Application of furochromone in preparation of medicine for treating triple negative breast cancer

Abstract

The invention discloses application of furochromone in preparation of a medicine for treating triple negative breast cancer. The invention relates to novel application of furochromone in treatment of triple negative breast cancer, research shows that furochromone can inhibit cell proliferation and migration of triple negative breast cancer, can significantly inhibit tumor growth of TNBC, reduce tumor size and weight and relieve symptoms of model animals, has a treatment effect superior to that of positive drugs, and has in-vivo safety. Meanwhile, the furochromone is relatively low in toxicity to normal mammary gland cells, and relatively high in selectivity and safety. Therefore, a foundation is laid for developing more medicines for treating the triple-negative breast cancer, the furochromone becomes a new choice of the medicines for treating the triple-negative breast cancer, and the furochromone has important significance for treating the triple-negative breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of furanocryocysteine ​​in the preparation of drugs for treating triple-negative breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors affecting women, with 2.261 million new cases and 685,000 deaths worldwide in 2020, making it the leading cause of cancer death globally and seriously threatening women's health and quality of life. Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer lacking targeted therapy. Because this cancer lacks the expression of estrogen receptors, progesterone receptors, and HER2 proteins, existing targeted therapies are often ineffective. Current treatments mainly rely on chemotherapy, but chemotherapy damages normal cells, leading to a series of toxic side effects, and patient survival rates remain low. Therefore, developing new therapeutic drugs or methods is an urgent task in current breast cancer research, possessing significant research value and aligning with the concept of building a global community of health for all.

[0003] Studies have revealed that traditional Chinese medicine (TCM) possesses the characteristics of multi-component, multi-target, and multi-pathway effects, exhibiting advantages in anti-tumor treatment such as low toxicity, reversal of anticancer drug resistance, and enhancement of the body's immunity. Among these, furanocoumarins, a subgroup of phenolic compounds belonging to the coumarin family, are a class of natural active ingredients widely found in plants of the Apiaceae (e.g., celery, angelica), Rutaceae (e.g., citrus, Sichuan pepper), and Moraceae families. Due to their diverse pharmacological activities, including anti-tumor, anti-inflammatory, antibacterial, and photosensitizing effects, they have become a hot topic in drug development. For example, psoralen can be used to treat cutaneous T-cell lymphoma and breast cancer; bergapten has anti-liver cancer effects and photosensitizing therapeutic effects; and imperatorin has anti-colon cancer effects and neuroprotective properties. It is evident that different furanochrome ketone compounds have completely different pharmacological activities. However, due to the low bioavailability of natural furanochrome ketone compounds and the fact that psoralen and other compounds can cause photosensitivity reactions in the skin, they are rarely used in the development of TNBC treatment drugs, thus limiting the application of furanochrome ketone compounds.

[0004] Khellin (chemical name: 4,9-dimethoxy-7-methylfurano[3,2-g]cryronone) is one of the effective active ingredients of the traditional Chinese medicine Cyperus rotundus. It possesses anti-inflammatory and antibacterial pharmacological effects and is mainly used to treat angina pectoris, bronchial asthma, and other diseases. Studies have shown that Khellin can inhibit the migration and invasion of melanoma cancer cells, exhibiting anti-melanoma effects. However, research on its application in triple-negative breast cancer is lacking, and its specific molecular mechanisms are rarely reported. Therefore, it is necessary to develop more drugs that can effectively treat TNBC, making khellin a promising new treatment option for triple-negative breast cancer. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing drugs for the treatment of TNBC. The present invention provides the application of furazolidone in the preparation of drugs for the treatment of triple-negative breast cancer.

[0006] The first objective of this invention is to provide the use of furanocrylone in the preparation of medicaments for treating triple-negative breast cancer.

[0007] A second objective of this invention is to provide the use of furazolidone in the preparation of drugs to alleviate triple-negative breast cancer.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a novel application of furazolidone in triple-negative breast cancer. In vitro cell experiments using CCK-8 and EdU assays show that furazolidone inhibits the proliferation and migration of TNBC cells, and does not affect the proliferation of normal breast cancer cells (Hs578bst), exhibiting low toxicity to normal breast cells and high selectivity and safety. Furthermore, studies in a triple-negative breast cancer mouse model demonstrate that furazolidone significantly inhibits tumor growth, reducing tumor size, weight, and volume, with effects superior to positive control drugs. Moreover, it does not affect the growth of model mice during treatment, demonstrating good biocompatibility. The furazolidone provided by this invention is a low-toxicity anticancer drug with great development potential, offering a more effective treatment method for breast cancer patients. It can be used to prepare more anticancer drugs for breast cancer and provides a theoretical basis for the treatment of invasive breast cancer.

