Application of sildilan cardiac glycoside in treatment of myelodysplastic syndrome

Abstract

The invention discloses application of sildilan cardiac glycoside in treatment of myelodysplastic syndrome, belongs to the technical field of medical treatment, and particularly relates to application of the sildilan cardiac glycoside in a medicine or a composition for treatment of myelodysplastic syndrome, preferably, the effective concentration of the sildilan cardiac glycoside is 0.1-3 mu M, and the effective concentration of the sildilan cardiac glycoside is 0.1-3 mu M. According to the medicine or the composition for treating the myelodysplastic syndrome, a diluting solvent of the cedilan is normal saline; according to the technical scheme, in-vivo and in-vitro efficacy verification of the sidilan cardiac glycoside for treating the myelodysplastic syndrome is provided; after myelodysplastic syndrome cells are treated by using the cedilan, mitochondrial dysfunction is caused, and the cells are finally induced to generate mitochondrial autophagy, so that the anti-tumor effect of the cedilan is exerted.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of deslanoside in the treatment of myelodysplastic syndrome. Background Art

[0002] Myelodysplastic Neoplasms (MDS) is a group of highly heterogeneous clonal hematopoietic system malignancies, mainly characterized by abnormal proliferation of hematopoietic cells in the bone marrow, ineffective hematopoiesis, and the potential risk of transformation into Acute Myeloid Leukemia (AML). Currently, about 80% of patients with myelodysplastic syndrome (MDS) are over 60 years old. In clinical treatment, the International Prognostic Scoring System (IPSS) is often used to evaluate the disease progression risk of MDS patients. This scoring system comprehensively considers factors such as cytopenia, the percentage of bone marrow blasts, and chromosome karyotype to guide the selection of treatment regimens for patients with different risk stratifications. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is currently the only method that can cure high-risk myelodysplastic syndrome (HR-MDS) patients. However, due to factors such as age, comorbidities, and the lack of suitable donors, most patients cannot receive Allo-HSCT treatment. The conversion rate of HR-MDS patients to acute myeloid leukemia (AML) exceeds 50%. Therefore, exploring new drugs, new targets, and new mechanisms is of great significance for the treatment of MDS. The present invention screens the FDA-approved compound library through cell anti-tumor activity screening, aiming to discover new drugs for the treatment of myelodysplastic syndrome (MDS). At the same time, in vitro and in vivo anti-tumor pharmacodynamic evaluations, safety assessments of the candidate drug deslanoside (Des), and a preliminary exploration of its potential mechanism for inducing cell death in myelodysplastic syndrome are carried out. Summary of the Invention

[0003] The present invention aims to provide an application of deslanoside in the treatment of myelodysplastic syndrome to solve the problems raised in the above background art.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] An application of deslanoside in a drug or composition for the treatment of myelodysplastic syndrome.

[0006] Preferably, the effective concentration of deslanoside is 0.1 - 3 μM.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] The present invention provides in vivo and in vitro pharmacodynamic verification of cedilanid in the treatment of myelodysplastic syndrome. After treating MDS cells with cedilanid, mitochondrial dysfunction is induced, and ultimately autophagy of the cells is induced, thereby exerting its anti-tumor effect. Brief Description of the Drawings

[0009] Figure 1 It shows the changes in cell viability after treating MUTZ-1 and MOLM-13 cells with different concentrations of cedilanid for different times;

[0010] Figure 2 It shows the changes in the DNA synthesis ability of MUTZ-1 and MOLM-13 cells after treating them with different concentrations of cedilanid for 24 h;

[0011] Figure 3 It shows the changes in the colony formation ability of MUTZ-1 and MOLM-13 cells after treating them with different concentrations of cedilanid for 24 h;

[0012] Figure 4 It shows the changes in mitochondrial morphology of MUTZ-1 and MOLM-13 cells after treating them with different concentrations of cedilanid for 24 h;

[0013] Figure 5 It shows the changes in intracellular reactive oxygen species and mitochondrial membrane potential of MUTZ-1 and MOLM-13 cells after treating them with different concentrations of cedilanid for 24 h (Note: The yellow arrow represents normal mitochondria, the red arrow represents abnormal mitochondria, and the blue arrow represents autophagolysosomes);

[0014] Figure 6 It shows the changes in the intracellular autophagy level of MUTZ-1 and MOLM-13 cells after treating them with cedilanid for 24 h;

[0015] Figure 7 It shows the changes in the expression of autophagy proteins in MUTZ-1 and MOLM-13 cells after treating them with different concentrations of cedilanid for 24 h;

[0016] Figure 8 It is a schematic diagram of the cardiac glycoside cedilanid. Detailed Embodiments

[0017] The present invention will be further described in detail below in conjunction with the drawings and embodiments:

[0018] An application of the cardiac glycoside cedilanid in the treatment of myelodysplastic syndrome, an application of the cardiac glycoside cedilanid in a drug or composition for the treatment of myelodysplastic syndrome, with an effective concentration of 0.1 - 3 μM. The cardiac glycoside cedilanid in the drug or composition induces mitochondrial dysfunction and ultimately leads to mitochondrial autophagy of the cells, thereby exerting its anti-tumor effect.

