Use of carvedilol in the preparation of drugs for reversing drug resistance of leukemia

Abstract

The application discloses a use of carvedilol in preparation of a medicine for reversing leukemia drug resistance, belongs to the technical field of medicines, and the medicine for reversing leukemia drug resistance refers to a medicine for reversing the drug resistance of leukemia to glucocorticoid drugs. The application of the carvedilol in preparation of the medicine for reversing leukemia drug resistance is provided, and it is found through the research of the application that the carvedilol can up-regulate the expression of TopoIIa by inhibiting beta3-AR / cAMP / PKA / STAT3, thereby reversing the resistance of T-ALL to dexamethasone.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of carvedilol in the preparation of drugs to reverse drug resistance in leukemia. Background Technology

[0002] Acute lymphoblastic leukemia (ALL) is a blood disorder characterized by abnormal differentiation and malignant proliferation of hematopoietic stem cells or lymphoid cells in the bone marrow. Currently, glucocorticoids, such as dexamethasone (DEX), are among the most important drugs in the treatment of acute T-lymphoblastic leukemia (T-ALL), but 20% of children develop resistance to this drug, which is a major cause of treatment failure and relapse. Therefore, reversing dexamethasone resistance in T-ALL would have significant clinical implications for leukemia treatment.

[0003] Beta-adrenergic receptors (β-ARs), classified as β1-AR, β2-AR, and β3-AR, have been found to be closely associated with cancer development and progression. Furthermore, most studies have reported the relationship between β-ARs and tumor proliferation, apoptosis, invasion, and angiogenesis. However, reports on the relationship between β-ARs and tumor drug resistance are limited. Calvani et al. found that β3-AR is associated with doxorubicin resistance in malignant tumors, but the specific mechanism remains unclear. Additionally, Feijun et al.'s research showed that β2-AR plays an important role in cisplatin resistance in ovarian cancer. This suggests that β-ARs may be related to cancer drug resistance. According to the Kyoto Encyclopedia of Genetics and Genomes (KEGG), activated β-ARs are recognized to directly or indirectly stimulate multiple signaling pathways, including the ras, PI3K / AKT / mTOR, and JAK2 / STAT3 signaling pathways, through cAMP / PKA or cAMP / Epac. Numerous studies have also shown that these signaling pathways are closely related to cancer drug resistance. Our previous studies have also confirmed that β-AR and STAT3 are highly expressed in T-ALL glucocorticoid-resistant cell lines. Therefore, the β3-AR / cAMP / PKA / STAT3 signaling pathway plays an important role in the resistance of T-ALL to DEX by regulating the expression of downstream drug resistance-related molecules. β-AR blockers reverse the resistance of T-ALL to DEX by blocking this signaling pathway.

[0004] Carvedilol (CVD), a third-generation β-adrenergic receptor blocker, is widely used in the treatment of heart failure and hypertension. It exhibits non-selective blocking activity against β-adrenergic receptors (β1-AR, β2-AR, and β3-AR). Currently, research on CVD focuses on its protective effect against chemotherapy-induced heart failure. However, some studies have found that CVD can reverse multidrug resistance (MDR) in tumors. Takara K and Kakumoto proposed that CVD may be a reversal agent for MDR in cervical cancer, while Jonsson O et al. suggested that it may act as an inhibitor of the resistance-associated transmembrane transporter p-glycoprotein (P-gp), reversing doxorubicin resistance in breast cancer. They also reported that CVD reversed doxorubicin resistance in bladder cancer cells. Therefore, it is well known that CVD can reverse tumor drug resistance. However, there are no reports on whether CVD can reverse resistance to DEX in T-ALL. Therefore, this invention provides the application of carvedilol in the preparation of drugs to reverse drug resistance in leukemia. Summary of the Invention

[0005] This invention provides the application of carvedilol in the preparation of drugs to reverse drug resistance in leukemia. Carvedilol upregulates the expression of TopoIIα by inhibiting β3-AR / cAMP / PKA / STAT3, thereby reversing the resistance of T-ALL to DEX.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the application of carvedilol in the preparation of drugs to reverse drug resistance in leukemia.

