The application of aspirin combined with abemaciclib in the treatment of breast cancer

Abstract

The present invention discloses the application of aspirin combined with abemacili or in combination with other drugs in the treatment of breast cancer, and belongs to the technical field of tumor therapeutic drugs. The present invention firstly finds that, compared with the single application of abemacili, the combined or coordinated use of aspirin and abemacili can significantly inhibit the proliferation of breast cancer MCF7 and MDA-MB-231 cells and promote the apoptosis of breast cancer MCF7 and MDA-MB-231 cells, and the effect of the combined use of aspirin and abemacili is significantly better than the effect of the single application of abemacili or aspirin; compared with the single application of abemacili, the toxicity to normal human breast MCF10A cells is not enhanced, and it is expected to be used for the treatment of breast cancer, and provide a new drug combination for reducing the cost of breast cancer treatment, improving the treatment effect, and alleviating the suffering of patients.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor therapeutic drugs, and in particular to the application of aspirin and abemaciclib in the treatment of breast cancer in combination or in collaboration. Background Art

[0002] Breast Cancer has become the most common type of cancer in women worldwide. According to the latest global cancer burden data for 2020 released by the World Health Organization's International Agency for Research on Cancer (IARC), the number of new breast cancer cases reached 2.26 million, officially replacing lung cancer as the cancer with the highest incidence rate worldwide. In the global female cancer statistics, the incidence rate of breast cancer is about 24.2%, also ranking first, seriously threatening human health and life. Although the incidence rate of breast cancer in my country is not the highest in the world, breast cancer still ranks first among female malignant tumors in my country, with approximately 300,000 new cases each year.

[0003] Studies have shown that cyclin-dependent kinase (CDK) can promote the proliferation of breast cancer cells. Based on this, the inhibitor of cyclin-dependent kinase 4 and 6, CDK4 / 6i, is a targeted therapy drug for a specific cell cycle regulatory mechanism. It is mainly used to treat hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer, and has achieved certain results in the treatment of HR+ / HER2- advanced breast cancer. Currently, several CDK4 / 6i have been approved for clinical treatment, including Abemaciclib, Palbociclib, Ribociclib and Dalpiclib. However, advanced breast cancer that is not hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) cannot benefit.

[0004] High expression of CDK in breast cancer cells can cause dysregulation of breast cancer cell division and promote abnormal proliferation of breast cancer cells. In recent years, targeting CDK4 / 6 protein has gradually become a hot topic in breast cancer treatment research. Abemaciclib (Herceptin, Abemaciclib) is a new oral targeted CDK4 / 6 inhibitor (CDK4 / 6i), developed by Eli Lilly and Company, which can selectively inhibit cyclin-dependent kinase 4 / 6 (CDK4 / 6), restore cell cycle regulation, and prevent breast cancer cell proliferation. Recent studies have shown that CDK4 / 6i makes animal model tumors (PDX tumors) more sensitive to HER 2 targeted therapy. CDK 4 / 6i combined with DNA damaging agents can inhibit tumor cell proliferation by blocking DNA repair after damage. However, this is far from enough to expand the therapeutic effect of abemaciclib, and new combination therapies are urgently needed. In addition, its high cost of treatment greatly limits the clinical application of abemaciclib. Therefore, it is of great clinical significance to find new therapeutic compositions for breast cancer and increase the efficacy of abemaciclib. Summary of the invention

[0005] The purpose of the present invention is to provide a combined or synergistic use of aspirin and abemaciclib in the treatment of breast cancer. The present invention first finds that the combined or synergistic use of aspirin and abemaciclib can significantly improve the inhibitory effect on breast cancer cell proliferation and increase the apoptosis rate of cancer cells.

[0006] To achieve the above object, the present invention provides the use of aspirin and abemaciclib in combination in the preparation of a drug for treating breast cancer.

[0007] Preferably, the breast cancer is ER-positive breast cancer or triple-negative breast cancer.

[0008] Preferably, the mass ratio of aspirin to abemaciclib is 1000:1-5.

[0009] Preferably, the mass ratio of aspirin to abemaciclib is 1000:1-2.

[0010] The application of aspirin and abemaciclib in the preparation of drugs for treating breast cancer.

