Application of actinomycin V in preparation of medicine for treating chronic myelogenous leukemia

Abstract

The invention relates to the technical field of medicines, in particular to application of actinomycin V in preparation of a medicine for treating chronic myelogenous leukemia. In the aspect of cell function, the Act-V shows comprehensive anti-CML activity, not only can inhibit K562 cell proliferation in a time and dose dependent manner, but also can effectively inhibit colony forming ability, reduce the number and diameter of formed tumor spheres and lower the sphere forming frequency. Experiments prove that the Act-V further weakens the dry characteristics of tumors by down-regulating the expression of dry markers such as CD133, CD44, ALDH1A1 and the like. Besides, the Act-V can significantly inhibit the invasion and migration ability of CML cells, and western blot experiment results prove that the Act-V down-regulates the expression of interstitial markers N-Cadherin and Vimentin, up-regulates the expression of an epithelial marker E-Cadherin and inhibits the invasion and migration ability, so that the Act-V has wide application scenarios.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of actinomycin V in the preparation of drugs for the treatment of chronic myeloid leukemia. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder driven by the BCR-ABL1 fusion gene, which originates from a genetic abnormality in hematopoietic stem cells. Leukemia stem cells (LSCs), especially chronic myeloid leukemia stem cells (CML-LSCs), play a central role in the occurrence, progression, and treatment resistance of CML. CML-LSCs not only possess the ability to self-renew and differentiate, but also relapse and progress by maintaining a stem state, evading immune surveillance, and resisting tyrosine kinase inhibitor (TKI) therapy. The survival and drug resistance mechanisms of CML-LSCs involve multiple levels. In the bone marrow microenvironment, factors secreted by mesenchymal stem cells provide a survival advantage. Simultaneously, CML-LSCs further enhance their drug resistance through epigenetic reprogramming mechanisms such as DNA methylation and histone modification. CML-LSCs are the root cause of disease occurrence, progression, and drug resistance relapse; their phenotype partially overlaps with normal hematopoietic stem cells, both expressing Lin. - CD34 + CD38 - The phenotype allows them to remain in a quiescent state for extended periods, evading the killing effects of TKIs on proliferating cells. CML-LSCs maintain clonal advantage through abnormal self-renewal and differentiation arrest, and their functional abnormalities are closely related to downstream signaling pathway disorders and the expression of specific biomarkers.

[0004] Actinomycin V (Act-V) is derived from Streptomyces and is a structural analog of the known drug actinomycin D (Act-D). Act-V exhibits both antitumor and antibacterial activities, and compared to Act-D, its hepatotoxicity and nephrotoxicity are significantly reduced, thus it is considered a promising anticancer drug candidate. Currently, research on Act-V is still in its early stages and has not yet entered clinical trials or practical applications. Summary of the Invention

[0005] In view of this, the present invention provides the use of actinomycin V in the preparation of a drug for treating chronic myeloid leukemia.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides the use of actinomycin V in the preparation of a drug for treating chronic myeloid leukemia. The structural formula of Actinomycin V is as follows:

[0007] In some embodiments, actinomycin V is the sole active ingredient in the drug.

[0008] In some implementations, actinomycin V is used alone.

[0009] In some implementations, the concentration of Act-V is 0.5-1.5 nM.

[0010] In some implementations, the Act-V treatment time for cells is 24-72 h.

[0011] In some implementations, Act-V can more significantly reduce the proliferative activity of CML cells compared to Act-D.

[0012] In some implementations, Act-V exhibits low cytotoxicity to normal hepatocytes while exerting its anti-CML effect.

[0013] In some implementations, the inhibition of CML cell proliferation by Act-V exhibits a time-dependent characteristic.

[0014] In some implementations, Act-V effectively inhibits the colony-forming ability of CML cells.