[0008] Preferably, the drug for reversing leukemia drug resistance refers to the drug that reverses the resistance of leukemia to glucocorticoids.

[0009] Preferably, the glucocorticoid drugs include dexamethasone and its derivatives, prednisone and its derivatives, and hydrocortisone and its derivatives.

[0010] Preferably, the leukemia is acute T-lymphoblastic leukemia.

[0011] Preferably, carvedilol reverses the resistance of acute T-lymphoblastic leukemia to glucocorticoids by upregulating the expression of TopoIIα through inhibition of β3-AR / cAMP / PKA / STAT3.

[0012] Preferably, the drug is in solid, semi-solid, or liquid form.

[0013] Preferably, the formulation of the drug includes aqueous solution, non-aqueous solution, suspension, lozenge, capsule, tablet, granule, pill or powder.

[0014] Preferably, the drug is administered via injection or oral administration.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The application of carvedilol provided by this invention in the preparation of drugs to reverse drug resistance in leukemia reveals that carvedilol upregulates the expression of TopoIIα by inhibiting β3-AR / cAMP / PKA / STAT3, thereby reversing the resistance of T-ALL to the glucocorticoid dexamethasone. Attached Figure Description

[0017] Figure 1 The present invention provides DEX treatment for the viability of CEM-C1 and CEM-C7 cells;

[0018] Figure 2 This invention demonstrates the induction of apoptosis in CEM-C1 and CEM-C7 cells by DEX.

[0019] Figure 3 To investigate the drug resistance of CEM-C1 cells treated with different concentrations of DEX using CVD according to this invention;

[0020] Figure 4 The present invention demonstrates the differential expression of β-AR in CEM-C1 and CEM-C7 cells;

[0021] Figure 5 This invention relates to the differential expression of JAK2 / STAT3 in CEM-C1 and CEM-C7 cells;

[0022] Figure 6 This invention illustrates the differential expression of different drug resistance proteins in CEM-C1 and CEM-C7 cells.

[0023] Figure 7 To enhance the viability of CEM-C1 and CEM-C7 cell lines treated with SR59230A of this invention;

[0024] Figure 8 The present invention provides sodium BRL 37344 treatment for the viability of CEM-C1 and CEM-C7 cell lines;

[0025] Figure 9 The expression differences between CEM-C1 and CEM-C7 were addressed using the 20μSR59230A of this invention;

[0026] Figure 10 To enhance the viability of CEM-C1 and CEM-C7 cell lines treated with the STAT3 inhibitor Static of this invention;

[0027] Figure 11To enhance the viability of CEM-C1 and CEM-C7 cell lines treated with Colivelin TFA according to this invention;

[0028] Figure 12 The present invention uses Colivelin TFA and Static to treat the expression differences of CEM-C1 and CEM-C7, respectively;

[0029] Figure 13 The present invention describes the treatment of CEM-C1 and CEM-C7 cell lines with bisoprolol hemifumarate, ICI 118551 hydrochloride, and Amonafide to enhance cell viability.

[0030] Figure 14 The present invention describes the resistance of CEM-C1 cells to bisoprolol hemifumarate, hydrochloride, SR59230A, and BRL 37344 sodium treatment by DEX.

[0031] Figure 15 This invention relates to the resistance of DEX cells to Static and Colivelin TFA treatments.

[0032] Figure 16 This invention relates to the resistance of DEX cells to Amonafide treatment.

[0033] Figure 17 The expression differences of β3-AR, cAMP, PKA, STAT3 and p-STAT3 after CVD treatment of CEM-C1 and CEM-C7 in this invention;

[0034] Figure 18 The images show the volume and weight of xenograft tumors in the DEX-C7 group and the CVD+DEX-C1 group of this invention.