[0009] The furanone used in this invention has the English name Khellin, CAS: 82-02-0, and molecular formula C. 14 H 12 O5, the structural formula is: .

[0010] Therefore, the present invention provides the use of furanocrylone in the preparation of drugs for treating triple-negative breast cancer.

[0011] Furthermore, the drug can inhibit the proliferation of triple-negative breast cancer cells.

[0012] Furthermore, the drug can inhibit the migration of triple-negative breast cancer cells.

[0013] Furthermore, the drug exerts its anticancer effect by regulating apoptosis-related signaling pathways.

[0014] Furthermore, the drug can reduce tumor size, weight, and volume.

[0015] This invention provides the use of furazolidone in the preparation of drugs to alleviate triple-negative breast cancer.

[0016] Preferably, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the drug is an oral preparation, an injectable preparation, or a topical preparation.

[0018] Preferably, the oral formulation includes tablets, capsules, granules, or oral liquid; the injectable formulation includes intravenous injection or intramuscular injection.

[0019] The present invention has the following beneficial effects: This invention discloses the application of furazolidone in the preparation of drugs for treating triple-negative breast cancer. This invention relates to a novel use of furazolidone in the treatment of triple-negative breast cancer. Studies show that furazolidone can inhibit the proliferation and migration of triple-negative breast cancer cells, and can significantly inhibit the growth of TNBC tumors, reduce tumor size and weight, and alleviate symptoms in model animals. Its therapeutic effect is superior to positive control drugs, and it has in vivo safety. Simultaneously, furazolidone has low toxicity to normal breast cells, exhibiting high selectivity and safety. Therefore, this invention lays the foundation for the development of more drugs for treating triple-negative breast cancer, making furazolidone a new option for the treatment of triple-negative breast cancer, which is of great significance for the treatment of triple-negative breast cancer. Attached Figure Description

[0020] Figure 1 The figure shows the effect of furazolidone on the proliferation of triple-negative breast cancer cells MDA-MB-231 and BT-549 (A in the figure is the effect of furazolidone on the proliferation of MDA-MB-231 detected by CCK-8 assay; B is the effect of furazolidone on the proliferation of BT-549 detected by CCK-8 assay; C is the effect of furazolidone on the proliferation of MDA-MB-231 and BT-549 detected by EdU assay; D is the effect of furazolidone on the cell cycle of MDA-MB-231 and BT-549 detected by flow cytometry).

[0021] Figure 2The figures show the Transwell and scratch assay results of furazolidone on triple-negative breast cancer cells MDA-MB-231 and BT-549 (A in the figure is the effect of Transwell assay on cell migration of MDA-MB-231 and BT-549; B is the effect of scratch assay on cell migration of MDA-MB-231; C is the effect of scratch assay on cell migration of BT-549).

[0022] Figure 3 The figure shows the effect of furazolidone on the proliferation of normal breast cancer cells Hs578bst (A in the figure is the effect of furazolidone on the proliferation of Hs578bst detected by CCK-8 assay; B is the effect of furazolidone on the migration of Hs578bst detected by cell scratch assay).

[0023] Figure 4 The figure shows the effects of furazolidone on tumor size and body weight in a triple-negative breast cancer mouse model (A in the figure is a schematic diagram of furazolidone administration; B is the anatomical result of tumor size in mice treated with furazolidone; C is the result of tumor body weight in mice treated with furazolidone; D is the result of tumor volume in mice treated with furazolidone). Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0025] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0026] Example 1: In vitro therapeutic effect of furazolidone on TNBC 1. Materials Triple-negative breast cancer cells (MDA-MB-231, BT-549) and normal breast cells (Hs578bst) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in a humidified incubator at 37°C and 5% CO2.

