Use of carvedilol in preparing leukemia treatment drugs

Abstract

The present invention discloses the use of carvedilol in preparing a drug for treating leukemia, belongs to the field of medical technology, and the use of carvedilol in preparing a drug for treating leukemia provided by the present invention is found through the research of the present invention: carvedilol has a strong inhibitory effect on the proliferation of leukemia cell lines, and carvedilol inhibits the cell cycle process by blocking the cell cycle of the leukemia cell line G0 / G1 phase, and promotes the apoptosis of leukemia cells. In summary, the third-generation β-adrenergic receptor blocker carvedilol has important potential significance in the treatment field of leukemia.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of carvedilol in preparing a drug for treating leukemia. Background Art

[0002] Leukemia is a malignant tumor of the hematopoietic system and the most common malignant disease in children. The incidence of leukemia is increasing year by year. However, the disease is associated with many factors and the cause is still unclear. β-adrenergic receptor blockers are mainly used to treat cardiovascular diseases such as heart failure and hypertension. HuangQ et al. proposed that abnormal activation of the sympathetic nervous system leads to increased secretion of adrenaline and norepinephrine, activating the β-AR signaling pathway, and then promoting the occurrence and development of tumors. Therefore, we speculate that β-adrenergic receptor blockers may become a new drug for the treatment of leukemia. The anti-tumor effect of β-adrenergic receptor blockers has been reported in some studies, however, most of these studies focus on the first-generation β-blocker, propranolol, which can inhibit the proliferation of some tumor cells and promote their apoptosis. For example, propranolol has anti-gastric cancer and anti-breast cancer effects, and propranolol is also effective in treating infantile hemangiomas. In addition, propranolol has been found to inhibit the proliferation of leukemia cell lines. However, propranolol can only have a dose-dependent cytotoxic effect on leukemia cell lines (Molt-4, BALL-1, and U937) at a concentration of ≥200 μM. Our previous studies have also confirmed that propranolol can inhibit the proliferation of the acute lymphoblastic leukemia cell line CEM-C1, but its half-inhibitory concentration IC50 is greater than 100 μM. The concentration of this drug that inhibits the proliferation of leukemia cells is much higher than the clinical dose of 2 mg / kg / d (2 mg / kg / d is equivalent to an in vitro cell experimental dose of 67.61 μM). Therefore, this result shows that propranolol has a weak inhibitory effect on the proliferation of leukemia cells.

[0003] As a non-selective third-generation β-adrenergic receptor blocker, carvedilol is mainly used for clinical treatment and prevention of heart failure after chemotherapy. However, there are few studies on the anti-tumor effect of carvedilol. For example, carvedilol has been shown to inhibit the proliferation of a few solid malignant tumors, such as melanoma and cervical cancer cell lines, with a half-inhibitory concentration IC50>30μM. However, the pathogenesis and development process of leukemia are different from those of other malignant solid tumors, and there is no report on whether carvedilol has an inhibitory effect on the proliferation activity of leukemia cells, whether it has an anti-leukemia effect, and how strong its effect is. To this end, the present invention provides the use of carvedilol in the preparation of a drug for treating leukemia. Summary of the invention

[0004] The invention provides a use of carvedilol in preparing a drug for treating leukemia, which effectively inhibits the proliferation of leukemia cells, blocks the cell cycle of leukemia cells in the G0 / G1 phase, promotes the apoptosis of leukemia cells (including human lymphocytic leukemia cell lines CEM-C1, CEM-C1, BALL-1 and human myeloid leukemia cell lines THP-1, MV4-11, OCI-AML2), and inhibits the cell development process thereof.

[0005] The invention provides use of carvedilol in preparing a medicine for treating leukemia.

[0006] Preferably, carvedilol is used in the preparation of a medicament for promoting apoptosis of leukemia cells.

[0007] Preferably, carvedilol is used in the preparation of a drug for blocking the G0 / G1 phase cell cycle of the leukemia cells.

[0008] Preferably, the leukemia cells include lymphocytic leukemia cell lines and myeloid leukemia cell lines.

[0009] Preferably, the lymphocytic leukemia cell lines include CEM-C1, CEM-C1, and BALL-1.

[0010] Preferably, the myeloid leukemia cell lines include THP-1, MV4-11, and OCI-AML2.

[0011] Preferably, the drug is in solid, semisolid or liquid form.

[0012] Preferably, the drug preparation is an aqueous solution, a non-aqueous solution, a suspension, a lozenge, a capsule, a tablet, a granule, a pill or a powder.

