Applications of MicrocolinA

Abstract

This invention relates to the field of biomedicine, and more particularly to the application of Microcolin A; it provides the application of Microcolin A in the preparation of drugs for treating osteosarcoma; this invention demonstrates that Microcolin A can effectively inhibit the proliferation of osteosarcoma cells, significantly promote osteosarcoma cell apoptosis, and promote the expression of the apoptosis-related protein p-H2AX in osteosarcoma cells, revealing the application of Microcolin A in the preparation of drugs for treating osteosarcoma; it also reveals that Microcolin A and cisplatin have good combined therapeutic effects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of Microcolin A. Background Technology

[0002] Osteosarcoma is the most common primary malignant bone tumor, originating from primitive mesenchymal cells in bone and soft tissue. It typically occurs in children and adolescents, accounting for approximately 5%-6% of all pediatric tumors. Osteosarcoma most commonly occurs in the metaphysis of the long bones of the limbs, primarily in areas of rapid bone growth, such as the distal femur, proximal tibia, proximal femur, and proximal humerus, with a smaller proportion appearing in the axial skeleton. The typical presentation of osteosarcoma is localized pain in the limb, accompanied by localized swelling and limited joint movement; pathological fractures occur in rare cases. Currently, the treatment principle for osteosarcoma is complete surgical resection combined with adjuvant chemotherapy. Chemotherapy drugs include doxorubicin, cisplatin, methotrexate, and ifosfamide, and the 5-year survival rate has increased to 60%-70%. However, osteosarcoma is a tumor that is particularly prone to developing resistance to chemotherapy drugs. Cisplatin remains the first-line chemotherapy drug for osteosarcoma; most cancer cells are initially sensitive to platinum, but sensitivity often decreases over time. Some patients develop resistance to chemotherapy, leading to tumor recurrence and progression. 40% of patients will experience local recurrence and distant metastasis, and the overall survival rate for patients with metastasis or recurrence is less than 20%. Tumor recurrence, metastasis, and drug resistance make the treatment of osteosarcoma still unsatisfactory and the prognosis poor.

[0003] The ocean covers approximately 71% of the Earth's surface and is home to over 80% of all life on Earth, forming a vast repository of biological resources. The uniqueness and diversity of the marine environment enable marine organisms to produce bioactive substances with special structures and functions, significantly different from terrestrial products. Drugs developed based on the active ingredients in marine organisms and minerals are called marine drugs. Marine drugs possess unique chemical structures, exhibiting strong specificity and unique biological activities. They are characterized by high activity, high efficacy, and good stability. Compared to traditional drug development, marine drug development has a higher success rate. It has been reported that the success rate of marine-derived drug development is approximately 0.03%, while the success rate of non-marine-derived drug development is only 0.01%-0.02%. The development of anti-tumor drugs is a key focus of marine drug research, with approximately 10% of marine products possessing anti-tumor activity. As of the end of 2020, 10 anti-tumor drugs based on marine natural products or their derivatives had been developed and marketed; 23 marine drugs had entered Phase III, Phase II, and Phase I clinical trials, of which 19 (83%) were being tested as anti-cancer drugs.

[0004] Microcolin A is a small-molecule chain-like lipopeptide compound isolated from the marine cyanobacterium Lyngbyamajuscula, with the molecular formula C0. 39 H 65 O9N5 has a molecular weight of 747. Due to its unique free hydroxyl functional group and pyrrolidone or hydroxyproline structure, Microcolin A exhibits good biological activity and is easily modified in various ways, making it a potential drug. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies and provide applications for Microcolin A.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention is to provide the use of Microcolin A in the preparation of a medicament for treating osteosarcoma.

[0008] Preferably, the drug for treating osteosarcoma inhibits the proliferation of osteosarcoma cells.

[0009] Preferably, the drug for treating osteosarcoma promotes osteosarcoma cell apoptosis.

[0010] A second aspect of the invention is to provide the use of Microcolin A in combination with a chemotherapy drug in the preparation of a medicament for treating osteosarcoma.

[0011] Preferably, the chemotherapy drug is cisplatin.

[0012] Preferably, the combination index of Microcolin A with the chemotherapy drug is less than 1.

[0013] A third aspect of the present invention is to provide the use of an agent that promotes p-H2AX expression in the preparation of a medicament for treating osteosarcoma, said agent that promotes p-H2AX expression includes: Microcolin A.

