Application of RSL3 combined adriamycin in preparation of medicine for treating breast cancer

Abstract

The invention is applicable to the technical field of medicines, and provides an application of RSL3 combined adriamycin in preparation of a medicine for treating breast cancer, and the RSL3 enhances the treatment sensitivity of breast cancer cells to adriamycin by inducing glycolytic dysfunction. The invention finds and verifies that RSL3 enhances the treatment sensitivity of breast cancer cells to adriamycin by inducing glycolytic dysfunction, so that the understanding of people on the molecular mechanism of ferroptosis agonists in chemotherapy sensitization is widened; and an innovative thought and a potential drug target are provided for reducing the toxic and side effects of the adriamycin and overcoming the drug resistance problem. Specifically, when the medicine for treating the breast cancer is prepared, the combined application of the RSL3 and the adriamycin shows huge application potential, and a new hope is brought to the field of breast cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of RSL3 combined with doxorubicin in the preparation of a drug for treating breast cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors in the world and is particularly common in women. Currently, the treatment of breast cancer usually includes surgery combined with radiotherapy and chemotherapy. However, in the actual treatment process, some patients will have poor treatment effects due to resistance to chemotherapy drugs. Studies have shown that the ability of breast cancer cells to maintain their rapid proliferation depends on a high level of glycolysis. Glycolysis not only provides ATP for cells, but also participates in the regulation of the cell proliferation cycle as a carbon source for synthesizing lipids and non-essential amino acids and other substances, and is the main cause of tumor cell metastasis and recurrence. In addition, the products of glycolysis can acidify the intracellular microenvironment, reduce drug absorption, reduce the production of oxygen free radicals, and resist oxidative damage of chemotherapy drugs, leading to drug resistance. Therefore, inducing glycolytic dysfunction and reshaping the metabolic pattern of breast cancer cells is expected to become a new strategy to reduce chemotherapy resistance and kill cancer cells.

[0003] Most chemotherapy drugs can change mitochondrial permeability and cause intrinsic apoptosis by interfering with glycolysis. There is a close connection between glycolysis rate and apoptosis resistance during tumor maintenance and progression. In addition, the expression of key proteins in the glycolysis process can also be used as a marker for the prognosis of breast cancer patients. Therefore, in-depth research on the metabolism of breast cancer cells can provide new molecular therapeutic targets and theoretical basis for the treatment and prognosis of breast cancer.

[0004] RSL3 was originally screened as an activator of ferroptosis, which can exert good anti-tumor effects by inducing ferroptosis of various tumor cells. In recent years, studies have found that RSL3 not only targets glutathione peroxidase 4 (GPX4) to activate ferroptosis, but also targets multiple signaling pathways to enhance the sensitivity of tumor cells to chemotherapeutic drugs, such as: as a radiotherapy sensitizer, it can synergize with radiotherapy to induce glioma cell death without increasing DNA damage; downregulate genes related to epithelial-mesenchymal transition, and enhance the therapeutic sensitivity of glioma cells to temozolomide (TMZ). Existing studies mainly focus on the good anti-tumor effect of inducing ferroptosis of breast cancer cells, and other small molecule compounds are selected to enhance the sensitivity of breast cancer cells to ferroptosis. However, there is no relevant report on the synergistic sensitization of RSL3 to the chemotherapeutic drug doxorubicin. Therefore, the application of RSL3 combined with doxorubicin in the preparation of drugs for the treatment of breast cancer is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an application of RSL3 combined with doxorubicin in the preparation of a drug for treating breast cancer, aiming to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Application of RSL3 combined with doxorubicin in the preparation of drugs for the treatment of breast cancer.

[0008] Furthermore, the RSL3 enhances the therapeutic sensitivity of breast cancer cells to doxorubicin by inducing glycolytic dysfunction.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention discovered and verified that RSL3 enhances the therapeutic sensitivity of breast cancer cells to doxorubicin by inducing glycolytic dysfunction. This discovery not only broadens our understanding of the molecular mechanism of ferroptosis agonists in chemotherapy sensitization, but also provides innovative ideas and potential drug targets for reducing the toxic side effects of doxorubicin and overcoming drug resistance. Specifically, in the preparation of drugs for the treatment of breast cancer, the combined use of RSL3 and doxorubicin has shown great application potential, bringing new hope to the field of breast cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a mechanism diagram of the present invention.

