Application of isogarcinol compounds combined with dexamethasone in the preparation of drugs for treating leukemia

Abstract

The present invention provides the use of isogaminol compounds in combination with glucocorticoids in the preparation of a leukemia treatment drug, belonging to the technical fields of biology and medicine. The isogaminol compounds described herein, when used in combination with glucocorticoids, can enhance the sensitivity of Jurkat cells to glucocorticoids, induce G1 cell cycle arrest in Jurkat cells, and induce apoptosis in Jurkat cells. The combined use of isogaminol compounds and glucocorticoids in the present invention provides a research foundation for overcoming glucocorticoid resistance in acute lymphoblastic leukemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and medicine, and in particular relates to the application of isogarcinol compounds combined with dexamethasone in the preparation of a drug for treating leukemia. Background Art

[0002] Leukemia is a malignant clonal disease of hematopoietic stem cells. It can be divided into acute leukemia and chronic leukemia based on the degree of differentiation and the length of its natural course. Acute leukemia includes acute lymphoblastic leukemia and acute myeloid leukemia. ALL is an aggressive hematologic malignancy and the most common childhood cancer. Despite improvements in cure rates for ALL with the increasing prevalence of personalized treatment options, it remains a leading cause of mortality in children. Current treatments for ALL in children have an event-free survival rate of approximately 75%, but relapses remain frequent. Challenges in managing relapses in ALL include leukemic cell resistance and decreased sensitivity to second-line therapy after first-line intensive treatment, resulting in lower remission rates, relapses, and decreased overall efficacy.

[0003] Currently, glucocorticoids are essential drugs for the treatment of acute lymphoblastic leukemia (ALL), but glucocorticoid resistance is a major challenge in the clinical treatment of ALL. Overcoming glucocorticoid resistance is crucial for improving the long-term survival of ALL patients. Therefore, there is an urgent need to develop a drug that can overcome glucocorticoid resistance for the treatment of ALL. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an application of an isogarcinol compound combined with dexamethasone in the preparation of a drug for treating leukemia, wherein the isogarcinol compound (Isogarcinol) can enhance the sensitivity of Jurkat cells to glucocorticoids and alleviate drug resistance.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The use of isogarcinol compounds combined with glucocorticoids in the preparation of drugs for treating leukemia, wherein the chemical formula of the isogarcinol compounds is shown in Formula I:

[0007]

[0008] The present invention also provides the use of isogarcinol compounds in the preparation of drugs for treating glucocorticoid-resistant leukemia. The chemical formula of the isogarcinol compounds is shown in Formula I:

[0009]

[0010] Preferably, the leukemia comprises acute lymphoblastic leukemia.

[0011] Preferably, the glucocorticoid comprises dexamethasone (DEX).

[0012] Preferably, the isogaminol compound combined with glucocorticoid can enhance the sensitivity of Jurkat cells to glucocorticoid.

[0013] Preferably, the isogaminol compound combined with glucocorticoid can induce G1 cell cycle arrest in Jurkat cells.

[0014] Preferably, the isogaminol compound combined with glucocorticoid can induce apoptosis of Jurkat cells.

[0015] The present invention also provides a pharmaceutical composition for treating acute lymphocytic leukemia, comprising an isogarcinol compound and a glucocorticoid; the molar ratio of the isogarcinol compound to the glucocorticoid is 15:50.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides the use of isoglucanol compounds in combination with glucocorticoids in the preparation of a drug for treating leukemia. The combination of isoglucanol compounds and glucocorticoids enhances the sensitivity of Jurkat cells to glucocorticoids, induces G1 cell cycle arrest, and induces apoptosis in Jurkat cells. The isoglucanol compounds described herein can serve as potential small molecules for enhancing glucocorticoid sensitivity, providing a research foundation for the development of drugs that enhance glucocorticoid sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 1. Validation results of Jurkat cells' resistance to DEX;

[0019] Figure 2 Schematic diagram of the inhibition of Jurkat cell viability by different concentrations of Isogarcinol combined with DEX, wherein the horizontal axis represents the concentration of Isogarcinol and DEX (μM); the vertical axis represents the inhibition rate (%); DMSO is the control group;