[0027] 2. CCK-8 Experiment Cells were evenly seeded onto 96 control plates, with a seeding density of at least 5000 cells / well (100 μL). The plates were pre-cultured in an incubator (37℃, 5% CO2) for 24 h to allow the cells to reach the exponential phase. Then, at 12 h and 24 h, 5 μL of different concentrations (0 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 80 μM) of khellin were added to the plates for intervention (experimental group e was treated with different concentrations of the drug, control group c (control) was treated without khellin (0 μM), with six wells per group; blank group b was treated with drug-free medium). After incubating for an appropriate time, 10 μL of CCK-8 reagent was added to the plates, and the plates were incubated for another 1 h.

[0028] The absorbance (OD value) at a wavelength of 450 nm was measured using an ELISA reader, and the effect of the drug on the proliferation of triple-negative breast cancer cells was calculated using the following formula.

[0029] Cell inhibition rate (%) = [(Ac-Ae) / (Ac-Ab)] × 100%; In the formula: Ac is the absorbance of the control group c; Ab is the absorbance of the blank group b; Ae is the absorbance of the experimental group e.

[0030] The results are as follows Figure 1 As shown in AB, for MDA-MB-231 and BT-549, 10 μM furazolidone had a significant inhibitory effect on cell proliferation after 24 h of intervention, and the effect increased after 48 h of intervention. The inhibitory effect on cells was most obvious when the drug intervention concentration was 80 μM.

[0031] 3. EdU Experiment First, MDA-MB-231 and BT-549 cells were co-incubated with EdU (5-ethynyl-2′-deoxyuridine) to incorporate it into the newly generated DNA of proliferating cells during DNA synthesis. Cells were then treated with 10 μM furanochrome, fixed with paraformaldehyde, and permeabilized using a Triton X-100 membrane. Next, a click chemistry reaction was used to covalently bind the ethynyl group of EdU to a fluorescently labeled azide under copper ion catalysis, achieving fluorescent labeling. Finally, the signal was detected by fluorescence microscopy or flow cytometry to quantitatively analyze the cell proliferation level.

[0032] The results are as follows Figure 1 As shown in C, compared with the control group, the proportion of EdU-positive cells in MDA-MB-231 and BT-549 cells treated with 10 μM furazolidone was significantly reduced. PThe value <0.01 indicates that furazolidone can inhibit the proliferation of triple-negative breast cancer cells MDA-MB-231 and BT-549.

[0033] 4. Cell flow cytometry experiment MDA-MB-231 and BT-549 cells were evenly seeded on 6-well plates. After the cell density reached about 80%, 10 μM furazolidone solution was added for drug intervention. Subsequently, the effect of furazolidone on the cell cycle of triple-negative breast cancer cells was detected by cell flow cytometry.

[0034] The results are as follows Figure 1 As shown in Figure D, compared with the control group, MDA-MB-231 and BT-549 cells were arrested in S phase after treatment with 10 μM furazolidone, indicating that furazolidone can inhibit the cell cycle progression of triple-negative breast cancer cells MDA-MB-231 and BT-549.

[0035] 5. Transwell experiment MDA-MB-231 and BT-549 cells were seeded in the upper chamber of a Transwell cell line (serum-free medium), while the lower chamber was treated with medium containing 10% FBS as a chemokine. The experimental group received 20 μM furazolidone (based on CCK8 results) for 24 h, while the control group received no treatment. After 24 h, non-migrated cells from the upper chamber were removed, fixed, stained, and photographed under a microscope. The number of cells that penetrated the membrane was counted, and the migration inhibition rate was calculated using the following formula. Statistical analysis (t-test / ANOVA) and visualization (bar chart / line chart) were performed using GraphPad Prism 8.0.* p <0.05 indicates a significant difference. This experiment assesses the inhibitory effect of drugs on cell migration by quantifying the number of cells that penetrate the membrane, which differs from the scratch closure rate determination in scratch assays.

[0036] Migration inhibition rate = [1 - number of migrating cells in the experimental group / number of migrating cells in the control group] × 100%; The results are as follows Figure 2 As shown in Figure A, treatment with 10 μM furazolidone resulted in decreased migration ability of MDA-MB-231 and BT-549 cells. Combined with cell scratch assays, this indicates that furazolidone can inhibit the migration of triple-negative breast cancer cells MDA-MB-231 and BT-549.