[0012] Figure 2 The combined use of RSL3 and doxorubicin to synergistically inhibit the proliferation of breast cancer cells was studied; (A) was the result of CCK8 detection in SKBR3 cells; (B) was the CI value of the synergistic drug-addition group on SKBR3 cells calculated by CompuSyn analysis software; (C) was the result of CCK8 detection in MCF7 cell line; (D) was the CI value of the synergistic drug-addition group on MCF7 cells calculated by CompuSyn analysis software; (E) was the cell morphological changes under the microscope in each drug-addition group; (F) was the effect of each drug-addition group on the proliferation of breast cancer cells SKBR3 and MCF7 detected by clone formation experiment.

[0013] Figure 3The study is about RSL3 enhancing the apoptosis sensitivity of breast cancer cells to doxorubicin; wherein, (A) is the cell cycle changes of each drug-added group detected by flow cytometry when acting on SKBR3 cell line; (B) is the statistical graph of the cell cycle detection results of SKBR3 cell line; (C) is the cell cycle changes of each drug-added group detected by flow cytometry when acting on MCF7 cell line; (D) is the statistical graph of the cell cycle detection results of MCF7 cell line; (E) is the number of apoptotic cells detected by flow cytometry when each drug-added group acts on SKBR3 cell line; (F) is the statistical graph of the number of apoptotic cells of SKBR3 cell line; (G) is the number of apoptotic cells detected by flow cytometry when each drug-added group acts on MCF7 cell line; (H) is the statistical graph of the number of apoptotic cells of MCF7 cell line.

[0014] Figure 4 This study is to investigate how RSL3-induced glycolytic dysfunction enhances the sensitivity of doxorubicin treatment; (A) is an ATP detection kit used to detect the effect of each drug group on the intracellular ATP content when acting on SKBR3 and MCF7 cell lines; (B) is a pyruvate detection kit used to detect the effect of each drug group on the intracellular pyruvate content when acting on SKBR3 and MCF7 cell lines.

[0015] Figure 5 This is a study on the role of RSL3 in synergizing with doxorubicin in inhibiting the proliferation of breast cancer in vivo; (A) is the experimental flow chart; (B) is a schematic diagram of the tumor volume of each group; (C) is the HE staining and Ki67 expression of the tumors in each group; (D) is the HE toxicity test of the heart, liver, spleen, lung, and kidney of each group of mice. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] The mechanism diagram of the present invention is as follows Figure 1 As shown, RSL3 induces glycolytic dysfunction in breast cancer cells, induces cell cycle arrest, enhances the sensitivity of doxorubicin to apoptosis, and reduces chemotherapy resistance.

[0018] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0019] Example 1: It is confirmed that the combined use of RSL3 and doxorubicin can synergistically inhibit the proliferation of breast cancer cells;

[0020] Breast cancer cells MCF-7 and SKBR3 were cultured to 70-80%, pretreated with RSL3 (0, 1, 2, 4 and 8 μM) for 1 h, and then cultured for 48 h after adding Doxorubicin (0, 0.25, 0.5, 1, 2 μM). The results of CCK8 detection showed that ( Figure 2 A and C), with the increase of drug concentration, the cell proliferation ability decreased, among which, the cell proliferation ability decreased most significantly when RSL3 was added with Doxorubicin. This result proves that RSL3 can promote the therapeutic sensitivity of breast cancer cells to Doxorubicin.

[0021] The appropriate drug dose was selected, and the experiment was divided into 4 groups: Control group, RSL3 group, Doxorubicin group, and RSL3+Doxorubicin synergistic administration group. CompuSyn software calculated the combination index (CI) based on the cell viability data after single drug and two drugs in combination, and further determined the concentration of the best drug coordination effect based on the CI value. Figure 2 As shown in Figure B, in the SKBR cell line, the CI value at the concentration of RSL3 (2 μM) + Doxorubicin (0.5 μM) was 0.60101. Figure 2 As shown in D, in the MCF7 cell line, the CI value at the concentration of RSL3 (4 μM) + Doxorubicin (0.5 μM) was 0.61164, indicating that the synergistic killing effect was the best. The changes in cell morphology of each drug treatment group were observed under a microscope, as shown in Figure 2 As shown in E, the cells in the synergistic drug group became significantly rounder and smaller in size, proving that the combined use of RSL3 and Doxorubicin synergistically inhibited the proliferation of breast cancer cells. The results of the clone formation experiment showed that compared with the Control group and the drug-treated group alone, the number of cells in the synergistic drug group was significantly reduced, further confirming that the combined use of RSL3 and Doxorubicin can synergistically inhibit the proliferation of breast cancer cells ( Figure 2 Middle F).