[0020] Figure 3 The effect of Isogarcinol combined with DEX on Jurkat cell morphology;

[0021] Figure 4The effect of Isogarcinol combined with DEX on the Jurkat cell cycle after 24 hours; A is the flow cytometry graph, and B is the statistical proportion of Jurkat cells in the G1, S, and G2 phases;

[0022] Figure 5 The effect of Isogarcinol combined with DEX on Jurkat cell apoptosis after 24h and 48h treatment; A is the flow cytometry graph of cell apoptosis, and B is the statistical graph of Jurkat cell apoptosis rate;

[0023] Figure 6 The effect of Isogarcinol combined with DEX on the expression levels of proteins related to cell cycle, apoptosis and proliferation inhibition in Jurkat cells; A is a cell cycle-related protein, B is an apoptosis-related protein, C is a c-Myc protein, and D is a protein related to the PI3K / AKT / mTOR pathway;

[0024] Figure 7 Real-time fluorescence quantitative PCR graph of the mRNA expression levels of glucocorticoid receptor target genes after the action of DEX, Isogarcinol, and Isogarcinol+DEX. DETAILED DESCRIPTION

[0025] The present invention provides the use of an isogarcinol compound combined with a glucocorticoid in the preparation of a drug for treating leukemia. The chemical formula of the isogarcinol compound is shown in Formula I:

[0026]

[0027] The present invention also provides the use of an isogarcinol compound in the preparation of a drug for treating glucocorticoid-resistant leukemia. The chemical formula of the isogarcinol compound is shown in Formula I:

[0028]

[0029] The leukemia of the present invention preferably includes acute lymphocytic leukemia; the glucocorticoid preferably includes dexamethasone.

[0030] The combination of the isogaminol compound of the present invention and glucocorticoid can enhance the sensitivity of Jurkat cells to glucocorticoid.

[0031] The isogaminol compounds of the present invention combined with glucocorticoids can induce G1 phase cell cycle arrest in Jurkat cells.

[0032] The present invention uses flow cytometry to determine the effect of Isogarcinol combined with DEX on the Jurkat cell cycle. The experimental results show that the combination of Isogarcinol and DEX can significantly arrest Jurkat cells in the G1 phase.

[0033] The isogaminol compounds of the present invention combined with glucocorticoids can induce apoptosis of Jurkat cells.

[0034] The present invention uses flow cytometry to determine the effect of isogarcinol combined with DEX on Jurkat cell apoptosis. The experimental results show that the combination of isogarcinol and DEX can significantly induce Jurkat cell apoptosis.

[0035] The present invention uses Western blot to detect the expression levels of cell apoptosis and cell cycle-related proteins after the action of isogarcinol combined with DEX. The experimental results show that the combination of isogarcinol and DEX induces G1 arrest of Jurkat cells by downregulating the expression of CDK4, CDK6 and CDK2, upregulating the expression of cleaved Caspase3, Caspase9, PARP, and BIM, and downregulating the expression of Survivin, thereby inducing apoptosis of Jurkat cells.

[0036] The present invention uses Western blot to detect the expression levels of cell proliferation-related proteins after the action of isogarcinol combined with DEX. The experimental results show that the combination of isogarcinol and DEX significantly downregulated the expression of p-PI3K, p-Akt, p-mTOR and c-Myc.

[0037] The present invention uses real-time fluorescence quantitative PCR to detect the mRNA expression levels of glucocorticoid receptor target genes after the action of DEX, isogarcinol, and isogarcinol+DEX. The experimental results show that isogarcinol combined with DEX can upregulate the expression of glucocorticoid receptor target genes NR3C1 and TSC22D3, thereby enhancing the sensitivity of Jurkat cells to DEX.

[0038] The present invention also provides a pharmaceutical composition for treating acute lymphocytic leukemia, comprising an isogarcinol compound and a glucocorticoid; the molar ratio of the isogarcinol compound to the glucocorticoid is 15:50.

[0039] The pharmaceutical composition of the present invention preferably further comprises pharmaceutically acceptable excipients and is formulated into a decoction, decoction paste, pill, granule, tablet, capsule or oral solution. The administration of the pharmaceutical composition preferably comprises injection, oral administration, inhalation spray or transdermal administration. The excipients preferably comprise one or more of autosolvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] In the following examples, unless otherwise specified, all methods are conventional.

