Application of Epigenetically Modified Nucleosides in the Treatment of Tumors

Abstract

The present invention provides the use of epigenetically modified nucleosides in the treatment of tumors, belonging to the technical field of biomedicine. The present invention provides the use of epigenetically modified nucleosides in the preparation of drugs for treating tumors, and the epigenetically modified nucleosides include: 5-formylcytidine (5fdC), 5-hydroxymethylcytidine (5hmdC), and 5-hydroxymethyluridine (5hmdU). The present invention provides that the inhibitory effects of epigenetically modified nucleosides on the proliferation of different acute myeloid leukemia cells are different. CDA highly expressed in acute myeloid leukemia tumor cells can effectively convert the epigenetically modified nucleoside 5fdC into toxic 5fdU, thereby achieving the specific killing effect of the epigenetically modified nucleoside on the tumor cells of acute myeloid leukemia and realizing targeted therapy. Therefore, the present invention also provides a targeted drug for inhibiting acute myeloid leukemia cells with high CDA expression.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of epigenetically modified nucleosides in the treatment of tumors. Background Art

[0002] Acute myeloid leukemia is a type of blood tumor with a high incidence and difficult to cure. At present, the clinical treatment of this disease mainly relies on chemotherapy, mainly using nucleoside analogs and base analogs to inhibit the proliferation of tumor cells so as to achieve the therapeutic effect. Among them, cytarabine (Ara-C) is the most commonly used nucleoside analog. It mainly acts on the S phase of cell division and inhibits the synthesis of cellular DNA by interfering with cell proliferation. As a classical chemotherapy drug for acute myeloid leukemia, after entering tumor cells, Ara-C is phosphorylated by phosphokinase to form Ara-CTP, which competes with endogenous dCTP (2'-deoxycytidine 5'-triphosphate) for DNA polymerase and then incorporates into the DNA strand, blocking DNA synthesis, thereby inhibiting cell proliferation. The rapid proliferation characteristics of tumor cells are accompanied by frequent DNA synthesis in cells, providing an opportunity for Ara-C to exert good therapeutic effects. However, the normal tissue cells of the body also need DNA replication for proliferation, which means that Ara-C will cause off-target toxicity to normal tissues and organs.

[0003] In addition, although cytarabine has achieved certain therapeutic effects in the chemotherapy of leukemia. However, during the treatment process, cytarabine faces the challenge of deamination and inactivation. It is reported that Ara-C entering cells will be recognized by the intracellular deaminase CDA (Cytidine deaminase) and deaminated into the non-cytotoxic deamination product Ara-U (uracil arabinoside). CDA in acute myeloid leukemia cells often shows high expression, which limits the application effect of Ara-C in clinical practice and also increases the treatment difficulty of acute myeloid leukemia. Therefore, finding nucleoside analogs that are specific to tumor cells and can avoid inactivation due to deamination reactions is crucial for the treatment of acute myeloid leukemia. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of epigenetically modified nucleosides in the treatment of tumors, which lays a foundation for the targeted treatment of acute myeloid leukemia with high CDA expression and expands the application potential of epigenetically modified nucleosides in the treatment of leukemia.

[0005] The present invention provides the application of epigenetically modified nucleosides in the preparation of drugs for treating tumors, and the epigenetically modified nucleosides include at least one of the following: 5-formylcytidine, 5-hydroxymethylcytidine, and 5-hydroxymethyluridine.

[0006] Preferably, the tumor includes blood cancer.

[0007] Preferably, treating the tumor includes inhibiting cancer cell proliferation.

[0008] Preferably, the cancer cells include acute myeloid leukemia cells.

[0009] Preferably, the acute myeloid leukemia cells include HL-60 cells and / or THP-1 cells.

[0010] The present invention also provides a tumor cell proliferation inhibitor, including at least one of the following: 5-formylcytidine, 5-hydroxymethylcytidine, and 5-hydroxymethyluridine;

[0011] Among them, 5-formylcytidine, 5-hydroxymethylcytidine, and 5-hydroxymethyluridine all have inhibitory effects on the proliferation of HL-60 cells;

[0012] 5-Hydroxymethyluridine has an inhibitory effect on the proliferation of THP-1 cells.

