqueous solution and fixed at -20°C overnight, and then centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and 500 μL of periodic dye solution (0.1 mg / mL RNase A, 50 μg / mL PI and 0.05% Triton X-100) was added after washing twice with pre-cooled 1×PBS, and stained for 15 minutes at room temperature in the dark, centrifuged at 1500 rpm for 3 minutes to discard the dye, resuspended with pre-cooled 1×PBS and filtered with a 200-mesh filter, and finally analyzed by flow cytometry. The results are shown in Figure 4 .

[0045] Depend on Figure 4 It can be seen that compared with the DMSO group, the use of DEX or DL504 alone could not significantly induce G1 phase arrest in Jurkat cells. However, the combination of DEX and DL504 significantly induced G1 phase 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 The cells were inoculated in 6-well plates. The DMSO control group (blank control), DEX (100 μM) single group, DL504 (20 μM) single group and combination group (100 μM DEX + 20 μM DL504) were set up respectively. Jurkat cells were treated for 48 hours, and the cells were collected, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed once with pre-cooled 1×PBS, resuspended with 1 mL pre-cooled 1×PBS and transferred to a 1.5 mL EP tube, centrifuged at 1000 rpm for 5 minutes to remove the supernatant, resuspended with 1×Binding Buffer, and 1.5 μL of FITC-Annexin V and PI were added and mixed, and stained at room temperature in the dark for 15 minutes, centrifuged at 1000 rpm for 5 minutes to remove the dye, resuspended with ×Binding Buffer, and finally analyzed by flow cytometry.

[0048] Depend on Figure 5 It can be seen that when Jurkat cells were treated with DMSO, DEX (100 μM), DL504 (20 μM) alone or in combination (100 μM DEX + 20 μM DL504) for 48 h, the cell apoptosis rates of each group were (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 of Jurkat cells. This suggests that DL504 can enhance the effect of DEX in inducing apoptosis of Jurkat cells and has the potential to become 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 in logarithmic growth were taken out and cultured at a rate of 1.5×10 6100 μM 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, and Jurkat cells were treated for 24 hours, and the cells were collected, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, washed once with pre-cooled 1×PBS, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and then resuspended with 1 mL of pre-cooled 1×PBS and transferred to a 1.5 mL EP tube, centrifuged at 1500 rpm for 5 minutes to remove the supernatant. According to the amount of cells, IP lysis buffer containing PMSF (10 μL PMSF per 1 mL IP lysis buffer) was added, lysed on ice for more than 30 minutes, centrifuged at 12000 rpm for 15 minutes, and the supernatant was taken to obtain the protein sample.

[0052] ②Isolation and extraction of nuclear and cytoplasmic proteins from Jurkat cells

[0053] Jurkat cells growing in logarithmic growth were taken out and cultured at a rate of 1.5×10 6 100μM DEX+20μM DL504) were inoculated in 60mm cell culture dishes. The DMSO control group (blank control), DEX (100μM) single group, DL504 (20μM) single group and combination group (100μM DEX+20μM DL504) were set up respectively, and Jurkat cells were treated for 24h and the cells were collected. 120μL of cytoplasmic protein extraction reagent A containing PMSF was added to the cell pellet, and the cells were resuspended by vortexing vigorously, lysed on ice for 12min, and 6μL of cytoplasmic protein extraction reagent B was added, vortexed vigorously for 10s, ice bathed for 2min, centrifuged at 13400rpm for 5min, and the supernatant (cytoplasmic protein) was taken. The supernatant was removed as much as possible and 40μL of nuclear protein extraction reagent containing PMSF was added to the pellet, and after vortexing vigorously for 20s, ice bathed for 30min (vortexed vigorously for 20s every 2min), centrifuged at 13400rpm for 5min after the end, and the supernatant (nuclear protein) was taken.

[0054] ③ Protein concentration determination

[0055] The protein concentration 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 well, boiled and denatured on a 95°C metal heating plate for 5 min, and the treated sample was obtained and stored at -20°C for later use.

[0058] ⑤SDS-PAGE electrophoresis

[0059] According to the molecular weight of the protein, prepare a polyacrylamide gel with a mass concentration of 8% to 12% as a separation gel and a polyacrylamide gel with a mass concentration of 5% as a concentration gel. After the gel is prepared, fix the laminated glass plate to the corresponding position in the electrophoresis tank, add electrophoresis buffer to the inner and outer storage tanks, remove the spotting comb, and use a micropipette to absorb 50μg of the treated sample. Turn on the power supply and perform electrophoresis at a constant voltage of 100V until the bromophenol blue indicator reaches 1cm before the bottom, and stop the electrophoresis.

