Use of hydroxyurea for the preparation of a medicament for the prevention or treatment of retinoblastoma

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of hydroxyurea in preparation of a drug for preventing or treating retinoblastoma, which can achieve the effect of effectively treating RB by specifically targeting ecDNA in retinoblastoma tumor cells, and has high effectiveness and high specificity. The hydroxyurea provided by the application can inhibit the expression of SUZ12 in retinoblastoma cell lines WERI-RB1 or Y79 cells, reduce the modification level of H3K27 trimethylation, and can inhibit the proliferation and tumorigenicity of retinoblastoma cell lines WERI-RB1 or Y79 cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of hydroxyurea in preparation of a medicine for preventing or treating retinoblastoma, which can specifically target ecDNA in retinoblastoma tumor cells, can inhibit expression of SUZ12 in retinoblastoma cell lines WERI-RB1 or Y79 cells, reduce modification level of H3K27 trimethylation, and can inhibit proliferation and tumorigenicity of retinoblastoma cell lines WERI-RB1 or Y79 cells. BACKGROUND

[0002] Retinoblastoma (RB) is the most common intraocular malignant tumor in children, which can cause blindness, disability and death. The annual new cases in China account for 20% of the world, and the incidence ranks first in the world. In 2019, RB was listed as one of the ten children's tumors that need to be treated as a priority by the National Health Commission and other four departments. RB is commonly seen in infants under 3 years old, about 1 / 3 of patients can be hereditary, and the incidence risk of offspring of hereditary patients is nearly 50%, and almost all of them involve both eyes. With the improvement of diagnosis technology and the popularization and application of new treatment methods, after the 1990s, the survival rate of RB children in developed countries is close to 95%. However, in developing countries with a large population and uneven medical resources, early diagnosis and treatment of RB are still a problem.

[0003] At present, chemotherapy is the main treatment method for RB, and chemotherapy is divided into systemic intravenous chemotherapy and ophthalmic artery interventional chemotherapy. Systemic intravenous chemotherapy can significantly improve the eye preservation rate and survival rate of children, reduce the application of radiotherapy in RB, and significantly reduce the incidence of secondary tumors. However, due to the insufficient bioavailability of chemotherapy drugs, systemic intravenous chemotherapy has the disadvantages of low effective rate of eye preservation treatment, serious systemic side effects, and intolerance of some children. Ophthalmic artery interventional chemotherapy has been used for eye preservation treatment of RB for more than ten years, and the ophthalmic artery can directly carry chemotherapy drugs to the intraocular tumor, which can improve the eye preservation rate of children and reduce systemic complications. It is worth noting that most patients still need multiple interventional treatment, and there are many eye complications. For some patients with high malignancy and tumor drug resistance, the treatment effect is still not ideal. In summary, it is urgent to solve the problem of revealing the pathogenesis of RB, finding new targets for effective treatment of RB, and reducing the mortality rate of RB. SUMMARY

[0004] In view of this, the purpose of the present application is to provide the application of hydroxyurea (HU) in the preparation of a medicine for preventing or treating retinoblastoma.

[0005] To achieve the above-mentioned purpose, the solution adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a biomarker targeting ecDNA in retinoblastoma tumor cells, which is hydroxyurea or a pharmaceutically acceptable salt thereof as shown in the following formula (I):

[0007]

[0008] In a second aspect, the present application also provides a pharmaceutical composition for preventing or treating retinoblastoma, which comprises the biomarker targeting ecDNA in retinoblastoma tumor cells as described above.

[0009] Preferably, the biomarker targeting ecDNA in retinoblastoma tumor cells is hydroxyurea or a pharmaceutically acceptable salt thereof.

[0010] Preferably, the pharmaceutical composition further comprises a medically acceptable adjuvant or carrier.

[0011] Preferably, the dosage form of the pharmaceutical composition is selected from tablets, powders, injections, capsules, suspensions, pastes, gels, coatings, drug films, sustained-release agents or microspheres.

[0012] In a third aspect, the present application also provides the use of hydroxyurea or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating retinoblastoma, which can target ecDNA in retinoblastoma tumor cells.

[0013] Preferably, the retinoblastoma tumor cells are human retinoblastoma cell lines WERI-RB1 or Y79 cells.

[0014] Preferably, the hydroxyurea or a pharmaceutically acceptable salt thereof can inhibit the expression of SUZ12 in retinoblastoma cell lines WERI-RB1 or Y79 cells, and reduce the modification level of H3K27 trimethylation.