[0013] The present invention also provides a targeted drug for inhibiting acute myeloid leukemia cells with high CDA expression, and the active ingredient includes 5-formylcytidine.

[0014] Preferably, it includes treating acute myeloid leukemia cells with high CDA expression with 5-formylcytidine.

[0015] Preferably, when treating, the concentration of 5-formylcytidine is not less than 10 μM and the time is not less than 48 h.

[0016] Preferably, the acute myeloid leukemia cells with high CDA expression include HL-60 cells.

[0017] Beneficial effects: In the present invention, 5-formylcytidine (5fdC), 5-hydroxymethylcytidine (5hmdC), 5-hydroxymethyluridine (5hmdU), 5-oxo-cytidine (5cadC), and 5-methylcytidine (5md) at different concentrations were co-cultured with acute myeloid leukemia cells HL-60 and THP-1 for 144 h, and subcultured and counted every 48 h to investigate the cell proliferation, so as to screen for epigenetic modification nucleosides with inhibitory effects on the proliferation of acute myeloid leukemia cells. The results showed that after treatment with 5fdC, the proliferation of HL-60 cells was significantly and continuously inhibited. In addition, with the prolongation of the treatment time, 5hmdC and 5hmdU also gradually showed inhibitory effects on the proliferation of HL-60 cells. Only 5hmdU showed an inhibitory effect on the proliferation of THP-1 cells, while 5fdC and 5hmdC did not show obvious inhibitory effects. It is indicated that epigenetic modification nucleosides have inhibitory effects on the proliferation of different acute myeloid leukemia cells, among which 5fdC has the strongest cytotoxicity to HL-60 cells, and THP-1 cells are the most sensitive to 5hmdU.

[0018] In the present invention, the epigenetic modification nucleoside 5fdC is used as a proliferation inhibitor for acute myeloid leukemia cells with high CDA expression, and the tumor killing effect is achieved by directly treating the cells. Compared with non-tumor cells, the highly expressed CDA in acute myeloid leukemia tumor cells can effectively convert the epigenetic modification nucleoside 5fdC into toxic 5fdU, so as to achieve the specific killing effect of the epigenetic modification nucleoside on the tumor cells of acute myeloid leukemia and realize targeted therapy. In addition, the characteristic of rapid proliferation of tumor cells can more effectively incorporate the deamination product of the epigenetic modification nucleoside 5fdC into the genome by DNA replication, trigger DNA damage of cells, and a large amount of DNA damage will cause cell proliferation inhibition and ultimately lead to the death of tumor cells; at the same time, the epigenetic modification nucleoside only has slight toxic and side effects on normal cells, but the toxic and side effects are much smaller than those of tumor cells, suggesting that the epigenetic modification nucleoside has specific killing ability for acute myeloid leukemia cells with high CDA expression. Description of the drawings

[0019] Figure 1 It is a graph showing the results of the analysis of the inhibitory effect of epigenetic modification nucleosides on the proliferation of HL-60 (A) and THP-1 (B) cells;

[0020] Figure 2 It is a graph showing the results of the correlation analysis between the inhibitory effect of 5fdC on cell proliferation and the expression level of cytidine deaminase (CDA); in the figure, A: analysis of the effect of β-ME in cell culture medium on the cytotoxicity of 5fdC; B: detection of CDA expression in HL-60 and THP-1 cells by qPCR; C: analysis of the inhibitory effect of 5fdC combined with THU on the proliferation of HL-60 cells; D: analysis of the inhibitory effect of 5fdC on the proliferation of THP-1 (CDAOE) cells;

[0021] Figure 3 Verification and construction verification diagrams of CDA overexpression amplification plasmids and THP-1 (CDA OE) cells; In the figure, A: Plasmid map of pLVX-EF1a-hCDA (NM_001785)-EGFP-IRES-Puro; B: Gel electrophoresis verification diagram of restriction enzyme digestion products; C: Plasmid sequencing alignment diagram; D: Fluorescence imaging diagram of THP-1 cells infected with CDA overexpressing lentivirus (100×); E: Product diagrams of THP-1 and its overexpressing cell line (Lane 1: CDA product in THP-1; Lanes 2, 3: CDA products in THP-1-NC; Lanes 4, 5: CDA products in THP-1 (CDA OE); Lane 6: CDA primer control; Lane 7: β-actin product in THP-1; Lanes 8, 9: β-actin products in THP-1-NC; Lanes 10, 11: β-actin products in THP-1 (CDA OE); Lane 12: β-actin primer control)