[0014] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0015] This invention demonstrates that Microcolin A can effectively inhibit the proliferation of osteosarcoma cells, significantly promote osteosarcoma cell apoptosis, and promote the expression of apoptosis-related protein p-H2AX in osteosarcoma cells, revealing the application of Microcolin A in the preparation of drugs for treating osteosarcoma; at the same time, it reveals that Microcolin A and cisplatin have good combined therapeutic effects. Attached Figure Description

[0016] Figure 1 The chemical formula of Microcolin A in one embodiment of the present invention is shown below.

[0017] Figure 2 shows the IC50 of Microcolin A in various osteosarcoma cell lines in one embodiment of the present invention; Figure 2A IC50 curves for various osteosarcoma cell lines; Figure 2B IC50 data for various osteosarcoma cell lines;

[0018] Figure 3 shows the osteosarcoma cell proliferation test results in one embodiment of the present invention; Figure 3A The proliferation status of 143B cells; Figure 3B The proliferation status of U2OS cells;

[0019] Figure 4 shows the formation result of osteosarcoma cell clone clusters in one embodiment of the present invention; Figure 4A These are the results of observations under a microscope; Figure 4B This is a graph showing the relative colony quantification of 143B cells; Figure 4C This is a graph showing the quantitative distribution of bacterial colonies in U2OS cells;

[0020] Figure 5 shows the migration ability test results of osteosarcoma cells in one embodiment of the present invention; Figure 5A These are the results of observations under a microscope; Figure 5B A quantitative map of relative migration of 143B cells; Figure 5C A quantitative map of relative migration of U2OS cells;

[0021] Figure 6 shows the results of a protein immunoblotting experiment in one embodiment of the present invention;

[0022] Figure 7 shows the apoptosis rate of osteosarcoma cells in one embodiment of the present invention; Figure 7A Scatter plot of osteosarcoma cells; Figure 7B A quantitative map of apoptosis in 143B cells; Figure 7C A quantitative map of apoptosis in U2OS cells;

[0023] Figure 8 shows the drug combination index of Microcolin A and cisplatin inhibiting osteosarcoma cells in one embodiment of the present invention. Figure 8A The combination drug index of Microcolin A and cisplatin inhibiting 143B cells; Figure 8B The combination drug index of Microcolin A and cisplatin inhibiting U2OS cells; Figure 8C The combination drug index of Microcolin A and cisplatin inhibiting HOS cells; Figure 8D The combination drug index of Microcolin A and cisplatin inhibiting MG63 cells;

[0024] Figure 9 shows the apoptosis rate of osteosarcoma cells in one embodiment of the present invention; Figure 9A Scatter plot of osteosarcoma cells; Figure 9B A quantitative map of apoptosis in 143B cells; Figure 9C A quantitative map of apoptosis in U2OS cells;

[0025] Figure 10 shows the tumor growth changes and tumor weight comparison of osteosarcoma-bearing mice treated with Microcolin A combined with cisplatin in one embodiment of the present invention. Figure 10A This is a schematic diagram illustrating the changes in tumor growth. Figure 10B Results of tumor weight comparison;

[0026] Figure 11 This is an HE staining image of the main organs of an osteosarcoma-bearing mouse after treatment with Microcolin A in one embodiment of the present invention. Detailed Implementation

[0027] 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.

[0028] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0029] 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.

[0030] Example

[0031] I. IC50 detection of osteosarcoma cells

[0032] Microcolin A was used to treat MG63, HOS, 143B, and U2OS cells, respectively, and the IC50 values ​​of each cell type were calculated using the CCK8 assay. The results are shown in Figure 2. The IC50 values ​​of MG63 cells were 3.07 nM, HOS cells were 3.80 nM, 143B cells were 2.84 nM, and U2OS cells were 4.55, indicating that Microcolin A has a significant inhibitory effect on the four osteosarcoma cell lines.

[0033] II. Osteosarcoma cell proliferative capacity detection

[0034] 1. Cell seeding: Use normal culture medium to fully precipitate and resuspend 143B cells and U2OS cells into single-cell state, and add 500-2000 cell suspensions to each well of a 96-well plate;

[0035] 2. Cells were cultured for 24h, 48h, 72h, and 96h, and then collected for testing.

[0036] 3. Incubation and color development: Add CCK8 solution (100 μL of free medium + 10 μL of CCK8 solution) to each well and incubate in an incubator for 1 hour;

[0037] 4. Select a wavelength of 450nm to measure the absorbance, analyze the results, and plot them as a graph.

[0038] As shown in Figure 3, Microcolin A significantly inhibited the proliferation of 143B and U2OS cells, and the inhibitory effect was more significant with increasing concentration.

[0039] III. Transwell Experiment

[0040] 1. Perform serum starvation on the cells before collecting them;

[0041] 2. Remove the cells from the incubator and observe their density and state under a microscope. Then digest the cells and resuspend them in an appropriate amount of serum-free culture medium.