[0035] Figure 19 The results of H&E staining of some xenografted cells in the DEX-C7 group and DEX+CVD-C1 group of this invention;

[0036] Figure 20 This invention relates to the bone marrow leukemia infiltration in mice in the DEX-C7 group and CVD+DEX-C1 group of the present invention.

[0037] Figure 21 This is an expression diagram of the CDs antigen of the present invention;

[0038] Figure 22 This is a flow cytometry analysis diagram of the present invention. Detailed Implementation

[0039] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.

[0040] Example

[0041] Materials and Methods

[0042] Material

[0043] Drugs and reagents:

[0044] Carvedilol (CVD) acquired TopScience (Shanghai, China) CAS No.: 72956-09-3, with the following structural formula:

[0045]

[0046] Bisoprolol hemifumarate (β1-AR blocker), ICI 118551 hydrochloride (β2-AR blocker), SR59230A (β3-AR blocker), BRL 37344 sodium (β3-AR activator), Static (STAT3 inhibitor), Colivelin TFA (STAT3 activator), Amonafide (TopoII inhibitor), and dexamethasone (DEX) were all purchased from MCE (Shanghai, China).

[0047] RPMI-1640 medium was purchased from Gibco (Grande Island, New York, USA);

[0048] Fetal bovine serum (FBS) was obtained from Livining Biotechnology Co., Ltd. (Beijing, China);

[0049] Cell-Counting Kit-8 (CCK8) was purchased from Dojindo (Beijing, China);

[0050] DNA content quantification analysis (cell cycle) was purchased from Solarbio (Beijing, China);

[0051] The apoptosis kit was purchased from US EVERBRIGHT (Suzhou, China);

[0052] The β1-AR, β2-AR, and β3-AR antibodies were obtained from Abbkine Scientific Co., Ltd (Santa Cruz, CA, USA).

[0053] cAMP, JAK2, STAT3, p-STAT3 and β-actin antibodies were purchased from Abcam (Cambridge, MA, USA);

[0054] The PKA, P-gp, MRP, TopoIIα, β-Tubulin, and GAPDH antibodies were all from Huabio (Hangzhou, China).

[0055] Experimental methods

[0056] Drug preparation: DEX and other drugs were dissolved in a small amount of DMSO and then diluted in RPMI-1640 medium and stored at -80℃. Before use, the drugs were diluted in the medium to prepare a concentration of 10-80 μM.

[0057] Cell culture: Human T-ALL cell lines CEM-C1 and CEM-C7 were obtained from West China Hospital of Sichuan University and were stored in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator.

[0058] Cell viability assay methods:

[0059] The inhibitory effect of carvedilol on leukemia cell viability was detected using the CCK8 assay. In 96-well plates (20,000 cells per well), the following assays were performed:

[0060] CVD (0, 10, 20, 40, 60 and 80 μM) treatment for 24 h and 48 h;

[0061] DEX (0, 0.5, 1, 5, 10, 20, 50, 100 and 200 μM) were applied for 24 h, 48 h and 72 h;

[0062] Bisoprolol hemifumarate, ICI 118551 hydrochloric acid, and SR59230A (0, 10, 20, 40, 60, and 80 μM) were used for 24 h and 48 h.

[0063] BRL 37344 sodium (0, 2.5, 5, 10, 20 and 50 μM) were applied for 6 h and 24 h;

[0064] Static and Colivelin TFA (0, 0.5, 1, 5, 10 and 20 μM) were applied for 24 h and 48 h;

[0065] Amonafide (0, 1, 5, 10, 20 and 50 μM) was applied for 24 h and 48 h;

[0066] Then, add 10 μL CCK8 to each well, incubate at 37°C for 2 hours in the dark, and then place the 96-well plate in a microplate reader (BIO-BRI Chengdu, China) for detection at a wavelength of 450 nm.