[0011] Preferably, the breast cancer is ER-positive breast cancer or triple-negative breast cancer.

[0012] Preferably, the mass ratio of aspirin to abemaciclib is 1000:1-2.

[0013] Use of aspirin in combination or synergistically with abemaciclib in the preparation of an inhibitor for inhibiting the proliferation of breast cancer cells, wherein the breast cancer is ER-positive breast cancer or triple-negative breast cancer.

[0014] The invention relates to a use of aspirin in combination or synergistically with abemaciclib in the preparation of a promoter for promoting apoptosis of breast cancer cells, wherein the breast cancer is ER-positive breast cancer or triple-negative breast cancer.

[0015] A drug combination for treating breast cancer, comprising aspirin and abemaciclib, wherein the breast cancer is ER-positive breast cancer or triple-negative breast cancer.

[0016] Aspirin has been used in clinical practice as an antipyretic and analgesic for hundreds of years. With its excellent anticoagulant and heart-protective effects, it has become a first-line and basic treatment for cardiovascular diseases.

[0017] Therefore, the combined or synergistic use of aspirin and abemaciclib in the treatment of breast cancer provided by the present invention has the following technical effects:

[0018] (1) The present invention first discovered that, compared with the administration of abemaciclib alone, the combined or synergistic use of aspirin and abemaciclib can significantly inhibit the proliferation of breast cancer MCF7 and MDA-MB-231 cells and promote the apoptosis of breast cancer MCF7 and MDA-MB-231 cells. The effect of the combined use of aspirin and abemaciclib is significantly better than the effect of abemaciclib or aspirin alone.

[0019] (2) Compared with the administration of abemaciclib alone, the drug combination provided by the present invention has less toxicity to normal cells, or no enhanced toxicity, and is expected to be used in the treatment of breast cancer, providing a new drug combination for reducing the cost of breast cancer treatment, improving the treatment effect, and alleviating the pain of patients.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0022] Figure 1 The results of the effects of different treatments on MCF7 cell proliferation in Example 5 of the present invention are as follows;

[0023] Figure 2 The results of the effects of different treatments on the proliferation of MDA-MB-231 cells in Example 6 of the present invention are as follows;

[0024] Figure 3The flow cytometric detection results and statistical results (E) of apoptosis of MCF7 cells in the control group (A), 5 mM aspirin group (B), 10 μM abemaciclib group (C) and 5 mM aspirin + 10 μM abemaciclib group (D) in Example 7 of the present invention;

[0025] Figure 4 The flow cytometric detection results and statistical results (E) of apoptosis of MDA-MB-231 cells in the control group (A), 5 mM aspirin group (B), 10 μM abemaciclib group (C) and 5 mM aspirin + 10 μM abemaciclib group (D) in Example 8 of the present invention;

[0026] Figure 5 The results are the effects of the pharmaceutical composition in Example 9 of the present invention on the cytotoxicity of normal human breast epithelial cells MCF10A. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0029] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0030] The breast cancer cell lines MCF7 and MDA-MB-231 used in the examples were both derived from the Shanghai Cell Bank of the Chinese Academy of Sciences and stored by the Experimental Center of Shenzhen Second People's Hospital. Aspirin (Acetylsalicylicacid) and MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) were purchased from Sigma. Abemaciclib was purchased from Selleckchem. Aspirin was dissolved in dimethyl sulfoxide (DMSO), and abemaciclib was dissolved in sterile water for injection.

[0031] Embodiment 1

[0032] The steps for preparing aspirin stock solution with a concentration of 1 M are as follows:

[0033] Dissolve 180.16 mg of aspirin in 1 mL of DMSO.

[0034] Embodiment 2

[0035] Prepare 1 mM abemaciclib stock solution as follows:

[0036] Dissolve 506.59 μg of abemaciclib in 1 mL of water for injection.

[0037] Embodiment 3

[0038] A drug combination for treating breast cancer is prepared, which contains 5 mM aspirin and 5 μM abemaciclib, and the mass ratio of aspirin to abemaciclib is 1000:1. The specific steps are as follows:

[0039] 10 μL of the aspirin stock solution prepared in Example 1 was taken, and added to 10 μL of the abemaciclib stock solution prepared in Example 2, and mixed well.