[0042] In a specific embodiment of the present invention, the Jurkat cells are a glucocorticoid-resistant acute lymphoblastic leukemia cell line, kindly provided by the University of Toronto. The resistant cell lines of the present invention are resistant to glucocorticoids. The resistant cell lines of the present invention are the same Jurkat cell lines used by Torres-López et al. (Torres-López, Liliana et al. Tamoxifen induces toxicity, causes autophagy, and partially reverses dexamethasone resistance in Jurkat T cells).

[0043] The present invention conducted DEX drug sensitivity experiments on glucocorticoid-sensitive strain CEMC7 and glucocorticoid-resistant strain Jurkat. Figure 1 It can be seen that DEX can significantly inhibit the proliferation of CEMC7 cells, but has no obvious effect on the proliferation of Jurkat cells, indicating that the Jurkat cells used in the examples of the present invention have significant resistance to DEX.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] Each experiment in the following examples was repeated three times independently, and all data were expressed as mean±SD. Student's t-test was performed using GraphPad Prism8, and P<0.05 was considered statistically significant.

[0046] Example 1

[0047] (1) Effect of Isogarcinol combined with DEX on Jurkat cell viability

[0048] Jurkat cells were cultured at a rate of 1 × 10 4 The cells were planted at a density of 100 μg / well in a 96-well plate, and 50 μM DEX, 5-20 μM Isogarcinol, and 5-20 μM Isogarcinol + 50 μM DEX were added for treatment. In addition, a control group was set up, and 0.1% DMSO was added to the control group. After culturing for 24 h, 48 h, and 72 h, 10 μL of 5 mg / mL MTT solution was added to each well, incubated at 37 ° C for 4 h, centrifuged and the supernatant was discarded, and 100 μL of triple solution was added to each well to dissolve. After the blue-purple crystalline formazan was completely dissolved, the absorbance values ​​at 570 nm of the different treatment groups and the control group were measured with an enzyme reader, and the corresponding inhibition rate was calculated. The inhibition rate was calculated as follows: inhibition rate = (control group OD570 nm - treatment group OD570 nm) / control group OD570 nm, and the result was obtained. Figure 2 .

[0049] Depend on Figure 2 It can be seen that Isogarcinol combined with DEX can significantly inhibit the proliferation of Jurkat cells, among which the combination of Isogarcinol (15μM) + DEX (50μM) has the best effect.

[0050] Example 2

[0051] (1) Effects of Isogarcinol combined with DEX on the number and morphology of Jurkat cells

[0052] Jurkat cells were cultured at a rate of 5 × 10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate and treated with 15 μM Isogarcinol, 50 μM DEX, and 15 μM Isogarcinol + 50 μM DEX. A control group was also set up, and 0.1% DMSO was added to the control group. After culturing for 24 hours, the morphological changes of the cells in each group were observed using an inverted microscope. Figure 3 .

[0053] Depend on Figure 3 It can be seen that the combination of Isogarcinol and DEX can significantly reduce the number of Jurkat cells and increase the number of fragmented cells.

[0054] Example 3

[0055] (1) Effects of Isogarcinol combined with DEX on the Jurkat cell cycle

[0056] Jurkat cells were cultured at a volume of 5 × 10 5 Cells were planted in 6-well plates at 1000 rpm / well and cultured with 15 μM Isogarcinol, 50 μM DEX, and 15 μM Isogarcinol + 50 μM DEX for 24 hours, and then the cells were collected. At the same time, a control group was set up, and 0.1% DMSO was added to the control group. The collected cells were centrifuged at 1000 rpm for 5 minutes, washed once with pre-cooled PBS, and resuspended with 70% pre-cooled ethanol, and fixed at -20°C overnight. After overnight, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and then washed once with PBS, centrifuged at 1000 rpm for 5 minutes, and the cell pellet was collected. The cell pellet was resuspended in 500 μL PBS, 5 μL RNaseA, 25 μL PI and 0.25 μL TritonX-1000 were added, and the mixture was mixed by flicking. After incubation at 37°C in the dark for 30 minutes, the mixture was centrifuged at 1000 rpm for 5 minutes, the dye was discarded, and the mixture was resuspended with 200 μL PBS. The cell cycle distribution was detected by flow cytometry, and the results are shown as follows. Figure 4 shown.