[0067] Experimental methods for apoptosis:

[0068] The effect of dexamethasone (1 μM and 5 μM) on apoptosis of leukemia cells was detected by Annexin V / PI fluorescence staining. Cells were cultured at 2 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of [number] cells / ml in 12-well plates and incubated with dexamethasone (1 μM and 5 μM) for 24 h and 48 h, respectively. Cells were then collected and gently washed three times with PBS. Finally, 2 × 10⁶ cells were collected. 5 Cells were resuspended in 100 μl of 1x Annexin V binding buffer, and then incubated with 5 μL of LYFR647A-Annexin V and 5 μL of propidium iodide (PI) at room temperature in the dark for 15 min. Apoptosis was detected by flow cytometry (BD FACSCanto, San Jose, CA, USA).

[0069] Western blot analysis method:

[0070] CEM-C1 and CEM-C7 cells were treated with 20 μM CVD, 20 μM SR59230, 20 μM MBRL 37344 sodium, 0.5 μM Static, and 5 μM Olivelin TFA for 24 h, respectively. After centrifugation at 1200 rpm for 5 min, the supernatant was removed, and RIPA lysis enhancement buffer containing PMSF (100:1) was added. Then, the cells were centrifuged at 12000 g for 30 min at 4 °C, and the supernatant was removed. Protein samples were quantified using the BCA method, adjusted to 2.3 μg / μl, and loading buffer was added.

[0071] Equal volumes of protein lysates were decomposed using 10% PAGE and transferred to an electrophoresis membrane using a BIO-RAD semi-dry transfer system. The membrane was then blocked with 5% skim milk at room temperature for 60 min and incubated overnight at 4°C with appropriate concentrations of primary antibodies (1:1000 GAPDH, β-actin, β-tubulin, β1-AR, β2-AR, β3-AR, cAMP, PKA, TopoIIα, P-gp, MRP; 1:2000 JAK2, STAT3, and P-STAT3). The membrane was then washed three times with TBST and incubated with secondary antibody (1:10000) for 60 min, followed by three washes. The membrane was then placed in ECL luminescent reagent for 10 seconds and scanned using a gel imaging system (Bio-Rad, Hercules, CA, USA).

[0072] Construction of the T-ALL in vivo model:

[0073] The animal testing protocol has been approved by Zunyi Medical University (ethics approval number: ZMU21-2204-001) and is conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and related guidelines, as well as EU Directive 2010 / 63 / EU.

[0074] Four-week-old female nude mice, weighing 17.0–20.0 g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (strain: BALB / c-nu, batch number: 202210375, license: SCXK(Su)2021-0013). These mice were housed for one week in the SPF laboratory of Zunyi Medical University. CEM-C1 and CEM-C7 cells in the logarithmic growth phase were washed twice with PBS, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 7 Cells / mL were administered by subcutaneous injection of 100 μl of cell suspension into the posterior right axilla of each mouse. One week after transplantation, xenograft tumors formed. Nude mice injected with CEM-C1 cells were randomly divided into four groups (n=3 per group): control group (CON-C1 group), carvedilol group (CVD-C1 group), dexamethasone group (DEX-C1 group), and combined treatment group (CVD+DEX-C1 group). Nude mice injected with CEM-C7 cells were randomly divided into two groups (n=3 per group): control group (CON-C7 group) and dexamethasone group (DEX-C7 group). When the leukemia xenograft reached approximately 0.5 cm, CVD was administered via gavage at 1 mg / kg / day; DEX was administered via intraperitoneal injection at 20 mg / kg / day. The control group received an equal volume of physiological saline. Two weeks after administration, we compared the size and weight of the leukemia xenograft, the percentage of CEM-C1 and CEM-C7 cells in peripheral blood, and the total white blood cell count.

[0075] Statistical analysis:

[0076] Statistical analysis was performed using SPSS 18.0 (Zunyi Medical University, China). The half-inhibitory concentration (WIC) of the drug was determined using Compusyn software. Data are expressed as mean ± standard deviation. One-way ANOVA or unpaired t-tests were used to assess differences. A p-value < 0.05 was considered statistically significant.