[0015] This invention uses the CCK8 assay to detect the effects of Act-V and Act-D on the proliferation activity of K562 chronic myeloid leukemia cells and QSG7701 normal hepatocytes 48 h after administration. The results showed that, compared to Act-D, Act-V significantly reduced the proliferation activity of leukemia cells and decreased the number of viable cells. More importantly, Act-V exhibited low cytotoxicity to normal hepatocytes while exerting its anti-leukemic effect. The results showed that after 48 h of treatment with Act-V, the cell viability of normal hepatocytes remained at a high level, while Act-D showed a more significant toxic effect on normal hepatocytes. Results at 24 h, 48 h, and 72 h after drug administration showed that Act-V had a significant inhibitory effect on K562 cell proliferation, exhibiting a time-dependent characteristic. This combination of potent tumor suppression and low normotoxicity gives Act-V a broader therapeutic window, ensuring sufficient therapeutic concentrations to exert its anti-leukemia effects while reducing damage to normal tissues such as the liver, thus lowering the risks associated with clinical use.

[0016] The ability of tumor cells to form colonies can also reflect their proliferative capacity. This invention investigates the effect of Act-V on the colony-forming ability of chronic myeloid leukemia (CML) cells using a suspension cell colony formation assay. Act-V (0, 0.5, 1, and 1.5 nM) was designed for K562 cell colony formation. Compared with the control group, the number of colonies in the Act-V treatment group was significantly reduced in a dose-dependent manner, indicating that Act-V can affect the self-renewal and proliferative capacity of leukemia cells and effectively inhibit the colony-forming ability of K562 cells.

[0017] In some implementations, Act-V has a significant inhibitory effect on the ability of CML cells to form tumor spheres.

[0018] In some implementations, Act-V can effectively target the stem phenotype of CML cells and inhibit the microsphere formation frequency.

[0019] In some implementations, Act-V can disrupt the stemness characteristics of tumor cells by downregulating the protein expression of core tumor stemness markers.

[0020] K562 cells were cultured using a serum-free culture method to simulate the stem cell microenvironment. Two weeks after drug administration, the diameter of tumor spheres at each concentration gradient was measured, and the number of tumor spheres was calculated. Compared with the control group, the diameter of individual tumor spheres in both cell types was significantly reduced and the number of tumor spheres formed was significantly decreased after Act-V treatment, and this inhibitory effect showed a concentration-dependent trend.

[0021] Then, this invention investigated its regulatory effect on the microsphere formation frequency of K562 cells using a limiting dilution experiment. Microsphere formation frequency, as a key indicator for assessing the self-renewal capacity and stemness characteristics of tumor cells, directly reflects the potential of cells to form clonal microspheres. After Act-V treatment, the microsphere formation frequency of K562 cells was significantly reduced compared to the control group, and showed a concentration-dependent inhibitory trend.

[0022] Finally, Western blotting was used to detect changes in the protein expression of tumor stem markers CD133, CD44, and ALDH1A1 after Act-V treatment. The expression levels of CD133, CD44, and ALDH1A1 proteins in K562 cells were significantly reduced in the Act-V treatment group, showing a concentration-dependent inhibitory trend.

[0023] In some implementations, Act-V can significantly inhibit the invasion and migration of CML cells in vitro.

[0024] In some implementations, Act-V concentration-dependently inhibits the invasion and migration of CML cells.

[0025] In some implementations, Act-V can significantly regulate the expression of key factors in the EMT pathway.

[0026] In this invention, we investigated the effects of Act-V on the invasion and metastasis of CML cells. In the experiment, K562 cells were treated with different concentrations of Act-V (0, 0.5, 1, and 1.5 nM) using Transwell assays and Matricella invasion assays, and changes in cell behavior were then examined. The Transwell assay results showed that, compared with the blank control group, the number of K562 cells treated with Act-V that migrated through the polycarbonate membrane was significantly reduced, and the number of cells that invaded through the Matricella was also significantly decreased, with statistically significant differences.

[0027] Epithelial-mesenchymal transition (EMT) is a biological process in which cells lose their epithelial phenotype and acquire mesenchymal characteristics. Its abnormal activation is associated with tumor invasion and metastasis. Western blotting was used to investigate the effects of Act-V on the expression of EMT-related molecules and genes. The results showed that Act-V significantly regulates the expression of key factors in the EMT pathway.