[0057] Depend on Figure 4 It can be seen that the combination of Isogarcinol and DEX can significantly arrest Jurkat cells in the G1 phase.

[0058] Example 4

[0059] (2) Effects of Isogarcinol combined with DEX on Jurkat cell apoptosis

[0060] Jurkat cells were cultured at a volume of 5 × 10 5 Cells were seeded in 6-well plates and cultured with 15 μM Isogarcinol, 50 μM DEX, and 15 μM Isogarcinol + 50 μM DEX for 24 h and 48 h, respectively. A control group was also set up, in which 0.1% DMSO was added. The collected cells were centrifuged at 1500 rpm for 3 min, washed once with PBS, and the supernatant was discarded. 50 μL 1× Binding Buffer was added to resuspend the cells. 2.5 μL Annexin-V and 2.5 μL PI were added and incubated at room temperature for 15 min. Flow cytometric analysis was then performed. The results are shown in Figure 2. Figure 5 shown.

[0061] Depend on Figure 5It can be seen that Isogarcinol combined with DEX can significantly induce apoptosis of Jurkat cells.

[0062] Example 5

[0063] Western blot was used to detect the expression levels of proteins related to cell cycle, apoptosis and proliferation inhibition after the treatment with DEX, Isogarcinol and Isogarcinol combined with DEX:

[0064] (1) Cell collection and lysis: Jurkat cells were plated at 1.5×10 6 Jurkat cells were seeded in 10 cm diameter culture dishes and cultured at 100 μM per well. After 24 hours of treatment with 15 μM isogarcinol, 50 μM DEX, or 15 μM isogarcinol plus 50 μM DEX, the cells were harvested, washed once with PBS, and lysed on ice for 45 minutes using RIPA lysis buffer containing PMSF. The cells were vortexed every 5 minutes, centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant collected.

[0065] (2) BCA protein quantification: Protein quantification was performed according to the instructions of the BCA protein quantification kit. After calculating the protein concentration, 5× loading buffer was added at a ratio of 4:1 based on the volume of the collected protein sample. The sample was denatured in a metal bath at 100°C for 5 min and stored at -80°C.

[0066] (3) SDS-PAGE electrophoresis: Prepare 8-12% separation gel and 5% stacking gel according to the molecular weight of the protein. After the gel is prepared, use a microsyringe to add 50 μg of protein sample to each well. Run electrophoresis at 80 V until the protein sample enters the separation gel. Adjust the voltage to 100 V and run until bromophenol blue reaches the bottom of the gel plate.

[0067] (4) Transfer: Activate the PVDF membrane with methanol. Use the sandwich method to arrange the glue, PVDF membrane and filter paper in sequence, place them in the transfer tank, add the wet transfer solution, and set the transfer conditions as follows: constant current 220mA, time 120min.

[0068] (5) Blocking: After transfer, immediately place the membrane in a Western blot washing box pre-filled with TBS washing buffer. Wash three times for 2 minutes each time to remove the transfer buffer from the membrane. Discard the washing buffer and add 3% BSA blocking buffer. Block at room temperature for 1 hour.

[0069] (6) Primary antibody incubation: Dilute the primary antibody with 3% BSA blocking solution in an appropriate ratio according to the instructions of the primary antibody. After recovering the blocking solution, immediately add the diluted primary antibody and incubate on a shaker at 4°C overnight.

[0070] (7) Secondary antibody incubation: Recover the primary antibody and wash the plate with TBS solution for 5 min on a rocking table for 3 times. Dilute the secondary antibody with 5% skim milk powder at a ratio of 1:30,000 according to the instructions of the secondary antibody. Aspirate the washing solution with a dropper and immediately add the diluted secondary antibody. Incubate the plate for 2 h at room temperature with slow shaking on a rocking table in the dark.