[0060] ⑥Transfer

[0061] Cut a PVDF membrane (8cm×5cm) of the same size as the electrophoresis gel and activate it with methanol for 30s. Place the gel and the pre-soaked PVDF membrane, filter paper, and sponge pad in the order of sponge pad-filter paper-polyacrylamide gel-PVDF membrane-filter paper-sponge pad from bottom to top to form a sandwich structure. Use a glass rod to remove the bubbles between the PVDF membrane or filter paper and the gel. Use a plexiglass support plate to clamp the "sandwich" structure, place the "sandwich" structure into the electrophoresis tank with the PVDF membrane facing the anode, add 1×Western Blot fast transfer solution, and transfer at 300mA constant current for 1h.

[0062] ⑦ Closed

[0063] After the transfer, the PVDF membrane was taken out 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] Take out the blocked PVDF membrane, wash it with 1×TBS for 5 minutes, pour out 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, and the diluted secondary antibody was added, incubated on a shaker at room temperature in the dark for 1.5 h, and washed three times with 1× TBS for 5 min each time.

[0068] ⑩ Target protein development

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

[0070] Depend on Figure 6As shown in the figure, DMSO, DEX (100μM), DL504 (20μM) single use group or combined use group (100μM DEX + 20μM DL504) were used to treat Jurkat cells for 24h. DL504 combined with DEX significantly downregulated the expression levels of c-Myc, CDK2 and Cyclin E1, which is consistent with the downregulation of cell cycle regulatory genes known to be involved in G1 / S transition. This shows that the combination of DL504 and DEX induces G1 phase 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 the combination group (100 μM DEX + 20 μM DL504) for 24 hours. Compared with DMSO, DEX or DL504 alone, the combination of DEX and DL504 can significantly downregulate the expression levels of anti-apoptotic proteins Bcl-XL and Bcl-2, and significantly upregulate the expression levels of apoptotic proteins CleavedCaspase9, CleavedCaspase3 and Cleaved PARP. This indicates that DL504 combined with DEX induces apoptosis of 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 the combination group had no significant effect on GR expression. Ser211 Activation of phosphorylation sites is a sign of GR activation. DEX alone and in combination upregulated the expression level of p-GR, indicating that GR was activated by DEX alone and in combination. GCs enter cells and bind to cytoplasmic GR. Once activated, GR can translocate to the nucleus and bind to GC response elements (GRE) to activate target genes. Western Blot detection of nuclear cytoplasmic proteins revealed that DEX significantly induced GR nuclear translocation, and DL504 enhanced DEX-induced GR translocation to the nucleus. GR transcription was active in DEX-resistant ALL xenografts, and its resistance was associated with the inability to induce the expression of the pro-apoptotic protein BIM. Western Blot results showed that DL504 combined with DEX significantly upregulated the expression level of BIM. The above results indicate that DL504 enhances DEX-induced GR nuclear translocation and BIM expression, thereby promoting Jurkat cell apoptosis and inhibiting cell proliferation.

[0073] As shown in Figure 9, abnormal stimulation of JAK2 / STAT3 is closely related to the survival and apoptosis of cancer cells. Western Blot results showed 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. The above results indicate that DL504 combined with DEX induces apoptosis of Jurkat cells by affecting the JAK2 / STAT3 pathway.

[0074] It can be seen from the above examples that the combination of DL504 and DEX in the present invention has a significant synergistic effect on Jurkat cells. DL504 enhances the sensitivity of Jurkat cells to DEX by inducing Jurkat cells to G1 phase arrest, and regulating GR / BIM and JAK2 / STAT3 signal transduction to activate Caspase cascade reaction, thereby inducing Jurkat cell apoptosis. Therefore, the DL504 described in the present invention has the potential to become a DEX sensitizer, and also provides a research basis for the development of drugs to overcome glucocorticoid resistance.

Claims

1. Application of 1,3-dicyclohexylbarbituric acid combined with dexamethasone in the preparation of drugs for the treatment of 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 leukemia is acute T-lymphocytic leukemia.

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

100.

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

100.

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

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