[0022] Figure 4 Diagram of apoptosis analysis results of HL-60 and THP-1 cells after treatment with 5fdC;

[0023] Figure 5 Diagram of analysis results of DNA damage in HL-60 and THP-1 cells after treatment with epigenetically modified nucleosides; In the figure, A and B are respectively the Western blot analysis and relative content statistical histogram of γ-H2AX protein in HL-60 cells after treatment with epigenetically modified nucleosides; C and D are respectively the Western blot analysis and relative content statistical histogram of γ-H2AX protein in THP-1 cells after treatment with epigenetically modified nucleosides; E and F are respectively the comet assay characterization of DNA damage and comet tail length statistical chart in HL-60 cells after treatment with epigenetically modified nucleosides; G and H are respectively the comet assay characterization of DNA damage and comet tail length statistical chart in THP-1 cells after treatment with epigenetically modified nucleosides;

[0024] Figure 6 Diagram of analysis results of damage recovery after inhibition of acute myeloid leukemia cells by epigenetically modified nucleosides; In the figure, A: Damage recovery analysis of 5fdC on HL-60 cells and THP-1 cells; B: Damage recovery analysis of 5hmC on HL-60 cells and THP-1 cells;

[0025] Figure 7Figure showing the specific analysis results of the inhibition of acute myeloid leukemia cell proliferation by epigenetically modified nucleosides; in the figure, A: Flow cytometry analysis of the effect of epigenetically modified nucleosides on the apoptosis of HEK293 cells; B and C are respectively the protein blotting characterization and relative content analysis of γ-H2AX protein in HEK293 cells treated with epigenetically modified nucleosides; D and E are respectively the comet assay characterization of DNA damage and the statistical column chart of comet tail length in HEK293 cells treated with epigenetically modified nucleosides. Detailed implementation mode

[0026] The present invention provides the use of epigenetically modified nucleosides in the preparation of drugs for treating tumors, and the epigenetically modified nucleosides include at least one of the following: 5-formylcytidine (5fdC), 5-hydroxymethylcytidine (5hmdC), and 5-hydroxymethyluridine (5hmdU).

[0027] The tumors of the present invention preferably include blood cancers, more preferably include leukemias, and most preferably include acute myeloid leukemia. In the examples, HL-60 cells (human promyelocytic cell line) and THP-1 cells (human leukemia monocytic cell line) are used as examples for illustration, but it cannot be considered as the entire protection scope of the present invention. The treatment of tumors in the present invention preferably includes inhibiting the proliferation of cancer cells and ultimately inducing apoptosis of cancer cells.

[0028] In the examples of the present invention, 5fdC, 5hmdC, 5hmdU, 5cadC, and 5md purchased from Jena Bioscience were formulated into solutions with different concentrations and co-cultured with acute myeloid leukemia cells HL-60 and THP-1 respectively. The results showed that after treatment with 5fdC, the proliferation of HL-60 cells was significantly and continuously inhibited; and with the prolongation of the treatment time, 5hmdC and 5hmdU also gradually showed inhibitory effects on the proliferation of HL-60 cells; and only 5hmdU showed an inhibitory effect on the proliferation of THP-1 cells, indicating that epigenetically modified nucleosides have inhibitory effects on the proliferation of different acute myeloid leukemia cells, among which 5fdC has the strongest toxicity to HL-60 cells, and THP-1 cells are the most sensitive to 5hmdU.

[0029] The present invention also provides a tumor cell proliferation inhibitor, including at least one of the following: 5-formylcytidine, 5-hydroxymethylcytidine, and 5-hydroxymethyluridine;

[0030] Among them, 5-formylcytidine, 5-hydroxymethylcytidine, and 5-hydroxymethyluridine all have inhibitory effects on the proliferation of HL-60 cells;

[0031] 5-Hydroxymethyluridine has an inhibitory effect on the proliferation of THP-1 cells.