[0071] (8) Protein detection: Recover the secondary antibody, add TBS solution, and wash three times in the dark, 5 minutes each time. Use Odyssey imager to detect the expression of cycle-related proteins CDK4, CDK2, CDK6, apoptosis-related proteins PARP, Caspase3, Caspase9, Bim, Survivin, proliferation-inhibitory proteins PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR, and c-Myc protein. The results are as follows. Figure 6 shown.

[0072] Depend on Figure 6 It can be seen that the combination of isogarcinol and DEX induces G1 cycle arrest in Jurkat cells by downregulating the expression of cycle-related proteins such as CDK4, CDK6 and CDK2, upregulating the expression of BIM and cleaved Caspase3, Caspase9, and PARP, downregulating the expression of Survivin to induce Jurkat cell apoptosis, inhibiting the expression of c-Myc and inhibiting the PI3K / Akt / mTOR signaling pathway to enhance the sensitivity of Jurkat cells to DEX.

[0073] Example 6

[0074] Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of glucocorticoid receptor target genes after the treatment of DEX, Isogarcinol, and Isogarcinol+DEX.

[0075] Extraction of total RNA:

[0076] (1) Jurkat cells were cultured at a rate of 1.5×10 6 Jurkat cells were seeded in 6-well plates with 100 μg of dapoxetine per well. After treatment with DEX (50 μM), isogarcinol (15 μM), or isogarcinol (15 μM) plus DEX (50 μM) for 24 hours, the cells were harvested. Total RNA was extracted according to the TRIzol kit instructions. An appropriate amount of dissolved total RNA was collected, and its quality was assessed by electrophoresis. RNA concentration was determined using a NanoDrop 2000 spectrophotometer. The extracted total RNA was stored at -80°C until use.

[0077] (2) Reverse transcription and synthesis of cDNA: TaKaRa reverse transcription kit was used to synthesize cDNA.

[0078] (3) According to the instructions of the qRT-PCR kit, the reaction system was prepared and qRT-PCR was performed to detect the expression levels of genes such as NR3C1 and TSC22D3. Figure 7 shown.

[0079] Depend on Figure 7 It can be seen that isogarcinol combined with DEX can upregulate the expression of glucocorticoid receptor target genes NR3C1 and TSC22D3, thereby enhancing the sensitivity of Jurkat cells to DEX.

[0080] The above examples demonstrate that the combination of isogarcinol and DEX described herein induces G1 cell cycle arrest in Jurkat cells by downregulating the expression of cycle-related proteins CDK4, CDK6, and CDK2, upregulating the expression of BIM and cleaved Caspase3, Caspase9, and PARP, downregulating Survivin expression to induce apoptosis, upregulating the expression of TSC22D3 and NR3C1 mRNA, and inhibiting the expression of c-Myc, p-PI3K, p-Akt, and p-mTOR, thereby enhancing the sensitivity of Jurkat cells to DEX. This indicates that isogarcinol described herein can be used as a potential small molecule to enhance DEX sensitivity, providing a research foundation for the development of drugs that enhance DEX sensitivity.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of isogarcinol compounds combined with glucocorticoids in the preparation of a drug for treating leukemia, characterized in that: The chemical formula of the isogarcinol compound is shown in Formula I: The molar ratio of the isogaminol compound to the glucocorticoid is 15:50; The leukemia is acute lymphocytic leukemia; the glucocorticoid is dexamethasone.

2. The use according to claim 1, characterized in that The isogaminol compound combined with glucocorticoid can enhance the sensitivity of Jurkat cells to glucocorticoid.

3. The use according to claim 1, characterized in that The isogaminol compound combined with glucocorticoid can induce G1 phase cell cycle arrest in Jurkat cells.

4. The use according to claim 1, characterized in that The isogaminol compound combined with glucocorticoid can induce Jurkat cell apoptosis.

5. A pharmaceutical composition for treating acute lymphoblastic leukemia, characterized in that: The invention comprises an isogarcinia phenol compound and a glucocorticoid; the molar ratio of the isogarcinia phenol compound to the glucocorticoid is 15:50; the glucocorticoid is dexamethasone; the chemical formula of the isogarcinia phenol compound is as shown in Formula I:

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