[0040] Embodiment 4

[0041] A drug combination for treating breast cancer is prepared, which contains 5 mM aspirin and 10 μM abemaciclib, and the mass ratio of aspirin to abemaciclib is 1000:2. The specific steps are as follows:

[0042] 10 μL of the aspirin stock solution prepared in Example 1 was taken, and added to 20 μL of the abemaciclib stock solution prepared in Example 2, and mixed well.

[0043] Embodiment 5

[0044] The MTT method was used to investigate the effects of the drug combination for treating breast cancer prepared in Examples 3 and 4 on the proliferation of ER (estrogen receptor) positive and PR (progesterone receptor) positive breast cancer cells MCF7. The specific steps are as follows:

[0045] S51, prepare 6 groups of culture medium:

[0046] ①Control group: DMEM culture medium.

[0047] ② 5 mM aspirin group: 10 μL of the aspirin stock solution prepared in Example 1 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0048] ③ 5 μM Abemaciclib group: 10 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0049] ④ 10 μM Abemaciclib group: 20 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0050] ⑤ 5 mM aspirin + 5 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 10 μL of the abemaciclib stock solution prepared in Example 2 were taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0051] ⑥ 5 mM aspirin + 10 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 20 μL of the abemaciclib stock solution prepared in Example 2 were added to 1970 μL of DMEM culture medium and mixed.

[0052] S52, the stored MCF7 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO 2 The normally growing MCF7 cells were cultured in an incubator and then subcultured and inoculated into a 96-well cell culture plate, with a volume of 200 μL per well and a cell number of 2×10 3 at 37°C and 5% CO 2 After culturing overnight in the incubator, the 6 groups of culture solutions prepared in step S51 were added respectively, and the cell proliferation was detected after 2 days. 20L of 5mg / mL MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) was added to each well and cultured for 4h. After that, the culture solution was discarded, 150μL of DMSO was added to each well, and the cells were shaken on a microplate reader for about 5min. The absorbance (OD) value was detected at 490nm, and a bar graph was drawn to represent the cell growth. Each group had 3 replicate wells and repeated 3 times. The results were expressed as (mean ± standard deviation). The results are shown in Figure 2. Figure 1 As shown, the vertical axis represents the cell proliferation rate, and the horizontal axis represents different drug groups.

[0053] Depend on Figure 1 It can be seen that compared with the control group cells, the application of 5mM aspirin alone can inhibit cell proliferation, and the application of 5μM abemaciclib or 10μM abemaciclib alone can also inhibit cell proliferation. However, 5mM aspirin + 5μM abemaciclib / + 10μM abemaciclib can more significantly inhibit cell proliferation.

[0054] Statistical analysis showed that the difference between cells treated with 5mM aspirin, 5μM abemaciclib, 10μM abemaciclib, and both aspirin and abemaciclib was statistically significant compared with cells in the control group ( P <0.05). Compared with the cells added with only 5mM aspirin, 5μM abemaciclib, and 10μM abemaciclib, the cell proliferation rate was more significantly inhibited by the addition of aspirin and abemaciclib at the same time, and the difference was statistically significant ( P<0.05). This shows that aspirin combined with abemaciclib can significantly inhibit the proliferation of MCF7 cells, and the inhibitory effect is better than that of abemaciclib and aspirin alone. Aspirin can promote, increase, amplify or synergize the inhibitory effect of abemaciclib on MCF7 breast cancer cells.

[0055] Embodiment 6

[0056] The MTT method was used to investigate the effect of the drug combination for treating breast cancer prepared in Examples 3 and 4 on the proliferation of MDA-MB-231 cells. The specific steps are as follows:

[0057] S61, prepare 6 groups of culture medium:

[0058] ①Control group: DMEM culture medium.

[0059] ② 5 mM aspirin group: 10 μL of the aspirin stock solution prepared in Example 1 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0060] ③ 5 μM Abemaciclib group: 10 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0061] ④ 10 μM Abemaciclib group: 20 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0062] ⑤ 5 mM aspirin and 5 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 10 μL of the abemaciclib stock solution prepared in Example 2 were taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0063] ⑥ 5 mM aspirin + 10 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 20 μL of the abemaciclib stock solution prepared in Example 2 were added to 1970 μL of DMEM culture medium and mixed.