[0019] The biomaterials, drugs, and experimental methods used in Examples 1-2 are as follows:

[0020] Cells: MUTZ-1 (human myelodysplastic syndrome cell line) and MOLM-13 (human acute myeloid leukemia cell line) can be obtained commercially, for example, from ATCC in the United States.

[0021] Drugs: Digoxin (from Selleck, USA) was accurately weighed, dissolved in dimethyl sulfoxide, and prepared into a stock solution with a digoxin concentration of 10 mmol / L, stored at -20°C, and diluted to an appropriate concentration with normal saline when used.

[0022] Experimental methods:

[0023] Cell resuscitation

[0024] (1) Prepare complete medium: Add 50 mL of FBS (10%) and 5 mL of penicillin-streptomycin double antibody (1%) to 450 mL of 1640 medium to prepare 1640 complete medium, and store it at 4°C for a long time;

[0025] (2) Warm the water bath to 37°C, take out the cells from the liquid nitrogen tank, and quickly shake them in the preheated water bath to completely dissolve them;

[0026] (3) Transfer the cells to a centrifuge tube containing complete medium in a laminar flow hood, centrifuge at 1200 rpm for 5 min, and discard the supernatant;

[0027] (4) Resuspend the cells with 5 mL of complete medium, and then transfer them to a T25 culture flask;

[0028] (5) Observe the cell size, morphology, and density under an ordinary optical microscope, and then transfer them to an incubator at 37°C and 5% CO2 for culture.

[0029] Cell passage

[0030] (1) Observe the cells every other day. When their density reaches 80-90%, aspirate the cell suspension in the T25 into a sterile 15 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, and discard the supernatant;

[0031] (2) Wash once with sterile 1×PBS;

[0032] (3) After adding 10 mL of medium to resuspend the cells, divide them equally into two T25s, and then place them in an incubator at 37°C and 5% CO2 for continued culture.

[0033] 3. Cell cryopreservation

[0034] (1) Prepare the cell cryopreservation solution in advance: Mix 9 mL of FBS with 1 mL of DMSO in a ratio of 9:1, and mix well for later use;

[0035] (2) Aspirate the cell suspension in the T25 into a sterile 15 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, and discard the supernatant;

[0036] (3) Resuspend the cells with 1 mL of the cell cryopreservation solution and transfer them to a sterile cryopreservation tube. Mark the cell name, passage number, date, name, etc. on the cryopreservation tube;

[0037] (4) Place the cell cryopreservation tube in a cryobox and incubate at -80 °C overnight;

[0038] (5) Take out the cells at -80 °C and transfer them to a liquid nitrogen tank for long-term storage.

[0039] 4. Cell viability assay

[0040] (1) Seed the cells at a density of 1×10 4 cells / well in a 96-well plate, add different concentrations of the drug and incubate for 24, 48, and 72 h respectively. Set 3 replicates for each group;

[0041] (2) Add 10 μL of CCK-8 reagent to each well, continue to culture at 37 °C for 2 h, and measure the absorbance at 450 nm [OD 450 using an enzyme-linked immunosorbent assay (ELISA) reader;

[0042] (3) Calculate the cell survival rate = [OD of the experimental group wells 450 - OD of the blank wells 450 / [OD450 of the control group - OD of the blank wells 450 × 100%.

[0043] 5. EdU detection

[0044] (1) Seed the cells at a density of 1×10 6 cells / well in a 6-well plate, add 0, 0.3, 1, 3 μM of Des and incubate for 24 h;

[0045] (2) Add the EdU working solution with a final concentration of 10 μM to the 6-well plate, incubate at 37 °C in 5% CO2 for 2 h, centrifuge at 1200 rpm for 5 min to collect the cells, and wash them once with 1×PBS;

[0046] (3) Add 1 mL of 4% paraformaldehyde to fix the cells at room temperature for 15 min, and wash them once with 1×PBS;

[0047] (4) Add 1 mL of PBS containing 0.3% Triton X-100, incubate at room temperature for 15 min, and wash them once with 1×PBS;

[0048] (5) Add 500 μL of Click reaction solution and incubate in the dark at room temperature for 30 min, then wash once with 1×PBS;

[0049] (6) Add 500 μL of Hoechst 33342 staining working solution with a final concentration of 10 μM, stain in the dark at room temperature for 30 min, then wash once with 1×PBS;

[0050] (7) Observe under an upright fluorescence microscope, randomly select 5 fields of view for photography, and calculate the positive cell rate.