[0077] result

[0078] 1. Identification of DEX resistance in CEM-C1 and CEM-C7 cell lines

[0079] CEM-C1 and CEM-C7 cells were treated with DEX at concentrations ranging from 0.5 to 200 μM for 24 h, 48 h, and 72 h, respectively. Figure 1As shown in the figure, CCK8 analysis revealed that dexamethasone significantly inhibited the activity of CEM-C7 cells compared to CEM-C1 cells (P<0.05). Flow cytometry was also used to analyze the apoptosis-inducing effects of 1 μM and 5 μM DEX on CEM-C1 and CEM-C7 cells. Figure 2 The effect of dexamethasone on inducing apoptosis in CEM-C1 and CEM-C7 cells. Figure 2 In Figure a, CEM-C1 and CEM-C7 induction for 24 hours was used; in Figure b, CEM-C1 and CEM-C7 induction for 48 hours was used. Figure 2 As shown in Figures a and b, compared with CEM-C1 cells, DEX did not significantly induce apoptosis in CEM-C1 cells, but significantly promoted apoptosis in CEM-C7 cells (P<0.05). Therefore, CEM-C1 cells are resistant to dexamethasone, while CEM-C7 cells are sensitive to dexamethasone.

[0080] 2. CVD reverses CEM-C1 cell resistance to DEX.

[0081] Two cell lines were treated with CVD at concentrations ranging from 10 to 80 μM for 24 h and 48 h, respectively. CVD reduced cell viability. The half-maximal inhibitory concentrations (IC50) of CVD on CEM-C1 and CEM-C7 cells at 24 h were 16.80 ± 4.51 μM and 12.35 ± 1.42 μM, respectively, and the IC50 at 48 h were 7.68 ± 1.35 μM and 7.27 ± 1.29 μM, respectively. Figure 2 (as shown in c).

[0082] CEM-C1 cells were then treated with non-inhibitory concentrations (less than IC50) of CVD combined with different concentrations of DEX for 24 h and 48 h. Figure 3 To investigate the effect of 2 μM and 5 μM CVD combined with different concentrations of DEX on the viability of CEM-C1 cells using the CCK8 assay. Figure 3 In the figures, a represents the fold change in resistance to DEX after 24 hours of CVD+DEX treatment, b represents the fold change after 24 hours of CVD+DEX treatment, c represents the fold change after 48 hours of CVD+DEX treatment, and d represents the fold change after 48 hours of CVD+DEX treatment. The results show that both 2 μM and 5 μM CVD can reverse the resistance of CEM-C1 to DEX, with reversal fold changes of 3.71 and 2.24 after 24 hours, and 2.32 and 2.28 after 48 hours, respectively.

[0083] 3. Differences in the levels of β3-AR, STAT3, and TopoIIα proteins between CEM-C1 and CEM-C7 cells.

[0084] To further explore the mechanisms by which CVD reverses DEX resistance in CEM-C1 cells, this invention preliminarily examined the expression differences of β-AR protein in CEM-C1 and CEM-C7 cells. Figure 4 The expression of β-AR differs between CEM-C1 and CEM-C7 cells. Figure 4 As shown, compared with CEM-C7 cells, β3-AR protein was significantly highly expressed in CEM-C1 cells (P<0.05), while there was no significant difference in the expression of β1-AR and β2-AR proteins between the two cell types (P>0.05).

[0085] The levels of JAK2 and STAT3 proteins were detected. Figure 5 The expression differences of JAK2 / STAT3 in CEM-C1 and CEM-C7 cells were observed. Figure 5 As shown, compared with CEM-C7 cells, STAT3 protein was significantly highly expressed in CEM-C1 cells (P<0.05), while JAK2 protein showed no significant difference between the two cell lines (P>0.05).

[0086] In addition, we also examined the expression levels of drug resistance-related proteins. Figure 6 The expression differences of different drug resistance proteins in CEM-C1 and CEM-C7 cells. For example... Figure 6 As shown, compared with CEM-C7 cells, we found that only TopoIIα protein expression was significantly reduced in CEM-C1 cells (P<0.05), while MRP and P-gp proteins showed no significant difference between the two cell lines (P>0.05).