[0064] S62, the normally growing MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO 2 After subculturing in the incubator, the cells were inoculated into 96-well cell culture plates with a volume of 200 μL per well and a cell number of 2×10 3 After culturing overnight, the six culture solutions prepared in step S61 were added respectively, and the cell proliferation was detected after 2 days.

[0065] MTT was prepared to a concentration of 5 mg / mL, 20 L was added to each well and culture was continued for 4 h. Then the culture medium was discarded and 150 μL of DMSO was added to each well. The cells were oscillated on a microplate reader for about 5 min. The absorbance (OD) value was detected at 490 nm. A bar graph was drawn to represent the cell growth. Each group had 3 replicate wells and the results were repeated 3 times. The results were expressed as (mean ± standard deviation).

[0066] The results are as follows Figure 2 As shown: Compared with the control group cells, the application of 5mM aspirin alone can inhibit cell proliferation, the application of 5μM abemaciclib or 10μM abemaciclib alone can also inhibit cell proliferation, but the simultaneous application of 5mM aspirin + 5μM abemaciclib / +10μM abemaciclib can more significantly inhibit cell proliferation.

[0067] Statistical analysis showed that compared with the control group, the proliferation of cells was inhibited by the addition of 5mM aspirin, 5μM abemaciclib, 10μM abemaciclib, and the addition of aspirin and abemaciclib at the same time, and the difference was statistically significant ( P <0.05). Compared with cells treated with 5mM aspirin, 5μM abemaciclib, and 10μM abemaciclib, the proliferation of cells treated with both aspirin and abemaciclib was significantly inhibited, and the difference was statistically significant ( P <0.05).

[0068] It can be seen that compared with the use of abemaciclib alone, the combined use of aspirin and abemaciclib has a better inhibitory effect on the proliferation of MDA-MB-231 cells, and aspirin can promote, increase, amplify or synergize the inhibitory effect of abemaciclib on triple-negative breast cancer cells MDA-MB-231.

[0069] Embodiment 7

[0070] The effects of the drug combination for treating breast cancer prepared in Examples 3 and 4 on apoptosis of ER-positive breast cancer cells MCF7 were investigated, and apoptosis was detected by flow cytometry. The specific steps were as follows:

[0071] S71. Prepare 4 groups of culture medium:

[0072] ①Control group: DMEM culture medium.

[0073] ② 5 mM aspirin group: 10 μL of the aspirin stock solution prepared in Example 1 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0074] ③ 10 μM Abemaciclib group: 20 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0075] ④5 mM aspirin+10 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 20 μL of the abemaciclib stock solution prepared in Example 2 were taken, added into 1970 μL of DMEM culture medium, and mixed well.

[0076] S72, MCF7 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO 2 The cells were subcultured in an incubator and then inoculated into a culture dish for further culture. When the cell density reached about 80%, the four culture solutions prepared in step S71 were added respectively for further culture. The cells were processed after 72 hours.

[0077] After digestion, resuspend the cells and adjust the cell number to 1×10 6 After washing three times with PBS, add 500 μL buffer to suspend the cells, then add 10 μL of Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and 10 μL of PI respectively, mix well, incubate at room temperature in the dark for 15 minutes, and perform dual-color fluorescent cell counting on a flow cytometer.

[0078] The results are as follows Figure 3 As shown, AD is the flow cytometric detection results of apoptosis of breast cancer cells MCF7 in different treatment groups; E is a bar graph of the effects of different treatments on apoptosis of breast cancer cells MCF7.

[0079] Depend on Figure 3 It can be seen that compared with the control group, the cell apoptosis rate increased with the addition of 5mM aspirin, and the cell apoptosis rate also increased with the addition of 10μM abemaciclib. However, compared with the groups adding 5mM aspirin and 10μM abemaciclib separately, the cell apoptosis rate of the group adding 5mM aspirin and 10μM abemaciclib at the same time was significantly increased.