[0032] The present invention also provides a targeted drug for inhibiting highly expressed CDA in acute myeloid leukemia cells, and the active ingredient includes the epigenetic modification 5-formylcytidine.

[0033] In the embodiments of the present invention, it was found that the presence of the reducing agent β-mercaptoethanol (2-Mercaptoethano, β-ME) does not affect the inhibitory effect of 5fdC on the proliferation of HL-60 cells; the high sensitivity of HL-60 cells to the epigenetically modified nucleoside 5fdC is very likely driven by highly expressed CDA in the cells, that is, after 5fdC is deaminated by intracellular CDA, cytotoxic uridine derivatives 5hmdU and 5fdU will be produced. After 5hmdU and 5fdU are inserted into genomic DNA, extensive DNA damage will be caused, leading to cell cycle arrest and cell death; then, the activity of highly expressed CDA in HL-60 cells was inhibited by the CDA inhibitor tetrahydrouridine (THU), and it was found that the inhibitory effect of 5fdC on the proliferation of HL-60 cells was significantly weakened after THU inhibited the activity of CDA, indicating that 5fdC may exert its cytotoxic effect depending on highly expressed CDA in acute myeloid leukemia cells; and the epigenetically modified nucleoside inhibits cell proliferation by triggering apoptosis of cells, that is, the epigenetically modified nucleoside will cause DNA breaks in acute myeloid leukemia cells, leading to apoptosis. The DNA damage of the epigenetically modified nucleoside to leukemia cells depends on the expression level of CDA in the cells, which lays a foundation for the targeted therapy of non-classical epigenetically modified nucleosides.

[0034] In the embodiments of the present invention, the targeted toxicity of the epigenetically modified 5fdC to acute myeloid leukemia cells with highly expressed CDA was also investigated. The results showed that the effect of the epigenetically modified nucleoside on the apoptosis of HEK293 cells was almost negligible, meaning that 5fdC only caused slight damage to normal cells and had no obvious toxicity, indicating that the epigenetically modified nucleoside only had slight side effects on normal cells, but the side effects were much smaller than those on tumor cells, suggesting that the killing of acute myeloid leukemia cells with highly expressed CDA by the epigenetically modified nucleoside is specific.

[0035] The present invention preferably uses 5-formylcytidine to treat acute myeloid leukemia cells with highly expressed CDA. When treating, the concentration of 5-formylcytidine is preferably not less than 10 μM and the time is not less than 48 h. The acute myeloid leukemia cells with highly expressed CDA of the present invention preferably include HL-60 cells.

[0036] In order to further illustrate the present invention, the application of the epigenetically modified nucleoside provided by the present invention in the treatment of tumors will be described in detail below with reference to embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0037] The test materials used in the embodiments of the present invention are commercial test materials obtained through conventional commercial channels in the art. For example, HL-60 (human promyelocytic cell line), THP-1 (human leukemia monocytic cell line), and HEK293 (Human embryonic kidney cell line) are all purchased from ATCC and cultured in an incubator with a cell culture environment of 37°C and 5% CO2. The complete culture medium for HL-60 consists of: a complete culture medium containing 20% FBS and 1% double antibody, that is, IMDM + 20% FBS + 1% double antibody. The complete culture medium for THP-1 and HEK293 cells consists of: RPMI 1640 complete culture medium containing 10% FBS and 1% double antibody. It should be noted that the culture of THP-1 also requires the addition of 5 μM β-mercaptoethanol (2-Mercaptoethano, β-ME) to the culture medium, that is, RPMI 1640 + 10% FBS + 1% double antibody + 5 μM β-mercaptoethanol, and the centrifugation conditions are 1000 rpm for 3 min.

[0038] The methods used in the embodiments of the present invention are all conventional methods in the art, such as:

[0039] (1) Proliferation experiment: 8.0×10 5 cells were seeded in a six-well plate, and the epigenetic modification nucleosides with final concentrations of 10 μM 5fdC, 100 μM 5hmdC, 50 μM 5hmdU, 100 μM 5mdC, 100 μM 5cadC and 1‰ DMSO were added and co-incubated with the cells. Passage was counted every 48 h. The culture medium in the six-well plate was collected, centrifuged at 1000 rpm for 3 min, the supernatant was aspirated with a pipette, and the cells were resuspended with 1 mL of fresh culture medium. 10 μL of the cell suspension was diluted 10 times and counted under a microscope, and the culture was continued for 144 h in total to draw a proliferation curve.