[0051] 6. Soft Agar cell soft clone formation assay

[0052] (1) Preheat the water bath to 37 °C and 42 °C in advance;

[0053] (2) Dissolve the low melting point Agar powder with ultrapure water, prepare 3.5% Agar solution and autoclave it. This concentration is the stock solution and place it in a 42 °C water bath;

[0054] (3) Prepare the lower layer gel: Dilute the stock solution 1:5 with 1640 medium to 0.7% Agar solution, 2 mL / well, and let it solidify at 4 °C for 5 min;

[0055] (4) Prepare the upper layer gel: Count the number of MUTZ-1 and MOLM-13 cells to be 4000 / well, resuspend them in 1640 medium with 0.35% Agar solution diluted 1:10 (at 37 °C), 1.5 mL / well, let it solidify at 4 °C for 1 - 2 h, and then place it in a 37 °C incubator;

[0056] (5) Observe after 2 - 3 weeks. After visible cell clones are observed with the naked eye, add 200 μL of MTT solution with a final concentration of 1 mg / mL to each well, incubate at 37 °C and 5% CO2 for 30 min, then take a photo and count the formed clones.

[0057] 7. Transmission electron microscopy for detecting cell mitochondrial morphology

[0058] (1) Seed cells at 1×10 6 cells / well in a 6-well plate, and add 0, 0.3, 1, 3 μM of Des and incubate for 24 h;

[0059] (2) Centrifuge at 1200 rpm for 5 min to collect cells, and wash once with 1×PBS;

[0060] (3) Add 1 mL of electron microscopy fixative and fix at room temperature for 2 h, then transfer to 4 °C for storage. The subsequent detection was completed by Wuhan Sevier Biotechnology Co., Ltd.

[0061] 8. Flow cytometry for detecting cell mitochondrial membrane potential and reactive oxygen species level

[0062] (1) Cells were seeded at 1×10 6 cells per well in a 6-well plate and treated with 0, 0.3, 1, 3 μM Des for 24 h;

[0063] (2) Cells were collected by centrifugation at 1200 rpm for 5 min and washed once with 1×PBS;

[0064] (3) 500 μL of TMRE working solution or DCFH-DA working solution with a final concentration of 10 μM was added, and the cells were stained in the dark at room temperature for 30 min. Then, the cells were centrifuged at 1200 rpm for 5 min and washed once with 1×PBS;

[0065] (4) The cells were resuspended in 500 μL of 1×PBS, filtered, and then detected by flow cytometry.

[0066] Detection of autophagy level in cells by DAPGreen

[0067] Cells were seeded at 1×10 6 cells per well in a 6-well plate and treated with 0, 0.3, 1, 3 μM Des for 24 h;

[0068] The working solution of DAPGreen with a final concentration of 0.1 μM was added to the 6-well plate, and the cells were incubated at 37 °C in 5% CO2 for 30 min. Then, the cells were collected by centrifugation at 1200 rpm for 5 min and washed once with 1×PBS;

[0069] 1 ml of the working solution of DeepRedFM with a final concentration of 500 nM was added, and the cells were incubated in the dark at 37 °C for 30 min and then washed once with 1×PBS; 1 ml of the staining working solution of Hoechst33342 with a final concentration of 10 μM was added, and the cells were stained in the dark at 37 °C for 30 min and then washed once with 1×PBS;

[0070]

[0071] Observation was performed under an upright fluorescence microscope. Five fields of view were randomly selected for photography, and the autophagy incidence rate of the cells was calculated.

[0072] Detection of autophagy protein expression in cells by Western Blot

[0073]

[0073] Cells were seeded at 1×10 6 cells per well in a 6-well plate. After treatment with different concentrations of Des (0.3, 1, 3 μM) for 24 h, the cells were collected;

[0074] Protein lysate was added, and the cells were lysed on ice for 30 min. After centrifugation at 12000 r / min at 4 °C for 10 min, the supernatant was taken;

[0075] The protein concentration was detected and quantified using a BCA protein quantification kit;

[0076] Add 5×loading buffer and heat in a metal bath at 100°C for 10 min for routine Western blot detection;

[0077] Prepare a 10% SDS-PAGE gel for electrophoresis, transfer the membrane to a PVDF membrane, block it quickly with a blocking solution for 15 min, incubate with primary antibodies LC3B, Atg7, Atg3, P62, LC3B, and β-actin overnight at 4°C at a dilution ratio of 1:1000, wash 3 times with 1×TBST for 10 min each time;

[0078] Incubate with the secondary antibody at room temperature for 2 h, wash 3 times with 1×TBST for 10 min each time. Prepare the developing solution, develop and take pictures with a chemiluminescence imager.