[0080] Statistical analysis showed that compared with the control group, the cell apoptosis in the 5mM aspirin group, 10μM abemaciclib group, and 5mM aspirin + 10μM abemaciclib group increased, and the difference was statistically significant ( P <0.05). Compared with the 5mM aspirin group and the 10μM abemaciclib group, the cell apoptosis in the 5mM aspirin + 10μM abemaciclib group was significantly increased, and the difference was statistically significant ( P <0.05). This shows that compared with the case of using abemaciclib alone, the combined use of aspirin and abemaciclib has a better effect on promoting apoptosis of MCF7 cells, indicating that aspirin can enhance or promote or amplify or synergize the effect of abemaciclib on promoting apoptosis of ER and PR positive breast cancer cells MCF7.

[0081] Embodiment 8

[0082] The effects of the drug combination for treating breast cancer prepared in Examples 3 and 4 on apoptosis of triple-negative breast cancer cells MDA-MB-231 were investigated, and apoptosis was detected by flow cytometry. The specific steps were as follows:

[0083] S81. Prepare 4 groups of culture medium:

[0084] ①Control group: DMEM culture medium.

[0085] ② 5 mM aspirin group: 10 μL of the aspirin stock solution prepared in Example 1 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0086] ③ 10 μM Abemaciclib group: 20 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0087] ④5 mM aspirin+10 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 20 μL of the abemaciclib stock solution prepared in Example 2 were taken, added into 1970 μL of DMEM culture medium, and mixed well.

[0088] S82. Normally grown MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO. 2 The cells were subcultured in an incubator and then inoculated into a culture dish. When the cell density reached about 80%, the four culture solutions prepared in step S81 were added respectively and continued to be cultured. The cells were processed after 72 hours.

[0089] After digestion, resuspend the cells and adjust the cell number to 1×10 6 After washing three times with PBS, add 500 μL buffer to suspend the cells, then add 10 μL of Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and 10 μL of PI respectively, mix well, incubate at room temperature in the dark for 15 minutes, and perform dual-color fluorescent cell counting on a flow cytometer.

[0090] The results are as follows Figure 4 As shown, AD is the flow cytometric detection results of apoptosis of triple-negative breast cancer cells MDA-MB-231 by different treatment groups; E is a bar graph of the effects of different treatment groups on apoptosis of triple-negative breast cancer cells MDA-MB-231.

[0091] Depend on Figure 4 It can be seen that compared with the control group, the cell apoptosis rate in the 5mM aspirin group was increased, and the cell apoptosis rate in the 10μM abemaciclib group was also increased. However, compared with the 5mM aspirin group and the 10μM abemaciclib group, the cell apoptosis rate in the 5mM aspirin + 10μM abemaciclib group was significantly increased.

[0092] Statistical analysis showed that compared with the control group, the cell apoptosis in the 5mM aspirin group, 10μM abemaciclib group, and 5mM aspirin + 10μM abemaciclib group increased, and the difference was statistically significant ( P <0.05). Compared with the 5mM aspirin group and the 10μM abemaciclib group, the cell apoptosis in the 5mM aspirin + 10μM abemaciclib group was significantly increased, and the difference was statistically significant ( P <0.05). This shows that compared with the case of using abemaciclib alone, the combined use of aspirin and abemaciclib has a better effect on promoting apoptosis of MDA-MB-231 cells, indicating that aspirin can enhance or promote or amplify or synergize the effect of abemaciclib on promoting apoptosis of triple-negative breast cancer cells MDA-MB-231.

[0093] Embodiment 9

[0094] The MTT method was used to investigate the effects of the drug combinations for treating breast cancer prepared in Examples 3 and 4 on the cytotoxicity of human normal breast epithelial cells MCF10A. The specific steps are as follows:

[0095] S91, prepare 6 groups of culture medium:

[0096] ①Control group: DMEM culture medium.

[0097] ② 5 mM aspirin group: 10 μL of the aspirin stock solution prepared in Example 1 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0098] ③ 5 μM Abemaciclib group: 10 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1990 μL of DMEM culture medium, and mixed well.