[0040] (2) Western Blot: The cell treatment was the same as that in the proliferation experiment. The total protein of the cells treated with epigenetic modification nucleosides was extracted with RIPA lysis buffer (Solarbio) and denatured at 100°C. TGX Stain-Free TMThe proteins in each group were electrophoresed using a 5 - 15% SDS - PAGE gel prepared with the FASTCASTTM Acrylamide Kit. After observing the total proteins in each lane by exposure, the proteins were transferred to a nitrocellulose membrane (NC membrane). The membrane was blocked with 5% non - fat milk at room temperature for 1 h, then incubated overnight with a 1:2000 anti - γ - H2AX primary antibody purchased from Cell Signaling Technology. After washing 3 times with PBST, 10 min each time, it was incubated with a 1:5000 horseradish peroxidase (HRP) - labeled goat anti - rabbit secondary antibody (Hua'an Biotech) at room temperature for 1 h. Then it was washed 3 times with PBST, 10 min each time. ECL chemiluminescent solution was used for development.

[0041] (3) Comet assay: After co - incubating cells with 10 μM 5fdC, 100 μM 5hmdC, 50 μM 5hmdU, 100 μM 5mdC, and 100 μM 5cadC for 144 h, the cells were collected and lysed with a lysis buffer (100 mM Na2EDTA, 1.2 M NaCl, 0.26 M NaOH, 0.1% sodium lauroyl sarcosinate, pH adjusted to > 13, stored at 4 °C) for standby. H2O2 is a strong oxidant, and the reactive oxygen species (ROS) it generates can directly act on DNA, causing DNA damage in cells. Therefore, cells treated with 0.001% H2O2 for 20 min were selected as the positive control group for this experiment. Subsequently, 100 μL of 0.8% normal melting point agarose after dissolution was added to the frosted surface of a glass slide and spread evenly. It was solidified at 4 °C for 10 min as the first layer of gel. Then, at room temperature, 300 μL of cell lysis buffer was mixed with 900 μL of 0.75% low melting point agarose, and 100 μL of the mixture was added to the first layer of gel. A coverslip was covered and solidified at 4 °C for 5 min. After solidification, the slide was washed and subjected to electrophoresis characterization (voltage 20 V, current 25 mA, electrophoresis for 20 min). Finally, the slide was washed with ultrapure water and placed in 30 mL of staining solution (3 μL of 10000×SYBR Green + 30 mL of ultrapure water) for staining at room temperature for 15 min. The results were observed under a fluorescence microscope and analyzed using Image J.

[0042] (4) RNA extraction by TRIzol method: Cells treated with the epigenetically modified nucleoside for 144 h were collected and washed once with PBS. An appropriate amount of TRIzol was added to the cell pellet and left standing at room temperature for 5 min to fully lyse the cells. Subsequently, chloroform was added (200 μL of chloroform was added to 1 mL of TRIzol), and the mixture was vigorously shaken for 15 s and left standing at room temperature for 2 - 3 min, followed by centrifugation at 12,000 g for 15 min at 4°C. After centrifugation, the aqueous phase was transferred to a new EP tube, and isopropanol was added (500 μL of isopropanol was added to 1 mL of TRIzol). After standing at room temperature for 10 min, it was centrifuged at 12,000 g for 10 min at 4°C. After centrifugation, the supernatant was taken, and 75% ethanol was added and mixed well (1 mL of 75% ethanol was added to 1 mL of TRIzol), and it was centrifuged at 7,500 g for 5 min at 4°C for a total of two washes. After washing, the supernatant was aspirated as completely as possible, and the precipitate was left standing at room temperature for 15 min. After drying the RNA, it was dissolved in an appropriate volume of DEPC-treated water. After measuring the concentration, it was stored at -80°C.