[0087] 4. Validation of the β3-AR / STAT3 / TopoIIα signaling pathway

[0088] First, to determine whether β3-AR can regulate STAT3, CEM-C1 and CEM-C7 cell lines were treated with SR59230A (β3-AR blocker) at concentrations of 10 μM to 80 μM for 24 h and 48 h, respectively, while CEM-C1 and CEM-C7 cell lines were treated with BRL37344sodium (β3-AR activator) at concentrations of 2.5 μM to 50 μM for 6 h and 24 h, respectively. Figure 7 Image showing the viability of CEM-C1 and CEM-C7 cell lines treated with SR59230A. Figure 7 In the diagram, a represents CEM-C1, and b represents CEM-C7; Figure 8 Image showing the cell viability of CEM-C1 and CEM-C7 cell lines treated with BRL 37344 sodium. Figure 8 In the diagram, a represents CEM-C1, and b represents CEM-C7. For example... Figure 7 and Figure 8As shown, CCK8 analysis revealed that SR59230A reduced the viability of both CEM-C1 and CEM-C7 cell lines, while BRL 37344 sodium promoted the viability of both cell lines. Subsequently, changes in STAT3 protein levels were detected after treating CEM-C1 and CEM-C7 cells with 20 μM SR59230A and BRL 37344 sodium for 24 h, respectively. Figure 9 As shown in the figure, in CEM-C1 cells, STAT3 was significantly decreased in the SR59230A group compared to the control group (P<0.05); while in CEM-C7 cells, STAT3 was increased in the BRL37344sodium group compared to the control group (P<0.05). This indicates that β3-AR can positively regulate STAT3 expression.

[0089] Secondly, the CEM-C1 and CEM-C7 cell lines were treated with the STAT3 inhibitor Static at concentrations of 0.5 μM to 20 μM for 24 h and 48 h, respectively, and with Colivelin TFA (STAT3 activator) at concentrations of 0.5 μM to 20 μM for 6 h and 24 h. Figure 10 A graph showing the cell viability of CEM-C1 and CEM-C7 cell lines treated with the STAT3 inhibitor Static. Figure 10 In the diagram, a represents CEM-C1, and b represents CEM-C7; Figure 11 Image showing the viability of CEM-C1 and CEM-C7 cell lines treated with Colivelin TFA. Figure 11 In the diagram, a represents CEM-C1, and b represents CEM-C7. For example... Figure 10 and Figure 11 As shown, CCK8 assay analysis indicated that Static inhibited the viability of both cell lines, while Colivelin TFA promoted viability. Subsequently, changes in TopoIIα protein were detected after treating CEM-C1 and CEM-C7 with 5 μM Colivelin TFA and Static for 24 h, respectively. Figure 12 As shown, Western blot analysis revealed that, compared with the control group, TopoIIα was significantly decreased in the Colivelin TFA group (P<0.05), while it was significantly increased in the Static group (P<0.05). This indicates that STAT3 can negatively regulate the expression of TopoIIα.

[0090] In summary, β3-AR positively regulates STAT3, and then negatively regulates Topo I Iα.

[0091] 5. CVD reverses CEM-C1 resistance to DEX by inhibiting β3-AR / cAMP / PKA / STAT3 and upregulating TopoII.

[0092] First, the CEM-C1 and CEM-C7 cell lines were treated for 24 h and 48 h with bisoprolol hemifumarate (β1-AR blocker) and ICI 118551 hydrochloride (β2-AR blocker) at concentrations of 10–80 μM and Amonafide (TopoII inhibitor) at concentrations of 1–50 μM, respectively. Figure 13 The images show the cell viability of CEM-C1 and CEM-C7 cell lines treated with bisoprolol hemifumarate, ICI 118551 hydrochloride, and Amonafide, respectively. Figure 13 In this context, a represents bisoprolol hemifumarate, b represents ICI 118551 hydrochloride, and c represents amonafide. (Example) Figure 13 As shown, the CCK8 results indicated that ICI 118551 hydrochloride and Amonafide inhibited the viability of both cell lines, while bisoprolol hemifumarate did not alter cell viability.