[0013] Preferably, the drug is administered by injection or oral administration.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The use of carvedilol provided by the present invention in preparing a drug for treating leukemia, through the research of the present invention, it is found that carvedilol has a strong inhibitory effect on the proliferation of leukemia cell lines, and carvedilol inhibits the cell cycle process by blocking the cell cycle of the leukemia cell line G0 / G1 phase, thereby promoting the apoptosis of leukemia cells. In summary, the third-generation β-adrenergic receptor blocker carvedilol has important potential significance in the field of leukemia treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1 to 6The carvedilol of the present invention inhibits the activity of CEM-C1, CEM-C7, BALL-1, THP-1, MV4-11, and OCI-AML2 cell lines respectively;

[0017] Figure 7 This is a graph showing the activity of propranolol in the present invention in inhibiting CEM-C1 cell lines;

[0018] Figure 8 The half-inhibitory concentration value of propranolol of the present invention on CEM-C1 is higher than that of carvedilol;

[0019] Fig. 9 This is a diagram showing the YFR647A-AnnexinV / PI staining results of CEM-C1, CEM-C7, and BALL-1 cell lines of the present invention;

[0020] Fig.10 This is a diagram showing the YFR647A-AnnexinV / PI staining results of THP-1, MV4-11, and OCI-AML2 cell lines of the present invention;

[0021] Fig.11 This is a flow cytometry result diagram of carvedilol promoting apoptosis of CEM-C1, CEM-C7 and BALL-1 cell lines;

[0022] Fig.12 The flow cytometry results of carvedilol promoting apoptosis of THP-1, MV4-11 and OCI-AML2 cell lines according to the present invention are shown;

[0023] Fig.13 The carvedilol of the present invention has a strong effect of promoting apoptosis of leukemia cells, and is concentration-dependent;

[0024] Fig.14 This is a flow cytometry result diagram of carvedilol blocking the G0 / G1 phase of the CEM-C1, CEM-C7 and BALL-1 cell cycles of the present invention;

[0025] Fig.15 The carvedilol of the present invention has a strong effect of blocking the cell cycle of lymphocyte leukemia;

[0026] Fig.16 This is a flow cytometry result diagram of carvedilol blocking THP-1, MV4-11, and OCI-AML2 cell cycles at the G0 / G1 phase;

[0027] Fig.17 The carvedilol of the present invention has a strong effect of blocking the cell cycle of myeloid leukemia;

[0028] Fig.18 This is a comparison of the volume and weight of leukemia transplanted tumors in nude mice between the CVD group and the CON group of the present invention;

[0029] Fig.19 This is the apoptosis picture of H&E staining of leukemia transplanted tumor in the CVD group of the present invention;

[0030] Fig. 20 This is a picture of leukemia infiltration in the bone marrow of mice in the CVD group of the present invention;

[0031] Fig.21 This is a comparison chart of the percentage of leukemia cells in nude mice between the CVD group and the CON group of the present invention;

[0032] Fig. 22 The CVD of the present invention can effectively inhibit the growth of leukemia cells in nude mice;

[0033] Fig.23 This is a comparison chart of the number of leukocytes in nude mice between the CVD group and the CON group of the present invention. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention. The following test methods and detection methods, unless otherwise specified, are conventional methods; the reagents and raw materials, unless otherwise specified, are commercially available.

[0035] Example

[0036] Materials and Methods

[0037] Material

[0038] Drugs and reagents: Fetal bovine serum (FBS) was from BI (Israel), RPMI-1640 medium was purchased from Gibco (USA), cell counting kit-8 (CCK8) was purchased from dojindo (China), cell culture plates were purchased from NEST (China), pipette tips were purchased from Biosharp (China), YFR647A-Annexin V / PI apoptosis kit was purchased from an American company (China), DNA content quantification analysis method (cell cycle) was purchased from Solarbio (China), and carvedilol and propranolol were purchased from Topological Science (China).

[0039] Cell lines: Human lymphocytic leukemia cell lines CEM-C1 and CEM-C7 were obtained from West China Hospital of Sichuan University, and BALL-1 was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Leibniz Institute, Germany). Human myeloid leukemia cell lines THP-1, MV-4-11, and OCI-AML2 were purchased from Fuheng Cell Center (Shanghai, China).

[0040] Experimental methods

[0041] Drug preparation: Carvedilol and propranolol were dissolved in a small amount of DMSO, and then dissolved in RPMI-1640 medium and stored at -80°C. The two drugs were diluted in the medium and prepared into concentrations of 0 to 80 μM.

[0042] Cell culture: The six cell lines were maintained in RPMI-1640 medium containing 10% fetal bovine serum and incubated with different concentrations of carvedilol (0-80 μM) for 24 h and 48 h at 37°C in a 5% carbon dioxide incubator.