[0043] (5) Detection of apoptosis by flow cytometry: Cells co-incubated with the epigenetically modified nucleosides of 10 μM 5fdC, 100 μM 5hmdC, 50 μM 5hmdU, 100 μM 5mdC, and 100 μM 5cadC for 144 h were collected and washed twice by centrifugation at 1000 rpm for 3 min with PBS containing 0.1% BSA (subsequent PBS was all prepared). Then, 500 μL of Binding Buffer was added and gently pipetted to form a single-cell suspension. Subsequently, 5 μL of Annexin V-FITC was added and mixed well, and then 5 μL of Propidium Iodide was added, mixed well, and reacted at room temperature in the dark for 5 - 10 min. Finally, the cells were washed with PBS and resuspended as a cell suspension, filtered through a 200-mesh filter, and detected on the machine.

[0044] Example 1

[0045] (1) Screening for epigenetically modified nucleosides with inhibitory effects on the proliferation of acute myeloid leukemia cells

[0046] In the present invention, different concentrations of 5-formylcytidine (5fdC), 5-hydroxymethylcytidine (5hmdC), 5-hydroxymethyluridine (5hmdU), 5-carbonylcytidine (5cadC), and 5-methylcytidine (5md) were co-cultured with acute myeloid leukemia cells HL-60 and THP-1 for 144 h, and passaged and counted every 48 h to examine the cell proliferation.

[0047] The results are as Figure 1As shown in A, after treatment with 5fdC, the proliferation of HL-60 cells was significantly and continuously inhibited. With the prolongation of the treatment time, 5hmdC and 5hmdU also gradually showed inhibitory effects on the proliferation of HL-60 cells. However, 5mdC and 5cadC never had obvious effects on the proliferation of HL-60 cells.

[0048] Epigenetically modified nucleosides that have inhibitory effects on the proliferation of another acute myeloid leukemia cell line, THP-1, are shown in Figure 1 As shown in B, only 5hmdU showed an inhibitory effect on the proliferation of THP-1 cells, while 5fdC and 5hmdC did not show obvious inhibitory effects.

[0049] It can be seen from Figure 1 that epigenetically modified nucleosides have inhibitory effects on the proliferation of different acute myeloid leukemia cells. Among them, 5fdC has the strongest cytotoxicity to HL-60 cells, while THP-1 cells are the most sensitive to 5hmdU.

[0050] (2) Mechanisms underlying the differences in the effects of epigenetically modified nucleosides on the proliferation of acute myeloid leukemia cells

[0051] Figure 1 As shown in, there were significant differences in the inhibition rates of 5fdC on two different acute myeloid leukemia cell lines after 144 hours of culture: the inhibition rate of HL60 proliferation was approximately 83%, and there was no obvious inhibitory effect on THP-1.

[0052] ① Design a control experiment to detect the proliferation of cells under different treatments. β-ME was also added to the culture medium of 5fdC co-cultured with HL-60 and compared with the group without addition. The results are shown in Figure 2 As shown in A, the presence of β-ME in the culture medium did not weaken the inhibitory effect of 5fdC on the expansion of HL-60 cells.

[0053] In addition, β-ME was removed from the culture environment of 5fdC and THP-1, and the proliferation was compared with that of the group without removing β-ME. The results are shown in Figure 2 As shown in B, even when the culture medium did not contain β-ME, the inhibitory effect of 5fdC on the proliferation of THP-1 did not increase, demonstrating that the presence of β-ME did not affect the toxicity of 5fdC.

[0054] ②In addition to undergoing oxidation reactions, bases in mammalian cells can also undergo deamination reactions to form corresponding deamination products. The epigenetic modification nucleosides 5hmdU, 5hmdC, and 5fdC cannot be phosphorylated by cytidine monophosphate kinase 1 (CMPK1), so they cannot be directly inserted into the genome and are not cytotoxic. However, after they are deaminated by intracellular CDA, they will produce cytotoxic uridine derivatives 5hmdU and 5fdU. After 5hmdU and 5fdU are inserted into genomic DNA, extensive DNA damage will be caused, leading to cell cycle arrest and cell death.