[0093] Secondly, the CCK8 assay was used to detect changes in DEX resistance in CEM-C1 cells after intervention with three β-AR blockers, and the β3-AR activator was used to detect changes in DEX sensitivity in CEM-C7 cells after intervention with β3-AR activator. Figure 14 In the given information, a is the β1-AR blocker bisoprolol hemifumarate, b is the β2-AR blocker ICI 118551 hydrochloride, c is the β3-AR blocker SR59230A, and d is the β3-AR activator BRL 37344 sodium. Figure 14 As shown in Figure 1, CEM-C1 cells were treated with 20 μM bisoprolol hemifumarate, 20 μM ICI 118551 hydrochloride, and 20 μM SR59230A for 24 hours, respectively, followed by treatment with 10 μM DEX for 24 h and 48 h. CCK8 results showed that only SR59230A treatment reduced CEM-C1 cell resistance to DEX (P<0.05), while bisoprolol hemifumarate and ICI 118551 hydrochloride did not reduce CEM-C1 cell resistance to DEX. Meanwhile, as shown in Figure 2, CEM-C1 cells were treated with 20 μM bisoprolol hemifumarate, 20 μM ICI 118551 hydrochloride, and SR59230A for 24 hours, respectively, followed by treatment with 10 μM DEX for 24 h and 48 h. Figure 14 As shown in Figure d, treatment with BRL 37344 sodium (β3-AR activator) slightly reduced the sensitivity of CEM-C7 cells to DEX. This indicates that β3-AR is positively correlated with CEM-C1 resistance to DEX.

[0094] Next, CEM-C1 cells were treated with 0.5 μM Static (STAT3 inhibitor) and CEM-C7 cells were treated with 1 μM Colivelin TFA (STAT3 activator) for 24 h, respectively, and then both cell lines were treated with 10 μM DEX. Figure 15 The changes in resistance and sensitivity to DEX after treating the two cell lines with Static and Colivelin TFA, respectively. Figure 15 In the table, 'a' represents Static processing of CEM-C1, and 'b' represents Colivelin TFA processing of CEM-C7. For example... Figure 15 As shown, Static treatment reduced the resistance of CEM-C1 cells to DEX (P<0.05), while Colivelin TFA treatment slightly reduced the sensitivity of CEM-C7 cells to DEX. This indicates that STAT3 is also positively correlated with the resistance of CEM-C1 cells to DEX.

[0095] Then, CEM-C1 and CEM-C7 cells were treated with 5 μM amonafide (a TopoII inhibitor) for 24 h, followed by treatment with 10 μM DEX. Figure 16 Changes in DEX resistance and sensitivity of CEM-C1 and CEM-C7 cells after treatment with Amonafide. Figure 16 In the table, 'a' represents Amonafide treatment of CEM-C1, and 'b' represents Amonafide treatment of CEM-C7. For example... Figure 16 As shown, CEM-C1 cells exhibited significantly increased resistance to DEX (P<0.05), while CEM-C7 cells showed significantly decreased sensitivity to DEX (P<0.05), indicating that they had developed resistance to DEX. This suggests that TopoII is a key target for DEX resistance in CEM-C1 cells.