[0028] Act-V effectively inhibited the expression of N-cadherin and vimentin, EMT-related mesenchymal markers, in K562 cells, while significantly promoting the expression of E-cadherin, an epithelial marker. Downregulation of E-cadherin, a core molecule in intercellular junctions, is a key marker of EMT initiation, while upregulation of N-cadherin and vimentin are important characteristics of cells acquiring mesenchymal properties and enhancing their migration and invasion capabilities. Act-V's regulatory effect on these three proteins directly reversed the EMT phenotype of K562 cells, suggesting that it may weaken the malignant biological behavior of leukemia cells by inhibiting the EMT process.

[0029] In a second aspect, the present invention provides a medicament for treating chronic myeloid leukemia, wherein the active pharmaceutical ingredient comprises actinomycin V.

[0030] In some implementations, the concentration of Act-V is 0.5-1.5 nM.

[0031] Compared with the prior art, the present invention has achieved the following beneficial effects: Act-V significantly reduced the proliferative activity of leukemia cells and decreased the number of viable cells, exhibiting a time-dependent effect. More importantly, while exerting its anti-leukemic effect, Act-V showed low cytotoxicity to normal hepatocytes. Act-V effectively inhibited the colony-forming ability of K562 cells, significantly suppressing the formation of tumor spheres in K562 cells. Act-V effectively targeted the stem phenotype of chronic myeloid leukemia cells, inhibiting the frequency of microsphere formation. The expression levels of CD133, CD44, and ALDH1A1 proteins in K562 cells treated with Act-V were significantly reduced, showing a concentration-dependent inhibitory trend. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 The effect of Act-V on the proliferation of K562 cells at 24 h, 48 h and 72 h after drug administration is shown in the figure. Figure 2 The figure shows the effect of Act-V on the colony-forming ability of K562 cells; (A) is a representative image of K562 cell colony formation experiment, and (B) is the statistical analysis of K562 cell colonies. Figure 3 The diagram shows the effect of Act-V on the spheroid formation ability of K562 cells; (A) is a representative diagram of the K562 cell spheroid formation experiment, (B) is a statistical analysis diagram of the diameter of K562 cell tumor spheres, and (C) is a statistical analysis diagram of the number of K562 cell tumor spheres. Figure 4 The effect of Act-V on the spheroidization frequency of K562 cells is shown in the figure. Figure 5 The diagram shows the effect of Act-V on stemness markers of K562 cells; (A) is a Western blot diagram of the expression levels of CD133, CD44, and ALDH1A1 proteins in K562 cells, and (B) is a gray-scale statistical diagram of CD133, CD44, and ALDH1A1 protein bands. Figure 6 The graph shows the effect of Act-V on the invasion and migration of K562 cells; where (A) is a statistical graph of K562 cell migration and (B) is a statistical graph of K562 cell invasion. Figure 7The diagram shows the effect of Act-V on EMT-related proteins in K562 cells; (A) is a Western blot diagram showing the expression levels of N-Cadherin, E-Cadherin, and Vimentin proteins in K562 cells; (B) is a gray-scale statistical diagram of N-Cadherin, E-Cadherin, and Vimentin protein bands. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0036] Example 1: Effect of Act-V on the Proliferative Capacity of Leukemia Cells CCK8 assay method: Cells in the logarithmic growth phase were collected, centrifuged, counted, resuspended, and seeded at an appropriate density in 96-well plates. After 3 h of incubation, Act-D, Act-V, and doxorubicin (ADR) were added, with 5 replicates for each concentration. Cells were incubated for 24 h, 48 h, and 72 h. 20 μL of CCK8 reagent was added to each well, and the cells were incubated at 37 ℃ for 1 h. OD values ​​were measured at 450 nm using a microplate reader to calculate cell viability, and data were analyzed using GraphPad Prism.

[0037] Experimental Results: Effects of Act-V on the proliferation activity of leukemia cells K562, BV173, and HL60, and normal hepatocytes QSG7701, 48 h after administration. The results are shown in Table 1. Compared to Act-D, Act-V significantly reduced the proliferation activity of leukemia cells and decreased the number of viable cells. More importantly, Act-V exhibited low cytotoxicity to normal hepatocytes while exerting its anti-leukemic effect. Tests showed that the cell viability of normal hepatocytes remained at a high level 48 h after Act-V treatment, while Act-D showed a more significant toxic effect on normal hepatocytes. Figure 1 The graph shows the effect of Act-V on the proliferation of K562 cells at 24 h, 48 h, and 72 h after drug administration. Figure 1 As shown, Act-V significantly inhibited the proliferation of K562 cells, and this inhibition was time-dependent.