[0079] Use ImageJ software to detect the gray value of protein bands. The relative expression level of the target protein is expressed as the ratio of the gray value of the target protein to that of the internal reference.

[0080] Example 1: Digoxin can significantly inhibit the proliferation of MDS cells in vitro and in vivo

[0081] Compared with the control group, the proliferation ability of MUTZ-1 and MOLM-13 cells was significantly inhibited after Des administration, showing dose- and time-dependence. Further fitting of the drug IC50 showed that the IC50 of Des in MUTZ-1 cells was 0.642 μM (24 h), 0.1876 μM (48 h), and 0.2046 μM (72 h), and the IC50 of Des in MOLM-13 cells was 1.384 μM (24 h), 0.3659 μM (48 h), and 0.2605 μM (72 h) ( Figure 1 ).

[0082] The effect of Des on the tumor cell cycle was detected by flow cytometry. The results showed that Des could significantly arrest the cell cycles of MUTZ-1 and MOLM-13 cells at the G0 phase, showing dose-dependence ( Figure 2 ).

[0083] The effect of Des on tumor cell DNA synthesis was detected by Edu. The results showed that Des could significantly arrest tumor cell DNA synthesis, showing dose-dependence ( Figure 3 ).

[0084] We also detected the effect of Des on tumor cell colony formation by the soft agar colony formation assay. The results showed that Des could significantly inhibit the colony formation of MDS cells, showing dose-dependence ( Figure 4 ).

[0085] The above results together indicate that Des can well inhibit the proliferation of MDS cells, showing dose-dependence.

[0086] Example 2: Cedilanid induces mitochondrial damage in MDS cells, thereby triggering autophagy and inhibiting cell proliferation

[0087] The mitochondrial morphology was observed by transmission electron microscopy. Compared with the control group, obvious abnormalities such as swelling and rupture occurred in the mitochondria after Des treatment, the mitochondrial cristae were broken or even disappeared, and autophagolysosomes were also observed in MDS cells after Des treatment. The above results indicate that Des may induce mitochondrial damage, leading to autophagy in cells( Figure 5 ).

[0088] Furthermore, flow cytometry was used to detect the changes in mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential in MDS cells after Des treatment. The results showed that compared with the control group, mitochondrial ROS increased in a dose-dependent manner and mitochondrial membrane potential decreased in a dose-dependent manner after Des treatment. The above results indicate that Des can induce mitochondrial oxidative damage, resulting in an increase in mitochondrial superoxide and a decrease in mitochondrial membrane potential( Figure 6 ).

[0089] The effect of Des on autophagy in tumor cells was detected by DAPGreen. The results showed that compared with the control group, the autophagy level in cells increased after Des treatment( Figure 7 A). The Western blot results showed that after Des treatment of MDS cells, the expressions of Atg3, Atg7, and LC3B were significantly up-regulated, and the expression of P62 was significantly down-regulated, indicating that Des can induce autophagy in MDS cells( Figure 7 B).

[0090] In summary, Des can significantly inhibit the proliferation of MDS cells in a dose-dependent manner. Further detection of mitochondrial ROS and membrane potential in MDS cells found that as the concentration of Des increased, mitochondrial ROS increased while mitochondrial membrane potential decreased. Transmission electron microscopy revealed obvious abnormalities in the mitochondrial morphology of MDS cells after Des administration, such as swelling and rupture. The above findings prove that Des can induce oxidative stress damage in the mitochondria of MDS cells. In addition, the generation of autophagolysosomes was observed by electron microscopy, indicating that the inhibition of MDS cell proliferation by Des may be related to mitochondrial oxidative damage-mediated autophagy.

[0091] The above are only embodiments of the present invention, and common general technical solutions and / or characteristics in the solutions are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. Use of digoxin in the treatment of myelodysplastic syndrome, characterized in that: Use of the cardiac glycoside cedilanid in a medicament or composition for treating myelodysplastic syndrome.

2. Use of a cardiotonic digoxin in the treatment of myelodysplastic syndrome according to claim 1, characterized in that: The effective concentration of the cardiac glycoside cedilanid is 0.1 - 3 μM.

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