Application of RSL3 in combination with chemotherapy drugs in the preparation of drugs for treating tumors

Abstract

This invention relates to the field of biomedicine, providing the application of RSL3 in combination with chemotherapy drugs in the preparation of drugs for treating tumors; this invention demonstrates that knocking out PSME3 downregulates important proteins SLC7A11 and GPX4 in the ferroptosis pathway, and that the ferroptosis inducer RSL3 has a significant inhibitory effect on chondrosarcoma cells HCS 2 / 8; this invention shows that the combination of RSL3 and cisplatin is effective in treating chondrosarcoma, and also has a synergistic effect in chondrosarcoma tumor-bearing animal models.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of RSL3 in the preparation of drugs for treating tumors in combination with chemotherapy drugs. Background Technology

[0002] Chondrosarcoma is the second most common primary malignant bone tumor, accounting for one-third of all bone tumors. Localized pain is the primary symptom in most patients, while some may present with pathological fractures as their first symptom. Chondrosarcoma commonly occurs in the pelvis and long bones, rarely affecting small bones such as the fingers and toes. Currently, surgical resection is the main treatment for chondrosarcoma, while traditional radiotherapy and chemotherapy have limited effectiveness.

[0003] In recent years, a large number of scholars have studied the pathogenesis of chondrosarcoma, aiming to solve the problem of highly malignant and metastatic chondrosarcoma with no available drugs. Ferroprelation, as a newly discovered cell death pathway in recent years, has a huge impact on radiotherapy, chemotherapy and immunotherapy of tumors. It is hoped that the ferroprelation pathway can solve the problem of drug resistance in chondrosarcoma. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing an application of ferroptosis-inducing agents in combination with chemotherapeutic drugs in the preparation of drugs for treating tumors.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A first aspect of the present invention is to provide the use of RSL3 in combination with chemotherapeutic drugs in the preparation of medicaments for treating tumors.

[0007] Preferably, the chemotherapy drug is cisplatin.

[0008] Preferably, the combination index of RSL3 with the chemotherapy drug is less than 1.

[0009] Preferably, the tumor is chondrosarcoma.

[0010] A second aspect of the invention is to provide the use of RSL3 in the preparation of medicaments for treating tumors.

[0011] Preferably, the tumor is chondrosarcoma.

[0012] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0013] This invention demonstrates that knocking out PSME3 downregulates the important proteins SLC7A11 and GPX4 in the ferroptosis pathway, and that the ferroptosis inducer RSL3 significantly inhibits the HCS2 / 8 cells of chondrosarcoma. This invention also shows that the combination of RSL3 and cisplatin is effective in treating chondrosarcoma and has a synergistic effect in chondrosarcoma-bearing animal models. Attached Figure Description

[0014] Figure 1 This is a graph showing the results of Western Blot detection of changes in key proteins in the ferroptosis pathway in one embodiment of the present invention;

[0015] Figure 2 This is a graph showing the change in GSH levels after HCS2 / 8 gene knockout of PSME3 in one embodiment of the present invention.

[0016] Figure 3 This is the combination drug index of RSL3 and cisplatin in inhibiting chondrosarcoma in one embodiment of the present invention;

[0017] Figure 4 shows the proliferation results of chondrosarcoma in one embodiment of the present invention; Figure 4A The image below shows the staining microscope. Figure 4B For analysis;

[0018] Figure 5 This is a graph showing the proportion of apoptotic cells in chondrosarcoma in one embodiment of the present invention;

[0019] Figure 6 This is a comparison chart of mouse tumor weights in one embodiment of the present invention;

[0020] Figure 7 This is a diagram showing tumor growth changes in one embodiment of the present invention;

[0021] Figure 8 This is a CT scan image of a tumor in one embodiment of the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0025] Example

[0026] 1. Western blot analysis to detect changes in key proteins in the ferroptosis pathway

[0027] (1) Sample preparation: Human chondrosarcoma cells HCS2 / 8 were taken and divided into two groups, one group was HCS2 / 8PSME3 WT and the other group was HCS2 / 8PSME3 KO with the PSME3 gene knocked out; the two groups of cells were lysed with 1×SDS loading and boiled in a 100℃ metal bath for 15 min to denature the proteins.