[0037] 6. Cell scratch test MDA-MB-231 and BT-549 cells were evenly seeded in 6-well plates. After 100% cell healing, a 20 μM furazolidone solution (as determined by CCK8) was added to the 6-well plates. After 24 h of drug intervention, cells were streaked using a 200 μL pipette tip, and observation time gradients were set at 0 h and 24 h. Microscopic observation and photography were performed during this period. The effect of the drug on cell migration rate at different time points was calculated using the following formula, and the final results were statistically analyzed and visualized using GraphPadPrism.

[0038] Cell migration rate = (initial scratch width - scratch width after 24 hours) / initial scratch width × 100%; The results are as follows Figure 2 As shown in BC, furanochrome significantly reduced the 24-h migration rate of both MDA-MB-231 and BT-549 in the experimental groups.

[0039] Example 2: Effects of furazolidone on normal breast cells Hs578bst Based on the experiment in Example 1, different concentrations of furazolidone were used to perform CCK-8 assays and cell scratch assays on normal breast cells Hs578bst. The methods and steps were the same as in Example 1, and the results were statistically analyzed.

[0040] The results are as follows Figure 3 As shown, treatment with 0 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 80 μM furazolidone did not significantly inhibit the proliferation and migration of normal breast cells Hs578bst. This indicates that furazolidone does not affect the proliferation of normal breast cells Hs578bst, and that furazolidone has low toxicity to normal breast cells, exhibiting high selectivity and safety.

[0041] Example 3: Effects of furanocrylone on a triple-negative breast cancer mouse model 1. Materials A BT-549 breast cancer mouse model was established using standard BALB / c Nude mice. The mice were housed in a specific pathogen-free (SPF) environment with a 12-hour light / dark cycle and adequate food and water.

[0042] 2. Model Building After the animals arrived, their condition was observed, ear tags were attached, and they were weighed. They were then acclimatized for 7 days to establish a BT-549 subcutaneous model. BT-549 breast cancer cells in the logarithmic growth phase were harvested, digested with trypsin, and then prepared into 2.5 × 10⁻⁶ cells using PBS. 6 / mL of cell suspension. Under aseptic conditions, 0.1mL was injected subcutaneously into the right axilla of nude mice. After several days of feeding, the mice were treated with the drug when the tumor was palpable.

[0043] 3. Drug administration The following groups were set up: (1) Control group A: 5 mice (no drug administered), injected with 0.9% saline; (2) Furantoin treatment group B: 5 mice (drug administered via tail vein at 10 mg / kg); (3) Paclitaxel positive control group C: 5 mice (drug administered via tail vein at 10 mg / kg). The drugs were administered for 15 days, once every other day, and the tumor volume was measured after each administration. Each mouse was treated with a 10 μL mixture (DMSO: vegetable oil, 10:1) as the drug carrier via tail vein injection. After 15 consecutive days of treatment, the mice were anesthetized with sodium pentobarbital at a dose of 50 mg / kg and then euthanized by cervical dislocation. The tumors were dissected and weighed to calculate the tumor inhibition rate.

[0044] Formula for calculating tumor volume: Tumor volume (mm) 3 ) = 0.5 × d1 × d2; In the formula: d1 is the longest diameter, and d2 is the shortest diameter.

[0045] The results are as follows Figure 4 As shown, furazolidone can reduce tumor size and weight, with significantly better effects than the positive control group. It can effectively inhibit the growth of triple-negative breast cancer, alleviate symptoms in model animals, and has good in vivo safety and therapeutic effect.

[0046] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of furanocrylone in the preparation of drugs for treating triple-negative breast cancer.

2. The application according to claim 1, characterized in that, The drug can inhibit the proliferation of triple-negative breast cancer cells.

3. The application according to claim 1, characterized in that, The drug can inhibit the migration of triple-negative breast cancer cells.

4. The application according to claim 1, characterized in that, The drug exerts its anti-cancer effect by regulating apoptosis-related signaling pathways.

5. The application according to claim 1, characterized in that, The drug can reduce tumor size, weight, and volume.

6. Application of furazolidone in the preparation of drugs to alleviate triple-negative breast cancer.

7. The application according to any one of claims 1 to 6, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

8. The application according to any one of claims 1 to 6, characterized in that, The drug is an oral preparation, an injectable preparation, or a topical preparation.

9. The application according to claim 8, characterized in that, The oral preparations include tablets, capsules, granules, or oral liquids.

10. The application according to claim 8, characterized in that, The injectable preparation includes intravenous injection or intramuscular injection.

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