[0015] Preferably, the hydroxyurea or a pharmaceutically acceptable salt thereof can inhibit the proliferation and tumorigenicity of retinoblastoma cell lines WERI-RB1 or Y79 cells.

[0016] Preferably, the effective dose of the hydroxyurea or a pharmaceutically acceptable salt thereof is not less than 150 μM, preferably 150-300 μM.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] (1) The hydroxyurea provided by the application can inhibit the expression of SUZ12 in retinoblastoma cell lines WERI-RB1 or Y79 cells, reduce the modification level of H3K27 trimethylation, and can significantly inhibit the growth of retinoblastoma cells, and the drug effect increases with the increase of concentration. The application aims to provide a new clinical treatment drug for retinoblastoma, improve the treatment effectiveness, prolong the life of patients, improve the quality of life, and thus open up a new field of hydroxyurea for retinoblastoma treatment.

[0019] (2) The hydroxyurea provided by the application can effectively treat RB by specifically targeting ecDNA in RB cells, and has high effectiveness and high specificity. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The results of the application example of the hydroxyurea inhibiting the expression of SUZ12 in retinoblastoma cells and reducing the modification level of H3K27me3 are shown in the figure.

[0021] Figure 2 The CCK8 result graph of the application example of the hydroxyurea significantly inhibiting the proliferation ability of retinoblastoma cells is shown. A is that the proliferation ability of retinoblastoma Y79 cell line is significantly reduced after being treated with 150 μM hydroxyurea; B is that the proliferation ability of retinoblastoma WERI-Rb-1 cell line is significantly reduced after being treated with 150 μM hydroxyurea, *p<0.01.

[0022] Figure 3 The soft agar colony formation experiment result graph of the application example of the hydroxyurea significantly inhibiting the tumorigenic ability of retinoblastoma cells is shown. A is that the tumorigenic ability of retinoblastoma cell line is significantly reduced after being treated with 150 μM hydroxyurea; B is the statistics of the relative number of colony formation, ***p<0.001. DETAILED DESCRIPTION

[0023] The application provides the use of hydroxyurea (Hydroxyurea) shown in the following chemical formula (I) in the preparation of a drug for preventing or treating retinoblastoma, which can inhibit the expression of SUZ12 in retinoblastoma cell lines WERI-RB1 or Y79 cells, reduce the modification level of H3K27 trimethylation, and can inhibit the proliferation and tumorigenic ability of retinoblastoma cell lines WERI-RB1 or Y79 cells.

[0024]

[0025] The present application also provides a biomarker targeting ecDNA in retinoblastoma tumor cells, which is hydroxyurea or a pharmaceutically acceptable salt thereof as shown in the above formula (I).

[0026] The present application provides hydroxyurea or a pharmaceutically acceptable salt thereof capable of targeting ecDNA in retinoblastoma tumor cells.

[0027] The present application also provides a pharmaceutical composition for preventing or treating retinoblastoma, which comprises the biomarker targeting ecDNA in retinoblastoma tumor cells as described above.

[0028] Preferably, the pharmaceutical composition further comprises a medically acceptable adjuvant or carrier.

[0029] Preferably, the dosage form of the pharmaceutical composition is selected from tablets, powders, injections, capsules, suspensions, pastes, gels, coatings, film agents, sustained-release agents, or microspheres.

[0030] Dosing and pharmaceutical compositions

[0031] In general, the pharmaceutical composition of the present application can be administered in an effective amount by any acceptable administration method for other similar purposes. For example, the pharmaceutical composition of the present application can be administered orally, parenterally, transdermally, topically, rectally, or intranasally. In the present application, the effective dose of the pharmaceutical composition of the present application is not less than 150 μM, and preferably 150-300 μM.

[0032] When used as a medicine, the present application is usually administered in the form of a pharmaceutical composition. These compositions can be prepared by methods well known in the pharmaceutical art, and comprise at least one active compound, which in the present application is hydroxyurea or a pharmaceutically acceptable salt thereof as shown in the above formula (I). In formulating the compositions provided by the present application, the active ingredient is typically combined with a medically acceptable adjuvant or carrier, diluted by a medically acceptable adjuvant or carrier, or enclosed within a capsule, sachet, paper, or other container by means of a medically acceptable adjuvant or carrier. When the medically acceptable adjuvant or carrier serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, sprays (as solids or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0033] Some typical pharmaceutically acceptable excipients or carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginic acid, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. Additionally, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweetening agents; and flavoring agents can be included. The pharmaceutical compositions of the present application can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing well-known methods in the art of pharmaceutical formulation.