[0038] Table 1. Effects of Act-D, Act-V, and ADR on the proliferation activities of K562, BV173, HL60, and QSG7701. <![CDATA[CCell Lines IC 50 (nM)]]> Act-V Act-D ADR K562 1.69±0.09 11.58±0.68 384±0.035 BV173 3.11±0.16 15.84±0.29 779±0.05 HL60 10.52±1.70 34.73±1.78 234±0.01 QSG7701 48.13±0.13 37.14±1.05 2170±0.12 Example 2: Act-V inhibits the formation of chronic myeloid leukemia cell colonies. Soft agar colony formation assay: Prepare agar in advance. Dissolve low-melting-point agarose powder in distilled water to make 3% and 1.5% agar solutions. Autoclave and maintain the temperature at approximately 42°C until liquid. If not used immediately, aliquot and store at low temperature; reheat to melt before use. For the lower gel layer, mix the 3% agar solution with RPMI-1640 medium supplemented with 20% FBS and 1% antibiotics at a 1:1 ratio. Spread 2 mL per well in a 6-well plate, taking care to avoid air bubbles during the process. Cool to room temperature in a clean bench until solidified. For cell treatment, collect cells in the logarithmic growth phase, centrifuge, wash once with PBS, count the cells, and resuspend. A top layer of gel was prepared. A 1:1 mixture of 1.5% agar solution and RPMI-1640 medium supplemented with 20% FBS and 1% antibiotics was added, and a suitable amount of cell suspension was added and mixed well. 2 mL of this mixture was seeded into each well of a 6-well plate, resulting in 1000 cells per well. The plate was then cooled to room temperature in a clean bench until solidified. Finally, 2 mL of medium containing 10% FBS was added to the top layer to prevent the agar from drying out, and a gradient of antibiotics was applied. After 24 h of incubation, the drug-treated top layer was removed, and 2 mL of medium containing 10% FBS was added again for 2 weeks of incubation, with the medium being changed periodically. Cells were stained with 0.005% crystal violet for 2 h, and visible cell colonies were counted and analyzed.

[0039] The results showed that the ability of tumor cells to form colonies can also reflect their proliferative capacity. The effect of Act-V on the colony-forming ability of chronic myeloid leukemia cells was investigated using a suspension cell colony formation assay. In the preliminary colony formation experiment, most tumor cell colonies failed to form in K562 cells when the Act-V concentration was greater than 2 nM. Therefore, Act-V (0, 0.5, 1, 1.5 nM) was designed for colony formation in K562 cells. Figure 2 The figure shows the effect of Act-V on the colony-forming ability of K562 cells; where (A) is a representative image of K562 cell colony formation experiment, and (B) is the statistical analysis of K562 cell colonies. "express P <0.01; "express P <0.001 vs. control group. (e.g.) Figure 2 As shown, compared with the control group, the number of colonies in the Act-V treatment group was significantly reduced in a dose-dependent manner, indicating that Act-V can affect the self-renewal and proliferation capacity of leukemia cells and effectively inhibit the colony formation capacity of K562 cells.

[0040] Example 3: Act-V's ability to inhibit spheroidization of chronic myeloid leukemia cells Spheroidization assay: Collect cells in the logarithmic growth phase, centrifuge, count and resuspend, and seed at an appropriate density in 6-well plates. Add sufficient culture medium (serum-free RPMI-1640), B27 (50X), FGFB (10 μg / mL), EGF (10 μg / mL), and insulin (5 μg / mL) to 3 mL. After culturing for 3 h, add the drugs and incubate statically for 2 weeks, strictly avoiding shaking. During this period, replace half of the culture medium with fresh complete medium every 3-4 days to prevent the spheroids from drying out. Observe the morphology and growth of the tumor spheroids under a microscope.