[0028] (2) Preparation of protein gel: Clean the protein gel plate and rinse it once with ddH2O; after cleaning, put it in the oven to dry, align the gel plates and clamp them parallel on the gel plate rack, prepare the lower separating gel according to the protein gel formula, flatten it with anhydrous ethanol, discard the anhydrous ethanol after the lower gel solidifies, prepare the upper stacking gel according to the formula, insert a comb, and after the upper gel solidifies, store it in a 4℃ refrigerator.

[0029] (3) Electrophoresis: The prepared protein samples are loaded sequentially from left to right according to the experimental design. Protein markers are left on both sides of the wells and filled with 1× loading buffer. After loading, 1× running buffer is added to the gel tank. Electrophoresis is started at 80V constant voltage. After the lower gel markers are separated, electrophoresis is continued at 120V constant voltage to the bottom.

[0030] (4) Transfer: Prepare 1× Transfer buffer (700mL ddH2O, 200mL methanol, 100mL 10× Transfer buffer) in advance, and prepare the transfer clamp, NC membrane and filter paper. Soak them in the Transfer buffer. After the protein gel electrophoresis, use a scraper to gently lift the plate, cut the upper gel, keep the lower gel, and transfer the lower gel to the transfer buffer. Clamp the transfer clamp in the following order: one layer of sponge, three layers of filter paper, lower gel, NC membrane, three layers of filter paper, and one layer of sponge. During the process, try to avoid air bubbles between the lower gel and the NC membrane. Place the transfer clamp in the transfer tank, pour in the transfer buffer, put it in an ice box, and transfer the membrane under the conditions of 200mA constant current and ice bath.

[0031] (5) Blocking: After the transfer is completed, the protein on the protein gel is successfully transferred to the NC membrane. Wash the NC membrane once with PBS buffer and block the NC membrane with 7% skim milk powder (prepared with PBS) for 60 min.

[0032] (6) Primary antibody incubation: After blocking, discard the skim milk powder, wash with PBS for 5 min, and cut the bands containing PSME3, SLC7A11, GPX4 and ACTIN proteins according to the protein marker, put them into the dark box, pour in the corresponding primary antibody, and incubate the dark box in a constant temperature shaker at 4℃ overnight.

[0033] (7) Fluorescent secondary antibody binding: The primary antibody was recovered, and the bands were washed three times with PBST (PBS buffer + 0.05% Tween-20) for 5 min each time. Then the corresponding fluorescent secondary antibody was added and incubated in a constant temperature shaker at 4℃ for 1 h.

[0034] (8) Membrane scanning: After the secondary antibody incubation, the bands were washed three times with PBST for 5 minutes each time, and the corresponding bands were scanned using an Oddesey membrane scanner to analyze the results.

[0035] The results are as follows Figure 1 As shown, with the knockout of the PSME3 gene, the key regulatory proteins SLC7A11 and GPX4 of the ferroptosis pathway decreased accordingly, proving that PSME3 can regulate ferroptosis.

[0036] 2. Detection of intracellular glutathione (GSH) levels

[0037] (1) HCS2 / 8PSME3 WT and HCS2 / 8PSME3 KO cells were seeded into 6-well plates and grown to a density of 80%-90% respectively;

[0038] (2) Preparation of glutathione working solution: Prepare working solution according to the following formula: 6.6 μL 5-fold diluted glutathione reductase, 6.6 μL DTNB stock solution, 150 μL total glutathione detection buffer;

[0039] (3) Digest and centrifuge the cells into a 1.5 mL EP tube, wash once with PBS and discard the supernatant; add 3 times the amount of protein removal reagent M solution according to the volume of cell pellet, mix by pipetting, and rapidly freeze and thaw the cells twice using a 37℃ water bath and liquid nitrogen to achieve cell lysis. Centrifuge at 4℃ and 1000g for 10 min and collect the supernatant into a new EP tube.