[0022] Example 2: It is clear that RSL3 enhances the apoptosis sensitivity of breast cancer cells to Doxorubicin;

[0023] In order to further study how the synergistic effect of RSL3 and Doxorubicin is exerted, we used flow cytometry to detect cells. The results showed that RSL3 alone can cause S phase arrest of breast cancer cells SKBR3 and MCF7, while Doxorubicin alone can partially increase G2 phase arrest. When RSL3 and Doxorubicin are used in combination, S phase arrest is significantly enhanced ( Figure 3AD), indicating that the combination of RSL3 and Doxorubicin can induce S phase cycle arrest in breast cancer cells, and the results are consistent in the two breast cancer cell lines. In addition, when RSL3 acts alone on breast cancer cells, it does not cause an increase in the number of apoptotic cells. However, compared with the group using Doxorubicin alone, the combination of RSL3 and Doxorubicin significantly enhances the apoptosis level of breast cancer cells ( Figure 3 EH), suggesting that RSL3 can enhance the apoptosis sensitivity of breast cancer cells to Doxorubicin, and the results are consistent in the two breast cancer cell lines.

[0024] Example 3: It is clear that RSL3 induces glycolytic dysfunction and enhances the sensitivity of Doxorubicin treatment;

[0025] In order to detect the effects of RSL3 and Doxorubicin on the glycolytic function of breast cancer cells, we detected the contents of glycolytic products ATP and pyruvate respectively. Figure 4 As shown in the figure, RSL3 can reduce the content of ATP and pyruvate, indicating that RSL3 can inhibit the glycolysis of breast cancer cells; Doxorubicin has no significant effect on the intracellular ATP content, but can slightly increase the pyruvate content, indicating that this concentration of Doxorubicin has no significant effect on the glycolysis rate; ATP and pyruvate levels in the synergistic administration group were significantly downregulated, indicating that RSL3 can enhance the sensitivity of cells to apoptosis by inducing glycolytic dysfunction.

[0026] Example 4: Clarify the regulatory effect of RSL3 on the sensitivity of Doxorubicin treatment in vivo;

[0027] In order to detect the role of RSL3 in synergistically enhancing the sensitivity of Doxorubicin treatment in vivo, a nude mouse breast cancer subcutaneous transplant tumor model was established using the breast cancer cell line 4T1. The drug was given for 21 days to observe the therapeutic effect of RSL3 on nude mouse breast cancer ( Figure 5 (A). Figure 5 As shown in Figure B, compared with the Control group and the single drug group, the synergistic drug group significantly inhibited the tumor volume and the expression of the proliferation marker Ki67 ( Figure 5 The above results suggest that RSL3 can enhance the therapeutic sensitivity of Doxorubicin to breast cancer in vivo. In addition, there was no obvious toxicity in the internal organs of mice in each group ( Figure 5 D), proving that the drug concentration had no obvious toxic side effects on nude mice.

[0028] The above experimental phenomena can be achieved in other breast cancer cell lines (MDA-MB-231, BT549, etc.).

[0029] In summary, RSL3 enhances the therapeutic sensitivity of breast cancer cells to doxorubicin by inducing glycolytic dysfunction. This discovery not only broadens our understanding of the molecular mechanism of ferroptosis agonists in chemotherapy sensitization, but also provides innovative ideas and potential drug targets for reducing the toxic side effects of doxorubicin and overcoming drug resistance. Specifically, in the preparation of drugs for the treatment of breast cancer, the combined use of RSL3 and doxorubicin has shown great application potential, bringing new hope to the field of breast cancer treatment.

[0030] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of RSL3 combined with doxorubicin in the preparation of drugs for the treatment of breast cancer.

2. The use according to claim 1, characterized in that: The RSL3 enhances the therapeutic sensitivity of breast cancer cells to doxorubicin by inducing glycolytic dysfunction.

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