[0041] Experimental Results: The tumor spheroid model is a key in vitro model for evaluating the self-renewal and tumorigenic potential of tumor stem cells. The number and size of the spheroids directly reflect the malignant proliferative activity of tumor cells. We simulated the stem cell microenvironment using a serum-free culture method, cultured K562 cells, and measured the diameter of tumor spheroids at each concentration gradient and calculated the number of tumor spheroids after 2 weeks. Figure 3 The graph shows the effect of Act-V on the spheroidization ability of K562 cells; (A) is a representative graph of the K562 cell spheroidization experiment, (B) is a statistical analysis graph of the diameter of K562 cell tumor spheres, and (C) is a statistical analysis graph of the number of K562 cell tumor spheres. "express P <0.05; "express P <0.01; "express P <0.001 vs. control group. (e.g.) Figure 3 As shown, compared with the control group, the diameter of individual tumor spheres in both cell types was significantly reduced and the number of tumor spheres formed was significantly decreased after Act-V treatment. Moreover, this inhibitory effect showed a certain concentration-dependent trend. Therefore, Act-V has a significant inhibitory effect on the tumor sphere formation ability of K562 cells.

[0042] Example 4: Act-V inhibits the spheroidization frequency of chronic myeloid leukemia cell microspheres. Limiting dilution assay: After collecting and centrifuging the cells for counting, wash them once with PBS, resuspend them in culture medium for counting, and then seed them into 96-well plates at concentrations of 500 cells / well, 100 cells / well, 20 cells / well, 4 cells / well, and 1 cell / well, with 10 replicates for each concentration. After culturing for 2 weeks, observe and record the formation of microspheres, and use the online software https: / / bioinf.wehi.edu.au / software / elda / to calculate and statistically analyze the microsphere frequency.

[0043] Experimental results: Spheroidization frequency, as a key indicator for assessing the self-renewal capacity and stemness characteristics of tumor cells, directly reflects the potential of cells to form clonal microspheres. Figure 4 This is a graph showing the effect of Act-V on the spheroidization frequency of K562 cells. (See figure.) Figure 4 As shown, after Act-V treatment, the spheroidization frequency of K562 cells was significantly reduced compared to the control group, and the inhibition trend was concentration-dependent. This result indicates that Act-V can effectively target the stem phenotype of CML cells and inhibit their spheroidization frequency.

[0044] Example 5: Effect of Act-V on stem cell markers in chronic myeloid leukemia cells Protein immunoblotting assay: Tumor stemness is a core characteristic of tumor cells in maintaining self-renewal, invasion, metastasis, and drug resistance. CD133, CD44, and ALDH1A1 are classic tumor stemness markers, and their expression levels are closely related to tumor malignancy. To investigate the regulatory role of Act-V on tumor stemness, this study used K562 cells as the research object and detected the changes in the protein expression of the above markers after Act-V treatment using Western blotting.

[0045] Figure 5 The graph shows the effect of Act-V on stem cell markers in K562 cells; (A) is a Western blot graph showing the expression levels of CD133, CD44, and ALDH1A1 proteins in K562 cells, and (B) is a gray-scale statistical graph of CD133, CD44, and ALDH1A1 protein bands. "ns" indicates no statistical significance; "express P <0.05; "express P <0.01; "express P <0.001 vs. control group. (e.g.) Figure 5 As shown, compared with the control group, the expression levels of CD133, CD44, and ALDH1A1 proteins in K562 cells treated with Act-V were significantly reduced, exhibiting a concentration-dependent inhibitory trend. This indicates that Act-V can disrupt the stemness characteristics of tumor cells by downregulating the protein expression of core tumor stemness markers.