[0055] Therefore, the present invention speculates that the difference in the response of two acute myeloid leukemia cells, HL-60 and THP-1, to 5fdC is caused by the difference in the expression of intracellular CDA. The present invention compared the expression of CDA in HL-60 and THP-1 cells by real-time fluorescence quantitative PCR (qPCR). The detection primers are respectively:

[0056] Forward primer (SEQ ID No.1): CCCTACAGTCACTTTCCTG,

[0057] Reverse primer (SEQ ID No.2): CGGGTAGCAGGCATTTTCTA.

[0058] The results are as Figure 2 shown in B below. The expression level of CDA in HL-60 cells is much higher than that in THP-1 cells (about 7 times). This result indicates that the high sensitivity of HL-60 cells to 5fdC epigenetic modification nucleosides is likely to be driven by the high expression of intracellular CDA.

[0059] ③To further verify the relationship between 5fdC and the intracellular CDA expression level, the present invention used the CDA inhibitor tetrahydrouridine (THU) to inhibit the activity of highly expressed intracellular CDA in HL-60, and investigated the effect of 5fdC on the proliferation of HL-60 cells with inhibited CDA activity.

[0060] The results are as Figure 2 shown in C below. After THU inhibited the activity of CDA, it significantly weakened the inhibitory effect of 5fdC on the proliferation of HL-60 cells, indicating that 5fdC may exert its cytotoxic effect depending on the highly expressed CDA in acute myeloid leukemia cells.

[0061] ④Construct THP-1 cells with high CDA expression (THP-1(CDAOE))

[0062] After designing and synthesizing the pLVX-EF1a-hCDA (NM_001785)-EGFP-IRES-Puro plasmid ( Figure 3In A), after double digestion with BamHΙ and EcoRΙ, the correctness of the plasmid was detected by agarose gel electrophoresis. The 1175bp small fragment and 8790bp large fragment shown in the enzyme digestion lane indicated the correctness of the plasmid. Figure 3 In B), the sequencing results further confirmed the correctness of the plasmid. Figure 3 In C), then, the plasmid was transfected into HEK293T cells to amplify the lentivirus overexpressing CDA. After the collected lentivirus was concentrated and titer determined, it was used to infect THP-1 cells. Green fluorescent protein expression was visible in some cells within 96h after infection. Figure 3 In D), finally, 6μg / mL of puro was added to screen the cells. The qPCR gel electrophoresis products were further detected to verify the expression of the overexpression plasmid in the cells. The results showed that the bands of the NC group and the wild type were lighter, while the band color of the CDA overexpression group was darker. Figure 3 In E), it indicated that the construction of the THP-1 cell line overexpressing CDA was successful.

[0063] The relationship between the cytotoxicity of 5fdC and the CDA expression level was verified by a cell proliferation inhibition experiment. The results were as Figure 2 shown in D. In THP-1 (CDA OE) cells with high CDA expression, the inhibitory effect of 5fdC on cell proliferation was significantly enhanced.

[0064] In summary, intracellular CDA is a key factor for 5fdC to exert its toxicity, laying the application potential of 5fdC in the treatment of acute myeloid leukemia with high CDA expression.

[0065] (3) Mechanism of action of epigenetically modified nucleosides on the proliferation inhibition of HL-60 and THP-1 cells

[0066] The present invention first detected the apoptosis of cells after continuous treatment with different epigenetically modified nucleosides by flow cytometry, using 5mdC with no obvious cytotoxicity as a control. The results were as Figure 4 shown. 5fdC could significantly induce apoptosis in HL-60 cells. However, in THP-1 cells, 5hmdU induced apoptosis in THP-1 cells. The data of cell apoptosis detection were highly consistent with the data of cell proliferation detection, indicating that epigenetically modified nucleosides inhibited cell proliferation by inducing apoptosis. Thus, it can be seen that epigenetically modified nucleosides inhibited cell proliferation by triggering apoptosis of cells.

[0067] (4) Content changes of γ-H2AX after treatment with different epigenetically modified nucleosides

[0068] When DNA damage occurs in cells, the serine residue at position 139 of histone variant H2AX is rapidly phosphorylated at the damaged DNA site, and then γ-H2AX is formed. Therefore, the protein content of γ-H2AX can be used as an indicator for judging the DNA damage situation.