[0096] Finally, after intervening CEM-C1 and CEM-C7 cells with 20 μM CVD and BRL 37344 Sodium, respectively, Western blot was used to detect the expression of key proteins in the β3-AR / cAMP / PKA / STAT3 / TopoII signaling pathway. Figure 17 To investigate the differences in the expression of β3AR, cAMP, PKA, STAT3, and p-STAT3 after CVD treatment of CEM-C1 and CEM-C7, Figure 17 In the image, a represents CVD intervention in CEM-C1 cells, and b represents BRL 37344 sodium intervention in CEM-C7 cells. Figure 17As shown, compared with the control group, β3-AR, cAMP, PKA, STAT3, and P-STAT3 were significantly decreased in CEM-C1 cells of the CVD group (P<0.05), while TopoIIα was significantly increased (P<0.05). Meanwhile, compared with the control group, β3-AR, cAMP, PKA, STAT3, and P-STAT3 were significantly increased in CEM-C7 cells of the BRL 37344sodium group (P<0.05), while TopoIIα was significantly decreased (P<0.05).

[0097] In summary, the β3-AR / cAMP / PKA / STAT3 / TopoII signaling pathway plays an important role in CEM-C1 resistance to DEX. CVD can reverse CEM-C1 resistance to DEX. The mechanism is that CVD upregulates TopoII expression after inhibiting β3-AR / cAMP / PKA / STAT3, thereby reversing CEM-C1 resistance to DEX.

[0098] 6. In vivo CVD reverses CEM-C1 resistance to DEX in mice

[0099] First, in vivo experiments were conducted to investigate the reversal of CEM-C1 resistance to DEX by CVD. Mice injected with CEM-C7 cells one week after leukemia xenograft formation were randomly divided into a control group (CON-C7 group) and a DEX group (DEX-C7 group). Mice injected with CEM-C1 cells were randomly divided into a control group (CON-C1 group), a CVD group (CVD-C1 group), a DEX group (DEX-C1 group), and a combination therapy group (CVD+DEX-C1). Two weeks after drug administration, Figure 18 Images showing the volume and weight of xenografts in each group, such as... Figure 18 As shown, compared with the DEX-C7 group, the volume and weight of xenografts increased rapidly in the CON-C7 group (P<0.05), and compared with the CVD+DEX-C1 group, the volume and weight of xenografts increased rapidly in the CON-C1, CVD-C1, and DEX-C1 groups (P<0.05). However, there was no difference among the CON-C1, CVD-C1, and DEX-C1 groups (P>0.05). Secondly, Figure 19 The H&E staining results for each group of xenograft tumors are shown. Figure 19 As shown, some xenografted cells in the DEX-C7 group and the DEX+CVD-C1 group underwent apoptosis and coagulative necrosis. Figure 20 The bone marrow (BM) leukemia infiltration status of each group of mice. For example... Figure 20 As shown, mice in the DEX-C7 group and the CVD+DEX-C1 group had lower myeloid leukemia infiltration.

[0100] at last, Figure 21 The expression diagram of CDs antigens, such as... Figure 21As shown, flow cytometry was used to detect the expression of CDs antigens in two cell lines, indicating that CD4 was the most highly expressed surface marker. Figure 22 This is a flow cytometry analysis diagram. Figure 22 In the diagram, a represents mouse peripheral blood, and b represents mouse bone marrow. Figure 22 As shown, flow cytometry analysis also confirmed that leukemia had infiltrated the peripheral blood and bone marrow (BM) of mice. The percentage of CEM-C7 cells in the DEX-C7 group was significantly lower than that in the CON-C7 group (P<0.05), and the percentage of CEM-C1 cells in the CVD+DEX-C1 group was significantly lower than that in the other groups (P<0.05). However, there was no significant difference in the percentage of leukemia cells between the DEX-C7 group and the CVD+DEX-C1 group.

[0101] In summary, this indicates that CVD can reverse CEM-C1 resistance to DEX in mice.

[0102] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of carvedilol in the preparation of drugs to reverse drug resistance in leukemia, characterized in that, The drug that reverses leukemia drug resistance refers to reversing the resistance of leukemia to glucocorticoid drugs, including dexamethasone, prednisone, and hydrocortisone, and the leukemia is acute T-lymphoblastic leukemia. Carvedilol reverses the resistance of acute T-lymphoblastic leukemia to glucocorticoid drugs by upregulating the expression of TopoIIα by inhibiting β3-AR / cAMP / PKA / STAT3.

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