[0042] 3. Gently add an appropriate amount of cell suspension to a Transwell chamber, and add 1.5 ml of normal culture medium to the lower chamber of a 12-well plate;

[0043] 4. After adding the cells, distribute them evenly in the chambers and incubate them in an incubator for 12-48 hours before removing the well plate.

[0044] 5. Remove the Transwell chamber and transfer it to a new well. Wash twice with an appropriate amount of PBS, fix with 4% paraformaldehyde at room temperature for 25 minutes, then hang the chamber at an angle in the well to air dry appropriately. Next, stain with 0.1% crystal violet for 15-20 minutes and observe the invasion status with the naked eye. If there are still many uninvaded cells inside the chamber, wipe them away with a cotton swab. Then observe and record under a microscope.

[0045] As shown in Figures 4-5, MicrocolinA significantly inhibited the scratch healing ability and pore migration ability of 143B and U2OS cells, and the inhibitory effect was more significant with increasing concentration.

[0046] IV. Western Blot analysis to detect the effect of Microcolin A on DNA damage repair pathways in osteosarcoma cells

[0047] 1. Sample preparation: 143B and U2OS cell samples were treated with different concentrations of Microcolin A, and the cells were lysed and collected using 1×SDS loading. The samples were then boiled in a 100℃ metal bath for 15 min to denature the proteins.

[0048] 2. Prepare protein gel: Clean the protein gel plate and rinse it once with ddH2O. After cleaning, dry it in an oven. Align the gel plates and clamp them parallel to each other on the gel plate holder. Prepare the lower separating gel according to the protein gel formula. Press it flat with anhydrous ethanol. After the lower gel solidifies, discard the anhydrous ethanol. Prepare the upper stacking gel according to the formula and insert a comb. After the upper gel solidifies, store it in a refrigerator at 4°C.

[0049] 3. Electrophoresis: Load the prepared protein samples from left to right in the order of the experimental design, leaving protein markers on both sides of the wells, and fill them with 1× loading buffer; after loading the samples, add 1× running buffer to the gel tank, start electrophoresis at a constant voltage of 80V, and after the lower gel markers separate, electrophoresis at a constant voltage of 120V to the bottom.

[0050] 4. Transfer: Prepare 1× Transfer buffer (700ml ddH2O, 200ml methanol, 100ml 10× Transfer buffer) in advance. Prepare the transfer clamps, NC membrane, and filter paper, and soak them in the Transfer buffer. After protein gel electrophoresis, gently lift the plate with a scraper, cut the upper gel layer, and keep the lower gel layer. Transfer the lower gel layer to the transfer buffer. Clamp the transfer clamps in the following order: one layer of sponge, three layers of filter paper, lower gel layer, NC membrane, three layers of filter paper, and one layer of sponge. Place the transfer clamps into the transfer tank, add the transfer buffer, place the tank in an ice bath, and perform the transfer at a constant current of 200mA under ice bath conditions.

[0051] 5. Blocking: After the transfer is complete, the protein on the protein gel has been 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.

[0052] 6. Primary antibody incubation: After blocking, discard the skim milk powder, wash with PBS for 5 minutes, and according to the protein marker, cut out the bands containing p-H2AX and ACTIN proteins, put them into a dark box, pour in the corresponding primary antibody, and incubate the dark box in a 4°C constant temperature shaker overnight.

[0053] 7. Fluorescent secondary antibody binding: The next day, recover the primary antibody, wash the band three times with PBST (PBS buffer + 0.05% Tween-20) for 5 min each time, then pour in the corresponding fluorescent secondary antibody and incubate in a 4℃ constant temperature shaker for 1 h;

[0054] 8. Membrane scanning: After the secondary antibody incubation, wash the bands three times with PBST for 5 minutes each time, and scan the corresponding bands using an Oddesey membrane scanner to analyze the results.

[0055] As shown in Figure 6, osteosarcoma cells showed increased expression of p-H2AX, a DNA damage repair-related protein, after treatment with Microcolin A.

[0056] V. Effects of Microcolin A on osteosarcoma cell apoptosis

[0057] 1. 143B cells and U2OS cells were seeded into six-well plates. When the cell density reached 50%, different concentrations of MCA (0 nM, 2.5 nM, 5 nM, 10 nM, 20 nM) were added and the cells were treated for 24 h.

[0058] 2. Collect the cultured cells in 15ml centrifuge tubes, then digest with trypsin to collect the cells into centrifuge tubes, centrifuge at 500rpm for 5min, discard the supernatant, resuspend the cell pellet in PBS, centrifuge again and discard the supernatant, repeat twice, leaving the cell pellet.