[0040] (4) Add 10 μL of sample and 150 μL of total glutathione working solution to each well, incubate at room temperature for 5 min, then add 50 μL of 0.5 mg / mL NADPH to each well and react in the dark for 25 min.

[0041] (5) After the reaction is complete, use an ELISA reader to detect the absorbance at 412 nm, collect and analyze the data.

[0042] The results are as follows Figure 2As shown, after PSME3 knockout, the intracellular GSH in HCS2 / 8 cells decreased significantly, indicating that the intracellular GSH synthesis was reduced and ferroptosis occurred.

[0043] 3. Combination Drug Index (CI) Test

[0044] (1) Digest HCS2 / 8, centrifuge and resuspend it, and spread it evenly in a 96-well plate to ensure 1500 cells per well;

[0045] (2) After the cells adhered, the cells were treated with RSL3 at concentrations of 0 μM, 0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM, and 4.0 μM and cisplatin at concentrations of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, and 10.0 μM.

[0046] (3) After 48 hours, remove the supernatant, add CCK-8 reagent and treat for 1 hour, and detect the absorbance at 450 nm.

[0047] (4) Calculate the drug combination index using Compusyn software based on absorbance. The drug combination index is an objective indicator of drug action. A value greater than 1 indicates that there is an antagonistic effect between drugs, and a value less than 1 indicates that there is a synergistic effect between drugs.

[0048] The results are as follows Figure 3 As shown, RSL3 and cisplatin have a synergistic effect in the treatment of chondrosarcoma, i.e., the combination drug index is less than 1.

[0049] 4. Animal model testing of the efficacy of combined RSL3 and cisplatin therapy

[0050] (1) Digest and centrifuge HCS2 / 8 cells in the logarithmic growth phase, resuspend the cells in PBS, and ensure a concentration of 5 × 10⁻⁶. 5 / 20μL;

[0051] (2) After preparing HCS2 / 8 cells, fix nude mice with a mouse fixation device, drill a hole in the tibia of the nude mouse through a clean insulin needle, and then inject tumor cells into the tibia through the hole using a needle with tumor cells.

[0052] (3) One week later, when the tumor has grown to 100mm 3 The mice were divided into four groups based on their size: PBS group, RSL3 group, Cisplatin group, and RSL3+Cisplatin group.

[0053] (4) The effects of combination therapy and monotherapy were compared by using tumor growth curves, tumor weight, and tumor CT images.

[0054] The results are shown in Figures 4-8. The combination of RSL3 and cisplatin showed a higher inhibitory effect on chondrosarcoma proliferation (Figure 4), and the combination of RSL3 and cisplatin significantly increased the proportion of apoptotic cells in chondrosarcoma. Figure 5 RSL3 combined with cisplatin significantly reduced tumor weight in chondrosarcoma-bearing mice. Figure 6 RSL3 combined with cisplatin for the treatment of chondrosarcoma tumors resulted in slow tumor growth. Figure 7 ), CT scan of chondrosarcoma-bearing tumors treated with RSL3 combined with cisplatin ( Figure 8 The results showed that RSL3 and cisplatin had a good combined therapeutic effect.

[0055] In summary, this invention demonstrates that knocking out PSME3 downregulates the important proteins SLC7A11 and GPX4 in the ferroptosis pathway, and that the ferroptosis inducer RSL3 significantly inhibits the HCS2 / 8 cells of chondrosarcoma. This invention also shows that the combination of RSL3 and cisplatin is effective in treating chondrosarcoma and has a synergistic effect in chondrosarcoma-bearing animal models.

[0056] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of RSL3 combined with cisplatin in the preparation of drugs for the treatment of chondrosarcoma.

2. The application according to claim 1, characterized in that, The combination index of RSL3 with the chemotherapy drug is less than 1.

3. Application of RSL3 in the preparation of drugs for treating chondrosarcoma.

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