[0046] Example 6: Act-V Inhibits the Invasion and Metastasis of Chronic Myeloid Leukemia Cells Transwell assay procedure: For the Transwell cell migration assay, sterilize the transwell chambers by immersion in alcohol and then sterilize them under UV light in a biosafety cabinet for 1 h. Place the Transwell chambers into 24-well plates. Collect cells, centrifuge, and resuspend in serum-free medium. Seed 100,000 cells / 200 μL / well in the upper chamber. Add 800 μL of medium containing 20% ​​FBS to the lower chamber and incubate for 48 h. Remove the chambers and photograph and count the cells in the lower chamber liquid from five randomly selected fields of view under a microscope, calculating the average value. For the Transwell cell invasion assay (the difference from the migration assay lies in the gel preparation), prepare the Matrigel by diluting Matrigel with serum-free medium at a 1:8 ratio. Take 80 μL and spread it evenly on the upper chamber membrane surface, then cure at 37 ℃ for 1-2 h. The remaining steps are the same as the migration assay.

[0047] Experimental Results: In this study, we investigated the effects of Act-V on the invasion and metastasis of CML cells. In the experiments, we used Transwell cell migration assays and Matrigel invasion assays to examine changes in cell behavior after treating K562 cells with different concentrations of Act-V. Figure 6 The graph shows the effect of Act-V on the invasion and migration of K562 cells; where (A) is a statistical graph of K562 cell migration, and (B) is a statistical graph of K562 cell invasion. "express P <0.05; "express P <0.01; "express P <0.001 vs. control group. Transwell results are as follows: Figure 6 As shown, compared with the blank control group, the number of K562 cells that migrated through the polycarbonate membrane and the number of cells that invaded through the matrix gel were significantly reduced after Act-V treatment, and the differences were statistically significant. P Act-V (<0.05) can significantly inhibit the invasion and migration of K562 cells in vitro, and this inhibitory effect shows a certain concentration-dependent characteristic.

[0048] Example 7: Act-V inhibits the EMT process in chronic myeloid leukemia cells. Experimental Methods: Epithelial-mesenchymal transition (EMT) is a biological process in which cells lose their epithelial phenotype and acquire mesenchymal characteristics. Its abnormal activation is associated with tumor invasion and metastasis. Using K562 cells as a model, we investigated the effect of Act-V on the expression of EMT-related molecules in cells through Western blotting experiments.

[0049] Figure 7 The diagram shows the effect of Act-V on EMT-related proteins in K562 cells; (A) is a Western blot diagram showing the expression levels of N-Cadherin, E-Cadherin, and Vimentin proteins in K562 cells; (B) is a gray-scale statistical diagram of N-Cadherin, E-Cadherin, and Vimentin protein bands. "express P <0.01; "express P <0.001 vs. control group. Experimental results are as follows: Figure 7 As shown, Act-V effectively inhibited the expression of N-cadherin and vimentin, EMT-related mesenchymal markers, in K562 cells, while significantly promoting the expression of E-cadherin, an epithelial marker. E-cadherin, a core molecule in intercellular connections, is downregulated, a key marker of EMT initiation, while the upregulation of N-cadherin and vimentin are important characteristics of cells acquiring mesenchymal properties and enhancing their migration and invasion capabilities. Act-V's regulatory effect on these three proteins directly reversed the EMT phenotype of K562 cells, suggesting that it may weaken the stem cell-like characteristics of leukemia cells by inhibiting the EMT process.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of actinomycin D in the preparation of a medicament for treating chronic myeloid leukemia.

2. Use according to claim 1, wherein Actinomycin D is the only active ingredient in the medicament.

3. The use according to claim 1, wherein Actinomycin D is used alone.

4. The use according to claim 1, wherein Actinomycin D can reduce the proliferation activity of CML cells; actinomycin D can effectively inhibit the colony formation ability of CML cells.

5. The use according to claim 1, wherein the compound is ###0002### Actinomycin D can inhibit the spheroid formation ability of CML cell tumor spheres.

6. The use according to claim 1, wherein Actinomycin D can regulate the expression of key factors of EMT pathway.

7. The use according to claim 1, wherein Actinomycin D can down-regulate the protein expression of tumor stemness markers CD133, CD44 and ALDH1A1.

8. The use according to claim 1, wherein The concentration of actinomycin D is 0.5-1.5 nM.

9. The use according to claim 1, wherein The time for treating cells with actinomycin D is 24-72 h.

10. An agent against chronic myeloid leukemia, characterized by, The active ingredient of the medicament comprises actinomycin D.

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