[0069] In the present invention, the changes of γ-H2AX after treatment with different epigenetically modified nucleosides were detected by Western Blot. The results are as Figure 5 shown: After treatment with 5fdC, 5hmdC and 5hmdU, the protein content of γ-H2AX in HL-60 cells increased to varying degrees, and the 5fdC treatment group was the most obvious ( Figure 5 A and B in it). On the contrary, in THP-1 cells, compared with the DMSO group, the protein content of γ-H2AX in the 5hmdU treatment group was significantly increased (P<0.01) ( Figure 5 C and D in it). The above results suggest that epigenetically modified nucleosides can cause DNA damage in leukemia cells, thereby triggering apoptosis.

[0070] (5) Degree of DNA damage of epigenetically modified nucleosides to acute myeloid leukemia cells

[0071] In the present invention, the DNA breakage of HL-60 and THP-1 cells after treatment with epigenetically modified nucleosides was observed by comet assay. The results are as Figure 5 shown. Compared with other epigenetically modified nucleoside treatment groups, trailing and significantly longer comet tails were observed in HL-60 cells treated with 5fdC and 5hmdC ( Figure 5 E and F in it). In THP-1 cells, compared with the DMSO group, trailing and obvious comet tails also appeared in the cells of the 5hmdU treatment group ( Figure 5 G and H in it), and the comet tails of the 5hmdC and 5fdC treatment groups of cells also increased. The above results together indicate that epigenetically modified nucleosides can cause DNA breakage in acute myeloid leukemia cells, thereby leading to apoptosis. And the DNA damage of epigenetically modified nucleosides to leukemia cells depends on the expression level of CDA in cells, which lays a foundation for the targeted therapy of non-classical epigenetically modified nucleosides.

[0072] (6) Proliferation recovery of cells after treatment with epigenetically modified nucleosides 5fdC and 5hmC

[0073] Severe DNA damage within cells can cause genomic fragmentation, and the resulting inhibition of cell proliferation may be an irreversible process. Accordingly, in the present invention, non-cytotoxic 5mdC was used as a control to further investigate the proliferation recovery of cells treated with epigenetically modified nucleosides 5fdC and 5hmC. After co-incubating HL-60 and THP-1 cells with 5fdC for 96 hours respectively, 5fdC showed obvious inhibition of the proliferation of both cell types. At this time, after removing the drug treatment and continuing the culture for 196 hours, it was found that the proliferation of both cell types did not recover after removing the drug ( Figure 6 in A and B), indicating that severe gene damage occurred in both cell types after treatment with 5fdC. The above results together indicate that the specific killing of acute myeloid leukemia cells by epigenetically modified nucleosides exerts an inhibitory effect on proliferation by causing DNA fragmentation in cells and ultimately triggering apoptosis.

[0074] (7) Targeted toxicity of epigenetically modified nucleosides to acute myeloid leukemia cells with high CDA expression

[0075] In the present invention, normal kidney cells HEK293 were selected as negative control cells to investigate the cytotoxicity of epigenetically modified nucleosides. For this purpose, the present invention first examined the apoptosis of HEK293 cells co-incubated with epigenetically modified nucleosides for 144 h by flow cytometry. The results are as Figure 7 shown in A and B, and the effect of epigenetically modified nucleosides on the apoptosis of HEK293 cells was almost negligible, indicating that 5fdC has no obvious toxicity to normal cells. Then, Western Blot and comet assays were also used to further verify that epigenetically modified nucleosides only caused slight damage to the DNA of HEK293 cells ( Figure 7 in C and D). The above experimental results together show that epigenetically modified nucleosides only have slight toxic side effects on normal cells, but the toxic side effects are much smaller than those on tumor cells, suggesting that the killing of acute myeloid leukemia cells with high CDA expression by epigenetically modified nucleosides is specific.

[0076] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of 5-formylcytidine in the preparation of a targeted drug for inhibiting acute myeloid leukemia cells with high CDA expression, characterized in that, Including treating acute myeloid leukemia cells with high CDA expression using 5-formylcytidine. When performing the treatment, the concentration of 5-formylcytidine is not less than 10 μM and the time is not less than 48 h.

2. The application according to claim 1, characterized in that, The acute myeloid leukemia cells with high CDA expression include HL-60 cells.

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