[0043] Inhibitory effect of carvedilol on proliferation of leukemia cells :

[0044] The cell counting kit (CCK8) method was used to detect the inhibitory effect of carvedilol on the viability of leukemia cells. Six types of cells were seeded in 96-well plates (20,000 cells per well) and then treated with carvedilol and propranolol. The concentrations of carvedilol were 0μM, 10μM, 20μM, 40μM, 60μM, and 80μM, respectively, and the concentrations of propranolol were 0μM, 20μM, 40μM, 80μM, 120μM, and 160μM, respectively. After incubation for 24h and 48h, 10μl of CCK8 activity detector was added to each well. The cells were incubated at 37℃ without light. After 2h, the plate was placed in an ELISA reader (BIO-BRI Chengdu, China) and detected at a wavelength of 450nm.

[0045] Cell apoptosis assay :

[0046] Annexin V / PI fluorescence staining was used to detect the effect of carvedilol (20 μM and 40 μM) on leukemia cell apoptosis. 5 The cells were seeded at a seeding density of 10 cells / ml in a 12-well plate and treated with carvedilol (20 μM, 40 μM) for 24 h. The cells were then collected and gently washed three times with PBS. 5 The cells were resuspended with 100uL 1x Annexin V binding buffer. Next, 5μl YFR647A Annexin V and 5μl propidium iodide (PI) were incubated with the cells at room temperature in the dark for 15min. Cell apoptosis was detected using an inverted microscope (Leica, Germany) and flow cytometer.

[0047] Cell cycle assay :

[0048] Flow cytometry was used to detect the effect of carvedilol (20 μM) on the leukemia cell cycle. 5The cells were seeded at a density of 10 cells / ml in a 12-well plate and treated with carvedilol (20 μM) for 24 h. The cells were then collected and gently washed twice with PBS. 6 Cells were resuspended in 500 μl of 75% precooled ethanol solution. After 2 h, the cells were washed once with precooled PBS and resuspended with 100 μl RNase A solution at 37°C for 0.5 h. Then, 400 μl of propidium iodide (PI) was added to the test tube and incubated at 4°C in the dark for 0.5 h. Finally, the cell cycle was determined using a flow cytometer (BD FACS Canto TM, USA).

[0049] Construction of in vivo leukemia model :

[0050] 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 raised in the SPF laboratory of Zunyi Medical University. One week later, 5 × 10 6 CEM-C1 cells (in the logarithmic growth phase) were subcutaneously injected into the posterior right axilla of each nude mouse. One week after transplantation, leukemia xenografts were formed, and the nude mice were randomly divided into two groups (3 mice in each group): a control group (CON group) and a carvedilol group (CVD group). When the leukemia xenografts reached about 0.5 cm, each nude mouse in the carvedilol group was gavaged with CVD (10 mg / kg / day) for two consecutive weeks; while the control group was given the same amount of water. The changes in the size of the xenografts were observed, and the apoptosis of leukemia cells in the xenografts and the degree of infiltration of leukemia cells in the bone marrow were detected by H&E staining. At the same time, the percentage of leukemia cells in the peripheral blood and the number of white blood cells were detected. This animal experiment protocol has been approved by Zunyi Medical University (Ethics Approval No.: ZMU21-2204-001)

[0051] Statistical analysis:

[0052] SPSS 18.0 (Zunyi Medical University) was used for statistical analysis. Data were expressed as mean ± standard deviation. One-way analysis of variance or unpaired t-test was used to analyze the differences, and P < 0.05 indicated that the differences were statistically significant.

[0053] Experimental Results

[0054] 1. Inhibitory effect of carvedilol on the proliferation activity of leukemia cells

[0055] Six human leukemia cells were treated with carvedilol for 24 h and 48 h. Figure 1-6As shown in the results, carvedilol can inhibit the proliferation of leukemia cells in a dose- and time-dependent manner. After 24 hours, the half-inhibitory concentrations of carvedilol for CEM-C1, CEM-C7 and BALL-1 lymphocytic leukemia cell lines were (13.80±4.51)μM, (7.93±0.91)μM and (23.78±2.32)μM, respectively. After 48 hours, the half-inhibitory concentrations of carvedilol for the above three lymphocytic leukemia cell lines were (3.58±1.02)μM, (1.76±0.40)μM and (15.84±2.00)μM, respectively. At the same time, after 24 hours of culture, the half-inhibitory concentrations of carvedilol on THP-1, MV4-11 and OCI-AML2 myeloid leukemia cell lines were (14.36±0.56)μM, (13.78±0.20)μM and (18.72±0.42)μM, respectively. After 48 hours, the half-inhibitory concentrations of carvedilol on the above three myeloid leukemia cell lines were (13.27±0.59)μM, (12.96±0.38)μM and (16.82±0.60)μM, respectively. It can be seen that the half-inhibitory concentration IC50 of carvedilol for all leukemia cells is less than 24μM, which is less than the maximum clinical dosage (1mg / kg / d, equivalent to a cell experimental dose of 24.6μM). Therefore, this shows that carvedilol has a strong inhibitory effect on leukemia proliferation activity. In addition, as a control, we also studied the inhibitory effect of propranolol (0, 20, 40, 80, 120 and 160 μM) on the proliferation of CEM-C1 cells. Figure 7 As shown in the figure, the half-inhibitory concentrations of propranolol on CEM-C1 after 24h and 48h were (100.17±23.95)μM and (114.54±25.73)μM, respectively. Figure 8 As shown in the results, carvedilol has a stronger inhibitory effect on the proliferation of leukemia cells than propranolol.