[0059] 3. Dilute the 5× Binding Buffer in the Annexin V-APC / PI apoptosis kit to 1× with PBS, resuspend the cell pellet from the previous step, 300 μL / tube, and gently pipette; then add APC and PI dye in sequence, and incubate at room temperature in the dark for 15 min.

[0060] 4. Perform analysis using a flow cytometer.

[0061] The results are as follows Figure 7A As shown in Figure B, flow cytometry results indicate that Microcolin A induces apoptosis in osteosarcoma cells, and this effect is concentration-dependent. After treatment of 143B and U2OS cells with Microcolin A, the proportion of apoptotic cells increased significantly, and the proportion increased with increasing concentration.

[0062] VI. Calculation of Combination Drug Index (CI) using the Chou-talalay method

[0063] 1. Digest, centrifuge and resuspend 143B cells, U2OS cells, HOS cells and MG63 cells respectively, and evenly seed them in 96-well plates to ensure 1500 cells per well;

[0064] 2. After the cells adhered, the cells were treated with Microcolin A at concentrations of 0 nM, 0.25 nM, 0.5 nM, 1.0 nM, 2.0 nM, and 4.0 nM and Cisplatin at concentrations of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, and 10.0 μM.

[0065] 3. After 48 hours, remove the supernatant, add CCK-8 reagent and treat for 1 hour, then measure the absorbance at 450 nm.

[0066] 4. Calculate the combination drug index using Compusyn software based on absorbance.

[0067] As shown in Figure 8, Microcolin A and cisplatin have a synergistic effect in the treatment of osteosarcoma, i.e., the combination drug index is less than 1.

[0068] VII. Effects of Microcolin A and Cisplatin on Apoptosis of Osteosarcoma Cells

[0069] Microcolin A and cisplatin were used as monotherapy or in combination to treat osteosarcoma cells, and the proportion of apoptotic cells was detected by the Chou-talalay assay.

[0070] As shown in Figure 9, the combined use of Microcolin A and cisplatin significantly increased the apoptosis rate of osteosarcoma cells and inhibited osteosarcoma cell growth.

[0071] VIII. Efficacy testing of combined treatment with Microcolin A and cisplatin in animal models

[0072] 1. Digest and centrifuge 143B cells in the logarithmic growth phase, resuspend the cells in PBS, ensuring a concentration of 5 × 10⁻⁶ cells / mL. 5 / 20μL;

[0073] 2. After preparing the cells, fix the nude mouse with a mouse restraint device, drill a hole in the tibia of the nude mouse through a clean insulin needle, and then use a needle with tumor cells to inject them into the tibia through the hole.

[0074] 3. One week later, when the tumor has grown to 100mm 3 Mice were divided into four groups based on their size: PBS (Vehicle group), Microcolin A (MCA group), Cisplatin group, and Microcolin A+Cisplatin group (DDP+MCA group).

[0075] 4. Dissect the mouse, remove the tissue, stain it with hematoxylin and eosin (HE), and then examine it under a microscope.

[0076] As shown in Figures 10-11, the combination therapy of Microcolin A and cisplatin can significantly inhibit the growth of osteosarcoma without causing damage to vital organs such as the heart, liver, spleen, lungs, and kidneys.

[0077] In summary, this invention demonstrates that Microcolin A can effectively inhibit the proliferation of osteosarcoma cells, significantly promote osteosarcoma cell apoptosis, and promote the expression of the apoptosis-related protein p-H2AX in osteosarcoma cells, revealing the application of Microcolin A in the preparation of drugs for treating osteosarcoma; at the same time, it reveals that Microcolin A and cisplatin have good combined therapeutic effects.

[0078] 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 Microcolin A in the preparation of drugs for treating osteosarcoma.

2. The application according to claim 1, characterized in that, The drug used to treat osteosarcoma inhibits the proliferation of osteosarcoma cells.

3. The application according to claim 1, characterized in that, The drug used to treat osteosarcoma promotes apoptosis in osteosarcoma cells.

4. The application of Microcolin A in combination with chemotherapy drugs in the preparation of drugs for treating osteosarcoma, characterized in that, The chemotherapy drug is cisplatin.

5. The application according to claim 4, characterized in that, The combination index of Microcolin A with the chemotherapy drug is less than 1.

6. The use of a formulation that promotes p-H2AX expression in the preparation of a drug for treating osteosarcoma, characterized in that, The formulation that promotes p-H2AX expression includes Microcolin A.

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