[0034] The amount of active ingredient, i.e., hydroxyurea or a pharmaceutically acceptable salt thereof, as represented by Formula (I) above, in the pharmaceutical composition and unit dosage form can be varied or adjusted depending upon the particular application and the particular compound's activity and desired concenfration.

[0035] "Treatment" means any treatment of a disease in a mammal, including: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop; (2) inhibiting the disease, i.e., arresting the development of clinical symptoms; and (3) relieving the disease, i.e., causing the clinical symptoms to regress.

[0036] The technical solutions of the present application are further described below in conjunction with specific examples, but the scope of protection of the present application is not limited to these examples. Any changes or equivalent replacements that do not depart from the concept of the present application are included in the scope of protection of the present application.

[0037] Example 1: Western Blot

[0038] Experimental procedure:

[0039] (1) When the RB cells in the T25 culture flask grow to about 70% to 80% density, the cell suspension is transferred to a 15 ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and then the supernatant is carefully discarded, followed by adding pre-cooled PBS to wash the cell precipitate. After thoroughly washing the residual culture medium, according to the amount of cells, 60-100 μl of cell lysis buffer RIPA containing phosphatase inhibitor and protease inhibitor is added for cell protein lysis, and the cells are incubated on ice for 30 minutes. Then, the cell lysate is transferred to a new 1.5 mL EP tube, centrifuged at 10000 rpm for 40 minutes at 4°C, and the supernatant is taken. The protein concentration is detected and quantified using the Bradford protein assay kit. According to the measured protein concentration, an appropriate amount of 5x SDS loading buffer is added to adjust the volume of the protein sample. After mixing well, heat at 95°C for 5 minutes. The obtained protein sample can be directly used for the following Western blotting experiment.

[0040] (2) The prepared gel is fixed on the electrophoresis device, the glass plate and the plastic clamp are tightened, and 900 ml of Tris-glycine electrophoresis buffer is added. 5-20 ul of protein Maker and protein sample are added to the channel using a pipette, and electrophoresis is carried out at a voltage of 80V for 30 minutes. After the protein sample enters the separation gel, the voltage is adjusted to 120V, and the electrophoresis is continued until the bromophenol blue reaches the bottom of the separation gel, and then the electrophoresis is stopped.

[0041] (3) After the electrophoresis is completed, the protein electrophoresis instrument is turned off, the gel is taken out, and the part of the gel without sample is cut off. According to the size of the remaining gel required, a polyvinylidene fluoride (PVDF) membrane of appropriate size is cut and activated by immersing it in 100% methanol for 3-5 minutes. A transfer buffer is prepared using Tris-base, glycine, methanol, and ddH2O, and then a transfer membrane special clamp is soaked in the transfer buffer. From the anode to the cathode, the sponge, filter paper, PVDF membrane, electrophoresed gel, filter paper, and sponge are placed in order, and the transfer clamp is installed and placed in the transfer device. 1000 ml of transfer buffer is added, and the transfer device is placed in ice. Transfer is carried out at a current of 200 mA.

[0042] (4) After the completion of the transfer film, the PVDF membrane was blocked with 5% BSA blocking solution at room temperature for 1 hour. After blocking, the membrane was washed with TBST solution for 3 times, each for 5 minutes. The primary antibody solution for Western blot was prepared according to the antibody instructions, and the PVDF membrane was immersed in it and incubated on a 4°C shaker overnight. The PVDF membrane was taken out and washed with TBST solution for 3 times, each for 5 minutes. Then the PVDF membrane was immersed in the secondary antibody solution prepared by diluting the antibody, and placed in a shaker at room temperature in the dark for 2 hours. The membrane was washed with TBST solution for 3 times, each for 5 minutes. Finally, the PVDF membrane was placed in the Odyssey scanning imaging system for scanning, and the gray value of the band was analyzed by Image J software.

[0043] Experimental results: as shown in Figure 1 , hydroxyurea can inhibit the expression of SUZ12 in RB cells and reduce the modification level of H3K27me3.