[0056] 2. Carvedilol promotes apoptosis of leukemia cells

[0057] Carvedilol can promote apoptosis of leukemia cells. Carvedilol (20 μM) was used to intervene in 6 human leukemia cell lines. After 24 hours, the YFR647A-AnnexinV / PI dual fluorescence method was used to study the effect of carvedilol on apoptosis of leukemia cells. All cells were collected according to the instructions of the YFR647A-AnnexinV / PI apoptosis kit. Leukemia cells were stained with AnnexinV and PI in the dark for 5 minutes. YFR647A-AnnexinV stains the cell membrane of early apoptotic cells, which is green fluorescence, and PI stains the nuclei of late apoptotic cells, which is red. Fig. 9 and Fig.10It can be seen that carvedilol promotes apoptosis of all leukemia cells. In addition, 6 human leukemia cell lines were intervened with carvedilol 20μM and 40μM, and their apoptosis rates were detected by flow cytometry after 24 hours. Fig.11 and Fig.13 It can be seen that carvedilol promotes apoptosis of all leukemia cells in a dose-dependent manner.

[0058] 3. Carvedilol blocks the cycle of leukemia cells

[0059] We performed cell cycle analysis on six human leukemia cell lines treated with 20 μM carvedilol for 24 h. Fig.14 , Fig.15 , Fig.16 and Fig.17 As shown, exposure of leukemia cells to carvedilol resulted in a prolongation of the G0 / G1 phase (P<0.05), which was associated with a decrease in the S phase. These data suggest that carvedilol inhibits the proliferation of leukemia cells by inhibiting the G0 / G1 phase of the cell cycle.

[0060] 4. Anti-leukemia effect of carvedilol in vivo

[0061] In the nude mouse leukemia transplantation model, CEM-C1 cells in the logarithmic growth phase were subcutaneously injected into the right axilla of 5-week-old nude mice; after the leukemia transplantation tumor was successfully modeled, the mice were divided into the CON group and the CVD group (10 mg / kg / d, continuous gavage for 2 weeks). Fig.18 As shown in Figure 2, compared with the carvedilol (CVD) group, the volume and weight of the transplanted tumors in the control group increased rapidly (P<0.05). Fig.19 As shown in Figure 2, H&E staining of the transplanted tumors showed that some leukemia cells in the CVD group underwent apoptosis and coagulative necrosis. Fig. 20 As shown in Figure 2, the leukemia infiltration in the BM of mice in the CVD group was low. At the same time, we identified the surface antigen of CEM-C1 and found that CD4 was the main antigen marker of CEM-C1 cells. We also detected the leukemia cells CEM-C1 in the peripheral blood of nude mice. The results are shown in Figure 2. Fig.21 and Fig. 22 As shown in Figure 2, the percentage of leukemia cells in the peripheral blood of nude mice in the CVD group was significantly reduced (P<0.05). Fig.23 As shown, the number of leukocytes in the peripheral blood of nude mice in the CVD group was also significantly decreased (P<0.05).

[0062] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of carvedilol in preparing a drug for treating leukemia, characterized in that: Carvedilol promotes apoptosis of leukemia cells, wherein the leukemia cells are lymphocytic leukemia cell lines and myeloid leukemia cell lines, and the leukemia cell lines are BALL-1, THP-1, MV4-11, and OCI-AML2.

2. The use according to claim 1, characterized in that The drug is in the form of solid, semisolid or liquid.

3. The use according to claim 1, characterized in that The drug preparation is an aqueous solution, a non-aqueous solution, a suspension, a lozenge, a capsule, a tablet, a granule, a pill or a powder.

4. The use according to claim 1, characterized in that The administration route of the drug is injection or oral administration.

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