[0099] ④ 10 μM Abemaciclib group: 20 μL of the Abemaciclib stock solution prepared in Example 2 was taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0100] ⑤ 5 mM aspirin + 5 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 10 μL of the abemaciclib stock solution prepared in Example 2 were taken, added to 1980 μL of DMEM culture medium, and mixed well.

[0101] ⑥ 5 mM aspirin + 10 μM abemaciclib group: 10 μL of the aspirin stock solution prepared in Example 1 and 20 μL of the abemaciclib stock solution prepared in Example 2 were added to 1970 μL of DMEM culture medium and mixed.

[0102] S92, MCF10A cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO 2 After subculturing in the incubator, the cells were inoculated into 96-well cell culture plates with a volume of 200 μL per well and a cell number of 2×10 3 After culturing overnight, the culture medium was replaced with the 6 culture mediums prepared in step S91, with a volume of 200 μL per well, and the cell proliferation was detected after 2 days.

[0103] MTT was prepared to a concentration of 5 mg / mL, 20 L was added to each well and culture was continued for 4 h. Then the culture medium was discarded and 150 μL of DMSO was added to each well. The cells were oscillated on a microplate reader for about 5 min. The absorbance (OD) value was detected at 490 nm. A bar graph was drawn to represent the cell growth. Each group had 3 replicate wells and the results were repeated 3 times. The results were expressed as (mean ± standard deviation).

[0104] The results are as follows Figure 5 As shown, compared with the 5mM aspirin group cells and the 5μM abemacili group cells, the simultaneous application of 5mM aspirin + 5μM abemacili did not increase the toxicity to normal mammary epithelial cells. Compared with the 10μM abemacili group cells, the simultaneous application of 5mM aspirin + 10μM abemacili did not increase the toxicity to normal mammary epithelial cells. Moreover, compared with the 10μM abemacili group cells, the simultaneous application of 5mM aspirin + 5μM abemacili reduced the toxicity to normal mammary epithelial cells.

[0105] Statistical analysis showed that there was no statistical difference in cytotoxicity between the 5mM aspirin group and the 5μM abemacili group. There was no statistical difference in cytotoxicity between the 5mM aspirin group and the 5μM abemacili group. Compared with the 10μM abemacili group, there was no statistical difference in cytotoxicity between the 5mM aspirin group and the 10μM abemacili group. Compared with the 10μM abemacili group, the simultaneous application of 5mM aspirin and 5μM abemacili reduced the toxicity to normal breast epithelial cells, and the difference was statistically significant ( P <0.05).

[0106] It can be seen that compared with the case of using 5μM abemaciclib alone, the combined use of 5mM aspirin + 5μM abemaciclib did not enhance the toxicity to normal mammary epithelial cells MCF 10A. Compared with the case of using 10μM abemaciclib alone, the combined use of 5mM aspirin + 5μM abemaciclib was less toxic to normal mammary epithelial cells MCF 10A, and the combined use of 5mM aspirin + 10μM abemaciclib did not enhance the toxicity to normal mammary epithelial cells MCF 10A. The combined use of aspirin and abemaciclib did not increase the toxicity of normal mammary epithelial cells MCF 10A.

[0107] Therefore, the present invention discovers for the first time that, compared with the administration of abemaciclib alone, the combined or synergistic use of aspirin and abemaciclib can significantly inhibit the proliferation of breast cancer MCF7 and MDA-MB-231 cells and promote the apoptosis of breast cancer MCF7 and MDA-MB-231 cells, and the effect of the combined use of aspirin and abemaciclib is significantly better than the effect of abemaciclib or aspirin alone; compared with the administration of abemaciclib alone, the toxicity to normal cells is smaller or not enhanced, and it is expected to be used for the treatment of breast cancer, providing a new drug combination for reducing the cost of breast cancer treatment, improving the treatment effect, and alleviating the suffering of patients.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The use of aspirin and abemaciclib in combination in the preparation of a drug for treating breast cancer, characterized in that: The mass ratio of aspirin to abemaciclib is 1000:1-5; the breast cancer is ER-positive breast cancer or triple-negative breast cancer.

2. The use of aspirin combined with abemaciclib according to claim 1 in the preparation of a drug for treating breast cancer, characterized in that: The mass ratio of aspirin to abemaciclib is 1000:1~2.

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