[0044] Example 2: CCK8 cell proliferation experiment

[0045] Experimental materials: human retinoblastoma cell line WERI-RB1, Y79. Hydroxyurea was purchased from Selleck (China), and CCK8 was purchased from Tongren Chemical (Japan).

[0046] Experimental steps:

[0047] (1) WERI-Rb-1, Y79 cells were cultured at 37°C in a 5% CO2 incubator, and were cultured with 20% FBS in 1640 medium.

[0048] (2) The cells were counted using a cell counting plate, and 3000 cells were seeded in each well of a 96-well plate, and 100 μl of complete culture medium was added to each well. Drug groups and control groups were set up, and 150 μM of hydroxyurea was added to each well of the drug group, and an equal amount of PBS was added to the control group. (3) After the cells were plated and incubated in the incubator for about 6 hours, 10 μL of CCK8 solution was added to each well, and the absorbance at 450 nm was measured using a microplate reader after incubation in the incubator for 4 hours. Detection was performed every 24 hours, and the growth curve of the cells was drawn according to the detection results at 0 days, 1 day, 2 days, 3 days, and 4 days.

[0049] Experimental results: as shown in Figure 2 , it can be seen that hydroxyurea can significantly inhibit the proliferation of retinoblastoma.

[0050] Example 3: Soft agar colony formation experiment (Soft Agar Assay)

[0051] Experimental materials: human retinoblastoma cell lines WERI-RB1, Y79. Low-melting agarose was purchased from beyotime (China), hydroxyurea was purchased from Selleck (China), CCK8 was purchased from Tongren Chemical (Japan).

[0052] Experimental steps:

[0053] (1) First, use powder medium to prepare 20% FBS 2x1640 culture solution, mix 2x1640 culture solution and 1.2% low-melting agarose in a volume ratio of 1:1, and note that the agarose should be cooled to below 70°C to avoid damaging the protein components.

[0054] (2) Use a Pasteur pipette to gently spread about 1ml of the mixed solution in a 6-well plate, and note that there should be no bubbles in the mixed solution. Wait for it to solidify at room temperature. Then place the 6-well plate in a 37°C incubator overnight, and observe whether there is contamination in the wells of the 6-well plate. If there is no contamination, count the cells and resuspend them in 2x1640 culture solution, resuspend them at a final density of 20000 cells / ml, set up drug groups and control groups, add hydroxyurea to each well of the drug group to make the drug concentration 150μM, and add an equal amount of PBS to the control group. Then reheat the low-melting agarose, add a PBS solution to prepare a 0.5% low-melting agarose solution, and then mix the cell suspension and the 0.5% low-melting agarose solution in a volume ratio of 1:1. Use a Pasteur pipette to take 1ml of the mixed solution and evenly spread it on the top layer of the 6-well plate. Wait for it to solidify at room temperature. After it solidifies, add a small amount of PBS solution to ensure that the PBS covers the agarose properly to prevent the agarose from drying and cracking.

[0055] (3) Culture for 3-4 weeks until obvious cell clonal masses are visible to the naked eye, then add methanol for fixation. After fixation, use crystal violet staining for 2h, remove the crystal violet, add ddH2O or use PBS to wash the background color in the culture medium, and take a photo for observation.

[0056] Experimental results: As shown in Figure 3 , hydroxyurea can significantly inhibit the tumorigenicity of retinoblastoma.

[0057] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. Use of hydroxyurea or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating retinoblastoma, wherein the hydroxyurea or a pharmaceutically acceptable salt thereof can target ecDNA in retinoblastoma tumor cells.

2. The use according to claim 1, characterized in that The retinoblastoma tumor cells are human retinoblastoma cell lines WERI-RB1 or Y79 cells.

3. The use according to claim 1, characterized in that The hydroxyurea or a pharmaceutically acceptable salt thereof can inhibit the expression of SUZ12 in retinoblastoma cell lines WERI-RB1 or Y79 cells and reduce the modification level of H3K27 trimethylation.

4. The use according to claim 1, characterized in that The hydroxyurea or a pharmaceutically acceptable salt thereof can inhibit the proliferation and tumorigenicity of retinoblastoma cell lines WERI-RB1 or Y79 cells.

5. The use according to claim 1, characterized in that The effective dose of hydroxyurea or a pharmaceutically acceptable salt thereof is not less than 150 μM.

6. The use according to claim 5, characterized in that The effective dose of hydroxyurea or a pharmaceutically acceptable salt thereof is 150-300 μM.

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